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Full text of "Preservation of bull semen at sub-zero temperatures"

XI B RARY 

OF THE 

UNIVERSITY 
OF ILLINOIS 

S3O/7 

U<ob 



AGRICULTURE 



CIRCULATING 



CHECK FOR UNBOUND 
CIRCULATING COPY 



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 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 to 79 C 24 

Use of higher glycerol levels and storage at 20 C 25 

THAWING 26 

Comparison of thawing at 5 C. and at 38 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 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* 

THE FIRST ANNOUNCEMENT in 1951 of the successful im- 
pregnation of a cow with bull semen that had been frozen stim- 
ulated 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 preserva- 
tion 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 gen- 
eral 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 * found that human sperm 
would withstand freezing. For thirty to forty years after that, little 

* 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. 

* These numbers refer to literature citations on page 38. 



6 BULLETIN No. 621 [October, 

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 sur- 
vive 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> T 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 decid- 
ing 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 freez- 
ability ? 

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, 



1957] PRESERVATION OF BULL SEMEN 7 

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 col- 
lecting semen, this difference between first and second ejaculates has 
been greatly reduced. Still it was noted that second ejaculates fre- 
quently 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 differ- 
ences 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 be- 
tween first and second ejaculates in their ability to withstand freezing 
(Table 1). 

Freezability of several consecutive ejaculates. The fact that sec- 
ond 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 con- 
Table 1. Comparison of the Freezability of First and Second 
Ejaculates Taken a Few Minutes Apart From the Same Bull 



Number 
of 
bulls 


Number 
of 
ejaculates 


Ejaculate 


Prefreezing 
motility 
(percent) 


Post-thawing 
motility 
(percent) 


Survival 
(percent) 


24 


58 


1st 


60 


39 


65 


26 


27 


2d 
1st 


62 
60 


45 
36 


74 
60 






2d 


65 


40 


61 



8 BULLETIN No. 621 [October, 

secutive ejaculates were collected from each of 6 bulls within a 4-hour 
period. The sperm in samples collected in this manner might be ex- 
pected 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 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.) 

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 in- 



50 



ORIGINAL MOTILITY 




POST-THAWING 
MOTILITY 



I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 

(6) (6) (5) (6) (6) (4) (5) (6) (6) (5) (4) (6) 14) (5) 14) (4) (5) (4) (3) (3) 

CONSECUTIVE EJACULATE NUMBER (AND NUMBER OF SAMPLES) 

Percent of motile sperm before and after freezing consecutive ejaculates 
collected within a 4-hour period from each of 6 bulls (Fig. 1) 



1957] PRESERVATION OF BULL SEMEN 

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

(Weighted averages for 6 bulls) 



Ejaculate 


Number 
of 
ejaculates 


Prefreezing 
motility 
(percent) 


Post-thawing 
motility 
(percent) 


Survival 
(percent) 


1st to 5th 


29 


53 3 


43 2 


81 


6th to 10th 


26 


43 8 


30 2 


69 


llth to 15th 


23 


28.6 


14.5 


50.7 


16th to 20th 


19 


18.1 


4.8 


26.5 













creased 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 per- 
cent on the last 5 (Table 2). 

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 ejacu- 
lates 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 acces- 
sory 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. 

To determine whether epididymal sperm would withstand freezing, 
the 12 epididymides (cauda only) of 6 slaughtered bulls were flushed 



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



Prefreezing Post-thawing 
Bull motility motility 

. (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 



10 BULLETIN No. 621 [October, 

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 cen- 
trifugation, removal of the supernatant plasma, and resuspension in a 
salt solution of known composition. Sometimes it is desirable to re- 
peat 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 per- 
cent sodium chloride solution withstood freezing well when finally 
resuspended and frozen in yolk-citrate diluent. The percentage of sur- 
vival 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 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 



1957] PRESERVATION OF BULL SEMEN 11 

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). 

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). 

Citrate level in the final diluent. The early work of the British 
indicated that a final citrate level near 2 percent in the diluent was 



Table 4. Effect of Egg- Yolk Level in Diluent 
on Freezability of Semen 

(Average of 20 semen samples) 



Medium pre , post _ 



Diluent Extending Glycerolating- . fi " al b fre ^f tha n S Survival 
B } & mixture b motihty motility (percent) 

yolk: citrate yolk : citrate (P ercei *) (percent) (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 


52 


26 


50 


9 


1:15 


1:15 


5 7 


49 


25 


51 

















a This mixture included 14 percent glycerol. 

b The average initial sperm concentration was 900 x 10 6 /ml. Sufficient extender was added 
to give 30 x lO'/ml. at the first extension. Thus the final concentration was 15 x 10 6 sperm/ml, 
after glycerolization. 



BULLETIN No. 621 [October, 



40 



a 30 
I 



20 

fc 

I- 

UJ 

ce 10 



X TRIAL I 
* TRIAL 2 



10 20 30 40 50 

PERCENT OF YOLK 

Percent of motile sperm after freezing and thawing semen in diluents con- 
taining various levels of egg yolk (Fig. 2) 



satisfactory for freezing bull sperm. Later, in a personal communica- 
tion, 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 Fig. 3. In these ex- 
periments, 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 Fig. 3. 
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 con- 
centration of 1.55 percent. 

From the results of these experiments, and from several reports 
in the literature, 5 - 6> T> 9> 10 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. 

Storing and freezing diluent. In some instances it would be ad- 
vantageous to have prepared diluent on hand for use at any time. The 



1957] PRESERVATION OF BULL SEMEN 13 



50 



40 



30 



/ - K LEVEL 

16% 

16% 

X- -X 24% 

X X 24% 

o o 32% 




095 1.15 1.35 1.55 1.75 1.95 2.15 335 

PERCENT OF CITRATE 

Percent of motile sperm after freezing and thawing semen in diluents con- 
taining various levels of egg yolk and various percentages of sodium citrate 

(Fig. 3) 



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. 

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. 

Other diluents. 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 Table 5. 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. 

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. 6 ' 7 



14 



BULLETIN No. 621 



[October, 



Table 5. Comparison of the Freezability of 4 Semen Samples 
in Different Extenders 





Dilution 


Pre- 


Post- 




Motility 


Extender 


rate 
(semen : 


freezing 
motility 


thawing 
motility 


Survival 
(percent) 


after 
storage" 




extender) 


(percent) 


(percent) 




(percent) 


Yolk-citrate 


1:1 


60 


49 


82 


46 




1:10 


53 


45 


85 


36 


Yolk-saline 


1:1 


57 


29 


51 


28 




1:10 


60 


31 


52 


24 


Yolk-phosphate 


1:1 


55 


35 


64 


25 




1:10 


60 


43 


72 


25 


Whole milk 


1:1 


60 


40 


67 


35 




1:10 


60 


35 


58 


16 



Stored at 5 C. for 7 hours after thawing. 



DILUTION RATES 

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 Table 6, 
show that the numbers of sperm between 10 and 90 million per 
milliliter did not influence the percentage of sperm that survived 
freezing. 

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. 11 ' 12 

During the early studies in the Illinois laboratory, the effects cf 
glycerol level were also tested. 13 These effects are discussed in the 
section on glycerol additions beginning on page 17. 

Effect of further dilution and refreezing after the initial freezing. 

Under some circumstances it might be advantageous to freeze semen 



1957] PRESERVATION OF BULL SEMEN 

Table 6. Effect of Sperm Numbers and Glycerol Level 

in Final Mixture on Freezability of Bull Sperm at 79 C. 

(Average of 10 ejaculates) 



15 



Glycerol level 
(percent) 


Post-thawing motility (percent) 8 


Number of sperm (millions/ml.) 


Average 


90 


30 


10 


5 


36 


34.0 
24.0 
0.9 
19.8 


36.0 
23.0 
0.2 
19.9 


35.0 
23.0 
1.4 
20.0 


10 


22 


15 


3 2 


Average 


20 3 







a Mean initial motility of sperm before freezing was 55 percent. 



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. 

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. 



Table 7. Effect of Further Dilution and Refreezing on Sperm Motility 
After the Initial Freezing of Bull Semen 





Dilution of 
semen 
(semen : 
extender) 


Pre- 
freezing 
motility 
(percent) 




Post-thawing 


motility 




After 
first 
freezing 


After storage 8 


After 


refreezing b 


No Diluted 
further to 15 
dilution million/ml. 


No 
further 
dilution 


Diluted 
to 15 
million/ml. 


1:1 

1:1 

15 




60 


First trial: 4 samples 
49 46 34 
45 36 30 

Second trial: 7 samples 
47 41 35 
30 32 


31 

25 

28 
18 


6 

5 

11 





. . 53 




67 


million/ml. . 


. . 67 







8 Stored at 5 C. for 3 to 7 hours after first thawing. 
b Refrozen following first thawing. 



16 



BULLETIN No. 621 



[October, 











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c 

rt 
J3 


percent r< 


CN CS CN CS CN O CN 

*O ^< tNI CN CN O PO 


1 




























































H 












2 
























-1 
















CD 












O) ' 


-*s 












C 










O 


U 










<u 


E 












a 










v 


^ 










O 






















































O bfl 












5 : i . : : s 












f R O "0 O O O 5 



7957] PRESERVATION OF BULL SEMEN 17 

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. 

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 Table 7. 

From Table 7 it can be seen that re freezing 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. 

GLYCEROL ADDITIONS 

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. 

Effect of glycerol on sperm survival at 5 C. 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 pro- 
gressive motility) are presented in Table 8. 

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 



18 BULLETIN No. 621 [October, 

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. 

Glycerol levels for freezing semen. 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 Table 6, 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 Table 9 it can be seen that 
glycerol levels of 6 and 8 percent in the final mixture resulted in max- 
imum 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 Table 10). 

The results shown in Tables 9 and 10 were confirmed also in later 
experiments. Thirty-six samples were subjected to various levels of 
glycerol and no significant difference in freezability was found be- 
tween 6 and 8 percent. Based on these findings, a glycerol level of 7 
percent was adopted for use in all experiments described in this bulle- 
tin, unless otherwise indicated. Results in a number of other labora- 
tories have agreed with our findings regarding the use of approximately 
7 percent glycerol with the yolk-citrate diluent. 5 - G> 7 ' 9> 10 With milk as 
the extender, 10 to 13 percent glycerol has been preferred by some. 5 ' 6> 7 

Rate, temperature, and method of adding glycerol. Closely associ- 
ated 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 



Table 9. Effect of Glycerol Level on Sperm Motility 
After Freezing to 79 C. and Thawing 







Pre- 


Post- 




Glycerol level 


Number 


freezing 


thawing 


Survival 


(percent) 


of samples 


motility 


motility 


(percent) 






(percent) 


(percent) 




2 . 


10 


53 


2 


4 


4 


19 


55 


29 


53 


6 


19 


55 


34 


62 


8 


19 


55 


35 


64 


10 


19 


55 


24 


44 


12.. 


10 


53 


13 


25 



1957] PRESERVATION OF BULL SEMEN 19 

Table 10. Effect of Glycerol Level and Storage at 5 C. 

After Thawing on Sperm Motility 

(Average of 13 ejaculates) 





Sperm 


motility (percent) 




Glycerol level 
(percent) 


Post- 


After storage at 


5 C. 




thawing 


1 day 3 


days 


4 


29 


22 


20 


6 


38 


34 


24 


8 


42 


33 


17 


10 


33 


18 


6 











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 in vitro 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 Table 11. 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 



Table 11. Effects of Temperature, Rate of Addition of Glycerol, 

and Equilibration Time on Sperm Motility 

(Average of 12 ejaculates) 

Post-thawing motility (percent) 
Temperature during addition Equilibration 



iraiure uuriiig iuui t ; Glycerol additions 

of glycerol ( C.) (h^, r ^ 

531 Average 



4.5 


2 


48 


48 


45 


47.4 




6 


49 


51 


47 


48.8 




18 


46 


47 


46 


46.3 




Average 


47.8 


48.6 


46.0 


47.5 


10.0 


2 


44 


43 


45 


43.9 




6 


48 


50 


46 


47.9 




18 


43 


46 


42 


44.0 




Average 


45.0 


46.5 


44.3 


45.3 


15.5 


2 


41 


38 


38 


39.1 




6 


42 


45 


43 


43.6 




18 


42 


43 


42 


42.5 




Average 


42.0 


41.8 


41.4 


41.7 



20 BULLETIN No. 621 [October, 

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. 

A comparison of the results from adding the glycerol in 5, 3, and 
1 equal portions is given also in Table 11. 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 sur- 
vival. 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. 

Allowing sperm to equilibrate with the glycerol. Allowing sperm 
to stand in the presence of glycerol is considered by some to be nec- 
essary 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 Table 11. 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 bet- 
ter than 2 or 18 hours. 

Sugar additions and equilibration time. 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. 5 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 Table 12. 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 



1957} 



PRESERVATION OF BULL SEMEN 



21 



Table 12. Effect of Adding Sugars to Yolk-Citrate Diluent 
on Sperm Motility During Equilibration With Glycerol" 



Stage when observed 


Sperm motility (percent) 


Glycerol 
only 


Glycerol 
and glucose 


Glycerol 
and rhamnose 


Fresh diluted semen 


56 


56 

54 

53 
52 
50 
46 


56 
54 

53 
53 
51 
46 


After glycerolization 


54 


After equilibration 
2 hours 


51 


6 hours 


48 


12 hours 


46 


18 hours 


40 







a Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level 
of 1.25 percent. 

of surviving sperm remaining after the various steps in the freezing 
procedure with and without the presence of these sugars are shown in 
Table 13. 

Three of the sugars glucose, arabinose, and rhamnose pro- 
tected 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 equili- 
bration, 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. 



Table 13. Effect of Adding Sugars to Yolk-Citrate Diluent 
on Sperm Motility During the Freezing Procedures" 

(Average of 10 ejaculates) 







Sperm 


motility (percent) 


Stage of 
observation 


Glycerol 
only 


Glycerol 
and 
glucose 


Glycerol 
and 
arabinose 


Glycerol 
and 
xylose 


Glycerol 
and 
rhamnose 


Fresh diluted semen 


63 


63 

55 
43 

34 
26 


63 

54 
44 

34 
26 


63 

57 
39 

29 

25 


63 
60 
46 

34 

27 


After glycerolization 


54 


After 18 hours equilibration . . 39 
After freezing to 79 C. and 
immediate thawing 28 


After 4 days at -79 C.. . . 


. 23 



Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level 
of 1.25 percent. 



22 BULLETIN No. 621 [October, 

Substitutes for glycerol. Since glycerol was so effective in protect- 
ing 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, trimethy- 
lene 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 inter- 
actions of experimental conditions, sufficient data were gathered to 
lead us to abandon further study until greater promise of success 
might be evident. 

FREEZING RATE 

Effect of freezing rate on sperm survival. 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. 

Some investigators have suggested that rapid cooling below 20 
C. is not detrimental to frozen semen. This idea was tested in conjunc- 
tion with other experiments. Twenty- five samples cooled slowly (2 



1957} 



PRESERVATION OF BULL SEMEN 



23 



-20 

o 

C, 
in 
-30 

s 



Z -40 




BATH 

GLASS 
PLASTIC 



Cooling rates of diluted 

semen samples in plastic 

vials and in glass ampules 

(Fig. 4) 



IS 20 

MINUTES 



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. 

Rate of cooling in plastic and in glass. 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 Figure 4. 

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 Figure 4. 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. 



24 



BULLETIN No. 621 



[October, 



STORAGE TEMPERATURE 

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 expen- 
sive. If warmer temperatures were suitable for storing frozen semen, 
the ordinary deep-freeze, which operates at 15 to 25 C., might 
be used. 

Storage at temperatures from 23 to 79 C. 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 (Fig. 5). 



40 



30 



20 



a 10 



-79C 




-37C 



12345 
DAYS STORED 

Effect of freezing and storing bull sperm at various temperatures on the 
sperm motility at thawing (average of 9 ejaculates) (Fig. 5) 



1957] PRESERVATION OF BULL SEMEN 25 

At the end of 1 day, samples stored at 79 C. exhibited approxi- 
mately 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 labora- 
tories indicate that storage temperatures much lower than 79 C. 
are just as satisfactory as 79 C. 

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: 

Day 2 9 16 51 

Percent of motile sperm 49 46 40 38 

Rate of motility 2.5 2.3 2.2 2.2 

The average pre freezing 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. 

The British and the Australians have both reported the successful 
maintenance of fertility in frozen semen stored at 79 C. for over 
two years. 5 

Use of higher glycerol levels and a 20 C. storage temperature. 

In 1953, a report from Arkansas suggested that warmer storage tem- 
peratures could be used if a high percentage of glycerol were included 
in the freezing mixture. 7 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. 



26 BULLETIN No. 621 [October, 

Table 14. Effect of Glycerol Level and Storage Temperature 

on Freezability of Semen 

(Average of 8 ejaculates) 



Storage 
temperature 
(C.) 


Glycerol 


Sperm motility after storage (percent) 


(percent) 


18 hours 


42 hours 


-79 
-20 


7 
7 
11 
15 
19 
23 
27 
31 


61 
2 
3 
14 
30 
29 
25 
21 


61 
1 
1 
10 
22 
19 
18 
12 



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 Table 14. 

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) con- 
tained 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 stor- 
ing, storage at ordinary deep-freeze temperatures is far inferior to 
storage at dry-ice temperatures. 

THAWING 

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 tem- 
perature might be more desirable so that cooling again will not be 
necessary. 



1957} 



PRESERVATION OF BULL SEMEN 



27 



Comparison of thawing temperatures of 5 C. and 38 C. 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 Figure 6. 

The 5 C. thawing temperature resulted in a higher percentage of 
sperm survival at all the glycerol levels than 38 C., with the differ- 
ence 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. (Table 15). It was also 
evident that the interaction between glycerol level and thawing tem- 
perature continued during storage (Fig. 7). 

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 



so 



40 



30 



20 



THAWED AT5C 




THAWED AT 38 C 




Effect of glycerol percent- 
age and thawing tempera- 
ture on sperm motility 
after freezing and thawing 
(Fig. 6) 



t 6 8 

PERCENT OF GLYCEROL 



28 



BULLETIN No. 621 



[October, 



40 



30 



20 



10 




THAWED AT 38 8 C 



Effect of thawing tempera- 
ture on sperm motility during 
storage at 5 C. following 
freezing and thawing (Fig. 7) 



DAYS STORED 

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 



Table 15. Effect of Glycerol Level, Thawing Temperature, 

and Storage at 5 C. After Thawing on Sperm Motility 

(Average of 13 ejaculates) 



Thawing 
temperature 
(C.) 


Glycerol 
level 
(percent) 


Sperm motility (percent) 


Post- 
thawing 


After storage at 5 C. 


Average 


1 day 


3 days 


38 


4 


28.5 


17.3 


5.1 


17.0 




6 


31.5 


22.4 


9.2 


21.1 




8 


33.1 


15.0 


4.6 


17.6 




10 


19.5 


3.6 


0.8 


8.0 




Average 


28.2 


14.6 


4.9 


12.2 


5 


4 


29.2 


21.7 


19.8 


23.9 




6 


37.7 


33.8 


23.5 


31.7 




8 


41.5 


33.1 


17.3 


30.6 




10 


33.1 


18.5 


6.0 


19.2 




Average 


35.4 


26.8 


16.6 


20.6 



1957} 



PRESERVATION OF BULL SEMEN 



29 



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. 11 If the semen is used immediately, a thawing tem- 
perature 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. 

Thawing rate in plastic and in glass. 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 Figure 8. 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. 



-60 



-70 




BATH 

GLASS 

PLASTIC 



15 20 

MINUTES 



Warming rates of diluted 
semen samples in plastic vials 
and in glass ampules (Fig. 8) 



30 BULLETIN No. 621 [October, 

EFFECTS OF FREEZING PROCEDURES ON METABOLIC 
ACTIVITY OF BULL SPERM 

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 measure- 
ment of oxygen-consumption and estimates of sperm motility during 
and after incubation at 37 C. in a Warburg apparatus. 

Effect of glycerol additions on oxygen uptake of diluted semen at 

37 C. The effect of adding glycerol to diluted semen on oxygen con- 
sumption 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 concentra- 
tion in 0.5 milliliter to 200 million to 500 million. An exact count was 
used to calculate the oxygen uptake per 10 8 sperm per hour (Z0 2 ). 

Glycerol in various percentages in 2.9 percent sodium citrate 
dihydrate solution was placed in the sidearm of the Warburg flasks. 



40 - 



I 

05 

K 

20 

i 

5 



NO ADDITIONS 
4% GLYCEROL 
8% GLYCEROL 
12% GLYCEROL 




^GLYCEROL ADDED 



20 40 60 80 100 120 140 160 180 

MINUTES 

Effect of glycerol additions on oxygen consumption of sperm at 37 C. 

(Fig. 9) 



1957} 



PRESERVATION OF BULL SEMEN 



31 



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. 

Oxygen uptake was increasingly stimulated during the first 20- 
minute interval by each increase in the amount of glycerol added 
(Fig. 9). After the first 20 minutes, the rate of oxygen utilization 
decreased at the two higher levels of glycerol but persisted at 4 per- 
cent. 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. 



60 



30 



20 




NO 
GLYCEROL 



NO CATALASE 



CATALASE ADDED 




4% 
GLYCEROL 



8% 
GLYCEROL 



12% 
GLYCEROL 



Effect of glycerol and glycerol-plus-catalase additions on sperm motility 
during incubation at 37 C. (Fig- 10) 



32 



BULLETIN No. 621 



[October, 



With each increase in glycerol level, motility was reduced during 
the incubation period. This is shown in Figure 10 along with the effect 
on motility of adding catalase, which is discussed in the next section. 

Effect of glycerol-plus-catalase on oxygen uptake of diluted 
semen. Certain bacteria have been shown to break glycerol down, 
forming hydrogen peroxide as follows: 

Glycerol + oxygen -> lactic acid -f- hydrogen peroxide. 
Hydrogen peroxide is known to be detrimental to sperm. The addition 
of glycerol to diluted semen first increased oxygen uptake and then re- 
duced 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 perox- 
ide 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. Com- 
parison of the resulting oxygen uptake with glycerol and with glycerol 
plus catalase is shown in Figure 11. 

Oxygen consumption was increased by the presence of added 



40 



30 



20 



NO ADDITIONS 
CATALASE ONLY 
4% GLYCEROL * CATALASE 
8% GLYCEROL + CATALASE 
13% GLYCEROL * CATALASE 




"GLYCEROL AND 
CATALASE ADDED 



20 



40 



60 



80 100 

MINUTES 



120 



140 



160 



ISO 



Effect of additions of glycerol-plus-catalase on oxygen consumption of 
sperm at 37 C. (Fig. 11) 



1957} PRESERVATION OF BULL SEMEN 33 

Table 16. Effect of Freezing Procedures on Oxygen Utilization 
of Bull Sperm in Yolk-Citrate Extender 

(Average of 5 ejaculates) 

Microliters of oxygen 
Semen sample tested utilized per 10* sperm 

First hour Second hour 



Fresh diluted semen . 


10 3 


8 1 


Fresh diluted semen glycerol tipped in at end of first hour 
Aged 20 to 24 hours at 5 C 


9.7* 
11.2 


12.9* 

8.3 


Aged 20 to 24 hours at 5 C. glycerol tipped in at end of first 
hour 


11.8* 


12.9* 


After 20 hours equilibration with glycerol 


11 7* 


7.8* 


After freezing and thawing 


9 7 


6.3 









* Average of 3 ejaculates. 

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 (Fig. 10). 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. 

Effect of freezing procedures on oxygen utilization by sperm. 

Limited data have been obtained on the effects of some of the freezing 
procedures on the oxygen utilization of bull sperm. The results ob- 
tained in these experiments confirmed the earlier findings that tipping 
glycerol directly into the diluted semen at 37 C. caused an increase in 
oxygen consumption (Table 16). All other steps in the freezing pro- 
cedure 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. 

Effect of freezing procedures on methylene-blue reduction time. 

The methylene-blue reduction test has been used as a means of meas- 
uring semen quality and is dependent on the metabolic activity of the 



34 



BULLETIN No. 621 



[October, 



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 X 10 6 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. 

A marked increase in the time required for the sperm to reduce 
methylene blue occurred when the glycerol was added (Table 17). 
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. 



Table 17. Effect of Freezing Procedures on the Methylene-Blue 

Reduction Time of Bull Semen With and Without 

the Addition of Sugars* 

(Average of 10 ejaculates) 



Methylene-blue reduction time (minutes) 




Glycerol 
only 


Glycerol 
and 
glucose 


Glycerol 
and 
arabinose 


Glycerol 
and 
xylose 


Glycerol 
and 
r ham nose 


Fresh semen 


5.2 
26.4 

7.4 

11.5 
14.3 


5.2 
25.2 
6.5 

10.5 
10.2 


5.2 
17.3 
6.4 

9.4 
11.3 


5.2 
14.3 
5.3 

9.0 
10.1 


5.2 
19.4 
6.2 

9.4 
9.5 


After glycerolization 


After 18 hours equilibration 
Thawed immediately after 
freezing 


Thawed 48 hours after 
freezing 





a Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level 
of 1.25 percent. 



1957] PRESERVATION OF BULL SEMEN 35 

PRACTICAL FREEZING PROCEDURE 

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 fol- 
lowing the directions and suggestions given below will usually pro- 
duce satisfactory results with semen samples that are of good quality 
at the start. 

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. 

Collection of the semen. 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. 

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 pro- 
tected from contamination and from sudden temperature drops (cold 
shock). 

Preparation of extender. 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 



36 BULLETIN No. 621 [October, 

then boiled or autoclaved. The citrate solution should be cooled be- 
fore 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. 

Dilution after collection. 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 (\Y2 to 2i/2 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 anti- 
biotics. 6 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.) 

Adding the glycerol. 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.) 

Equilibration. The results presented in this bulletin suggest that 



1957] PRESERVATION OF BULL SEMEN 37 

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. 

Freezing. 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 con- 
tainer 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. 

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 refrigerat- 
ing equipment. At no time prior to thawing should the samples be 
exposed to warmer temperatures. 

Thawing. The ampules of frozen semen can be thawed by remov- 
ing 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 breed- 
ing within a few minutes after thawing. 



38 BULLETIN No. 621 [October, 

LITERATURE CITED 

1. DAVENPORT, C. B. Effect of chemical and physical agents upon protoplasm. 

Macmillan and Co., New York. 1897. 

2. POLGE, C., and PARKES, A. S. Possibilities of long-term storage of sperma- 

tozoa at low temperatures. Anim. Breeding Abs. 20:1-5. 1952. 

3. EMMENS, C. W., and BLACKSHAW, A. W. The low temperature storage of 

ram, bull, and rabbit spermatozoa. Austral. Vet. Jour. 26:226. 1950. 

4. SMITH, AUDREY W. 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. 

5. EMMENS, C. W., and BLACKSHAW, A. W. Artificial insemination. Physiol. 

Rev. 36:277-306. 1956. 

6. Proceedings of the National Association of Artificial Breeders, 1953, 1954, 

and 1955. 

7. 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. 

8. BARKER, C. A. V. Low temperature preservation of bovine epididymal 

spermatozoa. Canad. Jour. Comp. Med. 18:390-393. 1954. 

9. SAROFF, JACK, and MIXNER, J. P. 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. 38:292- 
297. 1955. 

10. CKAGLE, R. G., MYERS, R. M., WAUGH, R. K., HUNTER, J. S., and ANDERSON, 

R. L. The effects of various levels of sodium citrate, glycerol, and equi- 
libration time on survival of bovine spermatozoa after storage at 79 C. 
Jour. Dairy Sci. 38:508-514. 1955. 

11. BRATTON, R. W., FOOTE, R. H., and CRUTHERS, JOAN C. Preliminary fertility 

results with frozen bovine spermatozoa. Jour. Dairy Sci. 38:40-46. 1955. 

12. HAFS, H. D., and ELLIOTT, F. I. The effects of methods of adding egg yolk 

and monosaccharides on the survival of frozen bull spermatozoa. Tour. 
Dairy Sci. 38:811-815. 1955. 

13. MILLER, W. J., and VANDEMARK, N. L. 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. 37:45-51. 
1954. 



1957] PRESERVATION OF BULL SEMEN 39 



TEMPERATURE CONVERSIONS 



+ 38 +100 -20 -4 

+ 35 +95 -25 -13 

+ 30 +86 -30 -22 

+ 25 + 77 -35 -31 

+ 20 +68 -40 -40 

+ 15 +59 -45 -49 

+ 10 +50 -50 -58 

+ 5 +41 -55 -67 

+32 -60 -76 

-5 +23 -65 -85 

-10 +14 -70 -94 

-15 +5 -75 -103 

-18 -79 -110 



5M 3-59 67331 






UNIVERSITY OF ILLINOIS-URBANA