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+The Project Gutenberg EBook of Preservation of Bull Semen at Sub-Zero
+Temperatures, by N. L. VanDemark and W. J. Miller and W. C. Kinney, Jr. and Carlos Rodriguez and M. E. Friedman
+
+This eBook is for the use of anyone anywhere at no cost and with
+almost no restrictions whatsoever.  You may copy it, give it away or
+re-use it under the terms of the Project Gutenberg License included
+with this eBook or online at www.gutenberg.org
+
+
+Title: Preservation of Bull Semen at Sub-Zero Temperatures
+
+Author: N. L. VanDemark
+        W. J. Miller
+        W. C. Kinney, Jr.
+        Carlos Rodriguez
+        M. E. Friedman
+
+Release Date: August 11, 2011 [EBook #37041]
+
+Language: English
+
+Character set encoding: ISO-8859-1
+
+*** START OF THIS PROJECT GUTENBERG EBOOK PRESERVATION OF BULL SEMEN ***
+
+
+
+
+Produced by Bryan Ness, Harry Lamé and the Online
+Distributed Proofreading Team at http://www.pgdp.net (This
+file was produced from images generously made available
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+
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+
+
+
+
+
+
+  +------------------------------------------------------------------+
+  |                       TRANSCRIBER'S NOTES:                       |
+  |                                                                  |
+  | * Transcription used in this e-text:                             |
+  |   _text_ indicates italics;                                      |
+  |   =text= indicates bold-face text;                               |
+  |   ^{text} indicates superscript text;                            |
+  |   _{text} indicates subscript text.                              |
+  | * Lettered footnotes (with anchors [A], [B], etc.), explaining   |
+  |   the text, have been moved to directly below the paragraph or   |
+  |   table they refer to. Footnotes with anchors [1], [2], etc.     |
+  |   refer to references, that are listed towards the end of the    |
+  |   text.                                                          |
+  | * The original text has not been modified, except that some minor|
+  |   typographical errors have been corrected silently.             |
+  |                                                                  |
+  +------------------------------------------------------------------+
+
+
+
+
+                              PRESERVATION
+                              OF BULL SEMEN
+                        AT SUB-ZERO TEMPERATURES
+
+
+     By N. L. VanDemark
+        W. J. Miller
+        W. C. Kinney, Jr.
+        Carlos Rodriguez
+        M. E. Friedman
+
+
+     Bulletin 621
+
+
+  UNIVERSITY OF ILLINOIS
+  AGRICULTURAL EXPERIMENT STATION
+
+
+
+
+CONTENTS
+
+
+                                                                Page
+
+  EARLY WORK ON FREEZING SEMEN                                     5
+
+  SELECTION OF SEMEN FOR FREEZING                                  6
+  Predicting freezability                                          6
+  Freezability of first and second ejaculates                      7
+  Freezability of several consecutive ejaculates                   7
+  Freezability of epididymal sperm                                 9
+  Freezability of washed sperm                                    10
+
+  EXTENDERS FOR FREEZING BULL SEMEN                               10
+  Proportion of egg yolk in final diluent                         10
+  Citrate level in final diluent                                  11
+  Storing and freezing diluent                                    12
+  Other diluents                                                  13
+
+  DILUTION RATES                                                  14
+  Effect of further dilution and refreezing                       14
+
+  GLYCEROL ADDITIONS                                              17
+  Effect of glycerol on sperm survival at 5° C.                   17
+  Glycerol levels for freezing semen                              18
+  Rate, temperature, and method of adding glycerol                18
+  Allowing sperm to equilibrate with glycerol                     20
+  Sugar additions and equilibration time                          20
+  Substitutes for glycerol                                        22
+
+  FREEZING RATE                                                   22
+  Effect of freezing rate on sperm survival                       22
+  Rate of cooling in plastic and in glass                         23
+
+  STORAGE TEMPERATURE                                             24
+  Storage at -23° to -79° C.                                      24
+  Use of higher glycerol levels and storage at -20° C.            25
+
+  THAWING                                                         26
+  Comparison of thawing at 5° C. and at 38° C.                    27
+  Thawing rate in plastic and in glass                            29
+
+  EFFECTS OF FREEZING PROCEDURES ON METABOLIC ACTIVITY OF SPERM   30
+  Effect of glycerol additions on oxygen uptake of diluted semen
+    at 37° C.                                                     30
+  Effect of glycerol-plus-catalase on oxygen uptake of diluted
+    semen                                                         32
+  Effect of freezing procedures on oxygen utilization             33
+  Effect of freezing procedures on methylene-blue reduction time  33
+
+  PRACTICAL FREEZING PROCEDURE                                    35
+  Collection of semen                                             35
+  Preparation of extender                                         35
+  Dilution after collection                                       36
+  Adding the glycerol                                             36
+  Equilibration                                                   36
+  Freezing                                                        37
+  Thawing                                                         37
+
+  LITERATURE CITED                                                38
+
+  TEMPERATURE CONVERSION TABLE                                    39
+
+
+
+
+  Urbana, Illinois         October, 1957
+
+Publications in the Bulletin series report the results of investigations
+made or sponsored by the Experiment Station
+
+
+
+
+ACKNOWLEDGMENT
+
+
+The authors gratefully acknowledge the financial assistance given to the
+Department of Dairy Science in support of these investigations by the
+Southern Illinois Breeding Association of Breese, Illinois, and the
+Northern Illinois Breeding Co-op of Hampshire, Illinois, through the
+Illinois Dairy Breeding Federation.
+
+
+
+
+THE PRESERVATION OF BULL SEMEN AT SUB-ZERO TEMPERATURES
+
+By N. L. VANDEMARK, W. J. MILLER, W. C. KINNEY, JR., CARLOS RODRIGUEZ,
+and M. E. FRIEDMAN[A]
+
+  [A] This publication was prepared by N. L. VANDEMARK, Professor of
+  Dairy Physiology. Research reported herein was carried out
+  cooperatively by the senior author and W. J. MILLER, W. C. KINNEY,
+  JR., CARLOS RODRIGUEZ, and M. E. FRIEDMAN, formerly members of the
+  Department of Dairy Science.
+
+
+The first announcement in 1951 of the successful impregnation of a cow
+with bull semen that had been frozen stimulated much interest and
+research in freezing as a method of preserving bull semen. Research
+during the years following 1951 resulted in considerable progress and
+success in using freezing as a means of holding semen for long periods
+of time without loss of fertility.
+
+Between 1952 and 1957, research on many aspects of the preservation of
+bull semen at sub-zero temperatures was carried out in the Department of
+Dairy Science at the University of Illinois. Many of these
+investigations have aided in perfecting the freezing technique that has
+been adapted for practical use. Some of these findings have been
+published, but many items have gone unreported except for general
+references at scientific meetings.
+
+It is the purpose of this bulletin to bring together the results of
+several experiments carried out in connection with the freezing of bull
+semen and to present a practical freezing procedure based on the results
+of these experiments and findings at other institutions. Persons
+interested in the development of the procedures and the reasons why
+certain steps are necessary in freezing semen will find the details in
+the first part of this bulletin. Those interested only in the freezing
+procedure may turn to page 35 where a practical method of freezing semen
+is described.
+
+
+
+
+EARLY WORK ON FREEZING SEMEN
+
+
+While it has been known for a long time that various types of tissues
+and organisms can withstand freezing and are even preserved by freezing,
+the first attempts at freezing sperm cells were made before the turn of
+the century. In 1897, Davenport[1][B] found that human sperm would
+withstand freezing. For thirty to forty years after that, little
+attention was paid to freezing as a possible means of preserving semen.
+An excellent review of the early attempts to freeze sperm has been
+assembled by Polge and Parkes.[2] These investigators also gave a good
+account of their work at the British National Institute of Medical
+Research in London, where in 1949 they demonstrated that glycerol would
+protect fowl sperm so that it would survive freezing. The next year they
+found that bull sperm and the sperm of several other species were
+protected by glycerol during freezing. During the same year, Emmens and
+Blackshaw[3] showed that ram and bull sperm would survive freezing. In
+1951 frozen semen was used to produce a calf in England and a lamb in
+Australia.
+
+  [B] These numbers refer to literature citations on page 38.
+
+The highlights in the development of frozen semen have been covered by
+other reviews and reports. Interested persons will find the articles of
+Polge and Parkes[2] and Smith[4] especially good on the early history
+and theoretical aspects of freezing sperm. Later progress on the
+freezing procedure has been reviewed and covered in a number of detailed
+reports.[5],[6],[7] Many items not covered in those articles have been
+assembled here.
+
+
+
+
+SELECTION OF SEMEN FOR FREEZING
+
+
+One of the first considerations in freezing semen is that of deciding
+which semen samples are to be frozen. Since preservation of the
+semen--the maintenance of the potential motility and especially the
+fertility of the sperm--is the primary aim, some attention should be
+directed to the kind of semen sample that will withstand freezing. Do
+the initial characteristics of the sample indicate whether the sperm
+will withstand freezing? Does maturity of the sperm affect their
+freezability?
+
+=Predicting freezability.= Estimates of semen quality in the past have
+been based in part on the numbers of sperm present in a fresh sample and
+on the percentage and rate of motility shown by the sperm. These
+characteristics were used to determine the relationship between the
+original concentration of sperm (in the fresh, undiluted sample), the
+percentage and rate of sperm motility in the diluted samples just prior
+to freezing, and the percentage and rate of sperm motility following
+freezing and thawing. From data collected before and after freezing and
+thawing 54 ejaculates, it was found that there was not a significant
+correlation between the number of sperm present in the original sample
+and the percent of motile sperm present after freezing and thawing (r =
+0.03). A highly significant correlation (r = 0.45) was found, however,
+between the percentages before freezing and after thawing. While this
+correlation coefficient was highly significant, its magnitude indicates
+that only about one fifth of the variation in percentage of motile sperm
+observed after freezing was accounted for by the motility of the sperm
+prior to freezing.
+
+=Freezability of first and second ejaculates.= In the early days of
+artificial breeding in this country, it was commonly believed that a
+second ejaculate collected a few minutes after the first resulted in a
+larger ejaculate containing more sperm. With the development of the
+procedure of stimulating sexual excitement by restraint prior to
+collecting semen, this difference between first and second ejaculates
+has been greatly reduced. Still it was noted that second ejaculates
+frequently withstood freezing better than first ejaculates, even though
+restraint and stimulation of the bull occurred prior to collection of
+the first ejaculate. During the course of a number of experiments, it
+was possible to compare the freezability of 2 ejaculates that were
+collected a few minutes apart from the same bull. Two consecutive
+ejaculates were obtained one or more times from 24 bulls so that a total
+of 58 comparisons could be made. The mean prefreezing and post-thawing
+percentages of motile sperm in first and second ejaculates are presented
+in Table 1.
+
+An analysis of variance showed that in this comparison the differences
+between first and second ejaculates in sperm survival during freezing
+were highly significant. A later comparison of 27 first and second
+ejaculates from 26 bulls did not show as great a difference between
+first and second ejaculates in their ability to withstand freezing
+(Table 1).
+
+Table 1.--Comparison of the Freezability of First and Second Ejaculates
+Taken a Few Minutes Apart From the Same Bull
+
+  =====================================================================
+  Number     Number               Prefreezing  Post-thawing
+    of         of      Ejaculate   motility      motility    Survival
+  bulls    ejaculates              (percent)     (percent)   (percent)
+  ---------------------------------------------------------------------
+  24           58         1st         60            39          65
+                          2d          62            45          74
+  26           27         1st         60            36          60
+                          2d          65            40          61
+  ---------------------------------------------------------------------
+
+=Freezability of several consecutive ejaculates.= The fact that second
+ejaculates sometimes withstood freezing better than first ejaculates
+suggested that the maturity of the sperm might be a factor affecting
+freezability. An opportunity to check this idea came when 20
+consecutive ejaculates were collected from each of 6 bulls within a
+4-hour period. The sperm in samples collected in this manner might be
+expected to be less mature with each additional collection.
+
+The results obtained in freezing several consecutive ejaculates are
+shown in Figure 1 as averages for the ejaculates from 6 bulls. In same
+instances, there was an insufficient quantity of semen available to test
+the freezability. (Procedure: Diluted to 30 × 10^{6} sperm per ml. with
+1:1 yolk-citrate, then cooled and glycerolated with an equal volume of
+14 percent glycerol (percent by volume) in 2.9 percent sodium citrate.
+Final sperm concentration, 15 × 10^{6}. Equilibration time, 15 hours.
+Freezing rate, 2° C. per minute from +5° to -19° C. then 4° C. per
+minute from -19° to -79° C. Held frozen for 5 or more hours then thawed
+in water at 5° C. and checked for motility.)
+
+[Illustration: Percent of motile sperm before and after freezing
+consecutive ejaculates collected within a 4-hour period from each of 6
+bulls (Fig. 1)]
+
+In general, the motility before freezing improved slightly from the
+first to the fourth to sixth ejaculate and then declined until about the
+12th or 14th ejaculate, at which point the prefreezing motility seemed
+to level off through the 20th ejaculate (Fig. 1). The percentage of
+motile sperm found after freezing and thawing followed the same trend at
+an average level 10 to 15 percent lower than the prefreezing level. As
+is readily seen from the trend lines in Fig. 1, the difference between
+the prefreezing motility and the post-thawing motility increased
+gradually after about the fifth ejaculate. Although the absolute
+difference did not increase greatly, the percentage of survival after
+freezing dropped from 81 percent on the first 5 ejaculates to 26.5
+percent on the last 5 (Table 2).
+
+Table 2.--Comparison of the Freezability of 20 Consecutive Ejaculates
+Collected Within a 4-Hour Period
+
+(Weighted averages for 6 bulls)
+
+  ===================================================================
+                   Number     Prefreezing   Post-thawing
+  Ejaculate          of        motility       motility     Survival
+                 ejaculates    (percent)     (percent)     (percent)
+  -------------------------------------------------------------------
+  1st to 5th         29           53.3          43.2          81.0
+  6th to 10th        26           43.8          30.2          69.0
+  11th to 15th       23           28.6          14.5          50.7
+  16th to 20th       19           18.1           4.8          26.5
+  -------------------------------------------------------------------
+
+=Freezability of epididymal sperm.= Since the freezability of bull semen
+seemed to be better in second than in first ejaculates and some
+improvement in freezability was evident through the first 4 to 6
+ejaculates taken consecutively, the question of whether epididymal sperm
+would withstand freezing seemed to be important. Although when 20
+collections were made, the later ejaculates no doubt contained fewer
+mature sperm, the lowered freezability could have been due to accessory
+gland secretions rather than changes in the sperm themselves. Removing
+sperm directly from the epididymis would eliminate any effect that the
+accessory gland secretions could be exerting. Further, if epididymal
+sperm could be frozen, obtaining and using semen from a bull shortly
+after his death should be possible.
+
+Table 3.--Freezability of Sperm in Samples Taken From the Epididymides
+
+  =================================================
+            Prefreezing   Post-thawing
+  Bull       motility      motility      Survival
+            (percent)     (percent)      (percent)
+  -------------------------------------------------
+  1            50            40             80
+  2            40            13             32
+  3            60            15             25
+  4            30            15             50
+  5+6          40            25             62
+  Average      44            22             50
+  -------------------------------------------------
+
+To determine whether epididymal sperm would withstand freezing, the 12
+epididymides (cauda only) of 6 slaughtered bulls were flushed with
+saline (0.9 percent) and the sperm obtained were frozen using the same
+procedure as was used with the 20 consecutive ejaculates discussed
+earlier. Averages of the 2 epididymides from each bull are given in
+Table 3; the samples from bulls 5 and 6 were combined. From the data in
+Table 3, it is obvious that motile sperm were present after freezing and
+thawing epididymal samples. It is likely that further experience in
+handling epididymal sperm may lead to improved results. Using frozen
+epididymal sperm from 2 bulls, Canadian workers have produced confirmed
+pregnancies in 8 out of 12 cows.[8]
+
+=Freezability of washed sperm.= In the laboratory it is frequently
+desirable to study sperm free of the seminal plasma in which they are
+ejaculated. Sperm can be separated from the seminal plasma by
+centrifugation, removal of the supernatant plasma, and resuspension in a
+salt solution of known composition. Sometimes it is desirable to repeat
+the process. This tends to wash the sperm with the salt solution and
+sperm handled in this way are called washed sperm.
+
+Sperm cells centrifuged three times and washed twice in 0.9 percent
+sodium chloride solution withstood freezing well when finally
+resuspended and frozen in yolk-citrate diluent. The percentage of
+survival in three samples subjected to this treatment was 60 percent.
+Thus it appears that the seminal plasma itself is not essential for
+ejaculated sperm to survive the rigors of freezing. This is not
+surprising, since it had already been found that epididymal sperm, which
+also are free of accessory gland secretions, can withstand freezing and
+thawing.
+
+
+
+
+EXTENDERS FOR FREEZING BULL SEMEN
+
+
+Both of the extenders that are widely used in routine storage of bull
+semen at 5° C. are used for freezing semen. These are the egg
+yolk-sodium citrate and whole or skimmilk extenders. Most of the
+research with extenders for freezing bull semen in this laboratory has
+been done with the yolk-citrate diluents.
+
+=Proportion of egg yolk in the final diluent.= Some early experiences
+with a diluent consisting of one part yolk and one part 2.9 percent
+sodium citrate dihydrate in distilled water showed poor sperm survival
+following freezing. The final mixture with this diluent consisted of
+about 45 percent yolk. In other attempts at adding glycerol in order to
+freeze semen, the final proportion of yolk was diminished and better
+sperm survival was obtained. Several experiments were carried out to
+test the effect of varying levels of egg yolk.
+
+In the first efforts to find the optimum level of egg yolk, the level
+of yolk in the final frozen mixture was varied from about 6 to 46
+percent. These levels were obtained by varying the proportion of yolk to
+2.9 percent citrate solution in the original extending media and also in
+the media added in glycerolating the samples.
+
+Split portions of 20 semen samples were frozen in each of the extender
+combinations indicated in Table 4. The mean percentages of motile sperm
+found before and after freezing and thawing are shown also. The highest
+percentages were found with extenders containing 23 and 24 percent yolk.
+The highest percentage of yolk, resulting when a 1:1 (yolk to citrate)
+extender was used for both extending and glycerolating, proved to be
+most detrimental to sperm survival during freezing. The lowest
+percentage of yolk used (6 percent) was not as effective in protecting
+sperm during freezing as the intermediate levels tested (Fig. 2).
+
+Table 4.--Effect of Egg-Yolk Level in Diluent on Freezability of Semen
+(Average of 20 semen samples)
+
+  =========================================================================
+                   Medium               Yolk in    Pre-      Post-     Sur-
+          ----------------------------   final    freezing  thawing   vival
+  Diluent  Extending   Glycerolating[C]   mix-    motility  motility  (per-
+          ------------ ----------------  ture[D] (percent) (percent)  cent)
+          yolk:citrate   yolk:citrate   (percent)
+  -------------------------------------------------------------------------
+  1          1:1             1:1          45.7       65        5        8
+  2          1:1             1:3          34.9       64       27       42
+  3          1:3             1:1          33.6       63       33       52
+  4          1:1             0:1          24.2       64       39       61
+  5          1:3             1:3          22.8       63       37       59
+  6          1:3             0:1          12.1       59       33       56
+  7          1:7             1:7          11.4       56       35       62
+  8          1:7             0:1           6.0       52       26       50
+  9          1:15            1:15          5.7       49       25       51
+  -------------------------------------------------------------------------
+
+  [C] This mixture included 14 percent glycerol.
+
+  [D] The average initial sperm concentration was 900 × 10^{6}/ml.
+  Sufficient extender was added to give 30 × 10^{6}/ml. at the first
+  extension. Thus the final concentration was 15 × 10^{6} sperm/ml.
+  after glycerolization.
+
+Since rather large changes in the percentages of yolk were used in this
+experiment, two further trials were conducted in which 16, 24, and 32
+percent yolk in the final mixture were compared, with the final citrate
+percentages held constant. In these tests, 16 and 24 percent yolk
+maintained sperm better at all citrate levels tried than 32 percent
+yolk. The 16 percent level was slightly better at most of the levels of
+citrate tested (Fig. 3).
+
+=Citrate level in the final diluent.= The early work of the British
+indicated that a final citrate level near 2 percent in the diluent was
+satisfactory for freezing bull sperm. Later, in a personal
+communication, Polge of the British group suggested that a citrate level
+of about 2.35 percent might be best with a final glycerol concentration
+of 7 percent. Some of the first attempts in this laboratory at
+establishing the optimum yolk-to-citrate ratios are shown in Fig. 3. In
+these experiments, the optimum levels of citrate appeared to be lower
+than anticipated from the British work. Thus a more complex experiment
+was set up to test a wider range of citrate levels using 16 and 24
+percent egg yolk in the final freezing mixture. The average percentages
+of motile sperm found after freezing 10 semen samples at each of the
+citrate and yolk levels in this experiment are shown also in Fig. 3.
+Little difference in freezability was found between citrate percentages
+of 1.55 and 1.95. When the rate of sperm motility following freezing and
+thawing was considered along with the percent of motile sperm, a slight
+advantage was found with 16 percent yolk and a citrate concentration of
+1.55 percent.
+
+[Illustration: Percent of motile sperm after freezing and thawing semen
+in diluents containing various levels of egg yolk (Fig. 2)]
+
+From the results of these experiments, and from several reports in the
+literature,[5],[6],[7],[9],[10] it appears that a diluting medium
+resulting in a final concentration of 16 to 25 percent yolk and 1.55 to
+2.2 percent sodium citrate dihydrate is highly satisfactory for
+freezing.
+
+=Storing and freezing diluent.= In some instances it would be
+advantageous to have prepared diluent on hand for use at any time. The
+suitability of stored diluent was tested with a yolk-citrate (equal
+parts yolk and citrate without antibiotics added) diluent prepared and
+stored at 5° C. for 0, 2, 5, 7, and 9 days. Seven semen samples were
+diluted and frozen in these diluents. No difference was noted in the
+survival of sperm that could be attributed to the age of the diluent.
+
+[Illustration: Percent of motile sperm after freezing and thawing semen
+in diluents containing various levels of egg yolk and various
+percentages of sodium citrate (Fig. 3)]
+
+In another trial, a similar diluent (1:1 yolk to citrate with 1000 units
+of penicillin and 5000 units of streptomycin) was prepared and stored in
+the freezer compartment of a refrigerator at -15° C. Upon thawing, it
+was whitish in color and more viscous than freshly prepared diluent.
+Except for the fact that the viscosity seemed to reduce the rate of
+sperm motility, this frozen diluent stored for 65 days compared
+favorably with freshly prepared diluent for freezing semen.
+
+=Other diluents.= Without the protective action of egg yolk or milk, few
+bull sperm will survive freezing. Several diluents were compared on a
+limited scale for freezing bull sperm. The results of these trials are
+compiled in Table 5. In this trial the yolk-citrate extender served best
+in maintaining sperm motility during freezing. Yolk-phosphate and
+homogenized whole milk were slightly less protective and yolk-saline
+seemed to furnish the least protection to sperm during freezing.
+
+A number of investigations in other laboratories have now proven that
+milk can be used as effectively as the yolk-citrate diluent for freezing
+bull sperm.[6],[7]
+
+Table 5.--Comparison of the Freezability of 4 Semen Samples in Different
+Extenders
+
+  ======================================================================
+                Dilution      Pre-       Post-                 Motility
+  Extender        rate      freezing   thawing     Survival    after
+                (semen:     motility   motility   (percent)   storage[E]
+                extender)   (percent)  (percent)              (percent)
+  ----------------------------------------------------------------------
+  Yolk-citrate    1:1          60         49          82          46
+                  1:10         53         45          85          36
+  Yolk-saline     1:1          57         29          51          28
+                  1:10         60         31          52          24
+  Yolk-phosphate  1:1          55         33          64          25
+                  1:10         60         43          72          25
+  Whole milk      1:1          60         40          67          35
+                  1:10         60         35          58          16
+  ----------------------------------------------------------------------
+
+  [E] Stored at 5° C. for 7 hours after thawing.
+
+
+
+
+DILUTION RATES
+
+
+The first trials by the British at freezing bull semen were made with
+samples containing many millions of sperm cells. In routine artificial
+breeding, it is common to add extenders to semen so that one milliliter
+of diluted semen may contain only 10 million living sperm cells. (This
+number still insures optimal fertility.) Frequently the addition of 100
+or more parts of the yolk extender to each part of the original semen
+sample is possible without reducing the sperm numbers below 10 million
+per milliliter. No one knew if this process of dilution would affect the
+resistance of bull sperm to freezing. The effect of various rates of
+dilution on the freezability of bull sperm was tested with 10 semen
+samples. The results, presented in Table 6, show that the numbers of
+sperm between 10 and 90 million per milliliter did not influence the
+percentage of sperm that survived freezing.
+
+In a later trial it was found that sperm survival was slightly better at
+lower dilution rates than in the same samples frozen following dilution
+to 15 million sperm per milliliter. However, field trials with frozen
+semen carried out by others, using sperm numbers as low as 15 million
+per milliliter of semen inseminated or even lower, have been highly
+satisfactory.[11],[12]
+
+During the early studies in the Illinois laboratory, the effects of
+glycerol level were also tested.[13] These effects are discussed in the
+section on glycerol additions beginning on page 17.
+
+=Effect of further dilution and refreezing after the initial freezing.=
+Under some circumstances it might be advantageous to freeze semen with
+a high concentration of sperm cells and then extend it further after
+thawing. With such a procedure less storage space is needed than when
+dilution is carried to the maximum before freezing. Two experiments were
+conducted to test the effects of dilution and storage at 5° C. and
+dilution and refreezing following an initial freezing of concentrated
+samples.
+
+Table 6.--Effect of Sperm Numbers and Glycerol Level in Final Mixture on
+Freezability of Bull Sperm at -79° C. (Average of 10 ejaculates)
+
+  ================================================================
+                         Post-thawing motility (percent)[F]
+                     ---------------------------------------------
+  Glycerol level       Number of sperm (millions/ml.)
+  (percent)          --------------------------------
+                           90       30       10         Average
+  ----------------------------------------------------------------
+  5                       36.0     34.0     36.0         35.0
+  10                      22.0     24.0     23.0         23.0
+  15                       3.2      0.9      0.2          1.4
+  Average                 20.3     19.8     19.9         20.0
+  ----------------------------------------------------------------
+
+  [F] Mean initial motility of sperm before freezing was 55 percent.
+
+Four semen samples were split and extended at rates of 1:1 (semen to
+extender) and 1:10. These were frozen, then thawed and halved. One half
+was further extended to a level of 15 million sperm per milliliter; the
+sperm numbers in the other remained unchanged. Each of these halves was
+split again, and one portion of each was stored at 5° C. for 3 to 7
+hours. The other two portions were refrozen.
+
+Table 7.--Effect of Further Dilution and Refreezing on Sperm Motility
+After the Initial Freezing of Bull Semen
+
+  ========================================================================
+                                Post-thawing motility
+  Dilution   Pre-      ---------------------------------------------------
+  of         freezing   After      After storage[G]    After refreezing[H]
+  semen      motility   first     ------------------- --------------------
+  (semen:   (percent)  freezing     No     Diluted        No     Diluted
+  extender)                      further    to 15      further    to 15
+                                 dilution million/ml   dilution million/ml
+  ------------------------------------------------------------------------
+                          First trial: 4 samples
+  1:1           60        49        46       34           31        6
+  1:10          53        45        36       30           25        5
+
+                          Second trial: 7 samples
+  1:9           67        47        41       35           28       11
+  15 million/ml 67        30        32       ..           18       ..
+  ------------------------------------------------------------------------
+
+  [G] Stored at 5° C. for 3 to 7 hours after first thawing.
+
+  [H] Refrozen following first thawing.
+
+Table 8.--Effect of Glycerol Level and Storage at 5° C. on Motility of
+Sperm in Yolk-Citrate Extender
+
+  ====================================================================
+                                   Sperm motility
+              --------------------------------------------------------
+                Post-        After storage at 5° C.
+  Glycerol     thawing    ----------------------------      Average
+   level                   1 day      3 days     7 days
+  (percent)   ---------   ---------  ---------  ---------  ---------
+              per- rate   per- rate  per- rate  per- rate  per- rate
+              cent        cent       cent       cent       cent
+  --------------------------------------------------------------------
+  Control[I]   56  2.5     55  1.9    46  1.8    38  1.4    48  1.90
+  0            54  2.4     44  1.9    46  1.8    36  1.4    45  1.87
+  5            52  2.2     50  1.9    46  1.7    32  1.4    45  1.80
+  10           52  2.3     46  1.8    42  1.7    28  1.6    42  1.85
+  20           52  2.1     50  1.7    44  1.6    38  1.1    46  1.62
+  30           50  0.7     44  0.5    42  0.4    30  0.4    42  0.51
+  Average      53  2.03    47  1.62   44  1.50   34  1.22   ..  ....
+  --------------------------------------------------------------------
+
+  [I] The control differed from the 0-glycerol treatment in that no
+  additional citrate or glycerol solution was added.
+
+A similar trial was carried out with seven samples; one portion was
+diluted 1:9; the other was extended at the outset to 15 million sperm
+per milliliter. Results for both tests are summarized in Table 7.
+
+From Table 7 it can be seen that refreezing following an initial
+freezing further reduced the number of surviving sperm. The second
+freezing was more detrimental to the portion of the samples extended to
+15 million sperm per milliliter than to the portion that was refrozen at
+a higher sperm concentration. The percentage of motile sperm remained
+fairly high in the portions that were diluted to 15 million sperm and
+stored at 5° C. However, in all cases, survival was best in the samples
+at the lower dilution levels.
+
+
+
+
+GLYCEROL ADDITIONS
+
+
+When the British procedure for freezing bull semen was first tried in
+this country, many of the refinements of the technique still had not
+been defined. It was known that glycerol worked well in protecting sperm
+during freezing. The effects of glycerol on sperm at 5° C., the
+appropriate levels to use in freezing, and the manner of adding it were
+not well established. Therefore, a number of trials were conducted in an
+attempt to establish the best procedures.
+
+=Effect of glycerol on sperm survival at 5° C.= Since early work
+indicated the need for adding glycerol to diluted semen in order to
+protect the sperm during freezing, it was considered important to
+determine the levels of glycerol that sperm would tolerate at 5° C. Ten
+semen samples were extended 1:9 (semen to diluent) in a 1:1 yolk-citrate
+diluent (yolk to 2.9 percent sodium citrate dihydrate). Each sample was
+then split into 6 portions and an equal volume of citrate solution
+containing glycerol was added slowly to each to bring the glycerol in
+the final mixture to 0, 5, 10, 20, or 30 percent (by volume). These
+samples were stored at 5° C. and examined for motile sperm after 1, 3,
+and 7 days. The effects of glycerol levels on the percentage of sperm
+surviving and the rate (or speed) of their forward motion (0 = no
+forward motion; 4 = extremely rapid progressive motility) are presented
+in Table 8.
+
+The percentage of motile sperm decreased slightly at the higher levels
+of glycerol. The most noticeable effect of the increase in glycerol
+level was the reduction in the rate of forward motion of the sperm. At
+the 30-percent level, the sperm moved slowly and could be seen to
+rotate as they moved forward. Some samples were checked after slowly
+bringing the diluent up to a level of 40 percent glycerol; the sperm
+seemed to be immobilized completely in this solution.
+
+=Glycerol levels for freezing semen.= The British procedure called for
+the use of 10 percent glycerol in the final mixture of semen and
+extender prior to freezing. Yet, as shown in Table 6, in our laboratory
+5 percent glycerol resulted in the survival of a higher percentage of
+sperm than did 10 or 15 percent. In order to define more clearly the
+optimum glycerol level, several ejaculates of semen were subsampled and
+portions were frozen after the addition of yolk-citrate extender and
+glycerol in varying quantities. From Table 9 it can be seen that
+glycerol levels of 6 and 8 percent in the final mixture resulted in
+maximum sperm survival during freezing. These results were confirmed in
+tests on the survival of sperm at 5° C. storage for 3 days following
+freezing and thawing with varying glycerol levels (see Table 10).
+
+The results shown in Tables 9 and 10 were confirmed also in later
+experiments. Thirty-six samples were subjected to various levels of
+glycerol and no significant difference in freezability was found between
+6 and 8 percent. Based on these findings, a glycerol level of 7 percent
+was adopted for use in all experiments described in this bulletin,
+unless otherwise indicated. Results in a number of other laboratories
+have agreed with our findings regarding the use of approximately 7
+percent glycerol with the yolk-citrate diluent.[5],[6],[7],[9],[10] With
+milk as the extender, 10 to 13 percent glycerol has been preferred by
+some.[5],[6],[7]
+
+Table 9.--Effect of Glycerol Level on Sperm Motility After Freezing to
+-79° C. and Thawing
+
+  ==========================================================
+  Glycerol    Number        Pre-       Post-      Survival
+   level        of        freezing    thawing     (percent)
+  (percent)   samples     motility    motility
+                         (percent)   (percent)
+  ----------------------------------------------------------
+      2         10          53           2            4
+      4         19          55          29           53
+      6         19          55          34           62
+      8         19          55          35           64
+     10         19          55          24           44
+     12         10          53          13           25
+  ----------------------------------------------------------
+
+Table 10.--Effect of Glycerol Level and Storage at 5° C. After Thawing
+on Sperm Motility
+
+(Average of 13 ejaculates)
+
+  ================================================
+                     Sperm motility (percent)
+  Glycerol      ----------------------------------
+    level        Post-     After storage at 5° C.
+  (percent)     thawing    -----------------------
+                               1 day   3 days
+  ------------------------------------------------
+     4            29             22      20
+     6            38             34      24
+     8            42             33      17
+    10            33             18       6
+  ------------------------------------------------
+
+Table 11.--Effects of Temperature, Rate of Addition of Glycerol, and
+Equilibration Time on Sperm Motility
+
+(Average of 12 ejaculates)
+
+  ===================================================================
+  Temperature                       Post-thawing motility (percent)
+    during       Equilibration     ----------------------------------
+   addition          time                 Glycerol additions
+  of glycerol       (hours)        ----------------------------------
+    (° C.)                           5       3       1       Average
+  -------------------------------------------------------------------
+     4.5               2            48      48      45        47.4
+                       6            49      51      47        48.8
+                      18            46      47      46        46.3
+                      Average       47.8    48.6    46.0      47.5
+
+    10.0               2            44      43      45        43.9
+                       6            48      50      46        47.9
+                      18            43      46      42        44.0
+                      Average       45.0    46.5    44.3      45.3
+
+    15.5               2            41      38      38        39.1
+                       6            42      45      43        43.6
+                      18            42      43      42        42.5
+                      Average       42.0    41.8    41.4      41.7
+  -------------------------------------------------------------------
+
+=Rate, temperature, and method of adding glycerol.= Closely associated
+with the question of how much glycerol should be added is that of how
+the additions should be made. Originally it was believed that the
+glycerol should be added in stages so that changes would occur
+gradually. However, there would be a saving in time if the entire amount
+could be added at once. Also, if the glycerol addition could be made
+soon after the dilution with egg yolk-citrate extender at room
+temperature, time would be gained in processing the semen for use. Since
+aging _in vitro_ is known to reduce the fertilizing ability of sperm,
+every effort should be made to keep the processing time at a minimum.
+The results of an experiment involving these items, along with that of
+how much time should be allowed after the additions before freezing
+(equilibration time), are presented in Table 11. One can see that sperm
+survived freezing better when the diluted semen was cooled to 4.5° C.
+before the glycerol was added. The survival at 10° and 15.5° C. was
+reduced with each rise in temperature. Thus, it appears that cooling to
+refrigerator temperature (4-5° C.) before adding the glycerol should be
+a part of the routine procedure.
+
+A comparison of the results from adding the glycerol in 5, 3, and 1
+equal portions is given also in Table 11. Little difference in survival
+during freezing was noted between the three rates of addition. Using 3
+equal additions resulted in slightly better results, but the advantage
+was not statistically significant. While little difference was evident
+from adding the glycerol in 3 portions as compared to 1, many still use
+3 additions in the hope of obtaining a slightly better sperm survival.
+In fact, some have gone to a procedure of adding the glycerol dropwise
+with constant gentle agitation. This method has not been tested in this
+laboratory.
+
+=Allowing sperm to equilibrate with the glycerol.= Allowing sperm to
+stand in the presence of glycerol is considered by some to be necessary
+in order that the glycerol penetrate the sperm heads before freezing.
+From the first successful attempts at freezing bull sperm came the
+practice of allowing 12 to 20 hours for this process of equilibration. A
+long equilibration time results in aging the sperm. Data from a number
+of sources indicate that a drop of approximately 5 percent in fertility
+in the field occurs with each 24 hours of aging in the test tube. Thus
+it would seem desirable to reduce the equilibration time to a minimum
+commensurate with good freezability in order to reduce the effects of
+aging (at 5° C.). Results of attempting to reduce equilibration time are
+given in Table 11. At 4.5° C., little variation in motility following
+freezing and thawing was found after equilibration times of 2, 6, and 18
+hours. At the higher temperatures of 10° and 15.5° C., the shortest
+equilibration time--2 hours--was slightly more detrimental with the
+differences significant at the 5-percent level at 15.5° C. For all
+temperatures combined, 6 hours was significantly better than 2 or 18
+hours.
+
+=Sugar additions and equilibration time.= Early in their experiences in
+freezing semen, the Australian workers found a short equilibration
+time--30 minutes--to be satisfactory if sugars were added to the
+diluent.[5] This protective action of sugars during the equilibration
+period was confirmed in our investigations. The results of one phase of
+this study are shown in Table 12. From these data it can be seen that
+the presence of glucose or rhamnose at a level of 1.25 percent improved
+sperm survival during the period of equilibration. In another trial
+these sugars and two others, arabinose and xylose, were tested for their
+protective action in freezing semen. The percentages of surviving sperm
+remaining after the various steps in the freezing procedure with and
+without the presence of these sugars are shown in Table 13.
+
+Table 12.--Effect of Adding Sugars to Yolk-Citrate Diluent on Sperm
+Motility During Equilibration With Glycerol[J]
+
+  ============================================================
+                               Sperm motility (percent)
+                         -------------------------------------
+  Stage when observed    Glycerol     Glycerol      Glycerol
+                           only     and glucose   and rhamnose
+  ------------------------------------------------------------
+  Fresh diluted semen       56           56            56
+  After glycerolization     54           54            54
+  After equilibration
+     2 hours                51           53            53
+     6 hours                48           52            53
+    12 hours                46           50            51
+    18 hours                40           46            46
+  ------------------------------------------------------------
+
+  [J] Glycerol level in the final frozen mixture was 7 percent. Sugars
+  were added to a level of 1.25 percent.
+
+Three of the sugars--glucose, arabinose, and rhamnose--protected the
+sperm during equilibration and freezing. Xylose was less effective, but
+its addition resulted in slightly better sperm survival than glycerol
+alone. It was found also that the methylene-blue reduction time
+(metabolic test for semen quality) was faster in samples to which the
+sugars had been added--after glycerolization, after equilibration, and
+after freezing the samples. This is confirming evidence for the presence
+of more living and actively metabolizing sperm in the portions to which
+sugars had been added.
+
+Table 13.--Effect of Adding Sugars to Yolk-Citrate Diluent on Sperm
+Motility During the Freezing Procedures[K]
+
+(Average of 10 ejaculates)
+
+  ======================================================================
+                                   Sperm motility (percent)
+                       -------------------------------------------------
+  Stage of              Glycerol  Glycerol  Glycerol  Glycerol  Glycerol
+  observation            only       and        and      and       and
+                                  glucose   arabinose  xylose   rhamnose
+  ----------------------------------------------------------------------
+  Fresh diluted semen      63        63        63        63        63
+  After glycerolization    54        55        54        57        60
+  After 18 hours
+    equilibration          39        43        44        39        46
+  After freezing to
+    -79° C. and
+    immediate thawing      28        34        34        29        24
+  After 4 days at -79° C.  23        26        26        25        27
+  -----------------------------------------------------------------------
+
+  [K] Glycerol level in the final frozen mixture was 7 percent. Sugars
+  were added to a level of 1.25 percent.
+
+=Substitutes for glycerol.= Since glycerol was so effective in
+protecting sperm during freezing, many have assumed that related
+compounds might be even better. Several compounds, some related to
+glycerol and some not, have been tried as substitutes for glycerol in
+the freezing procedure. They include ethylene glycol, propylene glycol,
+trimethylene glycol, mannitol, sorbitol, dextrans, and seminal-plasma
+proteins. None of these materials has been as effective as glycerol in
+protecting sperm during freezing. In fact, several of the materials
+proved to be injurious to sperm prior to attempts to freeze the samples.
+While the work in our laboratory with these substances as glycerol
+substitutes was by no means finally conclusive, because of the many
+possible interactions of experimental conditions, sufficient data were
+gathered to lead us to abandon further study until greater promise of
+success might be evident.
+
+
+
+
+FREEZING RATE
+
+
+=Effect of freezing rate on sperm survival.= Reports by one group of
+British workers in early trials on freezing bull semen indicated that
+the rate of cooling in freezing should not exceed 2° C. per minute
+between +5° and -15° C., although below -15° C. the rate could be
+faster. Another group expressed the view that semen could be plunged
+into dry ice at -79° C. after it had been cooled to -15° C. To clarify
+this part of the freezing procedure, 11 samples of semen were subdivided
+and portions of each were frozen at rates of 0.25°, 0.5°, 1.0°, 2.0°,
+and 4.0° C. drop per minute between +5° and -20° C. and then twice these
+rates between -20° and -79° C. Vials of each ejaculate at +5° C. were
+also plunged directly into an alcohol bath at -79° C. The samples which
+were cooled at the rates of 0.25°, 0.5°, 1.0°, 2.0°, and 4.0° C. per
+minute had the following percentages of motile sperm after thawing: 30,
+40, 46, 44, and 44. A mean of 32 percent of the sperm in the samples
+that were plunged directly into an alcohol bath at -79° C. were motile
+after thawing. There were no statistically significant differences among
+the samples frozen at 1.0°, 2.0° or 4.0° C. per minute. All of the
+others had significantly lower survival rates. Thus, it is obvious that
+too slow a cooling rate and plunging the samples directly into a -79° C.
+bath from a temperature of +5° C. cause greater harm to the sperm than
+cooling at a rate between 1.0° and 4.0° C. per minute.
+
+Some investigators have suggested that rapid cooling below -20° C. is
+not detrimental to frozen semen. This idea was tested in conjunction
+with other experiments. Twenty-five samples cooled slowly (2° C. per
+minute to -28° C., then 4° C. per minute to -79° C.) showed 62 percent
+sperm survival compared with only 45 percent when cooled rapidly below
+-28° C. (2° C. per minute to -28° C. then plunged into bath at -79° C.).
+Thus, rapid cooling was detrimental even after the critical temperature
+range of +5° C. to -20° C. had been passed.
+
+[Illustration: Cooling rates of diluted semen samples in plastic vials
+and in glass ampules (Fig. 4)]
+
+=Rate of cooling in plastic and in glass.= Plastic vials do not conduct
+the cold as rapidly as glass ampules do. The temperature in both glass
+and plastic containers tends to lag behind the change in the bath in
+which they are immersed as is shown in Figure 4.
+
+Temperatures in the immersion bath were recorded in a 2-milliliter glass
+ampule containing 1 milliliter diluted semen and in an 8-milliliter
+plastic vial containing 2.5 milliliters of diluted semen. A second
+plastic vial and glass ampule filled to capacity with diluted semen
+showed a cooling rate almost identical to that shown in Figure 4. It was
+obvious from the comparison that samples in the plastic vials cooled
+slower than those in glass and that the volume of semen (at least the
+small volumes used) in the vials had little effect on the rate of
+cooling. In another experiment, it was shown that the volume of diluted
+semen in the ampule to be frozen (0.2, 1.0 or 5.0 ml.) had little or no
+effect on the survival of the sperm.
+
+
+
+
+STORAGE TEMPERATURE
+
+
+In freezing and storing bull sperm, an alcohol bath containing dry ice
+at a temperature of -79° C. has been used as a cooling agent. In many
+areas, the availability of dry ice is limited and the cost is rather
+high. Mechanical means are available for obtaining temperatures as low
+as, or lower than, -79° C. but for the most part they are expensive. If
+warmer temperatures were suitable for storing frozen semen, the ordinary
+deep-freeze, which operates at -15° to -25° C., might be used.
+
+=Storage at temperatures from -23° to -79° C.= In testing the effects of
+storage temperatures on the survival of frozen bull sperm (in a diluent
+containing 7 percent glycerol), 9 ejaculates were frozen and kept at
+-23°, -37°, -51°, -65°, and -79° C. The desired temperatures were
+maintained by dropping pieces of dry ice into ethyl alcohol baths as
+needed. Samples were thawed after 1 hour, 1 day, 3 days, and 5 days.
+After 1 hour, the samples maintained at the various temperatures
+exhibited approximately equal motility (Fig. 5).
+
+[Illustration: Effect of freezing and storing bull sperm at various
+temperatures on the sperm motility at thawing (average of 9 ejaculates)
+(Fig. 5)]
+
+At the end of 1 day, samples stored at -79° C. exhibited approximately
+the same motility as did similar samples stored for 1 hour. The samples
+stored at -65° C. had declined slightly in motility and those maintained
+at -51° C. had only one-third the motility which they had displayed at 1
+hour. The samples at -23° and -37° C. exhibited practically no motility
+after 1 day in storage. After 5 days, only 3 of the 8 ejaculates stored
+at -51° C. showed motility upon thawing. Apparently detrimental changes
+take place more rapidly when the samples are stored at temperatures
+warmer than -65° C. The nature of these changes has not been determined.
+Reports from other laboratories indicate that storage temperatures much
+lower than -79° C. are just as satisfactory as -79° C.
+
+No tests of the effects of storage at -79° C. for periods longer than 51
+days have been conducted in this laboratory. Portions of 12 ejaculates
+were frozen and stored at -79° C. for various periods. One portion of
+each of these was examined on the second, ninth, 16th and 51st day of
+storage. The percent of motile sperm and rate of motility at each of
+these examinations were as follows:
+
+  Day                       2     9    16    51
+  Percent of motile sperm  49    46    40    38
+  Rate of motility          2.5   2.3   2.2   2.2
+
+The average prefreezing motility percentage for the above samples was
+58, with an average rate of motility of 2.9. It is apparent from these
+results that the loss in motility was greatest due to the initial
+freezing, and after that the drop was most pronounced during the first
+16 days of storage.
+
+The British and the Australians have both reported the successful
+maintenance of fertility in frozen semen stored at -79° C. for over two
+years.[5]
+
+=Use of higher glycerol levels and a -20° C. storage temperature.= In
+1953, a report from Arkansas suggested that warmer storage temperatures
+could be used if a high percentage of glycerol were included in the
+freezing mixture.[7] To test the effectiveness of various glycerol
+levels on protecting sperm stored at deep-freeze temperatures, glycerol
+levels of 3.5, 5.5, 7.5, and 9.5 percent were used with portions of 4
+semen samples. Survival in the portions frozen and stored at -20° C. was
+poor compared with the portions reduced and held at -79° C. In a second
+experiment, 4 samples were subdivided and frozen with a final
+concentration of 7, 11, 15, and 19 percent glycerol in the semen-diluent
+mixture. In this trial, poor results were obtained at -20° C. except
+that glycerol at a level of 19 percent protected the sperm more
+effectively than at lower levels. Maximal survival at -79° C. was
+obtained at the 7-percent glycerol level. A final trial was run, using
+glycerol levels of 7, 11, 15, 19, 23, 27, and 31 percent. The
+percentages of motile sperm present after storage at -79° C. and -20° C.
+are shown in Table 14.
+
+Table 14.--Effect of Glycerol Level and Storage Temperature on
+Freezability of Semen
+
+(Average of 8 ejaculates)
+
+  ================================================================
+  Storage       Glycerol    Sperm motility after storage (percent)
+  temperature   level       --------------------------------------
+  (°C.)         (percent)          18 hours      42 hours
+  ----------------------------------------------------------------
+    -79            7                 61            61
+    -20            7                  2             1
+                  11                  3             1
+                  15                 14            10
+                  19                 30            22
+                  23                 29            19
+                  27                 25            18
+                  31                 21            12
+  ----------------------------------------------------------------
+
+While survival was fair over a short period of time with 19 percent
+glycerol at -20° C., deterioration was rapid during storage. After 18
+hours of storage, the samples at -20° C. (19 percent glycerol) contained
+only one half as many motile sperm as were still present in the samples
+at -79° C. (7 percent glycerol). After 42 hours of storage, the best
+samples at -20° C. contained only one-third the number of motile sperm
+still present in the samples stored at -79° C. These trials leave little
+doubt that under the present system of freezing and storing, storage at
+ordinary deep-freeze temperatures is far inferior to storage at dry-ice
+temperatures.
+
+
+
+
+THAWING
+
+
+The importance of carefully controlled cooling and storage has been
+emphasized in the foregoing sections. The need for controlling thawing
+rates and the temperature of thawing was not clearly defined in the
+early work on freezing bull semen. The British used a thawing
+temperature of 40° C., which was satisfactory. If there is a need to
+hold the semen for a time after thawing, then a lower thawing
+temperature might be more desirable so that cooling again will not be
+necessary.
+
+=Comparison of thawing temperatures of 5° C. and 38° C.= The effects of
+thawing at temperatures of 38° (body temperature) and 5° C.
+(refrigerator temperature) were investigated. The first trial involved
+thawing as rapidly as possible by dropping glass ampules of frozen semen
+into water baths at the two temperatures. The frozen semen samples
+contained glycerol levels of 4, 6, 8, and 10 percent. The mean
+percentages of motile sperm found after thawing thirteen diluted semen
+samples treated in this manner are shown in Figure 6.
+
+[Illustration: Effect of glycerol percentage and thawing temperature on
+sperm motility after freezing and thawing (Fig. 6)]
+
+The 5° C. thawing temperature resulted in a higher percentage of sperm
+survival at all the glycerol levels than 38° C., with the difference in
+favor of 5° C. becoming greater as the glycerol level increased. The
+reason for the interaction between glycerol level and thawing
+temperature is not known. It may be that the presence of the higher
+levels of glycerol at 38° C. brought about harmful metabolic activity.
+The difference in survival of sperm in semen thawed at 5° C. and at 38°
+C. continued during storage at 5° C. (Table 15). It was also evident
+that the interaction between glycerol level and thawing temperature
+continued during storage (Fig. 7).
+
+[Illustration: Effect of thawing temperature on sperm motility during
+storage at 5° C. following freezing and thawing (Fig. 7)]
+
+Table 15.--Effect of Glycerol Level, Thawing Temperature, and Storage at
+5° C. After Thawing on Sperm Motility (Average of 13 ejaculates)
+
+  =====================================================================
+  Thawing      Glycerol            Sperm motility (percent)
+  temperature   level       -------------------------------------------
+    (° C.)     (percent)     Post-     After storage at 5° C.
+                            thawing    ----------------------   Average
+                                           1 day   3 days
+  ---------------------------------------------------------------------
+      38          4           28.5         17.3      5.1          17.0
+                  6           31.5         22.4      9.2          21.1
+                  8           33.1         15.0      4.6          17.6
+                 10           19.5          3.6      0.8           8.0
+                 Average      28.2         14.6      4.9          12.2
+
+       5          4           29.2         21.7     19.8          23.9
+                  6           37.7         33.8     23.5          31.7
+                  8           41.5         33.1     17.3          30.6
+                 10           33.1         18.5      6.0          19.2
+                 Average      35.4         26.8     16.6          20.6
+  ---------------------------------------------------------------------
+
+It is obvious that motility falls off rapidly after the semen is thawed.
+In a field trial in which the initial intent was to test the effect of
+glycerol levels on fertility of frozen semen, the semen was thawed in
+the morning and used during the same day. Survival of the sperm with 4
+percent glycerol was so poor that only a few breedings were made with
+these samples. Even at 7 and 10 percent, the fertility results were much
+lower than with semen that had not been subjected to freezing. At that
+time it was felt that thawing the samples and using them throughout the
+day may have caused the low fertility results. Since then, a
+large-scale experiment by Cornell University investigators, in
+cooperation with the New York Artificial Breeders' Cooperative, has
+shown definitely that thawing should be delayed until a few minutes
+prior to breeding.[11] If the semen is used immediately, a thawing
+temperature of either 5° or 38° C. appears to be suitable. However,
+there is less danger of cold shock due to recooling if 5° C is used.
+
+=Thawing rate in plastic and in glass.= Glass ampules transmit cold or
+heat more readily than plastic ones. The temperature rise is rapid in
+both glass and plastic when samples are taken from the storage box at
+-79° C. and placed in water at 5° C. However, complete thawing occurs
+more rapidly in glass than in plastic ampules. The changes in
+temperature that occurred when glass and plastic ampules were thawed in
+a water bath at 5° C. are shown in Figure 8. The initial temperature
+rise for the first minute or two was about the same, then the rate of
+warming in the plastic slowed and actual melting of the frozen sample
+occurred a little over a minute later in the plastic than it did in the
+glass. Both were thawed in less than four minutes.
+
+[Illustration: Warming rates of diluted semen samples in plastic vials
+and in glass ampules (Fig. 8)]
+
+
+
+
+EFFECTS OF FREEZING PROCEDURES ON METABOLIC ACTIVITY OF BULL SPERM
+
+
+By finding how methods of handling affect the sperm cells, one can
+sometimes improve the procedures to avoid harmful effects. Some attempts
+have been made in this laboratory to determine the effects of the
+freezing procedures on the metabolic activity of bull spermatozoa. These
+investigations have been limited in scope, involving the measurement of
+oxygen-consumption and estimates of sperm motility during and after
+incubation at 37° C. in a Warburg apparatus.
+
+=Effect of glycerol additions on oxygen uptake of diluted semen at 37°
+C.= The effect of adding glycerol to diluted semen on oxygen consumption
+of the sperm was tested in a Warburg apparatus, using semen diluted with
+an extender consisting of one part egg yolk and one part 2.9 percent
+sodium citrate dihydrate. The yolk-citrate extender was added to the
+semen at a rate which brought the sperm concentration in 0.5 milliliter
+to 200 million to 500 million. An exact count was used to calculate the
+oxygen uptake per 10^{8} sperm per hour (ZO_{2}).
+
+[Illustration: Effect of glycerol additions on oxygen consumption of
+sperm at 37° C. (Fig. 9)]
+
+Glycerol in various percentages in 2.9 percent sodium citrate dihydrate
+solution was placed in the sidearm of the Warburg flasks. The diluted
+semen was held in the main compartment. After a 60-minute preliminary
+run, in which the rate of oxygen uptake of the sperm in yolk-citrate
+diluent was determined, the contents of the sidearm were tipped into the
+main compartment. The resulting glycerol percentages after mixing the
+sidearm and main compartment contents were 0, 4, 8, and 12 percent. Ten
+samples of semen were subsampled and the oxygen uptake of each was
+determined at all four levels of glycerol.
+
+Oxygen uptake was increasingly stimulated during the first 20-minute
+interval by each increase in the amount of glycerol added (Fig. 9).
+After the first 20 minutes, the rate of oxygen utilization decreased at
+the two higher levels of glycerol but persisted at 4 percent. The rate
+of oxygen consumption for the first 20-minute period at the 4-percent
+glycerol level was 130 percent that of the control to which only sodium
+citrate had been added. At 8 and 12 percent the values for the period
+were 144 and 192 percent, respectively, of the control rate.
+
+[Illustration: Effect of glycerol and glycerol-plus-catalase additions
+on sperm motility during incubation at 37° C. (Fig. 10)]
+
+With each increase in glycerol level, motility was reduced during the
+incubation period. This is shown in Figure 10 along with the effect on
+motility of adding catalase, which is discussed in the next section.
+
+=Effect of glycerol-plus-catalase on oxygen uptake of diluted semen.=
+Certain bacteria have been shown to break glycerol down, forming
+hydrogen peroxide as follows:
+
+  Glycerol + oxygen --> lactic acid + hydrogen peroxide.
+
+Hydrogen peroxide is known to be detrimental to sperm. The addition of
+glycerol to diluted semen first increased oxygen uptake and then reduced
+it. Since a reduction in sperm survival followed, some harmful action
+must have taken place with the addition of glycerol at 37° C. To test
+whether this action could be due to the release of hydrogen peroxide as
+occurs in certain bacteria, glycerol with catalase--the enzyme which
+breaks down hydrogen peroxide--was added to a portion of 8 diluted semen
+samples and the oxygen uptake was recorded. Comparison of the resulting
+oxygen uptake with glycerol and with glycerol plus catalase is shown in
+Figure 11.
+
+[Illustration: Effect of additions of glycerol-plus-catalase on oxygen
+consumption of sperm at 37° C. (Fig. 11)]
+
+Oxygen consumption was increased by the presence of added catalase at
+all glycerol levels and in the control. Sperm survival during the 3-hour
+period at 37° C. also was improved by the presence of catalase (Fig.
+10). However, the general trend in oxygen consumption produced by the
+addition of glycerol was not changed greatly. The higher levels of
+glycerol still stimulated oxygen uptake during the first 20-minute
+period after the additions and then slowed the rate of oxygen
+utilization. The rate of utilization was generally higher during the
+test period in the presence of catalase than without added catalase. It
+appeared that a part of the harmful effect of glycerol might be due to
+the formation of hydrogen peroxide. Still, the detrimental effects of
+the higher levels of glycerol were not completely removed.
+
+Table 16.--Effect of Freezing Procedures on Oxygen Utilization of Bull
+Sperm in Yolk-Citrate Extender
+
+(Average of 5 ejaculates)
+
+  =====================================================================
+                                             Microliters of oxygen
+  Semen sample tested                        utilized per 10^{8} sperm
+                                             --------------------------
+                                             First hour    Second hour
+  ---------------------------------------------------------------------
+  Fresh diluted semen                            10.3         8.1
+  Fresh diluted semen glycerol tipped in
+    at end of first hour                          9.7[L]     12.9[L]
+  Aged 20 to 24 hours at 5° C.                   11.2         8.3
+  Aged 20 to 24 hours at 5° C. glycerol
+    tipped in at end of first hour               11.8[L]     12.9[L]
+  After 20 hours equilibration with glycerol     11.7[L]      7.8[L]
+  After freezing and thawing                      9.7         6.3
+  --------------------------------------------------------------------
+
+  [L] Average of 3 ejaculates.
+
+=Effect of freezing procedures on oxygen utilization by sperm.= Limited
+data have been obtained on the effects of some of the freezing
+procedures on the oxygen utilization of bull sperm. The results obtained
+in these experiments confirmed the earlier findings that tipping
+glycerol directly into the diluted semen at 37° C. caused an increase in
+oxygen consumption (Table 16). All other steps in the freezing procedure
+had little effect on oxygen consumption by the sperm. Except where
+glycerol was added during the determination, the rate of oxygen
+utilization was lower the second hour than during the first. The oxygen
+uptake of semen that had been frozen and thawed seemed to drop faster
+than that of unfrozen samples.
+
+=Effect of freezing procedures on methylene-blue reduction time.= The
+methylene-blue reduction test has been used as a means of measuring
+semen quality and is dependent on the metabolic activity of the sperm.
+The effects of various freezing procedures on the ability of samples to
+decolorize methylene blue were determined with 10 semen samples. Sperm
+numbers were standardized to 300 × 10^{6} cells per milliliter and the
+time required for these cells to reduce a 1:40,000 solution of methylene
+blue was determined on freshly diluted semen, after the addition of
+glycerol, after equilibration, and after freezing and thawing. Portions
+of each diluted sample were tested at these stages of the procedure with
+glycerol alone added and with glycerol and various sugars added.
+
+A marked increase in the time required for the sperm to reduce methylene
+blue occurred when the glycerol was added (Table 17). This increase was
+greatest in the portions with glycerol alone and with glycerol and
+glucose. The time increase was less pronounced in the presence of the
+three pentose sugars used. Following equilibration, the samples regained
+the ability to reduce methylene blue at a rate only slightly slower than
+when they were fresh. Freezing and storage of semen resulted in slower
+reduction of the methylene blue than was shown after equilibration with
+glycerol. Since freezing usually kills some of the sperm, a slowing of
+the reduction time after freezing would be expected.
+
+Table 17.--Effect of Freezing Procedures on the Methylene-Blue Reduction
+Time of Bull Semen With and Without the Addition of Sugars[M]
+
+(Average of 10 ejaculates)
+
+  ======================================================================
+                            Methylene-blue reduction time (minutes)
+                        ------------------------------------------------
+                        Glycerol  Glycerol  Glycerol   Glycerol Glycerol
+                        only      and       and        and      and
+                                  glucose   arabinose  xylose   rhamnose
+  ----------------------------------------------------------------------
+  Fresh semen             5.2       5.2       5.2        5.2       5.2
+  After glycerolization  26.4      25.2      17.3       14.3      19.4
+  After 18 hours
+    equilibration         7.4       6.5       6.4        5.3       6.2
+  Thawed immediately
+    after freezing       11.5      10.5       9.4        9.0       9.4
+  Thawed  48 hours
+    after freezing       14.3      10.2      11.3       10.1       9.5
+  ----------------------------------------------------------------------
+
+  [M] Glycerol level in the final frozen mixture was 7 percent. Sugars
+  were added to a level of 1.25 percent.
+
+
+
+
+PRACTICAL FREEZING PROCEDURE
+
+
+Good results usually can be obtained in freezing bull semen if care is
+taken in collecting, diluting and processing the semen. Occasionally the
+semen from certain bulls will not withstand freezing well. The reason
+for this is not understood at present. However, carefully following the
+directions and suggestions given below will usually produce satisfactory
+results with semen samples that are of good quality at the start.
+
+Experience in the field has shown that fertility results with frozen
+semen are usually slightly lower during the first few months than with
+liquid semen stored at 5° C. (41° F.). Most units that have worked with
+frozen semen over a period of a few months are able to improve and do
+get fertility results as good as, or better than, obtained in their
+liquid semen program.
+
+=Collection of the semen.= In order to obtain the best possible semen
+for freezing, care and cleanliness should be exercised in making the
+collection. The artificial vagina, and the glassware used should be
+clean and dry. The underline of the bull should also be clean and dry.
+The bull should be restrained near the teaser cow for a minute or two
+prior to collection in order to excite the flow of secretions prior to
+ejaculation. Allowing the bull to mount the teaser once without serving
+the artificial vagina is a good practice to use in properly stimulating
+the bull before collection of the semen.
+
+If the bull has not been used for three or four days, the collection of
+a second ejaculate for freezing may be advisable. The second ejaculate
+seems to withstand freezing better than the first in many instances. A
+clean, dry artificial vagina should be used for each ejaculate
+collected. Repeated collections in the same artificial vagina may result
+in contamination of the semen with bacteria, lubricating jelly and
+minute particles of dirt. The semen sample should be protected from
+contamination and from sudden temperature drops (cold shock).
+
+=Preparation of extender.= A suitable egg yolk-citrate extender for
+freezing bull semen can be prepared by the following procedure. One part
+egg yolk (free of egg white and the membrane surrounding the yolk) is
+mixed with 4 parts 2.4 to 2.9 percent sodium citrate dihydrate solution.
+The citrate is prepared with distilled water and then boiled or
+autoclaved. The citrate solution should be cooled before it is mixed
+with the egg yolk. After the egg and citrate are mixed, 1000 units of
+penicillin and 1000 micrograms of streptomycin are added per milliliter
+of extender. Sulfanilamide should not be added. This extender can be
+prepared 12 to 24 hours before use if it is stored at refrigerator
+temperature. The portion of the extender needed for the original
+dilution of the semen should be warmed to room temperature before it is
+mixed with the semen.
+
+=Dilution after collection.= As soon as possible after collection, the
+semen sample should be diluted with the extender. The extender must be
+at the same temperature as the semen (room temperature) when the two are
+mixed together. At this time the semen can be partially diluted (1 part
+semen to 4 parts of extender) or diluted to a sperm concentration twice
+the final desired concentration (later in adding the glycerol for
+freezing, the semen is diluted further with an equal volume of glycerol
+containing extender). The diluted semen is slowly cooled (1-1/2 to 2-1/2
+hours) to 5° C. (41° F.). Some units using frozen semen now allow the
+semen to stand at 5° C. for 5 to 6 hours before glycerolization to allow
+the antibiotics to be more effective against any vibrio fetus organisms
+that may be present. This step is taken because it has been shown that
+glycerol inhibits the effectiveness of the antibiotics.[6] After
+cooling, semen can be further diluted to twice the desired sperm
+concentration if that were not done at the start. (Caution: Be sure
+semen and diluent are at the same temperature.)
+
+=Adding the glycerol.= The glycerol solution is prepared by adding 14
+volumes of glycerol (reagent grade) to 86 volumes of yolk-citrate
+diluent (same as yolk-citrate used for original dilution). This solution
+may be added dropwise with constant gentle mixing to the already diluted
+semen, or one-third at a time at 10-minute intervals with gentle mixing
+during each addition. Either method should take about 20 to 30 minutes.
+The total volume of glycerol-yolk-citrate solution added should be equal
+to the volume of the original diluted semen. In this way a concentration
+of 7 percent glycerol is obtained in the final mixture that is to be
+frozen. Care must be taken to keep the temperature at 5° C. (41° F.)
+during the time the glycerol is being added. (A cold room is best for
+maintaining a temperature of 5° C., but with care the operation can be
+carried out at room temperature by using pans of ice water and a
+refrigerator.)
+
+=Equilibration.= The results presented in this bulletin suggest that
+little or no time need be allowed after the glycerol is added before
+freezing. However, results obtained by other workers show improved
+fertility with at least 12 hours equilibration. Some units getting good
+fertility results with frozen semen also are allowing the semen to stand
+at 5° C. for 12 to 18 hours before freezing. After the semen has
+equilibrated with the glycerol, 1-milliliter portions of the mixture are
+placed in 1.2- to 2-milliliter vials or ampules which are then sealed.
+Ampuling can be done with an automatic syringe or pipette, provided a
+large gage needle is used. Also, it is important not to force the fluid
+mixture rapidly through the syringe or the sperm may be injured.
+
+=Freezing.= The vials or ampules of diluted semen are placed in a bath
+of isopropyl alcohol which has been cooled to 5° C. (41° F.). This bath
+can be a wide-mouth thermos bottle or an insulated container of almost
+any sort with a large opening at the top. The size needed depends on the
+number of ampules being frozen. Some sort of convenient tray for holding
+the ampules in an orderly fashion and enabling the samples to be
+completely submerged is desirable. A few ampules can be kept together
+easily by placing them in a polyethylene freezer bag that has had many
+small holes cut in it to let the alcohol of the bath contact the
+ampules. The ampules must be completely covered by the alcohol to insure
+uniform cooling.
+
+The alcohol of the bath and the ampules of semen are cooled by adding
+chipped or ground dry ice in sufficient amounts to lower the temperature
+of the bath 2° C. (3.6° F.) per minute from +5° to -20° C. From -20°
+down to -79° C., the rate of cooling can be doubled (4° C. or 7.2° F.).
+Electrical equipment that regulates the cooling rate to the desired
+temperatures is available commercially, but the cost may be too high for
+some small operations. The samples should be held at -79° C. (-110° F.)
+until they are thawed. This can be done by using an alcohol bath and dry
+ice or by special mechanical refrigerating equipment. At no time prior
+to thawing should the samples be exposed to warmer temperatures.
+
+=Thawing.= The ampules of frozen semen can be thawed by removing them
+from the dry ice storage box and dropping them into a water bath at 5°
+C. (41° F.). Thawing temperatures up to body temperature, 38° C. (100°
+F.), can be used but extreme care must then be taken not to pass the
+semen through a cold inseminating tube; for this would subject the sperm
+to cold shock. The semen should be used for breeding within a few
+minutes after thawing.
+
+
+
+
+LITERATURE CITED
+
+
+     [1] DAVENPORT, C. B. Effect of chemical and physical agents upon
+     protoplasm. Macmillan and Co., New York. 1897.
+
+     [2] POLGE, C., and PARKES, A. S. Possibilities of long-term
+     storage of spermatozoa at low temperatures. Anim. Breeding Abs.
+     =20=:1-5. 1952.
+
+     [3] EMMENS, C. W., and BLACKSHAW, A. W. The low temperature
+     storage of ram, bull, and rabbit spermatozoa. Austral. Vet. Jour.
+     =26=:226. 1950.
+
+     [4] SMITH, AUDREY W. Effects of low temperatures on living cells
+     and tissues. In biological applications of freezing and drying.
+     Ed. R. J. C. Harris. Academic Press, Inc., New York, 1954.
+
+     [5] EMMENS, C. W., and BLACKSHAW, A. W. Artificial insemination.
+     Physiol. Rev. =36=:277-306. 1956.
+
+     [6] Proceedings of the National Association of Artificial
+     Breeders, 1953, 1954, and 1955.
+
+     [7] Proceedings of the American Dairy Science Association, 1953,
+     1954, and 1955. Published in the June issue of the Journal of
+     Dairy Science for each year.
+
+     [8] BARKER, C. A. V. Low temperature preservation of bovine
+     epididymal spermatozoa. Canad. Jour. Comp. Med. =18=:390-393.
+     1954.
+
+     [9] SAROFF, JACK, and MIXNER, J. P. The relationship of egg yolk
+     and glycerol content of diluters and glycerol equilibration time
+     to survival of bull spermatozoa after low temperature freezing.
+     Jour. Dairy Sci. =38=:292-297. 1955.
+
+     [10] CRAGLE, R G., MYERS, R. M., WAUGH, R. K., HUNTER, J. S., and
+     ANDERSON, R. L. The effects of various levels of sodium citrate,
+     glycerol, and equilibration time on survival of bovine
+     spermatozoa after storage at -79° C. Jour. Dairy Sci.
+     =38=:508-514. 1955.
+
+     [11] BRATTON, R. W., FOOTE, R. H., and CRUTHERS, JOAN C.
+     Preliminary fertility results with frozen bovine spermatozoa.
+     Jour. Dairy Sci. =38=:40-46. 1955.
+
+     [12] HAFS, H. D., and ELLIOTT, F. I. The effects of methods of
+     adding egg yolk and monosaccharides on the survival of frozen
+     bull spermatozoa. Jour. Dairy Sci. =38=:811-815. 1955.
+
+     [13] MILLER, W. J., and VANDEMARK, N. L. The influence of
+     glycerol level, various temperature aspects, and certain other
+     factors on the survival of bull spermatozoa at sub-zero
+     temperatures. Jour. Dairy Sci. =37=:45-51. 1954.
+
+
+
+
+TEMPERATURE CONVERSIONS
+
+
+  °C.   °F.
+
+  +38  +100
+  +35   +95
+  +30   +86
+  +25   +77
+  +20   +68
+  +15   +59
+  +10   +50
+   +5   +41
+    0   +32
+   -5   +23
+  -10   +14
+  -15    +5
+  -18     0
+  -20    -4
+  -25   -13
+  -30   -22
+  -35   -31
+  -40   -40
+  -45   -49
+  -50   -58
+  -55   -67
+  -60   -76
+  -65   -85
+  -70   -94
+  -75  -103
+  -79  -110
+
+
+
+
+
+End of the Project Gutenberg EBook of Preservation of Bull Semen at Sub-Zero
+Temperatures, by N. L. VanDemark and W. J. Miller and W. C. Kinney, Jr. and Carlos Rodriguez and M. E. Friedman
+
+*** END OF THIS PROJECT GUTENBERG EBOOK PRESERVATION OF BULL SEMEN ***
+
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+    "http://www.w3.org/TR/xhtml1/DTD/xhtml1-strict.dtd">
+
+<html xmlns="http://www.w3.org/1999/xhtml">
+  <head>
+    <meta http-equiv="Content-Type" content="text/html;charset=iso-8859-1" />
+    <title>
+      The Project Gutenberg eBook of Preservation of Bull Semen at Sub-Zero Temperatures, by N. L. VanDemark et al.
+    </title>
+    <style type="text/css">
+
+    .bb               {border-bottom: solid 1px;}
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+
+<pre>
+
+The Project Gutenberg EBook of Preservation of Bull Semen at Sub-Zero
+Temperatures, by N. L. VanDemark and W. J. Miller and W. C. Kinney, Jr. and Carlos Rodriguez and M. E. Friedman
+
+This eBook is for the use of anyone anywhere at no cost and with
+almost no restrictions whatsoever.  You may copy it, give it away or
+re-use it under the terms of the Project Gutenberg License included
+with this eBook or online at www.gutenberg.org
+
+
+Title: Preservation of Bull Semen at Sub-Zero Temperatures
+
+Author: N. L. VanDemark
+        W. J. Miller
+        W. C. Kinney, Jr.
+        Carlos Rodriguez
+        M. E. Friedman
+
+Release Date: August 11, 2011 [EBook #37041]
+
+Language: English
+
+Character set encoding: ISO-8859-1
+
+*** START OF THIS PROJECT GUTENBERG EBOOK PRESERVATION OF BULL SEMEN ***
+
+
+
+
+Produced by Bryan Ness, Harry Lamé and the Online
+Distributed Proofreading Team at http://www.pgdp.net (This
+file was produced from images generously made available
+by The Internet Archive/American Libraries.)
+
+
+
+
+
+
+</pre>
+
+
+<div class="bbox"><a name="Start" id="Start"></a>
+<p class="center">Please refer to the <a href="#TN">Transcriber's Notes</a> at the end of this document.</p>
+</div>
+
+<hr class="c25" />
+
+<p class='pagenum'><a name="Page_1" id="Page_1"></a></p>
+<h1>PRESERVATION<br />OF BULL SEMEN</h1>
+<h2>AT SUB-ZERO TEMPERATURES</h2>
+
+<table class="ind10" summary="Authors">
+<tr>
+<td class="padr1">By</td>
+<td class="left">N. L. VanDemark</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left">W. J. Miller</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left">W. C. Kinney, Jr.</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left">Carlos Rodriguez</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left">M. E. Friedman</td>
+</tr>
+
+</table>
+
+<p class="fsize150">Bulletin 621</p>
+
+<p class="fsize150">UNIVERSITY OF ILLINOIS<br />
+AGRICULTURAL EXPERIMENT STATION</p>
+
+<hr class="c25" />
+<p class='pagenum'><a name="Page_2" id="Page_2"></a></p>
+
+
+<h2>CONTENTS</h2>
+
+<table summary="ToC">
+
+<tr>
+<td colspan="3" class="right">Page</td>
+</tr>
+
+<tr>
+<td colspan="2" class="left"><a href="#Ch1">EARLY WORK ON FREEZING SEMEN</a></td>
+<td class="right"><a href="#Page_5">5</a></td>
+</tr>
+
+<tr>
+<td colspan="3">&nbsp;</td>
+</tr>
+
+<tr>
+<td colspan="2" class="left"><a href="#Ch2">SELECTION OF SEMEN FOR FREEZING</a></td>
+<td class="right"><a href="#Page_6">6</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;&nbsp;&nbsp;&nbsp;</td>
+<td class="left"><a href="#Sec2_1">Predicting freezability</a></td>
+<td class="right"><a href="#Page_6">6</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec2_2">Freezability of first and second ejaculates</a></td>
+<td class="right"><a href="#Page_7">7</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec2_3">Freezability of several consecutive ejaculates</a></td>
+<td class="right"><a href="#Page_7">7</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec2_4">Freezability of epididymal sperm</a></td>
+<td class="right"><a href="#Page_9">9</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec2_5">Freezability of washed sperm</a></td>
+<td class="right"><a href="#Page_10">10</a></td>
+</tr>
+
+<tr>
+<td colspan="3">&nbsp;</td>
+</tr>
+
+<tr>
+<td colspan="2" class="left"><a href="#Ch3">EXTENDERS FOR FREEZING BULL SEMEN</a></td>
+<td class="right"><a href="#Page_10">10</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec3_1">Proportion of egg yolk in final diluent</a></td>
+<td class="right"><a href="#Page_10">10</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec3_2">Citrate level in final diluent</a></td>
+<td class="right"><a href="#Page_11">11</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec3_3">Storing and freezing diluent</a></td>
+<td class="right"><a href="#Page_12">12</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec3_4">Other diluents</a></td>
+<td class="right"><a href="#Page_13">13</a></td>
+</tr>
+
+<tr>
+<td colspan="3">&nbsp;</td>
+</tr>
+
+<tr>
+<td colspan="2" class="left"><a href="#Ch4">DILUTION RATES</a></td>
+<td class="right"><a href="#Page_14">14</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec4_1">Effect of further dilution and refreezing</a></td>
+<td class="right"><a href="#Page_14">14</a></td>
+</tr>
+
+<tr>
+<td colspan="3">&nbsp;</td>
+</tr>
+
+<tr>
+<td colspan="2" class="left"><a href="#Ch5">GLYCEROL ADDITIONS</a></td>
+<td class="right"><a href="#Page_17">17</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec5_1">Effect of glycerol on sperm survival at 5&deg; C.</a></td>
+<td class="right"><a href="#Page_17">17</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec5_2">Glycerol levels for freezing semen</a></td>
+<td class="right"><a href="#Page_18">18</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec5_3">Rate, temperature, and method of adding glycerol</a></td>
+<td class="right"><a href="#Page_18">18</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec5_4">Allowing sperm to equilibrate with glycerol</a></td>
+<td class="right"><a href="#Page_20">20</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec5_5">Sugar additions and equilibration time</a></td>
+<td class="right"><a href="#Page_20">20</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec5_6">Substitutes for glycerol</a></td>
+<td class="right"><a href="#Page_22">22</a></td>
+</tr>
+
+<tr>
+<td colspan="3">&nbsp;</td>
+</tr>
+
+<tr>
+<td colspan="2" class="left"><a href="#Ch6">FREEZING RATE</a></td>
+<td class="right"><a href="#Page_22">22</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec6_1">Effect of freezing rate on sperm survival</a></td>
+<td class="right"><a href="#Page_22">22</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec6_2">Rate of cooling in plastic and in glass</a></td>
+<td class="right"><a href="#Page_23">23</a></td>
+</tr>
+
+<tr>
+<td colspan="3">&nbsp;</td>
+</tr>
+
+<tr>
+<td colspan="2" class="left"><a href="#Ch7">STORAGE TEMPERATURE</a></td>
+<td class="right"><a href="#Page_24">24</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec7_1">Storage at -23&deg; to -79&deg; C.</a></td>
+<td class="right"><a href="#Page_24">24</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec7_2">Use of higher glycerol levels and storage at -20&deg; C.</a></td>
+<td class="right"><a href="#Page_25">25</a></td>
+</tr>
+
+<tr>
+<td colspan="3">&nbsp;</td>
+</tr>
+
+<tr>
+<td colspan="2" class="left"><a href="#Ch8">THAWING</a></td>
+<td class="right"><a href="#Page_26">26</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec8_1">Comparison of thawing at 5&deg; C. and at 38&deg; C.</a></td>
+<td class="right"><a href="#Page_27">27</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec8_2">Thawing rate in plastic and in glass</a></td>
+<td class="right"><a href="#Page_29">29</a></td>
+</tr>
+
+<tr>
+<td colspan="3">&nbsp;</td>
+</tr>
+
+<tr class="top">
+<td colspan="2" class="left padr1"><span class='pagenum'><a name="Page_3" id="Page_3"></a></span><a
+href="#Ch9">EFFECTS OF FREEZING PROCEDURES ON METABOLIC ACTIVITY OF SPERM</a></td>
+<td class="right"><a href="#Page_30">30</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec9_1">Effect of glycerol additions on oxygen uptake of diluted semen at 37&deg; C.</a></td>
+<td class="right"><a href="#Page_30">30</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec9_2">Effect of glycerol-plus-catalase on oxygen uptake of diluted semen</a></td>
+<td class="right"><a href="#Page_32">32</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec9_3">Effect of freezing procedures on oxygen utilization</a></td>
+<td class="right"><a href="#Page_33">33</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec9_4">Effect of freezing procedures on methylene-blue reduction time</a></td>
+<td class="right"><a href="#Page_33">33</a></td>
+</tr>
+
+<tr>
+<td colspan="3">&nbsp;</td>
+</tr>
+
+<tr>
+<td colspan="2" class="left"><a href="#Ch10">PRACTICAL FREEZING PROCEDURE</a></td>
+<td class="right"><a href="#Page_35">35</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec10_1">Collection of semen</a></td>
+<td class="right"><a href="#Page_35">35</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec10_2">Preparation of extender</a></td>
+<td class="right"><a href="#Page_35">35</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec10_3">Dilution after collection</a></td>
+<td class="right"><a href="#Page_36">36</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec10_4">Adding the glycerol</a></td>
+<td class="right"><a href="#Page_36">36</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec10_5">Equilibration</a></td>
+<td class="right"><a href="#Page_36">36</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec10_6">Freezing</a></td>
+<td class="right"><a href="#Page_37">37</a></td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left"><a href="#Sec10_7">Thawing</a></td>
+<td class="right"><a href="#Page_37">37</a></td>
+</tr>
+
+<tr>
+<td colspan="3">&nbsp;</td>
+</tr>
+
+<tr>
+<td colspan="2" class="left"><a href="#Ch11">LITERATURE CITED</a></td>
+<td class="right"><a href="#Page_38">38</a></td>
+</tr>
+
+<tr>
+<td colspan="3">&nbsp;</td>
+</tr>
+
+<tr>
+<td colspan="2" class="left"><a href="#Ch12">TEMPERATURE CONVERSION TABLE</a></td>
+<td class="right"><a href="#Page_39">39</a></td>
+</tr>
+
+</table>
+
+<hr class="c25" />
+
+<table class="fsize80" style="width: 60%;" summary="Publishing info">
+
+<tr>
+<td class="left">Urbana, Illinois</td>
+<td class="right">October, 1957</td>
+</tr>
+
+<tr>
+<td colspan="2" class="center">Publications in the Bulletin series report the results of investigations made
+or sponsored by the Experiment Station</td>
+</tr>
+
+</table>
+
+<hr class="c25" />
+<p class='pagenum'><a name="Page_4" id="Page_4"></a></p>
+
+<h2>ACKNOWLEDGMENT</h2>
+
+<p>The authors gratefully acknowledge the financial
+assistance given to the Department of Dairy
+Science in support of these investigations by the
+Southern Illinois Breeding Association of Breese,
+Illinois, and the Northern Illinois Breeding Co-op
+of Hampshire, Illinois, through the Illinois Dairy
+Breeding Federation.</p>
+
+
+
+<hr class="c25" />
+<p class='pagenum'><a name="Page_5" id="Page_5">[5]</a></p>
+<h1>THE PRESERVATION OF BULL SEMEN<br />
+AT SUB-ZERO TEMPERATURES</h1>
+
+<p class="fsize80">By <span class="smcap">N. L. VanDemark</span>, <span class="smcap">W. J. Miller</span>,
+<span class="smcap">W. C. Kinney, Jr.</span>, <span class="smcap">Carlos Rodriguez</span>, and <span class="smcap">M. E.
+Friedman</span><a name="FNanchorA" id="FNanchorA"></a><a href="#FootnoteA" class="fnanchor">[A]</a></p>
+
+<div class="footnote"><p><a name="FootnoteA" id="FootnoteA"></a><a href="#FNanchorA"><span class="label">[A]</span></a> This
+publication was prepared by <span class="smcap">N. L. VanDemark</span>, Professor of Dairy
+Physiology. Research reported herein was carried out cooperatively by the
+senior author and <span class="smcap">W. J. Miller</span>, <span class="smcap">W. C. Kinney, Jr.</span>, <span class="smcap">Carlos
+Rodriguez</span>, and
+<span class="smcap">M. E. Friedman</span>, formerly members of the Department of Dairy Science.</p></div>
+
+<p>The first announcement in 1951 of the successful impregnation
+of a cow with bull semen that had been frozen stimulated
+much interest and research in freezing as a method of preserving
+bull semen. Research during the years following 1951 resulted in
+considerable progress and success in using freezing as a means of
+holding semen for long periods of time without loss of fertility.</p>
+
+<p>Between 1952 and 1957, research on many aspects of the preservation
+of bull semen at sub-zero temperatures was carried out in the
+Department of Dairy Science at the University of Illinois. Many of
+these investigations have aided in perfecting the freezing technique
+that has been adapted for practical use. Some of these findings have
+been published, but many items have gone unreported except for general
+references at scientific meetings.</p>
+
+<p>It is the purpose of this bulletin to bring together the results of
+several experiments carried out in connection with the freezing of bull
+semen and to present a practical freezing procedure based on the
+results of these experiments and findings at other institutions. Persons
+interested in the development of the procedures and the reasons why
+certain steps are necessary in freezing semen will find the details in
+the first part of this bulletin. Those interested only in the freezing
+procedure may turn to page 35 where a practical method of freezing
+semen is described.</p>
+
+
+<hr class="c25" />
+<h2><a name="Ch1" id="Ch1"></a>EARLY WORK ON FREEZING SEMEN</h2>
+
+<p>While it has been known for a long time that various types of
+tissues and organisms can withstand freezing and are even preserved
+by freezing, the first attempts at freezing sperm cells were made before
+the turn of the century. In 1897, Davenport<a name="FNanchor1" id="FNanchor1"></a><a href="#Footnote1"
+class="fnanchor">[1]</a> <a name="FNanchorB" id="FNanchorB"></a><a href="#FootnoteB" class="fnanchor">[B]</a>
+found that human sperm
+would withstand freezing. For thirty to forty years after that, little<span class='pagenum'><a name="Page_6" id="Page_6">[6]</a></span>
+attention was paid to freezing as a possible means of preserving semen.
+An excellent review of the early attempts to freeze sperm has been
+assembled by Polge and Parkes.<a href="#Footnote2" class="fnanchor">[2]</a>
+These investigators also gave a good account of their work at the British National Institute of Medical
+Research in London, where in 1949 they demonstrated that glycerol
+would protect fowl sperm so that it would survive freezing. The next
+year they found that bull sperm and the sperm of several other species
+were protected by glycerol during freezing. During the same year,
+Emmens and Blackshaw<a href="#Footnote3" class="fnanchor">[3]</a><a name="FNanchor3" id="FNanchor3"></a> showed
+that ram and bull sperm would survive freezing. In 1951 frozen semen was used to produce a calf in
+England and a lamb in Australia.</p>
+
+<div class="footnote"><p><a name="FootnoteB" id="FootnoteB"></a><a href="#FNanchorB"><span
+class="label">[B]</span></a> These numbers refer to literature citations on <a href="#Page_38">page 38</a>.</p></div>
+
+<p>The highlights in the development of frozen semen have been
+covered by other reviews and reports. Interested persons will find the
+articles of Polge and Parkes<a href="#Footnote2"
+class="fnanchor">[2]</a> and Smith<a name="FNanchor4" id="FNanchor4"></a><a href="#Footnote4"
+class="fnanchor">[4]</a> especially good on the early
+history and theoretical aspects of freezing sperm. Later progress on
+the freezing procedure has been reviewed and covered in a number of
+detailed reports.<a href="#Footnote5" class="fnanchor">[5],</a>
+<a href="#Footnote6" class="fnanchor">[6],</a>
+<a href="#Footnote7" class="fnanchor">[7]</a> Many items not covered in those articles have
+been assembled here.</p>
+
+
+<hr class="c25" />
+<h2><a name="Ch2" id="Ch2"></a>SELECTION OF SEMEN FOR FREEZING</h2>
+
+
+<p>One of the first considerations in freezing semen is that of deciding
+which semen samples are to be frozen. Since preservation of the
+semen&mdash;the maintenance of the potential motility and especially the
+fertility of the sperm&mdash;is the primary aim, some attention should be
+directed to the kind of semen sample that will withstand freezing. Do
+the initial characteristics of the sample indicate whether the sperm will
+withstand freezing? Does maturity of the sperm affect their freezability?</p>
+
+<p><a name="Sec2_1" id="Sec2_1"></a><b>Predicting freezability.</b> Estimates of semen quality in the past have
+been based in part on the numbers of sperm present in a fresh sample
+and on the percentage and rate of motility shown by the sperm. These
+characteristics were used to determine the relationship between the
+original concentration of sperm (in the fresh, undiluted sample), the
+percentage and rate of sperm motility in the diluted samples just prior
+to freezing, and the percentage and rate of sperm motility following
+freezing and thawing. From data collected before and after freezing
+and thawing 54 ejaculates, it was found that there was not a significant
+correlation between the number of sperm present in the original sample
+and the percent of motile sperm present after freezing and thawing
+(r = 0.03). A highly significant correlation (r = 0.45) was found,<span class='pagenum'><a name="Page_7" id="Page_7">[7]</a></span>
+however, between the percentages before freezing and after thawing.
+While this correlation coefficient was highly significant, its magnitude
+indicates that only about one fifth of the variation in percentage of
+motile sperm observed after freezing was accounted for by the motility
+of the sperm prior to freezing.</p>
+
+<p><a name="Sec2_2" id="Sec2_2"></a><b>Freezability of first and second ejaculates.</b> In the early days of
+artificial breeding in this country, it was commonly believed that a
+second ejaculate collected a few minutes after the first resulted in a
+larger ejaculate containing more sperm. With the development of the
+procedure of stimulating sexual excitement by restraint prior to collecting
+semen, this difference between first and second ejaculates has
+been greatly reduced. Still it was noted that second ejaculates frequently
+withstood freezing better than first ejaculates, even though
+restraint and stimulation of the bull occurred prior to collection of the
+first ejaculate. During the course of a number of experiments, it was
+possible to compare the freezability of 2 ejaculates that were collected
+a few minutes apart from the same bull. Two consecutive ejaculates
+were obtained one or more times from 24 bulls so that a total of 58
+comparisons could be made. The mean prefreezing and post-thawing
+percentages of motile sperm in first and second ejaculates are presented
+in <a href="#Tab1">Table 1</a>.</p>
+
+<p>An analysis of variance showed that in this comparison the differences
+between first and second ejaculates in sperm survival during
+freezing were highly significant. A later comparison of 27 first and
+second ejaculates from 26 bulls did not show as great a difference between
+first and second ejaculates in their ability to withstand freezing
+(<a href="#Tab1">Table 1</a>).</p>
+
+<h5><a name="Tab1" id="Tab1"></a>Table 1.&mdash;Comparison of the Freezability of First and Second<br />
+Ejaculates Taken a Few Minutes Apart From the Same Bull</h5>
+
+<table class="fsize80" summary="Table P 7">
+
+<tr class="bt2 bb">
+<td class="center padr1 padl1">Number<br />of<br />bulls</td>
+<td class="center padr1 padl1">Number<br />of<br />ejaculates</td>
+<td class="center padr1 padl1">Ejaculate</td>
+<td class="center padr1 padl1">Prefreezing<br />motility<br />(percent)</td>
+<td class="center padr1 padl1">Post-thawing<br />motility<br />(percent)</td>
+<td class="center padr1 padl1">Survival<br />(percent)</td>
+</tr>
+
+<tr>
+<td class="center">24</td>
+<td class="center">58</td>
+<td class="center">1st</td>
+<td class="center">60</td>
+<td class="center">39</td>
+<td class="center">65</td>
+</tr>
+
+<tr>
+<td colspan="2">&nbsp;</td>
+<td class="center">2d</td>
+<td class="center">62</td>
+<td class="center">45</td>
+<td class="center">74</td>
+</tr>
+
+<tr>
+<td class="center">26</td>
+<td class="center">27</td>
+<td class="center">1st</td>
+<td class="center">60</td>
+<td class="center">36</td>
+<td class="center">60</td>
+</tr>
+
+<tr class="bb">
+<td colspan="2">&nbsp;</td>
+<td class="center">2d</td>
+<td class="center">65</td>
+<td class="center">40</td>
+<td class="center">61</td>
+</tr>
+
+</table>
+
+<p><a name="Sec2_3" id="Sec2_3"></a><b>Freezability of several consecutive ejaculates.</b> The fact that second
+ejaculates sometimes withstood freezing better than first ejaculates
+suggested that the maturity of the sperm might be a factor affecting
+freezability. An opportunity to check this idea came when 20 consecutive<span class='pagenum'><a name="Page_8" id="Page_8">[8]</a></span>
+ejaculates were collected from each of 6 bulls within a 4-hour
+period. The sperm in samples collected in this manner might be expected
+to be less mature with each additional collection.</p>
+
+<p>The results obtained in freezing several consecutive ejaculates are
+shown in <a href="#Fig1">Figure 1</a> as averages for the ejaculates from 6 bulls. In
+same instances, there was an insufficient quantity of semen available
+to test the freezability. (Procedure: Diluted to 30 &times; 10<span class="expon">6</span> sperm per
+ml. with 1:1 yolk-citrate, then cooled and glycerolated with an equal
+volume of 14 percent glycerol (percent by volume) in 2.9 percent
+sodium citrate. Final sperm concentration, 15 &times; 10<span class="expon">6</span>. Equilibration
+time, 15 hours. Freezing rate, 2&deg; C. per minute from +5&deg; to -19&deg; C.
+then 4&deg; C. per minute from -19&deg; to -79&deg; C. Held frozen for 5 or
+more hours then thawed in water at 5&deg; C. and checked for motility.)</p>
+
+<div class="figcenter"><a name="Fig1" id="Fig1"></a><img src="images/illo001.png" alt="Fig 1" width="600" height="404" />
+
+<table class="cap600" summary="Caption Fig 1">
+
+<tr>
+<td colspan="2" class="left">Percent of motile sperm before and after freezing consecutive ejaculates collected
+within a 4-hour period</td>
+</tr>
+
+<tr>
+<td class="left">from each of 6 bulls</td>
+<td class="right">(Fig. 1)</td>
+</tr>
+
+</table>
+</div>
+
+<p>In general, the motility before freezing improved slightly from the
+first to the fourth to sixth ejaculate and then declined until about the
+12th or 14th ejaculate, at which point the prefreezing motility seemed
+to level off through the 20th ejaculate (<a href="#Fig1">Fig. 1</a>). The percentage of
+motile sperm found after freezing and thawing followed the same
+trend at an average level 10 to 15 percent lower than the prefreezing
+level. As is readily seen from the trend lines in <a href="#Fig1">Fig. 1</a>, the difference
+between the prefreezing motility and the post-thawing motility increased<span class='pagenum'><a name="Page_9" id="Page_9">[9]</a></span>
+gradually after about the fifth ejaculate. Although the absolute
+difference did not increase greatly, the percentage of survival after
+freezing dropped from 81 percent on the first 5 ejaculates to 26.5 percent
+on the last 5 (<a href="#Tab2">Table 2</a>).</p>
+
+<h5><a name="Tab2" id="Tab2"></a>Table 2.&mdash;Comparison of the Freezability of 20 Consecutive<br />
+Ejaculates Collected Within a 4-Hour Period</h5>
+
+<p class="tabhead">(Weighted averages for 6 bulls)</p>
+
+<table class="fsize80" summary="Table P 9 top">
+
+<tr class="bt2 bb">
+<td class="center padr1 padl1">Ejaculate</td>
+<td class="center padr1 padl1">Number<br />of<br />ejaculates</td>
+<td class="center padr1 padl1">Prefreezing<br />motility<br />(percent)</td>
+<td class="center padr1 padl1">Post-thawing<br />motility<br />(percent)</td>
+<td class="center padl1 padr1">Survival<br />(Percent)</td>
+</tr>
+
+<tr>
+<td class="left padl1 padr1">1st to 5th</td>
+<td class="center">29</td>
+<td class="center">53.3</td>
+<td class="right padr5">43.2</td>
+<td class="center">81.0</td>
+</tr>
+
+<tr>
+<td class="left padr1 padl1">6th to 10th</td>
+<td class="center">26</td>
+<td class="center">43.8</td>
+<td class="right padr5">30.2</td>
+<td class="center">69.0</td>
+</tr>
+
+<tr>
+<td class="left padr1 padl1">11th to 15th</td>
+<td class="center">23</td>
+<td class="center">28.6</td>
+<td class="right padr5">14.5</td>
+<td class="center">50.7</td>
+</tr>
+
+<tr class="bb">
+<td class="left padr1 padl1">16th to 20th</td>
+<td class="center">19</td>
+<td class="center">18.1</td>
+<td class="right padr5">4.8</td>
+<td class="center">26.5</td>
+</tr>
+
+</table>
+
+<p><a name="Sec2_4" id="Sec2_4"></a><b>Freezability of epididymal sperm.</b> Since the freezability of bull
+semen seemed to be better in second than in first ejaculates and some
+improvement in freezability was evident through the first 4 to 6 ejaculates
+taken consecutively, the question of whether epididymal sperm
+would withstand freezing seemed to be important. Although when 20
+collections were made, the later ejaculates no doubt contained fewer
+mature sperm, the lowered freezability could have been due to accessory
+gland secretions rather than changes in the sperm themselves.
+Removing sperm directly from the epididymis would eliminate any
+effect that the accessory gland secretions could be exerting. Further,
+if epididymal sperm could be frozen, obtaining and using semen from
+a bull shortly after his death should be possible.</p>
+
+<h5><a name="Tab3" id="Tab3"></a>Table 3.&mdash;Freezability of Sperm in Samples Taken<br />
+From the Epididymides</h5>
+
+<table class="fsize80" summary="Table P 9 bottom">
+
+<tr class="bt2 bb">
+<td class="left padr1 padl1">Bull</td>
+<td class="center padr1 padl1">Prefreezing<br />motility<br />(percent)</td>
+<td class="center padr1 padl1">Post-thawing<br />motility<br />(percent)</td>
+<td class="center padr1 padl1">Survival<br />(percent)</td>
+</tr>
+
+<tr>
+<td class="left padr1 padl1">1</td>
+<td class="center">50</td>
+<td class="center">40</td>
+<td class="center">80</td>
+</tr>
+
+<tr>
+<td class="left padr1 padl1">2</td>
+<td class="center">40</td>
+<td class="center">13</td>
+<td class="center">32</td>
+</tr>
+
+<tr>
+<td class="left padr1 padl1">3</td>
+<td class="center">60</td>
+<td class="center">15</td>
+<td class="center">25</td>
+</tr>
+
+<tr>
+<td class="left padr1 padl1">4</td>
+<td class="center">30</td>
+<td class="center">15</td>
+<td class="center">50</td>
+</tr>
+
+<tr>
+<td class="left padr1 padl1">5+6</td>
+<td class="center">40</td>
+<td class="center">25</td>
+<td class="center">62</td>
+</tr>
+
+<tr class="bb">
+<td class="left padr1 padl1">Average</td>
+<td class="center">44</td>
+<td class="center">22</td>
+<td class="center">50</td>
+</tr>
+
+</table>
+
+<p>To determine whether epididymal sperm would withstand freezing,
+the 12 epididymides (cauda only) of 6 slaughtered bulls were flushed<span class='pagenum'><a name="Page_10" id="Page_10">[10]</a></span>
+with saline (0.9 percent) and the sperm obtained were frozen using
+the same procedure as was used with the 20 consecutive ejaculates
+discussed earlier. Averages of the 2 epididymides from each bull are
+given in <a href="#Tab3">Table 3</a>; the samples from bulls 5 and 6 were combined. From
+the data in <a href="#Tab3">Table 3</a>, it is obvious that motile sperm were present after
+freezing and thawing epididymal samples. It is likely that further
+experience in handling epididymal sperm may lead to improved results.
+Using frozen epididymal sperm from 2 bulls, Canadian workers have
+produced confirmed pregnancies in 8 out of 12 cows.<a name="FNanchor8" id="FNanchor8"></a><a
+href="#Footnote8" class="fnanchor">[8]</a></p>
+
+<p><a name="Sec2_5" id="Sec2_5"></a><b>Freezability of washed sperm.</b> In the laboratory it is frequently
+desirable to study sperm free of the seminal plasma in which they are
+ejaculated. Sperm can be separated from the seminal plasma by centrifugation,
+removal of the supernatant plasma, and resuspension in a
+salt solution of known composition. Sometimes it is desirable to repeat
+the process. This tends to wash the sperm with the salt solution
+and sperm handled in this way are called washed sperm.</p>
+
+<p>Sperm cells centrifuged three times and washed twice in 0.9 percent
+sodium chloride solution withstood freezing well when finally
+resuspended and frozen in yolk-citrate diluent. The percentage of survival
+in three samples subjected to this treatment was 60 percent. Thus
+it appears that the seminal plasma itself is not essential for ejaculated
+sperm to survive the rigors of freezing. This is not surprising, since it
+had already been found that epididymal sperm, which also are free of
+accessory gland secretions, can withstand freezing and thawing.</p>
+
+
+<hr class="c25" />
+<h2><a name="Ch3" id="Ch3"></a>EXTENDERS FOR FREEZING BULL SEMEN</h2>
+
+<p>Both of the extenders that are widely used in routine storage of
+bull semen at 5&deg; C. are used for freezing semen. These are the egg
+yolk-sodium citrate and whole or skimmilk extenders. Most of the
+research with extenders for freezing bull semen in this laboratory has
+been done with the yolk-citrate diluents.</p>
+
+<p><a name="Sec3_1" id="Sec3_1"></a><b>Proportion of egg yolk in the final diluent.</b> Some early experiences
+with a diluent consisting of one part yolk and one part 2.9 percent
+sodium citrate dihydrate in distilled water showed poor sperm survival
+following freezing. The final mixture with this diluent consisted of
+about 45 percent yolk. In other attempts at adding glycerol in order
+to freeze semen, the final proportion of yolk was diminished and better
+sperm survival was obtained. Several experiments were carried out to
+test the effect of varying levels of egg yolk.</p>
+
+<p>In the first efforts to find the optimum level of egg yolk, the level<span
+class='pagenum'><a name="Page_11" id="Page_11">[11]</a></span>
+of yolk in the final frozen mixture was varied from about 6 to 46
+percent. These levels were obtained by varying the proportion of yolk
+to 2.9 percent citrate solution in the original extending media and also
+in the media added in glycerolating the samples.</p>
+
+<p>Split portions of 20 semen samples were frozen in each of the
+extender combinations indicated in <a href="#Tab4">Table 4</a>. The mean percentages of
+motile sperm found before and after freezing and thawing are shown
+also. The highest percentages were found with extenders containing
+23 and 24 percent yolk. The highest percentage of yolk, resulting when
+a 1:1 (yolk to citrate) extender was used for both extending and
+glycerolating, proved to be most detrimental to sperm survival during
+freezing. The lowest percentage of yolk used (6 percent) was not
+as effective in protecting sperm during freezing as the intermediate
+levels tested (<a href="#Fig2">Fig. 2</a>).</p>
+
+<h5><a name="Tab4" id="Tab4"></a>Table 4.&mdash;Effect of Egg-Yolk Level in Diluent on Freezability of Semen</h5>
+
+<p class="tabhead">(Average of 20 semen samples)</p>
+
+<table class="fsize80" summary="Table P 11">
+
+<tr class="bt2">
+<td rowspan="3" class="center padr1 padl1 bb">Diluent</td>
+<td colspan="7" class="center padr1 padl1 bb">Medium</td>
+<td rowspan="3" class="center padr1 padl1 bb">Yolk&nbsp;in<br />final<br />mixture<a name="FNanchorD"
+id="FNanchorD"></a><a href="#FootnoteD" class="fnanchor">[D]</a><br />(percent)</td>
+<td rowspan="3" class="center padr1 padl1 bb">Pre-<br />freezing<br />motility<br />(percent)</td>
+<td rowspan="3" class="center padr1 padl1 bb">Post-<br />thawing<br />motility<br />(percent)</td>
+<td rowspan="3" class="center padr1 padl1 bb">Survival<br />(percent)</td>
+</tr>
+
+<tr>
+<td colspan="3" class="center bb padr4 padl4">Extending</td>
+<td>&nbsp;</td>
+<td colspan="3" class="center bb padr1 padl1">Glycerolating<a name="FNanchorC" id="FNanchorC"></a><a
+href="#FootnoteC" class="fnanchor">[C]</a></td>
+</tr>
+
+<tr class="bb">
+<td class="center padl1">yolk</td>
+<td class="center">:</td>
+<td class="center">citrate</td>
+<td>&nbsp;</td>
+<td class="center padl1">yolk</td>
+<td class="center">:</td>
+<td class="center">citrate</td>
+</tr>
+
+<tr>
+<td class="left padl1">1</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">1</td>
+<td>&nbsp;</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">1</td>
+<td class="right padr4">45.7</td>
+<td class="right padr4">65</td>
+<td class="right padr4">5</td>
+<td class="right padr4">8</td>
+</tr>
+
+<tr>
+<td class="left padl1">2</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">1</td>
+<td>&nbsp;</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">3</td>
+<td class="right padr4">34.9</td>
+<td class="right padr4">64</td>
+<td class="right padr4">27</td>
+<td class="right padr4">42</td>
+</tr>
+
+<tr>
+<td class="left padl1">3</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">3</td>
+<td>&nbsp;</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">1</td>
+<td class="right padr4">33.6</td>
+<td class="right padr4">63</td>
+<td class="right padr4">33</td>
+<td class="right padr4">52</td>
+</tr>
+
+<tr>
+<td class="left padl1">4</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">1</td>
+<td>&nbsp;</td>
+<td class="right">0</td>
+<td class="center">:</td>
+<td class="left">1</td>
+<td class="right padr4">24.2</td>
+<td class="right padr4">64</td>
+<td class="right padr4">39</td>
+<td class="right padr4">61</td>
+</tr>
+
+<tr>
+<td class="left padl1">5</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">3</td>
+<td>&nbsp;</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">3</td>
+<td class="right padr4">22.8</td>
+<td class="right padr4">63</td>
+<td class="right padr4">37</td>
+<td class="right padr4">59</td>
+</tr>
+
+<tr>
+<td class="left padl1">6</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">3</td>
+<td>&nbsp;</td>
+<td class="right">0</td>
+<td class="center">:</td>
+<td class="left">1</td>
+<td class="right padr4">12.1</td>
+<td class="right padr4">59</td>
+<td class="right padr4">33</td>
+<td class="right padr4">56</td>
+</tr>
+
+<tr>
+<td class="left padl1">7</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">7</td>
+<td>&nbsp;</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">7</td>
+<td class="right padr4">11.4</td>
+<td class="right padr4">56</td>
+<td class="right padr4">35</td>
+<td class="right padr4">62</td>
+</tr>
+
+<tr>
+<td class="left padl1">8</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">7</td>
+<td>&nbsp;</td>
+<td class="right">0</td>
+<td class="center">:</td>
+<td class="left">1</td>
+<td class="right padr4">6.0</td>
+<td class="right padr4">52</td>
+<td class="right padr4">26</td>
+<td class="right padr4">50</td>
+</tr>
+
+<tr class="bb">
+<td class="left padl1">9</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">15</td>
+<td>&nbsp;</td>
+<td class="right">1</td>
+<td class="center">:</td>
+<td class="left">15</td>
+<td class="right padr4">5.7</td>
+<td class="right padr4">49</td>
+<td class="right padr4">25</td>
+<td class="right padr4">51</td>
+</tr>
+
+</table>
+
+<div class="footnote"><p><a name="FootnoteC" id="FootnoteC"></a><a href="#FNanchorC"><span
+class="label">[C]</span></a> This mixture included 14 percent glycerol.</p></div>
+
+<div class="footnote"><p><a name="FootnoteD" id="FootnoteD"></a><a href="#FNanchorD"><span
+class="label">[D]</span></a> The average initial sperm concentration was 900 &times; 10<span class="expon">6</span>/ml.
+Sufficient extender was added
+to give 30 &times; 10<span class="expon">6</span>/ml. at the first extension. Thus the final concentration was 15
+&times; 10<span class="expon">6</span> sperm/ml.
+after glycerolization.</p></div>
+
+<p>Since rather large changes in the percentages of yolk were used
+in this experiment, two further trials were conducted in which 16, 24,
+and 32 percent yolk in the final mixture were compared, with the final
+citrate percentages held constant. In these tests, 16 and 24 percent
+yolk maintained sperm better at all citrate levels tried than 32 percent
+yolk. The 16 percent level was slightly better at most of the levels of
+citrate tested (<a href="#Fig3">Fig. 3</a>).</p>
+
+<p><a name="Sec3_2" id="Sec3_2"></a><b>Citrate level in the final diluent.</b> The early work of the British
+indicated that a final citrate level near 2 percent in the diluent was<span class='pagenum'><a name="Page_12" id="Page_12">[12]</a></span>
+satisfactory for freezing bull sperm. Later, in a personal communication,
+Polge of the British group suggested that a citrate level of about
+2.35 percent might be best with a final glycerol concentration of 7
+percent. Some of the first attempts in this laboratory at establishing
+the optimum yolk-to-citrate ratios are shown in <a href="#Fig3">Fig. 3</a>. In these experiments,
+the optimum levels of citrate appeared to be lower than
+anticipated from the British work. Thus a more complex experiment
+was set up to test a wider range of citrate levels using 16 and 24
+percent egg yolk in the final freezing mixture. The average percentages
+of motile sperm found after freezing 10 semen samples at each of the
+citrate and yolk levels in this experiment are shown also in <a href="#Fig3">Fig. 3</a>.
+Little difference in freezability was found between citrate percentages
+of 1.55 and 1.95. When the rate of sperm motility following freezing
+and thawing was considered along with the percent of motile sperm,
+a slight advantage was found with 16 percent yolk and a citrate concentration
+of 1.55 percent.</p>
+
+<div class="figcenter"><a name="Fig2" id="Fig2"></a><img src="images/illo002.png" alt="Fig. 2" width="600" height="369" />
+
+<table class="cap600" summary="Caption Fig 2">
+
+<tr>
+<td colspan="2" class="left">Percent of motile sperm after freezing and thawing semen in diluents containing
+various levels of egg</td>
+</tr>
+
+<tr>
+<td class="left">yolk</td>
+<td class="right">(Fig. 2)</td>
+</tr>
+
+</table>
+</div>
+
+<p>From the results of these experiments, and from several reports
+in the literature, <a href="#Footnote5" class="fnanchor">[5],</a> <a href="#Footnote6" class="fnanchor">[6],</a>
+<a href="#Footnote7" class="fnanchor">[7],</a> <a name="FNanchor9"
+id="FNanchor9"></a><a href="#Footnote9" class="fnanchor">[9],</a> <a name="FNanchor10" id="FNanchor10"></a><a
+href="#Footnote10" class="fnanchor">[10]</a> it appears that a diluting medium resulting
+in a final concentration of 16 to 25 percent yolk and 1.55 to 2.2 percent
+sodium citrate dihydrate is highly satisfactory for freezing.</p>
+
+<p><a name="Sec3_3" id="Sec3_3"></a><b>Storing and freezing diluent.</b> In some instances it would be advantageous
+to have prepared diluent on hand for use at any time. The<span class='pagenum'><a name="Page_13" id="Page_13">[13]</a></span>
+suitability of stored diluent was tested with a yolk-citrate (equal parts
+yolk and citrate without antibiotics added) diluent prepared and stored
+at 5&deg; C. for 0, 2, 5, 7, and 9 days. Seven semen samples were diluted
+and frozen in these diluents. No difference was noted in the survival of
+sperm that could be attributed to the age of the diluent.</p>
+
+<div class="figcenter"><a name="Fig3" id="Fig3"></a><img src="images/illo003.png" alt="Fig. 3" width="600" height="372" />
+
+<table class="cap600" summary="Caption Fig. 3">
+
+<tr>
+<td colspan="2" class="left">Percent of motile sperm after freezing and thawing semen in diluents containing various levels of egg</td>
+</tr>
+
+<tr>
+<td class="left">yolk and various percentages of sodium citrate</td>
+<td class="right">(Fig. 3)</td>
+</tr>
+
+</table>
+</div>
+
+<p>In another trial, a similar diluent (1:1 yolk to citrate with 1000
+units of penicillin and 5000 units of streptomycin) was prepared and
+stored in the freezer compartment of a refrigerator at -15&deg; C. Upon
+thawing, it was whitish in color and more viscous than freshly prepared
+diluent. Except for the fact that the viscosity seemed to reduce the
+rate of sperm motility, this frozen diluent stored for 65 days compared
+favorably with freshly prepared diluent for freezing semen.</p>
+
+<p><a name="Sec3_4" id="Sec3_4"></a><b>Other diluents.</b> Without the protective action of egg yolk or milk,
+few bull sperm will survive freezing. Several diluents were compared
+on a limited scale for freezing bull sperm. The results of these trials
+are compiled in <a href="#Tab5">Table 5</a>. In this trial the yolk-citrate extender served
+best in maintaining sperm motility during freezing. Yolk-phosphate
+and homogenized whole milk were slightly less protective and yolk-saline
+seemed to furnish the least protection to sperm during freezing.</p>
+
+<p>A number of investigations in other laboratories have now proven
+that milk can be used as effectively as the yolk-citrate diluent for
+freezing bull sperm.<a href="#Footnote6" class="fnanchor">[6],</a> <a href="#Footnote7" class="fnanchor">[7]</a></p>
+
+<p class='pagenum'><a name="Page_14" id="Page_14">[14]</a></p>
+
+<h5><a name="Tab5" id="Tab5"></a>Table 5.&mdash;Comparison of the Freezability of 4 Semen Samples<br />
+in Different Extenders</h5>
+
+<table class="fsize80" summary="Table P 14">
+
+<tr class="bt2 bb">
+<td class="center padr1 padl1">Extender</td>
+<td class="center padr1 padl1">Dilution<br />rate<br />(semen:<br />extender)</td>
+<td class="center padr1 padl1">Pre-<br />freezing<br />motility<br />(percent)</td>
+<td class="center padr1 padl1">Post-<br />thawing<br />motility<br />(percent)</td>
+<td class="center padr1 padl1">Survival<br />(percent)</td>
+<td class="center padr1 padl1">Motility<br />after<br />storage<a name="FNanchorE" id="FNanchorE"></a><a
+href="#FootnoteE" class="fnanchor">[E]</a><br />(percent)</td>
+</tr>
+
+<tr>
+<td class="left padl1 padr1">Yolk-citrate</td>
+<td class="left padl3">1:1</td>
+<td class="center">60</td>
+<td class="center">49</td>
+<td class="center">82</td>
+<td class="center">46</td>
+</tr>
+
+<tr>
+<td class="left padl1 padr1">&nbsp;</td>
+<td class="left padl3">1:10</td>
+<td class="center">53</td>
+<td class="center">45</td>
+<td class="center">85</td>
+<td class="center">36</td>
+</tr>
+
+<tr>
+<td class="left padl1 padr1">Yolk-saline</td>
+<td class="left padl3">1:1</td>
+<td class="center">57</td>
+<td class="center">29</td>
+<td class="center">51</td>
+<td class="center">28</td>
+</tr>
+
+<tr>
+<td class="left padl1 padr1">&nbsp;</td>
+<td class="left padl3">1:10</td>
+<td class="center">60</td>
+<td class="center">31</td>
+<td class="center">52</td>
+<td class="center">24</td>
+</tr>
+
+<tr>
+<td class="left padl1 padr1">Yolk-phosphate</td>
+<td class="left padl3">1:1</td>
+<td class="center">55</td>
+<td class="center">33</td>
+<td class="center">64</td>
+<td class="center">25</td>
+</tr>
+
+<tr>
+<td class="left padl1 padr1">&nbsp;</td>
+<td class="left padl3">1:10</td>
+<td class="center">60</td>
+<td class="center">43</td>
+<td class="center">72</td>
+<td class="center">25</td>
+</tr>
+
+<tr>
+<td class="left padl1 padr1">Whole milk</td>
+<td class="left padl3">1:1</td>
+<td class="center">60</td>
+<td class="center">40</td>
+<td class="center">67</td>
+<td class="center">35</td>
+</tr>
+
+<tr class="bb">
+<td class="left padl1 padr1">&nbsp;</td>
+<td class="left padl3">1:10</td>
+<td class="center">60</td>
+<td class="center">35</td>
+<td class="center">58</td>
+<td class="center">16</td>
+</tr>
+
+</table>
+
+<div class="footnote"><p><a name="FootnoteE" id="FootnoteE"></a><a href="#FNanchorE"><span class="label">[E]</span></a>
+Stored at 5&deg; C. for 7 hours after thawing.</p></div>
+
+<hr class="c25" />
+<h2><a name="Ch4" id="Ch4"></a>DILUTION RATES</h2>
+
+
+<p>The first trials by the British at freezing bull semen were made
+with samples containing many millions of sperm cells. In routine
+artificial breeding, it is common to add extenders to semen so that
+one milliliter of diluted semen may contain only 10 million living
+sperm cells. (This number still insures optimal fertility.) Frequently
+the addition of 100 or more parts of the yolk extender to each part of
+the original semen sample is possible without reducing the sperm
+numbers below 10 million per milliliter. No one knew if this process
+of dilution would affect the resistance of bull sperm to freezing. The
+effect of various rates of dilution on the freezability of bull sperm
+was tested with 10 semen samples. The results, presented in <a href="#Tab6">Table 6</a>,
+show that the numbers of sperm between 10 and 90 million per
+milliliter did not influence the percentage of sperm that survived
+freezing.</p>
+
+<p>In a later trial it was found that sperm survival was slightly better
+at lower dilution rates than in the same samples frozen following
+dilution to 15 million sperm per milliliter. However, field trials with
+frozen semen carried out by others, using sperm numbers as low as
+15 million per milliliter of semen inseminated or even lower, have been
+highly satisfactory.<a name="FNanchor11" id="FNanchor11"></a><a href="#Footnote11" class="fnanchor">[11],</a>
+<a name="FNanchor12" id="FNanchor12"></a><a href="#Footnote12" class="fnanchor">[12]</a></p>
+
+<p>During the early studies in the Illinois laboratory, the effects of
+glycerol level were also tested.<a name="FNanchor13" id="FNanchor13"></a><a
+href="#Footnote13" class="fnanchor">[13]</a> These effects are discussed in the
+section on <a href="#Ch5">glycerol additions</a> beginning on <a href="#Page_17">page 17</a>.</p>
+
+<p><a name="Sec4_1" id="Sec4_1"></a><b>Effect of further dilution and refreezing after the initial freezing.</b>
+Under some circumstances it might be advantageous to freeze semen<span class='pagenum'><a name="Page_15" id="Page_15">[15]</a></span>
+with a high concentration of sperm cells and then extend it further
+after thawing. With such a procedure less storage space is needed
+than when dilution is carried to the maximum before freezing. Two
+experiments were conducted to test the effects of dilution and storage
+at 5&deg; C. and dilution and refreezing following an initial freezing of
+concentrated samples.</p>
+
+<h5><a name="Tab6" id="Tab6"></a>Table 6.&mdash;Effect of Sperm Numbers and Glycerol Level<br />
+in Final Mixture on Freezability of Bull Sperm at -79&deg; C.</h5>
+
+<p class="tabhead">(Average of 10 ejaculates)</p>
+
+<table class="fsize80" summary="Table P 15 top">
+
+<tr>
+<td rowspan="3" class="center bt2 bb padr1 padl1">Glycerol&nbsp;level<br />(percent)</td>
+<td colspan="4" class="center bt2 bb padr1 padl1">Post-thawing&nbsp;motility&nbsp;(percent)<a name="FNanchorF"
+id="FNanchorF"></a><a href="#FootnoteF" class="fnanchor">[F]</a></td>
+</tr>
+
+<tr>
+<td colspan="3" class="center bb padr1 padl1">Number&nbsp;of&nbsp;sperm&nbsp;(millions/ml.)</td>
+<td rowspan="2" class="center bb padr1 padl1">Average</td>
+</tr>
+
+<tr>
+<td class="center bb">90</td>
+<td class="center bb">30</td>
+<td class="center bb">10</td>
+</tr>
+
+<tr>
+<td class="left padl1">5</td>
+<td class="right padr3">36.0</td>
+<td class="right padr3">34.0</td>
+<td class="right padr3">36.0</td>
+<td class="right padr3">35.0</td>
+</tr>
+
+<tr>
+<td class="left padl1">10</td>
+<td class="right padr3">22.0</td>
+<td class="right padr3">24.0</td>
+<td class="right padr3">23.0</td>
+<td class="right padr3">23.0</td>
+</tr>
+
+<tr>
+<td class="left padl1">15</td>
+<td class="right padr3">3.2</td>
+<td class="right padr3">0.9</td>
+<td class="right padr3">0.2</td>
+<td class="right padr3">1.4</td>
+</tr>
+
+<tr class="bb">
+<td class="left padl1">Average</td>
+<td class="right padr3">20.3</td>
+<td class="right padr3">19.8</td>
+<td class="right padr3">19.9</td>
+<td class="right padr3">20.0</td>
+</tr>
+
+</table>
+
+<div class="footnote"><p><a name="FootnoteF" id="FootnoteF"></a><a href="#FNanchorF"><span
+class="label">[F]</span></a> Mean initial motility of sperm before freezing was 55 percent.</p></div>
+
+<p>Four semen samples were split and extended at rates of 1:1
+(semen to extender) and 1:10. These were frozen, then thawed and
+halved. One half was further extended to a level of 15 million sperm
+per milliliter; the sperm numbers in the other remained unchanged.
+Each of these halves was split again, and one portion of each was
+stored at 5&deg; C. for 3 to 7 hours. The other two portions were refrozen.</p>
+
+<h5><a name="Tab7" id="Tab7"></a>Table 7.&mdash;Effect of Further Dilution and Refreezing on Sperm Motility<br />
+After the Initial Freezing of Bull Semen</h5>
+
+<table class="fsize80" summary="Table P 15 bottom">
+
+<tr>
+<td rowspan="3" class="center bt2 bb padr1 padl1">Dilution&nbsp;of<br />semen<br />(semen:<br />extender)</td>
+<td rowspan="3" class="center bt2 bb padr1 padl1">Pre-<br />freezing<br />motility<br />(percent)</td>
+<td colspan="6" class="center bt2 bb padr1 padl1">Post-thawing motility</td>
+</tr>
+
+<tr>
+<td rowspan="2" class="center bb padr1 padl1">After<br />first<br />freezing</td>
+<td colspan="2" class="center bb padr1 padl1">After&nbsp;storage<a name="FNanchorG"
+id="FNanchorG"></a><a href="#FootnoteG" class="fnanchor">[G]</a></td>
+<td>&nbsp;</td>
+<td colspan="2" class="center bb padr1 padl1">After&nbsp;refreezing<a name="FNanchorH"
+id="FNanchorH"></a><a href="#FootnoteH" class="fnanchor">[H]</a></td>
+</tr>
+
+<tr>
+<td class="center bb padr1 padl1">No<br />further<br />dilution</td>
+<td class="center bb padr1 padl1">Diluted<br />to&nbsp;15<br />million/ml</td>
+<td class="bb">&nbsp;</td>
+<td class="center bb padr1 padl1">No<br />further<br />dilution</td>
+<td class="center bb padr1 padl1">Diluted<br />to&nbsp;15<br />million/ml</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td colspan="3" class="center">First&nbsp;trial:&nbsp;4&nbsp;samples</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+</tr>
+
+<tr>
+<td class="left padl1">1:1</td>
+<td class="right padr3">60</td>
+<td class="right padr3">49</td>
+<td class="right padr3">46</td>
+<td class="right padr4">34</td>
+<td>&nbsp;</td>
+<td class="right padr3">31</td>
+<td class="right padr4">6</td>
+</tr>
+
+<tr>
+<td class="left padl1">1:10</td>
+<td class="right padr3">53</td>
+<td class="right padr3">45</td>
+<td class="right padr3">36</td>
+<td class="right padr4">30</td>
+<td>&nbsp;</td>
+<td class="right padr3">25</td>
+<td class="right padr4">5</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td colspan="3" class="center">Second&nbsp;trial:&nbsp;7&nbsp;samples</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+</tr>
+
+<tr>
+<td class="left padl1">1:9</td>
+<td class="right padr3">67</td>
+<td class="right padr3">47</td>
+<td class="right padr3">41</td>
+<td class="right padr4">35</td>
+<td>&nbsp;</td>
+<td class="right padr3">28</td>
+<td class="right padr4">11</td>
+</tr>
+
+<tr class="bb">
+<td class="left padl1">15&nbsp;million/ml</td>
+<td class="right padr3">67</td>
+<td class="right padr3">30</td>
+<td class="right padr3">32</td>
+<td class="right padr4">..</td>
+<td>&nbsp;</td>
+<td class="right padr3">18</td>
+<td class="right padr4">..</td>
+</tr>
+
+</table>
+
+<div class="footnote"><p><a name="FootnoteG" id="FootnoteG"></a><a href="#FNanchorG"><span
+class="label">[G]</span></a> Stored at 5&deg; C. for 3 to 7 hours after first thawing.</p>
+
+<p><a name="FootnoteH" id="FootnoteH"></a><a href="#FNanchorH"><span
+class="label">[H]</span></a> Refrozen following first thawing.</p></div>
+
+<p class='pagenum'><a name="Page_16" id="Page_16">[16]</a></p>
+
+<h5><a name="Tab8" id="Tab8"></a>Table 8.&mdash;Effect of Glycerol Level and Storage at 5&deg; C. on Motility<br />
+of Sperm in Yolk-Citrate Extender</h5>
+
+<table class="fsize80" summary="Table P 16">
+
+<tr>
+<td colspan="2" rowspan="4" class="center bt2 bb padr1 padl1">Glycerol&nbsp;level<br />(percent)</td>
+<td colspan="19" class="center bt2 bb padr1 padl1">Sperm motility</td>
+</tr>
+
+<tr>
+<td colspan="3" rowspan="2" class="center bb padr1 padl1">Post-<br />thawing</td>
+<td>&nbsp;</td>
+<td colspan="11" class="center bb padr1 padl1">After storage at 5&deg; C.</td>
+<td>&nbsp;</td>
+<td colspan="2" rowspan="2" class="center bb padr1 padl1">Average</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td colspan="3" class="center bb padr1 padl1">1 day</td>
+<td>&nbsp;</td>
+<td colspan="3" class="center bb padr1 padl1">3 days</td>
+<td>&nbsp;</td>
+<td colspan="3" class="center bb padr1 padl1">7 days</td>
+<td>&nbsp;</td>
+</tr>
+
+<tr>
+<td class="center bb padr1 padl1">percent</td>
+<td colspan="2" class="center bb padr1 padl1">rate</td>
+<td class="bb">&nbsp;</td>
+<td class="center bb padr1 padl1">percent</td>
+<td colspan="2" class="center bb padr1 padl1">rate</td>
+<td class="bb">&nbsp;</td>
+<td class="center bb padr1 padl1">percent</td>
+<td colspan="2" class="center bb padr1 padl1">rate</td>
+<td class="bb">&nbsp;</td>
+<td class="center bb padr1 padl1">percent</td>
+<td colspan="2" class="center bb padr1 padl1">rate</td>
+<td class="bb">&nbsp;</td>
+<td class="center bb padr1 padl1">percent</td>
+<td class="center bb padr1 padl1">rate</td>
+</tr>
+
+<tr>
+<td colspan="2" class="left padl1">Control<a name="FNanchorI" id="FNanchorI"></a><a href="#FootnoteI" class="fnanchor">[I]</a></td>
+<td class="center">56</td>
+<td class="right padl1">2.5</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">55</td>
+<td class="right padl1">1.9</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">46</td>
+<td class="right padl1">1.8</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">38</td>
+<td class="right padl1">1.4</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">48</td>
+<td class="center">1.90</td>
+</tr>
+
+<tr>
+<td style="width: 1em;">&nbsp;</td>
+<td class="left">0</td>
+<td class="center">54</td>
+<td class="right padl1">2.4</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">44</td>
+<td class="right padl1">1.9</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">46</td>
+<td class="right padl1">1.8</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">36</td>
+<td class="right padl1">1.4</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">45</td>
+<td class="center">1.87</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left">5</td>
+<td class="center">52</td>
+<td class="right padl1">2.2</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">50</td>
+<td class="right padl1">1.9</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">46</td>
+<td class="right padl1">1.7</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">32</td>
+<td class="right padl1">1.4</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">45</td>
+<td class="center">1.80</td>
+</tr>
+
+<tr>
+<td class="right padl1">1</td>
+<td class="left">0</td>
+<td class="center">52</td>
+<td class="right padl1">2.3</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">46</td>
+<td class="right padl1">1.8</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">42</td>
+<td class="right padl1">1.7</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">28</td>
+<td class="right padl1">1.6</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">42</td>
+<td class="center">1.85</td>
+</tr>
+
+<tr>
+<td class="right padl1">2</td>
+<td class="left">0</td>
+<td class="center">52</td>
+<td class="right padl1">2.1</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">50</td>
+<td class="right padl1">1.7</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">44</td>
+<td class="right padl1">1.6</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">38</td>
+<td class="right padl1">1.1</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">46</td>
+<td class="center">1.62</td>
+</tr>
+
+<tr>
+<td class="right padl1">3</td>
+<td class="left">0</td>
+<td class="center">50</td>
+<td class="right padl1">0.7</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">44</td>
+<td class="right padl1">0.5</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">42</td>
+<td class="right padl1">0.4</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">30</td>
+<td class="right padl1">0.4</td>
+<td>&nbsp;</td>
+<td>&nbsp;</td>
+<td class="center">42</td>
+<td class="center">0.51</td>
+</tr>
+
+<tr class="bb">
+<td colspan="2" class="left padl1">Average</td>
+<td class="center">53</td>
+<td class="right padl1">2.0</td>
+<td class="left padr1">3</td>
+<td>&nbsp;</td>
+<td class="center">47</td>
+<td class="right padl1">1.6</td>
+<td class="left padr1">2</td>
+<td>&nbsp;</td>
+<td class="center">44</td>
+<td class="right padl1">1.5</td>
+<td class="left padr1">0</td>
+<td>&nbsp;</td>
+<td class="center">34</td>
+<td class="right padl1">1.2</td>
+<td class="left padr1">2</td>
+<td>&nbsp;</td>
+<td class="center padr1">..</td>
+<td class="center padr1">....</td>
+</tr>
+
+</table>
+
+<div class="footnote"><p><a name="FootnoteI" id="FootnoteI"></a><a href="#FNanchorI"><span
+class="label">[I]</span></a> The control differed from the 0-glycerol treatment in that
+no additional citrate or glycerol solution was added.</p></div>
+
+<p><span class='pagenum'><a name="Page_17" id="Page_17">[17]</a></span>A similar trial was carried out with seven samples; one portion was
+diluted 1:9; the other was extended at the outset to 15 million sperm
+per milliliter. Results for both tests are summarized in <a href="#Tab7">Table 7</a>.</p>
+
+<p>From <a href="#Tab7">Table 7</a> it can be seen that refreezing following an initial
+freezing further reduced the number of surviving sperm. The second
+freezing was more detrimental to the portion of the samples extended
+to 15 million sperm per milliliter than to the portion that was refrozen
+at a higher sperm concentration. The percentage of motile sperm
+remained fairly high in the portions that were diluted to 15 million
+sperm and stored at 5&deg; C. However, in all cases, survival was best in
+the samples at the lower dilution levels.</p>
+
+
+<hr class="c25" />
+<h2><a name="Ch5" id="Ch5"></a>GLYCEROL ADDITIONS</h2>
+
+
+<p>When the British procedure for freezing bull semen was first tried
+in this country, many of the refinements of the technique still had not
+been defined. It was known that glycerol worked well in protecting
+sperm during freezing. The effects of glycerol on sperm at 5&deg; C., the
+appropriate levels to use in freezing, and the manner of adding it were
+not well established. Therefore, a number of trials were conducted in
+an attempt to establish the best procedures.</p>
+
+<p><a name="Sec5_1" id="Sec5_1"></a><b>Effect of glycerol on sperm survival at 5&deg; C.</b> Since early work
+indicated the need for adding glycerol to diluted semen in order to
+protect the sperm during freezing, it was considered important to
+determine the levels of glycerol that sperm would tolerate at 5&deg; C.
+Ten semen samples were extended 1:9 (semen to diluent) in a 1:1
+yolk-citrate diluent (yolk to 2.9 percent sodium citrate dihydrate).
+Each sample was then split into 6 portions and an equal volume of
+citrate solution containing glycerol was added slowly to each to bring
+the glycerol in the final mixture to 0, 5, 10, 20, or 30 percent (by
+volume). These samples were stored at 5&deg; C. and examined for
+motile sperm after 1, 3, and 7 days. The effects of glycerol levels on
+the percentage of sperm surviving and the rate (or speed) of their
+forward motion (0 = no forward motion; 4 = extremely rapid progressive
+motility) are presented in <a href="#Tab8">Table 8</a>.</p>
+
+<p>The percentage of motile sperm decreased slightly at the higher
+levels of glycerol. The most noticeable effect of the increase in glycerol
+level was the reduction in the rate of forward motion of the sperm.
+At the 30-percent level, the sperm moved slowly and could be seen to<span class='pagenum'><a name="Page_18" id="Page_18">[18]</a></span>
+rotate as they moved forward. Some samples were checked after
+slowly bringing the diluent up to a level of 40 percent glycerol; the
+sperm seemed to be immobilized completely in this solution.</p>
+
+<p><a name="Sec5_2" id="Sec5_2"></a><b>Glycerol levels for freezing semen.</b> The British procedure called
+for the use of 10 percent glycerol in the final mixture of semen and
+extender prior to freezing. Yet, as shown in <a href="#Tab6">Table 6</a>, in our laboratory
+5 percent glycerol resulted in the survival of a higher percentage of
+sperm than did 10 or 15 percent. In order to define more clearly the
+optimum glycerol level, several ejaculates of semen were subsampled
+and portions were frozen after the addition of yolk-citrate extender
+and glycerol in varying quantities. From <a href="#Tab9">Table 9</a> it can be seen that
+glycerol levels of 6 and 8 percent in the final mixture resulted in maximum
+sperm survival during freezing. These results were confirmed in
+tests on the survival of sperm at 5&deg; C. storage for 3 days following
+freezing and thawing with varying glycerol levels (see <a href="#Tab10">Table 10</a>).</p>
+
+<p>The results shown in <a href="#Tab9">Tables 9</a> and <a href="#Tab10">10</a> were confirmed also in later
+experiments. Thirty-six samples were subjected to various levels of
+glycerol and no significant difference in freezability was found between
+6 and 8 percent. Based on these findings, a glycerol level of 7
+percent was adopted for use in all experiments described in this bulletin,
+unless otherwise indicated. Results in a number of other laboratories
+have agreed with our findings regarding the use of approximately
+7 percent glycerol with the yolk-citrate diluent.<a href="#Footnote5" class="fnanchor">[5],</a>
+<a href="#Footnote6" class="fnanchor">[6],</a> <a href="#Footnote7" class="fnanchor">[7],</a>
+<a href="#Footnote9" class="fnanchor">[9],</a> <a href="#Footnote10" class="fnanchor">[10]</a> With milk as
+the extender, 10 to 13 percent glycerol has been preferred by some.<a href="#Footnote5" class="fnanchor">[5],</a>
+<a href="#Footnote6" class="fnanchor">[6],</a> <a href="#Footnote7" class="fnanchor">[7]</a></p>
+
+<h5><a name="Tab9" id="Tab9"></a>Table 9.&mdash;Effect of Glycerol Level on Sperm Motility<br />
+After Freezing to -79&deg; C. and Thawing</h5>
+
+<table class="fsize80" summary="Table P 18">
+
+<tr class="bt2 bb">
+<td colspan="2" class="center padr1 padl1">Glycerol<br />level<br />(percent)</td>
+<td class="center padr1 padl1">Number<br />of<br />samples</td>
+<td class="center padr1 padl1">Pre-<br />freezing<br />motility<br />(percent)</td>
+<td class="center padr1 padl1">Post-<br />thawing<br />motility<br />(percent)</td>
+<td class="center padr1 padl1">Survival<br />(percent)</td>
+</tr>
+
+<tr>
+<td class="padr0" style="width: 1.5em;">&nbsp;</td>
+<td class="left padl0">2</td>
+<td class="right padr4">10</td>
+<td class="right padr4">53</td>
+<td class="right padr4">2</td>
+<td class="right padr4">4</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left padl0">4</td>
+<td class="right padr4">19</td>
+<td class="right padr4">55</td>
+<td class="right padr4">29</td>
+<td class="right padr4">53</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left padl0">6</td>
+<td class="right padr4">19</td>
+<td class="right padr4">55</td>
+<td class="right padr4">34</td>
+<td class="right padr4">62</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left padl0">8</td>
+<td class="right padr4">19</td>
+<td class="right padr4">55</td>
+<td class="right padr4">35</td>
+<td class="right padr4">64</td>
+</tr>
+
+<tr>
+<td class="right padr0">1</td>
+<td class="left padl0">0</td>
+<td class="right padr4">19</td>
+<td class="right padr4">55</td>
+<td class="right padr4">24</td>
+<td class="right padr4">44</td>
+</tr>
+
+<tr class="bb">
+<td class="right padr0">1</td>
+<td class="left padl0">2</td>
+<td class="right padr4">10</td>
+<td class="right padr4">53</td>
+<td class="right padr4">13</td>
+<td class="right padr4">25</td>
+</tr>
+
+</table>
+
+<p class='pagenum'><a name="Page_19" id="Page_19">[19]</a></p>
+
+<h5><a name="Tab10" id="Tab10"></a>Table 10.&mdash;Effect of Glycerol Level and Storage<br />at 5&deg; C.
+After Thawing on Sperm Motility</h5>
+
+<p class="tabhead">(Average of 13 ejaculates)</p>
+
+<table class="fsize80" summary="Table P 19 top">
+
+<tr>
+<td colspan="2" rowspan="3" class="center bt2 bb padr1 padl1">Glycerol<br />level<br />(percent)</td>
+<td colspan="3" class="center bt2 bb padr1 padl1">Sperm&nbsp;motility&nbsp;(percent)</td>
+</tr>
+
+<tr>
+<td rowspan="2" class="center bb padr1 padl1">Post-<br />thawing</td>
+<td colspan="2" class="center bb padr1 padl1">After&nbsp;storage&nbsp;at&nbsp;5&deg;&nbsp;C.</td>
+</tr>
+
+<tr>
+<td class="center bb padr1 padl1">1 day</td>
+<td class="center bb padr1 padl1">3 days</td>
+</tr>
+
+<tr>
+<td style="width:1.5em;">&nbsp;</td>
+<td class="left padl0">4</td>
+<td class="right padr4">29</td>
+<td class="right padr4">22</td>
+<td class="right padr4">20</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left padl0">6</td>
+<td class="right padr4">38</td>
+<td class="right padr4">34</td>
+<td class="right padr4">24</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="left padl0">8</td>
+<td class="right padr4">42</td>
+<td class="right padr4">33</td>
+<td class="right padr4">17</td>
+</tr>
+
+<tr class="bb">
+<td class="right padr0">1</td>
+<td class="left padl0">0</td>
+<td class="right padr4">33</td>
+<td class="right padr4">18</td>
+<td class="right padr4">6</td>
+</tr>
+
+</table>
+
+<h5><a name="Tab11" id="Tab11"></a>Table 11.&mdash;Effects of Temperature, Rate of Addition of Glycerol,<br />
+and Equilibration Time on Sperm Motility</h5>
+<p class="tabhead">(Average of 12 ejaculates)</p>
+
+<table class="fsize80" summary="Table P 19 bottom">
+
+<tr>
+<td colspan="2" rowspan="3" class="center bt2 bb padr1 padl1">Temperature&nbsp;during&nbsp;addition<br />of&nbsp;
+glycerol&nbsp;(&deg;&nbsp;C.)</td>
+<td rowspan="3" class="center bt2 bb padr1 padl1">Equilibration<br />time<br />(hours)</td>
+<td colspan="8" class="center bt2 bb padr1 padl1">Post-thawing&nbsp;motility&nbsp;(percent)</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td colspan="7" class="center bb padr1 padl1">Glycerol&nbsp;additions</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td colspan="2" class="center bb padr3 padl3">5</td>
+<td colspan="2" class="center bb padr3 padl3">3</td>
+<td colspan="2" class="center bb padr3 padl3">1</td>
+<td class="center bb padr1 padl1">Average</td>
+</tr>
+
+<tr>
+<td style="width: 1.5em;">&nbsp;</td>
+<td class="left padl0">4.5</td>
+<td class="center">2</td>
+<td>&nbsp;</td>
+<td class="right">48</td>
+<td>&nbsp;</td>
+<td class="right">48</td>
+<td>&nbsp;</td>
+<td class="right">45</td>
+<td>&nbsp;</td>
+<td class="right padr3">47.4</td>
+</tr>
+
+<tr>
+<td colspan="2">&nbsp;</td>
+<td class="center">6</td>
+<td>&nbsp;</td>
+<td class="right">49</td>
+<td>&nbsp;</td>
+<td class="right">51</td>
+<td>&nbsp;</td>
+<td class="right">47</td>
+<td>&nbsp;</td>
+<td class="right padr3">48.8</td>
+</tr>
+
+<tr>
+<td colspan="2">&nbsp;</td>
+<td class="center">18</td>
+<td>&nbsp;</td>
+<td class="right">46</td>
+<td>&nbsp;</td>
+<td class="right">47</td>
+<td>&nbsp;</td>
+<td class="right">46</td>
+<td>&nbsp;</td>
+<td class="right padr3">46.3</td>
+</tr>
+
+<tr>
+<td colspan="2">&nbsp;</td>
+<td class="center">Average</td>
+<td>&nbsp;</td>
+<td class="right padr0">47</td>
+<td class="left padl0">.8</td>
+<td class="right padr0">48</td>
+<td class="left padl0">.6</td>
+<td class="right padr0">46</td>
+<td class="left padl0">.0</td>
+<td class="right padr3">47.5</td>
+</tr>
+
+<tr>
+<td colspan="10" style="line-height: .5em;">&nbsp;</td>
+</tr>
+
+<tr>
+<td class="right padr0">1</td>
+<td class="left padl0">0.0</td>
+<td class="center">2</td>
+<td>&nbsp;</td>
+<td class="right">44</td>
+<td>&nbsp;</td>
+<td class="right">43</td>
+<td>&nbsp;</td>
+<td class="right">45</td>
+<td>&nbsp;</td>
+<td class="right padr3">43.9</td>
+</tr>
+
+<tr>
+<td colspan="2">&nbsp;</td>
+<td class="center">6</td>
+<td>&nbsp;</td>
+<td class="right">48</td>
+<td>&nbsp;</td>
+<td class="right">50</td>
+<td>&nbsp;</td>
+<td class="right">46</td>
+<td>&nbsp;</td>
+<td class="right padr3">47.9</td>
+</tr>
+
+<tr>
+<td colspan="2">&nbsp;</td>
+<td class="center">18</td>
+<td>&nbsp;</td>
+<td class="right">43</td>
+<td>&nbsp;</td>
+<td class="right">46</td>
+<td>&nbsp;</td>
+<td class="right">42</td>
+<td>&nbsp;</td>
+<td class="right padr3">44.0</td>
+</tr>
+
+<tr>
+<td colspan="2">&nbsp;</td>
+<td class="center">Average</td>
+<td>&nbsp;</td>
+<td class="right padr0">45</td>
+<td class="left padl0">.0</td>
+<td class="right padr0">46</td>
+<td class="left padl0">.5</td>
+<td class="right padr0">44</td>
+<td class="left padl0">.3</td>
+<td class="right padr3">45.3</td>
+</tr>
+
+<tr>
+<td colspan="10" style="line-height: .5em;">&nbsp;</td>
+</tr>
+
+<tr>
+<td class="right padr0">1</td>
+<td class="left padl0">5.5</td>
+<td class="center">2</td>
+<td>&nbsp;</td>
+<td class="right">41</td>
+<td>&nbsp;</td>
+<td class="right">38</td>
+<td>&nbsp;</td>
+<td class="right">38</td>
+<td>&nbsp;</td>
+<td class="right padr3">39.1</td>
+</tr>
+
+<tr>
+<td colspan="2">&nbsp;</td>
+<td class="center">6</td>
+<td>&nbsp;</td>
+<td class="right">42</td>
+<td>&nbsp;</td>
+<td class="right">45</td>
+<td>&nbsp;</td>
+<td class="right">43</td>
+<td>&nbsp;</td>
+<td class="right padr3">43.6</td>
+</tr>
+
+<tr>
+<td colspan="2">&nbsp;</td>
+<td class="center">18</td>
+<td>&nbsp;</td>
+<td class="right">42</td>
+<td>&nbsp;</td>
+<td class="right">43</td>
+<td>&nbsp;</td>
+<td class="right">42</td>
+<td>&nbsp;</td>
+<td class="right padr3">42.5</td>
+</tr>
+
+<tr class="bb">
+<td colspan="2">&nbsp;</td>
+<td class="center">Average</td>
+<td>&nbsp;</td>
+<td class="right padr0">42</td>
+<td class="left padl0">.0</td>
+<td class="right padr0">41</td>
+<td class="left padl0">.8</td>
+<td class="right padr0">41</td>
+<td class="left padl0">.4</td>
+<td class="right padr3">41.7</td>
+</tr>
+
+</table>
+
+<p><a name="Sec5_3" id="Sec5_3"></a><b>Rate, temperature, and method of adding glycerol.</b> Closely associated
+with the question of how much glycerol should be added is that
+of how the additions should be made. Originally it was believed that
+the glycerol should be added in stages so that changes would occur
+gradually. However, there would be a saving in time if the entire
+amount could be added at once. Also, if the glycerol addition could be
+made soon after the dilution with egg yolk-citrate extender at room
+temperature, time would be gained in processing the semen for use.
+Since aging <i>in vitro</i> is known to reduce the fertilizing ability of sperm,
+every effort should be made to keep the processing time at a minimum.
+The results of an experiment involving these items, along with that of
+how much time should be allowed after the additions before freezing
+(equilibration time), are presented in <a href="#Tab11">Table 11</a>. One can see that sperm
+survived freezing better when the diluted semen was cooled to 4.5&deg; C.
+before the glycerol was added. The survival at 10&deg; and 15.5&deg; C. was<span
+class='pagenum'><a name="Page_20" id="Page_20">[20]</a></span>
+reduced with each rise in temperature. Thus, it appears that cooling to
+refrigerator temperature (4-5&deg; C.) before adding the glycerol should
+be a part of the routine procedure.</p>
+
+<p>A comparison of the results from adding the glycerol in 5, 3, and
+1 equal portions is given also in <a href="#Tab11">Table 11</a>. Little difference in survival
+during freezing was noted between the three rates of addition. Using
+3 equal additions resulted in slightly better results, but the advantage
+was not statistically significant. While little difference was evident
+from adding the glycerol in 3 portions as compared to 1, many still
+use 3 additions in the hope of obtaining a slightly better sperm survival.
+In fact, some have gone to a procedure of adding the glycerol
+dropwise with constant gentle agitation. This method has not been
+tested in this laboratory.</p>
+
+<p><a name="Sec5_4" id="Sec5_4"></a><b>Allowing sperm to equilibrate with the glycerol.</b> Allowing sperm
+to stand in the presence of glycerol is considered by some to be necessary
+in order that the glycerol penetrate the sperm heads before
+freezing. From the first successful attempts at freezing bull sperm
+came the practice of allowing 12 to 20 hours for this process of
+equilibration. A long equilibration time results in aging the sperm.
+Data from a number of sources indicate that a drop of approximately
+5 percent in fertility in the field occurs with each 24 hours of aging in
+the test tube. Thus it would seem desirable to reduce the equilibration
+time to a minimum commensurate with good freezability in order to
+reduce the effects of aging (at 5&deg; C.). Results of attempting to reduce
+equilibration time are given in <a href="#Tab11">Table 11</a>. At 4.5&deg; C., little variation in
+motility following freezing and thawing was found after equilibration
+times of 2, 6, and 18 hours. At the higher temperatures of 10&deg; and
+15.5&deg; C., the shortest equilibration time&mdash;2 hours&mdash;was slightly more
+detrimental with the differences significant at the 5-percent level at
+15.5&deg; C. For all temperatures combined, 6 hours was significantly better
+than 2 or 18 hours.</p>
+
+<p><a name="Sec5_5" id="Sec5_5"></a><b>Sugar additions and equilibration time.</b> Early in their experiences
+in freezing semen, the Australian workers found a short equilibration
+time&mdash;30 minutes&mdash;to be satisfactory if sugars were added to the
+diluent.<a href="#Footnote5" class="fnanchor">[5]</a> This protective action of sugars during the equilibration
+period was confirmed in our investigations. The results of one phase
+of this study are shown in <a href="#Tab12">Table 12</a>. From these data it can be seen
+that the presence of glucose or rhamnose at a level of 1.25 percent
+improved sperm survival during the period of equilibration. In
+another trial these sugars and two others, arabinose and xylose, were
+tested for their protective action in freezing semen. The percentages
+of surviving sperm remaining after the various steps in the freezing
+procedure with and without the presence of these sugars are shown in
+<a href="#Tab13">Table 13</a>.</p>
+
+<p class='pagenum'><a name="Page_21" id="Page_21">[21]</a></p>
+
+<h5><a name="Tab12" id="Tab12"></a>Table 12.&mdash;Effect of Adding Sugars to Yolk-Citrate Diluent<br />
+on Sperm Motility During Equilibration With Glycerol<a name="FNanchorJ" id="FNanchorJ"></a><a href="#FootnoteJ"
+class="fnanchor">[J]</a></h5>
+
+<table class="fsize80" summary="Table P 21 top">
+
+<tr>
+<td colspan="3" rowspan="2" class="center bt2 bb padr4 padl4">Stage&nbsp;when&nbsp;observed</td>
+<td colspan="3" class="center bt2 bb padr1 padl1">Sperm&nbsp;motility&nbsp;(percent)</td>
+</tr>
+
+<tr>
+<td class="center bb padr1 padl1">Glycerol<br />only</td>
+<td class="center bb padr1 padl1">Glycerol<br />and<br />glucose</td>
+<td class="center bb padr1 padl1">Glycerol<br />and<br />rhamnose</td>
+</tr>
+
+<tr>
+<td colspan="3" class="left padl1">Fresh&nbsp;diluted&nbsp;semen</td>
+<td class="center">56</td>
+<td class="center">56</td>
+<td class="center">56</td>
+</tr>
+
+<tr>
+<td colspan="3" class="left padl1">After&nbsp;glycerolization</td>
+<td class="center">54</td>
+<td class="center">54</td>
+<td class="center">54</td>
+</tr>
+
+<tr>
+<td colspan="3" class="left padl1">After&nbsp;equilibration</td>
+<td colspan="3">&nbsp;</td>
+</tr>
+
+<tr>
+<td style="width: 2em;">&nbsp;</td>
+<td class="right" style="width: 1em;">2</td>
+<td class="left padl1">hours</td>
+<td class="center">51</td>
+<td class="center">53</td>
+<td class="center">53</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="right">6</td>
+<td class="left padl1">hours</td>
+<td class="center">48</td>
+<td class="center">52</td>
+<td class="center">53</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="right">12</td>
+<td class="left padl1">hours</td>
+<td class="center">46</td>
+<td class="center">50</td>
+<td class="center">51</td>
+</tr>
+
+<tr class="bb">
+<td>&nbsp;</td>
+<td class="right">18</td>
+<td class="left padl1">hours</td>
+<td class="center">40</td>
+<td class="center">46</td>
+<td class="center">46</td>
+</tr>
+
+</table>
+
+<div class="footnote"><p><a name="FootnoteJ" id="FootnoteJ"></a><a href="#FNanchorJ"><span class="label">[J]</span></a> Glycerol
+level in the final frozen mixture was 7 percent. Sugars were added to a level
+of 1.25 percent.</p></div>
+
+<p>Three of the sugars&mdash;glucose, arabinose, and rhamnose&mdash;protected
+the sperm during equilibration and freezing. Xylose was less
+effective, but its addition resulted in slightly better sperm survival than
+glycerol alone. It was found also that the methylene-blue reduction
+time (metabolic test for semen quality) was faster in samples to
+which the sugars had been added&mdash;after glycerolization, after equilibration,
+and after freezing the samples. This is confirming evidence
+for the presence of more living and actively metabolizing sperm in the
+portions to which sugars had been added.</p>
+
+<h5><a name="Tab13" id="Tab13"></a>Table 13.&mdash;Effect of Adding Sugars to Yolk-Citrate Diluent<br />
+on Sperm Motility During the Freezing Procedures<a name="FNanchorK" id="FNanchorK"></a><a href="#FootnoteK" class="fnanchor">[K]</a></h5>
+
+<p class="tabhead">(Average of 10 ejaculates)</p>
+
+<table class="fsize80" summary="Table P 21 bottom">
+
+<tr>
+<td rowspan="2" class="center bt2 bb padr1 padl1">Stage&nbsp;of<br />observation</td>
+<td colspan="5" class="center bt2 bb padr1 padl1">Sperm&nbsp;motility&nbsp;(percent)</td>
+</tr>
+
+<tr>
+<td class="center bb padr1 padl1">Glycerol<br />only</td>
+<td class="center bb padr1 padl1">Glycerol<br />and<br />glucose</td>
+<td class="center bb padr1 padl1">Glycerol<br />and<br />arabinose</td>
+<td class="center bb padr1 padl1">Glycerol<br />and<br />xylose</td>
+<td class="center bb padr1 padl1">Glycerol<br />and<br />rhamnose</td>
+</tr>
+
+<tr>
+<td class="left padl1">Fresh diluted semen</td>
+<td class="center">63</td>
+<td class="center">63</td>
+<td class="center">63</td>
+<td class="center">63</td>
+<td class="center">63</td>
+</tr>
+
+<tr>
+<td class="left padl1">After glycerolization</td>
+<td class="center">54</td>
+<td class="center">55</td>
+<td class="center">54</td>
+<td class="center">57</td>
+<td class="center">60</td>
+</tr>
+
+<tr>
+<td class="left padl1">After 18 hours equilibration</td>
+<td class="center">39</td>
+<td class="center">43</td>
+<td class="center">44</td>
+<td class="center">39</td>
+<td class="center">46</td>
+</tr>
+
+<tr>
+<td class="left padl1">After freezing to -79&deg; C. and<br />immediate thawing</td>
+<td class="center">28</td>
+<td class="center">34</td>
+<td class="center">34</td>
+<td class="center">29</td>
+<td class="center">24</td>
+</tr>
+
+<tr class="bb">
+<td class="left padl1">After 4 days at -79&deg; C.</td>
+<td class="center">23</td>
+<td class="center">26</td>
+<td class="center">26</td>
+<td class="center">25</td>
+<td class="center">27</td>
+</tr>
+
+</table>
+
+<div class="footnote"><p><a name="FootnoteK" id="FootnoteK"></a><a href="#FNanchorK"><span class="label">[K]</span></a> Glycerol
+level in the final frozen mixture was 7 percent. Sugars were added to a level
+of 1.25 percent.</p></div>
+
+<p><a name="Sec5_6" id="Sec5_6"></a><span class='pagenum'><a name="Page_22" id="Page_22">[22]</a></span><b>Substitutes
+for glycerol.</b> Since glycerol was so effective in protecting
+sperm during freezing, many have assumed that related compounds
+might be even better. Several compounds, some related to glycerol and
+some not, have been tried as substitutes for glycerol in the freezing
+procedure. They include ethylene glycol, propylene glycol, trimethylene
+glycol, mannitol, sorbitol, dextrans, and seminal-plasma proteins.
+None of these materials has been as effective as glycerol in protecting
+sperm during freezing. In fact, several of the materials proved to be
+injurious to sperm prior to attempts to freeze the samples. While the
+work in our laboratory with these substances as glycerol substitutes
+was by no means finally conclusive, because of the many possible interactions
+of experimental conditions, sufficient data were gathered to
+lead us to abandon further study until greater promise of success
+might be evident.</p>
+
+
+<hr class="c25" />
+<h2><a name="Ch6" id="Ch6"></a>FREEZING RATE</h2>
+
+<p><a name="Sec6_1" id="Sec6_1"></a><b>Effect of freezing rate on sperm survival.</b> Reports by one group
+of British workers in early trials on freezing bull semen indicated that
+the rate of cooling in freezing should not exceed 2&deg; C. per minute
+between +5&deg; and -15&deg; C., although below -15&deg; C. the rate could
+be faster. Another group expressed the view that semen could be
+plunged into dry ice at -79&deg; C. after it had been cooled to -15&deg; C.
+To clarify this part of the freezing procedure, 11 samples of semen
+were subdivided and portions of each were frozen at rates of 0.25&deg;,
+0.5&deg;, 1.0&deg;, 2.0&deg;, and 4.0&deg; C. drop per minute between +5&deg; and -20&deg;
+C. and then twice these rates between -20&deg; and -79&deg; C. Vials of
+each ejaculate at +5&deg; C. were also plunged directly into an alcohol
+bath at -79&deg; C. The samples which were cooled at the rates of 0.25&deg;,
+0.5&deg;, 1.0&deg;, 2.0&deg;, and 4.0&deg; C. per minute had the following percentages
+of motile sperm after thawing: 30, 40, 46, 44, and 44. A mean of 32
+percent of the sperm in the samples that were plunged directly into an
+alcohol bath at -79&deg; C. were motile after thawing. There were no
+statistically significant differences among the samples frozen at 1.0&deg;,
+2.0&deg; or 4.0&deg; C. per minute. All of the others had significantly lower
+survival rates. Thus, it is obvious that too slow a cooling rate and
+plunging the samples directly into a -79&deg; C. bath from a temperature
+of +5&deg; C. cause greater harm to the sperm than cooling at a rate
+between 1.0&deg; and 4.0&deg; C. per minute.</p>
+
+<p>Some investigators have suggested that rapid cooling below -20&deg;
+C. is not detrimental to frozen semen. This idea was tested in conjunction
+with other experiments. Twenty-five samples cooled slowly (2&deg;<span class='pagenum'><a name="Page_23" id="Page_23">[23]</a></span>
+C. per minute to -28&deg; C., then 4&deg; C. per minute to -79&deg; C.) showed
+62 percent sperm survival compared with only 45 percent when cooled
+rapidly below -28&deg; C. (2&deg; C. per minute to -28&deg; C. then plunged
+into bath at -79&deg; C.). Thus, rapid cooling was detrimental even after
+the critical temperature range of +5&deg; C. to -20&deg; C. had been passed.</p>
+
+<div class="figcenter"><a name="Fig4" id="Fig4"></a><img src="images/illo004.png" alt="Fig. 4" width="400" height="466" />
+
+<table class="cap400" summary="Caption Table 4">
+
+<tr>
+<td colspan="2" class="left">Cooling rates of diluted semen samples in plastic vials and in glass</td>
+</tr>
+
+<tr>
+<td class="left">ampules</td>
+<td class="right">(Fig. 4)</td>
+</tr>
+
+</table>
+</div>
+
+<p><a name="Sec6_2" id="Sec6_2"></a><b>Rate of cooling in plastic and in glass.</b> Plastic vials do not conduct
+the cold as rapidly as glass ampules do. The temperature in both glass
+and plastic containers tends to lag behind the change in the bath in
+which they are immersed as is shown in <a href="#Fig4">Figure 4</a>.</p>
+
+<p>Temperatures in the immersion bath were recorded in a 2-milliliter
+glass ampule containing 1 milliliter diluted semen and in an 8-milliliter
+plastic vial containing 2.5 milliliters of diluted semen. A second plastic
+vial and glass ampule filled to capacity with diluted semen showed a
+cooling rate almost identical to that shown in <a href="#Fig4">Figure 4</a>. It was obvious
+from the comparison that samples in the plastic vials cooled slower
+than those in glass and that the volume of semen (at least the small
+volumes used) in the vials had little effect on the rate of cooling. In
+another experiment, it was shown that the volume of diluted semen
+in the ampule to be frozen (0.2, 1.0 or 5.0 ml.) had little or no effect
+on the survival of the sperm.</p>
+
+<hr class="c25" />
+<p class='pagenum'><a name="Page_24" id="Page_24">[24]</a></p>
+<h2><a name="Ch7" id="Ch7"></a>STORAGE TEMPERATURE</h2>
+
+<p>In freezing and storing bull sperm, an alcohol bath containing dry
+ice at a temperature of -79&deg; C. has been used as a cooling agent. In
+many areas, the availability of dry ice is limited and the cost is rather
+high. Mechanical means are available for obtaining temperatures as
+low as, or lower than, -79&deg; C. but for the most part they are expensive.
+If warmer temperatures were suitable for storing frozen semen,
+the ordinary deep-freeze, which operates at -15&deg; to -25&deg; C., might
+be used.</p>
+
+<p><a name="Sec7_1" id="Sec7_1"></a><b>Storage at temperatures from -23&deg; to -79&deg; C.</b> In testing the
+effects of storage temperatures on the survival of frozen bull sperm
+(in a diluent containing 7 percent glycerol), 9 ejaculates were frozen
+and kept at -23&deg;, -37&deg;, -51&deg;, -65&deg;, and -79&deg; C. The desired
+temperatures were maintained by dropping pieces of dry ice into ethyl
+alcohol baths as needed. Samples were thawed after 1 hour, 1 day, 3
+days, and 5 days. After 1 hour, the samples maintained at the various
+temperatures exhibited approximately equal motility (<a href="#Fig5">Fig. 5</a>).</p>
+
+<div class="figcenter"><a name="Fig5" id="Fig5"></a><img src="images/illo005.png" alt="Fig 5" width="600" height="466" />
+
+<table class="cap600" summary="Caption fig. 5">
+
+<tr>
+<td colspan="2" class="left">Effect of freezing and storing bull sperm at various temperatures on the
+sperm motility at thawing</td>
+</tr>
+
+<tr>
+<td class="left">(average of 9 ejaculates)</td>
+<td class="right">(Fig. 5)</td>
+</tr>
+
+</table>
+</div>
+
+<p><span class='pagenum'><a name="Page_25" id="Page_25">[25]</a></span>At
+the end of 1 day, samples stored at -79&deg; C. exhibited approximately
+the same motility as did similar samples stored for 1 hour. The
+samples stored at -65&deg; C. had declined slightly in motility and those
+maintained at -51&deg; C. had only one-third the motility which they had
+displayed at 1 hour. The samples at -23&deg; and -37&deg; C. exhibited
+practically no motility after 1 day in storage. After 5 days, only 3 of
+the 8 ejaculates stored at -51&deg; C. showed motility upon thawing.
+Apparently detrimental changes take place more rapidly when the
+samples are stored at temperatures warmer than -65&deg; C. The nature
+of these changes has not been determined. Reports from other laboratories
+indicate that storage temperatures much lower than -79&deg; C.
+are just as satisfactory as -79&deg; C.</p>
+
+<p>No tests of the effects of storage at -79&deg; C. for periods longer
+than 51 days have been conducted in this laboratory. Portions of 12
+ejaculates were frozen and stored at -79&deg; C. for various periods. One
+portion of each of these was examined on the second, ninth, 16th and
+51st day of storage. The percent of motile sperm and rate of motility
+at each of these examinations were as follows:</p>
+
+<table summary="Table P 25">
+
+<tr>
+<td class="left">Day</td>
+<td class="right">2</td>
+<td>&nbsp;</td>
+<td class="right">9</td>
+<td>&nbsp;</td>
+<td class="right">16</td>
+<td>&nbsp;</td>
+<td class="right">51</td>
+<td>&nbsp;</td>
+</tr>
+
+<tr>
+<td class="left padr2">Percent of motile sperm</td>
+<td class="right">49</td>
+<td>&nbsp;</td>
+<td class="right">46</td>
+<td>&nbsp;</td>
+<td class="right">40</td>
+<td>&nbsp;</td>
+<td class="right">38</td>
+<td>&nbsp;</td>
+</tr>
+
+<tr>
+<td class="left">Rate of motility</td>
+<td class="right padr0">2</td>
+<td class="left padl0 padr2">.5</td>
+<td class="right padr0">2</td>
+<td class="left padl0 padr2">.3</td>
+<td class="right padr0">2</td>
+<td class="left padl0 padr2">.2</td>
+<td class="right padr0">2</td>
+<td class="left padl0 padr2">.2</td>
+</tr>
+
+</table>
+
+<p>The average prefreezing motility percentage for the above samples was
+58, with an average rate of motility of 2.9. It is apparent from these
+results that the loss in motility was greatest due to the initial freezing,
+and after that the drop was most pronounced during the first 16 days
+of storage.</p>
+
+<p>The British and the Australians have both reported the successful
+maintenance of fertility in frozen semen stored at -79&deg; C. for over
+two years.<a href="#Footnote5" class="fnanchor">[5]</a></p>
+
+<p><a name="Sec7_2" id="Sec7_2"></a><b>Use of higher glycerol levels and a -20&deg; C. storage temperature.</b>
+In 1953, a report from Arkansas suggested that warmer storage temperatures
+could be used if a high percentage of glycerol were included
+in the freezing mixture.<a href="#Footnote7" class="fnanchor">[7]</a> To test the effectiveness of various glycerol
+levels on protecting sperm stored at deep-freeze temperatures, glycerol
+levels of 3.5, 5.5, 7.5, and 9.5 percent were used with portions of 4
+semen samples. Survival in the portions frozen and stored at -20&deg; C.
+was poor compared with the portions reduced and held at -79&deg; C. In
+a second experiment, 4 samples were subdivided and frozen with a
+final concentration of 7, 11, 15, and 19 percent glycerol in the semen-diluent
+mixture. In this trial, poor results were obtained at -20&deg; C.<span class='pagenum'><a name="Page_26" id="Page_26">[26]</a></span>
+except that glycerol at a level of 19 percent protected the sperm more
+effectively than at lower levels. Maximal survival at -79&deg; C. was
+obtained at the 7-percent glycerol level. A final trial was run, using
+glycerol levels of 7, 11, 15, 19, 23, 27, and 31 percent. The percentages
+of motile sperm present after storage at -79&deg; C. and -20&deg; C. are
+shown in <a href="#Tab14">Table 14</a>.</p>
+
+<h5><a name="Tab14" id="Tab14"></a>Table 14.&mdash;Effect of Glycerol Level and Storage Temperature<br />
+on Freezability of Semen</h5>
+
+<p class="tabhead">(Average of 8 ejaculates)</p>
+
+<table class="fsize80" summary="Table P 26">
+
+<tr>
+<td rowspan="2" class="center bt2 bb padr1 padl1">Storage<br />temperature<br />(&deg;C.)</td>
+<td rowspan="2" class="center bt2 bb padr1 padl1">Glycerol<br />level<br />(percent)</td>
+<td colspan="2" class="center bt2 bb padr1 padl1">Sperm&nbsp;motility&nbsp;after&nbsp;storage&nbsp;(percent)</td>
+</tr>
+
+<tr>
+<td class="center bb padr1 padl1">18&nbsp;hours</td>
+<td class="center bb padr1 padl1">42&nbsp;hours</td>
+</tr>
+
+<tr>
+<td class="center">-79</td>
+<td class="right padr4">7</td>
+<td class="right padr8">61</td>
+<td class="right padr8">61</td>
+</tr>
+
+<tr>
+<td class="center">-20</td>
+<td class="right padr4">7</td>
+<td class="right padr8">2</td>
+<td class="right padr8">1</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="right padr4">11</td>
+<td class="right padr8">3</td>
+<td class="right padr8">1</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="right padr4">15</td>
+<td class="right padr8">14</td>
+<td class="right padr8">10</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="right padr4">19</td>
+<td class="right padr8">30</td>
+<td class="right padr8">22</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="right padr4">23</td>
+<td class="right padr8">29</td>
+<td class="right padr8">19</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="right padr4">27</td>
+<td class="right padr8">25</td>
+<td class="right padr8">18</td>
+</tr>
+
+<tr class="bb">
+<td>&nbsp;</td>
+<td class="right padr4">31</td>
+<td class="right padr8">21</td>
+<td class="right padr8">12</td>
+</tr>
+
+</table>
+
+<p>While survival was fair over a short period of time with 19 percent
+glycerol at -20&deg; C., deterioration was rapid during storage. After 18
+hours of storage, the samples at -20&deg; C. (19 percent glycerol) contained
+only one half as many motile sperm as were still present in the
+samples at -79&deg; C. (7 percent glycerol). After 42 hours of storage,
+the best samples at -20&deg; C. contained only one-third the number of
+motile sperm still present in the samples stored at -79&deg; C. These trials
+leave little doubt that under the present system of freezing and storing,
+storage at ordinary deep-freeze temperatures is far inferior to
+storage at dry-ice temperatures.</p>
+
+<hr class="c25" />
+<h2><a name="Ch8" id="Ch8"></a>THAWING</h2>
+
+
+<p>The importance of carefully controlled cooling and storage has
+been emphasized in the foregoing sections. The need for controlling
+thawing rates and the temperature of thawing was not clearly defined
+in the early work on freezing bull semen. The British used a thawing
+temperature of 40&deg; C., which was satisfactory. If there is a need to
+hold the semen for a time after thawing, then a lower thawing temperature
+might be more desirable so that cooling again will not be
+necessary.<span class='pagenum'><a name="Page_27" id="Page_27">[27]</a></span></p>
+
+<p><a name="Sec8_1" id="Sec8_1"></a><b>Comparison of thawing temperatures of 5&deg; C. and 38&deg; C.</b> The
+effects of thawing at temperatures of 38&deg; (body temperature) and
+5&deg; C. (refrigerator temperature) were investigated. The first trial
+involved thawing as rapidly as possible by dropping glass ampules of
+frozen semen into water baths at the two temperatures. The frozen
+semen samples contained glycerol levels of 4, 6, 8, and 10 percent. The
+mean percentages of motile sperm found after thawing thirteen diluted
+semen samples treated in this manner are shown in <a href="#Fig6">Figure 6</a>.</p>
+
+<div class="figcenter"><a name="Fig6" id="Fig6"></a><img src="images/illo006.png" alt="Fig. 6" width="400" height="444" />
+
+<table class="cap400" summary="Caption Fig. 6">
+
+<tr>
+<td colspan="2" class="left">Effect of glycerol percentage and thawing temperature on sperm</td>
+</tr>
+
+<tr>
+<td class="left">motility after freezing and thawing</td>
+<td class="right">(Fig. 6)</td>
+</tr>
+
+</table>
+</div>
+
+<p>The 5&deg; C. thawing temperature resulted in a higher percentage of
+sperm survival at all the glycerol levels than 38&deg; C., with the difference
+in favor of 5&deg; C. becoming greater as the glycerol level increased.
+The reason for the interaction between glycerol level and thawing
+temperature is not known. It may be that the presence of the higher
+levels of glycerol at 38&deg; C. brought about harmful metabolic activity.
+The difference in survival of sperm in semen thawed at 5&deg; C. and at
+38&deg; C. continued during storage at 5&deg; C. (<a href="#Tab15">Table 15</a>). It was also
+evident that the interaction between glycerol level and thawing temperature
+continued during storage (<a href="#Fig7">Fig. 7</a>).</p>
+
+<p class='pagenum'><a name="Page_28" id="Page_28">[28]</a></p>
+
+<div class="figcenter"><a name="Fig7" id="Fig7"></a><img src="images/illo007.png" alt="Fig. 7" width="400" height="462" />
+
+<table class="cap400" summary="Caption Fig. 7">
+
+<tr>
+<td colspan="2" class="left">Effect of thawing temperature on sperm motility during storage at 5&deg;</td>
+</tr>
+
+<tr>
+<td class="left">C. following freezing and thawing</td>
+<td class="right">(Fig. 7)</td>
+</tr>
+
+</table>
+</div>
+
+<h5><a name="Tab15" id="Tab15"></a>Table 15.&mdash;Effect of Glycerol Level, Thawing Temperature,<br />
+and Storage at 5&deg; C. After Thawing on Sperm Motility</h5>
+
+<p class="tabhead">(Average of 13 ejaculates)</p>
+
+<table class="fsize80" summary="Table P 28">
+
+<tr class="bt2">
+<td rowspan="3" class="center bb padl1 padr1">Thawing<br />temperature<br />(&deg; C.)</td>
+<td rowspan="3" class="center bb padl1 padr1">Glycerol<br />level<br />(percent)</td>
+<td colspan="4" class="center bb padl1 padr1">Sperm&nbsp;motility&nbsp;(percent)</td>
+</tr>
+
+<tr>
+<td rowspan="2" class="center bb padl1 padr1">Post-<br />thawing</td>
+<td colspan="2" class="center bb padl1 padr1">After&nbsp;storage&nbsp;at&nbsp;5&deg;&nbsp;C.</td>
+<td rowspan="2" class="center bb padl1 padr1">Average</td>
+</tr>
+
+<tr>
+<td class="center bb padl1 padr1">1 day</td>
+<td class="center bb padl1 padr1">3 days</td>
+</tr>
+
+<tr>
+<td class="right padr4">38</td>
+<td class="center">4</td>
+<td class="right padr3">28.5</td>
+<td class="right padr3">17.3</td>
+<td class="right padr4">5.1</td>
+<td class="right padr3">17.0</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="center">6</td>
+<td class="right padr3">31.5</td>
+<td class="right padr3">22.4</td>
+<td class="right padr4">9.2</td>
+<td class="right padr3">21.1</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="center">8</td>
+<td class="right padr3">33.1</td>
+<td class="right padr3">15.0</td>
+<td class="right padr4">4.6</td>
+<td class="right padr3">17.6</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="center">10</td>
+<td class="right padr3">19.5</td>
+<td class="right padr3">3.6</td>
+<td class="right padr4">0.8</td>
+<td class="right padr3">8.0</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="center">Average</td>
+<td class="right padr3">28.2</td>
+<td class="right padr3">14.6</td>
+<td class="right padr4">4.9</td>
+<td class="right padr3">12.2</td>
+</tr>
+
+<tr>
+<td class="right padr4">5</td>
+<td class="center">4</td>
+<td class="right padr3">29.2</td>
+<td class="right padr3">21.7</td>
+<td class="right padr4">19.8</td>
+<td class="right padr3">23.9</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="center">6</td>
+<td class="right padr3">37.7</td>
+<td class="right padr3">33.8</td>
+<td class="right padr4">23.5</td>
+<td class="right padr3">31.7</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="center">8</td>
+<td class="right padr3">41.5</td>
+<td class="right padr3">33.1</td>
+<td class="right padr4">17.3</td>
+<td class="right padr3">30.6</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="center">10</td>
+<td class="right padr3">33.1</td>
+<td class="right padr3">18.5</td>
+<td class="right padr4">6.0</td>
+<td class="right padr3">19.2</td>
+</tr>
+
+<tr class="bb">
+<td>&nbsp;</td>
+<td class="center">Average</td>
+<td class="right padr3">35.4</td>
+<td class="right padr3">26.8</td>
+<td class="right padr4">16.6</td>
+<td class="right padr3">20.6</td>
+</tr>
+
+</table>
+
+<p>It is obvious that motility falls off rapidly after the semen is
+thawed. In a field trial in which the initial intent was to test the effect
+of glycerol levels on fertility of frozen semen, the semen was thawed in
+the morning and used during the same day. Survival of the sperm with
+4 percent glycerol was so poor that only a few breedings were made
+with these samples. Even at 7 and 10 percent, the fertility results were
+much lower than with semen that had not been subjected to freezing.
+At that time it was felt that thawing the samples and using them
+throughout the day may have caused the low fertility results. Since<span class='pagenum'><a name="Page_29" id="Page_29">[29]</a></span>
+then, a large-scale experiment by Cornell University investigators, in
+cooperation with the New York Artificial Breeders&#8217; Cooperative, has
+shown definitely that thawing should be delayed until a few minutes
+prior to breeding.<a href="#Footnote11" class="fnanchor">[11]</a>
+If the semen is used immediately, a thawing temperature
+of either 5&deg; or 38&deg; C. appears to be suitable. However, there
+is less danger of cold shock due to recooling if 5&deg; C is used.</p>
+
+<p><a name="Sec8_2" id="Sec8_2"></a><b>Thawing rate in plastic and in glass.</b> Glass ampules transmit cold
+or heat more readily than plastic ones. The temperature rise is rapid in
+both glass and plastic when samples are taken from the storage box at
+-79&deg; C. and placed in water at 5&deg; C. However, complete thawing
+occurs more rapidly in glass than in plastic ampules. The changes in
+temperature that occurred when glass and plastic ampules were
+thawed in a water bath at 5&deg; C. are shown in <a href="#Fig8">Figure 8</a>. The initial
+temperature rise for the first minute or two was about the same, then
+the rate of warming in the plastic slowed and actual melting of the
+frozen sample occurred a little over a minute later in the plastic than
+it did in the glass. Both were thawed in less than four minutes.</p>
+
+<div class="figcenter"><a name="Fig8" id="Fig8"></a><img src="images/illo008.png" alt="Fig. 8" width="400" height="532" />
+
+<table class="cap400" summary="Caption Fig. 8">
+
+<tr>
+<td colspan="2" class="left">Warming rates of diluted
+semen samples in plastic vials
+and in glass</td>
+</tr>
+
+<tr>
+<td class="left">ampules</td>
+<td class="right">(Fig. 8)</td>
+</tr>
+
+</table>
+</div>
+
+<hr class="c25" />
+<p class='pagenum'><a name="Page_30" id="Page_30">[30]</a></p>
+
+<h2><a name="Ch9" id="Ch9"></a>EFFECTS OF FREEZING PROCEDURES ON METABOLIC
+ACTIVITY OF BULL SPERM</h2>
+
+<p>By finding how methods of handling affect the sperm cells, one can
+sometimes improve the procedures to avoid harmful effects. Some
+attempts have been made in this laboratory to determine the effects of
+the freezing procedures on the metabolic activity of bull spermatozoa.
+These investigations have been limited in scope, involving the measurement
+of oxygen-consumption and estimates of sperm motility during
+and after incubation at 37&deg; C. in a Warburg apparatus.</p>
+
+<p><a name="Sec9_1" id="Sec9_1"></a><b>Effect of glycerol additions on oxygen uptake of diluted semen at
+37&deg; C.</b> The effect of adding glycerol to diluted semen on oxygen consumption
+of the sperm was tested in a Warburg apparatus, using
+semen diluted with an extender consisting of one part egg yolk and one
+part 2.9 percent sodium citrate dihydrate. The yolk-citrate extender
+was added to the semen at a rate which brought the sperm concentration
+in 0.5 milliliter to 200 million to 500 million. An exact count was
+used to calculate the oxygen uptake per 10<span class="expon">8</span> sperm per hour (ZO<sub>2</sub>).</p>
+
+<div class="figcenter"><a name="Fig9" id="Fig9"></a><img src="images/illo009.png" alt="Fig. 9" width="600" height="404" />
+
+<table class="cap600" summary="Caption Fig. 9">
+
+<tr>
+<td class="left">Effect of glycerol additions on oxygen consumption of sperm at 37&deg; C.</td>
+<td class="right">(Fig. 9)</td>
+</tr>
+
+</table>
+</div>
+
+<p>Glycerol in various percentages in 2.9 percent sodium citrate
+dihydrate solution was placed in the sidearm of the Warburg flasks.<span class='pagenum'><a name="Page_31" id="Page_31">[31]</a></span>
+The diluted semen was held in the main compartment. After a 60-minute
+preliminary run, in which the rate of oxygen uptake of the
+sperm in yolk-citrate diluent was determined, the contents of the
+sidearm were tipped into the main compartment. The resulting
+glycerol percentages after mixing the sidearm and main compartment
+contents were 0, 4, 8, and 12 percent. Ten samples of semen were
+subsampled and the oxygen uptake of each was determined at all four
+levels of glycerol.</p>
+
+<p>Oxygen uptake was increasingly stimulated during the first 20-minute
+interval by each increase in the amount of glycerol added
+(<a href="#Fig9">Fig. 9</a>). After the first 20 minutes, the rate of oxygen utilization
+decreased at the two higher levels of glycerol but persisted at 4 percent.
+The rate of oxygen consumption for the first 20-minute period
+at the 4-percent glycerol level was 130 percent that of the control to
+which only sodium citrate had been added. At 8 and 12 percent the
+values for the period were 144 and 192 percent, respectively, of the
+control rate.</p>
+
+<div class="figcenter"><a name="Fig10" id="Fig10"></a><img src="images/illo010.png" alt="Fig. 10" width="600" height="479" />
+
+<table class="cap600" summary="Caption Fig. 10">
+
+<tr>
+<td colspan="2" class="left">Effect of glycerol and glycerol-plus-catalase additions on sperm motility
+during incubation at 37&deg; C.</td>
+</tr>
+
+<tr>
+<td>&nbsp;</td>
+<td class="right">(Fig. 10)</td>
+</tr>
+
+</table>
+</div>
+
+<p><span class='pagenum'><a name="Page_32" id="Page_32">[32]</a></span>With each increase in glycerol level, motility was reduced during
+the incubation period. This is shown in <a href="#Fig10">Figure 10</a> along with the effect
+on motility of adding catalase, which is discussed in the next section.</p>
+
+<p><a name="Sec9_2" id="Sec9_2"></a><b>Effect of glycerol-plus-catalase on oxygen uptake of diluted
+semen.</b> Certain bacteria have been shown to break glycerol down,
+forming hydrogen peroxide as follows:</p>
+
+<p class="center">Glycerol + oxygen &#8594; lactic acid + hydrogen peroxide.</p>
+
+<p>Hydrogen peroxide is known to be detrimental to sperm. The addition
+of glycerol to diluted semen first increased oxygen uptake and then reduced
+it. Since a reduction in sperm survival followed, some harmful
+action must have taken place with the addition of glycerol at 37&deg; C. To
+test whether this action could be due to the release of hydrogen peroxide
+as occurs in certain bacteria, glycerol with catalase&mdash;the enzyme
+which breaks down hydrogen peroxide&mdash;was added to a portion of
+8 diluted semen samples and the oxygen uptake was recorded. Comparison
+of the resulting oxygen uptake with glycerol and with glycerol
+plus catalase is shown in <a href="#Fig11">Figure 11</a>.</p>
+
+<div class="figcenter"><a name="Fig11" id="Fig11"></a><img src="images/illo011.png" alt="Fig. 11" width="600" height="401" />
+
+<table class="cap600" summary="Caption Fig. 11">
+
+<tr>
+<td class="left">Effect of additions of glycerol-plus-catalase on oxygen consumption of
+sperm at 37&deg; C.</td>
+<td class="right">(Fig. 11)</td>
+</tr>
+
+</table>
+</div>
+
+<p>Oxygen consumption was increased by the presence of added<span class='pagenum'><a name="Page_33" id="Page_33">[33]</a></span>
+catalase at all glycerol levels and in the control. Sperm survival during
+the 3-hour period at 37&deg; C. also was improved by the presence of
+catalase (<a href="#Fig10">Fig. 10</a>). However, the general trend in oxygen consumption
+produced by the addition of glycerol was not changed greatly. The
+higher levels of glycerol still stimulated oxygen uptake during the
+first 20-minute period after the additions and then slowed the rate of
+oxygen utilization. The rate of utilization was generally higher during
+the test period in the presence of catalase than without added catalase.
+It appeared that a part of the harmful effect of glycerol might be due
+to the formation of hydrogen peroxide. Still, the detrimental effects of
+the higher levels of glycerol were not completely removed.</p>
+
+<h5><a name="Tab16" id="Tab16"></a>Table 16.&mdash;Effect of Freezing Procedures on Oxygen Utilization<br />
+of Bull Sperm in Yolk-Citrate Extender</h5>
+
+<p class="tabhead">(Average of 5 ejaculates)</p>
+
+<table class="fsize80" summary="Table P 33">
+
+<tr class="bt2">
+<td rowspan="2" class="center bb padl1 padr1">Semen&nbsp;sample&nbsp;tested</td>
+<td colspan="4" class="center bb padl1 padr1">Microliters&nbsp;of&nbsp;oxygen<br />utilized&nbsp;per&nbsp;10<span
+class="expon">8</span>&nbsp;sperm</td>
+</tr>
+
+<tr>
+<td colspan="2" class="center bb padl1 padr1">First&nbsp;hour</td>
+<td colspan="2" class="center bb padl1 padr1">Second&nbsp;hour</td>
+</tr>
+
+<tr>
+<td class="left">Fresh&nbsp;diluted&nbsp;semen</td>
+<td class="right">10.3</td>
+<td>&nbsp;</td>
+<td class="right">8.1</td>
+<td>&nbsp;</td>
+</tr>
+
+<tr>
+<td class="left">Fresh&nbsp;diluted&nbsp;semen&nbsp;glycerol&nbsp;tipped&nbsp;in&nbsp;at&nbsp;end&nbsp;of&nbsp;first&nbsp;hour</td>
+<td class="right">9.7</td>
+<td class="left"><a href="#FootnoteL" class="fnanchor">[L]</a></td>
+<td class="right">12.9</td>
+<td class="left"><a href="#FootnoteL" class="fnanchor">[L]</a></td>
+</tr>
+
+<tr>
+<td class="left">Aged&nbsp;20&nbsp;to&nbsp;24&nbsp;hours&nbsp;at&nbsp;5&deg;&nbsp;C.</td>
+<td class="right">11.2</td>
+<td>&nbsp;</td>
+<td class="right">8.3</td>
+<td>&nbsp;</td>
+</tr>
+
+<tr>
+<td class="left">Aged&nbsp;20&nbsp;to&nbsp;24&nbsp;hours&nbsp;at&nbsp;5&deg;&nbsp;C.&nbsp;glycerol&nbsp;tipped&nbsp;in&nbsp;at&nbsp;
+end&nbsp;of&nbsp;first&nbsp;hour</td>
+<td class="right">11.8</td>
+<td class="left"><a href="#FootnoteL" class="fnanchor">[L]</a></td>
+<td class="right">12.9</td>
+<td class="left"><a href="#FootnoteL" class="fnanchor">[L]</a></td>
+</tr>
+
+<tr>
+<td class="left">After&nbsp;20&nbsp;hours&nbsp;equilibration&nbsp;with&nbsp;glycerol</td>
+<td class="right">11.7</td>
+<td class="left"><a href="#FootnoteL" class="fnanchor">[L]</a></td>
+<td class="right">7.8</td>
+<td class="left"><a href="#FootnoteL" class="fnanchor">[L]</a></td>
+</tr>
+
+<tr class="bb">
+<td class="left">After&nbsp;freezing&nbsp;and&nbsp;thawing</td>
+<td class="right">9.7</td>
+<td>&nbsp;</td>
+<td class="right">6.3</td>
+<td>&nbsp;</td>
+</tr>
+
+</table>
+
+<div class="footnote"><p><a name="FootnoteL" id="FootnoteL"></a><span class="label">[L]</span> Average of 3 ejaculates.</p></div>
+
+<p><a name="Sec9_3" id="Sec9_3"></a><b>Effect of freezing procedures on oxygen utilization by sperm.</b>
+Limited data have been obtained on the effects of some of the freezing
+procedures on the oxygen utilization of bull sperm. The results obtained
+in these experiments confirmed the earlier findings that tipping
+glycerol directly into the diluted semen at 37&deg; C. caused an increase in
+oxygen consumption (<a href="#Tab16">Table 16</a>). All other steps in the freezing procedure
+had little effect on oxygen consumption by the sperm. Except
+where glycerol was added during the determination, the rate of oxygen
+utilization was lower the second hour than during the first. The
+oxygen uptake of semen that had been frozen and thawed seemed to
+drop faster than that of unfrozen samples.</p>
+
+<p><a name="Sec9_4" id="Sec9_4"></a><b>Effect of freezing procedures on methylene-blue reduction time.</b>
+The methylene-blue reduction test has been used as a means of measuring
+semen quality and is dependent on the metabolic activity of the<span class='pagenum'><a name="Page_34" id="Page_34">[34]</a></span>
+sperm. The effects of various freezing procedures on the ability of
+samples to decolorize methylene blue were determined with 10 semen
+samples. Sperm numbers were standardized to 300 &times; 10<span class="expon">6</span> cells per
+milliliter and the time required for these cells to reduce a 1:40,000
+solution of methylene blue was determined on freshly diluted semen,
+after the addition of glycerol, after equilibration, and after freezing
+and thawing. Portions of each diluted sample were tested at these
+stages of the procedure with glycerol alone added and with glycerol
+and various sugars added.</p>
+
+<p>A marked increase in the time required for the sperm to reduce
+methylene blue occurred when the glycerol was added (<a href="#Tab17">Table 17</a>).
+This increase was greatest in the portions with glycerol alone and with
+glycerol and glucose. The time increase was less pronounced in the
+presence of the three pentose sugars used. Following equilibration, the
+samples regained the ability to reduce methylene blue at a rate only
+slightly slower than when they were fresh. Freezing and storage of
+semen resulted in slower reduction of the methylene blue than was
+shown after equilibration with glycerol. Since freezing usually kills
+some of the sperm, a slowing of the reduction time after freezing
+would be expected.</p>
+
+<h5><a name="Tab17" id="Tab17"></a>Table 17.&mdash;Effect of Freezing Procedures on the Methylene-Blue<br />
+Reduction Time of Bull Semen With and Without
+the Addition of Sugars<a name="FNanchor_M_13" id="FNanchor_M_13"></a><a href="#Footnote_M_13" class="fnanchor">[M]</a></h5>
+
+<p class="tabhead">(Average of 10 ejaculates)</p>
+
+<table class="fsize80" summary="Table P 34">
+
+<tr class="bt2">
+<td rowspan="2" class="bb">&nbsp;</td>
+<td colspan="5" class="center bb">Methylene-blue&nbsp;reduction&nbsp;time&nbsp;(minutes)</td>
+</tr>
+
+<tr>
+<td class="center padl1 padr1 bb">Glycerol<br />only</td>
+<td class="center padl1 padr1 bb">Glycerol<br />and<br />glucose</td>
+<td class="center padl1 padr1 bb">Glycerol<br />and<br />arabinose</td>
+<td class="center padl1 padr1 bb">Glycerol<br />and<br />xylose</td>
+<td class="center padl1 padr1 bb">Glycerol<br />and<br />rhamnose</td>
+</tr>
+
+<tr>
+<td class="left">Fresh&nbsp;semen</td>
+<td class="right padr3">5.2</td>
+<td class="right padr3">5.2</td>
+<td class="right padr3">5.2</td>
+<td class="right padr3">5.2</td>
+<td class="right padr3">5.2</td>
+</tr>
+
+<tr>
+<td class="left">After&nbsp;glycerolization</td>
+<td class="right padr3">26.4</td>
+<td class="right padr3">25.2</td>
+<td class="right padr3">17.3</td>
+<td class="right padr3">14.3</td>
+<td class="right padr3">19.4</td>
+</tr>
+
+<tr>
+<td class="left">After&nbsp;18&nbsp;hours&nbsp;equilibration</td>
+<td class="right padr3">7.4</td>
+<td class="right padr3">6.5</td>
+<td class="right padr3">6.4</td>
+<td class="right padr3">5.3</td>
+<td class="right padr3">6.2</td>
+</tr>
+
+<tr>
+<td class="left">Thawed&nbsp;immediately&nbsp;after&nbsp;freezing</td>
+<td class="right padr3">11.5</td>
+<td class="right padr3">10.5</td>
+<td class="right padr3">9.4</td>
+<td class="right padr3">9.0</td>
+<td class="right padr3">9.4</td>
+</tr>
+
+<tr class="bb">
+<td class="left">Thawed&nbsp;48&nbsp;hours&nbsp;after&nbsp;freezing</td>
+<td class="right padr3">14.3</td>
+<td class="right padr3">10.2</td>
+<td class="right padr3">11.3</td>
+<td class="right padr3">10.1</td>
+<td class="right padr3">9.5</td>
+</tr>
+
+</table>
+
+<div class="footnote"><p><a name="Footnote_M_13" id="Footnote_M_13"></a><a href="#FNanchor_M_13"><span class="label">[M]</span></a> Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level
+of 1.25 percent.</p></div>
+
+
+<hr class="c25" />
+<p class='pagenum'><a name="Page_35" id="Page_35">[35]</a></p>
+<h2><a name="Ch10" id="Ch10"></a>PRACTICAL FREEZING PROCEDURE</h2>
+
+<p>Good results usually can be obtained in freezing bull semen if care
+is taken in collecting, diluting and processing the semen. Occasionally
+the semen from certain bulls will not withstand freezing well. The
+reason for this is not understood at present. However, carefully following
+the directions and suggestions given below will usually produce
+satisfactory results with semen samples that are of good quality
+at the start.</p>
+
+<p>Experience in the field has shown that fertility results with frozen
+semen are usually slightly lower during the first few months than with
+liquid semen stored at 5&deg; C. (41&deg; F.). Most units that have worked
+with frozen semen over a period of a few months are able to improve
+and do get fertility results as good as, or better than, obtained in their
+liquid semen program.</p>
+
+<p><a name="Sec10_1" id="Sec10_1"></a><b>Collection of the semen.</b> In order to obtain the best possible semen
+for freezing, care and cleanliness should be exercised in making the
+collection. The artificial vagina, and the glassware used should be
+clean and dry. The underline of the bull should also be clean and dry.
+The bull should be restrained near the teaser cow for a minute or two
+prior to collection in order to excite the flow of secretions prior to
+ejaculation. Allowing the bull to mount the teaser once without serving
+the artificial vagina is a good practice to use in properly stimulating
+the bull before collection of the semen.</p>
+
+<p>If the bull has not been used for three or four days, the collection
+of a second ejaculate for freezing may be advisable. The second
+ejaculate seems to withstand freezing better than the first in many
+instances. A clean, dry artificial vagina should be used for each
+ejaculate collected. Repeated collections in the same artificial vagina
+may result in contamination of the semen with bacteria, lubricating
+jelly and minute particles of dirt. The semen sample should be protected
+from contamination and from sudden temperature drops (cold
+shock).</p>
+
+<p><a name="Sec10_2" id="Sec10_2"></a><b>Preparation of extender.</b> A suitable egg yolk-citrate extender for
+freezing bull semen can be prepared by the following procedure. One
+part egg yolk (free of egg white and the membrane surrounding the
+yolk) is mixed with 4 parts 2.4 to 2.9 percent sodium citrate
+dihydrate solution. The citrate is prepared with distilled water and<span class='pagenum'><a name="Page_36" id="Page_36">[36]</a></span>
+then boiled or autoclaved. The citrate solution should be cooled before
+it is mixed with the egg yolk. After the egg and citrate are
+mixed, 1000 units of penicillin and 1000 micrograms of streptomycin
+are added per milliliter of extender. Sulfanilamide should not be added.
+This extender can be prepared 12 to 24 hours before use if it is stored
+at refrigerator temperature. The portion of the extender needed for the
+original dilution of the semen should be warmed to room temperature
+before it is mixed with the semen.</p>
+
+<p><a name="Sec10_3" id="Sec10_3"></a><b>Dilution after collection.</b> As soon as possible after collection, the
+semen sample should be diluted with the extender. The extender must
+be at the same temperature as the semen (room temperature) when
+the two are mixed together. At this time the semen can be partially
+diluted (1 part semen to 4 parts of extender) or diluted to a sperm
+concentration twice the final desired concentration (later in adding the
+glycerol for freezing, the semen is diluted further with an equal
+volume of glycerol containing extender). The diluted semen is slowly
+cooled (1<span class="expon">1</span>&#8260;<span class="index">2</span> to
+2<span class="expon">1</span>&#8260;<span class="index">2</span> hours) to 5&deg; C. (41&deg; F.). Some units using frozen
+semen now allow the semen to stand at 5&deg; C. for 5 to 6 hours before
+glycerolization to allow the antibiotics to be more effective against any
+vibrio fetus organisms that may be present. This step is taken because
+it has been shown that glycerol inhibits the effectiveness of the antibiotics.<a href="#Footnote6" class="fnanchor">[6]</a>
+After cooling, semen can be further diluted to twice the
+desired sperm concentration if that were not done at the start.
+(Caution: Be sure semen and diluent are at the same temperature.)</p>
+
+<p><a name="Sec10_4" id="Sec10_4"></a><b>Adding the glycerol.</b> The glycerol solution is prepared by adding
+14 volumes of glycerol (reagent grade) to 86 volumes of yolk-citrate
+diluent (same as yolk-citrate used for original dilution). This solution
+may be added dropwise with constant gentle mixing to the already
+diluted semen, or one-third at a time at 10-minute intervals with
+gentle mixing during each addition. Either method should take about
+20 to 30 minutes. The total volume of glycerol-yolk-citrate solution
+added should be equal to the volume of the original diluted semen.
+In this way a concentration of 7 percent glycerol is obtained in the
+final mixture that is to be frozen. Care must be taken to keep the
+temperature at 5&deg; C. (41&deg; F.) during the time the glycerol is being
+added. (A cold room is best for maintaining a temperature of 5&deg; C.,
+but with care the operation can be carried out at room temperature by
+using pans of ice water and a refrigerator.)</p>
+
+<p><a name="Sec10_5" id="Sec10_5"></a><b>Equilibration.</b> The results
+presented in this bulletin suggest that<span class='pagenum'><a name="Page_37" id="Page_37">[37]</a></span>
+little or no time need be allowed after the glycerol is added before
+freezing. However, results obtained by other workers show improved
+fertility with at least 12 hours equilibration. Some units getting good
+fertility results with frozen semen also are allowing the semen to
+stand at 5&deg; C. for 12 to 18 hours before freezing. After the semen has
+equilibrated with the glycerol, 1-milliliter portions of the mixture are
+placed in 1.2- to 2-milliliter vials or ampules which are then sealed.
+Ampuling can be done with an automatic syringe or pipette, provided
+a large gage needle is used. Also, it is important not to force the
+fluid mixture rapidly through the syringe or the sperm may be injured.</p>
+
+<p><a name="Sec10_6" id="Sec10_6"></a><b>Freezing.</b> The vials or ampules of diluted semen are placed in a
+bath of isopropyl alcohol which has been cooled to 5&deg; C. (41&deg; F.).
+This bath can be a wide-mouth thermos bottle or an insulated container
+of almost any sort with a large opening at the top. The size
+needed depends on the number of ampules being frozen. Some sort of
+convenient tray for holding the ampules in an orderly fashion and
+enabling the samples to be completely submerged is desirable. A few
+ampules can be kept together easily by placing them in a polyethylene
+freezer bag that has had many small holes cut in it to let the alcohol
+of the bath contact the ampules. The ampules must be completely
+covered by the alcohol to insure uniform cooling.</p>
+
+<p>The alcohol of the bath and the ampules of semen are cooled by
+adding chipped or ground dry ice in sufficient amounts to lower the
+temperature of the bath 2&deg; C. (3.6&deg; F.) per minute from +5&deg; to -20&deg;
+C. From -20&deg; down to -79&deg; C., the rate of cooling can be doubled
+(4&deg; C. or 7.2&deg; F.). Electrical equipment that regulates the cooling rate
+to the desired temperatures is available commercially, but the cost may
+be too high for some small operations. The samples should be held at
+-79&deg; C. (-110&deg; F.) until they are thawed. This can be done by
+using an alcohol bath and dry ice or by special mechanical refrigerating
+equipment. At no time prior to thawing should the samples be
+exposed to warmer temperatures.</p>
+
+<p><a name="Sec10_7" id="Sec10_7"></a><b>Thawing.</b> The ampules of frozen semen can be thawed by removing
+them from the dry ice storage box and dropping them into a water
+bath at 5&deg; C. (41&deg; F.). Thawing temperatures up to body temperature,
+38&deg; C. (100&deg; F.), can be used but extreme care must then be taken
+not to pass the semen through a cold inseminating tube; for this would
+subject the sperm to cold shock. The semen should be used for breeding
+within a few minutes after thawing.</p>
+
+
+<hr class="c25" />
+<p class='pagenum'><a name="Page_38" id="Page_38">[38]</a></p>
+<h2><a name="Ch11" id="Ch11"></a>LITERATURE CITED</h2>
+
+<div class="footnote"><p><a name="Footnote1" id="Footnote1"></a><a href="#FNanchor1"><span class="label">[1]</span></a>
+<span class="smcap">Davenport, C. B.</span> Effect of chemical and physical agents upon protoplasm.
+Macmillan and Co., New York. 1897.</p></div>
+
+<div class="footnote"><p><a name="Footnote2" id="Footnote2"></a><span class="label">[2]</span>
+<span class="smcap">Polge, C.</span>, and <span class="smcap">Parkes, A. S.</span> Possibilities of long-term storage of spermatozoa
+at low temperatures. Anim. Breeding Abs. <b>20</b>:1-5. 1952.</p></div>
+
+<div class="footnote"><p><a name="Footnote3" id="Footnote3"></a><a href="#FNanchor3"><span class="label">[3]</span></a>
+<span class="smcap">Emmens, C. W.</span>, and <span class="smcap">Blackshaw, A. W.</span> The low temperature storage of
+ram, bull, and rabbit spermatozoa. Austral. Vet. Jour. <b>26</b>:226. 1950.</p></div>
+
+<div class="footnote"><p><a name="Footnote4" id="Footnote4"></a><a href="#FNanchor4"><span class="label">[4]</span></a>
+<span class="smcap">Smith, Audrey W.</span> Effects of low temperatures on living cells and tissues.
+In biological applications of freezing and drying. Ed. R. J. C. Harris.
+Academic Press, Inc., New York, 1954.</p></div>
+
+<div class="footnote"><p><a name="Footnote5" id="Footnote5"></a><span class="label">[5]</span>
+<span class="smcap">Emmens, C. W.</span>, and <span class="smcap">Blackshaw, A. W.</span> Artificial insemination. Physiol.
+Rev. <b>36</b>:277-306. 1956.</p></div>
+
+<div class="footnote"><p><a name="Footnote6" id="Footnote6"></a><span class="label">[6]</span>
+Proceedings of the National Association of Artificial Breeders, 1953, 1954,
+and 1955.</p></div>
+
+<div class="footnote"><p><a name="Footnote7" id="Footnote7"></a><span class="label">[7]</span>
+Proceedings of the American Dairy Science Association, 1953, 1954, and
+1955. Published in the June issue of the Journal of Dairy Science for
+each year.</p></div>
+
+<div class="footnote"><p><a name="Footnote8" id="Footnote8"></a><a href="#FNanchor8"><span class="label">[8]</span></a>
+<span class="smcap">Barker, C. A. V.</span> Low temperature preservation of bovine epididymal
+spermatozoa. Canad. Jour. Comp. Med. <b>18</b>:390-393. 1954.</p></div>
+
+<div class="footnote"><p><a name="Footnote9" id="Footnote9"></a><a href="#FNanchor9"><span class="label">[9]</span></a>
+<span class="smcap">Saroff, Jack</span>, and <span class="smcap">Mixner, J. P.</span> The relationship of egg yolk and glycerol
+content of diluters and glycerol equilibration time to survival of bull
+spermatozoa after low temperature freezing. Jour. Dairy Sci. <b>38</b>:292-297.
+1955.</p></div>
+
+<div class="footnote"><p><a name="Footnote10" id="Footnote10"></a><a href="#FNanchor10"><span class="label">[10]</span></a>
+<span class="smcap">Cragle, R G., Myers, R. M., Waugh, R. K., Hunter, J. S.</span>, and <span class="smcap">Anderson,
+R. L.</span> The effects of various levels of sodium citrate, glycerol, and equilibration
+time on survival of bovine spermatozoa after storage at -79&deg; C.
+Jour. Dairy Sci. <b>38</b>:508-514. 1955.</p></div>
+
+<div class="footnote"><p><a name="Footnote11" id="Footnote11"></a><a href="#FNanchor11"><span class="label">[11]</span></a>
+<span class="smcap">Bratton, R. W., Foote, R. H.</span>, and <span class="smcap">Cruthers, Joan C.</span> Preliminary fertility
+results with frozen bovine spermatozoa. Jour. Dairy Sci. <b>38</b>:40-46. 1955.</p></div>
+
+<div class="footnote"><p><a name="Footnote12" id="Footnote12"></a><a href="#FNanchor12"><span class="label">[12]</span></a>
+<span class="smcap">Hafs, H. D.</span>, and <span class="smcap">Elliott, F. I.</span> The effects of methods of adding egg yolk
+and monosaccharides on the survival of frozen bull spermatozoa. Jour.
+Dairy Sci. <b>38</b>:811-815. 1955.</p></div>
+
+<div class="footnote"><p><a name="Footnote13" id="Footnote13"></a><a href="#FNanchor13"><span class="label">[13]</span></a>
+<span class="smcap">Miller, W. J.</span>, and <span class="smcap">VanDemark, N. L.</span> The influence of glycerol level,
+various temperature aspects, and certain other factors on the survival of
+bull spermatozoa at sub-zero temperatures. Jour. Dairy Sci. <b>37</b>:45-51.
+1954.</p></div>
+
+<hr class="c25" />
+<p class='pagenum'><a name="Page_39" id="Page_39">[39]</a></p>
+<h2><a name="Ch12" id="Ch12"></a>TEMPERATURE CONVERSIONS</h2>
+
+<table summary="Table P 39">
+
+<tr>
+<td class="right padr2">&deg;C.</td>
+<td class="right padr2">&deg;F.</td>
+</tr>
+
+<tr>
+<td class="right padr2">+38</td>
+<td class="right padr2">+100</td>
+</tr>
+
+<tr>
+<td class="right padr2">+35</td>
+<td class="right padr2">+95</td>
+</tr>
+
+<tr>
+<td class="right padr2">+30</td>
+<td class="right padr2">+86</td>
+</tr>
+
+<tr>
+<td class="right padr2">+25</td>
+<td class="right padr2">+77</td>
+</tr>
+
+<tr>
+<td class="right padr2">+20</td>
+<td class="right padr2">+68</td>
+</tr>
+
+<tr>
+<td class="right padr2">+15</td>
+<td class="right padr2">+59</td>
+</tr>
+
+<tr>
+<td class="right padr2">+10</td>
+<td class="right padr2">+50</td>
+</tr>
+
+<tr>
+<td class="right padr2">+5</td>
+<td class="right padr2">+41</td>
+</tr>
+
+<tr>
+<td class="right padr2">0</td>
+<td class="right padr2">+32</td>
+</tr>
+
+<tr>
+<td class="right padr2">-5</td>
+<td class="right padr2">+23</td>
+</tr>
+
+<tr>
+<td class="right padr2">-10</td>
+<td class="right padr2">+14</td>
+</tr>
+
+<tr>
+<td class="right padr2">-15</td>
+<td class="right padr2">+5</td>
+</tr>
+
+<tr>
+<td class="right padr2">-18</td>
+<td class="right padr2">0</td>
+</tr>
+
+<tr>
+<td class="right padr2">-20</td>
+<td class="right padr2">-4</td>
+</tr>
+
+<tr>
+<td class="right padr2">-25</td>
+<td class="right padr2">-13</td>
+</tr>
+
+<tr>
+<td class="right padr2">-30</td>
+<td class="right padr2">-22</td>
+</tr>
+
+<tr>
+<td class="right padr2">-35</td>
+<td class="right padr2">-31</td>
+</tr>
+
+<tr>
+<td class="right padr2">-40</td>
+<td class="right padr2">-40</td>
+</tr>
+
+<tr>
+<td class="right padr2">-45</td>
+<td class="right padr2">-49</td>
+</tr>
+
+<tr>
+<td class="right padr2">-50</td>
+<td class="right padr2">-58</td>
+</tr>
+
+<tr>
+<td class="right padr2">-55</td>
+<td class="right padr2">-67</td>
+</tr>
+
+<tr>
+<td class="right padr2">-60</td>
+<td class="right padr2">-76</td>
+</tr>
+
+<tr>
+<td class="right padr2">-65</td>
+<td class="right padr2">-85</td>
+</tr>
+
+<tr>
+<td class="right padr2">-70</td>
+<td class="right padr2">-94</td>
+</tr>
+
+<tr>
+<td class="right padr2">-75</td>
+<td class="right padr2">-103</td>
+</tr>
+
+<tr>
+<td class="right padr2">-79</td>
+<td class="right padr2">-110</td>
+</tr>
+
+</table>
+
+<hr class="c25" />
+
+<div class="bbox"><a name="TN" id="TN"></a>
+
+<p class="center">Transcriber's Notes:</p>
+
+<p>The original text has not been modified, except that some minor typographical errors have been corrected silently.</p>
+
+<p>Lettered footnotes (with anchors [A], [B], etc.), explaining the text, have been moved to directly below the paragraph or table
+they refer to. Numbered footnotes ([1], [2], etc.) refer to references, that are listed towards the end of the text.</p>
+
+<p>Where a single footnote is referenced more than once, the backwards link to the anchor has not been implemented.</p>
+
+<p class="center">Back to <a href="#Start">top</a> of document.</p>
+
+</div>
+
+
+
+
+
+
+
+
+<pre>
+
+
+
+
+
+End of the Project Gutenberg EBook of Preservation of Bull Semen at Sub-Zero
+Temperatures, by N. L. VanDemark and W. J. Miller and W. C. Kinney, Jr. and Carlos Rodriguez and M. E. Friedman
+
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@@ -0,0 +1,1981 @@
+The Project Gutenberg EBook of Preservation of Bull Semen at Sub-Zero
+Temperatures, by N. L. VanDemark and W. J. Miller and W. C. Kinney, Jr. and Carlos Rodriguez and M. E. Friedman
+
+This eBook is for the use of anyone anywhere at no cost and with
+almost no restrictions whatsoever.  You may copy it, give it away or
+re-use it under the terms of the Project Gutenberg License included
+with this eBook or online at www.gutenberg.org
+
+
+Title: Preservation of Bull Semen at Sub-Zero Temperatures
+
+Author: N. L. VanDemark
+        W. J. Miller
+        W. C. Kinney, Jr.
+        Carlos Rodriguez
+        M. E. Friedman
+
+Release Date: August 11, 2011 [EBook #37041]
+
+Language: English
+
+Character set encoding: ASCII
+
+*** START OF THIS PROJECT GUTENBERG EBOOK PRESERVATION OF BULL SEMEN ***
+
+
+
+
+Produced by Bryan Ness, Harry Lame and the Online
+Distributed Proofreading Team at http://www.pgdp.net (This
+file was produced from images generously made available
+by The Internet Archive/American Libraries.)
+
+
+
+
+
+
+
+
+
+  +------------------------------------------------------------------+
+  |                       TRANSCRIBER'S NOTES:                       |
+  |                                                                  |
+  | * Transcription used in this e-text:                             |
+  |   _text_ indicates italics;                                      |
+  |   =text= indicates bold-face text;                               |
+  |   ^{text} indicates superscript text;                            |
+  |   _{text} indicates subscript text.                              |
+  | * Lettered footnotes (with anchors [A], [B], etc.), explaining   |
+  |   the text, have been moved to directly below the paragraph or   |
+  |   table they refer to. Footnotes with anchors [1], [2], etc.     |
+  |   refer to references, that are listed towards the end of the    |
+  |   text.                                                          |
+  | * The original text has not been modified, except that some minor|
+  |   typographical errors have been corrected silently.             |
+  |                                                                  |
+  +------------------------------------------------------------------+
+
+
+
+
+                              PRESERVATION
+                              OF BULL SEMEN
+                        AT SUB-ZERO TEMPERATURES
+
+
+     By N. L. VanDemark
+        W. J. Miller
+        W. C. Kinney, Jr.
+        Carlos Rodriguez
+        M. E. Friedman
+
+
+     Bulletin 621
+
+
+  UNIVERSITY OF ILLINOIS
+  AGRICULTURAL EXPERIMENT STATION
+
+
+
+
+CONTENTS
+
+
+                                                                Page
+
+  EARLY WORK ON FREEZING SEMEN                                     5
+
+  SELECTION OF SEMEN FOR FREEZING                                  6
+  Predicting freezability                                          6
+  Freezability of first and second ejaculates                      7
+  Freezability of several consecutive ejaculates                   7
+  Freezability of epididymal sperm                                 9
+  Freezability of washed sperm                                    10
+
+  EXTENDERS FOR FREEZING BULL SEMEN                               10
+  Proportion of egg yolk in final diluent                         10
+  Citrate level in final diluent                                  11
+  Storing and freezing diluent                                    12
+  Other diluents                                                  13
+
+  DILUTION RATES                                                  14
+  Effect of further dilution and refreezing                       14
+
+  GLYCEROL ADDITIONS                                              17
+  Effect of glycerol on sperm survival at 5 deg. C.                   17
+  Glycerol levels for freezing semen                              18
+  Rate, temperature, and method of adding glycerol                18
+  Allowing sperm to equilibrate with glycerol                     20
+  Sugar additions and equilibration time                          20
+  Substitutes for glycerol                                        22
+
+  FREEZING RATE                                                   22
+  Effect of freezing rate on sperm survival                       22
+  Rate of cooling in plastic and in glass                         23
+
+  STORAGE TEMPERATURE                                             24
+  Storage at -23 deg. to -79 deg. C.                                      24
+  Use of higher glycerol levels and storage at -20 deg. C.            25
+
+  THAWING                                                         26
+  Comparison of thawing at 5 deg. C. and at 38 deg. C.                    27
+  Thawing rate in plastic and in glass                            29
+
+  EFFECTS OF FREEZING PROCEDURES ON METABOLIC ACTIVITY OF SPERM   30
+  Effect of glycerol additions on oxygen uptake of diluted semen
+    at 37 deg. C.                                                     30
+  Effect of glycerol-plus-catalase on oxygen uptake of diluted
+    semen                                                         32
+  Effect of freezing procedures on oxygen utilization             33
+  Effect of freezing procedures on methylene-blue reduction time  33
+
+  PRACTICAL FREEZING PROCEDURE                                    35
+  Collection of semen                                             35
+  Preparation of extender                                         35
+  Dilution after collection                                       36
+  Adding the glycerol                                             36
+  Equilibration                                                   36
+  Freezing                                                        37
+  Thawing                                                         37
+
+  LITERATURE CITED                                                38
+
+  TEMPERATURE CONVERSION TABLE                                    39
+
+
+
+
+  Urbana, Illinois         October, 1957
+
+Publications in the Bulletin series report the results of investigations
+made or sponsored by the Experiment Station
+
+
+
+
+ACKNOWLEDGMENT
+
+
+The authors gratefully acknowledge the financial assistance given to the
+Department of Dairy Science in support of these investigations by the
+Southern Illinois Breeding Association of Breese, Illinois, and the
+Northern Illinois Breeding Co-op of Hampshire, Illinois, through the
+Illinois Dairy Breeding Federation.
+
+
+
+
+THE PRESERVATION OF BULL SEMEN AT SUB-ZERO TEMPERATURES
+
+By N. L. VANDEMARK, W. J. MILLER, W. C. KINNEY, JR., CARLOS RODRIGUEZ,
+and M. E. FRIEDMAN[A]
+
+  [A] This publication was prepared by N. L. VANDEMARK, Professor of
+  Dairy Physiology. Research reported herein was carried out
+  cooperatively by the senior author and W. J. MILLER, W. C. KINNEY,
+  JR., CARLOS RODRIGUEZ, and M. E. FRIEDMAN, formerly members of the
+  Department of Dairy Science.
+
+
+The first announcement in 1951 of the successful impregnation of a cow
+with bull semen that had been frozen stimulated much interest and
+research in freezing as a method of preserving bull semen. Research
+during the years following 1951 resulted in considerable progress and
+success in using freezing as a means of holding semen for long periods
+of time without loss of fertility.
+
+Between 1952 and 1957, research on many aspects of the preservation of
+bull semen at sub-zero temperatures was carried out in the Department of
+Dairy Science at the University of Illinois. Many of these
+investigations have aided in perfecting the freezing technique that has
+been adapted for practical use. Some of these findings have been
+published, but many items have gone unreported except for general
+references at scientific meetings.
+
+It is the purpose of this bulletin to bring together the results of
+several experiments carried out in connection with the freezing of bull
+semen and to present a practical freezing procedure based on the results
+of these experiments and findings at other institutions. Persons
+interested in the development of the procedures and the reasons why
+certain steps are necessary in freezing semen will find the details in
+the first part of this bulletin. Those interested only in the freezing
+procedure may turn to page 35 where a practical method of freezing semen
+is described.
+
+
+
+
+EARLY WORK ON FREEZING SEMEN
+
+
+While it has been known for a long time that various types of tissues
+and organisms can withstand freezing and are even preserved by freezing,
+the first attempts at freezing sperm cells were made before the turn of
+the century. In 1897, Davenport[1][B] found that human sperm would
+withstand freezing. For thirty to forty years after that, little
+attention was paid to freezing as a possible means of preserving semen.
+An excellent review of the early attempts to freeze sperm has been
+assembled by Polge and Parkes.[2] These investigators also gave a good
+account of their work at the British National Institute of Medical
+Research in London, where in 1949 they demonstrated that glycerol would
+protect fowl sperm so that it would survive freezing. The next year they
+found that bull sperm and the sperm of several other species were
+protected by glycerol during freezing. During the same year, Emmens and
+Blackshaw[3] showed that ram and bull sperm would survive freezing. In
+1951 frozen semen was used to produce a calf in England and a lamb in
+Australia.
+
+  [B] These numbers refer to literature citations on page 38.
+
+The highlights in the development of frozen semen have been covered by
+other reviews and reports. Interested persons will find the articles of
+Polge and Parkes[2] and Smith[4] especially good on the early history
+and theoretical aspects of freezing sperm. Later progress on the
+freezing procedure has been reviewed and covered in a number of detailed
+reports.[5],[6],[7] Many items not covered in those articles have been
+assembled here.
+
+
+
+
+SELECTION OF SEMEN FOR FREEZING
+
+
+One of the first considerations in freezing semen is that of deciding
+which semen samples are to be frozen. Since preservation of the
+semen--the maintenance of the potential motility and especially the
+fertility of the sperm--is the primary aim, some attention should be
+directed to the kind of semen sample that will withstand freezing. Do
+the initial characteristics of the sample indicate whether the sperm
+will withstand freezing? Does maturity of the sperm affect their
+freezability?
+
+=Predicting freezability.= Estimates of semen quality in the past have
+been based in part on the numbers of sperm present in a fresh sample and
+on the percentage and rate of motility shown by the sperm. These
+characteristics were used to determine the relationship between the
+original concentration of sperm (in the fresh, undiluted sample), the
+percentage and rate of sperm motility in the diluted samples just prior
+to freezing, and the percentage and rate of sperm motility following
+freezing and thawing. From data collected before and after freezing and
+thawing 54 ejaculates, it was found that there was not a significant
+correlation between the number of sperm present in the original sample
+and the percent of motile sperm present after freezing and thawing (r =
+0.03). A highly significant correlation (r = 0.45) was found, however,
+between the percentages before freezing and after thawing. While this
+correlation coefficient was highly significant, its magnitude indicates
+that only about one fifth of the variation in percentage of motile sperm
+observed after freezing was accounted for by the motility of the sperm
+prior to freezing.
+
+=Freezability of first and second ejaculates.= In the early days of
+artificial breeding in this country, it was commonly believed that a
+second ejaculate collected a few minutes after the first resulted in a
+larger ejaculate containing more sperm. With the development of the
+procedure of stimulating sexual excitement by restraint prior to
+collecting semen, this difference between first and second ejaculates
+has been greatly reduced. Still it was noted that second ejaculates
+frequently withstood freezing better than first ejaculates, even though
+restraint and stimulation of the bull occurred prior to collection of
+the first ejaculate. During the course of a number of experiments, it
+was possible to compare the freezability of 2 ejaculates that were
+collected a few minutes apart from the same bull. Two consecutive
+ejaculates were obtained one or more times from 24 bulls so that a total
+of 58 comparisons could be made. The mean prefreezing and post-thawing
+percentages of motile sperm in first and second ejaculates are presented
+in Table 1.
+
+An analysis of variance showed that in this comparison the differences
+between first and second ejaculates in sperm survival during freezing
+were highly significant. A later comparison of 27 first and second
+ejaculates from 26 bulls did not show as great a difference between
+first and second ejaculates in their ability to withstand freezing
+(Table 1).
+
+Table 1.--Comparison of the Freezability of First and Second Ejaculates
+Taken a Few Minutes Apart From the Same Bull
+
+  =====================================================================
+  Number     Number               Prefreezing  Post-thawing
+    of         of      Ejaculate   motility      motility    Survival
+  bulls    ejaculates              (percent)     (percent)   (percent)
+  ---------------------------------------------------------------------
+  24           58         1st         60            39          65
+                          2d          62            45          74
+  26           27         1st         60            36          60
+                          2d          65            40          61
+  ---------------------------------------------------------------------
+
+=Freezability of several consecutive ejaculates.= The fact that second
+ejaculates sometimes withstood freezing better than first ejaculates
+suggested that the maturity of the sperm might be a factor affecting
+freezability. An opportunity to check this idea came when 20
+consecutive ejaculates were collected from each of 6 bulls within a
+4-hour period. The sperm in samples collected in this manner might be
+expected to be less mature with each additional collection.
+
+The results obtained in freezing several consecutive ejaculates are
+shown in Figure 1 as averages for the ejaculates from 6 bulls. In same
+instances, there was an insufficient quantity of semen available to test
+the freezability. (Procedure: Diluted to 30 x 10^{6} sperm per ml. with
+1:1 yolk-citrate, then cooled and glycerolated with an equal volume of
+14 percent glycerol (percent by volume) in 2.9 percent sodium citrate.
+Final sperm concentration, 15 x 10^{6}. Equilibration time, 15 hours.
+Freezing rate, 2 deg. C. per minute from +5 deg. to -19 deg. C. then 4 deg. C. per
+minute from -19 deg. to -79 deg. C. Held frozen for 5 or more hours then thawed
+in water at 5 deg. C. and checked for motility.)
+
+[Illustration: Percent of motile sperm before and after freezing
+consecutive ejaculates collected within a 4-hour period from each of 6
+bulls (Fig. 1)]
+
+In general, the motility before freezing improved slightly from the
+first to the fourth to sixth ejaculate and then declined until about the
+12th or 14th ejaculate, at which point the prefreezing motility seemed
+to level off through the 20th ejaculate (Fig. 1). The percentage of
+motile sperm found after freezing and thawing followed the same trend at
+an average level 10 to 15 percent lower than the prefreezing level. As
+is readily seen from the trend lines in Fig. 1, the difference between
+the prefreezing motility and the post-thawing motility increased
+gradually after about the fifth ejaculate. Although the absolute
+difference did not increase greatly, the percentage of survival after
+freezing dropped from 81 percent on the first 5 ejaculates to 26.5
+percent on the last 5 (Table 2).
+
+Table 2.--Comparison of the Freezability of 20 Consecutive Ejaculates
+Collected Within a 4-Hour Period
+
+(Weighted averages for 6 bulls)
+
+  ===================================================================
+                   Number     Prefreezing   Post-thawing
+  Ejaculate          of        motility       motility     Survival
+                 ejaculates    (percent)     (percent)     (percent)
+  -------------------------------------------------------------------
+  1st to 5th         29           53.3          43.2          81.0
+  6th to 10th        26           43.8          30.2          69.0
+  11th to 15th       23           28.6          14.5          50.7
+  16th to 20th       19           18.1           4.8          26.5
+  -------------------------------------------------------------------
+
+=Freezability of epididymal sperm.= Since the freezability of bull semen
+seemed to be better in second than in first ejaculates and some
+improvement in freezability was evident through the first 4 to 6
+ejaculates taken consecutively, the question of whether epididymal sperm
+would withstand freezing seemed to be important. Although when 20
+collections were made, the later ejaculates no doubt contained fewer
+mature sperm, the lowered freezability could have been due to accessory
+gland secretions rather than changes in the sperm themselves. Removing
+sperm directly from the epididymis would eliminate any effect that the
+accessory gland secretions could be exerting. Further, if epididymal
+sperm could be frozen, obtaining and using semen from a bull shortly
+after his death should be possible.
+
+Table 3.--Freezability of Sperm in Samples Taken From the Epididymides
+
+  =================================================
+            Prefreezing   Post-thawing
+  Bull       motility      motility      Survival
+            (percent)     (percent)      (percent)
+  -------------------------------------------------
+  1            50            40             80
+  2            40            13             32
+  3            60            15             25
+  4            30            15             50
+  5+6          40            25             62
+  Average      44            22             50
+  -------------------------------------------------
+
+To determine whether epididymal sperm would withstand freezing, the 12
+epididymides (cauda only) of 6 slaughtered bulls were flushed with
+saline (0.9 percent) and the sperm obtained were frozen using the same
+procedure as was used with the 20 consecutive ejaculates discussed
+earlier. Averages of the 2 epididymides from each bull are given in
+Table 3; the samples from bulls 5 and 6 were combined. From the data in
+Table 3, it is obvious that motile sperm were present after freezing and
+thawing epididymal samples. It is likely that further experience in
+handling epididymal sperm may lead to improved results. Using frozen
+epididymal sperm from 2 bulls, Canadian workers have produced confirmed
+pregnancies in 8 out of 12 cows.[8]
+
+=Freezability of washed sperm.= In the laboratory it is frequently
+desirable to study sperm free of the seminal plasma in which they are
+ejaculated. Sperm can be separated from the seminal plasma by
+centrifugation, removal of the supernatant plasma, and resuspension in a
+salt solution of known composition. Sometimes it is desirable to repeat
+the process. This tends to wash the sperm with the salt solution and
+sperm handled in this way are called washed sperm.
+
+Sperm cells centrifuged three times and washed twice in 0.9 percent
+sodium chloride solution withstood freezing well when finally
+resuspended and frozen in yolk-citrate diluent. The percentage of
+survival in three samples subjected to this treatment was 60 percent.
+Thus it appears that the seminal plasma itself is not essential for
+ejaculated sperm to survive the rigors of freezing. This is not
+surprising, since it had already been found that epididymal sperm, which
+also are free of accessory gland secretions, can withstand freezing and
+thawing.
+
+
+
+
+EXTENDERS FOR FREEZING BULL SEMEN
+
+
+Both of the extenders that are widely used in routine storage of bull
+semen at 5 deg. C. are used for freezing semen. These are the egg
+yolk-sodium citrate and whole or skimmilk extenders. Most of the
+research with extenders for freezing bull semen in this laboratory has
+been done with the yolk-citrate diluents.
+
+=Proportion of egg yolk in the final diluent.= Some early experiences
+with a diluent consisting of one part yolk and one part 2.9 percent
+sodium citrate dihydrate in distilled water showed poor sperm survival
+following freezing. The final mixture with this diluent consisted of
+about 45 percent yolk. In other attempts at adding glycerol in order to
+freeze semen, the final proportion of yolk was diminished and better
+sperm survival was obtained. Several experiments were carried out to
+test the effect of varying levels of egg yolk.
+
+In the first efforts to find the optimum level of egg yolk, the level
+of yolk in the final frozen mixture was varied from about 6 to 46
+percent. These levels were obtained by varying the proportion of yolk to
+2.9 percent citrate solution in the original extending media and also in
+the media added in glycerolating the samples.
+
+Split portions of 20 semen samples were frozen in each of the extender
+combinations indicated in Table 4. The mean percentages of motile sperm
+found before and after freezing and thawing are shown also. The highest
+percentages were found with extenders containing 23 and 24 percent yolk.
+The highest percentage of yolk, resulting when a 1:1 (yolk to citrate)
+extender was used for both extending and glycerolating, proved to be
+most detrimental to sperm survival during freezing. The lowest
+percentage of yolk used (6 percent) was not as effective in protecting
+sperm during freezing as the intermediate levels tested (Fig. 2).
+
+Table 4.--Effect of Egg-Yolk Level in Diluent on Freezability of Semen
+(Average of 20 semen samples)
+
+  =========================================================================
+                   Medium               Yolk in    Pre-      Post-     Sur-
+          ----------------------------   final    freezing  thawing   vival
+  Diluent  Extending   Glycerolating[C]   mix-    motility  motility  (per-
+          ------------ ----------------  ture[D] (percent) (percent)  cent)
+          yolk:citrate   yolk:citrate   (percent)
+  -------------------------------------------------------------------------
+  1          1:1             1:1          45.7       65        5        8
+  2          1:1             1:3          34.9       64       27       42
+  3          1:3             1:1          33.6       63       33       52
+  4          1:1             0:1          24.2       64       39       61
+  5          1:3             1:3          22.8       63       37       59
+  6          1:3             0:1          12.1       59       33       56
+  7          1:7             1:7          11.4       56       35       62
+  8          1:7             0:1           6.0       52       26       50
+  9          1:15            1:15          5.7       49       25       51
+  -------------------------------------------------------------------------
+
+  [C] This mixture included 14 percent glycerol.
+
+  [D] The average initial sperm concentration was 900 x 10^{6}/ml.
+  Sufficient extender was added to give 30 x 10^{6}/ml. at the first
+  extension. Thus the final concentration was 15 x 10^{6} sperm/ml.
+  after glycerolization.
+
+Since rather large changes in the percentages of yolk were used in this
+experiment, two further trials were conducted in which 16, 24, and 32
+percent yolk in the final mixture were compared, with the final citrate
+percentages held constant. In these tests, 16 and 24 percent yolk
+maintained sperm better at all citrate levels tried than 32 percent
+yolk. The 16 percent level was slightly better at most of the levels of
+citrate tested (Fig. 3).
+
+=Citrate level in the final diluent.= The early work of the British
+indicated that a final citrate level near 2 percent in the diluent was
+satisfactory for freezing bull sperm. Later, in a personal
+communication, Polge of the British group suggested that a citrate level
+of about 2.35 percent might be best with a final glycerol concentration
+of 7 percent. Some of the first attempts in this laboratory at
+establishing the optimum yolk-to-citrate ratios are shown in Fig. 3. In
+these experiments, the optimum levels of citrate appeared to be lower
+than anticipated from the British work. Thus a more complex experiment
+was set up to test a wider range of citrate levels using 16 and 24
+percent egg yolk in the final freezing mixture. The average percentages
+of motile sperm found after freezing 10 semen samples at each of the
+citrate and yolk levels in this experiment are shown also in Fig. 3.
+Little difference in freezability was found between citrate percentages
+of 1.55 and 1.95. When the rate of sperm motility following freezing and
+thawing was considered along with the percent of motile sperm, a slight
+advantage was found with 16 percent yolk and a citrate concentration of
+1.55 percent.
+
+[Illustration: Percent of motile sperm after freezing and thawing semen
+in diluents containing various levels of egg yolk (Fig. 2)]
+
+From the results of these experiments, and from several reports in the
+literature,[5],[6],[7],[9],[10] it appears that a diluting medium
+resulting in a final concentration of 16 to 25 percent yolk and 1.55 to
+2.2 percent sodium citrate dihydrate is highly satisfactory for
+freezing.
+
+=Storing and freezing diluent.= In some instances it would be
+advantageous to have prepared diluent on hand for use at any time. The
+suitability of stored diluent was tested with a yolk-citrate (equal
+parts yolk and citrate without antibiotics added) diluent prepared and
+stored at 5 deg. C. for 0, 2, 5, 7, and 9 days. Seven semen samples were
+diluted and frozen in these diluents. No difference was noted in the
+survival of sperm that could be attributed to the age of the diluent.
+
+[Illustration: Percent of motile sperm after freezing and thawing semen
+in diluents containing various levels of egg yolk and various
+percentages of sodium citrate (Fig. 3)]
+
+In another trial, a similar diluent (1:1 yolk to citrate with 1000 units
+of penicillin and 5000 units of streptomycin) was prepared and stored in
+the freezer compartment of a refrigerator at -15 deg. C. Upon thawing, it
+was whitish in color and more viscous than freshly prepared diluent.
+Except for the fact that the viscosity seemed to reduce the rate of
+sperm motility, this frozen diluent stored for 65 days compared
+favorably with freshly prepared diluent for freezing semen.
+
+=Other diluents.= Without the protective action of egg yolk or milk, few
+bull sperm will survive freezing. Several diluents were compared on a
+limited scale for freezing bull sperm. The results of these trials are
+compiled in Table 5. In this trial the yolk-citrate extender served best
+in maintaining sperm motility during freezing. Yolk-phosphate and
+homogenized whole milk were slightly less protective and yolk-saline
+seemed to furnish the least protection to sperm during freezing.
+
+A number of investigations in other laboratories have now proven that
+milk can be used as effectively as the yolk-citrate diluent for freezing
+bull sperm.[6],[7]
+
+Table 5.--Comparison of the Freezability of 4 Semen Samples in Different
+Extenders
+
+  ======================================================================
+                Dilution      Pre-       Post-                 Motility
+  Extender        rate      freezing   thawing     Survival    after
+                (semen:     motility   motility   (percent)   storage[E]
+                extender)   (percent)  (percent)              (percent)
+  ----------------------------------------------------------------------
+  Yolk-citrate    1:1          60         49          82          46
+                  1:10         53         45          85          36
+  Yolk-saline     1:1          57         29          51          28
+                  1:10         60         31          52          24
+  Yolk-phosphate  1:1          55         33          64          25
+                  1:10         60         43          72          25
+  Whole milk      1:1          60         40          67          35
+                  1:10         60         35          58          16
+  ----------------------------------------------------------------------
+
+  [E] Stored at 5 deg. C. for 7 hours after thawing.
+
+
+
+
+DILUTION RATES
+
+
+The first trials by the British at freezing bull semen were made with
+samples containing many millions of sperm cells. In routine artificial
+breeding, it is common to add extenders to semen so that one milliliter
+of diluted semen may contain only 10 million living sperm cells. (This
+number still insures optimal fertility.) Frequently the addition of 100
+or more parts of the yolk extender to each part of the original semen
+sample is possible without reducing the sperm numbers below 10 million
+per milliliter. No one knew if this process of dilution would affect the
+resistance of bull sperm to freezing. The effect of various rates of
+dilution on the freezability of bull sperm was tested with 10 semen
+samples. The results, presented in Table 6, show that the numbers of
+sperm between 10 and 90 million per milliliter did not influence the
+percentage of sperm that survived freezing.
+
+In a later trial it was found that sperm survival was slightly better at
+lower dilution rates than in the same samples frozen following dilution
+to 15 million sperm per milliliter. However, field trials with frozen
+semen carried out by others, using sperm numbers as low as 15 million
+per milliliter of semen inseminated or even lower, have been highly
+satisfactory.[11],[12]
+
+During the early studies in the Illinois laboratory, the effects of
+glycerol level were also tested.[13] These effects are discussed in the
+section on glycerol additions beginning on page 17.
+
+=Effect of further dilution and refreezing after the initial freezing.=
+Under some circumstances it might be advantageous to freeze semen with
+a high concentration of sperm cells and then extend it further after
+thawing. With such a procedure less storage space is needed than when
+dilution is carried to the maximum before freezing. Two experiments were
+conducted to test the effects of dilution and storage at 5 deg. C. and
+dilution and refreezing following an initial freezing of concentrated
+samples.
+
+Table 6.--Effect of Sperm Numbers and Glycerol Level in Final Mixture on
+Freezability of Bull Sperm at -79 deg. C. (Average of 10 ejaculates)
+
+  ================================================================
+                         Post-thawing motility (percent)[F]
+                     ---------------------------------------------
+  Glycerol level       Number of sperm (millions/ml.)
+  (percent)          --------------------------------
+                           90       30       10         Average
+  ----------------------------------------------------------------
+  5                       36.0     34.0     36.0         35.0
+  10                      22.0     24.0     23.0         23.0
+  15                       3.2      0.9      0.2          1.4
+  Average                 20.3     19.8     19.9         20.0
+  ----------------------------------------------------------------
+
+  [F] Mean initial motility of sperm before freezing was 55 percent.
+
+Four semen samples were split and extended at rates of 1:1 (semen to
+extender) and 1:10. These were frozen, then thawed and halved. One half
+was further extended to a level of 15 million sperm per milliliter; the
+sperm numbers in the other remained unchanged. Each of these halves was
+split again, and one portion of each was stored at 5 deg. C. for 3 to 7
+hours. The other two portions were refrozen.
+
+Table 7.--Effect of Further Dilution and Refreezing on Sperm Motility
+After the Initial Freezing of Bull Semen
+
+  ========================================================================
+                                Post-thawing motility
+  Dilution   Pre-      ---------------------------------------------------
+  of         freezing   After      After storage[G]    After refreezing[H]
+  semen      motility   first     ------------------- --------------------
+  (semen:   (percent)  freezing     No     Diluted        No     Diluted
+  extender)                      further    to 15      further    to 15
+                                 dilution million/ml   dilution million/ml
+  ------------------------------------------------------------------------
+                          First trial: 4 samples
+  1:1           60        49        46       34           31        6
+  1:10          53        45        36       30           25        5
+
+                          Second trial: 7 samples
+  1:9           67        47        41       35           28       11
+  15 million/ml 67        30        32       ..           18       ..
+  ------------------------------------------------------------------------
+
+  [G] Stored at 5 deg. C. for 3 to 7 hours after first thawing.
+
+  [H] Refrozen following first thawing.
+
+Table 8.--Effect of Glycerol Level and Storage at 5 deg. C. on Motility of
+Sperm in Yolk-Citrate Extender
+
+  ====================================================================
+                                   Sperm motility
+              --------------------------------------------------------
+                Post-        After storage at 5 deg. C.
+  Glycerol     thawing    ----------------------------      Average
+   level                   1 day      3 days     7 days
+  (percent)   ---------   ---------  ---------  ---------  ---------
+              per- rate   per- rate  per- rate  per- rate  per- rate
+              cent        cent       cent       cent       cent
+  --------------------------------------------------------------------
+  Control[I]   56  2.5     55  1.9    46  1.8    38  1.4    48  1.90
+  0            54  2.4     44  1.9    46  1.8    36  1.4    45  1.87
+  5            52  2.2     50  1.9    46  1.7    32  1.4    45  1.80
+  10           52  2.3     46  1.8    42  1.7    28  1.6    42  1.85
+  20           52  2.1     50  1.7    44  1.6    38  1.1    46  1.62
+  30           50  0.7     44  0.5    42  0.4    30  0.4    42  0.51
+  Average      53  2.03    47  1.62   44  1.50   34  1.22   ..  ....
+  --------------------------------------------------------------------
+
+  [I] The control differed from the 0-glycerol treatment in that no
+  additional citrate or glycerol solution was added.
+
+A similar trial was carried out with seven samples; one portion was
+diluted 1:9; the other was extended at the outset to 15 million sperm
+per milliliter. Results for both tests are summarized in Table 7.
+
+From Table 7 it can be seen that refreezing following an initial
+freezing further reduced the number of surviving sperm. The second
+freezing was more detrimental to the portion of the samples extended to
+15 million sperm per milliliter than to the portion that was refrozen at
+a higher sperm concentration. The percentage of motile sperm remained
+fairly high in the portions that were diluted to 15 million sperm and
+stored at 5 deg. C. However, in all cases, survival was best in the samples
+at the lower dilution levels.
+
+
+
+
+GLYCEROL ADDITIONS
+
+
+When the British procedure for freezing bull semen was first tried in
+this country, many of the refinements of the technique still had not
+been defined. It was known that glycerol worked well in protecting sperm
+during freezing. The effects of glycerol on sperm at 5 deg. C., the
+appropriate levels to use in freezing, and the manner of adding it were
+not well established. Therefore, a number of trials were conducted in an
+attempt to establish the best procedures.
+
+=Effect of glycerol on sperm survival at 5 deg. C.= Since early work
+indicated the need for adding glycerol to diluted semen in order to
+protect the sperm during freezing, it was considered important to
+determine the levels of glycerol that sperm would tolerate at 5 deg. C. Ten
+semen samples were extended 1:9 (semen to diluent) in a 1:1 yolk-citrate
+diluent (yolk to 2.9 percent sodium citrate dihydrate). Each sample was
+then split into 6 portions and an equal volume of citrate solution
+containing glycerol was added slowly to each to bring the glycerol in
+the final mixture to 0, 5, 10, 20, or 30 percent (by volume). These
+samples were stored at 5 deg. C. and examined for motile sperm after 1, 3,
+and 7 days. The effects of glycerol levels on the percentage of sperm
+surviving and the rate (or speed) of their forward motion (0 = no
+forward motion; 4 = extremely rapid progressive motility) are presented
+in Table 8.
+
+The percentage of motile sperm decreased slightly at the higher levels
+of glycerol. The most noticeable effect of the increase in glycerol
+level was the reduction in the rate of forward motion of the sperm. At
+the 30-percent level, the sperm moved slowly and could be seen to
+rotate as they moved forward. Some samples were checked after slowly
+bringing the diluent up to a level of 40 percent glycerol; the sperm
+seemed to be immobilized completely in this solution.
+
+=Glycerol levels for freezing semen.= The British procedure called for
+the use of 10 percent glycerol in the final mixture of semen and
+extender prior to freezing. Yet, as shown in Table 6, in our laboratory
+5 percent glycerol resulted in the survival of a higher percentage of
+sperm than did 10 or 15 percent. In order to define more clearly the
+optimum glycerol level, several ejaculates of semen were subsampled and
+portions were frozen after the addition of yolk-citrate extender and
+glycerol in varying quantities. From Table 9 it can be seen that
+glycerol levels of 6 and 8 percent in the final mixture resulted in
+maximum sperm survival during freezing. These results were confirmed in
+tests on the survival of sperm at 5 deg. C. storage for 3 days following
+freezing and thawing with varying glycerol levels (see Table 10).
+
+The results shown in Tables 9 and 10 were confirmed also in later
+experiments. Thirty-six samples were subjected to various levels of
+glycerol and no significant difference in freezability was found between
+6 and 8 percent. Based on these findings, a glycerol level of 7 percent
+was adopted for use in all experiments described in this bulletin,
+unless otherwise indicated. Results in a number of other laboratories
+have agreed with our findings regarding the use of approximately 7
+percent glycerol with the yolk-citrate diluent.[5],[6],[7],[9],[10] With
+milk as the extender, 10 to 13 percent glycerol has been preferred by
+some.[5],[6],[7]
+
+Table 9.--Effect of Glycerol Level on Sperm Motility After Freezing to
+-79 deg. C. and Thawing
+
+  ==========================================================
+  Glycerol    Number        Pre-       Post-      Survival
+   level        of        freezing    thawing     (percent)
+  (percent)   samples     motility    motility
+                         (percent)   (percent)
+  ----------------------------------------------------------
+      2         10          53           2            4
+      4         19          55          29           53
+      6         19          55          34           62
+      8         19          55          35           64
+     10         19          55          24           44
+     12         10          53          13           25
+  ----------------------------------------------------------
+
+Table 10.--Effect of Glycerol Level and Storage at 5 deg. C. After Thawing
+on Sperm Motility
+
+(Average of 13 ejaculates)
+
+  ================================================
+                     Sperm motility (percent)
+  Glycerol      ----------------------------------
+    level        Post-     After storage at 5 deg. C.
+  (percent)     thawing    -----------------------
+                               1 day   3 days
+  ------------------------------------------------
+     4            29             22      20
+     6            38             34      24
+     8            42             33      17
+    10            33             18       6
+  ------------------------------------------------
+
+Table 11.--Effects of Temperature, Rate of Addition of Glycerol, and
+Equilibration Time on Sperm Motility
+
+(Average of 12 ejaculates)
+
+  ===================================================================
+  Temperature                       Post-thawing motility (percent)
+    during       Equilibration     ----------------------------------
+   addition          time                 Glycerol additions
+  of glycerol       (hours)        ----------------------------------
+    ( deg. C.)                           5       3       1       Average
+  -------------------------------------------------------------------
+     4.5               2            48      48      45        47.4
+                       6            49      51      47        48.8
+                      18            46      47      46        46.3
+                      Average       47.8    48.6    46.0      47.5
+
+    10.0               2            44      43      45        43.9
+                       6            48      50      46        47.9
+                      18            43      46      42        44.0
+                      Average       45.0    46.5    44.3      45.3
+
+    15.5               2            41      38      38        39.1
+                       6            42      45      43        43.6
+                      18            42      43      42        42.5
+                      Average       42.0    41.8    41.4      41.7
+  -------------------------------------------------------------------
+
+=Rate, temperature, and method of adding glycerol.= Closely associated
+with the question of how much glycerol should be added is that of how
+the additions should be made. Originally it was believed that the
+glycerol should be added in stages so that changes would occur
+gradually. However, there would be a saving in time if the entire amount
+could be added at once. Also, if the glycerol addition could be made
+soon after the dilution with egg yolk-citrate extender at room
+temperature, time would be gained in processing the semen for use. Since
+aging _in vitro_ is known to reduce the fertilizing ability of sperm,
+every effort should be made to keep the processing time at a minimum.
+The results of an experiment involving these items, along with that of
+how much time should be allowed after the additions before freezing
+(equilibration time), are presented in Table 11. One can see that sperm
+survived freezing better when the diluted semen was cooled to 4.5 deg. C.
+before the glycerol was added. The survival at 10 deg. and 15.5 deg. C. was
+reduced with each rise in temperature. Thus, it appears that cooling to
+refrigerator temperature (4-5 deg. C.) before adding the glycerol should be
+a part of the routine procedure.
+
+A comparison of the results from adding the glycerol in 5, 3, and 1
+equal portions is given also in Table 11. Little difference in survival
+during freezing was noted between the three rates of addition. Using 3
+equal additions resulted in slightly better results, but the advantage
+was not statistically significant. While little difference was evident
+from adding the glycerol in 3 portions as compared to 1, many still use
+3 additions in the hope of obtaining a slightly better sperm survival.
+In fact, some have gone to a procedure of adding the glycerol dropwise
+with constant gentle agitation. This method has not been tested in this
+laboratory.
+
+=Allowing sperm to equilibrate with the glycerol.= Allowing sperm to
+stand in the presence of glycerol is considered by some to be necessary
+in order that the glycerol penetrate the sperm heads before freezing.
+From the first successful attempts at freezing bull sperm came the
+practice of allowing 12 to 20 hours for this process of equilibration. A
+long equilibration time results in aging the sperm. Data from a number
+of sources indicate that a drop of approximately 5 percent in fertility
+in the field occurs with each 24 hours of aging in the test tube. Thus
+it would seem desirable to reduce the equilibration time to a minimum
+commensurate with good freezability in order to reduce the effects of
+aging (at 5 deg. C.). Results of attempting to reduce equilibration time are
+given in Table 11. At 4.5 deg. C., little variation in motility following
+freezing and thawing was found after equilibration times of 2, 6, and 18
+hours. At the higher temperatures of 10 deg. and 15.5 deg. C., the shortest
+equilibration time--2 hours--was slightly more detrimental with the
+differences significant at the 5-percent level at 15.5 deg. C. For all
+temperatures combined, 6 hours was significantly better than 2 or 18
+hours.
+
+=Sugar additions and equilibration time.= Early in their experiences in
+freezing semen, the Australian workers found a short equilibration
+time--30 minutes--to be satisfactory if sugars were added to the
+diluent.[5] This protective action of sugars during the equilibration
+period was confirmed in our investigations. The results of one phase of
+this study are shown in Table 12. From these data it can be seen that
+the presence of glucose or rhamnose at a level of 1.25 percent improved
+sperm survival during the period of equilibration. In another trial
+these sugars and two others, arabinose and xylose, were tested for their
+protective action in freezing semen. The percentages of surviving sperm
+remaining after the various steps in the freezing procedure with and
+without the presence of these sugars are shown in Table 13.
+
+Table 12.--Effect of Adding Sugars to Yolk-Citrate Diluent on Sperm
+Motility During Equilibration With Glycerol[J]
+
+  ============================================================
+                               Sperm motility (percent)
+                         -------------------------------------
+  Stage when observed    Glycerol     Glycerol      Glycerol
+                           only     and glucose   and rhamnose
+  ------------------------------------------------------------
+  Fresh diluted semen       56           56            56
+  After glycerolization     54           54            54
+  After equilibration
+     2 hours                51           53            53
+     6 hours                48           52            53
+    12 hours                46           50            51
+    18 hours                40           46            46
+  ------------------------------------------------------------
+
+  [J] Glycerol level in the final frozen mixture was 7 percent. Sugars
+  were added to a level of 1.25 percent.
+
+Three of the sugars--glucose, arabinose, and rhamnose--protected the
+sperm during equilibration and freezing. Xylose was less effective, but
+its addition resulted in slightly better sperm survival than glycerol
+alone. It was found also that the methylene-blue reduction time
+(metabolic test for semen quality) was faster in samples to which the
+sugars had been added--after glycerolization, after equilibration, and
+after freezing the samples. This is confirming evidence for the presence
+of more living and actively metabolizing sperm in the portions to which
+sugars had been added.
+
+Table 13.--Effect of Adding Sugars to Yolk-Citrate Diluent on Sperm
+Motility During the Freezing Procedures[K]
+
+(Average of 10 ejaculates)
+
+  ======================================================================
+                                   Sperm motility (percent)
+                       -------------------------------------------------
+  Stage of              Glycerol  Glycerol  Glycerol  Glycerol  Glycerol
+  observation            only       and        and      and       and
+                                  glucose   arabinose  xylose   rhamnose
+  ----------------------------------------------------------------------
+  Fresh diluted semen      63        63        63        63        63
+  After glycerolization    54        55        54        57        60
+  After 18 hours
+    equilibration          39        43        44        39        46
+  After freezing to
+    -79 deg. C. and
+    immediate thawing      28        34        34        29        24
+  After 4 days at -79 deg. C.  23        26        26        25        27
+  -----------------------------------------------------------------------
+
+  [K] Glycerol level in the final frozen mixture was 7 percent. Sugars
+  were added to a level of 1.25 percent.
+
+=Substitutes for glycerol.= Since glycerol was so effective in
+protecting sperm during freezing, many have assumed that related
+compounds might be even better. Several compounds, some related to
+glycerol and some not, have been tried as substitutes for glycerol in
+the freezing procedure. They include ethylene glycol, propylene glycol,
+trimethylene glycol, mannitol, sorbitol, dextrans, and seminal-plasma
+proteins. None of these materials has been as effective as glycerol in
+protecting sperm during freezing. In fact, several of the materials
+proved to be injurious to sperm prior to attempts to freeze the samples.
+While the work in our laboratory with these substances as glycerol
+substitutes was by no means finally conclusive, because of the many
+possible interactions of experimental conditions, sufficient data were
+gathered to lead us to abandon further study until greater promise of
+success might be evident.
+
+
+
+
+FREEZING RATE
+
+
+=Effect of freezing rate on sperm survival.= Reports by one group of
+British workers in early trials on freezing bull semen indicated that
+the rate of cooling in freezing should not exceed 2 deg. C. per minute
+between +5 deg. and -15 deg. C., although below -15 deg. C. the rate could be
+faster. Another group expressed the view that semen could be plunged
+into dry ice at -79 deg. C. after it had been cooled to -15 deg. C. To clarify
+this part of the freezing procedure, 11 samples of semen were subdivided
+and portions of each were frozen at rates of 0.25 deg., 0.5 deg., 1.0 deg., 2.0 deg.,
+and 4.0 deg. C. drop per minute between +5 deg. and -20 deg. C. and then twice these
+rates between -20 deg. and -79 deg. C. Vials of each ejaculate at +5 deg. C. were
+also plunged directly into an alcohol bath at -79 deg. C. The samples which
+were cooled at the rates of 0.25 deg., 0.5 deg., 1.0 deg., 2.0 deg., and 4.0 deg. C. per
+minute had the following percentages of motile sperm after thawing: 30,
+40, 46, 44, and 44. A mean of 32 percent of the sperm in the samples
+that were plunged directly into an alcohol bath at -79 deg. C. were motile
+after thawing. There were no statistically significant differences among
+the samples frozen at 1.0 deg., 2.0 deg. or 4.0 deg. C. per minute. All of the
+others had significantly lower survival rates. Thus, it is obvious that
+too slow a cooling rate and plunging the samples directly into a -79 deg. C.
+bath from a temperature of +5 deg. C. cause greater harm to the sperm than
+cooling at a rate between 1.0 deg. and 4.0 deg. C. per minute.
+
+Some investigators have suggested that rapid cooling below -20 deg. C. is
+not detrimental to frozen semen. This idea was tested in conjunction
+with other experiments. Twenty-five samples cooled slowly (2 deg. C. per
+minute to -28 deg. C., then 4 deg. C. per minute to -79 deg. C.) showed 62 percent
+sperm survival compared with only 45 percent when cooled rapidly below
+-28 deg. C. (2 deg. C. per minute to -28 deg. C. then plunged into bath at -79 deg. C.).
+Thus, rapid cooling was detrimental even after the critical temperature
+range of +5 deg. C. to -20 deg. C. had been passed.
+
+[Illustration: Cooling rates of diluted semen samples in plastic vials
+and in glass ampules (Fig. 4)]
+
+=Rate of cooling in plastic and in glass.= Plastic vials do not conduct
+the cold as rapidly as glass ampules do. The temperature in both glass
+and plastic containers tends to lag behind the change in the bath in
+which they are immersed as is shown in Figure 4.
+
+Temperatures in the immersion bath were recorded in a 2-milliliter glass
+ampule containing 1 milliliter diluted semen and in an 8-milliliter
+plastic vial containing 2.5 milliliters of diluted semen. A second
+plastic vial and glass ampule filled to capacity with diluted semen
+showed a cooling rate almost identical to that shown in Figure 4. It was
+obvious from the comparison that samples in the plastic vials cooled
+slower than those in glass and that the volume of semen (at least the
+small volumes used) in the vials had little effect on the rate of
+cooling. In another experiment, it was shown that the volume of diluted
+semen in the ampule to be frozen (0.2, 1.0 or 5.0 ml.) had little or no
+effect on the survival of the sperm.
+
+
+
+
+STORAGE TEMPERATURE
+
+
+In freezing and storing bull sperm, an alcohol bath containing dry ice
+at a temperature of -79 deg. C. has been used as a cooling agent. In many
+areas, the availability of dry ice is limited and the cost is rather
+high. Mechanical means are available for obtaining temperatures as low
+as, or lower than, -79 deg. C. but for the most part they are expensive. If
+warmer temperatures were suitable for storing frozen semen, the ordinary
+deep-freeze, which operates at -15 deg. to -25 deg. C., might be used.
+
+=Storage at temperatures from -23 deg. to -79 deg. C.= In testing the effects of
+storage temperatures on the survival of frozen bull sperm (in a diluent
+containing 7 percent glycerol), 9 ejaculates were frozen and kept at
+-23 deg., -37 deg., -51 deg., -65 deg., and -79 deg. C. The desired temperatures were
+maintained by dropping pieces of dry ice into ethyl alcohol baths as
+needed. Samples were thawed after 1 hour, 1 day, 3 days, and 5 days.
+After 1 hour, the samples maintained at the various temperatures
+exhibited approximately equal motility (Fig. 5).
+
+[Illustration: Effect of freezing and storing bull sperm at various
+temperatures on the sperm motility at thawing (average of 9 ejaculates)
+(Fig. 5)]
+
+At the end of 1 day, samples stored at -79 deg. C. exhibited approximately
+the same motility as did similar samples stored for 1 hour. The samples
+stored at -65 deg. C. had declined slightly in motility and those maintained
+at -51 deg. C. had only one-third the motility which they had displayed at 1
+hour. The samples at -23 deg. and -37 deg. C. exhibited practically no motility
+after 1 day in storage. After 5 days, only 3 of the 8 ejaculates stored
+at -51 deg. C. showed motility upon thawing. Apparently detrimental changes
+take place more rapidly when the samples are stored at temperatures
+warmer than -65 deg. C. The nature of these changes has not been determined.
+Reports from other laboratories indicate that storage temperatures much
+lower than -79 deg. C. are just as satisfactory as -79 deg. C.
+
+No tests of the effects of storage at -79 deg. C. for periods longer than 51
+days have been conducted in this laboratory. Portions of 12 ejaculates
+were frozen and stored at -79 deg. C. for various periods. One portion of
+each of these was examined on the second, ninth, 16th and 51st day of
+storage. The percent of motile sperm and rate of motility at each of
+these examinations were as follows:
+
+  Day                       2     9    16    51
+  Percent of motile sperm  49    46    40    38
+  Rate of motility          2.5   2.3   2.2   2.2
+
+The average prefreezing motility percentage for the above samples was
+58, with an average rate of motility of 2.9. It is apparent from these
+results that the loss in motility was greatest due to the initial
+freezing, and after that the drop was most pronounced during the first
+16 days of storage.
+
+The British and the Australians have both reported the successful
+maintenance of fertility in frozen semen stored at -79 deg. C. for over two
+years.[5]
+
+=Use of higher glycerol levels and a -20 deg. C. storage temperature.= In
+1953, a report from Arkansas suggested that warmer storage temperatures
+could be used if a high percentage of glycerol were included in the
+freezing mixture.[7] To test the effectiveness of various glycerol
+levels on protecting sperm stored at deep-freeze temperatures, glycerol
+levels of 3.5, 5.5, 7.5, and 9.5 percent were used with portions of 4
+semen samples. Survival in the portions frozen and stored at -20 deg. C. was
+poor compared with the portions reduced and held at -79 deg. C. In a second
+experiment, 4 samples were subdivided and frozen with a final
+concentration of 7, 11, 15, and 19 percent glycerol in the semen-diluent
+mixture. In this trial, poor results were obtained at -20 deg. C. except
+that glycerol at a level of 19 percent protected the sperm more
+effectively than at lower levels. Maximal survival at -79 deg. C. was
+obtained at the 7-percent glycerol level. A final trial was run, using
+glycerol levels of 7, 11, 15, 19, 23, 27, and 31 percent. The
+percentages of motile sperm present after storage at -79 deg. C. and -20 deg. C.
+are shown in Table 14.
+
+Table 14.--Effect of Glycerol Level and Storage Temperature on
+Freezability of Semen
+
+(Average of 8 ejaculates)
+
+  ================================================================
+  Storage       Glycerol    Sperm motility after storage (percent)
+  temperature   level       --------------------------------------
+  ( deg.C.)         (percent)          18 hours      42 hours
+  ----------------------------------------------------------------
+    -79            7                 61            61
+    -20            7                  2             1
+                  11                  3             1
+                  15                 14            10
+                  19                 30            22
+                  23                 29            19
+                  27                 25            18
+                  31                 21            12
+  ----------------------------------------------------------------
+
+While survival was fair over a short period of time with 19 percent
+glycerol at -20 deg. C., deterioration was rapid during storage. After 18
+hours of storage, the samples at -20 deg. C. (19 percent glycerol) contained
+only one half as many motile sperm as were still present in the samples
+at -79 deg. C. (7 percent glycerol). After 42 hours of storage, the best
+samples at -20 deg. C. contained only one-third the number of motile sperm
+still present in the samples stored at -79 deg. C. These trials leave little
+doubt that under the present system of freezing and storing, storage at
+ordinary deep-freeze temperatures is far inferior to storage at dry-ice
+temperatures.
+
+
+
+
+THAWING
+
+
+The importance of carefully controlled cooling and storage has been
+emphasized in the foregoing sections. The need for controlling thawing
+rates and the temperature of thawing was not clearly defined in the
+early work on freezing bull semen. The British used a thawing
+temperature of 40 deg. C., which was satisfactory. If there is a need to
+hold the semen for a time after thawing, then a lower thawing
+temperature might be more desirable so that cooling again will not be
+necessary.
+
+=Comparison of thawing temperatures of 5 deg. C. and 38 deg. C.= The effects of
+thawing at temperatures of 38 deg. (body temperature) and 5 deg. C.
+(refrigerator temperature) were investigated. The first trial involved
+thawing as rapidly as possible by dropping glass ampules of frozen semen
+into water baths at the two temperatures. The frozen semen samples
+contained glycerol levels of 4, 6, 8, and 10 percent. The mean
+percentages of motile sperm found after thawing thirteen diluted semen
+samples treated in this manner are shown in Figure 6.
+
+[Illustration: Effect of glycerol percentage and thawing temperature on
+sperm motility after freezing and thawing (Fig. 6)]
+
+The 5 deg. C. thawing temperature resulted in a higher percentage of sperm
+survival at all the glycerol levels than 38 deg. C., with the difference in
+favor of 5 deg. C. becoming greater as the glycerol level increased. The
+reason for the interaction between glycerol level and thawing
+temperature is not known. It may be that the presence of the higher
+levels of glycerol at 38 deg. C. brought about harmful metabolic activity.
+The difference in survival of sperm in semen thawed at 5 deg. C. and at 38 deg.
+C. continued during storage at 5 deg. C. (Table 15). It was also evident
+that the interaction between glycerol level and thawing temperature
+continued during storage (Fig. 7).
+
+[Illustration: Effect of thawing temperature on sperm motility during
+storage at 5 deg. C. following freezing and thawing (Fig. 7)]
+
+Table 15.--Effect of Glycerol Level, Thawing Temperature, and Storage at
+5 deg. C. After Thawing on Sperm Motility (Average of 13 ejaculates)
+
+  =====================================================================
+  Thawing      Glycerol            Sperm motility (percent)
+  temperature   level       -------------------------------------------
+    ( deg. C.)     (percent)     Post-     After storage at 5 deg. C.
+                            thawing    ----------------------   Average
+                                           1 day   3 days
+  ---------------------------------------------------------------------
+      38          4           28.5         17.3      5.1          17.0
+                  6           31.5         22.4      9.2          21.1
+                  8           33.1         15.0      4.6          17.6
+                 10           19.5          3.6      0.8           8.0
+                 Average      28.2         14.6      4.9          12.2
+
+       5          4           29.2         21.7     19.8          23.9
+                  6           37.7         33.8     23.5          31.7
+                  8           41.5         33.1     17.3          30.6
+                 10           33.1         18.5      6.0          19.2
+                 Average      35.4         26.8     16.6          20.6
+  ---------------------------------------------------------------------
+
+It is obvious that motility falls off rapidly after the semen is thawed.
+In a field trial in which the initial intent was to test the effect of
+glycerol levels on fertility of frozen semen, the semen was thawed in
+the morning and used during the same day. Survival of the sperm with 4
+percent glycerol was so poor that only a few breedings were made with
+these samples. Even at 7 and 10 percent, the fertility results were much
+lower than with semen that had not been subjected to freezing. At that
+time it was felt that thawing the samples and using them throughout the
+day may have caused the low fertility results. Since then, a
+large-scale experiment by Cornell University investigators, in
+cooperation with the New York Artificial Breeders' Cooperative, has
+shown definitely that thawing should be delayed until a few minutes
+prior to breeding.[11] If the semen is used immediately, a thawing
+temperature of either 5 deg. or 38 deg. C. appears to be suitable. However,
+there is less danger of cold shock due to recooling if 5 deg. C is used.
+
+=Thawing rate in plastic and in glass.= Glass ampules transmit cold or
+heat more readily than plastic ones. The temperature rise is rapid in
+both glass and plastic when samples are taken from the storage box at
+-79 deg. C. and placed in water at 5 deg. C. However, complete thawing occurs
+more rapidly in glass than in plastic ampules. The changes in
+temperature that occurred when glass and plastic ampules were thawed in
+a water bath at 5 deg. C. are shown in Figure 8. The initial temperature
+rise for the first minute or two was about the same, then the rate of
+warming in the plastic slowed and actual melting of the frozen sample
+occurred a little over a minute later in the plastic than it did in the
+glass. Both were thawed in less than four minutes.
+
+[Illustration: Warming rates of diluted semen samples in plastic vials
+and in glass ampules (Fig. 8)]
+
+
+
+
+EFFECTS OF FREEZING PROCEDURES ON METABOLIC ACTIVITY OF BULL SPERM
+
+
+By finding how methods of handling affect the sperm cells, one can
+sometimes improve the procedures to avoid harmful effects. Some attempts
+have been made in this laboratory to determine the effects of the
+freezing procedures on the metabolic activity of bull spermatozoa. These
+investigations have been limited in scope, involving the measurement of
+oxygen-consumption and estimates of sperm motility during and after
+incubation at 37 deg. C. in a Warburg apparatus.
+
+=Effect of glycerol additions on oxygen uptake of diluted semen at 37 deg.
+C.= The effect of adding glycerol to diluted semen on oxygen consumption
+of the sperm was tested in a Warburg apparatus, using semen diluted with
+an extender consisting of one part egg yolk and one part 2.9 percent
+sodium citrate dihydrate. The yolk-citrate extender was added to the
+semen at a rate which brought the sperm concentration in 0.5 milliliter
+to 200 million to 500 million. An exact count was used to calculate the
+oxygen uptake per 10^{8} sperm per hour (ZO_{2}).
+
+[Illustration: Effect of glycerol additions on oxygen consumption of
+sperm at 37 deg. C. (Fig. 9)]
+
+Glycerol in various percentages in 2.9 percent sodium citrate dihydrate
+solution was placed in the sidearm of the Warburg flasks. The diluted
+semen was held in the main compartment. After a 60-minute preliminary
+run, in which the rate of oxygen uptake of the sperm in yolk-citrate
+diluent was determined, the contents of the sidearm were tipped into the
+main compartment. The resulting glycerol percentages after mixing the
+sidearm and main compartment contents were 0, 4, 8, and 12 percent. Ten
+samples of semen were subsampled and the oxygen uptake of each was
+determined at all four levels of glycerol.
+
+Oxygen uptake was increasingly stimulated during the first 20-minute
+interval by each increase in the amount of glycerol added (Fig. 9).
+After the first 20 minutes, the rate of oxygen utilization decreased at
+the two higher levels of glycerol but persisted at 4 percent. The rate
+of oxygen consumption for the first 20-minute period at the 4-percent
+glycerol level was 130 percent that of the control to which only sodium
+citrate had been added. At 8 and 12 percent the values for the period
+were 144 and 192 percent, respectively, of the control rate.
+
+[Illustration: Effect of glycerol and glycerol-plus-catalase additions
+on sperm motility during incubation at 37 deg. C. (Fig. 10)]
+
+With each increase in glycerol level, motility was reduced during the
+incubation period. This is shown in Figure 10 along with the effect on
+motility of adding catalase, which is discussed in the next section.
+
+=Effect of glycerol-plus-catalase on oxygen uptake of diluted semen.=
+Certain bacteria have been shown to break glycerol down, forming
+hydrogen peroxide as follows:
+
+  Glycerol + oxygen --> lactic acid + hydrogen peroxide.
+
+Hydrogen peroxide is known to be detrimental to sperm. The addition of
+glycerol to diluted semen first increased oxygen uptake and then reduced
+it. Since a reduction in sperm survival followed, some harmful action
+must have taken place with the addition of glycerol at 37 deg. C. To test
+whether this action could be due to the release of hydrogen peroxide as
+occurs in certain bacteria, glycerol with catalase--the enzyme which
+breaks down hydrogen peroxide--was added to a portion of 8 diluted semen
+samples and the oxygen uptake was recorded. Comparison of the resulting
+oxygen uptake with glycerol and with glycerol plus catalase is shown in
+Figure 11.
+
+[Illustration: Effect of additions of glycerol-plus-catalase on oxygen
+consumption of sperm at 37 deg. C. (Fig. 11)]
+
+Oxygen consumption was increased by the presence of added catalase at
+all glycerol levels and in the control. Sperm survival during the 3-hour
+period at 37 deg. C. also was improved by the presence of catalase (Fig.
+10). However, the general trend in oxygen consumption produced by the
+addition of glycerol was not changed greatly. The higher levels of
+glycerol still stimulated oxygen uptake during the first 20-minute
+period after the additions and then slowed the rate of oxygen
+utilization. The rate of utilization was generally higher during the
+test period in the presence of catalase than without added catalase. It
+appeared that a part of the harmful effect of glycerol might be due to
+the formation of hydrogen peroxide. Still, the detrimental effects of
+the higher levels of glycerol were not completely removed.
+
+Table 16.--Effect of Freezing Procedures on Oxygen Utilization of Bull
+Sperm in Yolk-Citrate Extender
+
+(Average of 5 ejaculates)
+
+  =====================================================================
+                                             Microliters of oxygen
+  Semen sample tested                        utilized per 10^{8} sperm
+                                             --------------------------
+                                             First hour    Second hour
+  ---------------------------------------------------------------------
+  Fresh diluted semen                            10.3         8.1
+  Fresh diluted semen glycerol tipped in
+    at end of first hour                          9.7[L]     12.9[L]
+  Aged 20 to 24 hours at 5 deg. C.                   11.2         8.3
+  Aged 20 to 24 hours at 5 deg. C. glycerol
+    tipped in at end of first hour               11.8[L]     12.9[L]
+  After 20 hours equilibration with glycerol     11.7[L]      7.8[L]
+  After freezing and thawing                      9.7         6.3
+  --------------------------------------------------------------------
+
+  [L] Average of 3 ejaculates.
+
+=Effect of freezing procedures on oxygen utilization by sperm.= Limited
+data have been obtained on the effects of some of the freezing
+procedures on the oxygen utilization of bull sperm. The results obtained
+in these experiments confirmed the earlier findings that tipping
+glycerol directly into the diluted semen at 37 deg. C. caused an increase in
+oxygen consumption (Table 16). All other steps in the freezing procedure
+had little effect on oxygen consumption by the sperm. Except where
+glycerol was added during the determination, the rate of oxygen
+utilization was lower the second hour than during the first. The oxygen
+uptake of semen that had been frozen and thawed seemed to drop faster
+than that of unfrozen samples.
+
+=Effect of freezing procedures on methylene-blue reduction time.= The
+methylene-blue reduction test has been used as a means of measuring
+semen quality and is dependent on the metabolic activity of the sperm.
+The effects of various freezing procedures on the ability of samples to
+decolorize methylene blue were determined with 10 semen samples. Sperm
+numbers were standardized to 300 x 10^{6} cells per milliliter and the
+time required for these cells to reduce a 1:40,000 solution of methylene
+blue was determined on freshly diluted semen, after the addition of
+glycerol, after equilibration, and after freezing and thawing. Portions
+of each diluted sample were tested at these stages of the procedure with
+glycerol alone added and with glycerol and various sugars added.
+
+A marked increase in the time required for the sperm to reduce methylene
+blue occurred when the glycerol was added (Table 17). This increase was
+greatest in the portions with glycerol alone and with glycerol and
+glucose. The time increase was less pronounced in the presence of the
+three pentose sugars used. Following equilibration, the samples regained
+the ability to reduce methylene blue at a rate only slightly slower than
+when they were fresh. Freezing and storage of semen resulted in slower
+reduction of the methylene blue than was shown after equilibration with
+glycerol. Since freezing usually kills some of the sperm, a slowing of
+the reduction time after freezing would be expected.
+
+Table 17.--Effect of Freezing Procedures on the Methylene-Blue Reduction
+Time of Bull Semen With and Without the Addition of Sugars[M]
+
+(Average of 10 ejaculates)
+
+  ======================================================================
+                            Methylene-blue reduction time (minutes)
+                        ------------------------------------------------
+                        Glycerol  Glycerol  Glycerol   Glycerol Glycerol
+                        only      and       and        and      and
+                                  glucose   arabinose  xylose   rhamnose
+  ----------------------------------------------------------------------
+  Fresh semen             5.2       5.2       5.2        5.2       5.2
+  After glycerolization  26.4      25.2      17.3       14.3      19.4
+  After 18 hours
+    equilibration         7.4       6.5       6.4        5.3       6.2
+  Thawed immediately
+    after freezing       11.5      10.5       9.4        9.0       9.4
+  Thawed  48 hours
+    after freezing       14.3      10.2      11.3       10.1       9.5
+  ----------------------------------------------------------------------
+
+  [M] Glycerol level in the final frozen mixture was 7 percent. Sugars
+  were added to a level of 1.25 percent.
+
+
+
+
+PRACTICAL FREEZING PROCEDURE
+
+
+Good results usually can be obtained in freezing bull semen if care is
+taken in collecting, diluting and processing the semen. Occasionally the
+semen from certain bulls will not withstand freezing well. The reason
+for this is not understood at present. However, carefully following the
+directions and suggestions given below will usually produce satisfactory
+results with semen samples that are of good quality at the start.
+
+Experience in the field has shown that fertility results with frozen
+semen are usually slightly lower during the first few months than with
+liquid semen stored at 5 deg. C. (41 deg. F.). Most units that have worked with
+frozen semen over a period of a few months are able to improve and do
+get fertility results as good as, or better than, obtained in their
+liquid semen program.
+
+=Collection of the semen.= In order to obtain the best possible semen
+for freezing, care and cleanliness should be exercised in making the
+collection. The artificial vagina, and the glassware used should be
+clean and dry. The underline of the bull should also be clean and dry.
+The bull should be restrained near the teaser cow for a minute or two
+prior to collection in order to excite the flow of secretions prior to
+ejaculation. Allowing the bull to mount the teaser once without serving
+the artificial vagina is a good practice to use in properly stimulating
+the bull before collection of the semen.
+
+If the bull has not been used for three or four days, the collection of
+a second ejaculate for freezing may be advisable. The second ejaculate
+seems to withstand freezing better than the first in many instances. A
+clean, dry artificial vagina should be used for each ejaculate
+collected. Repeated collections in the same artificial vagina may result
+in contamination of the semen with bacteria, lubricating jelly and
+minute particles of dirt. The semen sample should be protected from
+contamination and from sudden temperature drops (cold shock).
+
+=Preparation of extender.= A suitable egg yolk-citrate extender for
+freezing bull semen can be prepared by the following procedure. One part
+egg yolk (free of egg white and the membrane surrounding the yolk) is
+mixed with 4 parts 2.4 to 2.9 percent sodium citrate dihydrate solution.
+The citrate is prepared with distilled water and then boiled or
+autoclaved. The citrate solution should be cooled before it is mixed
+with the egg yolk. After the egg and citrate are mixed, 1000 units of
+penicillin and 1000 micrograms of streptomycin are added per milliliter
+of extender. Sulfanilamide should not be added. This extender can be
+prepared 12 to 24 hours before use if it is stored at refrigerator
+temperature. The portion of the extender needed for the original
+dilution of the semen should be warmed to room temperature before it is
+mixed with the semen.
+
+=Dilution after collection.= As soon as possible after collection, the
+semen sample should be diluted with the extender. The extender must be
+at the same temperature as the semen (room temperature) when the two are
+mixed together. At this time the semen can be partially diluted (1 part
+semen to 4 parts of extender) or diluted to a sperm concentration twice
+the final desired concentration (later in adding the glycerol for
+freezing, the semen is diluted further with an equal volume of glycerol
+containing extender). The diluted semen is slowly cooled (1-1/2 to 2-1/2
+hours) to 5 deg. C. (41 deg. F.). Some units using frozen semen now allow the
+semen to stand at 5 deg. C. for 5 to 6 hours before glycerolization to allow
+the antibiotics to be more effective against any vibrio fetus organisms
+that may be present. This step is taken because it has been shown that
+glycerol inhibits the effectiveness of the antibiotics.[6] After
+cooling, semen can be further diluted to twice the desired sperm
+concentration if that were not done at the start. (Caution: Be sure
+semen and diluent are at the same temperature.)
+
+=Adding the glycerol.= The glycerol solution is prepared by adding 14
+volumes of glycerol (reagent grade) to 86 volumes of yolk-citrate
+diluent (same as yolk-citrate used for original dilution). This solution
+may be added dropwise with constant gentle mixing to the already diluted
+semen, or one-third at a time at 10-minute intervals with gentle mixing
+during each addition. Either method should take about 20 to 30 minutes.
+The total volume of glycerol-yolk-citrate solution added should be equal
+to the volume of the original diluted semen. In this way a concentration
+of 7 percent glycerol is obtained in the final mixture that is to be
+frozen. Care must be taken to keep the temperature at 5 deg. C. (41 deg. F.)
+during the time the glycerol is being added. (A cold room is best for
+maintaining a temperature of 5 deg. C., but with care the operation can be
+carried out at room temperature by using pans of ice water and a
+refrigerator.)
+
+=Equilibration.= The results presented in this bulletin suggest that
+little or no time need be allowed after the glycerol is added before
+freezing. However, results obtained by other workers show improved
+fertility with at least 12 hours equilibration. Some units getting good
+fertility results with frozen semen also are allowing the semen to stand
+at 5 deg. C. for 12 to 18 hours before freezing. After the semen has
+equilibrated with the glycerol, 1-milliliter portions of the mixture are
+placed in 1.2- to 2-milliliter vials or ampules which are then sealed.
+Ampuling can be done with an automatic syringe or pipette, provided a
+large gage needle is used. Also, it is important not to force the fluid
+mixture rapidly through the syringe or the sperm may be injured.
+
+=Freezing.= The vials or ampules of diluted semen are placed in a bath
+of isopropyl alcohol which has been cooled to 5 deg. C. (41 deg. F.). This bath
+can be a wide-mouth thermos bottle or an insulated container of almost
+any sort with a large opening at the top. The size needed depends on the
+number of ampules being frozen. Some sort of convenient tray for holding
+the ampules in an orderly fashion and enabling the samples to be
+completely submerged is desirable. A few ampules can be kept together
+easily by placing them in a polyethylene freezer bag that has had many
+small holes cut in it to let the alcohol of the bath contact the
+ampules. The ampules must be completely covered by the alcohol to insure
+uniform cooling.
+
+The alcohol of the bath and the ampules of semen are cooled by adding
+chipped or ground dry ice in sufficient amounts to lower the temperature
+of the bath 2 deg. C. (3.6 deg. F.) per minute from +5 deg. to -20 deg. C. From -20 deg.
+down to -79 deg. C., the rate of cooling can be doubled (4 deg. C. or 7.2 deg. F.).
+Electrical equipment that regulates the cooling rate to the desired
+temperatures is available commercially, but the cost may be too high for
+some small operations. The samples should be held at -79 deg. C. (-110 deg. F.)
+until they are thawed. This can be done by using an alcohol bath and dry
+ice or by special mechanical refrigerating equipment. At no time prior
+to thawing should the samples be exposed to warmer temperatures.
+
+=Thawing.= The ampules of frozen semen can be thawed by removing them
+from the dry ice storage box and dropping them into a water bath at 5 deg.
+C. (41 deg. F.). Thawing temperatures up to body temperature, 38 deg. C. (100 deg.
+F.), can be used but extreme care must then be taken not to pass the
+semen through a cold inseminating tube; for this would subject the sperm
+to cold shock. The semen should be used for breeding within a few
+minutes after thawing.
+
+
+
+
+LITERATURE CITED
+
+
+     [1] DAVENPORT, C. B. Effect of chemical and physical agents upon
+     protoplasm. Macmillan and Co., New York. 1897.
+
+     [2] POLGE, C., and PARKES, A. S. Possibilities of long-term
+     storage of spermatozoa at low temperatures. Anim. Breeding Abs.
+     =20=:1-5. 1952.
+
+     [3] EMMENS, C. W., and BLACKSHAW, A. W. The low temperature
+     storage of ram, bull, and rabbit spermatozoa. Austral. Vet. Jour.
+     =26=:226. 1950.
+
+     [4] SMITH, AUDREY W. Effects of low temperatures on living cells
+     and tissues. In biological applications of freezing and drying.
+     Ed. R. J. C. Harris. Academic Press, Inc., New York, 1954.
+
+     [5] EMMENS, C. W., and BLACKSHAW, A. W. Artificial insemination.
+     Physiol. Rev. =36=:277-306. 1956.
+
+     [6] Proceedings of the National Association of Artificial
+     Breeders, 1953, 1954, and 1955.
+
+     [7] Proceedings of the American Dairy Science Association, 1953,
+     1954, and 1955. Published in the June issue of the Journal of
+     Dairy Science for each year.
+
+     [8] BARKER, C. A. V. Low temperature preservation of bovine
+     epididymal spermatozoa. Canad. Jour. Comp. Med. =18=:390-393.
+     1954.
+
+     [9] SAROFF, JACK, and MIXNER, J. P. The relationship of egg yolk
+     and glycerol content of diluters and glycerol equilibration time
+     to survival of bull spermatozoa after low temperature freezing.
+     Jour. Dairy Sci. =38=:292-297. 1955.
+
+     [10] CRAGLE, R G., MYERS, R. M., WAUGH, R. K., HUNTER, J. S., and
+     ANDERSON, R. L. The effects of various levels of sodium citrate,
+     glycerol, and equilibration time on survival of bovine
+     spermatozoa after storage at -79 deg. C. Jour. Dairy Sci.
+     =38=:508-514. 1955.
+
+     [11] BRATTON, R. W., FOOTE, R. H., and CRUTHERS, JOAN C.
+     Preliminary fertility results with frozen bovine spermatozoa.
+     Jour. Dairy Sci. =38=:40-46. 1955.
+
+     [12] HAFS, H. D., and ELLIOTT, F. I. The effects of methods of
+     adding egg yolk and monosaccharides on the survival of frozen
+     bull spermatozoa. Jour. Dairy Sci. =38=:811-815. 1955.
+
+     [13] MILLER, W. J., and VANDEMARK, N. L. The influence of
+     glycerol level, various temperature aspects, and certain other
+     factors on the survival of bull spermatozoa at sub-zero
+     temperatures. Jour. Dairy Sci. =37=:45-51. 1954.
+
+
+
+
+TEMPERATURE CONVERSIONS
+
+
+   deg.C.    deg.F.
+
+  +38  +100
+  +35   +95
+  +30   +86
+  +25   +77
+  +20   +68
+  +15   +59
+  +10   +50
+   +5   +41
+    0   +32
+   -5   +23
+  -10   +14
+  -15    +5
+  -18     0
+  -20    -4
+  -25   -13
+  -30   -22
+  -35   -31
+  -40   -40
+  -45   -49
+  -50   -58
+  -55   -67
+  -60   -76
+  -65   -85
+  -70   -94
+  -75  -103
+  -79  -110
+
+
+
+
+
+End of the Project Gutenberg EBook of Preservation of Bull Semen at Sub-Zero
+Temperatures, by N. L. VanDemark and W. J. Miller and W. C. Kinney, Jr. and Carlos Rodriguez and M. E. Friedman
+
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