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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° 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[A]

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


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

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

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




EARLY WORK ON FREEZING SEMEN


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

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

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




SELECTION OF SEMEN FOR FREEZING


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

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

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

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

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

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

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

The results obtained in freezing several consecutive ejaculates are
shown in Figure 1 as averages for the ejaculates from 6 bulls. In same
instances, there was an insufficient quantity of semen available to test
the freezability. (Procedure: Diluted to 30 × 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.)

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

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

(Weighted averages for 6 bulls)

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

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

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

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

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

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

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




EXTENDERS FOR FREEZING BULL SEMEN


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

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

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

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

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

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

  [C] This mixture included 14 percent glycerol.

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

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

=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
satisfactory for freezing bull sperm. Later, in a personal
communication, 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 experiments, 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 concentration of
1.55 percent.

[Illustration: Percent of motile sperm after freezing and thawing semen
in diluents containing various levels of egg yolk (Fig. 2)]

From the results of these experiments, and from several reports in the
literature,[5],[6],[7],[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
advantageous to have prepared diluent on hand for use at any time. The
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.

[Illustration: Percent of motile sperm after freezing and thawing semen
in diluents containing various levels of egg yolk and various
percentages of sodium citrate (Fig. 3)]

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]

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

  ======================================================================
                Dilution      Pre-       Post-                 Motility
  Extender        rate      freezing   thawing     Survival    after
                (semen:     motility   motility   (percent)   storage[E]
                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         33          64          25
                  1:10         60         43          72          25
  Whole milk      1:1          60         40          67          35
                  1:10         60         35          58          16
  ----------------------------------------------------------------------

  [E] 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 of
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 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.

Table 6.--Effect of Sperm Numbers and Glycerol Level in Final Mixture on
Freezability of Bull Sperm at -79° C. (Average of 10 ejaculates)

  ================================================================
                         Post-thawing motility (percent)[F]
                     ---------------------------------------------
  Glycerol level       Number of sperm (millions/ml.)
  (percent)          --------------------------------
                           90       30       10         Average
  ----------------------------------------------------------------
  5                       36.0     34.0     36.0         35.0
  10                      22.0     24.0     23.0         23.0
  15                       3.2      0.9      0.2          1.4
  Average                 20.3     19.8     19.9         20.0
  ----------------------------------------------------------------

  [F] Mean initial motility of sperm before freezing was 55 percent.

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. 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.

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

  ========================================================================
                                Post-thawing motility
  Dilution   Pre-      ---------------------------------------------------
  of         freezing   After      After storage[G]    After refreezing[H]
  semen      motility   first     ------------------- --------------------
  (semen:   (percent)  freezing     No     Diluted        No     Diluted
  extender)                      further    to 15      further    to 15
                                 dilution million/ml   dilution million/ml
  ------------------------------------------------------------------------
                          First trial: 4 samples
  1:1           60        49        46       34           31        6
  1:10          53        45        36       30           25        5

                          Second trial: 7 samples
  1:9           67        47        41       35           28       11
  15 million/ml 67        30        32       ..           18       ..
  ------------------------------------------------------------------------

  [G] Stored at 5° C. for 3 to 7 hours after first thawing.

  [H] Refrozen following first thawing.

Table 8.--Effect of Glycerol Level and Storage at 5° C. on Motility of
Sperm in Yolk-Citrate Extender

  ====================================================================
                                   Sperm motility
              --------------------------------------------------------
                Post-        After storage at 5° C.
  Glycerol     thawing    ----------------------------      Average
   level                   1 day      3 days     7 days
  (percent)   ---------   ---------  ---------  ---------  ---------
              per- rate   per- rate  per- rate  per- rate  per- rate
              cent        cent       cent       cent       cent
  --------------------------------------------------------------------
  Control[I]   56  2.5     55  1.9    46  1.8    38  1.4    48  1.90
  0            54  2.4     44  1.9    46  1.8    36  1.4    45  1.87
  5            52  2.2     50  1.9    46  1.7    32  1.4    45  1.80
  10           52  2.3     46  1.8    42  1.7    28  1.6    42  1.85
  20           52  2.1     50  1.7    44  1.6    38  1.1    46  1.62
  30           50  0.7     44  0.5    42  0.4    30  0.4    42  0.51
  Average      53  2.03    47  1.62   44  1.50   34  1.22   ..  ....
  --------------------------------------------------------------------

  [I] The control differed from the 0-glycerol treatment in that no
  additional citrate or glycerol solution was added.

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 refreezing 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 progressive 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
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
maximum 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 between
6 and 8 percent. Based on these findings, a glycerol level of 7 percent
was adopted for use in all experiments described in this bulletin,
unless otherwise indicated. Results in a number of other laboratories
have agreed with our findings regarding the use of approximately 7
percent glycerol with the yolk-citrate diluent.[5],[6],[7],[9],[10] With
milk as the extender, 10 to 13 percent glycerol has been preferred by
some.[5],[6],[7]

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

  ==========================================================
  Glycerol    Number        Pre-       Post-      Survival
   level        of        freezing    thawing     (percent)
  (percent)   samples     motility    motility
                         (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
  ----------------------------------------------------------

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        Post-     After storage at 5° C.
  (percent)     thawing    -----------------------
                               1 day   3 days
  ------------------------------------------------
     4            29             22      20
     6            38             34      24
     8            42             33      17
    10            33             18       6
  ------------------------------------------------

Table 11.--Effects of Temperature, Rate of Addition of Glycerol, and
Equilibration Time on Sperm Motility

(Average of 12 ejaculates)

  ===================================================================
  Temperature                       Post-thawing motility (percent)
    during       Equilibration     ----------------------------------
   addition          time                 Glycerol additions
  of glycerol       (hours)        ----------------------------------
    (° C.)                           5       3       1       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
  -------------------------------------------------------------------

=Rate, temperature, and method of adding glycerol.= Closely associated
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
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
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 survival.
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 necessary
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 better 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 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.

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

  ============================================================
                               Sperm motility (percent)
                         -------------------------------------
  Stage when observed    Glycerol     Glycerol      Glycerol
                           only     and glucose   and rhamnose
  ------------------------------------------------------------
  Fresh diluted semen       56           56            56
  After glycerolization     54           54            54
  After equilibration
     2 hours                51           53            53
     6 hours                48           52            53
    12 hours                46           50            51
    18 hours                40           46            46
  ------------------------------------------------------------

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

Three of the sugars--glucose, arabinose, and rhamnose--protected 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 equilibration, 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[K]

(Average of 10 ejaculates)

  ======================================================================
                                   Sperm motility (percent)
                       -------------------------------------------------
  Stage of              Glycerol  Glycerol  Glycerol  Glycerol  Glycerol
  observation            only       and        and      and       and
                                  glucose   arabinose  xylose   rhamnose
  ----------------------------------------------------------------------
  Fresh diluted semen      63        63        63        63        63
  After glycerolization    54        55        54        57        60
  After 18 hours
    equilibration          39        43        44        39        46
  After freezing to
    -79° C. and
    immediate thawing      28        34        34        29        24
  After 4 days at -79° C.  23        26        26        25        27
  -----------------------------------------------------------------------

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

=Substitutes for glycerol.= Since glycerol was so effective in
protecting 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,
trimethylene 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 interactions 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 conjunction
with other experiments. Twenty-five samples cooled slowly (2° 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.

[Illustration: Cooling rates of diluted semen samples in plastic vials
and in glass ampules (Fig. 4)]

=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.




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 expensive. 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).

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

At the end of 1 day, samples stored at -79° C. exhibited approximately
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 laboratories 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 prefreezing 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 temperatures
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. 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.

Table 14.--Effect of Glycerol Level and Storage Temperature on
Freezability of Semen

(Average of 8 ejaculates)

  ================================================================
  Storage       Glycerol    Sperm motility after storage (percent)
  temperature   level       --------------------------------------
  (°C.)         (percent)          18 hours      42 hours
  ----------------------------------------------------------------
    -79            7                 61            61
    -20            7                  2             1
                  11                  3             1
                  15                 14            10
                  19                 30            22
                  23                 29            19
                  27                 25            18
                  31                 21            12
  ----------------------------------------------------------------

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) contained
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 storing, 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
temperature might be more desirable so that cooling again will not be
necessary.

=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.

[Illustration: Effect of glycerol percentage and thawing temperature on
sperm motility after freezing and thawing (Fig. 6)]

The 5° C. thawing temperature resulted in a higher percentage of sperm
survival at all the glycerol levels than 38° C., with the difference 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 temperature
continued during storage (Fig. 7).

[Illustration: Effect of thawing temperature on sperm motility during
storage at 5° C. following freezing and thawing (Fig. 7)]

Table 15.--Effect of Glycerol Level, Thawing Temperature, and Storage at
5° C. After Thawing on Sperm Motility (Average of 13 ejaculates)

  =====================================================================
  Thawing      Glycerol            Sperm motility (percent)
  temperature   level       -------------------------------------------
    (° C.)     (percent)     Post-     After storage at 5° C.
                            thawing    ----------------------   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
  ---------------------------------------------------------------------

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 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 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
temperature 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.

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




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 measurement 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 consumption
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 concentration 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 (ZO_{2}).

[Illustration: Effect of glycerol additions on oxygen consumption of
sperm at 37° C. (Fig. 9)]

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

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

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 + hydrogen peroxide.

Hydrogen peroxide is known to be detrimental to sperm. The addition of
glycerol to diluted semen first increased oxygen uptake and then reduced
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 peroxide 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. Comparison of the resulting
oxygen uptake with glycerol and with glycerol plus catalase is shown in
Figure 11.

[Illustration: Effect of additions of glycerol-plus-catalase on oxygen
consumption of sperm at 37° C. (Fig. 11)]

Oxygen consumption was increased by the presence of added 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.

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^{8} sperm
                                             --------------------------
                                             First hour    Second hour
  ---------------------------------------------------------------------
  Fresh diluted semen                            10.3         8.1
  Fresh diluted semen glycerol tipped in
    at end of first hour                          9.7[L]     12.9[L]
  Aged 20 to 24 hours at 5° C.                   11.2         8.3
  Aged 20 to 24 hours at 5° C. glycerol
    tipped in at end of first hour               11.8[L]     12.9[L]
  After 20 hours equilibration with glycerol     11.7[L]      7.8[L]
  After freezing and thawing                      9.7         6.3
  --------------------------------------------------------------------

  [L] Average of 3 ejaculates.

=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 obtained
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 procedure
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 measuring
semen quality and is dependent on the metabolic activity of the 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 × 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[M]

(Average of 10 ejaculates)

  ======================================================================
                            Methylene-blue reduction time (minutes)
                        ------------------------------------------------
                        Glycerol  Glycerol  Glycerol   Glycerol Glycerol
                        only      and       and        and      and
                                  glucose   arabinose  xylose   rhamnose
  ----------------------------------------------------------------------
  Fresh semen             5.2       5.2       5.2        5.2       5.2
  After glycerolization  26.4      25.2      17.3       14.3      19.4
  After 18 hours
    equilibration         7.4       6.5       6.4        5.3       6.2
  Thawed immediately
    after freezing       11.5      10.5       9.4        9.0       9.4
  Thawed  48 hours
    after freezing       14.3      10.2      11.3       10.1       9.5
  ----------------------------------------------------------------------

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




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 following the
directions and suggestions given below will usually produce 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 protected 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 then boiled or
autoclaved. The citrate solution should be cooled before 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 (1-1/2 to 2-1/2
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 antibiotics.[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
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 container 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 refrigerating 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 removing 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 breeding within a few
minutes after thawing.




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 spermatozoa 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] CRAGLE, 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 equilibration 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. Jour. 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.




TEMPERATURE CONVERSIONS


  °C.   °F.

  +38  +100
  +35   +95
  +30   +86
  +25   +77
  +20   +68
  +15   +59
  +10   +50
   +5   +41
    0   +32
   -5   +23
  -10   +14
  -15    +5
  -18     0
  -20    -4
  -25   -13
  -30   -22
  -35   -31
  -40   -40
  -45   -49
  -50   -58
  -55   -67
  -60   -76
  -65   -85
  -70   -94
  -75  -103
  -79  -110