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PRESERVATION OF BULL SEMEN AT SUB-ZERO TEMPERATURES

By N. L. VanDemark W. J. Miller W. C. Kinney, Jr. Carlos Rodriguez M. E. Friedman

Bulletin 621

UNIVERSITY OF ILLINOIS AGRICULTURAL EXPERIMENT STATION

CONTENTS

Page

EARLY WORK ON FREEZING SEMEN 5

SELECTION OF SEMEN FOR FREEZING 6 Predicting freezability 6 Freezability of first and second ejaculates 7 Freezability of several consecutive ejaculates 7 Freezability of epididymal sperm 9 Freezability of washed sperm 10

EXTENDERS FOR FREEZING BULL SEMEN 10 Proportion of egg yolk in final diluent 10 Citrate level in final diluent 11 Storing and freezing diluent 12 Other diluents 13

DILUTION RATES 14 Effect of further dilution and refreezing 14

GLYCEROL ADDITIONS 17 Effect of glycerol on sperm survival at 5 deg. C. 17 Glycerol levels for freezing semen 18 Rate, temperature, and method of adding glycerol 18 Allowing sperm to equilibrate with glycerol 20 Sugar additions and equilibration time 20 Substitutes for glycerol 22

FREEZING RATE 22 Effect of freezing rate on sperm survival 22 Rate of cooling in plastic and in glass 23

STORAGE TEMPERATURE 24 Storage at -23 deg. to -79 deg. C. 24 Use of higher glycerol levels and storage at -20 deg. C. 25

THAWING 26 Comparison of thawing at 5 deg. C. and at 38 deg. C. 27 Thawing rate in plastic and in glass 29

EFFECTS OF FREEZING PROCEDURES ON METABOLIC ACTIVITY OF SPERM 30 Effect of glycerol additions on oxygen uptake of diluted semen at 37 deg. C. 30 Effect of glycerol-plus-catalase on oxygen uptake of diluted semen 32 Effect of freezing procedures on oxygen utilization 33 Effect of freezing procedures on methylene-blue reduction time 33

PRACTICAL FREEZING PROCEDURE 35 Collection of semen 35 Preparation of extender 35 Dilution after collection 36 Adding the glycerol 36 Equilibration 36 Freezing 37 Thawing 37

LITERATURE CITED 38

TEMPERATURE CONVERSION TABLE 39

Urbana, Illinois October, 1957

Publications in the Bulletin series report the results of investigations made or sponsored by the Experiment Station

ACKNOWLEDGMENT

The authors gratefully acknowledge the financial assistance given to the Department of Dairy Science in support of these investigations by the Southern Illinois Breeding Association of Breese, Illinois, and the Northern Illinois Breeding Co-op of Hampshire, Illinois, through the Illinois Dairy Breeding Federation.

THE PRESERVATION OF BULL SEMEN AT SUB-ZERO TEMPERATURES

By N. L. VANDEMARK, W. J. MILLER, W. C. KINNEY, JR., CARLOS RODRIGUEZ, and M. E. FRIEDMAN[A]

[A] This publication was prepared by N. L. VANDEMARK, Professor of Dairy Physiology. Research reported herein was carried out cooperatively by the senior author and W. J. MILLER, W. C. KINNEY, JR., CARLOS RODRIGUEZ, and M. E. FRIEDMAN, formerly members of the Department of Dairy Science.

The first announcement in 1951 of the successful impregnation of a cow with bull semen that had been frozen stimulated much interest and research in freezing as a method of preserving bull semen. Research during the years following 1951 resulted in considerable progress and success in using freezing as a means of holding semen for long periods of time without loss of fertility.

Between 1952 and 1957, research on many aspects of the preservation of bull semen at sub-zero temperatures was carried out in the Department of Dairy Science at the University of Illinois. Many of these investigations have aided in perfecting the freezing technique that has been adapted for practical use. Some of these findings have been published, but many items have gone unreported except for general references at scientific meetings.

It is the purpose of this bulletin to bring together the results of several experiments carried out in connection with the freezing of bull semen and to present a practical freezing procedure based on the results of these experiments and findings at other institutions. Persons interested in the development of the procedures and the reasons why certain steps are necessary in freezing semen will find the details in the first part of this bulletin. Those interested only in the freezing procedure may turn to page 35 where a practical method of freezing semen is described.

EARLY WORK ON FREEZING SEMEN

While it has been known for a long time that various types of tissues and organisms can withstand freezing and are even preserved by freezing, the first attempts at freezing sperm cells were made before the turn of the century. In 1897, Davenport[1][B] found that human sperm would withstand freezing. For thirty to forty years after that, little attention was paid to freezing as a possible means of preserving semen. An excellent review of the early attempts to freeze sperm has been assembled by Polge and Parkes.[2] These investigators also gave a good account of their work at the British National Institute of Medical Research in London, where in 1949 they demonstrated that glycerol would protect fowl sperm so that it would survive freezing. The next year they found that bull sperm and the sperm of several other species were protected by glycerol during freezing. During the same year, Emmens and Blackshaw[3] showed that ram and bull sperm would survive freezing. In 1951 frozen semen was used to produce a calf in England and a lamb in Australia.

[B] These numbers refer to literature citations on page 38.

The highlights in the development of frozen semen have been covered by other reviews and reports. Interested persons will find the articles of Polge and Parkes[2] and Smith[4] especially good on the early history and theoretical aspects of freezing sperm. Later progress on the freezing procedure has been reviewed and covered in a number of detailed reports.[5],[6],[7] Many items not covered in those articles have been assembled here.

SELECTION OF SEMEN FOR FREEZING

One of the first considerations in freezing semen is that of deciding which semen samples are to be frozen. Since preservation of the semen--the maintenance of the potential motility and especially the fertility of the sperm--is the primary aim, some attention should be directed to the kind of semen sample that will withstand freezing. Do the initial characteristics of the sample indicate whether the sperm will withstand freezing? Does maturity of the sperm affect their freezability?

=Predicting freezability.= Estimates of semen quality in the past have been based in part on the numbers of sperm present in a fresh sample and on the percentage and rate of motility shown by the sperm. These characteristics were used to determine the relationship between the original concentration of sperm (in the fresh, undiluted sample), the percentage and rate of sperm motility in the diluted samples just prior to freezing, and the percentage and rate of sperm motility following freezing and thawing. From data collected before and after freezing and thawing 54 ejaculates, it was found that there was not a significant correlation between the number of sperm present in the original sample and the percent of motile sperm present after freezing and thawing (r = 0.03). A highly significant correlation (r = 0.45) was found, however, between the percentages before freezing and after thawing. While this correlation coefficient was highly significant, its magnitude indicates that only about one fifth of the variation in percentage of motile sperm observed after freezing was accounted for by the motility of the sperm prior to freezing.

=Freezability of first and second ejaculates.= In the early days of artificial breeding in this country, it was commonly believed that a second ejaculate collected a few minutes after the first resulted in a larger ejaculate containing more sperm. With the development of the procedure of stimulating sexual excitement by restraint prior to collecting semen, this difference between first and second ejaculates has been greatly reduced. Still it was noted that second ejaculates frequently withstood freezing better than first ejaculates, even though restraint and stimulation of the bull occurred prior to collection of the first ejaculate. During the course of a number of experiments, it was possible to compare the freezability of 2 ejaculates that were collected a few minutes apart from the same bull. Two consecutive ejaculates were obtained one or more times from 24 bulls so that a total of 58 comparisons could be made. The mean prefreezing and post-thawing percentages of motile sperm in first and second ejaculates are presented in Table 1.

An analysis of variance showed that in this comparison the differences between first and second ejaculates in sperm survival during freezing were highly significant. A later comparison of 27 first and second ejaculates from 26 bulls did not show as great a difference between first and second ejaculates in their ability to withstand freezing (Table 1).

Table 1.--Comparison of the Freezability of First and Second Ejaculates Taken a Few Minutes Apart From the Same Bull

===================================================================== Number Number Prefreezing Post-thawing of of Ejaculate motility motility Survival bulls ejaculates (percent) (percent) (percent) --------------------------------------------------------------------- 24 58 1st 60 39 65 2d 62 45 74 26 27 1st 60 36 60 2d 65 40 61 ---------------------------------------------------------------------

=Freezability of several consecutive ejaculates.= The fact that second ejaculates sometimes withstood freezing better than first ejaculates suggested that the maturity of the sperm might be a factor affecting freezability. An opportunity to check this idea came when 20 consecutive ejaculates were collected from each of 6 bulls within a 4-hour period. The sperm in samples collected in this manner might be expected to be less mature with each additional collection.

The results obtained in freezing several consecutive ejaculates are shown in Figure 1 as averages for the ejaculates from 6 bulls. In same instances, there was an insufficient quantity of semen available to test the freezability. (Procedure: Diluted to 30 x 10^{6} sperm per ml. with 1:1 yolk-citrate, then cooled and glycerolated with an equal volume of 14 percent glycerol (percent by volume) in 2.9 percent sodium citrate. Final sperm concentration, 15 x 10^{6}. Equilibration time, 15 hours. Freezing rate, 2 deg. C. per minute from +5 deg. to -19 deg. C. then 4 deg. C. per minute from -19 deg. to -79 deg. C. Held frozen for 5 or more hours then thawed in water at 5 deg. C. and checked for motility.)

In general, the motility before freezing improved slightly from the first to the fourth to sixth ejaculate and then declined until about the 12th or 14th ejaculate, at which point the prefreezing motility seemed to level off through the 20th ejaculate (Fig. 1). The percentage of motile sperm found after freezing and thawing followed the same trend at an average level 10 to 15 percent lower than the prefreezing level. As is readily seen from the trend lines in Fig. 1, the difference between the prefreezing motility and the post-thawing motility increased gradually after about the fifth ejaculate. Although the absolute difference did not increase greatly, the percentage of survival after freezing dropped from 81 percent on the first 5 ejaculates to 26.5 percent on the last 5 (Table 2).

Table 2.--Comparison of the Freezability of 20 Consecutive Ejaculates Collected Within a 4-Hour Period

(Weighted averages for 6 bulls)

=================================================================== Number Prefreezing Post-thawing Ejaculate of motility motility Survival ejaculates (percent) (percent) (percent) ------------------------------------------------------------------- 1st to 5th 29 53.3 43.2 81.0 6th to 10th 26 43.8 30.2 69.0 11th to 15th 23 28.6 14.5 50.7 16th to 20th 19 18.1 4.8 26.5 -------------------------------------------------------------------

=Freezability of epididymal sperm.= Since the freezability of bull semen seemed to be better in second than in first ejaculates and some improvement in freezability was evident through the first 4 to 6 ejaculates taken consecutively, the question of whether epididymal sperm would withstand freezing seemed to be important. Although when 20 collections were made, the later ejaculates no doubt contained fewer mature sperm, the lowered freezability could have been due to accessory gland secretions rather than changes in the sperm themselves. Removing sperm directly from the epididymis would eliminate any effect that the accessory gland secretions could be exerting. Further, if epididymal sperm could be frozen, obtaining and using semen from a bull shortly after his death should be possible.

Table 3.--Freezability of Sperm in Samples Taken From the Epididymides

================================================= Prefreezing Post-thawing Bull motility motility Survival (percent) (percent) (percent) ------------------------------------------------- 1 50 40 80 2 40 13 32 3 60 15 25 4 30 15 50 5+6 40 25 62 Average 44 22 50 -------------------------------------------------

To determine whether epididymal sperm would withstand freezing, the 12 epididymides (cauda only) of 6 slaughtered bulls were flushed with saline (0.9 percent) and the sperm obtained were frozen using the same procedure as was used with the 20 consecutive ejaculates discussed earlier. Averages of the 2 epididymides from each bull are given in Table 3; the samples from bulls 5 and 6 were combined. From the data in Table 3, it is obvious that motile sperm were present after freezing and thawing epididymal samples. It is likely that further experience in handling epididymal sperm may lead to improved results. Using frozen epididymal sperm from 2 bulls, Canadian workers have produced confirmed pregnancies in 8 out of 12 cows.[8]

=Freezability of washed sperm.= In the laboratory it is frequently desirable to study sperm free of the seminal plasma in which they are ejaculated. Sperm can be separated from the seminal plasma by centrifugation, removal of the supernatant plasma, and resuspension in a salt solution of known composition. Sometimes it is desirable to repeat the process. This tends to wash the sperm with the salt solution and sperm handled in this way are called washed sperm.

Sperm cells centrifuged three times and washed twice in 0.9 percent sodium chloride solution withstood freezing well when finally resuspended and frozen in yolk-citrate diluent. The percentage of survival in three samples subjected to this treatment was 60 percent. Thus it appears that the seminal plasma itself is not essential for ejaculated sperm to survive the rigors of freezing. This is not surprising, since it had already been found that epididymal sperm, which also are free of accessory gland secretions, can withstand freezing and thawing.

EXTENDERS FOR FREEZING BULL SEMEN

Both of the extenders that are widely used in routine storage of bull semen at 5 deg. C. are used for freezing semen. These are the egg yolk-sodium citrate and whole or skimmilk extenders. Most of the research with extenders for freezing bull semen in this laboratory has been done with the yolk-citrate diluents.

=Proportion of egg yolk in the final diluent.= Some early experiences with a diluent consisting of one part yolk and one part 2.9 percent sodium citrate dihydrate in distilled water showed poor sperm survival following freezing. The final mixture with this diluent consisted of about 45 percent yolk. In other attempts at adding glycerol in order to freeze semen, the final proportion of yolk was diminished and better sperm survival was obtained. Several experiments were carried out to test the effect of varying levels of egg yolk.

In the first efforts to find the optimum level of egg yolk, the level of yolk in the final frozen mixture was varied from about 6 to 46 percent. These levels were obtained by varying the proportion of yolk to 2.9 percent citrate solution in the original extending media and also in the media added in glycerolating the samples.

Split portions of 20 semen samples were frozen in each of the extender combinations indicated in Table 4. The mean percentages of motile sperm found before and after freezing and thawing are shown also. The highest percentages were found with extenders containing 23 and 24 percent yolk. The highest percentage of yolk, resulting when a 1:1 (yolk to citrate) extender was used for both extending and glycerolating, proved to be most detrimental to sperm survival during freezing. The lowest percentage of yolk used (6 percent) was not as effective in protecting sperm during freezing as the intermediate levels tested (Fig. 2).

Table 4.--Effect of Egg-Yolk Level in Diluent on Freezability of Semen (Average of 20 semen samples)

========================================================================= Medium Yolk in Pre- Post- Sur- ---------------------------- final freezing thawing vival Diluent Extending Glycerolating[C] mix- motility motility (per- ------------ ---------------- ture[D] (percent) (percent) cent) yolk:citrate yolk:citrate (percent) ------------------------------------------------------------------------- 1 1:1 1:1 45.7 65 5 8 2 1:1 1:3 34.9 64 27 42 3 1:3 1:1 33.6 63 33 52 4 1:1 0:1 24.2 64 39 61 5 1:3 1:3 22.8 63 37 59 6 1:3 0:1 12.1 59 33 56 7 1:7 1:7 11.4 56 35 62 8 1:7 0:1 6.0 52 26 50 9 1:15 1:15 5.7 49 25 51 -------------------------------------------------------------------------

[C] This mixture included 14 percent glycerol.

[D] The average initial sperm concentration was 900 x 10^{6}/ml. Sufficient extender was added to give 30 x 10^{6}/ml. at the first extension. Thus the final concentration was 15 x 10^{6} sperm/ml. after glycerolization.

Since rather large changes in the percentages of yolk were used in this experiment, two further trials were conducted in which 16, 24, and 32 percent yolk in the final mixture were compared, with the final citrate percentages held constant. In these tests, 16 and 24 percent yolk maintained sperm better at all citrate levels tried than 32 percent yolk. The 16 percent level was slightly better at most of the levels of citrate tested (Fig. 3).