would survive freezing. In 1951 frozen semen was used to produce a calf in England and a lamb in Australia.

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

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 × 106 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 × 106. 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.)