Sep 9, 1981 - loss of DNA (Ackerman and Sod-Moriah, 1968; Quinn et al.,. 1969). ...... College of Agriculture, Kyoto University, March, 1961. p. 97-104. (Anim.
EFFECT OF SEVERAL PROCESSING VARIABLES ON MOTILITY AND GLUTAMIC OXALACETIC TRANSAMINASE LEVELS FOR FROZEN GOAT SEMEN
A Thesis Presented to the Faculty of California State Polytechnic University, Pomona
In Partial Fulfillment of
the Requirements for the Degree Master of Science in Agriculture
by Erma Zimmerman Drobnis 1981
SIGNATURE PAGE THESIS:
EFFECT OF SEVERAL PROCESSING VARIABLES ON MOTILITY AND GLUTAMIC OXALACETIC TRANSAMINASE LEVELS FOR FROZEN GOAT SEMEN
AUTHOR:
Erma Zimmerman Drobnis
DATE SUBMITTED:
September 9, 1981
DEPARTMENT OF ANIMAL SCIENCE Dr. Edward A. Nelson Thesis committee chairman, Animal Science Dr. Edward A. Cogger Animal Science Dr. Daniel F. Stiffler Biological Sciences
ACKNOWLEDGMENT Special thanks to Dr. Michael P. Gross for making available to me the tools necessary to undertake this work and to meet many other challenges.
iii
ABSTRACT The effects of washing, diluent and glycerol level on motility and extracellular GOT levels of goat semen were investigated.
For semen processed
then incubated 6 hr at 37 C, motility was higher (p
for Tris-citric acid-yolk diluent than for
< .01)
TEST-yolk with both yolk diluents maintaining higher (p
motility than skim milk.
< .01)
In thawed samples,
motility was highest in the Tria-citric acid-yolk diluent (p (p
< .01)
< .05),
but the GOT levels were higher
than for the other diluents.
Skim milk
and TEST-yolk were not different for post-thaw motility or GOT.
Though motility was higher (p
< .05)
for
unwashed semen before freezing, incubation and freezing resulted in lower (p semen.
< .01)
motility for unwashed
Extracellular GOT was higher (p
unwashed semen after incubation.
< .01)
for
Interactions between
washing and diluent indicated that, for incubated semen, washing had less effect for skim milk than for yolk diluents.
However, for thawed semen,
washing was more beneficial with milk than with yolk diluents.
Motility was maintained best (p
< .01)
in
thawed samples containing 4 percent glycerol, while iv
7 percent and 10 percent levels better protected cell membranes and conserved intracellular GOT. Thaw motility was highest (p < .05) for Tris-citric acid-yolk at 4 percent glycerol, while skim milk had similar motility for 4, 7 and 10 percent glycerol. Overall, the best treatment combination included washing once, dilution with Tris-citric acid-yolk and a final glycerol level of 4 percent.
v
TABLE OF CONTENTS Page
Abstract
. .... . ....... ...... . ...... . . .. .......
. .
Introduction
.................................
Literature Review
. • .. . . . . . . . . • . .. .. . . . . . . . . .
Artificial Insemination for the Goat Washing
...
..................................
iv 1
3 3 12
Diluent
44
Cryoprotection
56
Evaluation
75
. .. . .. . .... ... ...... . . .. . . . . 100 Collection ............ . . ..................... . 100 Washing . ..................................... . 101 Diluents and Dilution ....................... 103
Materials and Methods
.
Cooling, Glycerolization and Freezing .. 107 Evaluation
.. . . ..... ... .. ... .. ... .......... . 109
Results and Discussion Diluent Washing Glycerol Conclusions Literature Appendix I
. .
.........................
.
...................................... ............................ ' ......... . ................................
111 111 118 130
..................................... 140 Cited ...................................... 142 Analyses of Variance . . .. . . . . . . . . . 178
Appendix II Complete Means and Standard Deviations
....................... vi
187
LIST OF TABLES Page
Table 1
Goat Semen Washing Solution ................. 102
2
Skim Milk Diluent Preparation ............
104
3
Tris-citric acid Buffer Preparation ......
105
4
TEST Buffer Preparation.
106
5
Effect of Three Diluents on Progressive Motility of Goat Semen ..........•.......
112
6
Effect of Three Diluents on Extracellular GOT Levels of Goat Semen .................
113
7
Comparison of Post-thaw Motility Evaluations Made With Heated and Unheated Microscope Stages ...............
116
8
Interaction between Diluent and Thawing Rate for Progressive Motility of Goat Semen..
9 10
..........................
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Effect of Washing on Progressive Motility
of Goat Semen. .. .. . . . . . . . . .. . . . .. . . . . .. . . . . . . .
Effect of Washing on GOT Levels of
118
Goat Semen . ............................ , . . . .
120
11
Interaction Between Washing and Diluent for Prefreeze Motility of Goat Semen .....
121
12
Interaction Between Washing and Diluent for Motility of Incubation-Stressed
Goat Spermatozoa ........... .. . . . . . . . . . . . . . . . . .
13
Interaction Between Washing and Diluent for GOT Release from Incubation-Stressed
124
Goat Spermatozoa. . . . . . . . . . .. .. .. .. . . . . .. .. . . .
125
14
Interaction Between Washing and Diluent for Post-thaw Motility of Goat Semen .....
126
15
Interaction Between Washing and Diluent for GOT Release from Frozen/Thawed Goat Spermatozoa. . . . . .. . . . .. . . . .. . . . . .. . . . . . . .. . .
vii
127
16
Effect of Storage Time on Post-thaw Motility and GOT Release of Goat
Spermatozoa. . ................. . . . . ... ... . . . . 131
17
Effect of Glycerol Level on Post-thaw Motility and GOT Levels of Goat Semen.
....
132
18 Interaction Between Glycerol Level and Diluent for Post-thaw Motility
of Goat Semen . ............................. ... 133
19
Interaction Between Glycerol Level and Washing for Post-thaw Motility of
Goat Semen . .................................. 136
20
Results for the Best Two Treatment Combinations.
. ................ ............. . 139
viii
LIST OF FIGURES Figure 1
Page
Lecithinases and the release of lvsolecithin, acid-soluble phosphate
and fatty acids ................................
2
3 4
Diagrammatic representation of a sperma tozoon..
. ............................... .
Comparison between the acrosome reaction
and acrosome degeneration . ............... .
14 83
92
Interaction between washing and diluent for motility and GOT release of thawed
goat spermatozoa. . ......................... , 129 5
Interaction between glycerol level and diluent for post-thaw motility of thawed
goat semen . .. , ............................ .. 135
6
Interaction between glycerol level and washing for post-thaw motility of thawed goat semen. ......... "
.
................... . 137
ix
INTRODUCTION Prior to the development of techniques for freezing goat semen, difficulty in storing chilled and frozen semen for artificial insemination was encountered. It was discovered that storage time was maximized by washing the semen to remove deleterious substances present in the seminal plasma.
One enzyme-like factor found
in the fluid fraction of buck semen acts on egg yolk phospholipids to form toxic products, and it has been suggested by some researchers that egg yolk may not be suitable for use in goat semen extenders.
Nonetheless,
excellent fertility results were reported using semen frozen in a Tris-citric acid-yolk diluent (Fougner, 1979} as compared to the extensively tested skim milk diluent (Corteel, 1976a) which is yolk-free.
In this study,
the value of washing goat semen was investigated and the effect of once washing and twice washing were compared. Also, the two diluents most extensively used for goat semen freezing were compared and an additional buffer was investigated.
It has been shown that the zwitter-
ionic extender TEST-yolk is superior to conventional buffers for bull, boar and ram semen and there has been some indication that a TEST-yolk diluent may be suitable for goat semen (Graham et al., 1972}. 1
2
Since optimal glycerol level for semen freezing depends on diluent employed, this was included as a factor, The effect of each treatment was evaluated using percent progressive motility as a measure of middle piece condition and cell metabolism and extracellular glutamic oxalacetic transaminase (GOT) level as a measure of spermatozoal membrane permeability and damage (Graham and Pace, 1967).
LITERATURE REVIEW Artificial Insemination for Goats History.
Artificial Insemination techniques were used
for goats soon after their development for commercial use in dairy cattle (Lunca, 1964).
The formulation of
an egg yolk extender for bovine semen, which allowed for chilling to 5 C without loss of fertility (Phillips, 1939), provided the necessary means for decreasing the spermatozoal metabolic rate and thus extending its lifespan (Mann, 1964).
By using diluted and chilled semen,
it was then possible to maintain a smaller number of improved males at a central location, for use by the producers in a community.
Unfortunately, while refriger-
ated bull semen maintains its fertility for up to a week (Salisbury et al., 1943), the fertility of chilled goat semen is greatly reduced after the first day of storage (Baudet et al., 1954;
Setinski et al., 1956; Blokhuis,
1957; Tewari et al., 1968; Sahni and Roy, 1969; Grüttemeir, 1969; John and Raja, 1973; Corteel, 1973).
Although good
conception rates have been obtained with chilled goat semen used soon after collection (Rosenberger, 1941; Wagner, 1949; Dauzier, 1956; Blokhuis, 1957, 1959; Singh, 1961; Blokhuis, 1962; Lunca, 1964; Patel, 1967; 3
4
Kupferschmied, 1969; Vlachos, 1975;
Schindler et al.,
1979), longer storage periods are desirable in order to maximize the advantages of artificial insemination. Outstanding
progeny-tested bucks may be located at a
distance (Kupferschmied, 1972) or, due to their short breeding life may have died before their genetic potential was realized (Corteel, 1973).
The frozen preservation
of goat spermatozoa has overcome these difficulties. Frozen storage of goat semen has proved to be more difficult than for bull semen.
Originally, the
processing methods employed were borrowed from those which were successful for the bovine species, but poor initial results were obtained (Liess and Ostrowski, 1960; Tierzuchter, 1960; Weissflog, 1961; Lyngset 1965; Goffaux and Corteel, 1967).
et a al.,
These were due in
part to an enzyme found in goat seminal plasma which decomposes egg yolk lecithins, producing spermicidal lysolecithin (Roy, 1957; Iritani and Nishikawa, 1961; Fossum et al., 1965).
In addition, González Stagnaro
(1975) has pointed out that these reports have in common that the semen was frozen to -79 C (dry ice) while recent successful techniques utilize liquid nitrogen and freeze to -196 C. As techniques have been developed specifically for freezing goat semen, its fertility has improved giving conception rates of 50-85 percent, after insemination
5
during one estrus period (Waide and Niwa, 1961; Fraser, 1962; Herman, 1963; Bonfert, 1964, 1971; Vlachos and Karagiannida, 1968; Andersen, 1959; Corteel and Surcin, 1969; Hahn, 1972; Kupferschmied, 1969; Samoulidis and Hahn, 1972; Reskovatskow et al., 1974; Tsakalov et al., 1974;
González
Stagnaro, 1976).
The semen used in many
of these insemination trials was diluted with egg yolkcontaining media, and none of these researchers report washing the spermatozoa free of seminal plasma.
It is
probable that semen showing egg yolk lysis and poor sperm motility were discarded and not used in the insemination trials. The semen processing techniques developed for goats are similar to those used for bull and ram semen with minor modifications.
Semen is collected using an artifi-
cial vagina (Eaton and Simmons, 1952; Austin --. et al., 1968)
because the alternative technique, electroejaculation,
results in semen with large differences in pH, fructose, citric acid and total protein content, relative to naturally ejaculated goat semen (Iritani and Nishikawa, 1964b).
Following collection, semen is maintained at
30 C during initial evaluation and dilution; this temperature reduction having little or no negative effect on the cell (Wagner, 1949). is
cooled
After dilution, the semen
slowly to 5 C and a second volume of glycerol-
containing diluent is added over a period of time
6
(González Stagnaro, 1975).
After this glycerolization,
some investigators allow a further period of time, termed equilibration, prior to packaging.
Equilibration
is commonly 30 min to 3 hr (Corteel, 1981).
Prior to
freezing, semen is packaged, usually in .5 ml or .25 ml plastic semen straws (Andersen, 1969).
Freezing is
accomplished in liquid nitrogen vapor, requiring 2 to 5 min to cool to -196 C (Waide et al., 1977). Problems.
There are a number of studies involving
artificial insemination in which variable results were obtained with frozen goat semen.
Corteel (1981) pointed
out that most reports of high fertility have not been followed by reports of further success.
This is due
to some inherent problems for this species which add to variability, and make interpretation of results and development of techniques difficult.
This includes
difficulties in insemination of the doe as well as poor results with frozen semen. Among the greatest problems facing both goat and sheep artificial insemination programs is the current inefficiency involved.
The number of motile prefreeze
spermatozoa required for optimal fertility with frozen bull semen is some 10 x 106 /dose (Stewart and Bennett, 1968), while 350 x 106 motile cells/dose are required in sheep (Salamon, 1977) and 100 to 150 x 10 6 /dose yields
7 optimal conception rates in goats (Corteel et al., 1972; Corteel and De Montigny, 1975; Corteel, 1976a; Fougner, 1974).
This means that while the average bull
ejaculate produces some 200 insemination doses, a goat ejaculate yields only about 20 doses.
Also, since the
freezability of goat semen is variable, and researchers often evaluate samples just prior to insemination, a sizeable percentage of the frozen semen may be discarded. Gonzáles Stagnaro (1975) reported discarding over half of the semen evaluated just before insemination. Further reduction of artificial insemination efficiency results from double insemination.
In order
to achieve adequate conception rates, most workers inseminate each doe twice during estrus using a separate dose for each insemination (Vlachos and Tsakalov, 1964; Corteel
et a al.,
Muther, 1977).
1972; Corteel, 1976b; Kupferschmied and Since ovulation occurs near the end of
estrus, and fertilization depends on the presence of a substantial population of viable spermatozoa at this time, the time of insemination is critical.
Sperm
viability is also critical and may be the most limiting factor. Detection of estrus is also a problem in goats. Optimal conception rates are usually obtained following insemination between 12 and 30 hr after the onset of estrus (Bonfert and
Thier,
1963; Dauzier, 1966; Vlachos
8
and Karagiannida, 1968).
Failure to inseminate during
this period has resulted in lower fertility.
Corteel
(1976b) reported that for 1247 does inseminated with frozen semen, conception rates were 69 percent when estrus was detected by a vasectomized buck, and only 58 percent when detection was by the owner of the doe. Kupferschmied (1972) had similar problems with owner estrus detection.
Bonfert and Thier (1963) found that
insemination of does showing strong estrus symptoms, including an open cervix, resulted in 78.8 percent nonreturn to estrus, while only 59.7 percent non-return rate was obtained for those showing little symptoms of estrus, and in which the cervix was closed. To overcome the problems involved in estrus detection and daily insemination, synchronization of estrus involving hormone therapy is often practiced in conjunction with artificial insemination.
In general, these synchro-
nization methods lower the conception rate somewhat (Corteel, 1976a; Montigny, 1977) and make the results· of different trials difficult to compare. The insemination technique itself is much more complicated in the doe than in the cow.
The cervix is
difficult to penetrate with the insemination pipette and conception rates are dependent on site of semen deposition. Highest conception rates are obtained for uterine deposition, lowest for vaginal and intermediate of midcervical
9
(Setinsky 1976).
et a al.,
1956; Corteel, 1975, 1976b, Fougner,
Insemination skill is essential for this species,
and low conception rates have been attributed to unskilled inseminators (Bonfert, 1964; Kupferschmied, 1972), while inseminator differences in conception rate of 10 to 25 percent have been reported (Bonfert and Thier, 1963; Vlachos and Karagiannida, 1968; Vlachos, 1975).
Fougner
(1976) found that after three years of insemination trials, trained inseminators achieved intrauterine deposition for 85 percent of 916 does, resulting in 71 percent conception rate to first insemination with frozen semen.
A final complication brought about by cervical
anatomy was first observed by Chang (1946) who reported that the cervix of the rabbit could accommodate only a limited volume, and in consequence, the number of motile sperm required for good fertility, must be concentrated into a small volume.
In the goat, several workers have
observed that the cervix holds .1 to .2 ml, and additional semen deposited in the cervix will drip back out into the vagina (Dauzier, 1966; Andersen, 1969; Surcin, 1971). The smaller .25 ml straws are used by most workers, however, in the United States, goat semen is packaged in the larger .5 ml straws which are in use in the dairy cattle industry. All of the above factors have increased the variability of results with frozen goat semen, but a problem which is more closely related to actual semen technology
10
is the poor storage time attained with frozen goat semen. While bull semen retains its fertility for twelve years with little reduction (Mixner and Wiggin, 1964; Mixner, 1968), and ram semen maintains its full post-thaw revival for three to five years (Salamon, 1972; Salamon and Visser, 1974), the post-thaw motility and fertility of goat semen is seriously reduced within one year of storage at -196 C (Corteel, 1975; Waide et al., 1977) or two years (Waide and Niwa, 1961).
Corteel (1975)
claims that this rapid decrease in semen quality is a function of the toxicity of goat seminal plasma.
For
semen washed prior to freezing, there was no difference in fertility for storage of one to fourteen months (Corteel, 1976b).
Storage time differences explain
some of the variability observed in results of insemination trials with frozen goat semen. An additional problem with goat semen quality is
its seasonality.
The goat is a seasonal breeder with
the doe showing estrus only during a limited breeding season except in the tropics where seasonal variation is slight (Phillips et al., 1943).
In the male goat
also there is a seasonal trend in semen characteristics such as volume and concentration (Eaton and Simmons, 1952; Shukla and Bhattacharya, 1952; Yao and Eaton, 1954; Dziuk et al., 1954; Sharma et al., 1957), including variations in semen chemistry (Iritani et al., 1964;
11 Iritani and Nishikawa, 1964a; Mohri et al., 1970) and semen quality (Corteel, 1973; Kang and Chung, 1976, Corteel, 1977; Muhuyi et al., 1982).
Poorer fertility
has been reported for goat semen frozen out of season for use during the breeding season (Kupferschmied, 1972; Cortee1, 1976a).
However, washing the goat semen prior
to freezing may minimize these variations in post-thaw semen quality (Corteel et a1., 1980a, 1981). Solutions.
Modifications of the techniques employed in
processing bovine semen have somewhat improved the prospects for more extensive use of artificial insemination for goats.
This includes washing the spermatozoa free
of seminal plasma prior to dilution and chilling in a yolk diluent (Nishikawa et al., 1961).
Even for bull
semen, storage time is generally poorer in milk diluents unless yolk is included (Salisbury, 1978).
Washing has
improved the quality of frozen semen by increasing its storage time and reducing seasonal variation (Corteel, 1981).
There is also some indication that the number of
sperm required to obtain good fertility may be reduced by washing.
Fougner (1976) reported 48 percent kidding rate using only S x 106 washed frozen spermatozoa per dose, in contrast to 100 x 10 6 normally used for goats.
In a previous report (Fougner, 1974) 150 x 106 sperm per dose gave no higher fertility than 50 x 106 /dose. Washing has also allowed for development of a yolk
12 diluter for goat semen (Fougner, 1974), which may provide better protection to spermatozoa during freezing and thawing than does skim milk. In addition to washing, optimal semen processing techniques for goat semen differ from those for other species in their time requirements.
Dilution with
washing solution must occur within two minutes after ejaculation (Corteel, private communication).
Dilution,
cooling and glycerolization techniques have been developed to allow for rapid semen freezing (Fraser, 1962; González Stagnaro, 1976).
Goat semen can be frozen 2 to 3 hr
after collection.
Washing Goat Semen.
The egg yolk-coagulating property of goat
semen was first investigated following problems with chilled storage of goat semen.
--
Roy et al.
(1959)
determined that Cowper's gland was the source of the factor responsible.
They further characterized the
factor as calcium dependent and as being inactivated by heating for 2 min at 100 C.
Further investigation
by Iritani et al. (1961) revealed that egg yolk coagulation by goat seminal plasma was accompanied by a drop in pH which was not explained by the lactic acid production of spermatozoal glycolysis.
These researchers did
not find egg yolk coagulation with ram, bull or boar
13
semen.
Iritani and Nishikawa (1961) studied the optimum
conditions of temperature, substrate concentration and pH for yolk coagulation and observed that the factor responsible resembles an enzyme in its activity. Studies with fractionated yolk disclosed that this factor acts to cleave the ester linkage of acyl groups in yolk phospholipids, releasing palmitic and stearic acids as well as unsaturated fatty acids and lysolecithin (Iritani and Nishikawa, 1963b,c).
These activities are
similar to those of phospholipase A (figure 1) which is found in snake venom, bacteria and some animal tissues (Metzler, 1977). Phospholipase A is only one of several lecithinases which have been suggested as participants in egg yolk coagulation.
Two such phosphodiesterases,are phospho-
lipase C and D (figure 1) which occur in bacteria and plant tissues respectively.
These cleave the two sides
of the lecithin phosphodiester.
A third lecithinase,
phospholipase B, has activity very similar to that of phospholipase A except it removes the second fatty acid from lysolecithin yielding glyceryl phosphorylcholine and acid-soluble phosphate. Aamdal et al. (1965) proposed that the substance found in goat semen, and to a lesser extent in boar semen, was not phospholipase A, but lysolecithin itself which Hartree and Mann (1960a,b) found to inhibit sperm motility
14
8 GLYCERLPHOSPHORYL CHOLINE
(Acid soluble Phosphate)
FATTY ACID
P PHOSPHATIDYL CHOLINE (LECITHIN)
N
+
+ CHOLINE
Figure 1.
Lecithinases and the release of lysolecithin, acid soluble phosphate and fatty acids.
15 and metabolism.
The presence of lysolecithin alone,
however, would not explain the drop in pH which accompanies yolk coagulation.
The action of phospholipase
A on yolk phospholipid to release lysolecithin and fatty acids would inhibit the spermatozoa and also increase the acidity.
The production of lysolecithin and the
drop in pH has been reproduced by treating semen with snake venom which is one of the richest sources of phospholipase A
(Dawson et
al., 1957; Hartree and Mann,
1960a,b). Mann (1964) later suggested that the enzyme found in goat semen was similar to the calcium-activated phospholipase C found in bacteria. with the report by Roy
This explanation is consistent
al. (1959)
et al
coagulation was calcium dependent.
that egg yolk However, phospholipase
C does not produce lysolecithin which is released during yolk coagulation by goat seminal fluids, therefore more than one enzyme may be involved. This conclusion was also reached by Iritani and Nishikawa (1963a) who found that goat seminal plasma, like phospholipase C, caused dephosphorylation of the yolk phospholipids and release of acid-soluble phosphorus. This phospholipase C-like activity, however, was not significant in Cowper's gland extract.
In addition,
the increase in liberated fatty acids during coagulation was greater with whole seminal plasma than with Cowper's gland extract, suggesting that phospholipase B activity
16 is present.
Apparently not all of the egg yolk lysis
activity originates in Cowper's gland and more than one enzyme may be involved. does
The phospholipase A-like factor
originate in Cowper's gland, and this factor is
responsible for spermicidal lysolecithin formation. This is the most deleterious of the lecithinase activities relative to spermatozoa. The concept that phospholipase A is present in semen is not new.
Sea urchin spermatozoa metabolize
intracellular phospholipids and liberate lysolecithin and acid-soluble glycerylphosphoylcholine (GPC) compounds (Maggio and Monroy, 1955).
Mann (1964) proposed
that the reason that mammalian epididymal secretions, hence semen, were so high in GPC was because of a lecithin breakdown by phospholipase A and B in the epididymis. Mammalian sperm have also been shown to utilize the intracellular phospholipid phosphotidylcholine or plasmologen. Phospholipase A has also been shown to act on plasmologen to produce lysoplasmologen which is just as inhibitory to spermatozoa as lysolecithin (Mann, 1964}. Another indication of phospholipase A activity in semen production is the presence of the prostaglandin PGF2a in seminal fluid (Bygdeman and Holmberg, 1966}. PGFza causes uterine contraction believed to be important to sperm transport in the female reproductive tract (Edqvist et al., 1975).
The first step in prostaglandin
synthesis is thought to be the breakdown of the phospholipids
17 phosphatidylinositol or phosphatidylcholine (lecithin) by phospholipase A releasing arachidonic acid, the 20-carbon polyenoic acid precursor (Eliasson, 1958, Metzler, 1977).
Therefore, this type of enzyme may be
present in the male reproductive tract.
Perhaps the
function of phospholipase A-like enzyme in goat seminal fluid is to cause the production of protaglandin precursors in the female reproductive tract since the uterine tissue is capable of PGF 2a synthesis. The washing of goat semen has proven to be beneficial even for extending media not containing egg yolk. It has been consistently demonstrated that goat seminal plasma exerts a debilitating effect upon spermatozoa.
Fougner (1976) stated that Cowper's gland, which
is the source of the egg yolk coagulating factor, might also contribute other factors which damage the sperm even in the absence of yolk.
However, Cowperectomy
does not completely remove the ill-effect of seminal fluids (Corteel, 1977).
This evidence indicates that
a toxic substance, not contributed by Cowper's gland, is present in goat seminal plasma.
Since this effect
is independent of diluent used, it probably acts directly on the spermatozoa rather than on the extender components. Washing, thus removing the seminal plasma, has many beneficial effects for goat semen.
Oxygen uptake
by spermatozoa is stimulated (Fukuhara and Nishikawa, 1973a), a better percent motility and a greater sperm
18 velocity have been reported following thawing and 2 hr post-thaw incubation at 37 C (Corteel, 1974: Vander Westhuysen, 1978). loss
Also, it was determined that cell
during freezing was positively correlated to the
initial volume of the ejaculate (r = .9) and negatively to the cell concentration (r = -.9), therefore, freezability was improved for ejaculates with less seminal plasma (Corteel, 1974). Additional evidence of the toxic nature of goat seminal plasma is the increased storage life of washed spermatozoa at -196 C.
As discussed in the preceeding
section, goat semen has a very poor storage life compared with that of the bull and ram, and deterioration begins soon after freezing.
Corteel (1975) has shown that this
is related to the seminal plasma.
In his study, washed
goat semen evaluated from 3 to 90 days after freezing did not change in post-thaw motility, while unwashed semen lost 16.6 percent of its original motility·.
After
91-181 days, unwashed samples had lost 22 percent of their original post-thaw motility of 43 percent compared to washed samples which decreased only 1.3 percent.
In
an insemination trial, 873 does were inseminated with washed semen stored 1 to 14 months with no difference in fertilizing ability due to storage time (Corteel, 1976c). These results emphasize the value of washing goat semen prior to freezing.
19 The toxicity of goat seminal plasma is apparently a species specific problem and generally washing is not beneficial for the spermatozoa of other species. Corteel
(1976a) has suggested that toxic factors may
be present for other species as well, but the influence is more pronounced for the goat for which high sperm concentrations are required to obtain good fertility. The depressive effect of buck seminal plasma may be negligible at the dilutions used when processing bull semen. There is a tremendous quantity of literature in which the washing of mammalian or fowl spermatozoa is discussed.
In most cases the authors were studying
the effect of various treatments on the isolated spermatozoa, or the spermatozoal metabolic processes were under investigation.
In this type of work, the seminal
plasma is a nuisance variable and studies were undertaken to wash the cells free of seminal fluids with minimal cellular damage.
Although these reports are
not geared to developing washing techniques as a routine semen processing step, they include results which may add valuable information to the refinement of goat semen processing methods.
20 The Dilution Effect.
The in depth study of washing
spermatozoa was first undertaken in conjunction with research into the dilution effect.
This phenomenon
is characterized by a change in spermatozoal activity with varying dilution rates when all other factors are held constant. The term "dilution effect" was first used to describe the intense activity of sea urchin spermatozoa when they are diluted with sea water (Gray, 1928).
This
term when applied to mammalian or fowl spermatozoa usually describes the depression observed in dilute sperm suspensions.
However, Rothschild (1950) explained that the
reaction of sea urchin spermatozoa to dilution is related to that of higher species, involving a stimulation of motility and metabolism by mild dilution but depression upon further dilution. The first published report of the adverse effect of dilution was an index developed by Milovanov (1934) using the dilution with physiological saline required to immobilize spermatozoa as an assay of semen quality. Milovanov proposed that the toxicity of chloride was responsible for the loss of motility (Mann, 1964). Winchester and McKenzie (1941) concentrated samples of ram and boar semen then resuspended them to varying concentrations with either seminal plasma or a phosphate buffer diluent.
They found that oxygen consumption was greater as sperm concentration decreased to 1 x 10 9 /ml,
21 but results were variable for higher dilution.
This
represents a dilution of approximately 1:3 for ram semen but is more concentrated than whole boar semen. In the classic dilution effect paper, Salisbury
al. (1943)
et a
published evidence that the number of
days bull semen could be stored was greatly reduced by increasing the dilution rate.
Since this was in
citrate diluent, chloride toxicity was not involved. Chang (1946) reported that dilution reduced the motility and in vivo fertilizing rate of rabbit spermatozoa. In this study, the dilution rate was statistically confounded with the total number of spermatozoa inseminated, i.e., as the dilution was increased, the number of germ cells per insemination dose was decreased. It was also proposed that a decrease in sperm fertility may occur with increased dilution (Chang, 1946).
In a
subsequent study, a correlation was determined (r
=
.43)
between motility and fertility of diluted samples by Cheng and Casids (1948) which also reflects a concomitant decrease of motility and fertility with dilution.
The
damage caused by dilution was irreversible, and when immobilized bovine spermatozoa were reconcentrated, no return of forward motility was observed
(Cheng et
al.,
1949). Further studies indicate that optimal dilution rates exist for other species and dilution above these limits results in damage to spermatozoa of the bull
22
(Salisbury et al., 1945; Bishop and Salisbury, 1955a,b), boar (Pursel et al., 1973; Paquignon and Courot, 1975), stallion (Pickett et al., 1975), ram (Sahni and Roy, 1969; Visser, 1974) and man (White, 1954).
The dilution
effect for goat semen has not been shown to be as great as in other species (Sahni and Roy, 1969) and no dilution effect for the buck was found by Fukuhara and Nishikawa (1973a), with dilution from 1:10 to 1:40 as measured by oxygen uptake.
These findings are probably the result
of diluting the toxic seminal plasma of goat semen. The dilution effect is not simply the toxic effect of diluent components upon the spermatozoa.
Depression
occurs when dilute suspensions are produced by dilution with any medium including Baker's solution (Emmens and Swyer, 1948), calcium-free Krebs-Ringer- Phosphate (Cheng and Casida, 1948), sodium citrate (Cheng et al., 1949; Blackshaw, 1953) and phosphate (Bishop and Salisbury, 1955b).
The dilution effect was even produced by dilution
with seminal plasma for the bull (Rottensten et al., 1960) and man (Freund and Wiederman, 1966).
Since the
role of the seminal plasma during dilution was questionable, washing has been used to study sperm suspensions with and without seminal fluids. Washing Damage.
Emmens and Swyer (1948) reported no
effect of washing rabbit spermatozoa twice, even though this removed most of the seminal plasma and a sample
23
diluted to a similar percent seminal plasma showed severely depressed motility.
They suggested that the
damage was caused by leakage of intracellular substances, and cell concentration was more important than inclusion of seminal plasma in diluent.
This was true for bull
semen as well; a marked depressive effect of dilution on motility and sperm metabolism was observed even when all samples were washed twice prior to dilution (Lodge et al., 1963a). There have been numerous reports supporting the thesis that the damage caused by washing does not result from centrifugation alone.
White (1953c) found that
four washing (300 g, 10 min) seriously reduced motility of bull or ram spermatozoa after storage for 5 hr at 37 C, but centrifugation/resuspension four times had no effect.
Further, total oxygen uptake by ram and bull
spermatozoa, incubated 3 hr at 37 C, was not changed by four washings or centrifugations, but lactic acid production was sharply reduced by washing and unaffected by centrifugation alone (White, 1953c).
For six washings
of ram sperm (350 g, 15 min), metabolic activity during 6 hr dialysis at 30 C was decreased by each successive washing but was not affected by six centrifugations (Dott and Walton, 1960).
Similar results for motility
were reported by Blackshaw (1953).
Fertility of washed
rabbit sperm was depressed by washing but not by centrifugation (Brackett, 1969).
Ultrastructrual evaluation
24 using transmission electron microscopy showed that centrifugation resulted in none of the damaged aerosomes and middle pieces that were observed in washed (100 g, 10 min) samples (Jones and Holt, 1974).
Washing
bull spermatozoa once before freezing (270 g, 3 min) caused lower conception rates than centrifugation alone; the best fertility was obtained with uncentrifuged controls (Picket et al., 1975). Centrifugation of semen is desirable in some species to concentrate the required number of motile spermatozoa into the desired inseminate volume or, in the case of swine, to reduce the frozen storage space required. For boar semen centrifugation and resuspension along with one washing yielded samples with poorer motility than uncentrifuged controls (Salamon, 1973).
Although
centrifugation was not detrimental to prefreeze or post-thaw motility of stallion spermatozoa, one washing decreased motility
(Picket et al.,
1975).
Fertility of
frozen ram semen was not reduced by centrifugation (Visser and Salamon, 1974). Also in favor of the hypothesis that the damage from washing semen is not simply mechanical, is the fact that when centrifugal force has been included as a treatment, it has rarely caused measurable differences. Centrifugal forces of 650, 1000, and 1700 g resulted in increased recovery of ram sperm numbers, but had no effect on the percent unstained (live) cells {O'Shea, 1969).
25 Forces of 370 g or 829 g
were not detrimental to
prefreeze or post-thaw motility of stallion semen (Pickett et al., 1975).
A force of 12,000 g was
required to cause significant damage to the membranes of bull spermatozoa as measured by GOT release, whereas centrifugation for 10 min
at 1470 and 3900 g caused
no measurable damage; these forces greatly exceed those normally used for sperm washing (Pace and Graham, 1970). Corteel (1981) has suggested that for washing goat semen centrifugal forces between 500 and 600 g are essential to obtain separation and maintain semen quality.
Lesser
centrifugal forces resulted in incomplete sedimentation and the most highly motile cells remain in the supernatant . HHowever, cooling prior to washing allowed for partial immobilization hence allowing more gentle centrifugation for complete separation. Researchers have also studied the effect of multiple washings.
For ram semen, one washing (1:3, 300 g, 10 min)
had little effect but a second washing depressed motility, oxygen consumption and lactic acid production.
The same
study showed no reduction of motility for rabbit or bull semen and no lower oxygen uptake by bull sperm when washed twice (White, 1953a).
Dott and Walton (1960) found a
negative linear relationship between times washed and sperm activity (impedence change frequency) for up to six washings of ram spermatozoa.
Additionally these
investigators found a positive correlation between dilution
26 rate of washing solution and ram sperm activity. Although the motility of ram spermatozoa was not changed by additional washings, electron micrographs indicated that two washings increased the number of cells with damage to the middle piece, plasma membrane and acrosome, and four washings further increased the incidence of broken plasma membranes (Jones and Holt, 1974). Washing Solution.
Although the chloride in Milovanov's
diluting medium was not in itself responsible for the dilution effect, the effects of the diluent components are clearly confounded with the effect of dilution and/ or washing.
This fact has made it difficult to compare
the results obtained by different workers. Calcium ion (Ca++) was excluded from Baker's phosphate solution (Baker, 1931) because it was "quite unnecessary" to spermatozoa.
Calcium has since been
demonstrated to be detrimental to sperm when included in the storage or extending medium.
Even low calcium
levels cause decreased respiration and motility as well as agglutination of the cells (Lardy and Phillips, 1943; Lardy et al., 1945; Wallace and Wales, 1964; Mann, 1964). Citrate, which binds calcium, improves the motility of bovine spermatozoa (Lardy and Phillips, 1943).
Manganese
(Mn++) and potassium (K+) were required for optimal sperm
activity (Lardy and Phillips, 1943) .
27
Phosphate, which has been the most widely used buffer for semen research, has subsequently been reported to strongly inhibit oxygen consumption by bovine spermatozoa (Bishop and Salisbury, 1955b).
These researchers
claimed that many eariler results indicating depressed oxygen uptake by washed spermatozoa were actually due to the inhibitory effect of phosphate in the washing and extension media.
Furthermore, phosphate diluent
has been shown to inhibit motility, depress oxygen uptake, increase fructose utilization and greatly increase lactic acid accumulation by bovine spermatozoa {Salisbury and Nakabayashi, 1957); indicating inability of sperm to oxidize lactic acid produced by glycolysis (Lodge et al., 1963b).
However, some phosphate is required and twice
washed bovine sperm were less able to utilize fructose unless some phosphate was included in the incubation medium (Lodge et al., 1963b).
Ram semen was also stimu-
lated by addition of phosphate to a saline diluent (Wallace and Wales, 1964). Dott and White {1964) reported that bicarbonate is apparently responsible for head-to-head agglutination of motile ram sperm previously observed after washing in calcium-free bicarbonate diluent {Dott and Walton, 1960).
This agglutination could be reversed by agitation
of the sperm suspension and did not occur if a calciumfree Krebs-Ringer-Phosphate solution was used.
--
However,
28 agglutination of spermatozoa has been reported to result from washing ram semen in phosphate solutions {Blackshaw, 1953) and from washing human semen with a tris buffer (Eliassen, 1971).
Jones and Holt (1974)
said the increased percentage of eosinophilic (dead) ram sperm cells and the tendency to agglutinate suggest increased membrane permeability associated with the removal of surface antiagglutins. Oxygen has also been considered as a contributor to dilution and washing damage.
It was thought that,
in dilute suspensions, spermatozoa are damaged by increased oxygen resulting in oxidation of the cell membranes or of intracellular reserves (Chang, 1946; Salisbury, 1946).
However, Cheng et al. (1949) found
that even when dilution was performed under a vacuum, bull spermatozoa were still immobilized by dilution. Increased oxygen near spermatozoa during the actual washing procedure has been suggested as the cause of the subsequent damage observed (Brackett, 1969). Shannon (1965a) proposed that the deleterious effect of dilution was due to increased oxygen supply resulting in peroxide formation of which spermatozoa are capable (Tosic and Walton, 1950).
Shannon found that saturation
of diluents with nitrogen removed the effect of dilution on livability of bovine sperm and oxygenation reduced the life span of concentrated samples.
29
Leakage of Intracellular Substances.
Most recent
studies involving the effects of washing and dilution have indicated leakage of or
intracellular
substances
removal of extracellular substances as causing damage
to spermatozoa.
In the case of readily diffusible sub-
stances, removal from the diluting medium results in leaching of intracellular reserves.
The most obvious
example is seminal fructose which is utilized by spermatozoa to promote motility either anaerobically or aerobically {Mann, 1946).
Washed cells will "starve"
if no substrate is replaced and this was a factor in early washing studies where samples were incubated in media without glucose. The first investigation of the possibility that washing caused spermatozoa to lose vital material involved rabbit spermatozoa (Emmens and Swyer, 1948). After two washings, resuspension in the first supernatant resulted in no better livability at 20 C than resuspension in fresh diluent.
In fact, there was no
effect of washing on motility, though the concentration of seminal plasma in washed samples was similar to that of a dilution to 0.4 x 106 cells/ml. In such dilute suspensions serious immobilization occurred.
Six washings
immobilized cells unless some seminal plasma was restored. These scientists concluded that the material lost is intracellular requiring rigorous washing or dilution for removal.
It was also noted that the necessary cell
30 constituents were reabsorbed by spermatozoa if provided in the suspension medium. Another approach to demonstrate the leakage of substances from spermatozoa was to dilute semen with '•
seminal plasma or diluent which had previously contained sperm stressed by incubation, cold shock or heating. Thus the leached substances would be present at higher levels.
In general,
while this reduced the effect of
dilution and washing, depression was still produced by further dilution with this medium (Emmens and Swyer, 1948; Blackshaw, 1953; Rottensten et al., 1960). Washing has been demonstrated to result in the loss of intracellular cytochrome C (Mann, 1951), plasmalogen (Hartree and Mann, 1959), protein,lipoprotein (Mann, 1964), and hyaluronidase (Emmens and Swyer, 1948). Washing also affects the cell metabolism indicating an upset in the balance of metabolic coenzymes, enzymes, and activators.
White (1953c) reported that oxygen
uptake by ram and bull semen was not influenced by four washings while lactic acid production was sharply reduced. He concluded that washing inhibits the glycolytic process rather than the oxidative process. Smith
et a al.
This is supported by
(1957) who found no effect of four washings
on the aldolase and malic dehydrogenase activity of bull spermatozoa whereas glyceraldehyde-3-phosphate dehydrogenase activity was depressed.
It was suggested
that the latter be added to washed semen.
The activity
of succinic dehydrogenase was strongly stimulated by
___________
31 washing due to the removal of the inhibitor diphosphopyridinenucleotide (DPN) which is present in seminal plasma.
Jones and Holt (1974) chose two enzymes
to, study the effect of washing on membrane-bound and soluble enzymes.
Washing did not remove succinic
dehydrogenase bound to the mitochondrial membrane from the middle piece of ram spermatozoa but
glucose-6~phosphate
dehydrogenase was removed easily by dilutions as low as 1:10. The most extensively studied component which is leached from spermatozoa is potassium.
Lardy
et a al.
(1943) first described the stimulatory effect of potassium on respiration and glycolysis of twice washed ram spermatozoa and further reported that at least .005 M potassium ion was necessary to maintain optimal motility, while higher concentrations further stimulated respiration and glycolysis of bull and ram sperm.
This explained
the advantage shown for Baker's and Krebs-Ringer solutions containing potassium over physiological saline (Emmens and Swyer, 1948; Cheng and Casida, 1948).
The stimulation
by potassium of washed spermatozoa was demonstrated for bull, ram, dog, and fowl spermatozoa (White, 1953a,b,c; Wallace and Wales, 1954; O'Shea and Wales, 1964; O'Shea, 1969), but it was discovered that potassium had a depressive effect on unwashed and epididymal sperm (Bishop and Salisbury, 1955a,b; Cragle and Salisbury, 1959; Salisbury
et al.
et al., 1950; O'Shea and Wales, 1964; Wallace and Wales,
32
1964).
In fact, potassium in high levels as are found
in the epididymus act as a natural inhibitor to spermatozoal metabolism. half
Seminal plasma contains only about
the concentration found inside the spermatozoa,
so even dilution with seminal plasma to dilute suspension will leach the cells of this electrolyte (Cragle
et al., 1958a, b; Dott and White, 1964; Quinn et al., --1966). High seminal plasma potassium levels are correlated with high fertility for frozen bovine spermatozoa which may be associated with improved maintenance of acquiescence or with resistance to dilution (Graham, 1966}. Potassium depletion is accompanied by accumulation of sodium, and loss of calcium and magnesium.
This
syndrome results from dilution and washing (Quinn et al., 1966). Although leaching of potassium is certainly involved in the dilution effect, inclusion of this electrolyte in diluents and washing media is important but later studies have demonstrated that this alone will not completely curb the negative influence of dilution and washing (Blackshaw, 1953; White, 1953b,c; Dott and Walton, 1960; Wales and White, 1963; O'Shea and Wales, 1964). Carbon dioxide is another natural inhibitor of sperm metabolism and in high tensions it promotes acquiescence of spermatozoa and prevents rapid aging (Salisbury et al., 1960; Lodge et al., l963c).
However,
33
co2
also is apparently excessively removed by washing
and dilution and replacement decreased the negative
effect of dilution and washing on glycolysis (Lodge and Salisbury, 1963;
Lodge et al.,
1963a; Bracket,
1969). Protective Substances.
Many high molecular weight sub-
stances have been used to protect spermatozoa against the damage resulting from dilution and washing.
Harrison
and White (1972) hypothesized that washing removes lipoprotein from the plasma membrane.
The resulting loss of
membrane integrity may drastically alter membrane permeability to essential or detrimental molecules.
Protective
agents either protect or replace membrane constituents. Among these compounds are bovine alumin, bovine gammaglobulin and casein for fowl spermatozoa (Wales and White, 1961); egg albumin, bovine plasma albumin, potato starch and gelatin for ram and bull spermatozoa (Blackshaw, 1953); crystalline plasma albumen for ram and bull spermatozoa (White 1953b); catalase in yolk extender for bull spermatozoa (Foote and Dunn, 1962); egg albumin, casein, acacia, serine and alanine for washed dog spermatozoa (Wales and White, 1963); and casein for washed swine spermatozoa
(Pursel et
al., 1973).
However,
most of these materials were only moderately protective and other studies showed no protection.
Human spermatozoa
are particularly susceptible to dilution, and no protective
34 action was shown for high molecular weight substances beneficial to sperm of other species (White, 1954). Overall results are variable. The additive which has given the most consistent protection to spermatozoa against dilution damage has been egg yolk.
Salisbury (1946) theorized that in
dilute suspension yolk protects spermatozoa from excessive damage by oxygen by maintaining a relatively anaerobic environment near the cells.
Lodge
et a al.
(1963c) proposed that egg yolk was protective because of the high solubility of carbon dioxide in lipid. The yolk lipids insure that cells.
co 2
is maintained near the
Yolk has been reported as successful in increasing
the livability of diluted spermatozoa of the turkey, ram, stallion (Lardy and Phillips, 194la), bull
(Emmens and
Swyer, 1948; Cheng et al., 1949), and man (Norman et al., 1960).
This has complicated studies in which egg yolk
was included in the extender.
For example, Ohms and
Willett (1955) reported no difference in the post-thaw motility of bull semen which was unwashed, twice centrifuged and resuspended, and twice washed then resuspended in seminal plasma or fresh diluent.
Since yolk-citrate
was used for all dilutions, the protection of the yolk may have reduced treatment differences to insignificant levels.
35 Seminal plasma, as previously mentioned, was beneficial to washed or diluted spermatozoa after it had been in contact with stressed sperm cells but even freshly
ejaculated seminal plasma has afforded protec-
tion in some studies.
For washed bull semen motility
was almost completely restored when resuspension occurred in seminal plasma (Blackshaw, 19531 Nakabayashi and Salisbury, 1959).
However, only partial protection was
obtained for dilute rabbit sperm (Emmens and Swyer, 1948), washed ram spermatozoa (Blackshaw, 1953) and washed bull sperm (Albright et al., 1958; Salisbury and Nakabayashi, 1959).
Conversely, dialyzed seminal plasma did not
protect the motility of dilute fowl spermatozoa (Wales and White, 1961), oxygen uptake by washed ram spermatozoa (Wallace and Wales, 1964), nor metabolism of washed bull semen (Wales and Wallace, 1965). Seminal plasma addition has stimulated the metabolism of sperm in some studies by replacing substrates.
Since
the report by Mann (1946) in which it was shown that spermatozoa readily utilize glucose, fructose, or mannose for anaerobic glycolysis, one of these sugars is normally included in semen di1uents.
This energy
source is essential for washed spermatozoa from which seminal fructose has been removed (Hartree and Mann, 1959) and is also necessary in dialysis media for spermatozoa and seminal plasma (Dott and Walton, 1960).
As with
other species, washed goat spermatozoa require fructose,
36 mannose, or glucose to maintain motility (Fukuhara and Nishikawa, 1973b; Corteel, 1974).
Oxidation of fructose
by bull sperm was unimpaired by washing but oxidation of_ other substrates was depressed resulting in overall decreased oxygen uptake by washed sperm (Wales and Wallace, 1965).
These authors proposed that substrates,
other than fructose, present in seminal plasma had been removed by washing. Cold Shock.
Another consideration when washing semen is
the role of seminal plasma during cooling and freezing. There is a dilution effect related to cold shock.
Dilute
sperm suspensions are more susceptible to cold shock than concentrated samples for the bull (Choong and Wales, 1962), ram (O'Shea and Wales, 1964; Quinn and White, 1966; Quinn et al., 1968), and boar (Purcel et al., 1973). Washed semen is also more readily damaged by subsequent cooling and freezing.
O'Shea and Wales (1964) found that
washed ram spermatozoa were more severely depressed by cooling to 5 C than unwashed samples but replacement of seminal plasma after washing decreased this effect. Dialyzed seminal plasma was just as effective as nondialyzed indicating that high molecular weight substances were responsible for the protection afforded. Washed swine semen was more susceptible to cold shock than centrifuged/resuspended samples, assayed by lower motility and percent normal acrosomes (Purcel et al.,
37 1973).
These studies indicate that seminal plasma
protects the sperm cells against some of the
conse-
quences of cooling to 5 C. Actually the damage caused by dilution, washing, cold shock and freezing are similar in that they all result in leakage of vital intracellular substances. Washing increases the leakage from subsequently cold shocked spermatozoa.
Washing ram semen three times
(2000 g, 15 min) caused no loss of DNA or phospholipid but resulted in loss of
acid~soluble
phosphorus and
protein after subsequent cold shock and freezing (Quinn et al., 1969).
Washed spermatozoa also lost more protein
from the middle piece
during freezing than did the
unwashed. Washing apparently potentiates cold shock since washing after cooling instead of before cooling results in less damage (Blackshaw, 1953).
O'Shea (1969) found
that though washing caused a large depression in oxygen uptake, substrate oxidized and percent unstained (live) ram spermatozoa during freezing, washing after freezing/ thawing did not reduce semen quality.
If goat semen is
to be washed prior to freezing, adequate cryoprotective· substances must be used to replace the protection of high molecular weight substances normally contributed by seminal plasma. protection.
Egg yolk affords this type of cryo-
As stated previously, Ohms and Willett
(1955) reported no difference in the post-thaw motility
38
of bovine semen which was unwashed, twice centrifuged/ resuspended, or twice washed and resuspended in diluent with or without seminal plasma.
Since egg yolk-citrate
was used for all dilutions, the yolk may have minimized the detrimental effect of seminal plasma removal on post-thaw revival. Cytoplasmic Droplets. semen is the fluid.
A final consideration when washing
removal of materials other than seminal
Semen is composed of spermatozoa, fluid from the
accessory glands and testes, and cellular debris.
This
latter group includes sperm fragments, epithelial cells and cytoplasmic droplets.
The latter are membrane-
bound bodies originally formed in a process termed spermateliosis during which the sperm cell sheds a major portion of its cytoplasm.
Droplets may remain
attached to the spermatozoa when it leaves the testis but they are normally shed during passage through the epididymus (Dott and Dingle, 1968).
Free cytoplasmic
droplets contain material rich in enzymes and, in fact, a considerable fraction of the total enzyme activity of the non-fluid portion of semen.
For bovine semen
the cytoplasmic droplets contain 96 percent of the acid phosphatase, 44 percent of the a-glucuronidase, 87 percent of the RNAse, 97 percent of the acid protease and 50 percent of the hyaluronidase (Dott and Dingle, 1968).
39 In this same study, ram cytoplasmic droplets were reported to contain an even larger fraction of the total activity of these enzymes, and the ratio of spermatozoa to droplets was 2:1, while a much higher ratio of 5.7:1 was counted for ejaculated bull semen. Even washed suspensions of spermatozoa may contain cytoplasmic droplets which, if damaged, release enzymes having an adverse effect on spermatozoa or diluent components.
But, since these bodies are smaller than sperm-
atozoa, the washing process itself can be utilized to remove them.
Allison and Hartree (1970) outlined a
washing procedure which produced droplet-free washed semen.
The bulk of the droplets remained in the super-
natant when bovine semen was diluted then centrifuged at 254 g for 15 min.
Further washing was required to
remove the seminal plasma. A further complication in dilution and washing studies is the action of Tris buffer on cytoplasmic droplets.
This buffer makes the droplets more fragile
and susceptible to breakage.
Dott and Dingle (1968)
found that Tris buffer reduced the number of droplets per spermatozoa in some samples to zero.
Thus, damage.
to spermatozoa reportedly resulting from dilution, may have been due to the action of enzymes released from cytoplasmic droplets.
40
Negative Effect of Seminal Plasma for Other Species.
The
only species for which washing of the semen has consistently been reported as beneficial is the goat.
However,
for, other species dilution and washing have not always been damaging to spermatozoa.
There is some indication
that the seminal fluids produced in species other than the goat are detrimental when included in the storage diluent. The adverse effect of dilution is caused not only by synthetic diluents but by seminal plasma as well, e.g., depressed motility of dog semen (Wales and White, 1963), lowered motility for bovine semen (Rottensten et al., 1960) and inhibited livability of human spermatozoa (Freund and Wiederman, 1966).
Although these reports may
indicate that seminal plasma dilution is accompanied by the same spermatozoal changes as dilution with other media, it is also possible that a dilution effect is not involvedbutthe seminal fluids contain harmful substances as is the case for the goat. There have been other reports of spermatozoal depression upon addition of seminal plasma.
Washed
bovine spermatozoa, resuspended in seminal plasma, showed the same motility as unwashed controls after 4 hr at 37 C, however, lactic acid accumulation increased after seminal plasma addition (Salisbury and Nakabayashi, 1959). The authors proposed inhibition of lactic acid utilization bv seminal plasma.
Shannon (l965a) found that the
41
addition of seminal plasma to extended bovine semen further depressed motility in a number of diluents. Suspension of washed human sperm cells in seminal plasma
depressed metabolism, and centrifugation and
resuspension in buffer increased oxygen consumption. This indicates that human seminal plasma has a strongly depressive effect on spermatozoa (Eliasson, 1971). Sexton and Fewlass (1978) report that poultry seminal plasma has a negative effect on the fertilizing capacity of stored poultry sperm. Less direct evidence of a negative influence of seminal fluids on spermatozoa are the scattered reports of no damage resulting from washing or reports that washing has been advantageous.
White (1953a) found
tbz.t washing ram semen once had little effect on motility, oxygen consumption or lactic acid production, and bull or rabbit spermatozoa could be washed twice with minimal injury.
He felt that the washing damage reported earlier
may have been due to the respiration of microbial contaminants. factor.
Inclusion of antibiotics controlled this
Dott and White (1964) observed minimal reduction
of activity (impedence change frequency) of washed ram spermatozoa and Hoskins and Patterson (1967) reported that twice-washing of primate semen resulted in little loss of motility.
Washing has also been shown to maintain
respiration of ram sperma.tozoa for a longer period at 37 C
42
(Lardy
et al al.,
1945b), stimulate uptake of glucose by
bull sperm (Flipse, 1954), increase oxygen consumption of bull spermatozoa (Bishop and Salisbury, 1955b) and greatly increase oxygen consumption by human spermatozoa (Eliasson, 1971). One explanation for stimulation of semen by washing is the removal of inhibitory substances which may be important in maintaining spermatozoal quiescence in the epididymis.
Such inhibitors do not cause irreversible
damage to the cells and may in fact suppress activity thus extending the spermatozoal life span.
These inhi-
bitors may explain the stimulation of spermatozoa at low dilution rates.
Potassium which is quite high in goat
seminal plasma (Iritani and Nishikawa, 1964a) has been implicated in this capacity, as well as carbon dioxide (Salisbury et
al.,
1960;
Lodge et
al., 1963c).
Smith et
al. (1957) found that the activity of succinic dehydrogenase was strongly stimulated by washing which they demonstrated was due to the removal of the inhibitor diphosphopyridinenucleotide (DPN).
Another inhibitor
known to be found in semen binds the enzyme acrosin which is required by the sperm for fertilization (Polakoski et al al.,
1971).
It is possible that other inhibitory
substances are present in seminal fluid which would help explain the stimulation by washing spermatozoa which has been observed, however, removal of these inhibitors does not necessarily improve semen quality.
43
It is also possible that the semen of species other than the goat contains toxic factors which irreversibly decrease semen quality.
The egg yolk
coagulating enzyme in goat seminal plasma is also found in very low levels in swine semen (Fossum et al., 1965). Goat seminal plasma is also considerably higher than that of the ram, bull, boar or dog in proteolytic enzymes, tributyrase and an egg yolk lysis factor.
Bull semen is
high in a haemolyzing factor (Mann, 1964) which Fossum et al. (1965) found in the semen of only one of four goats tested but in allof 17 bulls. The only actual report of a toxic enzyme-like factor in semen other than the goat was by Shannon (1965a,b).
He found that the addition of bull seminal
plasma to diluted semen depressed livability.
The
factor responsible was heat labile (1 min. 90 C) and acted directlv on the spermatozoa rather than bv degradation of diluent components.
Generally, it may
be the heterogeneous nature of seminal plasma that causes the varied results observed when it is removed.
Removal
of deleterious factors, particularly for goat semen, and of cytoplasmic droplets improves semen quality but care is required to replace substrates and metabolic cofactors and to provide the large molecular weight substances which protect cells during cooling and freezing.
Washing also involves the removal of the
2
PGF a
44 found in semen (Bygdeman and Holmberg, 1966) which aides sperm transport in the female reproductive tract (Edqvist
et al., 1975). There has been no study com-
paring the fertility of washed and unwashed goat semen, but
PGF
2a and other fertility factors may be required
by washed semen to obtain optimal fertilizing capacity. Diluent Introduction.
Semen diluents are used to extend the
ejaculated volume producing a less concentrated sperm suspension for insemination of multiple females per ejaculate.
If the semen is to be used immediately after
collection, physiological saline is an adequate extending medium, but in order to maximize the advantages of artificial insemination, diluents must be formulated to protect spermatozoa during processing and storage. Important considerations include maintaining the optimal pH, osmotic pressure, electrolyte balance and gas phase, as well as providing substrate and inhibiting microorganism proliferation (Mann, 1964; Salisbury et al., 1978).
All of these functions were fulfilled by early
buffer systems which were used to study spermatozoal activity and metabolism including Baker's solution (Baker, 1939) and calcium-free Krebs-Ringer-Phosphate, or "sperm-Ringer" (Mann, 1946).
However, for long-term
storage of spermatozoa, it is necessary to cool semen
45
below ambient temperatures in order to inhibit metabolic processes and prolong the cellular life-span (Mann, 1964). Diluents must therefore contain substances which protect against the damage resulting from cooling spermatozoa. Certain high molecular weight components of milk and egg yolk have proven so well suited to fulfill this function that virtually all extenders in commercial use today contain one or both of these natural substances (Salisbury et al., 1978; Graham et al., 1978). Phillip's (1939) classic report of the protection afforded by egg yolk during refrigerated storage resulted in the widespread use of his egg yolk-phosphate diluent for chilled bovine semen (Mann, 1964).
Since
this initial account, numerous buffered egg yolk extenders have been formulated for the bull and other species, and the results of their use have been extensively reviewed (Salisbury, 1957; Maule, 1962; Salisbury et al., 1977, Graham et al., 1978).
The major buffer systems developed
have been egg yolk-phosphate, egg yolk-citrate (Salisbury et al., 1941; Melrose and Stewart, 1956). Carbon dioxide and bicarbonate (VanDemark and Sharma, 1957; Bartlett and VanDemark, 1962) and zwitterionic buffers, particularly Tris (Davis et al., 1963a,b; Foote, 1972; Graham et al., 1972; Jones and Foote, 1972). The other major class of semen extenders is that based on milk.
Whole milk, skim milk and cream have
46
buffering properties and require only the addition of limited glucose and antibiotics to become complete extenders for semen.
All milk preparations however,
must be heated to 95 C for 10 min to destroy lactenin which is spermicidal (Flipse et al., 1954).
Since this
heating is more rigorous than that of pasteurization, only boiled, canned or powdered milk is suitable for use in diluents.
In general, milk-diluted semen has produced
similar fertility results to that extended in properly formulated egg yolk diluents (Salisbury, 1957; Mann, 1964; Salisbury et al., 1978).
Dried skim milk is usually
used because microscopic observation is difficult in whole milk and large batches of powdered milk can be obtained in order to minimize variability (Salisbury et
al.,
1978). The use of the natural substances egg yolk and
milk in semen diluents is not ideal, because they are by nature heterogeneous and variable.
Although they contain
protective substances, they also contain damaging substances such as enzymes and electrolytes (Powrie, 1977). Efforts have been made to determine the specific components of milk and yolk responsible for their protective activity, in order to develop defined media (Gebauer et al., 1970). Egg yolk was fractionated by several investigators, and the ability of the fractions to extend the viability of spermatozoa was reported (Lardy and Phillips, 194la; Mayer and Lasley, 1945; Phillips and Spitzer, 1946;
47
Blackshaw, 1954).
Though both a lipoprotein fraction
and a phospholipid fraction protected against cold shock, only the lipoprotein increased livability at low temperature (Kampschmidt et al., 1953).
The low-
density lipoprotein responsible (Watson and Martin, 1975) remains in the supernatant after centrifugation at 35,000 g for 30 min, which removes the yolk granules (Watson and Martin, 1973).
The protective agent in milk is in the
protein content (Choong and Wales, 1962) notably casein (O'Shea and Wales, 1966). Diluents for Goat Semen.
All of the basic types of diluents
developed for bull or ram semen have been used with varying success to extend goat semen.
A tremendous amount of
the variability in results can be accounted for by the egg yolk-coagulating property of goat seminal plasma. This activity is strongly influenced by many factors including the buffer used (Roy et al., 1959), dilution of semen and seminal plasma (Corteel, 1976b) and the individual buck (Iritani
et a al.,
1964;
Fossum et
al., 1965).
Some
investigators have achieved conception rates exceeding SO percent using unwashed goat semen frozen in yolkcontaining diluents, including egg yolk-citrate (Waide and Niwa, 1951; Vlachos and Tsakalov, 1963, 1964; Vlachos and Karagiannida, 1968; Tsakalov
et a al.,
1974), egg yolk-
--
lactose (Nagase, 1966; Peskovatskov et al., 1974) egg
48
yolk-Tris (Hahn, 1972; Tsakalov et al., 1974) and yolkspermasol, which contains citrate, phosphate and gelatin (Gruttemeir, 1969).
While other workers reported concep-
tion rates of less than 40 percent for unwashed semen frozen in these yolk-containing extenders (Liess and Ostrowski, 1960; Weissflog, 19611 Kalev andVenkof, 1951; Lyngset et al., 1965; Goffaux and Corteel, 1967).
There
have also been reports of superior survival for unwashed goat semen extended and chilled in egg yolk diluents rather than milk (Blokhuis, 1962; Schindler et al., 1979) and reports of superior results for freezing in egg yolk diluents when in vitro evaluation was used rather than actual conception rates (Jelam and Manbier, 1965; Sahni and Roy, 1969; Rossouw, 1974; Koh and Ong, 1976). The variable results obtained using egg yolkcontaining extenders for freezing goat semen are not reflected in the conception rates achieved when whole milk or skim milk diluents were used, which consistently exceed 50 percent (Fraser, 1962; Bonfert, 1964, 1965, 1969; Bonfert and Thier, 1963; Herman, 1964; Wettke, 1964; Barker, 1966; Goffaux and Corteel, 1967; Kupferschmied, 1969, 1972; Andersen, 1969; Corteel et al., 1970; 1972; Corteel, 1974, 1976a,b,c; Tsakalof et al., 1974; González Stagnaro, 1975).
The discrepancy in consistency between
milk and egg yolk diluents is explained by the toxic egg yolk coagulation caused by goat seminal fluids.
49
There is no indication that egg yolk-containing media are unsuitable for washed spermatozoa and, in fact, the coagulating enzyme is removed by washing (Iritani and Nishikawa, 1961).
Some of the best fertility
results obtained for goat semen followed washing, extending and freezing in an egg yolk-Tris diluent. A conception rate of 71 percent was achieved for 916 does inseminated during one estrus (Fougner, 1976) and over three years and 3240 first inseminations using this method 63.4 percent of the does kidded (Fougner, 1979). Buffers and pH.
Spermatozoa utilize fructose, glucose
or mannose via glycolysis producing lactic acid (Mann, 1946).
During processing and storage this may result
in decreasing pH and irreversible immobilization of the sperm cell.
Semen diluents therefore contain buffers
which maintain the pH in a biological range, preferably close to 7.0.
In addition, an ideal buffer should resist
enzymatic degradation, resist participation or interference with cellular metabolism, produce minimal toxic salt effects, refrain from penetration of the sperm membranes and effectively bind toxic ions (Graham et al., 1972).
The buffers which have been commonly included in
semen extenders do not conform with all of these criteria. Phosphate buffers are effective in a biological range and have been used extensively for tissue culture, including semen storage (Umbreit et al., 1949, Mann 1946).
50
While they effectively precipitate or bind most polyvalent cations which interfere with metabolic processes (Graham et
al., 1972), phosphate itself acts as a metabo-
lite or an inhibitor in many systems
(Good et al., 1966).
Phosphate has been shown to inhibit sperm motility, oxygen uptake (Bishop and Salisbury, 1955a; Salisbury and Nakabayashi, 1957) and lactic acid oxidation (Lodge et al., 1963b).
In addition, for ram spermatozoa, the
inhibition by phosphate of lactate utilization was considerably greater for washed samples (Wallace and Wales, 1964).
This has not been shown for goat semen but may
be important since washing is suggested in this species. Citrate buffers are particularly useful for semen extenders since citrate binds calcium, which is inhibitory to spermatozoa (Lardy and Phillips, 1943), and dissolves the yolk granules which clears the diluent, allowing for observation of sperm motility.
Egg yolk-citrate buffers
have been preferentially used for goat semen since the report by Roy (1957) that egg yolk coagulation by goat seminal fluids is calcium dependent, and that calcium binding by citrate minimized this phenomenon.
Although
citric acid is involved in the Krebs cycle and is thus a metabolite, it is not readily utilized by spermatozoa (Mann, 1949) and has little effect on spermatozoal metabolism
(Salisbury et al., 1978).
51 Diluents buffered with'bicarbonate or carbonated by gasing with carbon dioxide inhibit sperm motility and metabolism (Salisbury et al., 1960), but this effect is
at
least partially reversible and bicarbonate inhibi-
tion is a factor in maintenance of epididymal sperm quiescence (Mann, 1964).
In contrast, low levels of
carbon dioxide stimulate sperm respiration (Salisbury and Kinney, 1957; Lodge and Salisbury, 1963) particularly for washed spermatozoa.
It has been suggested that carbon
dioxide is one of the essential substances removed from spermatozoa by washing, and that the protective activity of egg yolk is due partly to the high solubility of carbon dioxide in lipid (Lodge et al., 1964a).
High levels of
bicabonate buffer have been reported to cause head agglutination of washed sperm (Dott and White, 1964). Overall, bicarbonate is suitable for semen diluents only at controlled levels such as are included in the two most common carbonated diluents:
Cornell University extender or CUE and Illinois
variable temperature diluent or IVT (Bartlett and VanDemark, 1962). Because of some of the problems with conventional biological buffers, Good et al. {1966) developed a new group of zwitterionic buffers for biology studies.
Prior
to this report, only Tris (tris (hydroxymethyl)amino methane) had been successfully used in diluents, giving particularly good results for goat semen as Tris-citric acid-yolk {Hahn, 1972; Fougner, 1974, 1976, 1979;
52
Vander Westhuysen, 1978), however, it does have several problems as a buffer.
Tria has poor buffering properties
below a pH of 7.5, notably below room temperature.
Its
most effective buffering points (pKa) at 37 C, 20 C, and 0 C respectively are 7.9, 8.3 and 8.9 (Good et al., 1966).
Tris is also a primary aliphatic amine of con-
siderable reactivity and consequently is often inhibitory in biological systems.
Mitochondrial oxygen uptake is
seriously depressed by Tris in some tissues (Good et al., 1966).
Amines have been shown to competitively interfere
with potassium uptake by bacteria
(MacLeod and Onofrey,
1954) and there is some indication that this is true in spermatozoa as well (O'Shea and Wales, 1964).
Tris
may penetrate the spermatozoal membrane acting as an intracellular buffer as occurs for erythrocytes (Omachi £! al., 1961). Dilution with this buffer also caused accumulation of calcium by ram spermatozoa, though this was not shown for bull semen (Quinn et al., 1966).
Not
all of these activities are negative, however, they indicate interference with cellular function.
Of great
concern for semen is the action of Tria buffer on cytoplasmic droplets.
These membrane-bound bodies contain
large quantities of hydrolytic enzymes, and Tris increases their susceptibility to breakage (Dott and Dingle, 1968). Other zwitterionic buffers have recently been tested for semen extenders including Mes, Pipes, Bes, Mops, Tes, Heoes, and Tricine (Graham et al., 1972).
Of these seven
53
Tes (N-tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid) combined with Tris to form the complete buffer TEST, was the most effective for bull semen (Graham et al., 1972) and boar semen (Graham et al., 1971; Crabo £! al., 1972), and was suitable for freezing ram semen (Graham et al., 1978).
Tes and Tria do not bind
divalent cations such as calcium, but Tes is very active in binding toxic heavy metals such (Graham et al., 1972).
as copper
Tes crosses cell membranes
with difficulty, has little or no ion effects and is non-reactive with respect to cell metabolism (Good et al., 1966).
These are of interest as possibly
superior buffers for semen extenders, but there is little fertility data available and no
studies in the
literature testing their suitability for goat semen. The initial pH of ejaculated goat semen is between 6.3 and 7.2 with most reports showing a mean value of 6.5 (Iritani and Nishikawa, 1964a; Kurian and Raja, 1965; Patel, 1967; Kang and Chung, 1976; Dhillon et al., 1977; Varshney et al., 1977).
But seminal fluids are not
necessarily the ideal diluting medium for sperm storage (Mann, 1964).
For most species a pH just above 7 best
maintains sperm motility and metabolism.
Sperm metabolism
is optimal for pH ranges of 6.9 to 7.0 for the bull (Salisbury and Kinney, 1957; Foote, 1964; Steinback and Foote, 1967), 7.0 to 7.5 for the ram (Lardy et al., 1954b;
54
Winchester and McKenzie, 1941; Graham et al., 1978) and 6.8 for the rabbit (tardy and Phillips, 1943). For goat semen maximum oxygen uptake occurred at pH
7.2 to 7.5 while motility at
37 C was best maintained
at pH 7.0 and 7.2 (Blockhuis, 1962; Fukuhara and Nishikawa, 1973a). Osmolality.
Osmolality of the diluent is also very
important to sperm survival (Mann, 1964).
A medium
which is hypotonic to the spermatozoa may increase leakage of intracellular substances (Mann, 1951), and hypertonicity of the medium reduced the detrimental effects of high dilution of turkey spermatozoa, perhaps by limiting leaching of vital materials (Wales and White, 1961).
However, freezing semen involves removal
of water as ice and consequent dehydration of spermatozoa.
Under these conditions suggested osmolalities
have been 300 to 325 milliosmoles/kg for turkey semen (Graham and Brown, 1971), bull semen (Martin, 1963a; Yassen and Foote, 1967; Rao (Graham et
al., 1978).
et a al.,
1968) and ram semen
These diluents are isotonic with
mammalian seminal plasma.
Some reports however, suggest
freezing ram semen in hypertonic diluents (Jones, 1965; ~ightfoot
and Salamon, 1969).
Similar osmolality is
apparently appropriate for chilled storage of goat semen as well (Koh and Ong, 1976).
Although for freezing goat
55
semen, there have been no reports investigating the optimum diluent osmotic pressure required. Substrate.
Spermatozoa utilize fructose, glucose,
and mannose aerobically or anaerobically to maintain motility (Mann, 1946).
Seminal plasma contains fructose
at levels close to 1 percent in the goat (Iritani and Nishikawa, 1964a; Barakat et al., 1972; Patil and Raja, 1974; Varshney et al., 1977).
Substrate must
be added to diluents of washed sperm in particular, since seminal contributions have been removed (Salisbury, 1946; Corteel, 1974).
Goat spermatozoa are also capable
of utilizing pyruvate, acetate and lactate to promote motility (Fukuhara and Nishikawa, 1973b).
A level of
5 x 10-2 M sodium pyruvate gave the best stimulation of oxygen uptake by washed goat spermatozoa (Fukuhara and Nishikawa, 1973a). Antibiotics.
Semen diluents provide all of the nutrients
and protective substances required to preserve microorganisms along with the spermatozoa during processing and freezing.
Consequently, antibiotics are included
in extenders to protect spermatozoa and to prevent
infec-
tions in inseminated females {Salisbury et al., 1978). Sulfanilamide is toxic to spermatozoa during freezing and is not recommended for use in diluents for frozen
56
semen (Dunn et al., 1953).
Streptomycin and penicillin
have no effect on post-thaw motility or fertility of bull semen (Foote and Bratton, 1950; Erickson et 1954; Willett and Ohms, 1955).
al.,
Many other antibiotics
have been tested for bull semen and might be routinely included in extenders in the future (Salisbury et al., 19 78) . Egg Yolk Level.
Early egg yolk-containing diluents for
bull semen were 50 percent yolk and reducing this to 20 percent had little effect on fertility (Stewart et al., 1950; Almquist, 1951; Olds et al., 1951).
As
buffer formulation improved, reduction in egg yolk content resulted in better results with bovine semen (Foote and Bratton, 1960).
Results for optimal egg
yolk level for ram semen are variable (Graham et al., 1978).
For goat semen, the optimal egg yolk content
was reported as 20 percent in a citrate diluent (Waide et al., 1977). Cryoprotection Cooling and Cold Shock.
Sudden cooling of semen to 5 C
results in irreversible cellular damage termed cold shock.
Spermatozoa thus treated exhibit reduced motility
and metabolic rate (Blackshaw and Salisbury, 1957; Blackshaw, 1958; Choong and Wales, 1962), and an increased proportion
57 of tail
abnormalities (Salisbury et al., 1942).
Although
the mechanism is not completely understood, cooling apparently causes differential changes in membrane constituents resulting in increased permeability. This is evidenced by an increased staining with eosin or Congo red (Lasley et al., 1942b; Hancock, 1951), and loss of vital intracellular substances including cations, phospholipids, protein and DNA (Hartree and Mann, 1959; Quinn and White, 1966; Pickett and Komarek, 1967; Quinn et al., 1969).
This increased permeability
and leakage of substances is similar to the damage caused by washing spermatozoa (Hartree and Mann, 1959; Quinn et al., 1969), and cold shock damage is increased by washing the spermatozoa prior to cooling (O'Shea and Wales, 1964; Quinn et
al.,
1969; Pursel et al., 1973).
Washing
potentiates cold shock in this manner, not because of mechanical damage, but because of substances in the seminal plasma which protect against this phenomenon. Semen diluents are normally formulated to contain egg yolk and/or milk because they contain substances which protect spermatozoa against cold shock, including lecithin and other phospholipids, lipoproteins and casein (Lasley et al., 1942a; Mayer and Lasley, 1956; Bogart and Mayer, 1950; Kampschmidt et al., 1953; Blackshaw, 1954; Quinn and White, 1966; Graham et al., 1971). Including these protective agents, and cooling semen
58 slowly to 5 C prevents cold shock damage.
The rate
of temperature decrease is an important factor.
For
goat semen, cooling over 60 min has resulted in better
post-thaw motility than shorter periods, and
longer
periods have not improved spermatozoal revival
(Waide et al., 1977; Vander Westhuysen, 1978).
This
is in contrast to ram and bull semen for which cooling periods up to 4 hr have increased storage life, fertility and post-thaw revival (Martin, 1965; Patt and Nath, 1969). In general rapid processing is beneficial for goat semen (González
Stagnaro, 1975), and prolonged contact with
seminal plasma, even that remaining after washing, is avoided by allowing only 1 hr for cooling to 5 C. Freezing Spermatozoa.
The freezing of cell suspensions,
including semen, involves the dehydration of the cells. At relatively slow cooling rates the cells tend to super cool to -10 or -15 C, while ice is forming in the extracellular medium.
As ice crystals form, the remaining
extracellular solution increases in concentration creating an osmotic gradient across the cell membrane.
To com-
pensate, water leaves the cell resulting in cellular dehydration (Mazur, 1963). If slow freezing rates are employed, this gradual process results in cells being exposed to high solute concentrations over relatively long periods of time.
In his classic work with human
erythrocytes, Lovelock (1953) proposed that these elevated
59
intracellular salt levels have an adverse "solutioneffect" on the cell, causing irreversible damage.
In addition,
the precipitation of buffers may cause changes in pH, and toxic levels of organic solutes may develop (Karow, 1969).
Dehydration also causes osmotic damages.
Once
cells reach a specific minimum volume, they are no longer able to compensate for the continued increase in extracellular osmolality as water is removed as ice.
At this
stage erythrocytes will lose their membrane integrity and will take in electrolytes to reduce the osmotic gradient (Meryman, 1968).
Irreversible changes in
membrane permeability are produced.
These problems
are minimized by using faster freezing rates such that cells are subject to these stresses at lower temperatures for shorter periods of time. Rapid cooling has an upper limit also, as intracellular ice formation occurs if the temperature is lowered too quickly for cells to compensate by outflux of water (Mazur, 1963).
Intracellular ice crystals
cause extensive damage and cellular death (Sherman, 1962). The critical rate at which internal ice is formed depends on the ratio of cell volume to surface area and membrane permeability to water.
Intracellular ice forms for sea
urchin ova, yeast and erythrocytes at rates exceeding 1 C/min, 10 C/min, and 5000 C/min respectively.
This
very rapid rate for red blood cells is due to their low volume and high water permeability (Mazur, 1970).
60
The optimal freezing rate for spermatozoa has not been determined, but until quite recently slow cooling rates were generally employed.
This was after the
first successful revival of frozen spermatozoa by Polge et a al.
(1949) in which a slow freezing rate was used.
In a subsequent study he reported a critical range was determined between -15 C and -20 C, and rapid cooling of 3 C/min was suggested to -25 C (Polge, 1953).
Erickson
et al. (1954) developed a freezing protocol for bull semen involving rates of 3 to 5 C/min and requiring some 25 min to lower the semen to -79 C (dry ice).
The
standard rate for cooling ram semen required 32 min and used cooling rates of 1, 2, 4, and 5 C/min (First et al., 1961; Patt and Nath, 1969).
These relatively slow
freezing processes were developed for semen packaged in 1 to 2 ml glass or plastic ampules.
With the development
of new packaging methods and freezing to -196 C in liquid nitrogen, more rapid freezing rates were used.
Semen
pellets are produced by dripping small volumes of semen directly onto indentations on the surface of a block of dry ice (Nagase and Niwa, 1964).
This method involves
extremely rapid freezing, but results in excellent revival rates for bull, ram, and boar semen (Salamon, 1970; Graham et al., 1978).
The modern .25 or .5 ml plastic
straws (Jondet, 1964; Cassou, 1968), which are currently used for commercial artificial insemination of dairy cattle, are also frozen rapidly, usually by holding the
61 straws horizontally in the vapor over liquid nitrogen. Cooling from 5 to -100 C is accomplished in 4 to 8 min for ram and bull semen (Colas, 1975; Rodriquez et al., 1975). Goat semen technology has followed the same trend as that in other species.
Fraser (1962) reported
best revival with an extremely slow freeze requiring 30 min to cool to 0 C, followed by .5 C/min to -5 C, 1 C/min to -10 C, 2 C/min to -17 C and 4 C/min to -79 C. The 2 ml plastic vials used for this study have now been replaced with the modern semen straw and freezing in liquid nitrogen vapor is accomplished in 2 to 8 min (Gonzalez Stagnaro, 1976; Waide et al., 1977). Cryoprotectants.
An optimal freezing rate for some cells
does not exist because rates slow enough to prevent intracellular ice formation may result in tremendous dehydration damage.
Protection is afforded to cells during
freezing by a group of substances called cryoprotectants, the first of which was glycerol.
In their classic work
Polge et al. (1949) reported that frozen fowl spermatozoa were revived upon thawing when glycerol was included in the diluting medium.
Virtually all subsequent
methods developed for freezing mammalian spermatozoa for artificial insemination have included addition of glycerol to semen prior to freezing.
The optimum levels
of glycerol for freezing semen will be discussed in the next section.
62
Other cryoprotective substances have been developed for freezing diverse tissues and cells, the most widely used of which is dimethyl sulfoxide or DMSO.
Although
these other compounds are often superior to glycerol for cryopreservation, none of them have proven as consistently beneficial for semen.
DMSO has been tried
with limited success for freezing the semen of the bull (Lovelock and Bishop, 1956; Page et al., 1968; Snedeker and Gaunya, 1970), ram (Jones, 1965), and man (Zimmerman et al., 1964).
However, other reports have indicated
that DMSO is suitable for preserving human semen (Sherman, 1964; Karow, 1972; Graham and Crabo, 1978).
Numerous
other substances have been tested for cryopreservation of mammalian semen, including sugar solutions, particularly lactose and raffinose (Nagase et al., 1964; Berndtson and Foote, 1972), propylene glycol and ethylene glycol (Polge et al., 1949; Waide and Niwa, 1961), polyvinylpyrrolidone or PVP (Menge and Sullivan, 1964) and 1,3 butanediol, 1,5 pentanediol and 1,7 heptanediol (Graham et a al.,
1974), however, these have not been as efficacious
as glycerol. The mechanism of action of the cryoptotectants is not completely understood.
Several differing models
are still widely accepted, and have been reviewed (Merryman, 1968; Karow, 1969; Mazur, 1970). Basically, since freezing damage is the result of intracellular ice formation for
63 fast freezing or is brought about by dehydration, concentration of solutes and osmotic stress during slow cooling, cyroprotectants must emeliorate these consequences of freezing. The earliest model, which is still considered useful, was proposed by Lovelock (1953) in the classic work in which solution effects and cellular dehydration were described.
This investigator proposed a colligative
action by glycerol, by which it acts as a non-electrolyte solute.
Assuming that glycerol penetrates the cell and
is in equal concentration inside and outside the cell, then the increasing intracellular solute concentrations resulting from formation of extracellular ice and consequent outflux of water, will be only partly electrolytes.
The concentration of these deleterious salts
will be lower as they make up only part of the solutes, the rest being glycerol. This postpones high levels of electrolytes and buffer precipitation until the temperature is lower and the reaction rates of damaging chemical processes are reduced.
Although this model
was tested by its successful prediction of DMSO cryoprotection (Lovelock and Bishop, 1956), it alone could not completely explain all of the relationships between penetrating cryoprotectants (Karow, 1969). The hydrogen-bonding properties of these substances were found to be important to their success as cryoprotectants, and effectiveness increases with number of hydrogen-
64 bonding sites available (Doebbler, 1966).
Merryman
(1956) had suggested that glycerol may form hydrogenbonds with water, thus holding water inside the cell during
extracellular ice formation.
This would limit
solution effects to lower temperatures and prevent osmotic stress and "leaky cells".
This theory is con-
sistent with the fact that as the glycerol level used increases, the optimal freezing rate decreases (Mazur, 1970).
Glycerol holds water inside the cell decreasing
cell shrinkage, which for rapid freezing will increase the likelihood of intracellular ice formation (Morris and Farrant, 1972).
This may explain the results of
Rodriguez et al. (1975) who reported that as the glycerol content of bull semen diluents was decreased, the optimal freezing rate increased.
Freezing rates are thus dependent
upon the diluent composition.
These interaction between
glycerol level and freezing rate are not consistent with a second model which explains hydrogen-bonding of water as preventing intracellular ice crystal formation (Doebbler, 1966), since high glycerol levels should then be best for fast freezing where internal ice is a problem. Hydrogen bonding has also been described as protecting intracellular proteins from denaturation by solution effects or dessication.
This may result from formation of
a water lattice around proteins (Karow and Webb, 1965) or from direct hydrogen-bonding of the cryoprotectant to the protein (Levitt, 1966).
65 All of the above models of cryprotection assume that glycerol rapidly penetrates cell membranes and acts intracellularly. that
However, Sherman (1963) showed
for mouse ova, penetration of glycerol was not
only unnecessary for cryoprotection, but was quite toxic.
He further proposed that glycerol penetration
of spermatozoa at 5 C is probably negligible and that its cryoprotective activity is extracellular, probably involving prefreezing dehydration.
Berndtson and Foote
(1972) found that glycerol does penetrate bull spermatozoa at 5 C, however, best revival was obtained when freezing was accomplished prior to significant penetration.
Using direct pellet freezing on dry ice they found
that results were optimal for freezing 10 to 60 sec after glycerolization.
Revival decreased over 2, 3, 4,
5, and 6 min, at which point glycerol penetration was apparently complete.
Further increasing the length
of exposure to glycerol did not decrease or increase post-thaw semen quality.
These investigators agreed
with Sherman that dehydration and subsequent rapid freezing prevented intracellular ice formation.
Actually, these
results are consistent with the penetrating cryoprotectant model for glycerol, which describes intracellular glycerol as protecting primarily during slower freezing, and promoting intracellular ice formation for rapid freezing (Rodriquez et al., 1975}.
Low glycerol or
66 glycerol-free diluents have been successfully used for pelleted semen (Nagase and Graham, 1964; Graham et al., 1972), which involves extremely rapid freezing. Of considerable interest in freezing of spermatozoa is the significant cryoprotective properties of egg yolk, which is a component of many semen diluents.
Yolk is
cryoprotective for the semen of the bull, boar, ram and goat (Graham et al., 1972); the lipoprotein fraction apparently being the agent responsible (Pace and Graham, 1974).
Egg yolk competes for sperm membrane binding
sites with the fluorescent membrane marker, 1-anilinonaphthalene-8-sulphonate or ANS (Watson, 1975). When the ram semen diluent contained 10 percent yolk, no ANS could be detected on the membranes, but attached egg yolk could be removed by washing in yolk-free media. It was proposed that egg yolk cryoprotection was provided by coating the cell membrane.
Watson and Martin (1974)
found that yolk protected the acrosome of ram spermatozoa during freezing which is consistent with the report of Graham et al. (1972) that egg yolk prevented release of the intracellular enzyme glutamic oxalacetic transaminase or GOT from bull, boar, ram, and goat sperm during freezing.
Yolk was particularly protective for
the goat, and semen pellet-frozen in a yolk extender without glycerol maintained a post-thaw motility as high as that obtained when glycerol was included.
67
For bull semen, yolk is synergistic with glycerol, with better post-thaw revival when both are present than the
combined improvement when either is used alone
(Graham et al., 1972).
Egg yolk alone was more bene-
ficial than glycerol alone in a buffered diluent, and GOT released from the acrosome during freezing was nbt influenced by glycerol level as long as egg yolk was included in the medium. Glycerol Level.
The proper level of glycerol to include
in semen extenders is extremely species specific, and is governed by the degree of glycerol toxicity to spermatozoa.
The toxicity of glycerol was first reported by
Polge et al. (1949) after they observed its negative effect on rabbit spermatozoa.
More recent investiga-
tions indicate that glycerol is toxic to spermatozoa on addition, and is further inhibitive to sperm fertility in fowl (Polge, 1951; Brown and Graham, 1971) and particularly in swine (Polge, 1956; Paquignon and Courot, 1975).
Graham et al., 1971;
Bower et al., (1973a)
found that addition of glycerol to swine semen caused a large outflux of GOT from spermatozoa, perhaps indicating a change in membrane permeability.
It was proposed that
the increased extracellular osmolarity resulting from glycerol addition, causes water to leave the cell while glycerol enters the cell.
~--·····
A final influx of water
-----------------------------
68
completes the process.
These investigators concluded
that flexing of the membrane may account for the damage observed.
Alternatively, the problem may involve
decreased sperm transport, fertilization or implantation. In contrast, the semen of other species tolerate a wide range of glycerol levels.
Bovine spermatozoa in
particular (Berndtson and Foote, 1972), with ram sperm also able to withstand concentrations up to 10 percent of the diluent without reduced revival (Hill et al., 1959; First et al., 1961). Optimal glycerol level is also influenced by other diluent components.
In general, optimal glycerol
concentrations for egg yolk-containing extenders were lower than that for milk extenders (Erickson, et al., 1954).
As higher glycerol proportions are included in
the extender, yolk becomes less beneficial (First et al., 1961). Seminal plasma is also a component of the semen extender, unless it has been removed by washing.
As
discussed previously, washed semen is more susceptible to cold shock than samples left in their seminal fluids. Seminal plasma acts to protect spermatozoa from cold shock (Purcel et al., 1973), and washed spermatozoa lose more protein from the midpiece and other intracellular substances when they are subsequently frozen (Quinn et al., 1969).
Substances found in undiluted mammalian seminal
plasma which may have cryoprotective activities include
69
free glycerol (Clark et al., 1967) and phospholipids (Jain and Anand, 1975).
This factor has not been
adequately investigated and may be significant for goat semen which is washed during processing. All of the factors involved in semen processing influence the optimal glycerol concentration, including diluent composition, washing, dilution, cooling rate, equilibration time, packaging, freezing rate and thawing rate.
Consequently, there is considerable variation
in the glycerol levels suggested for a given species. Still from the large number of investigations of semen freezing, definite patterns have emerged.
Bull semen
extenders usually contain 7 percent and 9 to 11 percent for yolk and milk diluents respectively (Salisbury et al., 1978).
Swine semen is so sensitive to glycerol that levels
exceeding 3 percent glycerol are rarely used (Paquignon and Courot, 1975).
For man, yolk-containing diluents are
not in common use, and semen may be frozen with negligible extension.
Usually 7 to 10 percent glycerol or a similar
level of DMSO is used for cryopreservation (Graham and Crabo, 1978). Goat semen is similar to that of the ram relative to glycerol tolerance.
Glycerol is tolerated well with
no high toxic effect (Graham et al., 1972).
Most reports
suggest levels close to 7 percent for slow freezing in milk (Fraser, 1962), slow freezing in yolk-containing
70
diluents (Waide and Niwa, 1961; Waide et al., 1977), fast freezing in yolk containing diluent (Hahn, 1972; Tsakalof et al., 1974) and for rapid freezing in skim milk
diluents (Corteel, 1974;
Gonzáles
Stagnaro, 1976).
Only three reports advocate the use of only 3 to 4 percent glycerol, and these involve yolk-containing extenders and fast freezing (Andersen, 1969; Rossouw, 1974; Fougner, 1976).
No difference in the fertility
of frozen goat semen has been shown due to glycerol level.
Evaluation has been based on in vitro techniques.
Glycerolization.
Polge (1953) reported higher revival
rates for bull spermatozoa if glycerol was added at 5 C rather than before cooling.
Further reports for bull
semen have been variable, with some reports of an advantage to glycerol addition after cooling (Miller and VanDemark, 1954; Dunn and Hafs 1943; Choong and Wales, 1964) while others found no difference due to temperature (Graham et a al.,
1958).
Ram semen is apparently somewhat more
sensitive and though most differences reported have been small (Graham et al., 1978), Colas (1975) reported substantially superior results for glycerolization after cooling.
Graham et al. (1978) further found that for
ram spermatozoa, better post-thaw motility was obtained if glycerol was added after holding at 5 C for 3 hr. This was consistent with recent reports that glycerol
71
penetration occurs within 10 min, and holding spermatozoa in contact with glycerol is not beneficial (Berndtson and Foote, 1972).
In general, glycerolization of semen
is performed after cooling, as there have been no reports of superior results for higher temperatures. Glycerolization rate has also been investigated with variable results.
Slightly better results have
been obtained by stepwise glycerol addition to cooled bull semen (Salisbury et al., 1978), but other investigators found no difference between gradual glycerolization and one step addition of glycerol (Miller and Van Demark, 1954; Graham et al., 1958).
Results for ram semen are
also variable (Graham et al., 1978). Goat semen is also not tremendously sensitive to sudden incorporation of high concentrations of glycerol at higher temperatures
(González
Stagnaro, 1975), although
Graham and Crabo (1978) made the statement that, "turkey, ram, and bull semen can be added directly to the glycerolated buffer at 37 C with no apparent effect on fertility. It must, however, be added at a lower temperature (5 to 10 C) to goat or boar spermatozoa".
As this was not
referenced it must be based on work in the author's laboratory.
Most studies on freezing goat semen have
used stepwise addition of glycerol over a period of time, usually by addition of three to five aliquots of glycerolcontaining diluent at 10 to 20 min intervals at 5 C (González
Stagnaro, 1975).
72
Equilibration.
Early experiences with preparing spermatozoa
for freezing indicated that after cooling and glycerolization, storing semen for additional time prior to freezing was beneficial to revival and fertility on thawing. This period of t:ime was termed "glycerol equilibration", and 12 to 24 hr yielded superior fertility for bull semen than shorter periods (Emmens and Martin, 1956; 1961; Blackshaw et al., 1957; Graham et al., 1957).
Shorter
equilibration is required by ram spermatozoa, usually 1 to 3 hr (Hill et al., 1959; Jones and Martin, 1965; Salamon, 1968; Patt and Nath, 1969; Colas, 1975; Hinshelwood et al. , 1981). This benefit of holding semen before freezing is not actually related to 'glycerol equilibration', or adjusting to glycerol.
More recent studies indicate
that addition of the glycerol after the holding period, just prior to freezing, provides equal or superior cryoprotection (Berndtson and Foote, 1972; Jondet, 1972; Karow, 1972).
The mechanism for the maturation process
which prepares the spermatozoa for freezing is unknown. Holding semen at 37 C prior to dilution (Rajamannon et al., 1971; Graham and Crabo, 1972; Pursel et al., 1973), increasing the cooling rate, and prolonging the period before freezing increases the apparent quality and the fertility of spermatozoa.
73
This requirement for spermatozoal maturation prior to freezing is complicated, for goat semen, by the benefit of rapid processing and freezing. studies
Many
have been complicated by the use of egg yolk-
containing diluents during the holding treatment. The toxic effect of egg yolk decomposition by goat seminal plasma may have led to underestimation of the optimal holding time.
In Fraser's classic report (1962)
storage for 8 to 24 hr in his skim milk diluent gave superior post-thaw motility to storage for 4 hr.
González
Stagnaro (1976), also using a milk-based diluent, found that stepwise glycerolization to 4 percent glycerol with a 60 min equilibration produced superior fertility results to one step glycerolization to 7 percent followed by a 3 hr equilibration; 68.6 percent and 42.9 percent conception rates respectively to first insemination. Although he proposed that the difference observed resulted from more
rapid
processing, the variables of glycerol
level, method of glycerolization and equilibration are confounded as only two treatments are used.
Another facet
involved is an interaction between washing and equilibration time determined by Van der Westhuysen (1978) for Angora goat semen.
In this study using a Tris-citric acid-yolk
diluent, an optimal equilibration of 2 hr was found for post-thaw motility, while semen held for
or 1 hr before
freezing exhibited severely reduced revival.
For washed
74
samples, however, there was no difference between these three holding periods and all washed samples were equivalent in percent motility to the best unwashed sample.
Apparently some effect of the washing process
removed the necessity for the 2 hr maturation required by unwashed spermatozoa.
Perhaps a slight change in
membrane permeability, as is seen upon washing sperm of other species, promotes dehydration and prevents an increased loss of membrane integrity.
The inability
of cellular volume to decrease past a given limit, and the resulting uptake of electrolytes producing a "leaky cell", is one prevalent theory of damage during freezing (Meryman, 1968).
In our lab we also found little
difference between 0, 1, and 2 hr equilibration in a Tris-citric acid-yolk diluent (Drobnis et al., 1982). Thawing Rate.
Glass ampules were used extensively for
semen packaging prior to the development of straws and pelleting methods.
For ampules, ice water was the tradi-
tional thawing bath, however, widely varying optimal thawing temperatures have been reported .(Salisbury et al., 1978). For semen frozen in straws, rapid thawing rates are usually superior with increases in semen revival even up to 100 C for some studies. Thawing baths of 75 C and 95 C are optimal for ram and bull semen (Aamdal and Andersen, 1968a,b; Andersen and Aamdal,l972; Rodriquez et al., 1975).
75
Theoretically, rapid thawing rates should follow the fast freezing currently used for straws.
Rapid
cooling produces small crystals which will recrystalize to form fewer larger crystals during warming.
Recrystal-
ization involves tremendous mechanical damage to the frozen cell, therefore rapid thawing is used (Mazur, 1970). The thawing rates suggested for obtaining optimal post-thaw revival of goat semen are produced by holding frozen straws in a water bath at 75 C (Andersen, 1969; González
Stagnaro, 1975; Fougner, 1974).
care involved in not over
Although the
the cells at
heating
these
high temperatures makes for more uniform results for 30 to 35 C for field work (Corteel, 1976b; Fougner, 1976). Evaluation Semen Fertility.
The only true measure of the post-
thaw quality of semen is the conception rates obtained with its use.
However, this is not practical for the
evaluation of all treatment effects as large numbers of females must be inseminated in order to determine small differences in semen fertility.
While breeding studies
with dairy cattle have commonly included ten thousand cows inseminated with each treatment level, such trials in dairy goats rarely have a total of a thousand does, and often involve less than fifty.
Only in France
(Montigny, 1977) and in Norway (Fougner, 1979) have
76 recent trials been conducted with comparatively large numbers of inseminations per breeding season. The situation with only small numbers of does available
for fertility studies is further complicated
by the great variability in insemination results for this species.
Problems of estrus detection, estrus
synchronization, double or single insemination during estrus, and differences in site of semen deposition may result in poor fertility even when semen quality was adequate (Andersen, 1969; Vlachos, 1975; Corteel, 1976a,b; Fougner, 1976).
These problems are even greater for
insemination trials in the ewe (Lightfoot and Restall, 1971; Andersen et al., 1973; Gustafsson, 1978; Graham et a al.,
1978) and similar types of problems exist for
using fertility trials in cattle (Bishop et al., 1954). In order to investigate the numerous variables in the processing of frozen semen, it is essential that preliminary studies be evaluated using in vitro techniques. The classic work of Fraser (1962) with goat semen was conducted in this way.
After evaluating numerous treat-
ment combinations in the laboratory, he reported his results along with the results of an insemination trial with a few does to test his results.
The next year this
Canadian semen was used for large scale trials in the United States, Germany, and the Netherlands with excellent results (Herman, 1963).
77 Unfortunately, none of the widely used methods of bull semen evaluation, is adequately correlated with final fertility, and their use for the prediction of conception rates is unwarranted (Linford et al., 1976). Still the relationships are adequate to give lower limits for several evaluation methods, providing for the identification of subfertile frozen ejaculates by commercial semen producers.
Better correlations would simply
prevent the destruction of valuable semen of outstanding genetic merit, which does not satisfy all of the established quality criteria, but is perhaps even above average in fertility.
The correlations of fertility and in vitro
evaluation are, in general, much poorer for species other than cattle, particularly for swine (Graham and Crabo, 1972). Although these results are individually poor, this is not as big a problem for the researcher as it is for commercial purposes. mined for a
given
In general, the correlation deter-
evaluation method may be quite good,
but the statistical removal of individual bull effect results in an insignificant result (Bishop et al., 1954). Work with pooled ejaculates, which have been split and subjected to various treatments, allows for the evaluation of treatment effect, even if fertility correlations for the measures used are poor.
To illustrate this point,
the fact that a straw of bull semen has been carefully evaluated and is shown to have 65 percent progressive
78 motility upon thawing, does not allow us to predict, with 95 percent confidence, what the final conception rate will be for the 200 straws in that ejaculate. However, if pooled ejaculates are split into several samples each and processed using various freezing rates the significantly different motility rates produced indicate trends in the relative efficacy of the freezing methods employed, since a significant correlation of .412 (Linford, 1976) does exist between motility and fertility.
This is particularly true if consistent
results are obtained using several evaluation methods. The major methods available which include motility, resistance to stress, metabolic activity, vital staining, structural integrity and enzyme release, measure the effect of treatment on different vital structures and properties of the spermatozoa. Evalution of Undiluted Semen.
The evaluation of neat
semen cannot be used to evaluate semen processing techniques, yet the study of semen characteristics has ellucidated some aspects of semen fertility.
The classic
study of Williams (1920) was the first in which a significant correlation between a semen characteristic and fertility was determined.
If the percentage of
abnormal spermatozoa was high, the fertility of that bull was low for natural mating.
This stimulated interest
79
in sperm morphology and it was determined that bulls with impaired fertility or sterile bulls had high proportions of abnormal spermatozoa (Lagerlof, 1936; Hancock, 1949).
Subfertility has been related to
abnormal nucleus, tailless sperm, abnormal midpieces, immature sperm with protoplasmic droplet attached, and bent and coiled tailes (Sullivan, 1978).
Overall percent
abnormal cells is highly correlated to the fertility of fresh and frozen semen and is used as a routine quality control parameter by bull semen producers (Linford, 1976; Saake, 1972).
The normal range of these morphological
characteristics of goat semen has been reported (Eaton and Simons, 1952; Lunca, 1964; Kurian and Raja, 1965; Patel, 1967; Mohri et al., 1970;
Prasad et
al., 1970).
A second widely used technique for evaluating neat semen, mass movement, is less closely related to fertility.
With this subjective evaluation procedure,
a drop of neat semen is observed under a light microscope with a heated stage (37 C).
A scale of 1 to 5 or 1 to 10
is normally used to assess the degree of swirling, wavelike motion of the sample.
It is difficult to see
individual cells, and thus the surface of a drop from a highly motile and concentrated sample appears as a swirling mass (Walton, 1952).
Initial attempts to
correlate this evaluation method to conception rates in cattle were unsuccessful (Bishop et al., 1954; Stewart et al., 1972).
80 However Linford et al. correlation with r
=
(1976) has determined a positive
.672 (p < .001) for frozen semen,
this being one of the best three of 12 evaluation methods studied. Attempts have been made to make more objective determinations of mass movement, notably the motility meter of Glover (1968) and impedence change frequency of Rothschild (1948).
The motility meter apparatus
involves applying shear force to a microscope slide to stop mass movement.
When released, movement resumes
and the rate of return of motility is measured.
A photo-
cell detects motility since the immobilized sample is opaque, but as movement begins the field darkens.
This
technique has given good correlations to fertility of subsequently frozen bull semen (Stewart, et al., 1972; Linford et al., 1976).
Impedence change frequency was
also developed to measure the activity of neat semen. Two electrodes are positioned in the semen sample and the frequency of impedence change is determined. Rothschild (1948) proposed that the electrical changes observed were caused by variations in the position of the biological system with respect to the electrodes, or in other words, by wave motion in the semen mass. This characteristic was correlated with fertility of chilled bull semen (Bishop et al., 1954), and was later developed for use with diluted semen.
This will be
81
further discussed relative to evaluation of sperm motility and velocity.
These evaluation techniques
have not been used with goat semen. Further tests of semen quality involve chemical and enzyme analyses of the seminal plasma.
Variations
in the concentration of several ions have been related to fertility'of rams or bulls including pH (Anderson, 1945; Van Duijn and Rikmenspoel, 1960), calcium, potassium, sodium, and chloride (Graham 1966; Graham et al., 1970. Concentrations of fructose and citric acid in seminal fluids is under tight androgen control (Mann, 1964), and it thus is related to fertility.
The initial level
of the enzyme glutamic oxalacetic transaminase (GOT) in seminal plasma is negatively correlated to bull fertility (Breeuwsma, 1972) and fertility of frozen bull semen (Linford et al., 1976).
Levels of phenylalanine-a-
ketoglutarate transaminase in boar seminal plasma is also related to fertilization rate (Van Gemert et al., 1972). An interesting property of goat seminal plasma is its melanizing activity.
In semen samples of rabbits,
bulls, rams or goats a dark yellow color indicates stronger melanizing activity.
In the bull this property
is unrelated to any other semen evaluation method (Bishop et a al.,
1954).
However, the semen of some individual
bucks, having stronger melanizing activity, also displays
82
lower metabolic rate, reduced middle piece area and fewer live spermatozoa (percent unstained) than samples with
weak activity of where melanizing activity is
absent (Mukherjee, 1964; Misra and Mukherjee, 1979). No attempts have been made to correlate this characteristic directly to semen fertility. Motility.
Evaluation of diluted semen is better suited
to the needs of semen-processing research, allowing comparison between initial, prefreeze and post-thaw results.
In order to assess the ability of a spermatozoon
to fulfill its role in fertilization, it is necessary to evaluate the structures responsible for its function. The morphologically normal spermatozoon (figure 2) includes three important structures which are required for fertility. The nucleus containing the cell's genetic material is the most essential, but is apparently least damaged by frozen storage, requiring serious shock to produce loss of DNA (Ackerman and Sod-Moriah, 1968; Quinn et al., 1969). The acrosome, a membrane-bound organelle covering the anterior 60 percent of the nucleus, contains enzymes thought to be responsible for penetration of ovum vestments, as will be discussed below. with its mitochondrial
sheath, is
The middle piece,
the site of the exten-
sive metabolic processes of the cell which are geared primarily to motility.
Motility of spermatozoa has been
83
HEAD
NECK
Figure 2.
Diagrammatic representation of a spermatozoon (After Mann, 1964 Fig. 3, p. 20) •
84
used as an evaluation technique from the early studies in artificial insemination, without questioning its relationship to fertility and its role in fertilization. ~e
report that even completely immotile spermatozoa
reached the ovarian portion of the oviduct within 4.3 min in the cow (Van Demark and Moeller, 1951), demonstrated clearly that motility is not required for sperm to transport themselves to the site of fertilization. The female reproductive tract is largely responsible. Further questioning of the value of this evaluation method was raised by the observation that boar and turkey spermatozoa can be completely immobilized by cooling or storage, yet recover motility if storage time is not prolonged (Polge, 1956; Schindler and Nevo, 1962).
This
means that samples from these species which appear dead, may actually yield good fertility.
Although these reports
and studies showing no correlation between motility and fertility
have cast doubt on the role of sperm activity
in fertilization, recent studies indicate that motility is an important factor. Observations of in vitro fertilization indicate that the spermatozoon becomes activated, exhibiting intense motility which appears to be active in propelling the cell through the zona oellucida material (Yanigimachi, 1966; Yang et al., 1972; Sato and Blandau, 1979).
85
The most common methods of fertility evaluation have been rapid estimations of the proportion of the cells motile.
in a field of diluted semen which are progressively This may be given a percentage value or be
rated on a scale of one to five.
Many attempts to
correlate this value with fertility data have failed, including the sperm of cattle Stewart et
(Graham et al.,
1958;
al., 1972), sheep (Smorag and Kareta, 1974)
and humans (Behrman and Sawada, 1966). Cheng and Casida (1948) reported a correlation between motility and fertility in rabbits with r
= .43, but they also reported
fertility using nonmotile spermatozoa. et al.
Recently Linford
(1976) determined a correlation with conception
rates in cattle, with r = .698 (p < .001) for initial motility and r = .596 (p
