Storage of Bovine Semen in Liquid and Frozen State: R. Vishwanath, P. Shannon

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Animal Reproduction Science 62 2000.

2353
www.elsevier.comrlocateranireprosci

Storage of bovine semen in liquid and frozen state


R. Vishwanath) , P. Shannon
Liestock Improement Corporation, Priate Bag 3016, Hamilton, New Zealand

Abstract

This review describes the historical and current methods used for storage of bovine semen. The
essential physiological differences between liquid and frozen semen, their relative advantages and
disadvantages are addressed, and the current state of technology, the procedures used, their merits
and future possibilities are also discussed. q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Liquid semen; Spermatozoa numbers; Frozen bovine semen

1. Introduction

Genetic advancement in the cattle population, particularly in the dairy sector has
relied on two processes, namely, the use of bulls of high genetic merit and the selective
rearing of calves of high breeding merit as replacements. Artificial insemination has
remained the main vehicle for the rapid dissemination of valuable genes and the method
of choice for dairy farmers worldwide to improve the genetic quality of their stock. This
steady level of genetic progress in dairy cattle is primarily due to advances in semen
technology. For the purposes of this review, the reference will be mostly with the dairy
industry, for this is where technical advancement in semen technology has been captured
most successfully Chupin and Schuh, 1993; Chupin and Thibier, 1995; Cunningham,
1998; Foote, 1998..
The potential genetic contribution of a sire is described by Foote 1998. as:

Contribution of a sire to genetic improvements Number of progeny per sire


= Genetic superiority of the sire.

)
Corresponding author. Tel.: q64-7-8560867; fax: q64-7-8582758.
E-mail address: rvish@lic.co.nz R. Vishwanath..

0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 4 3 2 0 0 0 . 0 0 1 5 3 - 6
24 R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353

The number of progeny per sire will be determined by: i. total sperm output of the
bull; ii. the number of sperm used per insemination; and iii. the percentage of cows
calving to a single insemination. This can be represented as:
Number of progeny per sire
s Number of sperm per sirernumber of sperm inseminated per cow .
= Proportion of cows calving to a single insemination.
These principles effectively determine the number of bulls required for a dairy cow
population. The equation becomes particularly attractive when very few bulls are needed
to service a large population of dairy cows thereby significantly raising the selection
intensity Shannon, 1978..
The requirement of semen from bulls of high genetic merit has been the main impetus
for developing and refining storage technologies for cattle semen. In a 1980 survey, it
was believed that the total number of inseminations worldwide exceeded 130 million
Bonadonna and Succi, 1980.. A more recent survey Chupin and Thibier, 1995. showed
that the total number of doses of semen produced exceeded 200 million, with more than
95% of this processed as frozen product. About 4 million doses were used as liquid
semen, and this was restricted primarily to New Zealand with smaller amounts in
France, The Netherlands, Australia and Eastern Europe. In this review, the main focus
will be on the well-known storage technologies, which include liquid and frozen stored
semen along with a brief discussion on emerging technologies for semen storage in
cattle.

2. Liquid storage of semen

There have been many reports since the turn of the century on diluting media for
semen of livestock with much of this work originating in the former Soviet Union
Anderson, 1945.. Each diluent, ranging from a simple salt solution to the more complex
buffered medium had its own merits. Perhaps the accepted principle of semen dilution
technology was that survival of spermatozoa for extended periods was inversely related
to their metabolic activity. To be useful for artificial insemination, diluted semen had to
have a minimum shelf-life of between 2 and 4 days to provide for easy transport and use
in distant locations. It was this guiding principle that led to the initial storage tempera-
ture of 58C Salisbury and Van Demark, 1961.. The predominant effect of storage at 58C
was a lowering of metabolic rate of spermatozoa, which contributed to extended
survival. The discovery that egg yolk was a useful additive in increasing the preserving
properties of the various media, added further impetus to this work Phillips, 1939..
Many extenders have been developed for liquid storage of semen, and this chapter only
provides a brief description of some of the major developments. Detailed discussions on
some of the early diluents are available in a comprehensive review by Foote 1978..

2.1. Diluents for storage of semen at refrigerated temperatures

The early diluents for storage at refrigerated temperatures were significantly influ-
enced by the discovery of egg yolk as a useful additive, and the primary buffering
R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353 25

Table 1
Comparison of 60- to 90-day non-returns % NRR. to inseminations with bovine semen diluted to 8 million
spermatozoarml in CUE and Tris media. Data from Foote 1978.. Diluent compositions are shown in Table 2
Diluent No. 1st inseminations % NRR
CUE 5981 73.0
Tris 5673 73.3

component in these diluents was phosphate Phillips, 1939.. Subsequently, citrate was
found to have adequate buffering capacity with an enhanced period of survival of
spermatozoa stored at 58C Willett and Salisbury, 1942.. Citrate then became the salt of
choice, as its chelating properties improved the solubility of protein fractions in egg
yolk. Egg yolk has been a more common additive, but homogenised whole milk, fresh or
reconstituted skim milk and coconut milk have also been used to preserve fertility of
bovine spermatozoa Melrose, 1962; Norman et al., 1962..
Many of the zwitterionic buffers Good et al., 1966. provided good buffering capacity
over a wide pH range. Tris, TES, MES, HEPES, PIPES, MOPS and BES titrated with
hydrochloric acid were tested at various pH levels as semen diluents and all of them
were similar in suitability Foote, 1972. cited by Foote 1978.. However, the Tris-based
diluent has become more universal, and this buffering medium combined with egg yolk
and glycerol has been tested extensively Davis et al., 1963a,b.. Motility of spermatozoa
was slightly lower in Tris medium buffered to lower pH 6.25 vs. 6.75. than in Cornell
University Extender CUE., but good results were obtained in a fertility trial with both
media Table 1..
A further field trial demonstrated that a lower pH 6.5. and the presence of glycerol
in Tris medium improved fertility, compared with higher pH and absence of glycerol
Foote, 1970.. The results were comparable with semen diluted with CUE. The
conclusion from these series of studies was that the Tris diluent was suitable for storage
of semen at refrigerated or ambient temperature and also in frozen state. The diluents
shown in Table 2, and some subsequent modifications to these have been used in field
trials with varying success Bartlett and Van Demark, 1962; Van Demark and Bartlett,
1962; Shannon, 1965, 1978; Foote, 1978.. In the case of the Illini Variable Temperature
IVT. diluent, the main changes have been in the ratio of bicarbonate to citrate and the
concentration of glucose increasing from 0.3% to 1.2%.

2.1.1. Milk-based diluents


The historical origin of milk as a diluent for bull semen has been described by
Salisbury and Van Demark 1961.. The first report on the use of milk originated in
Germany Koelliker, 1856. cited by Salisbury and Van Demark 1961.. This observation
went unnoticed until Underbjerg et al. 1942. compared fertility of bull semen stored in
egg yolkphosphate and autoclaved milk diluents. The results were similar for both
media. The methods of preparation of milk diluents and the various combinations have
been reviewed by Melrose 1962. and Foote 1978.. The important aspect of storage of
semen in milk-based extenders was the necessity to inactivate the lactenin a toxic
26
Table 2

R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353


Composition of diluents for storage of bovine semen at low 58C. and ambient temperatures. Ingredients are in gr100 ml of medium
Ingredients Egg yolk Egg yolkcitrate Original IVT diluent CUE Tris medium for CAPROGEN w
phosphate Salisbury Van Demark Foote et al., 1960. ambient storage Shannon, 1965.
Phillips, 1939. et al., 1941. et al., 1957. Foote, 1970.
Temperature of 58C 58C 58C 58C and ambient 58C and ambient Ambient
storage
Tris 3.028
Potassium hydrogen 0.2
phosphate
Sodium hydrogen 2
phosphate
Sodium citrate 3.6 2 1.45 2
Sodium bicarbonate 0.21 0.21
Potassium chloride 0.04 0.04
Glucose 0.3 0.3 1.25 0.3
Citric acid 0.087 1.675 1
Glycine 20 0.014
Glycerol 8 1.25
Catalase 0.003
Caproic acid 0.025
Egg yolk %. 50 50 10 20 25 5
Antibiotics Yes Yes Yes Yes
Gas phase CO 2 self carbonating N2
R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353 27

factor. by heating. Different preparations of milk needed different heating requirements


to selectively inactivate the lactenin, but still maintain the integrity of the protein and
sugar moieties in milk. Glycerol added to the milk-based diluents was advantageous and
maintained fertility of bull spermatozoa for 4 days OConnor and Smith, 1959;
Almquist, 1962.. In general, milk-based extenders have been successful in maintaining
fertility and gave comparable results to egg yolkcitrate diluents Foote, 1978.. Egg
yolk 510%. added to milk-based extenders seemed to inactivate the toxic factor in
milk. This process has overcome the problem of heating the skim or fortified milk
before addition to semen, and also adequately maintained the fertility of diluted bovine
semen stored at 58C.

2.2. Storage of semen at ambient temperature

It has been the maxim of semen dilution technology that survival of spermatozoa in
diluted state is inversely related to their metabolic activity. The pursuit of this principle
led to the development of egg yolk diluents for storage of semen at 58C Phillips, 1939.
and deep freeze techniques for total immobilisation of spermatozoa at very low
temperatures Polge et al., 1949; Polge and Rowson, 1952.. In order to store semen at
temperatures above 58C and achieve satisfactory results, alternative methods of metabolic
inhibition were attempted. Thus, Van Demark and Sharma 1957., proposed CO 2
narcosis, Norman et al. 1958. suggested lowering the pH and Shannon 1965. proposed
N2 gassing as methods to inhibit metabolic activity of spermatozoa.
Storage at 58C reduces metabolic activity, but not all changes associated with lower
temperatures are beneficial to spermatozoa. For example, the activity of the NaqrKq
pump decreases with reduced temperatures such as 58C, but is unable to cope with
diffusion of ions across the cell membrane Quinn and White, 1966, 1967; Sweadner
and Goldin, 1980.. A consequent increase in the intracellular concentration of Naq is
detrimental to survival of spermatozoa Makler et al., 1981.. It was then postulated that
storage at ambient temperature may be superior to storage at 58C, provided the medium
in which spermatozoa are suspended inhibits those pathways that are detrimental to their
survival at higher temperatures Shannon and Curson, 1972b, 1982a, 1984.. The
temperature of storage is an important consideration. The optimum temperature range is
considered to be 188C to 248C Shannon and Curson, 1984.. Storage at temperatures
above this results in lower fertility, compared to storage at 58C Foote and Bratton,
1960; Bartlett and Van Demark, 1962..
Another significant development in ambient temperature diluent technology has been
the change in the level of egg yolk in the diluent. The ratio of egg yolk to buffer became
particularly important at higher storage temperatures than it was with storage at 58C
Foote and Bratton, 1960; Shannon and Curson, 1983.. Historically, semen diluents have
included anywhere from 12.5% to 50% egg yolk in the medium. Efforts to decrease the
egg yolk concentration in the simple phosphate and citrate buffers gave no advantage in
survival of spermatozoa or fertility Foote and Bratton, 1960.. Egg yolk does protect the
sperm cells from the toxic effects of seminal plasma; however, it also provides
substrates aromatic amino acids such as L-phenylalanine. for H 2 O 2 production by an
aromatic amino acid oxidase AAAO. released from dead cells to the detriment of live
28 R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353

Table 3
Percentage of NRRs 49 day. for semen diluted in CAPROGEN w media containing differing levels of egg
yolk. Data from Shannon and Curson 1983.
Trial Egg yolk %.
20 5 2 1
1 66.5"0.4 67.6"0.4
2 67.4"0.3 67.9"0.3
3 67.6"0.7 67.4"0.7 66.1"0.7

spermatozoa Shannon and Curson, 1972b, 1982b.. The amount of egg yolk required in
semen diluents to provide protection against seminal plasma toxins is proportional to the
amount of seminal plasma in diluted semen Shannon and Curson, 1972a.. Thus, when
semen is diluted to a high rate, and the seminal plasma concentration is consequently
reduced, there would be some advantage in reducing the egg yolk concentration. This
was borne out in a large fertility trial, where a decrease in egg yolk concentration from
20% to 5% had no detrimental effect on fertility. Further decrease of egg yolk
concentration affected fertility of some sires, suggesting that the level of egg yolk was
insufficient in some cases to be completely protective Table 3..

2.2.1. Coconut milk extender


Coconut milk extenders have been equivalent to skim milkglycerol, CUE or
CAPROGEN w in maintaining motility and survival of spermatozoa Norman and Rao,
1972; Foote, 1978.. This extender is quite simple and contains 15% decanted coconut
milk boiled for 10 min, 2.2% sodium citrate, antibiotics and 5% egg yolk. The presence
of egg yolk was essential to provide a lipid component to the medium. In some cases,
the medium has been supplemented with 0.1% calcium carbonate Norman et al., 1958..
A recent report on the use of Coconut extract and Coconut milk on ram semen showed
dramatic maintenance of motility over a 48 h period of 308C but no fertility trials have
been reported Chairussyuhur et al., 1993.. There have been no recent reports on
significant advances in this front in the literature.

2.2.2. Immobilisation of spermatozoa by low pH


An early observation in 1924 by Krshyshkovsky and Pavlov, cited by Norman et al.
1958., showed that spermatozoa were immobilised when placed in sealed tubes at room
temperature, but subsequent exposure to air produced a resumption of activity. Inhibition
of sperm motility was due to the decrease of pH by the accumulation of lactic acid in the
medium. Further studies by Norman et al. 1958. confirmed the finding and suggested
the decrease of pH as an effective method to inhibit metabolic activity of spermatozoa.
In this study, conclusive evidence was obtained that lowering the pH substantially
reduced metabolic activity measured by O 2 consumption, lactate production, and
motility of live spermatozoa. This effect was reversible, as activity resumed when the
old diluting medium was replaced after 150 h incubation with fresh medium at neutral
pH. By merely altering the pH from 5.76 to 7.45, motile activity of spermatozoa could
R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353 29

be altered from relative senescence to rapid movement Norman et al., 1958.. The effect
of acidic conditions on sperm metabolism was identified by Koellikker 1856., cited by
Norman et al. 1958. and Guenther 1907., who concluded that activity of spermatozoa
is a function of hydrogen ion concentration. A decrease in pH down to 5.5 was well
tolerated by spermatozoa, and the effect could be reversed by alkaline conditions, but a
pH below 5.5 was spermicidal and caused irreversible enzyme denaturation.

2.2.3. Immobilisation of spermatozoa in diluents gassed with carbon dioxide


Carbon dioxide was found to be a very effective inhibitor of sperm motility Shettles,
1940.. Early experiments showed that sperm motility was reversibly inhibited when
exposed to CO 2 for short periods, but prolonged exposure to this gas proved toxic to
sperm cells. Van Demark and Sharma, 1957 proposed CO 2 narcosis as an effective
means to maintain viability and fertilising ability of bovine spermatozoa for 6 to 7 days
at room temperatures. The first diluent designed on the basis of CO 2 immobilisation of
spermatozoa was the IVT diluent Van Demark et al., 1957.. The IVT diluent contained
a mixture of salts, sugar and antibacterial agents, and was saturated with CO 2 until the
pH decreased to 6.35. The final mixture contained 10% egg yolk. Bovine semen
extended with this diluent and stored at room temperature maintained fertility for over 3
days Bartlett and Van Demark, 1962.. Several diluents were then formulated using this
concept to optimise sperm survival and to extend the shelf-life of diluted semen Table
2.. By optimising the concentration of bicarbonate in the medium 0.1 M., and at higher
concentrations of glucose 0.067 M. in the IVT diluent, sperm motility was maintained
at high levels ) 45%. for over 90 days at 58C. While motility was maintained for
extended periods, fertility decreased sharply after 2 days of storage at 58C, Bartlett and
Van Demark, 1962.. This inconsistency in fertility result was evident also in other
studies, where CO 2 narcosis was used to maintain motility and fertility of spermatozoa
for extended periods Dunn and Foote, 1958; McFee et al., 1958; Foote et al., 1960..
The main difference between the IVT diluent and the CUE was that CUE was self
carbonating and relied on the action of citric acid on bicarbonate to release CO 2 with no
major effect on the pH of the medium Table 2.. The CUE was a substantial
improvement on the IVT diluent in this regard. Numerous field trials have confirmed
that CUE along with CAPROGEN w are the most successful liquid semen diluents to
date Table 4; Shannon, 1964; Foote, 1978..

Table 4
Fertility of bull spermatozoa stored in CUE and CAPROGEN w diluents. Data from Shannon 1964. and
expressed as % NRR at 49 days
Diluent Day of collection Day after collection
No. of inseminations % NRR No. of inseminations % NRR
CUE 59,091 62.8 25,309 65.2
CUEqglycerol 30,058 63.1 13,909 64.9
CAPROGEN w 73,993 62.8 37,758 63.5
CAPROGEN w qglycerol 35,351 62.7 17,957 64.9
30 R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353

Fig. 1. Incubated life of spermatozoa h. stored at 12.5 millionrml and 200 millionrml at 58C. The samples
were incubated at 378C at 12.5 million spermrml. Data from Shannon 1965..

2.2.4. Metabolic inhibition by nitrogen gassing


Shannon 1964, 1965. developed the CAPROGEN w diluent for bovine semen, which
replaced the self-carbonating concept with an ingenious method to reduce the dissolved
O 2 levels in the medium with N2 gas. This had no effect on the pH, but substantially
reduced the metabolic activity of spermatozoa. An additional effect of N2 gassing was
that it nullified the adverse effect of high dilution on sperm survival. Nitrogen saturation
halted the decline in incubation life of stored spermatozoa. This effect was more
noticeable when spermatozoa were stored in a dilute form 12.5 millionrml., compared
to storage in a concentrated state over 200 millionrml, Fig. 1.. At the same time,
inclusion of volatile saturated fatty acids Shannon, 1962. and catalase Shannon, 1972,
1973; Macmillan et al., 1978. significantly improved the length of time that diluted
bovine semen could be stored. The diluent was originally developed for use at 58C, but
proved superior for semen stored at temperatures between 158C and 278C Shannon and
Curson, 1984.. The conception rates with semen stored in CAPROGEN w were 0.9%
better at the elevated temperatures on the day of collection and 2.3% higher on the
following day compared to semen stored at 58C Table 5; Shannon, 1971..
2.3. Requirements for storage of bull semen at ambient temperatures
Storage of semen at 58C was not always easy or convenient, therefore, methods to
store semen at ambient or room temperatures were actively investigated. The require-

Table 5
Fertility of semen diluted and stored in CAPROGEN w at 58C or ambient temperature 18248C. and used on
day of or day after collection. Data from Shannon 1971.. Ejaculates were split between the two treatments
and fertility measured as % NRR at 49 days
Storage times Storage at 58C Ambient temperature
No. of inseminations % NRR No. of inseminations % NRR
612 h 268,201 65.3 44,354 66.2
1224 h 172,663 66.6 29,772 68.9 )
)
Significantly different to storage at 58C p- 0.05..
R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353 31

ments regarding diluting media that could accommodate the decrease in metabolic
activity of spermatozoa when stored at ambient temperature are the following.
i. An energy source in the form of simple sugars glucose, fructose.. Bovine
spermatozoa function adequately under both aerobic and anaerobic situations and the
rate of respiration can be controlled by the level of substrates Hammerstedt et al., 1988;
Krzyzosiak et al., 1999. and temperature of storage Hammerstedt and Hay, 1980;
Inskeep and Hammerstedt, 1985..
ii. A buffered medium to accommodate pH changes, usually in the form of citrate,
Tris, HEPES, MOPS, or bicarbonate buffers.
iii. Glycerol, a dual purpose additive, to provide osmolality to the medium and as an
invasive thermal protectant. In liquid semen diluents, glycerol has been shown to reduce
the decline in fertility associated with the aging of spermatozoa Shannon, 1964.. In
some cases, however, it did decrease fertility with semen used shortly after collection
OConnor and Smith, 1959; Almquist, 1962.. The effect of glycerol may depend on the
basic diluent to which it has been added. The addition of 1% glycerol to an egg
yolkcitrateglycine diluent increased the conception rate by 1.4 " 0.7% for semen
stored for 24 h at ambient temperature Shannon, 1964.. The beneficial effect of
glycerol was also noticed when added to milk and Tris diluents with an increase in
conception rates of 1.4% and 2.5%, respectively Foote, 1970; Bratton and Foote, 1973,
cited by Foote, 1978 and Stewart 1964..
iv. Egg yolk or other macromolecular substances such as skim milk, whole milk, or
coconut milk provide thermal protection to spermatozoa. The inclusion of macromolecu-
lar substances, particularly egg yolk has benefit even when sperm cells are not subjected
to cold shock. Perhaps the most important effect of egg yolk is its ability to bind seminal
plasma fractions that have a detrimental effect on survival of spermatozoa Shannon and
Curson, 1972a; Al-Somai et al., 1994; Prendergast et al., 1995..
v. A combination of antibiotics to provide both bacteriostatic and bactericidal
protection.
Other additives to diluents for storage of semen at ambient temperature have also
influenced the keeping quality of bovine semen with some positive effects on conception
rates. Some of these additives are listed below.

2.3.1. Glycine
The reason why glycine has been used as an additive to semen diluents is still not
clear. Many plants, unicellular organisms and marine elasmobranchs, accumulate com-
patible solutes, which provide protection during stress situations Yancey and Somero,
1979; Aspinall and Paleg, 1981.. Some of these compounds such as free amino acids
and quaternary nitrogen containing compounds retard thermal denaturation of enzymes
Paleg et al., 1981., provide thermal protection Heber et al., 1971. and maintain
enzyme structure and function Bowlus and Somero, 1979.. Conflicting fertility results
have been reported with diluents containing glycine Strom, 1956; Stower and Bud
Hasaim, 1957; Foote et al., 1958; Salisbury and Van Demark, 1961; Shannon, 1964.. A
fertility trial with 20% egg yolkcitrateglucose diluent containing 1% glycine gave a
fertility advantage of 2.1% on the day of collection and 2.7% with semen stored for 24
h, compared to a simple 20% egg yolkcitrate diluent Shannon, 1964.. The advantage
32 R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353

appears to be directly attributable to the glycineglucose moiety. Presence of glycine in


diluents for frozen semen has been detrimental to post-thaw survival of spermatozoa
Martin, 1965..

2.3.2. Caproic acid


Volatile fatty acids included in semen diluents is presumed to aid in maintaining
membrane integrity and fluidity Shannon, 1962.. While this feature has not been
investigated in detail, it is well known that lower-order fatty acids will prolong the
respiratory activity of spermatozoa for extended periods Mann and Lutwak-Mann,
1981.. Caproic acid enhances cell survival and fertility of diluted bovine semen. In a
large-scale fertility trial, addition of caproic acid had no significant effect on fertility
with semen used on the day of collection, but a significant increase q1.3%. was seen
on the day after collection Table 6; Shannon, 1962..

2.3.3. Catalase
The medium for dilution and storage of semen has significant effects on the integrity
of spermatozoa. Within an aerobic or even a partially aerobic system, the production of
reactive oxygen species is inevitable Mann and Lutwak-Mann, 1975; Fridovich, 1978,
1981; Mann et al., 1980.. The most damaging are the superoxide anion Oy 2 , peroxide
.
H 2 O 2 . and the hydroxyl free radical OH.. The reactions producing these free radicals
are more active when semen is stored at ambient temperatures than in frozen state.
Peroxide is the more pernicious of the pro-oxidants formed. The main source of
peroxide during storage of bovine semen at ambient temperature is the oxidative
de-amination of aromatic amino acids by a membrane bound AAAO as shown in the Eq.
1..

R P CH 2 CH NH 2 . P COOHq O 2 q H 2 O
Rsphenyl, p-hydroxyphenyl or indolyl groups .
R P CH COCOOHq H O q NH
AAAO
2 2 2 3

1.
The reaction was first described for bovine spermatozoa by Tosic and Walton 1946,
1950.. Subsequently, Shannon and Curson 1972b. demonstrated that AAAO activity
was restricted to the dead sperm population. The properties of AAAO are well
established. The enzyme is heat and acid labile: it is inactive in live spermatozoa and is

Table 6
Conception rates of semen diluted in 14G a diluent containing caproic acid 0.025%.. Data from Shannon
1962. and expressed as % NRR at 49 days
Age of semen 14G control. 14Gqcaproic acid
No. of inseminations % NRR No. of inseminations % NRR
618 h 224,275 64.1 41,997 63.7
2436 h 95,587 64.3 16,831 66.9 )
a
The 14G diluent is the precursor to the current CAPROGEN w medium containing all the basic
ingredients except caproic acid.
)
Significantly different to control p- 0.05..
R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353 33

Table 7
Effect of catalase in CAPROGEN w diluent on fertility of bovine semen stored at ambient temperature and
used on day of collection 618 h. and day after collection 2448 h.. Data from Shannon 1968. and fertility
was measured as % NRR at 49 days
Age of semen CAPROGEN w CAPROGEN w qcatalase
No. of inseminations % NRR No. of inseminations % NRR
Trial 1 618 h 94,671 63.9 16,117 64.7
2448 h 148,286 68.1 27,584 69.7 )
Trial 2 618 h 30,233 63.5 146,337 64.5
2448 h 44,126 66.2 230,107 68.1)
)
Significantly different to CAPROGEN w p- 0.05..

released from dead cell membranes in the presence of citrate. It is confined to the tail
region of bull spermatozoa and the activity of AAAO is also affected by the oxygen
tension in the diluent. It was established from kinetic studies that the response of this
enzyme increased with increasing temperature and duration of storage Shannon and
Curson, 1972b, 1982a,b.. The enzyme is specific for L-aromatic amino acids and in
particular, L-phenylalanine was significantly more toxic than L-tyrosine or L-tryptophan
Macmillan et al., 1972.. Egg yolk is an excellent source of aromatic amino acids,
particularly L-phenylalanine.
The levels of peroxide generated in the storage medium can be minimised by the
presence of catalase Shannon, 1968.. The significance of this reaction is that in the
breakdown of peroxide, half the amount of oxygen is returned back to the system Eq.
2..

2H 2 O 2 2H O q O
Catalase
2 2 2.
Two separate field trials substantiated the effect of catalase and the results are shown
in Table 7. With extended storage times, the beneficial effect of added catalase became
evident and the field trial clearly emphasises the need to either reduce or abolish
endogenous peroxide production during liquid storage of semen.

2.4. Factors influencing fertility after liquid storage

2.4.1. The effect of duration of storage on fertility of spermatozoa


Bovine spermatozoa stored at ambient temperature in CAPROGEN w diluent show a
slow decrease in motility over an extended period about 34 weeks., but the concomi-
tant decrease in fertility is significantly higher Fig. 2.. Fertility, as measured by
non-return rate NRR., is maintained for the first 3 to 5 days after dilution 69.9 " 1.2%.,
followed by an accelerating decrease in NRR until day 10 of storage 41.5 " 3.7%.
Vishwanath and Shannon, 1997.. Bartlett and Van Demark 1962. showed a similar
dichotomy in response to storage at 58C. Semen stored in a medium containing
bicarbonate, catalase and glucose and gassed with CO 2 had 56% motile spermatozoa
after 40 days of storage. However, fertility decreased in the first few days of storage and
was down on average by 814% by day 3 Bartlett and Van Demark, 1962..
34 R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353

Fig. 2. The effect of ambient temperature 18218C. storage in CAPROGEN w on sperm motility and % NRR
at 24 days. Data from Vishwanath and Shannon 1997..

The physiological reasons for this rapid decline in fertility of spermatozoa stored at
ambient temperature is presumed to be due to three factors: extracellular oxidative
stress, effects of seminal plasma and endogenous free radical production. The combined
effects of these three factors could lead to significant damage to spermatozoa. This is
perhaps the main reason why semen stored at ambient temperature is restricted in
shelf-life with a declining fertilising potential. Vishwanath and Shannon, 1997..

2.4.2. Effect of number of spermatozoa inseminated on fertility of liquid stored semen


An important factor that contributes to the fertility of diluted semen is the dose rate or
absolute sperm numbers in a given inseminate. Salisbury and Van Demark 1961. have
described the relationship between semen quantity sperm numbers. and semen quality
fertilising potential.. The central point in this concept is that up to a threshold level,

Fig. 3. NRRs 24 day. for different sperm concentrations in liquid semen inseminated on days 13 after
collection. Day 1 is the day after collection of semen. Data from Vishwanath et al. 1996..
R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353 35

fertility is under the influence of sperm numbers. This threshold varies significantly
between bulls. Once this level is attained, fertility is unaffected by further increases in
sperm concentration. This effect has been adequately demonstrated in field trials where
some of the inherent differences in the fertility of different bulls can be compensated for
by altering the absolute numbers of spermatozoa in the inseminate den Daas, 1992;
Shannon and Vishwanath, 1995.. The effect of sperm numbers being able to influence
fertility is a defined compensable element and increasing sperm concentration does alter
the probability of fertilisation Saacke et al., 1994; Shannon and Vishwanath, 1995..
However, increasing sperm numbers does not alter the rate of decrease in fertility after 5
to 6 days of storage at ambient temperature 10218C, Vishwanath, unpublished
information.. The change in NRR for different sperm concentrations in liquid semen is
shown in Fig. 3. The probability of fertilisation is quite high on the first day of use at all
three sperm concentrations 4, 6 and 8 millionrml., and they are not significantly
different from each other. On the contrary, with storage, the NRR is lower on days 2 and
3 at the lowest sperm concentration of 4 millionrml p - 0.05.. The reasons could be:

probability of fertilisation decreased due to lower sperm numbers;


effect of dilution compounding the sperm aging effect; and
bull = dose rate interaction.

3. Frozen storage of semen

Effective storage of semen in frozen state implies a complete arrest in the develop-
mental process of sperm cells that began in the testis and continued through the
epididymis and after ejaculation. The logical conclusion for this process is the presence
of spermatozoa in the female tract preparing for fertilisation. Watson 1995. described
the frozen preservation as a hiatus in the process that demarcates this continuum of
sperm development with a period of suspended animation that eventually leads to
successful fertilisation. There is an argument that spermatozoa stored at y798C in dry
ice. or at y1968C in liquid nitrogen. retain their fertilising potential indefinitely. Mazur
1980. proposed a time period of between 3000 and 10,000 years before genome
destruction, while insemination trials with frozen stored semen Salisbury and Hart,
1970. suggest a far shorter time period for survival at the above-mentioned low
temperatures with no change in fertilising potential. However, other evidence suggests
that this could have been due to inadequate maintenance of temperatures. With more
advanced cryogenic vessels, it is possible to maintain spermatozoa in a frozen state for a
very long period.
The fortuitous discovery of glycerol as an effective cryoprotective agent Polge et al.,
1949; Polge and Rowson, 1952. introduced a completely new system of semen storage,
a method which is widely in practice today. An axiom of semen freezing is that even
with the best preservation techniques and all the developments that have occurred over
the years, the best cell recovery after thawing is just over 50%.
36 R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353

3.1. Effects of freezing


The stresses associated with freezing are primarily the temperature changes that
spermatozoa are subjected to during the process of cooling, the injurious effects of the
media components and cryoprotectants themselves during the process, and finally the
effects of thawing. The basic principles of freezing were established quite early and
several authors have reviewed the physical effects of each of the processes mentioned
above Mazur, 1963, 1965; Watson, 1979, 1995.. The physical effects of cooling and
then freezing a cell suspension results in a number of changes in the external environ-
ment in terms of water and solute movement. The rate at which water moves out of the
cell during this process plays an important role in determining cooling rates, which have
to be optimised for cell survival after thawing Mazur, 1963, 1977.. The curves for
survival vs. cooling rate show a biphasic response. The optimum cooling rate is between
808Crmin and 1208Crmin with faster and slower cooling rates resulting in lower
survival Watson, 1979, 1995; Woelders, 1997.. This also has bearing on a period of
withholding times at different temperatures to allow for equilibration before freezing.
The purpose of this equilibration time at certain temperatures is to allow for the
translocation of water and thereby reduce the injurious effect of ice nucleation during
the freezing and thawing process.
3.2. Diluents and cryoprotectants for freezing of semen
The basic components of the diluents for freezing of semen are essentially the same
as those used for ambient temperature storage. The general requirements are:
ionic or non-ionic substances to maintain the osmolarity and to buffer the medium;
a source of lipoprotein or high molecular weight material to prevent cold shock, such
as egg yolk or milk;
glycerol, propane-diol or DMSO to offer cryoprotection;
glucose or fructose as an energy source; and
other additives such as enzymes and antibiotics.
Phillips 1939. made a crucial discovery when he found that egg yolk added to the
semen diluent had a beneficial effect on fertility. Phillips and Lardy 1940. recom-
mended the use of a diluent containing equal parts of phosphate buffer and egg yolk as a
protective agent against cold shock. Salisbury et al. 1941. presented conception results
from field trials with bovine semen diluted in an egg yolksodium citrate diluent EYC..
Since then, egg yolk in combination with Tris and citrate has been the most common
constituent in most freezing diluents for bovine semen. The reason why citrate is a better
ionic substitute than phosphate is because phosphate inhibits the oxidation of lactic acid
causing its accumulation White, 1956.. Davis et al. 1963a,b. developed a 0.2 M
Tris-buffered 20% egg yolk extender TRIS-EY.. When compared with EYC and skim
milk, TRIS-EY was found to be significantly better at maintaining post-thaw motility.
Steinbach and Foote 1964. confirmed this observation. Field trials with cooled but
unfrozen semen at sperm concentrations of 4 to 8 million rml failed to show evidence
of improved fertility with TRIS-EY compared with EYC Foote, 1970.. Compositions of
these diluents and their modifications are shown in Table 8.
R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353 37

Table 8
Composition of Tris- and citrate-based diluents for freezing bovine semen. All diluents contain antibiotics at
approximately 100,000 units of penicillin and dihydrostreptomycinr100 ml of the diluent. All values are given
in gr100 ml of the medium, unless otherwise mentioned
Diluent Trisyolk Trisfructose Citrateyolk Netherlands
component glycerol yolkglycerol fructoseglycerol extender
Davis et al., Steinbach and Steinbach and De Leeuw
1963a. Foote, 1964. a Foote, 1964. a et al., 1993.
Tris b 3.03 2.4 3.15 c
Glycerol 14 ml 8.8 ml 7.2 ml 6 ml
Fructose 1 1 1
Citric acid 1.69 1.354 1.23
monohydrate
Sodium citrate 2.32
dihydrate
Egg yolk 20 ml 20 ml 20 ml 20 ml
a
Original diluent modified by Foote 1970. to include fructose.
b
Tris hydroxymethyl. aminomethane.
c
Tris hydroxymethyl. aminomethane hydrochloride.

3.2.1. Egg yolk


There have been many attempts to find the protective component in egg yolk with the
aim to prepare a chemically defined diluent, and thus reduce the possibility of
transmission of harmful pathogens, the likely inconsistencies between different batches
of egg yolk and the yolk globules which interfere with microscopic examination of
diluted semen. Early experiments on the protective action of egg yolk soon led to
research in the specific components of egg yolk that might be responsible for the
cryoprotective action, namely, phosphatidylcholine lecithin., phosholipids, lipid ex-
tracts, lipoprotein fractions and specific lipoproteins Mayer and Lesley, 1945; Black-
shaw, 1954; Blackshaw and Salisbury, 1957; Martin, 1963; Lanz et al., 1965; Gebauer et
al., 1970.. There was a consensus among these studies that lecithin provides some
protection to spermatozoa during cold shock and freezing. One of the major problems
with lecithin is that it is insoluble in aqueous solutions, forming an unstable suspension.
It partly precipitates in the absence of vigorous stirring and can break down to form
lysolecithin which is toxic to spermatozoa Jones, 1976.. Synthetic liposomes made up
of dioleoyl phosphatidyl choline, phosphatidyl choline, phosphatidyl serine and combi-
nations thereof with cholesterol were effective in preventing cooling damage to sperma-
tozoa, but were a poor substitute compared to egg yolk in preventing freezethaw
damage De Leeuw et al., 1993..
The major component of egg yolk offering protection is a phospholipid moiety of a
low density lipoprotein fraction Pace and Graham, 1974; Foulkes, 1977; Watson, 1981..
Further analysis of the type of lipoprotein extracted from whole egg yolk by water,
saline and citrate revealed that the major extract is a cationic lipoprotein fraction that
preferentially bound to the sperm membrane and afford far greater protection. This
protection is assumed to result from the lipoprotein binding to the sperm membrane via
the charged protein moiety, which aids in stabilising the plasma membrane through the
38 R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353

Table 9
Recovery of spermatozoa and survival at 378C after cold shock in CAPROGEN w containing 5% whole egg
yolk or an equivalent amount of water-based lipoprotein extract WTM. of egg yolk. Data from Vishwanath et
al. 1992.
Diluent Recovery %. Survival h.
w
CAPROGEN no egg yolk. -5 )
2.5a
CAPROGEN w 5% egg yolk. 80 104 b
Water extract WTM. 80 116 c

Values with different superscripts differ, p- 0.05.

critical temperature zones, with the lipid portion acting as thermal insulation Vishwanath
et al., 1992.. This water-based fraction was significantly better than egg yolk in
maintaining survival of spermatozoa recovered after cold shock Table 9; Vishwanath et
al., 1992.. Field fertility with the same fraction in ambient temperature stored semen
where it was used as a thermal protectant also revealed a significant advantage Table
10..
Evidence suggests that the whole lipoprotein molecule is required to protect sperma-
tozoa during cold shock. Selective digestion of either the protein or the lipid fractions
did not offer the same protection against cold shock Watson, 1981; Prendergast et al.,
1994.. It is possible that the protein or the lipid fractions are unable to bind to the
membrane and offer thermal protection on their own.
The amount of egg yolk commonly used in freezing media is between 15% and 30%
on a volume to volume basis. This range in egg yolk concentration is based on results
obtained by Van Demark et al. 1957. where the percentage of motile spermatozoa after
freezing and thawing was similar for 15% and 30%, but was significantly lower at egg
yolk concentrations above and below this range.
3.2.2. Ions
The ions in the storage medium are not mainly for ionic strength, but more for
maintaining osmolarity Watson, 1979.. This purpose is adequately served by zwitteri-
onic buffers, amino acids, a-keto acids and a combination of salts and carbohydrates
Miller and Van Demark, 1954..
3.2.3. Cryoprotectants

3.2.3.1. Glycerol. The discovery of the protective action of glycerol Polge et al., 1949.
was an important technological breakthrough, as it reduced the mechanical damage to

Table 10
Fertility of semen from five sires diluted in CAPROGEN w containing 5% egg yolk or an equivalent amount of
water-based egg yolk extract WTM.. Data from Vishwanath et al. 1992.. Ejaculates were split between the
two treatments and fertility measured as % NRR at 24 days
Diluent No. of inseminations % NRR
w
CAPROGEN 5% egg yolk. 8849 66.6
Water extract WTM. 6195 68.5 )
)
Indicates significantly different to CAPROGEN w p- 0.05..
R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353 39

spermatozoa during the freezing process. Glycerol and other penetrating cryoprotectants
such as DMSO reduce cell damage by preventing the concentration effects of extracellu-
lar media. Colloids such as sucrose and dextran on the other hand, are non-penetrating
cryoprotectants inducing the formation of ice crystal lattices, which are thought to form
external shields around sperm membranes Karow and Webb, 1965; Schmehl et al.,
1986; Nicolajsen and Hvidt, 1994.. The role of cryoprotectants is a matter of substantial
debate and the current thinking on the nature of the protective effects of penetrating and
non-penetrating cryoprotectants are discussed by Watson 1995..
Glycerol is the most widely used cryopreservative for bull spermatozoa. Conventional
methods of freezing in 0.25- or 0.5-ml straws have used approximately 7% glycerol as
the optimum concentration for citrateyolk and Trisegg yolk extenders Pickett and
Berndtson, 1974, 1978; Rodriguez et al., 1975; Watson, 1979, 1990; De Leeuw et al.,
1993.. The optimum concentration of glycerol for freezing bovine semen is also
influenced by other components in the diluent Woelders et al., 1997.. Suffice to say,
glycerol concentrations for freezing have been between 0.25 M 2.25%. and 1 M 9%.
with many studies demonstrating toxicity beyond this concentration Fahy, 1986..

3.2.3.2. Sugars and polyols. Nagase et al. 1964. found that xylose, fructose, glucose,
galactose, maltose, sucrose and raffinose were all effective for rapid freezing of bull
semen. Sugars perform several functions in the diluent. They add osmotic pressure to the
medium and act as cryoprotectants Watson, 1979.. Polyols, like glycerol and several
sugars, form hydrogen bonds with the polar head groups of membrane lipids Crowe and
Crowe, 1984; Crowe et al., 1985.. The main effect of sugars and polyols is their ability
to replace the water molecule in the normally hydrated polar groups and this property
helps to stabilise the membrane during transition through the critical temperature zones
Woelders, 1997.. Sugars, like glycerol, alter the mechanical properties of the diluent by
increasing its viscosity. This prevents the eutectic crystallisation of solutes and increases
the glass-forming tendency of the medium, a property used increasingly in vitrification
media Nicolajsen and Hvidt, 1994.. A recent study Woelders et al., 1997., using
trehalose and sucrose, demonstrated a significant interaction between cooling rates and
the presence of sugars. In this study, an isotonic sugar medium where the Triscitrate
components were substituted with sucrose and trehalose was significantly superior to a
Triscitrateegg yolk medium in preserving acrosome integrity. The suggestion was
that by using a cooling rate in the optimum range of 761408Crmin a further
improvement in viability after thawing could be achieved. Trehalose is a common
substitute for water molecules in the dehydration process and is the sugar found in many
organisms that survive anhydrobiosis Woelders et al., 1997..

3.2.4. Milk diluents


Milk proved to be a suitable diluent for storage of bull semen both in the liquid and
frozen state Salisbury and Van Demark, 1961; Melrose, 1962.. A 10% whole milk or
skim milk diluent in combination with 7% glycerol and antibiotics has been very
successfully used as a diluent for freezing semen for many years. Along with the
Trisegg yolkcitrate diluent developed by Davis et al. 1963a., milk diluents have been
routinely used in many AI centres to freeze semen Almquist et al., 1954; Ahmad and
40 R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353

Foote, 1985.. Various studies have attempted to improve the whole milk or skim milk
extenders, but have generally been unsuccessful Foote et al., 1993.. The main disadvan-
tage with milk extenders is the poor visibility of spermatozoa under a microscope, which
makes post-thaw evaluations difficult. It is for this reason that egg yolk diluents in
combination with Tris and citrate are preferred.

3.3. Freezing protocols: processing, cooling rates, freezing and thawing

3.3.1. Initial processing and temperature equilibration


Methods of processing of semen immediately after collection vary considerably. It is
mostly guided by the methods of use that have suited the organisation, requirements for
disease control and semen export protocols. Based on these requirements, procedures
vary between organisations that are actively engaged in the international trade of bovine
semen. Semen is usually diluted at 308C to 378C with an equilibrating medium
containing all the basic constituents of the freezing diluent. In most cases, glycerol is
omitted from this medium. The ratio of dilution is approximately 1:5 semenrdiluent.
This procedure is necessary to provide a buffering medium for spermatozoa, some
antibiotic protection and to provide thermal insulation during the subsequent cooling.
The Certified Semen Services CSS, USA. procedure for entry of all imported semen
into the USA requires undiluted semen to be treated with antibiotics before initial
dilution Lorton et al., 1988.. It is generally considered that the slow cooling of diluted
semen to 58C before freezing has beneficial effects Ennen et al., 1976; Gilbert and
Almquist, 1978.. This period, during which glycerol is usually added is sometimes
erroneously referred to as the glycerol equilibration period. Glycerol equilibrates quite
rapidly across the cell membrane at 58C and can be added at any time during the cooling
period Martin, 1965; De Leeuw et al., 1993.. Once the diluted semen attains the
holding temperature of 58C, the mixture is extended to the final sperm concentration
with the same medium containing glycerol. All subsequent manipulations such as filling
and sealing into straws are usually conducted at 58C. The final sperm concentration per
milliliter of diluted semen differs between organisations. It also depends on stipulations
by importing countries. The relationship between the total number of spermatozoa per
breeding unit and threshold fertility is discussed in a later section. The average sperm
concentration is between 10 and 20 million per breeding unit, depending on the demand
of semen from individual sires.

3.3.2. Effect of cooling rates


A two factor theory accounts for the effects of cooling rates on spermatozoa Mazur,
1965; Mazur et al., 1972.. A slow cooling rate exposes spermatozoa to the solution
effects such as increasing salt concentration, increasing osmolality and changing pH.
Fast cooling may not allow intracellular water to pass out, leading to intracellular ice
nucleation Mazur, 1963, 1977; Mazur et al., 1972.. In practice, the cooling rate at
which freezing damage occurs in spermatozoa is several orders lower than the calculated
rate at which ice nucleation should occur. A recent report Woelders, 1997. shows a
clear relationship between survival of bull spermatozoa and cooling rate Fig. 4.. The
R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353 41

Fig. 4. Relation between % motile bull spermatozoa post-thaw and the cooling rate. Values are means"se for
five bulls. Data from Woelders 1997..

optimum cooling rate was 1008Crmin, and this is greater than the optimum of
26528Crmin suggested earlier for bovine spermatozoa Robbins et al., 1976.. A
further detailed study by Woelders et al. 1997. suggests a significant interaction
between the osmolality of the medium and cooling rate. Supra-optimum cooling rates
are tolerated at higher osmolalities where the medium contains sucrose or trehalose.

3.3.3. Methods of freezing


The technique of freezing semen in 0.25- or 0.5-ml straws in liquid N2 is now
universal Cassou, 1964.. This technology is well established and all the equipment
required for filling semen in straws and racking them up for freezing is readily available.
The appliances used for freezing of semen range from static freezers to controlled
programmable freezers. Although there has been considerable research in determining
optimum cooling rates and adapting cooling curves for individual bulls, there has been
very little progress in the practical sense. The programmable freezers currently available
cannot reliably alter or allow customisation of cooling rates for a large batch of straws in
a production environment Wagtendonk, personal communication.. Many semen pro-
cessing organisations continue to use static vapour freezers where the straws containing
semen are subjected to uncontrolled freezing conditions, depending on the distance of
the straw chamber from the liquid N2 layer. In these freezers, the rate of decrease in
temperature is between 1508Crmin and 3008Crmin. The advantage of the static vapour
freezer is that all the straws in any given freezing cycle are subjected to the same
cooling rates, because the straws are placed in one layer above the liquid N2 level. In
programmable freezers, the straws are placed in more than one layer, and this con-
tributes to considerable variation in cooling rates in individual layers within a freezing
cycle Wagtendonk, personal communication..

3.4. Thawing of semen

The general theory is that rapid thawing of semen is advantageous to prevent injury
during rewarming. Mazur 1984. observed that rapid thawing prevents the possibility of
re-crystallisation of water molecules, which could be injurious to cell membranes. A
42 R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353

Table 11
Fertility of frozen semen from nine bulls thawed at 358C or at ambient temperature. Data from Vishwanath
1998. and fertility expressed as % NRR at 24 days
Temperature Number of % NRR
of thawing inseminations
Ambient 1182 72.7"1.3
14"38C.
35"18C 1105 71.4"1.4

potential problem in slow thawing is the osmotic change due to ingress of water during
the process, as this is considered to be more damaging than water egress during freezing
Curry and Watson, 1994.. Studies by Holt and North 1994. have also confirmed this
observation with ram spermatozoa. There are some practical considerations regarding
the temperature at which semen used in field conditions should be thawed to obtain
maximum fertility. This is a question often asked with many anecdotal references to
higher fertility with semen thawed at higher temperatures ) 258C.. There are some
earlier studies evaluating fertility of semen thawed at temperatures of 308C, 158C and
48C Arnott, 1961.. The 6090 day NRR followed the trend of 48C ) 158C ) 308C in
thawing temperature. Other studies have also suggested a lower temperature of around
48C as favourable for thawing, compared to higher temperatures of around 158C Pickett
et al., 1965; Pickett and Berndtson, 1978.. A recent field trial to determine the effect of
thawing semen at 358C compared with ambient temperature 14 " 38C. showed no
difference in NRR between the two thawing temperatures Table 11; Vishwanath, 1998..

3.5. Re-dilution of bulk frozen semen

The concept of re-dilution of frozenthawed semen was first explored by James and
Fyvie 1955.. The idea was to develop it as a convenient method to harness the
out-of-season production potential of a bull. The initial trials were not very successful
and the fertilising ability of re-diluted semen could be retained for only a few hours. The
technique was later successfully modified for field use Shannon, 1972; Macmillan et
al., 1978.. In this system, the semen was frozen in straws at high sperm concentrations
in an egg yolkcitrateglycerol diluent and re-diluted with CAPROGEN w upon thaw-
ing Shannon, 1965. and before using as liquid semen. This technology was used very
efficiently for a number of years in many isolated areas in New Zealand. The AI
technician diluted the contents of a 0.25-ml straw in a test tube containing 5 ml of
CAPROGEN w and completed the days inseminations. Inseminations of 0.5 ml volumes
were made by using a syringe pipette. At the end of the day, the remaining diluted
semen was discarded, as earlier trials had shown a significant decrease in fertility of the
re-diluted semen stored overnight Shannon, 1978.. The process has been improved so
that large volumes of concentrated semen 525 ml. can be bulk frozen patent pending.
and re-diluted in the production laboratory on the days required for use. This is then
dispatched as standard liquid semen and used within 60 h of re-dilution. The sperm
concentration of the re-diluted material range between 6 and 10 million spermatozoarin-
R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353 43

Fig. 5. NRRs 24 day. for re-diluted frozen RDF. semen and liquid semen on days 13 after thawingrre-dilu-
tion and collection, respectively. Data from Vishwanath et al. 1996..

seminate. The results with re-diluted frozen semen are shown in Fig. 5. The percentage
NRR for the first 2 days of use was not significantly different for the two types of
semen, but was significantly lower on day 3 of use with re-diluted semen p - 0.05..
Two aspects that could potentially affect this system are the significant bull = sperm
concentration interaction, which determines the final sperm concentration in the insemi-
nate and, secondly, the bull = age of semen interaction, which limits the number of days
semen can be used in a diluted state in the field.

3.6. Effect of sperm numbers on fertility of semen stored in the liquid or frozen state

Bulls differ in their inherent fertility and follow similar fertility trends whether their
semen is diluted and stored in liquid or frozen state. This effect is shown in Fig. 6. The
percentage NRR of liquid and frozen semen for the same 11 bulls at optimum sperm
concentration liquid 2.5 millionrinseminate, frozen 20 millionrinseminate. range
between 62% and 72% Shannon and Vishwanath, 1995.. When sperm concentrations
were decreased to sub-optimum levels liquid 0.5 millionrinseminate, frozen 5
millionrinseminate., fertility declined by an average of 7% for liquid semen and by
7.9% for frozen semen. There was a significant bull = dose rate interaction in the frozen
semen system, which was not seen, in the liquid system p - 0.01.. This clearly
highlights the fact that semen ejaculates from different bulls inherently differ in their
susceptibility to freezing as measured by their NRR. Further evidence suggests that this
could be due to a reduced probability of fertilisation or an altered pattern of survival of
frozenthawed spermatozoa in the female reproductive tract Shannon and Vishwanath,
1995.. A similar bull = dose rate interaction for frozen semen was observed by den Daas
1992.. Much greater differences between bulls regarding NRR was seen at lower than
at higher sperm concentrations.
The true relationship between the number of spermatozoa inseminated and fertility
was first proposed by Salisbury and Van Demark 1961.. This was further explained by
a model proposed by Schwartz et al. 1981., which relates the number of spermatozoa to
the probability of conception, based on a Poisson distribution. The validity of the model
44 R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353

Fig. 6. Variation in NRR between bulls at optimum '. and suboptimum v . sperm concentrations in the
liquid A. and frozen B. treatments. Data from Shannon and Vishwanath, 1995..

was reaffirmed by the results of a series of field trials den Daas, 1992.. Bulls differ in
their maximum NRR value, and this is reached as the number of spermatozoa per
inseminates increases. The rate at which this maximum value is approached also differs
between bulls. The added variation to this is the inherent fertility difference between
sires that occur depending on time of insemination. There is a significant interaction
between insemination time, sperm concentration, stage of oestrus of the cow pre- or
post-oestrus. and fertility of individual sires Macmillan and Curnow, 1977..
The differences in sire fertility have been illustrated in two separate studies by
Macmillan and Watson 1975., and Macmillan and Curnow 1977.. The stage of oestrus
at insemination did not significantly influence the results for sires of above average
fertility, but below average sires had reduced conception rates with mid and early
oestrus inseminations. The observation suggests that there could be an in vivo decline in
fertilising ability of spermatozoa, which differs between sires.
R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353 45

The reasons for the differences in the response to dilution rate are not known. It is
possible that individual bulls react differently to a single freezing protocol that is
normally used by semen processing companies. Perhaps, customising freezing protocols
for individual bulls could help in reducing the dose rates of frozen semen Parkinson and
Whitfield, 1987; den Daas, 1992.. The fact that higher numbers of spermatozoa are
required for insemination with frozen semen due to losses in the freezing process is not
unexpected. The number of live spermatozoa required for insemination is far lower
when using liquid than frozen semen; at the same dose rate of 2 million motile sperm,
NRRs for liquid and frozen semen were 68.1% and 59.7%, respectively.

3.7. Adantages and disadantages of liquid stored and frozenthawed semen

The relative advantages and disadvantages in the field usage of liquid stored and
frozenthawed semen are listed in Table 12. The most obvious advantage of liquid
stored semen is one of sperm numbers, and this has been the main impetus in further
refining liquid semen technology for extensive use in New Zealand and other countries.
An average ejaculate of 5 ml containing 1.5 billion spermatozoarml will yield between
350 and 500 frozen semen straws at a dose rate of 15 to 20 million spermatozoarin-
seminate. On the other hand, the same ejaculate as liquid semen will yield 7500 straws
at a dose rate of 1 millionrinseminate and 3750 straws at the higher sperm concentra-
tion. The storage costs of liquid semen are very low. If semen utilisation is high within
the shelf-life period after dilution, the economic advantages are quite significant
Shannon, 1978; Curson et al., 1991.. The obvious disadvantage with liquid stored
semen is the limited shelf-life. By contrast, the most important advantage of frozen
thawed semen is the possibility of long-term storage capability. This allows for
extensive testing of the processed semen, and is a reliable method for genetic insurance
of valuable bulls. The biggest disadvantage in a high demand situation is the high sperm
numbers required for insemination to maintain the same level of fertility as for liquid
stored semen. In New Zealand, a unique system of seasonal breeding operates. More
than 2.5 million inseminations are performed in a 16- to 20-week period during the
Spring months of September to Summer Fig. 7.. More than 85% of these inseminations

Table 12
Relative advantages and disadvantages of liquid-stored and frozenthawed semen
Liquid-stored semen Frozenthawed semen
Adantages
Low sperm numbers. Long term storage.
High sire utilisation. Flexibility of use.
Inexpensive storage.
Ease of use in the field.

Disadantages
Limited shelf life. High sperm numbers.
Expensive to store.
46 R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353

Fig. 7. The distribution of inseminations in New Zealand during the spring mating period 1st September31st
December.. Data from Vishwanath et al. 1996..

are with liquid stored semen, which has a 4-day shelf-life Curson et al., 1991;
Vishwanath et al., 1996..

3.8. Semen packaging

The current methods for semen packaging are largely based on the French mini-straw,
the 0.25-ml pailette Cassou, 1964.. The procedure has been in operation for many years
and has generally worked well for packaging frozen and liquid semen in many countries
Chupin and Schuh, 1993.. The straw allows essential details of the sire to be recorded
and this has become mandatory for semen traded internationally. The packaging method
for liquid semen in New Zealand is the minitub straw sealed at both ends with glass
Germany..
beads Minitub,
Freezing of semen in pellets was first described by Japanese workers Nagase and
Niwa, 1964; Nagase et al., 1964. and is still used in some countries for storing semen
from young sires and for local use. This procedure is not widely practised because
details of the sire cannot be easily recorded on a pellet.

4. Commercially available diluents

Most AI companies prepare their own semen diluents with minor modifications to
suit their requirements. In the last few years, a few proprietary brands have been
available and they are listed below. The move to eliminate egg yolk from the system has
been quite strong because of a perceived health risk associated with a biological material
being included in the diluting media. However, suitable alternatives have not been as
successful as either milk or egg yolk van Wagtendonk-de Leeuw et al., 2000.. Some of
the diluents listed below are dual purpose and recommended for both liquid and frozen
storage of semen. The list is by no means exhaustive; nor has a detailed market survey
R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353 47

been conducted on the relative merits of all the media available. The common ones cited
in literature are the following.
1. Biladyl supplied by Minitub Germany and used with frozen semen. It consists
of two fractions A and B for step-wise dilution of semen with addition of egg yolk,
water and antibiotics as prescribed by CSS.
2. Triladyl supplied by Minitub Germany and used with frozen semen. This is
similar to Biladyl and is used as a one-step dilution medium.
3. Biociphos supplied by IMV, LAigle, France. It is a one-step dilution medium
containing soy bean extract, replacing the egg yolk fraction.
4. Laciphos supplied by IMV. It is a skim milk-based powder medium requiring
addition of water. Both Biociphos and Laciphos are supplied with antibiotic fractions
that satisfy either the USA CSS-certified antibiotic. or the EEC antibiotic formulae.
5. Tris concentrate Gibco BRL, Manufactured by Holland Genetics, The
Netherlands. This is a 5 = concentrate of the popular Tris-based medium Davis et al.,
1963a. and modified by De Leeuw et al. 1993.. It is the most common diluent for
freezing semen. The Tris diluent has been used also for liquid semen Foote, 1978..
6. CAPROGEN w concentrate concentrated liquid semen diluent for storage at
ambient temperatures 188C to 248C. supplied by Livestock Improvement, New Zealand.
It is a diluent extensively used for liquid storage of semen with no drop in fertility over
a 4-day period Curson et al., 1991..

5. Future directions
5.1. Liquid stored semen
Major gains can be made with liquid semen technology if the decline in fertility upon
storage at ambient temperature is halted or reduced. To this end, the physiological
processes that contribute to aging of spermatozoa upon storage in vitro need to be
understood. Reactive oxygen species, which are more likely to be generated in an
ambient temperature system have to be contained in the media in which the spermatozoa
are suspended. Intracellular activity could also contribute to the production of these free
radicals and the slowing down of respiratory activity could be beneficial.
5.2. Frozen semen
The future direction of research on freezing semen should be directed towards
improvement of freezing protocols to allow a lower number of spermatozoa to be
included per breeding unit. The current freezing protocols require from 40 to 100 million
spermatozoarml of diluting medium and each inseminate contains between 10 and 25
million spermatozoa. For any substantial gain to be made in this direction, future work
needs to concentrate on diluent composition and repeatable cooling rates to protect the
sperm cells during the freezing process.
5.3. Alternatie storage technologies
Storage of semen in frozen state is expensive, as liquid N2 and well-insulated
containers are essential. The on-going cost of storing semen in liquid N2 can be
48 R. Vishwanath, P. Shannonr Animal Reproduction Science 62 (2000) 2353

prohibitive. There has always been an interest in developing alternative strategies for
long-term storage of bovine spermatozoa, and experiments on desiccation, vitrification
and freeze drying have been attempted in the past with limited success Larson and
Graham, 1976; Jeyendran et al., 1981, 1983.. A recent review by Holt 1997. discusses
the future requirements for preservation of germplasm and how these strategies need to
be re-visited in the light of modern knowledge on the response of sperm membranes to
severe manipulations. The next breakthrough in semen storage technology will almost
certainly involve some of these strategies.

6. Concluding remarks
The use of liquid stored semen allows high utilisation of individual sires. This is
possible because of the low sperm numbers required to maintain fertility and the
extended shelf-life of up to 4 days of use in the field. If the effects of semen age can be
overcome, the efficiency and utilisation of liquid stored semen could be increased quite
significantly. The essential difference between liquid and frozenthawed semen lies in
the response of bulls to sub-optimum dose rates. While all bulls respond similarly to
sub-optimum dose rates with liquid stored semen, the response varies greatly when
frozenthawed semen is used. This means that higher than required dose rates are
recommended for frozenthawed semen because of the possibility of a bull = dose rate
interaction. The use of bulk-frozen semen re-diluted and used as liquid semen is an
option to combine the convenience of frozen semen technology with the efficiency of
liquid semen.

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