Preservation of Bull Semen At Sub-Zero Temperatures

Part 1

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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

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.

Part 2

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 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. 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 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.

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 and Smith 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.,, 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.

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