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diff --git a/37041.txt b/37041.txt new file mode 100644 index 0000000..c8b0083 --- /dev/null +++ b/37041.txt @@ -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 + +*** END OF THIS PROJECT GUTENBERG EBOOK PRESERVATION OF BULL SEMEN *** + +***** This file should be named 37041.txt or 37041.zip ***** +This and all associated files of various formats will be found in: + http://www.gutenberg.org/3/7/0/4/37041/ + +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.) + + +Updated editions will replace the previous one--the old editions +will be renamed. + +Creating the works from public domain print editions means that no +one owns a United States copyright in these works, so the Foundation +(and you!) can copy and distribute it in the United States without +permission and without paying copyright royalties. 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