Preservation of Bull Semen at Sub-Zero Temperatures Part 1

Preservation of Bull s.e.m.e.n at Sub-Zero Temperatures.

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

ACKNOWLEDGMENT

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

THE PRESERVATION OF BULL s.e.m.e.n 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 s.e.m.e.n that had been frozen stimulated much interest and research in freezing as a method of preserving bull s.e.m.e.n. Research during the years following 1951 resulted in considerable progress and success in using freezing as a means of holding s.e.m.e.n for long periods of time without loss of fertility.

Between 1952 and 1957, research on many aspects of the preservation of bull s.e.m.e.n 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 s.e.m.e.n and to present a practical freezing procedure based on the results of these experiments and findings at other inst.i.tutions. Persons interested in the development of the procedures and the reasons why certain steps are necessary in freezing s.e.m.e.n 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 s.e.m.e.n is described.

EARLY WORK ON FREEZING s.e.m.e.n

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 s.e.m.e.n.

An excellent review of the early attempts to freeze sperm has been a.s.sembled by Polge and Parkes.[2] These investigators also gave a good account of their work at the British National Inst.i.tute 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 s.e.m.e.n 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 s.e.m.e.n 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 a.s.sembled here.

SELECTION OF s.e.m.e.n FOR FREEZING

One of the first considerations in freezing s.e.m.e.n is that of deciding which s.e.m.e.n samples are to be frozen. Since preservation of the s.e.m.e.n--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 s.e.m.e.n 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 s.e.m.e.n 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 e.j.a.c.u.l.a.t.es, 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 e.j.a.c.u.l.a.t.es.= In the early days of artificial breeding in this country, it was commonly believed that a second e.j.a.c.u.l.a.t.e collected a few minutes after the first resulted in a larger e.j.a.c.u.l.a.t.e containing more sperm. With the development of the procedure of stimulating s.e.xual excitement by restraint prior to collecting s.e.m.e.n, this difference between first and second e.j.a.c.u.l.a.t.es has been greatly reduced. Still it was noted that second e.j.a.c.u.l.a.t.es frequently withstood freezing better than first e.j.a.c.u.l.a.t.es, even though restraint and stimulation of the bull occurred prior to collection of the first e.j.a.c.u.l.a.t.e. During the course of a number of experiments, it was possible to compare the freezability of 2 e.j.a.c.u.l.a.t.es that were collected a few minutes apart from the same bull. Two consecutive e.j.a.c.u.l.a.t.es 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 e.j.a.c.u.l.a.t.es are presented in Table 1.

An a.n.a.lysis of variance showed that in this comparison the differences between first and second e.j.a.c.u.l.a.t.es in sperm survival during freezing were highly significant. A later comparison of 27 first and second e.j.a.c.u.l.a.t.es from 26 bulls did not show as great a difference between first and second e.j.a.c.u.l.a.t.es in their ability to withstand freezing

=Freezability of several consecutive e.j.a.c.u.l.a.t.es.= The fact that second e.j.a.c.u.l.a.t.es sometimes withstood freezing better than first e.j.a.c.u.l.a.t.es suggested that the maturity of the sperm might be a factor affecting freezability. An opportunity to check this idea came when 20 consecutive e.j.a.c.u.l.a.t.es 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 e.j.a.c.u.l.a.t.es are shown in Figure 1 as averages for the e.j.a.c.u.l.a.t.es from 6 bulls. In same instances, there was an insufficient quant.i.ty of s.e.m.e.n available to test the freezability. (Procedure: Diluted to 30 10^{6} sperm per ml. with 1:1 yolk-citrate, then cooled and glycerolated with an equal volume of 14 percent glycerol (percent by volume) in 2.9 percent sodium citrate.

Final sperm concentration, 15 10^{6}. Equilibration time, 15 hours.

Freezing rate, 2 C. per minute from +5 to -19 C. then 4 C. per minute from -19 to -79 C. Held frozen for 5 or more hours then thawed in water at 5 C. and checked for motility.)

[Ill.u.s.tration: Percent of motile sperm before and after freezing consecutive e.j.a.c.u.l.a.t.es collected within a 4-hour period from each of 6 bulls (Fig. 1)]

In general, the motility before freezing improved slightly from the first to the fourth to sixth e.j.a.c.u.l.a.t.e and then declined until about the 12th or 14th e.j.a.c.u.l.a.t.e, at which point the prefreezing motility seemed to level off through the 20th e.j.a.c.u.l.a.t.e (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 e.j.a.c.u.l.a.t.e. Although the absolute difference did not increase greatly, the percentage of survival after freezing dropped from 81 percent on the first 5 e.j.a.c.u.l.a.t.es to 26.5 percent on the last 5 (Table 2).

Table 2.--Comparison of the Freezability of 20 Consecutive e.j.a.c.u.l.a.t.es Collected Within a 4-Hour Period

(Weighted averages for 6 bulls)

=================================================================== Number Prefreezing Post-thawing e.j.a.c.u.l.a.t.e of motility motility Survival e.j.a.c.u.l.a.t.es (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 s.e.m.e.n seemed to be better in second than in first e.j.a.c.u.l.a.t.es and some improvement in freezability was evident through the first 4 to 6 e.j.a.c.u.l.a.t.es taken consecutively, the question of whether epididymal sperm would withstand freezing seemed to be important. Although when 20 collections were made, the later e.j.a.c.u.l.a.t.es 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 s.e.m.e.n 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 e.j.a.c.u.l.a.t.es 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 e.j.a.c.u.l.a.t.ed. 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 e.j.a.c.u.l.a.t.ed 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 s.e.m.e.n

Both of the extenders that are widely used in routine storage of bull s.e.m.e.n at 5 C. are used for freezing s.e.m.e.n. These are the egg yolk-sodium citrate and whole or skimmilk extenders. Most of the research with extenders for freezing bull s.e.m.e.n 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 s.e.m.e.n, 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 s.e.m.e.n 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).