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THE SEMEN OF THE BULL WITH
SPfiCtAL REFERENCE TO ITS STOR AGE
AND TRANSPORT FOR ARTEHCIAL
lNiSEMINATION

Thesis fee the Dagm :35 M. S.
NUCHIGAN STATE COLLEGE
Ma-immad Sum-mar Pirfiux Buriro
19433

THE-5‘5

M-795

This is to certify that the

thesis entitled

"The Semen of the Bull with Special
Reference to its Storage and
TranSport for Artificial Insemination"

presented bg

Mohamed Buriro

has been accepted towards fulfillment

of the requirements for

_JL9__§_L _ _degree in _A_n.i.rna_lL figsbandr'y

_ -—— R; ajor professor

Date_JA_aI__‘2._5 : 133% __

THE SEMEN OF THE BULL
WITH SPECIAL REFERENCE TO ITS
STORAGE AND TRANSPORT
FOR
ARTIFICIAL INSEMINATION

BY

Mohamed Soomar Pirhux Buriro

A THESIS

Submitted to the School of Graduate Studies of Michigan
‘ State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Animal Husbandry

1948

Inesus
\

 

é/i/ets'

ACKNOWLEDGMENTS

The writer thanks and expresses his sincere
appreciationto Professor R. H. Nelson of Animal Hus-
bandry Department for planning and directing the pro-
cedure of this work and for making the study possible
by his continuous guidance and advice.

He also takes this Opportunity to thank Mr. A.
C. Baltzer of dairy extension who supplied the neces-

sary material in connection with semen study.

5301245

TABLE OF CONTENTS

Page
I. INTRODUCTION ............................. 1
Advantages and Limitations .................... 4
II. REVIEW OF LITERATURE ...................... 10

A. Properties of Semen ...................... 10
Technical procedures — Viability of sperm in female
reproductive tract - Bacterial contamination of
semen - Metabolism - Effects of temperature on
viability of sperm in storage - Effects of exercise
on semen production.

B. Factors Affecting Semen Production ........... 20
HypOplasia or Testicular degeneration - Endocrine
functions - Nutrition - Temperature and season -
Frequency of copulation - Sexual behaviour.

C. Clinical Examination of Semen ............... 26
Appearance and physical properties - The volume
of the semen - The motility of the sperm - Hydro-
gen-ion concentration - Concentration and enumera-
tion of spermatozoa - Preparation of slides -

Staining methods.

D. Technical Procedures for Collection of Semen ..... 39
Control of the bull and cow - Preparation of appa-
ratus - Artificial vagina technique - Points to be

ill

Page

taken into consideration while using artificial
vagina - Rectal massage of ampullae - Collection
of semen from vagina.

E. Storage and Transport of Semen .............. 47
Care and storage of semen - Preparation of dilu-
ting fluids - Egg yolk-citrate diluter - Egg yolk-
phosphate diluter — Chick-Embryo diluter - Glucose
diluter — Temperature control, packing and tran—
sport.

F. Factors Affecting Storage of Semen ........... 57
Interaction of storage temperatures - Effects of
diluting fluids on storage and fertility of semen -
Effect of dilution on fertility.

III. STUDY OF SEASONAL AND BREED DIFFERENCES IN

SEMEN PRODUCTION OF DAIRY BULIS ........... 67
IV. SUMMARY ............................... 83
V. BIBLIOGRAPHY ............................ 85

iv

1. INTRODUCTION

Although the practical application of artificial insemination is
comparatively new, the concept is a very old one. Arabs are reported
to have used artificial insemination successfully, in about 1322. An
Arab chief is said to have inseminated his prize mare from the semen
collected from a stallion belonging to his enemy. Scientific research
in artificial insemination of domestic animals was conducted by the
Italian physiologist, L. Spallanzani, in 1780 who conducted successful
experiments in bitches. Soon after the beginning of the present century,
the practical potentialities of artificial insemination began to be recog—
nized. It was Russia who initiated and took up the method of insemina-
tion seriously, as a practical means of obtaining improvement in farm
animals by using the sperm of valuable sires. The next drive towards
artificial insemination was initiated by Danish people who organized
first c00perative artificial breeding association in 1937.

The first cooperative artificial breeding in the United States was
organized in 1938 by the Extension Service of the New Jersey State
College of Agriculture. Since this new era, interest in the artificial
insemination of farm animals has increased tremendously, eSpecially
in dairy cattle. New and better techniques have been devised for collec-
tion of semen, its evaluation, preservation and dilution, storage and
transportation, preservation after tranSportation till used for effective

insemination. Hand in hand with these deveIOpments, many facts have
been established concerning the biology of sperm cells, the secretions
of the glands of the reproductive tract of the male, and of oestrus and
its related phenomena in the female. The growing realization of its
genetic and economic possibilities is now increasing and its use will be
made in various cormtries as time goes on. Thus the key to success in
artificial insemination is the use of superior bulls and a high conception
rate. Its main purpose is to promote herd improvement by getting
semen from good bulls and enough cows be made available to use it upon.
Therefore it is a great task to keep a sufficient quantity and quality of
semen in the field.

To accomplish this, correct measures should be adopted in
techniques in collection of semen, its evaluation and injection of the
semen into the female.

Techniques in collection of semen have improved very greatly
during the past few years. The invention of artificial vagina marked a
new mile-stone in the practice of artificial insemination; and most of
the methods contrived for semen collection have been abandoned in
favor of its use. It has greatly contributed to the deveIOpment of large
scale breeding by mechanical means. In this method practically the
entire ejaculate is collected and the semen is free from extraneous
secretions, thus danger of disease spread is minimized. Besides that
the volume of the ejaculate is easily measured and its characteristics
easily determined; and the viability of the spermatozoa in semen so

obtained is higher than when other methods of collection are used. The

only limitation of this way of making collections is that a few males
refuse to serve the vagina. Therefore collection of semen must be en-
gineered with considerable degree of judgment and patience.

Research and experience have revealed a lot of information about
insemination. In practice it has been shown that relatively small amounts
of viable semen effect better conception than large quantities. In this
connection Salisbury continues to extend the frontier where five years
ago a dilution rate of one part of semen to 5 parts diluter was thought to
be considerable, a rate of 1:100 is now found by Salisbury to give results
as satisfactory as lower rates of dilution. The matter of concern is the
required number of viable healthy Sperm per implantation rather than
the amount of semen to use.

The use of egg yolk as a main constituent of diluting medium
remains one of the most important advances in sperm storage. Many
diluters have been tried and discarded. The most satisfactory diluters
to date are yolk-citrate and yolk-phosphate. The fertility of the semen
preserved in the phosphate and in the citrate diluent is similar but
citrate diluent is recommended for use in the artificial breeding associ-
ations as it disperses the substances in the egg yolk and enables the
spermatozoa to be seen readily under microscOpe, which can only be
done with difficulty in case of phosphate buffer. Addition of dyes to egg
yolk diluter have been found to help in identifying semen of bulls of
different breeds. Sufficient neutral red in 5% solution has been used
with no adverse effects on motility of spermatozoa. Penicillin, sul-

phanilamide and streptomycin, to the diluted semen has been found to

4
control bacterial and fungal growth without harming the sperm. Methy-
lene blue reduction test have been found successful in semen evalution.

Much progress has been made as to the maintenance of storage
temperatures after collection of semen and during shipment. Today as
a routine practice the semen is not only shipped long distances but is
often used for at least four days and occasionally longer. This change
has been brought about by appropriate diluters, proper cooling of

semen, and deve10pment of satisfactory shipping containers.

Advantages and Limitations

Uses.

 

Artificial insemination has been used relatively very little in
the past and it is only during the last few years that the practice has
been expanded to a large extent and its real potentialities for animal
improvement have been discussed by many writers. The deve10pments
of improved techniques, the percentage of successful inseminations is
increasing. The following are the uses in comparison with natural
mating.

1. Increasing the Use of Valuable Sires.

 

It provides means of impregnating large numbers of females
with semen from one male whose influence can thus be greatly extended.
Full utilization of sires that have been proved can be advanced for
transmission of desirable characteristics as butterfat and milk pro-
duction in case of dairy cows. With the improvement in the technique

of preservation of semen and the rapid extension of air transportation,

5

the stocks of cattle having best genetic make-up may be spread widely
throughout various countries at a very small expense compared with the
system of conveyance of breeding animals by rail or sea. 1,000 cows
can be bred by a single bull during the season.

2. Making Advantageous Use of Young Sires.

The reproductive life of sires in most species of live stock is
short. Therefore one of the problems in animal breeding is to prove
sires in early life so that information can be used to discard the poor
sires and to recognize and use the best sires as soon as possible. In
artificial insemination young bulls might be proved at an earlier age
than with natural mating due to the progeny tests on a more adequate
'scale.

3. Extending the period of usefulness of valuable sires.

A male is sometimes unable to mate normally, or may tire
quickly, because of age and crippled condition and may serve only a
very limited number of females. Thus artificial insemination may
serve as an effective means of extending the period of usefulness of
valuable sires which would otherwise have to be slaughtered.

4. Overcoming deficiencies due to difference in size.

 

If natural service is impossible because of marked difference in
size or weight, artificial insemination can be used for effecting concep-
tion, especially in the breeding of young females to old heavy bulls.
Seasonal production can be controlled better by artificial insemination
than by the practice of allowing males to run with the herds or flocks

and to mate at will.

5. An effective aid in disease control.

The dissemination of certain diseases has been one of the chief
handicaps in breeding problems. Artificial insemination preperly used,
can be instrumental in preventing exposure of sires to genital disease
and thus minimizing the spread of the disease. The indiscriminate use
of bulls among several herds encourages the spread of genital disease
and sterility. In herds of cows there are several important diseases
such as contagious abortion, Trichomoniasis, Contagious vaginitis,
Corynebacterium pyogenes, which can be prevented by artificial insem-
ination.

6. Maintenance of satisfactory fertility.

Artificial insemination can only be expected to give best results
when compared to natural breeding, and to achieve this above object
certain procedures must be adopted which assist in obtaining and main-
taining a satisfactory conception rate. These include: (a) Regular
microscopic examination of the semen and use of high quality semen
for insemination; (b) Keeping of good records and close attention to
breeding performance as it makes more accurate control of dates and
prevents the overwork of males; (c) Examination of cows for preg-
nancy; (d) Early detection of cases of sterility and their treatment.

7. Effectigg Conception in females that do not conceive after capulation
or that refuse the male.

 

In cases of temporary disturbances of function in valuable
females artificial insemination brings about conception when normal

mating proves ineffective. Care should be taken in cases where the

7
impaired fertility may have an inherent basis. In such cases breeding
of these females should be avoided, or their offspring should be used
for purposes other than breeding.

8. Benefits to small herds.

 

The greatest immcdiate value of artificial insemination lies
with its use, particularly in small herds, where a distinct saving can be
effected. Thus it makes possible for individual farmers to diapense
with their own bulls, and to many dairy farmers it is of great advantage
because dairy bulls are frequently very dangerous and always require
some barn Space and labor. Therefore additional income can be so-
cured by using that barn space for housing productive females.

9. Obtaining species crosses when natural mating is impracticable.

In cross breeding where it is sometimes desirable to use breeds
quite dissimilar in size, anatomical structure or psychological charac-
teristics, natural mating has proved unpracticable. In this way it has
proved useful as a tool for hybridization experiments.

IQ. Use in breeding associations and in large herds.

 

The greatest promise artificial insemination holds for livestock
improvement is through the large scale use of valuable sires; and
makes possible the deve10pment of large families of superior animals
within a particular area which attracts the outside buyers due to uni-
form merits of the animals.

11. General.

 

It eliminates the need for taking females to the sire and vice-
versa, thus matings may be made between animals located at distant

points at less expense or hazard. Participation in a better breeding
program and mutual study of problems brings forth the best thought and
spirit for advancement of breeding business.

Limitations of artificial insemination.

 

1. Artificial insemination is harmful if the influence of low
quality males is widely disseminated.

2. Problem of disease transmission always remains as a result
of careless technique. To be successful the method must be practiced
by skilled persons such as veterinarians who have had special, adequate
training. A good knowledge of the reproductive organs and of the pr0per
methods of collection and handling of semen are fundamental to the
efficient execution of the job.

3. There is likelihood of the injury to the reproductive system
of both males and females therefore greatest care should be taken to
avoid injury to the genital organs.

4. Instances are said to be existing where insemintors are not
pr0perly trained and where instruments are not pr0perly cleaned and
are roughly or improperly used. These above instances will probably
always tend to limit the full usefulness of artificial insemination.

5. A large financial loss to farmers may occur if artificial
insemination organizations are imprOperly organized or managed. The
individual farmer is interested in improving his herd and getting his
cows settled and if the herd owner’s cows are not settled within the
limit of normal expectations, the resulting financial loss may offset the

benefits of the farmer. Therefore it is imperative that each step in

9
artificial insemination program should be done accurately and with the
utmost care.

6. Due to carelessness of the inseminator exchange of semen
may occur and he may inseminate a cow of one breed with the semen of

some other breed or vice-versa.

II. REVIEW OF LITERATURE

A. Properties of Semen

Technical procedures. Changes in technical procedures of artificial

 

insemination have had an important bearing on its deve10pments in the .
period under review.

More progress has been made in the preparations and use of
diluters than in any other aspect of the work. Previous to 1939 the
diluents available served only to increase the bulk of ejaculate and
storage for periods over twenty—four hours was accomplished by dilu-
tion. Phillips as stated by Henderson (17) introduced egg yolk-phosphate
medium, which increased both bulk and storage life of Sperm samples.
The egg yolk-citrate introduced by Salisbury (27) has the important
advantage, that samples could be examined microscopically with great
ease. An addition of glucose and gelatin has been found to increase
Sperm survival (19).

Chick embryo diluter by Frank and others described by Ander-
son (1, p. 72) has significantly increased the survival time of Sperm.
The rate of dilution has been increasing steadily, and in the field the
work of Salisbury and his associates (31) has been outstanding. Citrate
buffer excelled the phosphate buffer when used with egg yolk—citrate
for the preservation of semen (36). It has been shown that the egg yolk-
citrate maintained motility at a higher level for extended periods of

10

11
storage than did the egg yolk-phosphate diluent. One serious disad-
vantage of the egg yolk-phosphate diluent is the presence of fat globules
which makes it impossible to see the individual spermatozoa under the
microscope. Salisbury (1941) introduced the diluent in which micro-
scepic examination was done satisfactorily, and they were also able to
Show no significant decrease in fertility from the use of semen diluted up
to 1:50 parts of egg yolk-citrate diluter. Salisbury (1946) found no dif-
ference in fertility between dilution rates of one part semen and up to
100 parts of egg yolk—citrate diluter. A recent method used by Danes is
interesting and is apparently very successful. It consists of placing
semen diluted in a gelatinous base in small soluble wax tubes which are
slowly cooled, until the gelatin base is firm. In the field the technician
cuts off the solid portion of the tube equivalent to 1 cc. of semen and
introduces it into the vagina by ejector. This cool gelatin warms up
slowly in the uterine tract and thus allows the sperm to return to
normal activity rather slowly. It is reported that high conception rate
is attained (9).

Addition of various dyes to egg yolk diluter in coloring samples
for identification purposes have been reported by Phillips (23). This
procedure does not affect the livability and fertility of the sperm.

Survival of Sperm on storage is dependent on appropriate stor-
age temperatures and on the avoidance of temperature shock shown by
Davis (7).

The storage temperature for bull semen is 5° C. Reduction of

temperature to 15° C. can be accomplished rapidly, but further

12
reduction to storage temperature Should cover about one hour. If
semen is to be stored at temperatures below 5° C., a Slow cooling
method Should be adapted (17). While Rawson’s observations by Hen-
derson (17) observed that using eg yolk—phosphate diluter and storing
the semen at 5° C. found little difference between slow and rapid
cooling. Slow cooling is an inexpensive precaution and is worth while
particularly if storage over three days is contemplated.

In general it can be said that good quality semen if properly
cooled and diluted can be preserved up to four days, and gradual
warming of semen before use is not thought to be beneficial ( 17).

Citrate buffer containing sulphanilamide was protected from
direct sun light rays and used. An increase in the fertility by the use
of sulphanilamide was obtained. These results are said to be due to the
beneficial effect of the drug due to metabolic changes rather than due to
bacterial control alone (18). Schultz and Davis (1947) showed that six
out of twenty-seven samples showed retardation of oxygen consumption
by the addition of thyroxine. Salisbury (31) showed that semen handled
in the usual way decreases in ability to live under low temperature
storage conditions with each increase in dilution rate, this so-called
“Dilution effect” has been observed under a variety of handling tech-
niques, and a‘method of treatment has not yet been devised to prevent it.

Methods of collection of semen have improved very greatly
during the past few years. In the early days the practice of withdrawing
the semen from the vagina of a mated cow was followed. Only a small
portion of the ejaculate could be recovered from the vagina. It was

13
mixed with vaginal secretions which were injurious to the Sperm, if it
was to be stored (9).

Mechanical massaging of the ampullae by hand through the
rectum has also been practiced. This method is useful for injured or
lame sires and is still being practiced.

For collection of semen the artificial vagina has almost sup—
planted other methods. Great care is taken to insure that the semen is
not subjected to a sudden drop in temperature due to contact with a
cold collecting tube. A longer type of vagina with the collecting test
tube enclosed in the water jacket has been introduced by Salisbury and
Willett (37). Walton observed by Henderson (17) has designed a double
wall collecting cup for use with vagina. Both models serve the purpose.
Sufficient care is taken to protect the test tube and collecting cone.

Dummy cows are said to be satisfactory by some Operators;
while others find certain bulls unresponsive to them (3). It has been
recognized that stages of the process, as time and place of collection
and temperature and pressure of artificial vagina Should be standardized
as much as possible for each individual bull. Variety has been found to
be necessary in some cases.

Maintenance of storage temperatures during shipment is accom-
plished by utilization of ice-filled balloons or thermos flasks well
packed in cardboard boxes (35). For actual transport any form of con-
veyance suitable can be used. Airlines are commonly used for long
distances. Russians in 1938 were delivering semen by plane to out-

lying stations, the packages were drOpped without landing. The

14
packages were drOpped from a distance Of 300 meters and this did not
harm the sperm cells (17).

In Russia and England pigeon carriers have been used for trans-
port Of semen for short distances.

In actual insemination, the method made popular in the United
States by Larson, whereby the cervix is fixed through the rectal wall,
has almost entirely supplanted the use of speculums, but some indiv—
idual Operators do use Speculum method. In Russia the speculum
method is still being used (17).

The practice is to place the semen through the cervix into the
body Of the uterus and this can only be accomplished by fixing the
cervix either through the rectal wall or directly through the vagina.

In this connection results have been reported from New Zealand
(1945) which say over—all conception rate of 24.6 per cent on 4226
inseminations using a speculum, where as in one herd, where the semen
was deposited through the cervix, the conception rate was 60 per cent
on 141 inseminations. In one English association, two inseminators used
different methods, a difference of about 15 per cent in their conception
rate was Observed in favour of cervix method as compared to the use of
Speculum (19). Yet the success Of the Operation depends upon the Opera-
tor as to how far he is efficient in his technical job.

Various inseminating instruments have been devised but the
ordinary glass capillary tube attached to a 2 cc. glass syringe by a
rubber connection is still in general use. The diluting semen is drawn
only into the inseminating tube and has no contact with the rubber

15
connection. The syringe being used as a pump and having no contact
with the semen, can be used for different bulls without danger Of mixing
semen, and problem to clean rubber becomes unimportant.

Viability Of sperm in female reproductive tract.

The life of the Sperm is generally regarded short, inside the
vagina, especially in higher animals, about 36 to 48 hours in most
cases, while it differs widely in human beings and lower animals. Live
Sperm have been recovered in a woman 7 days after coition. In bats
sperms are capable of fertilizing the egg even after some months. In
fowls fertilization is known to occur 32 days after insemination of
semen (26). Observations of Brewster, May, and Cole by Anderson
(1, p. 83) found that the minimum time required for Sperm to reach the
upper third of fallopian tube was 5% hours for mature cows and 4-;- hours
for heifers. Experiments conducted with dead sperms, indicated that
the uterine contractions have no influence on sperm travel in the
genital tract. Sergin and cO-workers Observations by Anderson (1, p.
83) state that the life of sperm depends upon the conditions prevailing
in the female genital tract of the cow. The alkaline reaction of the
genital tract and high action Of vaginal secretions stimulate the activity
of the sperm and usually have unfavorable action on the survival of the
Sperm. Therefore they die Off rapidly in vagina and uterus. The cervix
is the most favorable place for sperm life due to weakly acid reaction
and absence of leucocytes. The sperm can survive for two to three
days in cervix and from here constantly pass towards the horns of

uterus. Counts Of sperms in the cervix after mating showed that the

airfieldeaum -51.. 14‘ .v...v.l ... Humiaflgfi
. t .

 

16
number reached maximum in 2% to 3 hours and then decreased at 5
hours to about half the number.
Bacterial contamination Of semen.

Several investigations have been made into the significance of
bacteria contained in bovine semen. Gilman’s observations by Anderson
(1, p. 66) that twenty-five years ago Gilmain isolated a number of
organisms including streptococci and Brncella abortus, and demon-
strated that, in the bulls studied, the lowered fertility was associated
with increased number Of bacteria in their semen. He also isolated
pseudomonas pyocyaneous and unidentified rods and showed that the
genital tracts of normal healthy bulls were either free from bacteria or
contained very low numbers. Gunsalus, Salisbury, and Willett (16)
showed number of organisms except streptococci. Diptheroid organisms
were found in large numbers. Presence of heavy number of contami-
nants did not seem to have any marked adverse effect on semen storage
at the temperatures now employed. Anyhow contamination should be
kept to a minimum by strict attention in collection, dilution and cleaning
equipment. Slight mrulent discharge which sometimes occurs after
insemination may be due to semen contamination of the inseminating
tube from the vulva. It has also been shown that trichomonads can be
transmitted by artificial insemination from infected bulls and the im-
portance of using bulls free from contagious abortion has been empha-
sized, which indicates high percentage of positive blood reaction follow-
ing insemination to an infected bull ( 17). A method for preparation of
sterile yolk-phOSphate diluent have been described (16). Unless rigid

17
precautions are taken, diluents may be responsible for large number of
bacteria tO semen samples. During storage bacterial growth is held at
minimum by storing at 5° C. or lower.

Sulpha, penicillin, and streptomycin have recently been added to
reduce bacterial growth during storage. Sulphanilamide 1:300 of
semen and penicillin 1:1200 seems to be effective (15). A lot of work
is yet to be done in this connection of using these drugs in preservation
of semen against bacteria.

 

Metabolism.

The most important biological reactions Of spermatozoa are
glycolysis and respiration. Spermatozoa derive energy through both
respiration and glycolysis, but glycolysis is chief source of energy for
it. Although motility of bovine spermatozoa is not dependent on a
supply Of oxygen, there is ample evidence to Show that reapiration rate
is important as a measure of potential fertility (17). High quality semen
contains a high percentage of vigorous, active, spermatozoa and strong
spermatozoa Show a definitely higher glycolytic and respiratory rate
than do weak or immotile sperm (15). Glycolysis Of spermatozoa is the
reduction Of glucose, thus changes in the pH of semen have been looked
upon as a rough measure Of the metabolic activity of the spermatozoa.
The increase in the acidity of semen during storage is a good example
of the effect of accumulated metabolic production (15).

Effects of temperature on viability of Sperm in storage.
Spermatozoa carry certain biological reactions during the time

they are in storage. These reactions depend upon the concentration and

18
activity of the sperm. There are several factors which are responsible
for the decrease of Sperm motility. Such factors are temperature,
vigor Of sperm, food supply to sperm (15). Salisbury (31) Observed
that the usual handling of semen decreased the ability under low tem-
perature storage conditions with each increase in dilution rate called
“dilution effect.” Dilution rate of 1 part of semen to 8, 12, 16, 24, 50
parts Of yolk-citrate diluter were compared following number of
spermatozoa were contained in 1 ml. Of diluted semen used in each
insemination. 150, 104, 80, 54, 26 million.

Willet and Salisbury (36) found the effects of low temperatures
from sudden cooling had a harmful effect on the subsequent reSpiratory
activity Of the sperm, and lower the temperature to which the semen is
cooled, the greater is the harm done, and also it was shown that the
sperm suffered if ejaculation was received in a cold receiver even at a
temperature of 15° C. Spermatozoa, whether in undiluted semen or in
yolk-phosphate diluent, suffer from temperature shock caused by rapid
cooling Of the semen. Anderson (1) also Shows that the sperm cooled '
suddenly at 5, 10, 15° C. showed further reduction of activity. Sperm
which had been subjected to temperature shock perished rapidly on
further storage of 38° C. to 39° C. Green and Winters (14) showed that
Sperm are sensitive to low as well as high temperatures. They are
usually more sensitive to high temperatures and are injured at tem-
peratures above body heat. Outside body they live relatively short
periods.

19

Effects of exercise on semen production.
Very little work has been done in this connection. Bartlett and

Perry referred to by Anderson (1, p. 8) found that the regular exercise
of bulls for one hour at the rate of 2% miles per hour increased the out-
put 51 per cent. Anderson also gives reference of Kelly (1940) and
Hamilton and Symington (1939) who showed a great increase in the
quality of semen with exercise. The motility was 5 to 45 per cent
before exercise, it was 60 per cent to 100 per cent after exercise.
Without exercise motility ceased at 3 to 24 hours. They also showed
that daily exercise increased the volume of the ejaculate, sperm concen-

tration and motility.

 

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_.

 

B. FACTORS AFFECTING SEMEN PRODUCTION

Hypoglasia or Testicular degeneration.

In testicular hypOplasia normal spermatogenesis does not occur.
The epithelial layers of the seminal ducts are less developed with low
percentage Of fertilization occurring. It has been noted by several
workers that it is usually unilateral and of a permanent nature. But
when it is of bilateral nature the animal is completely sterile producing
no viable gametes while one-sided hypoplasts may have irregular pro-
duction from very good to sterile. The hypothesis of two recessive
genes has been said to be the cause of hypoplasia, which when separate
cause Single sided hypoplasia and together double hypOplasia.

It is probable that scrotal temperatures and disturbance in the
heat regulating function of the scrotum may adversely affect sperm
production. It has also been observed that under colder conditions bulls
are less keen to serve and produced poorer semen. Certain diseases
affect the testicular substance. It has been noted by Anderson (1) that
decrease in semen quality in the bull occurs after anaplasmosis and
foot and mouth disease; this may be due to increased body temperature
which affect spermatogenesis.

Endocrine functions.

 

The function of the testis is controlled by the reciprocal action
of the pituitary and the testis. The internal secretion Of testis controls

20

21
the deve10pment and activity of spermatozoa and a fluid vehicle for their
transport. The accessory glands and functions of the ducts is quickly
affected if the secretion from the testis fails to occur (1). Spermato-
zoan motility test is the physiological indicator of the hormone activity.
Moore’s observations of Anderson (1, p. 24) that when both testes are
removed motility is lost in 20 to 23 days in guinea pigs. Thus the
hormone of testes is responsible for prolonging the life of spermatozoa
within the epididymis. While the degree to which the testes hormone
and secretion from epididymis, may be responsible for poor motility of
spermatozoa in ejaculates, and perhaps to some extent for variation in
different ejaculates is not known as yet.

There is general relationship between the production of germ
cells and hormone secretion but in case of some males that mate
throughout the year, there is constant rate of gonadrotrOphic hormone
secretion, while in seasonal breeders the rate of secretion of the gona-
drotropic hormone ceases rapidly after the breeding season and remains
low till the next breeding season.

Nutrition.

It is believed that ordinary bull ration fed at slightly more than
maintenance level is adequate for the special demands of reproduction,
but in order to maintain fertility it is suggested that with increased
protein feeding an increase in concentration, volume and sexual interest
results, while low protein feed, frequently exerts a depressing influence.
It has also been suggested by Branton (5) that dairy bulls should be kept

22
in vigorous physical condition but not too fat, since high finish is
believed to make them sluggish.

An experiment was performed on 19 bulls, 10 Holstein-Friesians
and 9 Guernseys. Studies were conducted with regard to semen charac-
teristics, relative fertility, and body weight changes to total digestible
nutrients. Concentrate mixtures containing 12, 16 and 20 per cent total
protein were fed simultaneously. Under the condition of experiment, 12
per cent protein in the concentrate mixture fed with mixed hay con-
taining approximately 10 per cent of legumes, supplied enough protein
for semen production and maintenance of fertility.

Deficiency of vitamin A is said to cause degeneration of the
germinal epithelium with consequent depression of fertility. This
usually results from poor feeding. Subcutaneous administration of
ascorbic acid improves semen quality. Vitamin E is said to be of
doubtful importance in reproduction in the bull. An experiment was con-
ducted by Salisbury in which 10 bulls were each fed one ounce daily of
wheat germ oil in addition to standard ration for a period of one year.
No beneficial effect was found. Pasture has a beneficial effect on
fertility, it corrects amino acids and vitamin deficiencies, adds suc-
culence, and provides exercise. Various minerals are important in
reproductive requirements.

Temperature and season.

 

High atmospheric temperature, humidity and quality and quan-
tity of light, has a deleterious effect on the reproductive processes of
the male. It has been demonstrated that external heat applied to the

23
scrotum can affect both spermatogenesis and mature sperm. Studies of
the influence of season upon reproduction in dairy cattle made at the
Louisiana Experimental Station (12) show that more services per con—
ception were required during the summer months than at any other period
of the year. Phillips (23) noted a high abnormal count consisting mainly
of tailless and coiled tail spermatozoa during the first few collections
after a period of some months of sexual inactivity; after a few services
these degenerate spermatozoa were cleared out and normal spermatozoa
were ejaculated. It thus seems that heat can induce morphological
changes in spermatozoa in the epididymis, as shown by coiling of tails
and separation of tails from heads.

Frequency of copulation.

 

There is much individual variation in the number of Mes that
males can be used without impairing their fertility. It has been shown
that when ejaculates were taken from the bull once daily for 57 days,
the volume of the semen was well maintained, but sperm concentration
varied from 2,179 million sperm per cc. in the fourth week to none at
the beginning of the ninth week. The longevity of the sperm increased
slightly until the fifth week and then declined rapidly (3). Collections after
about 10 days, taking two ejaculations are found to give best results, '
although some individual bulls may be capable of heavier service. Ex-
tremely long periods between services may adversely affect semen
quality (17). It has been observed that too many valuable sires have been
rendered infertile by over use. They will last longer in a program of

moderate service at reasonably regular intervals than under a system

24
of intensive artificial or natural service for short periods.

Bogart’s observations by Anderson (1, p. 137) that, as the interval
between the collections increased from one up to 10 days, semen volume,
total Sperm per ejaculate, percentage of abnormal Sperm, and fertility
increased, sperm concentration increased for five days, then decreased.

Sexual behaviour.

 

Among the most perplexing problems which have to be faced in
artificial insemination is impotence or inability to serve in the bull.

It is therefore necessary to distinguish between inability and unwilling-
ness to serve, or between pathological and psychological causes.
Anderson (1) states that different types and degrees of sexual activity
may be on the following lines: (1) Normal service, (2) mounts female,
complete erection and intromission, but failure at ejaculation, (3)
mounts female but failure of intromission, (4) mounts female, no erec-.
tion, (5) failure to mount female, (6) no interest other than standing near
female, (7) fails to approach female.

Most common condition affecting abthy to serve is foot infec-
tion. Either injury or infection affecting the hind feet may soon inhibit
sexual desire, and, if accompanied by an increase in temperature, may
adversely affect Spermatogenesis. Contraction of extensor penis muscle
can make mating impossible. Trichomonad infection is said to cause
slowness in serving.

Anderson (1) has noted that bulls of all grades of fertility
exhibit great variation in their ability and desire to serve, and some-

times a completely sterile bull will be much keener and more capable

25
of service than a highly fertile one. Slowness in service may be due to
strange surroundings and to the nature of the control to which the animal
is subjected. Over-exertion, or inability to capulate or when the bull
mounts the cow feels pain, may cause diminished sexual desire. Acute
infection of testes and seminal vesicles may incapacitate the bull to

capulate.

C. CLINICAL EXAMINATION OF SEMEN

Great variation exists in the quantity and quality of the semen
obtained from different males and also from same males at different
times. Therefore, when examining an ejaculate, the typical character-
istics of the particular species must be kept in mind. Consequently the
semen of any male should be carefully examined before it is used for
insemination. No single method is entirely adequate for evaluation of
semen, therefore a combination of methods is employed for thorough
examination. The following points are considered in examination of
semen: (1) appearance, as color and cloudiness, (2) the volume of the
semen, (3) concentration or consistency, (4) the motility of the Sperm,
(5) the number of normal Sperm per cubic millimeter of semen, (6)
freedom from bacteria, parasites, or cells that would indicate the
pathological condition of the genital organs of the male from which the
sample is obtained, (7) the respiration rate of the Sperm, (8) proportion
of abnormal spermatozoa, (9) hydrogen-ion concentration of the semen,
(10) resistence of sperm, (11) longevity of Sperm. In males intended
for extensive use it is desirable that semen examination should be
complete, for it has been shown that there is a relationship between
certain of the above characteristics and fertility. In large breeding
programs sires should be subjected to such examinations prior to and
at intervals throughout the breeding season, because without fertile
sperm the whole program for better breeding will break down. An
examination based on one particular ejaculate refers to that ejaculate

26

27
only. It is therefore desirable to base the estimate of fertility on
examination of as many ejaculates as possible, in order to obtain the
average and range of variations in semen characteristics for the par-
ticular male. As considerable time is required for the collection of
number of ejaculates, it may be said that a single good specimen would
indicate that the male is of good fertility, but a poor ejaculate by no
means condemns the male. However, succession of poor ejaculates
from the same animal for over a period of time would certainly indicate
a low breeding efficiency.

In extreme cases the Sperm may be dead or absent in which case
the animal would be declared completely sterile. While in other cases
it can only be said the animal’s fertility is “reduced” or “low.” Thus
the reproductive capacity of the male may be measured not on a single
ejaculate, but on the total ejaculate produced in a given interval of

time upon several different occasions.

 

Appearance and physical properties.

The general appearance of the semen, the color, consistency,
opaqueness, and any abnormal qualities should be noted. The color
and consistency varies with the concentration of spermatozoa; the
thicker the semen, the higher the concentration. Normal semen of most
species has the appearance of whole milk and its Opacity is due to the
dense mass of sperm. Samples of semen of lesser density are character-
ized by a more watery appearance and in such an ejaculate spermatozoa
may be few or absent. A yellowish colour may indicate the presence

of pus or urine, which may often be also detected by smell. Pinkish

28
or redish color indicates an admixture of fresh blood, while a deeper red
or brownish coloration probably indicates the presence of degenerating
blood and tissues, and greenish coloration may indicate purulent degen-
eration. Normal semen has little or no smell, but in abnormal condition
of the male genital organs it may have a highly offensive smell. Such
abnormal coloration of the semen may indicate abnormal conditions in
the genital organs of the male, or possibly of the female in case the
sample was collected directly from vagina. A sire from which abnormal
semen is conected should not be used for insemination or breeding
purposes until the cause of the abnormalities has been established and
removed. If semen is collected from vagina care should be taken to see
that the female is healthy. Because of admixture with vaginal secre-
tions semen so collected will have somewhat different appearance than
that collected in other ways. Early investigators have found that the
cloudy formation of bull semen, due to masses of sperms, was usually
associated with high rate of conception. Experiments conducted (1)
showed that out of 206 inseminations, 86 per cent conceptions resulted
when marked cloudiness was marked in the semen. When there was
little cloudiness, 55 per cent conception occurred in 47 inseminations.
When cloudiness was slight, 25 per cent conceptions were obtained in
20 cases; and when there was no cloudiness, one pregnancy in 19 insem-
inations was obtained, i.e., 5.3 per cent conception.

The volume of the semen.

 

As the number of inseminations that can be made from any one

male is dependent upon the volume of semen produced, it is therefore

29
important to use males that produce large quantities of semen containing
large number of active, viable spermatozoa. Large volume of semen
does not necessarily mean a high sperm count, there is a reasonably
high correlation for most animals between the total quantity of semen
in the ejaculate and the number of spermatozoa contained therein,
provided that the ejaculate is complete. In attempting the measurement
of the volume of semen production for any given sire, one should not
rely on a single ejaculate but upon a total ejaculate produced in a
given interval of time upon several different occasions. In some species
semen production varies slightly with the seasons. It is usually lowest
in July, August, and September for dairy bulls. The amount of semen
per ejaculate is roughly proportionate to age up to maturity, but varies
markedly between the sires of the same age and size. Frequency of
collection influences volume to some extent in individual males,
especially those which are regularly used throughout the year. While
some sires yield a constant amount under even heavy service. Little
difference is found in the volume of first, second, and third ejaculates.
Difference in volume is sometimes due to the thrust at the time of
collection. It is therefore important that the artificial vagina be at
the right temperature and suited to the male handled. Precautions
should also be taken about the noises or other interferences when
collection is made.

The average volume of the ejaculate in bulls is about 4 m1.,
and it may range from approximately 0.5 to 12.0 ml. Accessory glands

greatly contribute to the volume of semen in (1) efficiency of the

30

ejaculatory reflex, (2) concentration of spermatozoa, (3) the amount of
accessory secretions. Pathological as well as physiological conditions
may influence the above three factors which may alter the volume of
the ejaculate. The bulk of the semen is formed by the accessory secre-
tions but if these accessory glands are abnormal the volume may be
reduced.

There are indications of breed differences, of dairy bulls giving
larger ejaculates than beef bulls. (17).
The motility of the sperm.

 

Microscopical examination of semen for motility is highly im-
portant, because without active movement spermatozoa cannot reach
the fallopian tube for fertilization of ova. The essential points to be
observed in the estimation of motility are ( 1) examination of semen
immcdiately after collection of semen, (2) maintenance of optimum
temperature, and (3) prevention of drying of the semen. If the semen
is not examined immediately after collection a fall in temperature may
reduce the activity of the sperm. Initial motility is believed to be the
best evidence of viability of semen of a fertile male. Determination
of motility, number of Spermatozoa per cubic millimeter of ejaculate,
abnormalities of the sperm, and examination for pathological cells
should be conducted immediately after collection of semen. Samples
should be taken and care exercised to see that the sample is represent-
ative of the whole ejaculate. The examination for motility is best made
by means of hanging drop method. This is accomplished by placing a

well mixed drop of semen on a cover slip with a pipette and a hollow

31
ground slide inverted over the cover slip. The slide is then turned over
and placed on a microscope stage. If cover slips and hollow glass slides
are not available, a drop of semen can be placed on an ordinary glass
slide and dr0p of medicinal paraffin placed on the semen to prevent it
drying up. For routine examination hollow—ground slides should be
used. To accomplish this, the examination should be made in a warm
room or by providing some means for keeping the sample at a constant
temperature of 38° C. to 40° C. ; such as slide incubator, a heating pad
or hot water bottle. But at the same time care should be taken that
the temperature should not be high as the sperm is killed at 46° C. A
fairly satisfactory examination can be made by placing a drop of semen
on a cover slip, and then inverted on the glass slide and cover slip
supported by other cover slip to provide space for the semen drop with
pressure on the sperm and examined immediately for motility using
the low power objective on the microscope. Care should be taken to
avoid the use of cold cover slips and slides. While examining the semen
under the microscope, observations should be made at the edge of the
semen to ascertain an approximation of the percentage of live, active
spermatozoa. This should be done before the edges have become dry.

The areas near the center of the drOp should be examined to
observe the waves and all the movement of the drop. If the motility
is poor on the first examination a fresh dr0p of semen should be examined
after insuring that the temperature conditions are Optimum.

Different types of motilityhave been investigated by different

investigators. As the progressive motion by which the sperm move in

32
in a straight line, a rotary motion by which the Sperm move in circles
with diameters of about their own length, and an oscillatory movement
which are convulsive in nature and without progress.

The relative number of Sperm showing progressive movement
may be expressed, as a percentage of the total number of Sperm.

The method recommended by Herman (15) for classifying
motility is as follows:

5 = Excellent motility - About 80 per cent or more of the Sperm-
atozoa exhibit energetic progressive motion. Movement is so vigorous
that it is impossible to observe individual Spermatozoa in undiluted semen.

4 2 Very good motility - About 70 to 80 per cent of the sperma-
tozoa are in vigorous rapid motion.

3 = Good motility - about 50 to 75 per cent of the Spermatozoa
are in progressive motion.

2 == Fair motility - From 20 to 50 per cent of the Sperm are
in motion. Movements are vigorous but no waves.

1 = Poor motility - Less than 30 per cent of the Sperm are in
motion. Motion is mostly weak and less progressive.

0 = No sperm are present.

Some investigators use the plan of estimating the motility by following
classification: Very actively motile, actively motile, motile, sluggishly
motile, and non—motile, and are given values of 100, 75, 50, 25, 0,
respectively. It is very necessary to rate the motility with some

degree of accuracy.

33

Another method of microscOpic motility examination is the eval-
uation of semen by dead-alive staining of Sperms. This is based upon
the difference between dead cells and live cells in absorbing certain
dyes. In this examination the sperm that are dead at the time of making
slides will absorb the stain and appear blue, and those that were alive
will remain white.

Preparation of stains: Stain A - 2% water soluble eosin in M/ 8
phosphate buffer. Stain B - 1 part opal blue, 1 part 14/ 8 buffer. Equal
parts of above status to be mixed, heated and filtered. One to two draps
of this stain to be placed on clean glass slide and then small amount of
mixed semen Spread on the slide. Second slide Should be drapped over
the smear and excess of the stain removed by wiping edges of slides
with the cloth. The slides then should be made apart and placed on a
hot place at about 150° to 200° F. till it gets dried. Sperm may be counted
by oil immersion objective of microscope. At the most 333 sperms in all
are generally counted on the slide.

Percentage of live Sperm is computed by the following formula:

Percentage = Number of live sgerm counted 1: 3

 

Suppose 260 live Sperms are counted on the Slide; percentage of live

sperm will be:

260x3

34

Hydrogen-ion concentration.

 

The fact that spermatozoa viability is limited to narrow pH range
has emphasized the need for the consideration of hydrogen—ion concentra-
tion semen studies. However little investigation has been conducted to
determine the relationship of pH to other pertinent data on semen char—
acteristics. Webster’s observation by flaps and others (24) that they
first reported in 1932 that hydrogen-ion concentration of bull semen
varied from pH 7.0 to 7.5. Milovanov’s early observations quoted by
Anderson (1, p. 4) that if the semen of the bull be Slightly acid, pH
usually would range from 6.5 to 6.8, but in one case it was 5.5. Presence
of abnormal amounts of necessary gland secretions may be responsible
for an alkaline reaction (pH 7.0 to 7.5). Davis (7) found that collection
techniques modified the hydrogen-ion concentration of samples to a
marked degree. Rectal massage of the ampullae and the accessory
organs yielded semen of pH 7.5 to 8.0, while the use of artificial
vagina produced semen whose pH usually was below 7.0. Sergen’s
observation by Anderson (1, p. 4) reported an average pH of 6.74 when
using quino-hydrone electrode for the determination. Anderson (1)
from the studies of 11 fertile bulls reported the mean pH of semen to
be 6.73 i .02. He further observed that in conditions involving patho-
logical conditions of the genital organs, the reaction was characteristically
alkaline. This observation was constant in hypogonadism and epididy—
mitis, where consecutive samples have been studied, he states that,
second ejaculate was more acid and was of better quality than the first.

Anderson (1) further showed that Sperm concentration up to 1.8 billion

2115

 

35
Sperm per cubic millimeter existed in ejaculates having a pH of 5.8 to
6.0 while samples of pH 7.0 contained an average of .6 billion Sperm per
cubic millimeter. Relation between long survival time and more acid
pH has been described. Willett and Salisbury (36) showed that the effect
of initial pH level on Spermatozoa motility during storage indicated that
the range was not narrow. Equal results were obtained when semen yolk
phosphate mixture was adjusted to a pH level of 6.5 to 6.75 or 7.0 before
storage; they further showed that the pH of highly buffered semen ranges
from 4.0 to 5.0 and 9.5 to 10. Storage for three days increased the buffer
capacity of semen in the region of pH 8.0 to 10.0 due to increase in
carbonates present. Raps, Greg and Cannon (1947) studied the pH of 371
semen samples from 66 bulls in five studies. Highly significant correla-
tion was found between the pH levels of individual studs. Conditions of
the stud exerted marked effect upon the pH level of semen produced. They
showed that the semen of individual bulls, irrespective of breeds, tended
to maintain constant pH level and definite seasonal fluctuations were
recognized during the study.

Concentration and enumeration of spermatozg.

 

The concentration of sperm, both among the ejaculates of the
Same male and from male to male of the same Species varies widely.
It is very important to know the approximate number of Sperm in a cubic
centimeter as this helps to guage its quality and determine the rate for
dilution for routine examination. There are several methods of calculating
sperm count. One is by means of a Haemocytometer, or cell counting

apparatus. For this examination semen is diluted 100 or more times

36
with 0.9 per cent physiological saline solution by means of a Special '
pipette. After diluting and thorough mixing, a drop of solution is placed
on the counting chamber, and spermatozoa are counted for a definite
number of squares on the counting chamber. When this number is obtained,
the number per cubic millimeter may be calculated.

The apparatus used for this examination must be clean, dry and
free from grease, and the semen Should be well mixed, before a sample
is taken. After the semen and saline solution are drawn up in the pipette,
they should be well shaken and first few draps discarded. Procedures
in enumeration of Spermatozoa: (1) Sample should be stirred well. (2)
Take 0.1 cc. of sample in a graduated pipette. (3) Add 9.9 cc. of 0.9 per
cent solution of sodium chloride to make dilution of 1 in, 100. (4) Again
add 9.9 cc. of 0.9 per cent solution of sodium chloride to 0.1 cc. of 1:100
solution to make dilution of 1 in 10,000. (5) This should be mixed well
and a dr0p placed on the slide and wait till the sperm settle. (6) Three
such counting slides Should be made. (7) In each Slide 16 to 64 squares
should be counted. The number of squares counted depending upon
whether or not the sample is concentrated.

Calculation: 0.2 mm. (marked on slide) x 1/ 16 sq. m. = 1/80
cubic mm. Number of Sperm per Square = a/b where a = number of
sperm, and b = number of squares counted. Number of Sperm per cubic
millimeter = a/(b x 1/80) = (a x 80)/b. This should be multiplied by
10,000 to get the number of Sperm per cubic millimeter, because dilu-
tion is 1 in 10,000.

37

Now to calculate the exact number of sperm per c.mm.:

Slide Number I there are 25 sperm in 16 squares.
Slide Number H there are 28 sperm in 16 squares.
Slide Number III there are 30 sperm in 16 squares.
"8'3 48
Therefore, the concentration will be equal to: (83 x 80)/48 x 10,000 =
1,380,000 sperm per cubic millimeter.
Preparation of slides.

The slide Should be well cleaned and sterilized. A small drOp of
well mixed undiluted semen is placed at one end of the glass Slide. The
edge of another glass slide is applied to the semen on the first Slide
which lies on the flat smooth surface. The semen is Spread across the
edge of second slide, which is held at an angle of 45°, and is pushed along
the first slide, drawing the semen after it, in the same manner as the
blood smears are made.

Another method consists in placing the dr0p of semen on the
slide and gently drapping another slide on to the semen and then sepa-
rating the two slides, the slide is then labeled and set aside in a clean,
dust-free place to dry, after which it is stained for examination.

When using the two Slides for Spread of semen care should be
taken that the layer of semen is sufficiently left over the Slide to pro-
tect the sperm from maceration in which case the spermatozoa will be
damaged, for the tails break very easily.

38

Staining methods.

 

Herman and Swanson (1941) introduced the Rose Bengal method of
staining bull semen which is as follows: Methods: (1) 2 or 3 drops of
buffer solution to be placed on the clean slide and then 1 dr0p of mixed
semen is added and spread over the slide with second slide. The slide
should be dried thoroughly and then stained with Rose Bengal stain
and dried for 5—10 minutes, then rinse off excess stain by dipping in
distilled water. Slide is dried and examined under microscope. Rose
Bengal stain is made up as follows: 3 grams powdered Rose Bengal,

99 cc. distilled water, 1 cc. 40% formalin. (2) Dried semen smears may
be stained satisfactorily with 0.5 per cent alcohol solution of gentian
violet for three minutes. (3) The slide is air dried, and put in a saturated
solution of chlorazene for 5 to 10 minutes, rinsed in distilled water,

fixed in 10 per cent formalin for 3 to 5 minutes, stained with Ziehl’s
carbolfuchsin for two minutes, washed gently in running tap water,

dried and examined.

D. TECHNICAL PROCEDURES FOR COLLECTION OF SEMEN

Equipment required.

 

The equipment and supplies for collection of semen and insem-
ination vary considerably for different Species but the procedures to be
followed are generally the same.

The following is the complete list of equipment needed for col-
lection of semen, semen examination, and insemination regardless of
the methods used:

( 1) Supply of 65 per cent alcohol (not denatured).

(2) Several rolls of absorbent cotton in sterile packages.

(3) A good lubricant.

(4) Rubber gloves.

(5) A flashlight and heifer Size speculum.

(6) Good quality hand soap.

(7) Supply of test tubes and hand bottles with rubber stoppers.

(8) A stand for bottles and test tubes.

(9) Glass test tube holder.

(10) Insulated container in which to put the semen after collection.

(11) A glass (wax) pencil or gummed labels for labeling the tubes.

(12) An enameled tray and pan for keeping equipment after use.

(13) Supply of distilled water and sodium chloride tablets for normal
saline solution.

(14) Two graduated beakers or flasks of 100 cc. and 500 cc. for
preparing solutions, and several others ungraduated.

39

40

(15) Box of glass slides with cover glasses.

(16) One hollow-ground slide for making motility observations.

(17) Slide box for storing slides after preparation.

(18) Thermometer for measuring the temperature of water for
the artificial vagina.

(19) About a dozen of medicinal drOpperS.

(20) Two glass funnels, preferably of pyrex.

(21) A test tube brush with medium soft bristles.

(22) A 2 cc. graduated glass syringe.

(23) An 18” ebonite nozzle equipped for attaching to the syringe.
A glass tube with 1/8” inner diameter may be substituted for the nozzle.

(24) Artificial vagina.

(25) It is well to have a surgical and first aid kit for use in case
of injury to man or animals.

(26) A micrOSOOpe with magnifications up to 300 to 400 diameter
and Hemocytometer, pipette and a supply of stains are needed for
clinical examination of the semen.

Control of the bull and cow.

 

For collection Of semen a cow in heat should be made available,
preferably an old cow which may have been used several times and
stands quietly. A nymphomaniac cow may be used for the purpose. The
cow should be tethered to the post with halter or with a rape around the
horns. The ground should be level and not slippery. The hull should be
handled as for normal mating. He should be led up to the cow with a
staff or halter. The safety of the Operator Should always be assured.

41
The paddock Should be sufficiently large to allow plenty Of room for
turning and preferably the same yard should be used for all services
as a bull soon learns to anticipate service when led into a yard which is
used for that purpose.

Preparation aggrig.

 

As spermatozoa are easily injured or killed by the toxic agents
of many sorts, it is necessary that all the apparatus be free from
harmful chemical agents as well as free from harmful bacteria and
molds. Therefore, all apparatus must be thoroughly washed and disin-
fected before and after each use; and exacting sanitary precautions must
be taken or artificial insemination may prove more of a liability than
an asset to the live stock industry.

Rigid adherence to sanitary practice will insure the best viability
of spermatozoa.

All new apparatus should be washed thoroughly with hot water
and soap in order to remove all grease and other chemical substances
that may be adhering to it. After being washed it should be carefully
rinsed in several changes of clean water, preferably distilled water and
then allowed to stand for about 10 minutes in 70 per cent ethyl alcohol.
It is then kept in a clean, dust-free place, or, if required for immediate
use, it may be rinsed in several changes of normal saline solution to
remove the alcohol. Dry heat and flaming are excellent means for
sterilizing glassware where facilities are available, but precautions are
taken against too rapid heating or cooling, which may result in break-

age.

42

The use of soap subsequent to the cleaning of new apparatus is
not recommended, for traces of soap are injurious to spermatozoa and
rubber apparatus. Rubber material is scrubbed with a paste of sodium
bicarbonate and water, rinsed with distilled water, and dried well between
periods of use.

All vials and centrifuge tubes should be well-corked.
Artificial vagina technique.

 

This is the most satisfactory method for collection of semen
from the bull. The artificial vagina consists of a rubber cylinder 16%”
long and 2-3/4” in diameter, fitted with an inner rubber lining, the ends
of which are turned back over the ends of cylinder and held in place with
heavy rubber bands. Near one end of this cylinder is a hole to admit
warm water. The preper pressure is obtained by filling the outer jacket
about one-half to two-thirds full of water at a temperature of 125° to 160° F.
The whole inner lining is evenly and thoroughly smeared with a thin coat
of lubricant as vaseline, mineral oil, or gum tragacanth to prevent the
semen from contacting the rubber. The temperature is determined by
inserting a thermometer in the Open end of the artificial vagina. The
temperature on the inside of vagina should be 105° to 110° F. when the
apparatus is ready for use. If the water is too warm, time should be
allowed for cooling; if the water is cold, some of it is emptied out and
replaced with hot water. After the prOper temperature is attained
pressure is adjusted by the quantity of water used or by the application
of air pressure in addition to water pressure. Then the bull is led up

slowly behind the cow and the collector stands on the right side, holding

43
the apparatus in the right hand. The bull is allowed to mount the cow,
the artificial vagina raised close to the flank of the cow behind the
bull’s fore leg with the Opening directed toward the penis at an angle
of about 45°. The penis is directed into the opening of the artificial
vagina by applying the left hand to the sheath. When the penis comes
into contact with the warm lubricated surface of the artificial vagina,
the bull thrusts upward and ejaculates.
Points to be taken into consideration while using artificial vagina.

 

(1) Care should be taken, not to touch the penis as it may cause
the bull to retract the organ and dismount.

(2) Too much lubricant should not be used. It may cause contam-
ination of semen.

(3) Semen should be protected against shock after collection.

(4) After use water should be removed from vagina and the vagina
cleaned by a thorough scrubbing with a paste made of sodium bicarbonate
and water. It is then rinsed well, disinfected and allowed to dry.

(5) Care should be used to collect semen aseptically.

(6) Semen is labeled as soon as collected with regard to name
and number of the bull, breed, date, and volume of semen.

fieetal massage of ampullae.

 

A trained Operator can readily collect semen from bulls by
rectal massage of ampullae of the sperm duct. In this method knowledge
Of the bull’s anatomy, or instruction in the technique, is necessary. This
method has the advantage of being rapid and requires little special

equipment, and is practiced when a male cannot use the artificial vagina

44
due to lameness or some other condition. If proper care is taken a bull
can be used by this method regularly over a long period Of time without
injury to the bull or his reproductive tract. The sire used for this
purpose should be trained, as some difficulty may be encountered in
securing samples from some untrained animals. Details Of the methods
are as follows:

The hull is tied in a place in a stall in such a manner that he
cannot shift from side to side, and the sheath is carefully clipped and
stroked two or three times towards the opening to “strip” out any
residual urine to prevent urine contamination Of semen. Then the sheath
is washed with soft brush and warm water. After finishing this cleaning
procedure, the assistant should hold a funnel, leading to the test tube
at the end Of sheath of penis. Several test tubes should be made available
during the collection of the sample so that tubes may be substituted if
one tube becomes dirty. The finger nails of the Operator should be
clipped very short. A rubber glove is placed on the hand and the arm,
and anus of the hull lubricated with vaseline, mineral oil or mild
soap. The hand is then inserted in the bull’s rectum and all feces
removed and the hand inserted enough forward into the rectum so that
the fingers drOp out the anterior border of the pelvic bone; then the hand
‘ is pulled backward with a gentle downward pressure by the hand, the
fingers will come into a triangular space formed by the two ampullae.

At the apex of this triangle the two seminal vesicles join the urethra.
The ampullae are massaged with “stripping” action and backward strokes
and the second finger of the hand is run between the ampullae and the

45
index and third fingers are placed on the outer side of the respective
ampullae. A slow, rhythmic motion is used while massaging, and, if
done correctly a sperm sample free from accessory secretions may be
secured. Sometimes the ejaculate is retained in the sigmoid flexure of
the penis. To avoid this, the Operator should straighten this flexure
with his other hand after massaging the ampullae. The semen is
collected from the end Of the penis or sheath by means of a funnel and
test tube held by an assistant and after collection it is handled in the
same manner as in other methods.

Collection Of semen from vagna.

 

I no: in! ,‘.'-h~'¥;‘-7 I‘m; £ua~2m9 fulfil:
4‘

This is the Oldest and simplest method for collection of semen
from the vagina of a cow that has been served by the bull. The vagina
is washed free from mucous by repeated flushings. The semen is
collected from the floor of vagina, following copulation by means of a
pipette, syringe, or special vaginal spoon. The use of speculum facili-
tates more complete collection of the ejaculate and there is less chance
of injury to the cow from insertion of the nozzle, and the chances of the
cows breaking the tube by sudden movement are minimized. In case of
pipette coating of lubricant should be applied prior to use to prevent
irritation of the lining Of the vagina.

It has to be ascertainedthat the male and female used in this
method are free from disease. It is necessary to wash the vagina
thoroughly between services of different bulls, if the pedigrees Of the

Offspring are desired.

This above method is not recommended by several authors
because the semen becomes contaminated with mucous and urine and

there is an increased danger of Spreading genital diseases.

46

E. STORAGE AND TRANSPORT OF SEMEN

Care and storage of semen.

 

It is absolutely necessary that the temperature Of semen be
carefully regulated. Sperm are injured at temperatures above body
heat and during the time they are in storage, carry on certain biological
reactions. The rates of these reactions are largely dependent upon the
concentration and the activity of the sperm. Therefore it is of utmost
importance to estimate the length of time the sperm will remain viable
under prOper storage conditions.

Two essential conditions for the successful storage of bull
semen are:

(1) Gradual cooling.

(2) The additions of prOper diluents as egg yolk-phosphate or
egg yolk-citrate buffer.

After collection of semen its temperature should be carefully
checked. The semen bottle and all glassware used for handling the
semen, as well as liquid paraffin, should be warmed to a temperature of
30° C. Semen remains in the bottle at room temperature for few
minutes during the estimation of motility. All the glassware that holds
the semen must be at the same temperature as the semen; and when
mixed with diluters, the two fluids must be at the same temperature.
Then the semen is diluted with the preservative and mixed by gently
inverting or shaking the container. A wide range Of dilution rates have

47

48
been used. Dilutions of one part of semen to 100 parts of diluter have
been used with satisfactory results, but as a general rule such large
volumes of diluted semen are not necessary and dilutions Of 1:10 to
1:50 are most commonly practiced. The diluted semen is placed in
test tubes which are wrapped in cotton wool. Liquid paraffin is added
to the tubes which are corked and sealed with candle wax.

The temperature of 40° F. is considered ideal for storage of
semen. This is not cold enough to freeze the semen but it is suffi-
ciently cool to keep the cells in a dormant state, thus preventing the
exhaustion of their energy. The gradual cooling to this temperature
may be accomplished by immersing the tube of semen in a body of
water Of 75° F. to 80° F. After this the semen should be so handled that
the gradual cooling should take place at the approximate rate of 10 F.
per minute up to 40° F. If the temperature is to be reduced to 33 to 35°
F. great care is to be taken for cold shock to the spermatozoa. The
cooling should be so gradual that it should take two additional hours to
get the above temperatures. The sperm will die if they are subjected
to mmperatures below 32° F.

Preparation of diluting fluids.

 

The main object Of diluting fluids for semen is to increase the
volume of the ejaculate so that many cows can be inseminated from a
single collection from a healthy and valuable bull than would be possible
in natural mating.

Diluents, are used not only for dilution but also for providing

favorable media which should aid in the preservation and longevity of

49
the spermatozoa. Diluters should improve the media surrounding the
sperm by supplying nutritive requirements and undergo as little change
as possible during storage and protect spermatozoa against adverse
conditions and changes. Such chemical and physical characteristics as

(1) Buffering capacity: to protect against marked changes in
the hydrogen-ion concentration.
(2) Osmotic pressure: The diluting fluid must be isotonic with

the sperm of given species.

'~ A ”12“.“ “.04
u

(3) The diluter must not be toxic to spermatozoa.
Pure salts have an injurious effect on vitality Of sperm, for in such

as.um‘mmfi-

1’

solutions sperm move very actively, but rapidly lose motility and die.
Physiological saline solution is said to have destructive action on the
capsule.

(4) It should be inexpensive and easy to prepare. Economy must
be considered in formulating a diluter. It should be readily available.

(5) It should contain sufficient food nutrients and resistant
factors for sperm viability. Diluters are being improved as more in-
formation in that connection becomes available.

The most widely used diluters for bull semen to date are yolk-
citrate, yolk-phosphate and chick embryo; and investigators do not
agree as to which of these is the most desirable for storage and
breeding (15). Many other diluters are being tried and discarded, many
other diluters are not satisfactory but are still being tried with various
modifications.

50

mik-citrate diluter

 

Salisbury Method as prepared at the Michigan Artificial

Breeding Co-operative at East Lansing.
Equipment Required:

A. (1) Sodium citrate - Na2C6H507 . 2H20. Chemically pure
reagent must be used. Other grades as technical or commercial are
not satisfactory. fr :

(2) Double distilled water should be used. It must be free ‘
from traces of acids or mineral salts.
(3) Balance and weights.

 

(4) Clean weighing pans.
(5) Volumetric flasks (such as 1 or ml or 1000 ml).

(6) Funnels.

(7) Clean graduated bottles with screw tops to store
diluter.

(8) Auto-clave or pressure cooker.

(9) Refrigerator

B. Procedure

(1) Exact amount of citrate needed should be weighed to
prepare 3.6% solution Of Na2C6350-7 - 21120 or 36 grams in' 1000 ml of
redistilled water.

(2) Transfer weighed citrate to volumetric flask and fill to
mark with redistilled water. Mix till all traces of sodium citrate have
have disappeared. Shake for 5 minutes more to insure prOper disper-
sion of citrate molecule.

51

(3) Pour citrate solution in suitable bottle. It should not be
filled too full, about 4/ 5 from tOp for maximum.

(4) Heat solution in bottle under 15 pounds pressure for 20
minutes in auto -clave or pressure cooker. Use precautions to prevent
loss of liquid or breaking of bottles.

(5) Remove from sterilizer. Close bottle securely. Dis-
card any bottle if too much liquid has been lost. Store in refrigerator
at 40° F. The pH of the buffer solution should be 6.7 to 6.8.

C. Precautions

 

(1) Prepare sodium citrate solution Often enough so that it
is not stored for more than three months when autoclaved.

(2) No attempt should be made to use un-autoclaved solution
for more than two weeks. Solution must be kept refrigerated at 40° F.
Even under low temperatures some mold may grow in citrate solution
which will produce toxins that are harmful to sperm. More molds will
grow at higher temperature and there would be more chance Of toxin to
be present.

D. Preparation Of dilution as used for diluting semen.

Taking all aseptic precautions, equal volumes of fresh egg yolk
and prepared sodium citrate solution should be mixed at the same tem-
perature. Desired volume of citrate solution is poured into graduated
cylinder and warmed at room temperature when preparing egg yolk.

(2) Fresh sterile eggs should be cleaned in water and
alcohol and allowed to dry. White should be separated from the yolk by

means of clean half shell and yolk held just over top of second graduated

52

cylinder, then puncture egg yolk membrane with sterile glass rod. All
yolk contents should be allowed to flow into graduated cylinder and be
kept absolutely free from white of egg or yolk membrane. Egg yolk
should be poured into citrate solution to equalize the volumes. Clean
cylinder or test tubes should be used for pouring the contents from one
container tO another until thoroughly mixed. The temperature of this
diluent is adjusted to the temperature of semen and mixed and kept in
the refrigerator at 40° F. Great care should be taken to avoid tem-
perature shock. Sample semen may be diluted with saline on a micro-
scOpe slide in order to check motility.

Fertilizing capacity of stored semen is estimated to be four
days. The main advantages of the citrate buffer are; first, that it is
easily prepared as it requires the weighing of only one chemical,
secondly it disperses the globules in the egg yolk and improves micro-
scophic examination, and the sperm tends to live longer than in other
diluents. Dilution should be made as soon as possible after the semen
sample is secured.
walk phosphate diluter.

This diluter was first developed by Phillips in 1939 and at
present is known as Phillips’ Buffer or Wisconsin buffer.

Formulae for the solution of phosphate buffer. All the equipment
used for citrate buffer is required.

0. 2 gram KHZ P133 (Pot: Dihydrogen phosphate)

2.0 @ Naz 2H 12H2 0 (Sodium phosphate)
100 cc. of H220 dou e distilled.

53
The pH Of the solution should be 6.7 to 6.8. All precautions should be
taken as in preparation of citrate buffer. If the pH is lower than 6.75,
more of the buffer solution should be added. Crystalline material should
be dissolved and mixed in boiling water and then kept in cool place at
150 C. Fresh egg yolk should be prepared by taking all aseptic precau-
tions. All white from the egg is removed and the yolk poured into a
sterile container. Equal volumes of fresh egg yolk and phosphate
buffer are mixed thoroughly. It should be diluted with semen when both
are at the same temperature and then kept in refrigerator and cooled
at 40° F. till used.

This diluter is also used at present and has proved to give a
good conception rate. It aids in protecting the sperm against adverse
conditions. Its high viscosity, dark color and presence Of fat globules
from the egg yolk, oppose convenient microscopical examination until
and unless Na2804, S. G. C.-2 or glucose solution is added to it.
Chick-Embryofidiluter

 

10 to 12 days incubated egg should be used. Contents mixed in
waring blender for 15 minutes and centrifuged at 2750 revolutions per
minute for 1% to 2 hours. Then supernatant fluid removed with pipette
and used for diluter.

Many investigators vary as to its better usefulness than citrate
and phosphate buffer. Most trials have shown that the chick embryo is
very unsatisfactory and far inferior to both the phosphate and citrate
diluters.

54

Glucose diluter.

 

This diluter was introduced by Swanson and Herman in 1941 in
which 3% and 5% glucose solutions were first deveIOped to provide a
diluent which would supply the spermatozoa with food nutrients, but the
later investigations have shown that there is no beneficial factor in this
diluter. Hence it is not widely used at present time because of its
inferiority to egg yolk buffer diluters. It is prepared by mixing 3 grams
of glucose in 100 cc. of glass-distilled water or 5 grams glucose in 100
cc. of glass-distilled water. It has been shown that dilutions of 1:3 were
definitely harmful to Sperm as measured by their effect upon motility
and maintenance.

Temperature control, packing and transport.

 

When the semen of the bull or any other animal is to be shipped
a long distance more elaborate precautions are taken. Cooling takes
place promptly after examination and then ultimately cooled to 40° F.
Cooling should be gradual prior to packing by changing the semen from
one container to another. To maintain prOper temperature while in
transit, generally, each shipment is accompanied by a balloon or tin
can Of ice as a refrigerant. The vial containing the semen is wrapped
in a layer Of cotton and placed along a small can filled with solidly
frozen ice or the package should be placed in quart vacuum bottle and
tightly packed with chipped ice without salt. To avoid danger of
breakage, it is best to place a layer of cotton in the bottom of the vacuum
bottle before packing with ice and to add layer of cotton before stOppering.
The can or the vacuum bottle containing the vial and the ice are wrapped

a.
«\m r
n...
r
«a
Ltd
. a.

.4
.3
3+
.I.

.‘
u.

 

54

Glucose diluter.

 

This diluter was introduced by Swanson and Herman in 1941 in
which 3% and 5% glucose solutions were first deveIOped to provide a
diluent which would supply the spermatozoa with food nutrients, but the
later investigations have shown that there is no beneficial factor in this
diluter. Hence it is not widely used at present time because of its
inferiority to egg yolk buffer diluters. It is prepared by mixing 3 grams
of glucose in 100 cc. of glass-distilled water or 5 grams glucose in 100
cc. of glass-distilled water. It has been shown that dilutions of 1:3 were
definitely harmful to Sperm as measured by their effect upon motility
and maintenance.
Temperature control, Maud transport.

When the semen Of the bull or any other animal is to be shipped
a long distance more elaborate precautions are taken. Cooling takes
place promptly after examination and then ultimately cooled to 40° F.
Cooling should be gradual prior to packing by changing the semen from
one container to another. To maintain proper temperature while in
transit, generally, each shipment is accompanied by a balloon or tin
can Of ice as a refrigerant. The vial containing the semen is wrapped
in a layer of cotton and placed along a small can filled with solidly
frozen ice or the package should be placed in quart vacuum bottle and
tightly packed with chipped ice without salt. To avoid danger of
breakage, it is best to place a layer of cotton in the bottom of the vacuum
bottle before packing with ice and to add layer of cotton before stOppering.
The can or the vacuum bottle containing the vial and the ice are wrapped

54

Glucose diluter.

 

This diluter was introduced by Swanson and Herman in 1941 in
which 3% and 5% glucose solutions were first developed to provide a
diluent which would supply the spermatozoa with food nutrients, but the
later investigations have shown that there is no beneficial factor in this
diluter. Hence it is not widely used at present time because of its
inferiority to egg yolk buffer diluters. It is prepared by mixing 3 grams
of glucose in 100 cc. of glass-distilled water or 5 grams glucose in 100
cc. of glass-distilled water. It has been shown that dilutions of 1:3 were
definitely harmful to Sperm as measured by their effect upon motility
and maintenance.

Temperature conuolechgm transport.

 

When the semen Of the bull or any other animal is to be shipped
a long distance more elaborate precautions are taken. Cooling takes
place promptly after examination and then ultimutely cooled to 40° F.
Cooling should be gradual prior to packing by changing the semen from
one container to another. To maintain prOper temperature while in
transit, generally, each shipment is accompanied by a balloon or tin
can of ice as a refrigerant. The vial containing the semen is wrapped
in a layer Of cotton and placed along a small can filled with solidly
frozen ice or the package should be placed in quart vacuum bottle and
tightly packed with chipped ice without salt. To avoid danger of
breakage, it is best to place a layer of cotton in the bottom of the vacuum
bottle before packing with ice and to add layer of cotton before stOppering.
The can or the vacuum bottle containing the vial and the ice are wrapped

55
tightly in a heavy insulating paper commonly referred to as "‘I iffy
paper.” The wrapped package is then inserted in a shipping container
and sufficient paper is used in packing to hold the whole container
firmly. Under these above conditions semen may be kept at the re-
quired temperature of 40° F. for about 30 hours. For long shipments
for 3 or 4 days, it is very important that adequate refrigeration of
package should be maintained. The semen should be protected from
intense cold of the refrigerant and insulation of the refrigerant against
the external temperature.

Transport

In about the last seven or eight years considerable interest has
been developed in the transportation of semen to distant herds. It is
therefore very necessary to have careful co-ordination between the
persons shipping the semen and those doing the insemination. It is
necessary to plan the shipments so that a minimum of time will elapse
between collection and insemination; it is also necessary that the
females to be inseminated be in heat; and at the proper stage of heat,
if large percentage of conception is to be obtained. The shipment may
be mailed daily by special delivery to assure arrival in the field the
morning before collection, at the latest. In case of short distances,
semen may be transported by car, bus, or rail. Before shipment it
should be placed in the insulated vials in a thermos bottle filled with
cracked ice, to maintain the constant temperature, package should be
labeled “Glass - handle with care.”

 

56

The 40° F. temperature should be maintained until the semen is
used. Immediately upon its arrival the temperature should. be checked.
1: it is found to be 40° F., it should be immediately transferred to a
refrigerator or vacuum bottle of the same temperature. If the tem-
perature is higher, it should be immersed in a body of water of equal
temperature and exposed to 40° F. allowing it to cool gradually to this
degree.

Long distance shipment of semen seems practical, and several
foreign governments as well as individual breeders are attempting to
secure the inheritance of valuable sires by this practice. Thus the
wide spread deve10pment of air services for transport of semen would
be more effective at large experimental stations, on ranches, or in

organized groups to spread the services of a sire over a large number
of females.

F. FACTORS AFFECTING STORAGE OF SEMEN

Interaction of storage temperatures:

 

Sperm are sensitive to both high and low temperatures. The fall
of motility is associated with a fall of temperature and it may be re-
versible, partially reversible or irreversible in accordance with the
rate of cooling. Rapid cooling causes more or less irreversible changes
which might lead to death of sperm. This harmful effect of sudden
cooling, called “Temperature Shock,” is Of great practical importance
and necessitates careful regulation of temperature of artificial insemi-
nation laboratories. It has been shown that a fall in temperature from
40° c. to 0° c. within 30 to 60 minutes caused rapid decrease of resis-
tance and the loss of ability to be reactivated on being reheated to body
temperature (36). If cooling was continued over two hours, the tem-
perature shock was less severe and all samples reactivated on re-
heating; thus the susceptibility of bull sperm to temperature shock
depends on the degree and rate of cooling. It was also noted by Ander-
son (1) that on rapid cooling from body temperature to 5° to 10° C.,
activity decreased and was not restored on re-heating.

Temperature shock is more severe at low temperatures than at
higher ones, and while sudden cooling is harmful at all temperatures
and the effect is more marked at the lower ones. Rapid warming has
no harmful effect upon the subsequent respiratory activity of the

sperm.

57

58

An experiment at Cornell University, in connection with tem-
perature shock from too rapid cooling was conducted by Salisbury and
others (36) to check the findings.

After collection semen was transported to laboratory in water
at 37° C. Each sample consisted of 0.5 cc. of undiluted semen. One-
half Of the samples were cooled from room temperature in un-insulated
test tubes by placing them directly in the water bath at 5° 0.; this
treatment cooled the semen from the room temperature to the storage
temperature of 5° C. in less than 1 minute.

The other half of the samples, in large insulated test tubes were
cooled from room temperature at the rate of 10 degrees per 10
minutes. The semen was covered with mineral oil.

The results are given as below in table Nos. 1 and 2.

Table NO. 1 - Comparison Of motility of spermatozoa cooled

slowly before storage at 5° C. under mineral Oil, and those cooled

 

 

 

 

 

 

 

 

 

 

rapidly.
Motility - mean percentage Probabili-
Number leter storage * ties for
Number of Rate~10 RateTG Differences
days Sample before deg. / 10 deg. / 1 between
Stored Pairs Btoragi min. min. means
2 9 79 29 14 4 0.01
8 L 79 24 12 40.01
6 8 79 24 7 40.01
8 6 78 17 8 7 0.05

59
It is seen that the rapid cooling was detrimcntal for the sperma-
tozoa maintained greater motility after being cooled at the slower rate.
In addition there was highly significant difference in pH values after
storage with the two rates of cooling as is shown in table No. 2.
Table NO. 2. Comparison of pH of Semen Cooled Slowly Before
Storage at 5° C. under Mineral oil, and of that Cooled Rapidly.

 

 

 

 

 

pH 11% Profifili-
Number er sto e ties for
Number of L—1Tate 10 %te 30 Differences
days Sample Before deg. / 10 deg. /1 between
Stored Pairs Storage min. minL means
2 9 6.27 6.10 6.33 40.01
4 8 6.27 6.07 6.32 40.01
6 8 6.28 t 6.01 6.29 4 0.01
8 6 6.26 6.00 6.33 4. 0.01

 

 

 

 

 

 

Apparently the spermatozoa in the semen that were cooled more
rapidly were rather inactive during storage, as indicated by the constant
pH level. From the above figures it is seen that the injury to the
spermatozoa due to rapid cooling was severe and retarded metabolic

activity as concluded from the pH values.

 

Effects of diluting fluids on staggefiand fertility of semen.

Several studies have been made by various investigators in
which the semen have been diluted with yolk phosphate and yolk citrate
ciluters composed of 1 part fresh egg yolk and 1 part of a buffer solution.
.2 grams of KH2P04 and 2 grams of NaZHPO4 - 12H20; and 3.6 grams
Na3C6H507 - 2H20 per 100 ml of distilled water. It was suggested by

60

Phillips and Lardy (2 3) that the rate Of cooling had no influence on the
fertility of bull semen when the semen is stored in a nutrient buffer
solution such as the yolk-phosphate or yolk-citrate diluent.

Studies at the Cornell University were made to test the effect of
the rate of cooling on the survival of spermatozoa diluted with the yolk-
phosphate and yolk-citrate diluents.

In the experiment with yolk-phosphate, semen was diluted in
the ratio of 1:4. One-third Of the samples were cooled directly from
the incubator into the 5° C. water bath without using the insulated
tubes while other two samples were cooled at the rate Of 5° C. per 5

minutes and 5° C. per 20 minutes respectively and taking about one

hour to cool from room temperature to 5° C. Mineral oil was not used.

The results are given on the next page in (Table NO. 3).

’23:; '3:.l:_.:~s n.5‘l

m.

__._.__..._
I .. “3,...

it." I» D'Illli‘ .

all"!

I 434....

61

 

 

 

 

 

 

 

 

 

 

 

 

 

850 Se 85% a S a 8 e S
no... A See 1 856. S S a a. m 5
85% 8.3 as.» as 2 N . E. e S
850 850 85% 8 a e 2. S m
85A 85% Se 5 «a 2 s. 5 e
.58 .58 .58 5.8 .58- .58 emflssm End Beam
Q as. a Q .mee m e\ .mnu n .5 .meu n\ .meu < one chasm e385 use. as
e5 .58 use .58 use .58 m sum meow on some. 8 unease
m\ .uov Q .mov Q .wou c Sun new: song
Ibsen Prozac o e um , l {
condemn—a. «o nouzfianoam t

 

so 88o58 82:5 .0 on on omsnoom 3&8 seem cannons— oeafi. 8 380 8.55 noses
need 5e» 2: 5 «Seosaeam 8 5:52 8 sontsano .m ease

62
From the figure on the preceding page it will be seen that the
significant differences are found in the motility percentage in all the
three samples treated. This indicates that even in nutrient medium the
spermatozoa suffered from temperature shock and that the slowest rate
of cooling, 5 degrees per 20 minutes, was the most satisfactory.
Similar experiment was conducted for pH. It was shown that there was

no significant difference between the average of pH values for the

..‘4‘i

samples cooled.

' ‘42. 1 O J'm—f R ‘v 3..

Therefore, it can be concluded that spermatozoa, whether in

undiluted semen or in yolk phosphate diluent, suffer from temperature it.

 

shock caused by rapid cooling of semen. The rate of chemical reaction Ms
doubles with increase of 10° C. Salisbury (Personal Discussions)
recommends that the semen may be stored at 0° C. to 1° C. but

greater care is to be taken with rate of cooling between 5° C. to 0° or

1° C. At least 2 hours time should elapse to keep the temperature from
5° C. to 0° or 1° C. Such studies were carried out at Cornell Univer-
sity with egg yolk citrate diluent by keeping the samples at various tem-
peratures at the dilution rate of 1:4. Samples consisting Of 0.5 cc. of the
mixture were placed in small vials and kept in large insulated tubes.
One-fourth Of these samples were stored at 5° C. and were cooled at the
rate of-5° C. per 10 minutes. The remaining three-fourth of samples
were stored at 1° C. but were cooled at different rates: 5° C. per 10
minutes; 5° C. per 20 minutes; 5° C. per 30 minutes. Mineral Oil was
not used. The results Of motility are shown in (Table No. 4).

63
Table No. 4. Comparison of Motility of Spermatozoa in the yolk

citrate diluent in stored supply. Mineral oil was not used.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Probabilities
Motility Means (per cent) for
Differences
Number Number Alter stor e between
of days of Before Treatmen __ means
Stored Ejaculates Storage :4 B C D A and B
2 6 77 40 24 33 28 L 0.01 f |
4 6 77 30 18 l 27 26 4 0.01
6 6 77 19 15 23 22 7 0.05
8 6 77 13 10 . 18 16 7 0.05 ».
I
10 6 77 11 9 . 10 15 7 0.05 1...;
Note: ' ' ’
A = Stored at 5° C. and cooled at the rate of 5 degrees per 10
minutes.
B = Stored at 1° c. and cooled at the rate of 5 degrees per 10
minutes.

C = 5 degrees per 20 minutes.
D = 5 degrees per 30 minutes.
From the figures it will be seen that significant differences were

found only in favor of treatment 5 degrees per 10 min. over 5° C. per 10
minutes at 1° C. In addition it has been suggested that for the longest
storage interval the storage temperature of 1° C. was the most satisfac-
tory at the slowest rate of cooling, 5 degrees per 30 minutes. Not much
difference was noticed in pHvalues.

Effect Of dilution on fertility.

 

The rate Of diluting bull semen has ranged all the way from one
part semen and one part diluter to one part semen and one hundred parts

diluter. Early studies conducted by Willett and others (3 6) showed a high

64
rate of conception for diluted semen used during the first 24 hours after
collection, which rate was continued without important deviation up to
96 hours. Later Salisbury (9) studied to determine if dilution effect
would influence the fertility of semen, testing dilution rates Of 1:4, 1:8,
1L10, 1:12, and 1:16, 1:24, and 1:50 parts of egg yolk-citrate-diluent.
All semen samples containing motile spermatozoa were used regard-
less of concentration. The latest investigation on this problem by
Salisbury (32) has been made with dilution rates of 1:40, 1:60, 1:80, and
1:100 parts Of the yolk citrate diluent. The results of all the above

experiments are given on the next page in Table No. 5. t7

 

65

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as. use are use is use ”.8 3:. won «.8 one «.5 seoaooseo
«coo Mom

32 some 52 3e as: ea. 8m 8m 8... in an as unoEom
8 888:2

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8 seem

 

 

 

 

 

 

 

 

 

 

 

 

 

858838 8:55 e8 8 and sadness e5 8 .8883. .8 62 uses

66

From the figure on the preceding page no difference in fertility
was found between all the dilution rates. At the highest rate of dilution
on the average 12,836,000 spermatozoa were introduced in each 1.0 ml.
Of diluted semen used for insemination. And also it will be seen from
the figures that the highest average efficiency of conception was ob-
tained with the highest dilution rate. 1:100; and the small differences

shown between dilution rates are not significant.

K - J Okdf'ni'ie. ‘L’IIA. Ff“

 

III. STUDY OF SEASONAL AND BREED DIFFERENCES IN SEMEN
PRODUCTION OF DAIRY BULLS

It has long been recognized that in many animals spermatogenesis
is not continuous but tends to occur at one rather definite period Of the
year as in case Of man-oestrus animals. Cyclic spermatogenic activity
is characteristic of some animals. Domesticated animals such as the
bull, stallion, ram, etc., have been classified as continuous breeders, but
the evidence is now accumulating (3) to show that although spermatozoa
are produced throughout the year certain seasonal trends in the rate Of
spermatogenesis and the degree of fertility are evident.

It was therefore decided to study and determine the influence of
season and breeds upon semen quality in the dairy bull with regard to its
volume, motility and concentration.

Review of literature.

 

Moore’s Observation by Anderson (1) that the testis is regulated
by the gonadrotropic hormone of the anterior pituitary gland and to some
extent by other endocrine glands, by light, by environmental testicular
temperatures and the plane of body nutrition. In extensive studies of
semen production of sheep was reported (12) that although definite
seasonal trends in semen. quality were apparent, maxinium quality semen
was observed from October to January and maximum efficiency was said
to have been obtained in May. Studies made by Erb (13) showed that

except for a noticeable increase in concentration in the spring season,

67

 

68
seasons other than summer had little effect on semen quality. During
July, August and September motility and semen volume was lowered
and the percentage of abnormal forms was increased. Bartlett (3)
reported monthly averages of semen volume, concentration and longev-
ity for six bulls and found it to be correlated with fertility. Anderson
(1) presented monthly average semen volumes and motility observations
made on bulls in Kenya which indicated distinct seasonal tendency.

Materials and Methods

The bulls were all pure bred animals used as regular sires by
the Michigan Artificial Breeding Cooperative unit at Michigan State
College. Four Holstein-Friesian and four Guernsey bulls were used in
this study. They varied in age from 3 to 8 years but the majority of
them were aged bulls, five years old or Older. The animals had no
access to pastures and were maintained on usual grain rations con-
taining 15-16% protein composed of 1000 pounds oats, 250 pounds wheat
middlings, 350 pounds wheat, 300 pounds soybean oat meal, 100 pounds
meat scrap, 100 pounds poultry concentrate, 36 pounds salt. Of this
mixture 4 to 5 pounds were fed to each hull with one ounce of mixed
minerals as cobalt, iron, copper, etc. All the animals were in good
breeding condition. First cutting of alfalfa and bromegrass was fed at
liberty.

Semen was collected from all bulls with an artificial vagina
once each week and with few exceptions after two weeks. Two ejacu-
lates were obtained within a ten minute interval at each collection

period.

69

The methods of semen evaluation which are commonly used in
investigations Of this type and have been fully described under the
chapter “Clinical Examination of Semen.” Semen volume, initial
motility, and concentration of spermatozoa were immediately recorded.

The degree of motility of the spermatozoa have already been
expressed.

Concentration of spermatozoa per unit volume of semen was
determined by haemocytomter using the technique which is ordinarily
applied for the counting Of red blood cells; and total motility rating
score was Obtained by summation of the individual motility ratings
during the period for examination of smears of each ejaculate. Average
records of semen examination of 8 bulls for winter and summer are

shown in Tables 6 and 7.

Table No. 6. Average Record of Semen Examination
of 8 Bulls During January, February and March.

 

 

 

 

7 fiftiil ficentration

Name Of Volume Motility Of

Bulls in mi chenm§§_ sperm /m1n3
Hoisteins
KbOp 6.8 80 1312
Silver 5.4 74 1100
Rainbow 7.7 70 1053
Barber 7.3 71 1330
Guernseys
Maxim 6.2 77 1283
Master 8.3 71 766
Neptune 8.2 80 1050
Mars 3.3 79

 

 

 

1511

 

70

Table NO. 7. Average Record of Semen Examination
of 8 Bulls During July, August and September.

 

 

 

 

fl Initial Concentration

Name of Volume Motility of

Bulls in mi Percentage - sperm /mm3
Holsteins
Kbop 5.9 71 1333
Silver 5.1 71 1100
Rainbow 6.7 61 1464
Barber 5.9 68 686
Guernseys
Maxim 6.5 75 1328
Master 7.7 74 1022
Neptune 10.0 72 900
Mars 3.1 71 1590

 

 

 

 

71

72

Results

The result of examination of 179 ejaculates produced by 8 bulls
between January, February, March and July, August and September
are presented in this study. Significance of variation has been tested,
and analysis of variance on samples collected from bulls during winter
and summer has been performed and the details are given on the
following tables. These bulls were normal, young, healthy animals
with good breeding records and were maintained under the same sys-

tem of management throughout the period.

Bull Age Breed
1. K. Bop 8 years Holstein
2. Silver 7 years ”
3. Rainbow 6 years ”
4. Barber 3 years ”
5. Maxim 6 years Guernsey
6. Master 6 years ”
7. Neptune 8 years ”
8. Mars 4 years ’ ’

Summary of factors studied is shown in Table Nos. 8, 9, and 10.
Significant differences occurred in initial motility between seasons only
while no significant difference occurred in semen volume and concentra-
tion with regard to season and breeds. These results suggest that
seasonal trends in the total motility rating show significant variation
while no significant variation occurred in volume and concentration.

:mpxmmm-h ”A. huh—x

73
Data shows that semen of lower motility was produced during the
months of July, August and September. NO appreciable differences in
semen quality occurred between the seasons and the breeds.
Initial Motility.

 

Two bulls produced spermatozoa with the lowest initial motility
during the summer months.

The variation of these two bulls, Rainbow and Barber, may be due
to individual differences. The average volume Of semen per ejaculate
did not show much variation with the exception of Mars, whose average
semen volume was least in the summer.

Concentration Of sperm.

 

NO difference in Sperm concentration in winter and summer and
no difference between Holsteins and Guernseys was noticed.

Temperature as a factor in seasonal variation.

 

Although temperature, humidity and light vary during the seasons
of the year and the conditions during the same months of different years
are often dissimilar, it is believed that temperature changes are at least
partially responsible for the differences in semen quality which were
Observed in total motility at the times when maximum temperatures were
observed. Results of the Analysis of Variance are shown on table No. 11.

74
Table 8. Average Volume of Semen Collected from Eight Holstein and

Guernsey Bulls During Winter and Summer.

 

 

 

 

 

 

 

 

 

 

 

 

Winter Summer
Guernseys
1 6.2 6 5
2 8.3 7 7
3 8.2 10.0
4 3.3 3 1
Total . 26.0 t 27.3 53.3
Holsteins
1 6.8 5.9
2 5.4 5.1
3 ' 7.7 6.7
4 - 7.3 5.9
Total 27.2 23.6 50.8
Grand Total 53.2 50.9 104.1
. 2 w
Interaction S.S - (Tfifill = 725.11 - 677.3 = 47.81

2 2
Between Seasons = (£322 + 1°%°_) - 677.3 = 0.33
53.3)2 50.8)2
Between Breeds = + LT- - 677.3 = 0.43
Sub Classes = 679.52 - 677.3 = 2.22
Interaction = 2.22 - (0.33 + .39) = 1.50

f S“t.§3.}-t“d (fiat-r “47.1"“. inf—“m

75

Analysis of Variance

Source of Variation D/ F iof Squares Mean Squares
Total 15 47.81
Between Seasons 1 0.33 0.33
Between Breeds 1 0.39 0.39
Breed 8: Season

Interaction 1 1.50 1.50
Within Sub Classes 12 44.59 3.72
Season F = $73; = 0.09 Non Significant
Breeds F = 337°2- = 0.1 Non Significant

1.50

0.4 Non Significant

BICBGS Ct Season F = m

76
Table 9. Average Initial Motility of Semen Collected from Eight Holstein
and Guernsey Bulls During Winter and Summer.

 

 

 

 

 

 

Winter Summer
Guernseys
1 ' 77 75
2 71 74
3 80 72
4 79 71
a I 1
Total 307 292 599
Holsteins
1 80 71
2 74 71
3 70 6 1
4 71 68
Total 295 271 566
Grand Total 602 563 1165

 

 

 

 

 

 

2
Interaction = 1165 = 84827

2
8.8. - E—‘i-te-al-z = 85181 - 84827 = 354 (8.3. = Summution of Squares)

 

 

2 2

Between Seasons = (602) g (563) = 84922 84922 - 84827 = 95
2 2

Between Breeds = Q99) 3 (566) = 84895 84895 - 84827 = 68

2 2 2 2
Sub Classes = L307) + (29°) 4 (29°; + Q11) = 84995
84995 - 84827 = 168

 

77

Analysis of Variance

Source of Variation D/ F fiof Squares Mean Squares
Total 15 354
Between Seasons 1 95 95*
Between Breeds 1 68 68
Breed & Season

Interaction 1 5 5 . .
Within Sub Classes 12 186 15.5 ( 1
Season F = 35% = 6.13 Significant
Breeds F = T6583 = 4.39 Non Significant

5 e...

 

Breeds & Season F = -5-.-5 = 0.32 Non Significant

J

a;

78
Table 10. Average Concentration of Sperm /mm3 from Semen Collected
from Eight Holstein and Guernsey Bulls During Winter and Summer.

 

 

 

 

 

 

 

 

 

 

 

Winter Summer
Guernseys
1 12 83 1328
2 7 66 1022
3 1050 900
4 151 1 1590
Total 4610 4840 9450
Holsteins
1 13 12 1333
2 1 100 1100
3 1053 1464
4 1330 686
, Total 4795 4583 9378
Grand Total 9405 9423 18828

 

 

2
Interaction s.s - T‘l’tal) =.= 23123468 - 22155849 = 967619
2 2
Between Seasons = 9405) + 9:23 - 22155849 = 17

2 2
Between Breeds = 945° + 9378 - 22155849 = 300
2 2 2 2
_ (4610) (4840) 4795 4583 _
Sub Classes .. 4 + 4 + 57—1 + L44 - 22155849 .. 12554

Interaction = 12554 - (17 + 300) = 12237

 

57.-.

.1 fl.

79

Analysis of Variance

 

 

Source of Variation D/ F inf Squares Mean Squares
Total 15 967619
Between Season 1 17 17
Between Breeds 1 300 300
Breeds and Season

Interaction 1 12237 12237
Sub Classes 12 955065 79589

.. 17 ._..
Season F - 79 589 0.0002 Non Significant
300 9

Interaction $3; = 0.15 Non Significant

80

Table 11. Summary of the Analysis Of Variance of the Semen

Characteristic of Eight Bulls.

 

Source of Variation

 

Factor
Analyzed
for
’ Variance

Between
season

Between

Breed and
Season

 

Volume in m1

Breeds

Interaction

 

ilnitial motility
percentages

 

1Concentration‘3 of
sperm /mm

 

 

 

 

 

- = Non Significant
*+ = Significant

 

‘Ukijrl ,

:-

81

Discussion

The cause of sterility and the factors which influence it are
numerous and the investigators continue in that line of research.

Those species which are strictly seasonal breeders are sterile
during the non-breeding season, whereas such species as man, cow,
horse, fowl and others, although classified as continuous breeders, tend
to produce at different intensities throughout the year.

Some critical studies Of the influence of season upon reproduc-

 

tion in dairy cattle have already been made, evidence is that such influence '
does exist. It has been reported at Louisiana Experimental Station (2)

that more services per conception were required during the summer
months than at any other period of the year.

Since the management and plane of nutrition of the bulls were
similar throughout the period, it is concluded that the changes observed
as in motility rating were the result of those factors which characterize
the season, temperature, and relative humidity.

Seasonal changes in semen volume and concentration were
not significant as Observed by Erb and others (12). Environmental
temperature may be of considerable importance; as it has been shown that
the direct application of heat to the testis, the insulation of the scrotum,
the replacement of the testis in the abdominal cavity or continued febrile
condition cause partial or complete inhibition of spermatogenesis.
However, the number of bulls used in this study was too small to draw

any definite conclusions. Results are merely an indication.

82

Summary

1. Analysis Of variance in characteristics of semen production
between seasons and breeds did not reveal highly significant differences.

2. Average semen volume did not show much difference except
in one bull in which it was least in July, August, and September.

3. Average initial motility was least in July, August, and
September.

4. Average concentration and volume of semen showed no

significant differences between seasons and breeds.

'- ml“:

IV. SUMMARY

The various advantages of artificial insemination over natural
mating, mentioned, lead one to be too enthusiastic about its use to
speed up the rate of live stock improvement. It provides means for
increased use of sires, for evaluation of young sires, for overcoming
difficulties due to size difference in males and females, for maintenance
of satisfactory conception rate, for control Of genital diseases and
economic service, particularly to small herds.

There has been considerable progress with regard to semen
collection and its evaluation, dilution, storage and transport. As to the
collection of the semen, artificial vagina technique remains outstanding.

The accurate evaluation of the semen samples at the time of
collection is important for Obtaining satisfactory conception rate.

Progress has been made in preparation and use Of diluters. In
this connection, Salisbury’s work is outstanding. Dilution rates varying
from 1:4 up to 1 part of semen to 100 parts Of yolk-citrate have had no
influence on the fertility of bull semen stored for a period of four days.

The highest average efficiency of conception was obtained with
the highest dilution rate of 1:100.

Refrigeration is the most effective method of preserving bull
semen. A temperature of 40° F. is considered ideal, as it is sufficiently
cool to keep the cells in dormant state without harming spermatozoa.

The Optimum rate of cooling for storage at 5° C. is found to be
5 degrees per 10 minutes.

83

84
The Optimum rate of cooling for storage at 10 C. is found to be
5 degrees per 2 hours.
Rapid cooling is found to be detrimental to the viability of Sperm-

atozoa.

. .‘m'j.’

1. Anders
1945

2. Ashev?
1946

BIBLIOGRAPHY

1. Anderson, James
1945 The Semen of Animals and Its Use for Artificial Insemina-
tion. Imperiaf Bureau of Animal Breeding and Genetics,
King’s Buildings, Edinburgh. 151 pp.

 

 

 

2. Asheville, N. C.
1946 Artificial Breeding of a Dairy Cattle as a Practical Proce-
dure for the General Practitioner. North American Veter-
inarian, 21 (1): 17-21.

 

3. Bartlett, W., and Tayler, George E.
1847 The Artificial InseminAtion 2f Farm Animals. Rutgers Uni-
versity - Press, New BrunswiE, N. J. 235 pp.

 

4. Beck, G. H., and Salisbury, G. W.
1943 The High Correlation of Any Tests with Viability Should
Indicate its Correlation with Semen Fertility. Journal of
Dairy Science, _2___6: 483-494.

 

5. Ceecil Branton, and Others
1947 Total Digestible Nutrients and Protein Levels for Dairy
Bulls Usedin Artificial Breeding. Journal of __Daigy Science,
_3_0 (12): 1003-1013.

 

6. Cole, C. L.
1940 Artificial Insemination of Dairy Cattle. Journal of ___Dairy
Science, _2___2 (2): 107- 113.

7. Davis, H. P., and Williams, N. K.
1938 Evaluating Bovine Semen. Proc. Amer. Soc.
1939 Anim. Prod. 31 and 32: 232, 2.42

~#*

 

 

8. Edward, J.
1947 Artificial Insemination of tmCattle; AReview. Empire Journal

_o__f Experimental Agcul ture, 15. 149- 159.

9. Engle,E ..T, and Others
1946 The Problem of Ferti 1ity_, Princeton University Press, N. J.
253*“ PP

10. Erb, R. E.
1941 Variation in Bull Semen and Its Relation to Fertility. J our-
na____l_ of Dairy Science, 24 (4): 321- 331.

 

 

85

86

11. Erb, R. E., and Others
1941 Storage of Dairy Bull Spermatozoa. Journal 9; Dairy Science,
_2_4 (7): 579-587. ’

12. Erb, R. E., and Others
1942 Seasonal Variations in Semen Quality of Dairy Bulls. J our-
_n_a1_ _o_f_ Dairy Scienge, 25 (9): 815-826.

 

13. Ernest, M., and Salisbury, G. W.
1947 Fertility Level in Artificial Breeding Associated with Sea-
son, Hours of Daylight and the Age of Cattle, Journal _o_f_ Dali-1
Science, 32 (11): 817-826.

14. Green, W. W., and Winters, L. M.
1946 Artificial Insemination of Farm Animals. University of
Minnesota, Agricultural Experiment Station, Bulletin 326.

15. Herman, H. A. and Madden, F. W.
1947 The Artificial Insemination of Dairy Cattle. Lucas Brothers,
Columbia, Missouri. 95m.

16. Gunsalues, L. C. , and Others
1941 Bacteriology of Bull Semen. Journal _o_f_ Dairy Science, 23:
911-919.

17. Henderson, J. A.
1946 Developments in Artificial Insemination of Dairy Cattle.
Cornell Veterinarian, §_6_ (2): 118-137.

 

18. Knodt, C. B., and Salisbury, G. W.
1946 The Effect of Sulphanilamide upon the Livabilityand Metabo-
lism of Bovine Spermatozoa. Journal of Dairy Science, _2_9:
285-291.

19. Knoop, C. E.
1941 A New Diluentfor Bovine Semen. Journal 91 Dairy Science,
24: 891. I

20. Lambert, W. V.
1941 Artificial Insemination in Live Stock Breeding. U. S. De-
partment of Agriculture, Circular 567.

21. Lardy, H. A., and Phillips, P. H.
1941 The Interrelation of Oxidative and Glycolytic Processes as
Sources of Energy for Bull Spermatozoa. American Journal
9_f_ Physiolgy, 133: 602-609.

 

 

87

22. Mercier, E., and Salisbury, G. W.
1947 Light VariationAssociated with Conception Rate in Artificial
Breeding. Journal _o_f Dairy Science, _3_9: 547.

 

23. Phillips, R. W., and Others
1943 Variation in Semen of Bulls, American Journal o__f V___e_t_e___ri-
naryResearch, 4: 115- 119. '

 

 

24. Raps, Greg, and Cannon, C. Y.
1947 The Influence of Management, Breed and Season upon the
pH of Bull Semen. _J_______ournal 9_f Dairy Science, 3__Q (12): 933-
938.

 

25. Reid, J. T., and Others
1947 The Relationship of Semen Production, Age, Plasma Cal-
cium, and Plasma Phosphorus of Dairy Bulls. Journal of
Dairy Science, 3___(_l_ (8): 546-547.

 

26. Rice, V. A.
1942 Bre and Improvement of Farm Animals. McGraw Hill
Boo Company, New York, 75? pp.

 

 

 

27. Salisbury, G. W., and Fuller, H. K.
1941 Preservation of Bovine Spermatozoa inYolk-Citrate Diluent
and Field Results from its Use. _J_______ournal of Dairy Science,
24: 205-210.

 

28. Salisbury, G. W.
1943 Storage, Packing and Shipping of Semen. American Veteri—
nary Medical Association Journal, 103: 1.6 7

 

 

29. Salisbury, G. W., and Others
1943 Effect of Dilution Rate on the Livability and the Fertility of
of BullSpermatozoa Used for Artificial Insemination. J our-
nal of Dairy Science, 26 (11): 1057- 1069.

 

30. Salisbury, G. W., and Others
1945 Livability and Glycolysis of Bovine Spermatozoa in Yolk-
Citrate, Incubated Eggs and Chick Embryo. Journal of ___Dairz
§________cience, 2___8_ (4): 270-276.

31. Salisbury, G. W., and Others
1945 Further Studies on the Effect of Dilution Rate on the Fertili-
ty of Bull Semen Used for Artificial Insemination. Journal
of Dairy Science, 28: 233-241.

 

32. Salisbury, G. W.
1946 Fertility of Bull Semen Diluted to 1: 100. Journal ofD Daigy
SLence, _2___9 (10): 295-297.

 

88

33. Swanson, E. W., Herman, H. A.
1944 Seasonal Variation in Semen Quality of Some Missouri
Dairy Bulls. Journal _o_f Dairy Science, 21 (4): 303-310.

 

33A. Trimber, H. G., and Davis, H. P.
1942 PresentDay Techniques of Artificial Insemination. Journal

of Dairy Science, 25: 671- 692.

 

34. Vandemark, N. L. , and Others.
1946 The Methylene Blue Test and Its Relation of Other Measures
of Quality in Bull Semen. Journal of Dairy Science, 2___8_ (2):
121- 128.

35. VanHooser, F. L.
1947 Artificial Insemination Problems. Rural New Yorker, April

1947.

36. Willett, E. L., and Salisbury, G. W.

1942 The Effect of Various Diluters, Cooling Rate, Temperatures
of Storage, and Some other Factors on the Livability of
Spermatozoain StoredSamples of Bull Semen. ' Cornell Uni-
versity - Memoir, 249.

 

37. Willett, E. L., and Others
1940 Preservation of Bovine, Spermatozoa in Yolk-Phosphate
Diluent and Field Results from its Use. Cornell Veterina-

rian 32: 25.

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