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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> </td> +<td class="left">W. J. Miller</td> +</tr> + +<tr> +<td> </td> +<td class="left">W. C. Kinney, Jr.</td> +</tr> + +<tr> +<td> </td> +<td class="left">Carlos Rodriguez</td> +</tr> + +<tr> +<td> </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"> </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> </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> </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> </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> </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> </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"> </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> </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> </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> </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> </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"> </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> </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"> </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> </td> +<td class="left"><a href="#Sec5_1">Effect of glycerol on sperm survival at 5° C.</a></td> +<td class="right"><a href="#Page_17">17</a></td> +</tr> + +<tr> +<td> </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> </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> </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> </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> </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"> </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> </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> </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"> </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> </td> +<td class="left"><a href="#Sec7_1">Storage at -23° to -79° C.</a></td> +<td class="right"><a href="#Page_24">24</a></td> +</tr> + +<tr> +<td> </td> +<td class="left"><a href="#Sec7_2">Use of higher glycerol levels and storage at -20° C.</a></td> +<td class="right"><a href="#Page_25">25</a></td> +</tr> + +<tr> +<td colspan="3"> </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> </td> +<td class="left"><a href="#Sec8_1">Comparison of thawing at 5° C. and at 38° C.</a></td> +<td class="right"><a href="#Page_27">27</a></td> +</tr> + +<tr> +<td> </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"> </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> </td> +<td class="left"><a href="#Sec9_1">Effect of glycerol additions on oxygen uptake of diluted semen at 37° C.</a></td> +<td class="right"><a href="#Page_30">30</a></td> +</tr> + +<tr> +<td> </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> </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> </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"> </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> </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> </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> </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> </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> </td> +<td class="left"><a href="#Sec10_5">Equilibration</a></td> +<td class="right"><a href="#Page_36">36</a></td> +</tr> + +<tr> +<td> </td> +<td class="left"><a href="#Sec10_6">Freezing</a></td> +<td class="right"><a href="#Page_37">37</a></td> +</tr> + +<tr> +<td> </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"> </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"> </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—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?</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.—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"> </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"> </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 × 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 × 10<span class="expon">6</span>. 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.)</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.—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.—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° 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.—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 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> </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> </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> </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> </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> </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> </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> </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> </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> </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> </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> </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 × 10<span class="expon">6</span>/ml. +Sufficient extender was added +to give 30 × 10<span class="expon">6</span>/ml. at the first extension. Thus the final concentration was 15 +× 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° 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° 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.—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"> </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"> </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"> </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"> </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° 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° C. and dilution and refreezing following an initial freezing of +concentrated samples.</p> + +<h5><a name="Tab6" id="Tab6"></a>Table 6.—Effect of Sperm Numbers and Glycerol Level<br /> +in Final Mixture on Freezability of Bull Sperm at -79° 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 level<br />(percent)</td> +<td colspan="4" class="center bt2 bb padr1 padl1">Post-thawing motility (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 of sperm (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° C. for 3 to 7 hours. The other two portions were refrozen.</p> + +<h5><a name="Tab7" id="Tab7"></a>Table 7.—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 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 storage<a name="FNanchorG" +id="FNanchorG"></a><a href="#FootnoteG" class="fnanchor">[G]</a></td> +<td> </td> +<td colspan="2" class="center bb padr1 padl1">After 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 15<br />million/ml</td> +<td class="bb"> </td> +<td class="center bb padr1 padl1">No<br />further<br />dilution</td> +<td class="center bb padr1 padl1">Diluted<br />to 15<br />million/ml</td> +</tr> + +<tr> +<td> </td> +<td> </td> +<td colspan="3" class="center">First trial: 4 samples</td> +<td> </td> +<td> </td> +<td> </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> </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> </td> +<td class="right padr3">25</td> +<td class="right padr4">5</td> +</tr> + +<tr> +<td> </td> +<td> </td> +<td colspan="3" class="center">Second trial: 7 samples</td> +<td> </td> +<td> </td> +<td> </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> </td> +<td class="right padr3">28</td> +<td class="right padr4">11</td> +</tr> + +<tr class="bb"> +<td class="left padl1">15 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> </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° 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.—Effect of Glycerol Level and Storage at 5° 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 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> </td> +<td colspan="11" class="center bb padr1 padl1">After storage at 5° C.</td> +<td> </td> +<td colspan="2" rowspan="2" class="center bb padr1 padl1">Average</td> +</tr> + +<tr> +<td> </td> +<td colspan="3" class="center bb padr1 padl1">1 day</td> +<td> </td> +<td colspan="3" class="center bb padr1 padl1">3 days</td> +<td> </td> +<td colspan="3" class="center bb padr1 padl1">7 days</td> +<td> </td> +</tr> + +<tr> +<td class="center bb padr1 padl1">percent</td> +<td colspan="2" class="center bb padr1 padl1">rate</td> +<td class="bb"> </td> +<td class="center bb padr1 padl1">percent</td> +<td colspan="2" class="center bb padr1 padl1">rate</td> +<td class="bb"> </td> +<td class="center bb padr1 padl1">percent</td> +<td colspan="2" class="center bb padr1 padl1">rate</td> +<td class="bb"> </td> +<td class="center bb padr1 padl1">percent</td> +<td colspan="2" class="center bb padr1 padl1">rate</td> +<td class="bb"> </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> </td> +<td> </td> +<td class="center">55</td> +<td class="right padl1">1.9</td> +<td> </td> +<td> </td> +<td class="center">46</td> +<td class="right padl1">1.8</td> +<td> </td> +<td> </td> +<td class="center">38</td> +<td class="right padl1">1.4</td> +<td> </td> +<td> </td> +<td class="center">48</td> +<td class="center">1.90</td> +</tr> + +<tr> +<td style="width: 1em;"> </td> +<td class="left">0</td> +<td class="center">54</td> +<td class="right padl1">2.4</td> +<td> </td> +<td> </td> +<td class="center">44</td> +<td class="right padl1">1.9</td> +<td> </td> +<td> </td> +<td class="center">46</td> +<td class="right padl1">1.8</td> +<td> </td> +<td> </td> +<td class="center">36</td> +<td class="right padl1">1.4</td> +<td> </td> +<td> </td> +<td class="center">45</td> +<td class="center">1.87</td> +</tr> + +<tr> +<td> </td> +<td class="left">5</td> +<td class="center">52</td> +<td class="right padl1">2.2</td> +<td> </td> +<td> </td> +<td class="center">50</td> +<td class="right padl1">1.9</td> +<td> </td> +<td> </td> +<td class="center">46</td> +<td class="right padl1">1.7</td> +<td> </td> +<td> </td> +<td class="center">32</td> +<td class="right padl1">1.4</td> +<td> </td> +<td> </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> </td> +<td> </td> +<td class="center">46</td> +<td class="right padl1">1.8</td> +<td> </td> +<td> </td> +<td class="center">42</td> +<td class="right padl1">1.7</td> +<td> </td> +<td> </td> +<td class="center">28</td> +<td class="right padl1">1.6</td> +<td> </td> +<td> </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> </td> +<td> </td> +<td class="center">50</td> +<td class="right padl1">1.7</td> +<td> </td> +<td> </td> +<td class="center">44</td> +<td class="right padl1">1.6</td> +<td> </td> +<td> </td> +<td class="center">38</td> +<td class="right padl1">1.1</td> +<td> </td> +<td> </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> </td> +<td> </td> +<td class="center">44</td> +<td class="right padl1">0.5</td> +<td> </td> +<td> </td> +<td class="center">42</td> +<td class="right padl1">0.4</td> +<td> </td> +<td> </td> +<td class="center">30</td> +<td class="right padl1">0.4</td> +<td> </td> +<td> </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> </td> +<td class="center">47</td> +<td class="right padl1">1.6</td> +<td class="left padr1">2</td> +<td> </td> +<td class="center">44</td> +<td class="right padl1">1.5</td> +<td class="left padr1">0</td> +<td> </td> +<td class="center">34</td> +<td class="right padl1">1.2</td> +<td class="left padr1">2</td> +<td> </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° 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° 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° 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° 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 <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° 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.—Effect of Glycerol Level on Sperm Motility<br /> +After Freezing to -79° 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;"> </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> </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> </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> </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.—Effect of Glycerol Level and Storage<br />at 5° 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 motility (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 storage at 5° 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;"> </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> </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> </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.—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 during addition<br />of +glycerol (° 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 motility (percent)</td> +</tr> + +<tr> +<td> </td> +<td colspan="7" class="center bb padr1 padl1">Glycerol additions</td> +</tr> + +<tr> +<td> </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;"> </td> +<td class="left padl0">4.5</td> +<td class="center">2</td> +<td> </td> +<td class="right">48</td> +<td> </td> +<td class="right">48</td> +<td> </td> +<td class="right">45</td> +<td> </td> +<td class="right padr3">47.4</td> +</tr> + +<tr> +<td colspan="2"> </td> +<td class="center">6</td> +<td> </td> +<td class="right">49</td> +<td> </td> +<td class="right">51</td> +<td> </td> +<td class="right">47</td> +<td> </td> +<td class="right padr3">48.8</td> +</tr> + +<tr> +<td colspan="2"> </td> +<td class="center">18</td> +<td> </td> +<td class="right">46</td> +<td> </td> +<td class="right">47</td> +<td> </td> +<td class="right">46</td> +<td> </td> +<td class="right padr3">46.3</td> +</tr> + +<tr> +<td colspan="2"> </td> +<td class="center">Average</td> +<td> </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;"> </td> +</tr> + +<tr> +<td class="right padr0">1</td> +<td class="left padl0">0.0</td> +<td class="center">2</td> +<td> </td> +<td class="right">44</td> +<td> </td> +<td class="right">43</td> +<td> </td> +<td class="right">45</td> +<td> </td> +<td class="right padr3">43.9</td> +</tr> + +<tr> +<td colspan="2"> </td> +<td class="center">6</td> +<td> </td> +<td class="right">48</td> +<td> </td> +<td class="right">50</td> +<td> </td> +<td class="right">46</td> +<td> </td> +<td class="right padr3">47.9</td> +</tr> + +<tr> +<td colspan="2"> </td> +<td class="center">18</td> +<td> </td> +<td class="right">43</td> +<td> </td> +<td class="right">46</td> +<td> </td> +<td class="right">42</td> +<td> </td> +<td class="right padr3">44.0</td> +</tr> + +<tr> +<td colspan="2"> </td> +<td class="center">Average</td> +<td> </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;"> </td> +</tr> + +<tr> +<td class="right padr0">1</td> +<td class="left padl0">5.5</td> +<td class="center">2</td> +<td> </td> +<td class="right">41</td> +<td> </td> +<td class="right">38</td> +<td> </td> +<td class="right">38</td> +<td> </td> +<td class="right padr3">39.1</td> +</tr> + +<tr> +<td colspan="2"> </td> +<td class="center">6</td> +<td> </td> +<td class="right">42</td> +<td> </td> +<td class="right">45</td> +<td> </td> +<td class="right">43</td> +<td> </td> +<td class="right padr3">43.6</td> +</tr> + +<tr> +<td colspan="2"> </td> +<td class="center">18</td> +<td> </td> +<td class="right">42</td> +<td> </td> +<td class="right">43</td> +<td> </td> +<td class="right">42</td> +<td> </td> +<td class="right padr3">42.5</td> +</tr> + +<tr class="bb"> +<td colspan="2"> </td> +<td class="center">Average</td> +<td> </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° C. +before the glycerol was added. The survival at 10° and 15.5° 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° 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° C.). Results of attempting to reduce +equilibration time are given in <a href="#Tab11">Table 11</a>. 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.</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—30 minutes—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.—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 when observed</td> +<td colspan="3" class="center bt2 bb padr1 padl1">Sperm motility (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 diluted 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 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 equilibration</td> +<td colspan="3"> </td> +</tr> + +<tr> +<td style="width: 2em;"> </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> </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> </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> </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—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.</p> + +<h5><a name="Tab13" id="Tab13"></a>Table 13.—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 of<br />observation</td> +<td colspan="5" class="center bt2 bb padr1 padl1">Sperm motility (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° 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° 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° 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.</p> + +<p>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°<span class='pagenum'><a name="Page_23" id="Page_23">[23]</a></span> +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.</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° 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.</p> + +<p><a name="Sec7_1" id="Sec7_1"></a><b>Storage at temperatures from -23° to -79° 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°, -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 (<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° 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.</p> + +<p>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:</p> + +<table summary="Table P 25"> + +<tr> +<td class="left">Day</td> +<td class="right">2</td> +<td> </td> +<td class="right">9</td> +<td> </td> +<td class="right">16</td> +<td> </td> +<td class="right">51</td> +<td> </td> +</tr> + +<tr> +<td class="left padr2">Percent of motile sperm</td> +<td class="right">49</td> +<td> </td> +<td class="right">46</td> +<td> </td> +<td class="right">40</td> +<td> </td> +<td class="right">38</td> +<td> </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° 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° 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° 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.<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° 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 <a href="#Tab14">Table 14</a>.</p> + +<h5><a name="Tab14" id="Tab14"></a>Table 14.—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 />(°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 motility after storage (percent)</td> +</tr> + +<tr> +<td class="center bb padr1 padl1">18 hours</td> +<td class="center bb padr1 padl1">42 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> </td> +<td class="right padr4">11</td> +<td class="right padr8">3</td> +<td class="right padr8">1</td> +</tr> + +<tr> +<td> </td> +<td class="right padr4">15</td> +<td class="right padr8">14</td> +<td class="right padr8">10</td> +</tr> + +<tr> +<td> </td> +<td class="right padr4">19</td> +<td class="right padr8">30</td> +<td class="right padr8">22</td> +</tr> + +<tr> +<td> </td> +<td class="right padr4">23</td> +<td class="right padr8">29</td> +<td class="right padr8">19</td> +</tr> + +<tr> +<td> </td> +<td class="right padr4">27</td> +<td class="right padr8">25</td> +<td class="right padr8">18</td> +</tr> + +<tr class="bb"> +<td> </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° 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.</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° 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° C. and 38° C.</b> 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 <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° 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. (<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°</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.—Effect of Glycerol Level, Thawing Temperature,<br /> +and Storage at 5° 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 />(° 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 motility (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 storage at 5° 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> </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> </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> </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> </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> </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> </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> </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> </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’ 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° or 38° C. appears to be suitable. However, there +is less danger of cold shock due to recooling if 5° 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° 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 <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° 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° 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° 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° C.</td> +</tr> + +<tr> +<td> </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 → 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° 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 <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° 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° 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.—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 sample tested</td> +<td colspan="4" class="center bb padl1 padr1">Microliters of oxygen<br />utilized per 10<span +class="expon">8</span> sperm</td> +</tr> + +<tr> +<td colspan="2" class="center bb padl1 padr1">First hour</td> +<td colspan="2" class="center bb padl1 padr1">Second hour</td> +</tr> + +<tr> +<td class="left">Fresh diluted semen</td> +<td class="right">10.3</td> +<td> </td> +<td class="right">8.1</td> +<td> </td> +</tr> + +<tr> +<td class="left">Fresh diluted semen glycerol tipped in at end of first 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 20 to 24 hours at 5° C.</td> +<td class="right">11.2</td> +<td> </td> +<td class="right">8.3</td> +<td> </td> +</tr> + +<tr> +<td class="left">Aged 20 to 24 hours at 5° C. glycerol tipped in at +end of first 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 20 hours equilibration with 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 freezing and thawing</td> +<td class="right">9.7</td> +<td> </td> +<td class="right">6.3</td> +<td> </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° 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 × 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.—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"> </td> +<td colspan="5" class="center bb">Methylene-blue reduction time (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 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 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 18 hours 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 immediately after 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 48 hours after 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° 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.</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>⁄<span class="index">2</span> to +2<span class="expon">1</span>⁄<span class="index">2</span> 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.<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° 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.)</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° 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° 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.</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° 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.</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° 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.</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° 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">°C.</td> +<td class="right padr2">°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. 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