Animal Reproduction Science 172 (2016) 1–9

Contents lists available at ScienceDirect

Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Review article

Cryopreservation of bull semen: Evolution from egg yolk

based to soybean based extenders
S.S. Layek a , T.K. Mohanty a , A. Kumaresan a , J.E. Parks b,∗
a
Livestock Research Centre, National Dairy Research Institute, Karnal, 132 001 Haryana, India
b
Department of Animal Science, Cornell University, Ithaca, NY 14850, USA

a r t i c l e i n f o a b s t r a c t

Article history: Since the inception of bovine semen cryopreservation, egg yolk and milk based extenders
Received 12 January 2015 have been used to protect sperm from the detrimental effects of cooling and freezing. In
Received in revised form 19 April 2016 recent years, demand for alternatives to conventional commercial extenders has arisen
Accepted 29 April 2016
as the risk of introducing exotic diseases through transporting egg yolk based products
Available online 3 May 2016
has been recognized. Egg yolk can also interfere with sperm evaluation and the presence
of particulate material in the extender may reduce fertility. Soybeans contain lecithin, a
Keywords:
phospholipid fraction that can substitute for high molecular weight lipoprotein and phos-
Bovine semen
pholipids from egg yolk and prevent or ameliorate damage to the sperm plasma membrane
Sperm
Cryopreservation that occurs during extension, cooling, and cryopreservation. Soy lecithin based extenders
Extender have been evaluated for processing and freezing bovine semen, although extender from soy-
Egg yolk bean milk has not been studied as extensively. Commercially available soy lecithin based
Soybean extenders are used increasingly but remain under scrutiny and are not universally accepted.
Lecithin With these observations in mind, this review is intended to examine effects of conventional
cryopreservation procedures, methods of assessment, and potential for developing soybean
extract as an acceptable alternative to traditional egg yolk and milk based extenders for bull
sperm cryopreservation.
© 2016 Published by Elsevier B.V.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Factors affecting fertility of cryopreserved sperm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Role of extender in reducing cryoinjury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
4. Implications of using animal protein based extenders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
5. The evolution of soybean-based extenders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
6. Soybean extraction methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
7. Soybean extract as extender for semen cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
8. Soybean based extender as an alternative to animal product based extender . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

∗ Corresponding author at: 131 Morrison Hall, Animal Science Department, Cornell University, Ithaca, NY 14850, USA.
E-mail address: jep5@cornell.ed (J.E. Parks).

http://dx.doi.org/10.1016/j.anireprosci.2016.04.013
0378-4320/© 2016 Published by Elsevier B.V.
2 S.S. Layek et al. / Animal Reproduction Science 172 (2016) 1–9
1. Introduction
In modern cattle breeding, where artificial insemina-
tion (AI) is the most widely applied tool for facilitating
extensive utilization and distribution of semen from genet-
ically superior sires, cryopreservation is a critical part of
the process. The potential of artificial insemination can be
fully explored and attained only with frozen semen which
permits indefinite storage and distribution. With advances
in frozen semen technology, post-thaw semen quality has
improved significantly. Though the relationship between
semen quality and fertility remains difficult to ascertain,
lower conception with frozen compared to fresh semen is
generally assumed. A popular notion is that approximately
50% of sperm are rendered immotile by cryopreservation
and sperm that remain motile are compromised due to cry-
oinjury, leading to decreased fertilizing capacity (Watson,
1995; Watson, 2000). Advances in cryopreservation tech-
niques for bull semen have progressed slowly over the past
several decades and are currently fairly standardized.
Effects of cryopreservation on sperm function and fer-
tility have been studied extensively in many species,
particularly in the bull. Sperm ultrastructure, particularly
the plasma membrane and outer acrosomal membranes,
are compromised during cryopreservation. Induction of
premature capacitation (cryo-capacitation) and acroso-
mal reaction, altered mitochondrial function and thereby
reduced motility are among the most significant influ-
ences of cryopreservation on the sperm (Parks and Graham,
1992; Hinkovska-Galcheva et al., 1989; Muller et al., 1999;
Bailey et al., 2003). Cholesterol efflux from sperm and
influx of Na+ and Ca2+ ions are major contributors to cryo-
capacitation (Breitbart et al., 1984; Langlais and Roberts,
1985; Robertson et al., 1990; Bailey and Buhr, 1994; Bailey
et al., 1994; Lin and Kan, 1996; Parrish et al., 1999). Reactive
oxygen species (ROS) released from dead or dying sperm
are also of concern due to their adverse effects on the live
sperm subpopulation in the frozen semen dose (Shannon
and Curson, 1972; Kodama et al., 1996; de Lamirande et al.,
1998; Upreti et al., 1998).
Numerous approaches have been attempted to reduce
sperm damage by modifying extenders and freezing pro-
tocols. Addition of cryoprotectant agents such as glycerol,
and other components such as egg yolk, milk, bovine serum
albumin, polyvinyl alcohol and liposomes to extenders
have been reported to reduce the detrimental effects of
freezing and thawing sperm (Batellier et al., 2001). Exten-
ders protect sperm from cold shock, osmotic stress, and
alterations in membrane fluidity and permeability plus
provide energy substrates for sperm metabolism (Batellier
et al., 2001; Medeiros et al., 2002). A variety of extenders
have been used successfully for bull semen cryopreserva-
tion such as citrate-sugar-, lactose-, saccharose-milk-, egg
yolk- and some plant base extenders. Variants of Tris-egg
yolk- and milk-based extenders have emerged as universal
extenders for bull semen cryopreservation (Medeiros et al.,
2002; Barbas and Mascarenhas, 2009; Singh et al., 2012).
However, with increasing emphasis on biosecurity
issues and controlling disease with international semen
shipment, egg yolk extenders have become suspect for
facilitating the transmission of diseases. Escherichia coli,
Staphylococcus, Streptococcus, Pseudomonus, Haemophilus,
Salmonella, Avian influenza, Campylobacter, Listeria as well
as Mycoplasma can be transmitted by egg yolk (Thibier
and Guerin, 2000). Besides the risk of disease transmis-
sion, particulates in yolk and milk pose difficulty in semen
evaluation and quality control (Stradaioli et al., 2007).
Growing concern over issues with egg yolk as a semen
extender components motivated consideration of alter-
nate plant-based extenders that effectively maintain sperm
viability and fertility while minimizing the risk of dis-
ease transmission. Among plant based extenders, soybean
contains lecithin, a substitute for high molecular weight
lipoprotein in egg yolk that can prevent or repair damage to
the sperm plasma membrane during cryopreservation. The
potential of soybean based extenders for replacing egg yolk
extenders has been investigated by several researchers.
Most studies have tested the efficacy of commercially avail-
able soy lecithin based extenders (Hinsch et al., 1997;
Bousseau et al., 1998; Van Wagtendonk-de Leeuw et al.,
2000; Aires et al., 2003; Muiño et al., 2007; Arifiantini and
Yusuf, 2010; Akhter et al., 2011) but only sporadic stud-
ies have been reported so far using total soybean extract
as a base medium for bull semen extender (Pankaj, 2006;
Kasimanickam et al., 2011; Singh et al., 2012). Further, no
standard protocol had been developed to date for extract-
ing soybean milk for use in bull semen extender or other
applications.
The present review intends to elaborate upon different
facets of cryopreservation including the effects, assessment
and control of cryoinjury, and then consider the effec-
tiveness of extenders currently used in commercial semen
processing and the potential for soybean milk based exten-
der.
2. Factors affecting fertility of cryopreserved sperm
Successful sperm cryopreservation depends upon sev-
eral interrelated factors, including initial quality of sperm,
extender composition, cryoprotectant, cooling protocol,
packaging, thawing rate, and interaction of these compo-
nents, as well as individual animal variation (Cooter et al.,
2005; Andrabi, 2007; Clulow et al., 2008). Some loss in
sperm viability is inevitable due to the cumulative effects of
semen processing procedures prior to and during the actual
freezing process. Moreover, reduced fertility with cryopre-
served semen suggests that post-thaw sperm function is
compromised even in the viable subpopulation post-thaw
(Samper et al., 1991; Watson, 1995; Watson, 2000).
To attain successful fertilization, sperm must remain
progressively motile, able to produce energy in the form
of ATP for cellular processes, maintain plasma membrane
and acrosomal integrity and retain enzymes necessary for
oocyte penetration. Disruption of any of these sperm fea-
tures during semen processing and cryopreservation will
likely compromise fertilizing ability. A well-established
effect on sperm membrane integrity and function is
through the fate of intracellular water during semen freez-
ing. As the temperature of extended sperm is reduced
to below freezing, the extender undergoes rapid cooling.
As the temperature is reduced further, extracellular ice
crystals begin to form from the supercooled water in the
 S.S. Layek et al. / Animal Reproduction Science 172 (2016) 1–9
surrounding medium. Ice formation increases the concen-
tration of solutes in the extender, creating an osmotic
gradient across the membrane and thus dehydration of
sperm (Woelders, 1997; Watson, 2000; Andrabi, 2007). The
extent of water efflux from the sperm depends upon the
cooling rate. The slower the rate, the greater the efflux
of water, and hence cellular dehydration. At more rapid
freezing rates, the probability of intracellular ice formation
(IIF) increases, causing physical damage that is generally
lethal (Hammerstedt et al., 1990). Cell injury due to dehy-
dration is reduced with rapid cooling. Therefore, optimal
cell survival during cryopreservation requires an optimal
cooling rate that balances the effects due to slow versus
rapid cooling.
Coincident with sperm damage due to IIF and solute
effects are cryo-capacitation like changes that include tyro-
sine phosphorylation (Leyton and Saling, 1989; Breitbart
and Naor, 1999; Bailey et al., 2000; Tardif et al., 2001;
Kumaresan et al., 2011, 2012a,b), formation of reactive
oxygen species (specifically superoxide anion (O2 − ), hydro-
gen peroxide (H2 O2 ), and nitric oxide (NO) production),
(Slaweta and laskowska-klita, 1985; Parks and Lynch,
1992; de Lamirande et al., 1997; Mazur et al., 2000;
Chatterjee et al., 2001; Aitken and Baker, 2004) and sperm
plasma membrane lipid scrambling and destabilization
(Hammerstedt et al., 1990; Medeiros et al., 2002; Guthrie
and Welch, 2005) which can lead to loss of membrane
integrity and premature acrosome reaction.
3. Role of extender in reducing cryoinjury
The role of extenders in semen preservation is primarily
two-fold—to extend the fertile life of sperm (indefinitely
in the case of frozen semen) and to greatly extend the
genetic potential of individual males through artificial
insemination (A. I.) (Foote and Bratton, 1950). An exten-
der must first meet the basic physiological requirements
of sperm— iso-osmotic, near-neutral pH with appropri-
ate buffer, metabolizable substrate (Salamon and Maxwell,
2000). For low temperature storage, addition of cryoprotec-
tive agents to semen is essential to protect sperm from cold
shock during cooling to near 0 ◦ C and from solute effects
and intracellular ice formation during freezing. The for-
mer damage appears to be a function of the thermotrophic
phase transition of sperm membrane phospholipids from
a fluid to more rigid state which alters the membrane
environment for various transport proteins and enzymes
(Harrison and White, 1972; Hammerstedt et al., 1990;
Parks and Graham, 1992; Noiles et al., 1993; Holt and North,
1994; Watson, 1995; Noiles et al., 1995; Maxwell and
Johnson 1997; Collin et al., 2000). The latter includes both
thermotrophic and lyotropic (dehydration) phase transi-
tions and is even more disruptive (Mazur et al., 1970;
Mazur, 1984; Drobnis et al., 1993).
Numerous extenders combining various components
(sugars, electrolytes, buffers, egg yolk, milk and milk prod-
ucts, soy lecithin, and permeating cryoprotectants) have
been formulated, evaluated, and utilized with various suc-
cess for bull semen cryopreservation (Batellier et al., 2001).
The more typical extenders for freezing sperm include the
following:
3
a non-permeating cryoprotectant (milk or milk prod-
ucts; egg yolk or yolk derivatives such as low density
lipoproteins or phospholipids); a permeating cryoprotec-
tant (most commonly glycerol); an organic buffer (typically
Tris-hydroxymethylaminomethane); one or more sugars
as energy substrate or osmoticum (glucose, fructose, lac-
tose, raffinose, saccharose, or trehalose); solutes to adjust
pH and osmolarity (sodium citrate, citric acid); and antibi-
otics (penicillin, streptomycin).
Egg yolk based extenders with glycerol were among the
first to provide adequate post-thaw motility and fertility
for commercial success (Phillips and Lardy, 1940, Salisbury
et al., 1941; Almquist et al., 1949; Foote and Bratton, 1950;
Foote et al., 1960; Davis et al., 1963; Martin, 1963, 1965;
Gebauer et al., 1970) and which evolved as a universal
extender for cryopreservation of bovine semen (Iritani,
1980). While essential for successful sperm freezing (Noiles
et al., 1995; Liu and Foote, 1998), glycerol did pose some
detrimental effects on sperm including osmotic stress,
changes in membrane organization, and altered membrane
fluidity and permeability which compromised sperm func-
tion (Hammerstedt and Graham, 1992; Watson, 1995).
Thus, determining an optimum glycerol concentration and
equilibration time was crucial in extender development
to realize its benefit and ameliorate these side effects.
(Rodriguez et al., 1975; Cochran et al., 1984; De Leeuw et al.,
1993).
Numerous semen extenders have been developed over
the past half century based on the discoveries and princi-
ples mentioned above and have been used with variable
success over a wide range of species (Table 1)
4. Implications of using animal protein based
extenders
For the last 65 years, egg yolk (alone or in combina-
tion with milk) with added glycerol has formed the basis
for the most commonly used extenders for sperm cryop-
reservation (Phillips and Lardy, 1940; Salisbury et al., 1941;
Almquist et al., 1949; Foote and Bratton, 1950; Foote et al.,
1960; Davis et al., 1963; Martin, 1963, 1965; Gebauer et al.,
1970; Rodriguez et al., 1975; Iritani, 1980; Cochran et al.,
1984; De Leeuw et al., 1993; Vishwanath and Shannon,
2000; Holt, 2000; Stradaioli et al., 2007). However, con-
cerns have been raised in recent years over the use of egg
yolk in particular as a semen extender component for the
following reasons:
a Egg yolk represents a risk of bacterial or xenobiotic
contamination and as a consequence, the potential for
introducing exotic diseases through semen distribution
domestically and internationally.
b Endotoxins produced by such contaminants reduce
potential fertilizing capacity of sperm (Bousseau et al.,
1998; Aires et al., 2003).
c Presence of lipid vacuoles and other particulates in egg
yolk extenders interfere with microscopic evaluation and
biochemical and metabolic assays of extended semen
(Wall and Foote 1999; Vishwanath and Shannon, 2000;
Amirat et al., 2004; Singh et al., 2012; Singh et al., 2013).
4 S.S. Layek et al. / Animal Reproduction Science 172 (2016) 1–9
Table 1
Different constituents used in extenders for cryopreservation of bovine semen.
Component Constituents
Buffering system Tris (hydroxymethyl) amino-methane (Tris), Citric
acid, Sodium phosphate
Cold shock protection and cryoprotectant Glycerol, Egg yolk, Milk, Low density lipoproteins,
Soy-lecithin
Metabolizable substrates Fructose, Glucose
Antibiotics Different combinations of following: Streptomycin,
Penicillin, Polymixin B, Gentamycin, Tylosin,
Lincomysin and Spectinomycin
d The diverse and variable composition of particulates in
egg yolk makes quality control of yolk-based extenders
difficult. (Bousseau et al., 1998).
e Apart from sanitary concerns, egg yolk and milk-
based extenders may alter the structural and functional
integrity of sperm post-thaw (Kampshmidt et al., 1953;
Pace and Graham, 1974; Watson and Martin, 1975;
Karabinus et al., 1991; Cheng et al., 1998; Möstl et al.,
2001; Pugliesi et al., 2012).
Based on these issues and others, concern over the
use of animal-based products and the need for a defined,
pathogen-free alternative in semen extenders have gained
increasing attention in the artificial breeding indus-
try (Hinsch et al., 1997; Van Wagtendonk-de Leeuw
et al., 2000; Gil et al., 2000; Aires et al., 2003; Muiño
et al., 2007; Arifiantini and Yusuf, 2010; Akhter et al.,
2011; Kasimanickam et al., 2011; Singh et al., 2012;
Kasimanickam et al., 2012; Akhter et al., 2012; Singh et al.,
2013).
5. The evolution of soybean-based extenders
Soybean (Glycine max) is an annual leguminous
plant that originated in northeastern China (Singh and
Hymowitz, 1999) and has become a major source of dietary
protein in domestic animals and humans. Soybean seeds
contain an average of 36–38% protein, 30% carbohydrate,
References
Salisbury et al. (1941), Phillips and Lardy
(1940), Foote et al. (1960), Davis et al. (1963),
Martin (1963, 1965), Gebauer et al. (1970),
Rodriguez et al. (1975), Cochran et al. (1984),
De Leeuw et al. (1993)
Phillips and Lardy (1940), Salisbury et al.
(1941), Polge et al. (1949), Foote et al. (1960),
Michajilov (1950), Thacker and Almquist
(1953), O’Dell and Almquist (1957), Davis et al.
(1963), Martin (1963,1965), Gebauer et al.
(1970), Rodriguez et al. (1975), Milovanov and
Sokolovskaja (1980), Cochran et al. (1984),
Hammerstedt et al. (1990), De Leeuw et al.
(1993), Van Wagtendonk-de Leeuw et al.
(2000), Moussa et al. (2002), Aires et al. (2003),
Amirat et al. (2004), Muiño et al. (2007),
Stradaioli et al. (2007), Barbas and
Mascarenhas (2009), Vera Munoz et al. (2009),
Arifiantini and Yusuf, (2010), Amirat-Briand
et al., (2010), Akhter et al. (2011),
Kasimanickam et al. (2011), Akhter et al.
(2012), Kasimanickam et al. (2012), Singh et al.
(2012), Singh et al. (2013)
Salisbury et al. (1941), Foote et al. (1960),
Martin (1963), Davis et al. (1963), Martin
(1965), Gebauer et al. (1970), De Leeuw et al.
(1993)
Almquist et al. (1949), Foote and Bratton
(1949), Foote and Bratton (1950), Howard et al.
(1982), Ahmad and Foote (1985), Shin et al.
(1988), Lorton et al. (1988), Eaglesome and
Garcia (1995), Hasan et al. (2001)
19% oil, 9% crude fibre and 5% ash on a dry weight basis
(Brumm and Hurburgh, 2002).
Soybean proteins are a complex mixture of polypeptides
broadly categorized as metabolic enzymes, structural pro-
teins including both ribosomal and chromosomal, intrinsic
membrane proteins and storage proteins (Zarkadas et al.,
2007). The two major multi-subunit storage proteins of
soybeans are the salt soluble glycinin (360,000 Da) and ␤-
conglycinin (180,000 Da), which account for 40% and 25%
of the total seed endosperm protein, respectively (Zarkadas
et al., 2007, Speroni et al., 2010). The genetic background
of each variety is extremely important in determining seed
composition (Nielsen et al., 1997). The composition of stor-
age proteins also varies with maturity, nutrient supply
from the soil, fertilizer treatment (Krishnan, 2000) and
with environmental factors. In addition to these two stor-
age proteins, there are other known bioactive proteins
including the Kunitz trypsin, Bowman–Birk and related
protease inhibitors, ␤-amylases, lipoxygenases, urease and
seed lectins, which each account for about 2–5% of the total
seed protein in soybeans (Liener, 1994a,b; Speroni et al.,
2010).
Soybean lipids are present mainly in the form of
triglycerides in oil bodies. Oil bodies in soybeans are rel-
atively homogenous in size ranging between 0.2–0.5 ␮ in
diameter. Refined soybean oil contains more than 99%
triglycerides. Most of the fatty acids in soybean are unsat-
urated with linoleic species followed by oleic, palmitic,
linolenic and stearic acid. Phospholipid content of soybean
 S.S. Layek et al. / Animal Reproduction Science 172 (2016) 1–9
is only 1–3%. About 35% of total phospholipid is phos-
phatidylcholine (lecithin), 25% phosphatidylethanolamine,
15% phosphatidylinositol, 5–10% phosphatidic acid with
other minor phospholipids. The term lecithin generally
refers to the entire phospholipid fraction. Lecithin can eas-
ily be extracted from soybean chemically (using hexane) or
mechanically (by grinding and extraction). Lecithin has low
solubility in aqueous solutions, forming multilamellar lipo-
somes, bilayer sheets, micelles or other lamellar structures
depending on hydration and temperature.
Mature soybean contains small amount of di- and
oligosaccharides (2.5–8.2% sucrose, 0.1–0.9% raffinose and
1.4–4.1% stachyose) and trace amounts of monosaccha-
rides, such as glucose and arabinose.
Soybean seeds are known to contain different anti-
nutritive factors, such as trypsin inhibitors, phytic acid,
raffinose and stachyose, many of which lose their effects
after processing (Becker-Ritt et al., 2004; Kumar et al.,
2006). Soybean contain lipoxygenase, an iron containing
dioxygenase that catalyses the oxidation of unsaturated
fatty acids resulting in the formation of aldehyde and
ketone compounds (Rackis et al., 1979). Lipoxygenase in
soybean seeds is present in the form of three isozymes
i.e., Lox-I, Lox-II and Lox-III. Though trypsin inhibitor is
heat-labile, heat-treatment insolubilizes the much-valued
proteins (Anderson, 1992) and, more importantly, exces-
sive heat-treatment can cause loss of essential amino acids
in soy proteins (Rios-Iriarte and Barnes, 1996; Kumar et al.,
2003).
6. Soybean extraction methods
Soy milk is an aqueous extract of soybeans, first used in
China during the second century B. C. (Shi and Ren, 1993)
and closely resembling dairy milk in appearance and com-
position. Soy milk is composed of 90.8 gm water, 3.6 gm
protein, 2 gm fat, 2.9 gm carbohydrate, 0.5 gm ash in 100 gm
of extract. Unsaturated fatty acid comprises 52–60% of the
total fatty acid. In the traditional Chinese method, whole
soybeans are soaked overnight, then washed and ground
with 8–10 vols of fresh water. The slurry is then filtered
through muslin cloth. A slightly modified Japanese version
of the extraction method includes heating the slurry prior
to filtration (Shurtleff and Aoyagi, 1979)
Wilkens et al. (1967) at Cornell University, developed
a method for soybean extraction referred to as the Cornell
or hot grinding method, in which unsoaked, dehulled soy-
beans were ground in a pre-heated grinder with hot water.
The temperature of the slurry was maintained between
80 and 100 ◦ C in the grinder to deactivate lipoxygenase
activity, and then the slurry was boiled for 10 min with stir-
ring. Solids are then removed by centrifugation or filtered
through a filter press. To completely control the enzyme
action during soybean grinding, Nelson et al., 1976 at the
University of Illinois introduced the pre-blanch or Illinois
method in which presoaked soybeans were blanched for
10 min in boiled water or dry beans were placed in to hot
water for 20 min. This method or its variants are most com-
monly used for soy milk production. The beans are then
drained and ground with sufficient cold water to make
12% bean solids; the slurry is then heated to 93.3 ◦ C and
5
homogenized. In some cases 0.25–0.50% sodium bicarbon-
ate solution is used instead of water during soaking and
blanching and pH is later neutralized with HCl.
Johnson et al. (1981) published an alternative method
for producing soy milk in which soybeans are first ground
into flour, which is then mixed with hot water to form
a slurry. The slurry is rapidly subjected to direct steam
infusion at 154 ◦ C for 30 s to inactivate lipoxygenases. The
cooked slurry is finally cooled, adjusted to 10% solids with
water and centrifuged.
7. Soybean extract as extender for semen
cryopreservation
Several modifications for using soybean extract as an
extender for semen cryopreservation have been reported.
Singh et al. (2012) has modified the method first described
by Nelson et al. (1976) in which 50 gm of soybeans were
weighed and soaked overnight in 0.5% NaHCO3 solution.
Soaked beans were then ground in a high-speed homoge-
nizer and filtered through muslin cloth. The final volume of
stock soybean extract was adjusted to 300 ml with distilled
water. Soybean extract was autoclaved and stored at 4 ◦ C.
This method was further modified by baking 50 gm of soy-
bean in microwave before processing. Kasimanickam et al.
(2011) in his experiment in ram semen prepared soybean
extract by dissolving 11 gm of commercially available soy-
bean extract powder in 80 ml distilled water. Twenty ml of
Tris-fructose-citric acid solution was added and the final
extender was centrifuged at 4324g for 20 min and then fil-
tered through a sterile bottle top filter with a 0.4 ␮m pore
size filter.
8. Soybean based extender as an alternative to
animal product based extender
Soybean by-products, especially soy lecithin, have
emerged as a suitable substitute for the egg yolk in the
conventional egg yolk based extenders in the quest to
develop non-animal based alternative commercial semen
extenders. Numerous studies have been reported uti-
lizing soy based extenders with varied success (Hinsch
et al., 1997; Bousseau et al., 1998; Van Wagtendonk-de
Leeuw et al., 2000; Gil et al., 2000; Aires et al., 2003;
Muiño et al., 2007; Arifiantini and Yusuf, 2010; Akhter
et al., 2011; Kasimanickam et al., 2011; Singh et al., 2012;
Kasimanickam et al., 2012; Akhter et al., 2012; Singh et al.,
2013) primarily centered on the commercially available soy
lecithin based extenders (Biociphos, Bioexcell, Andromed
and others). Very few studies using a crude soybean extract
have been reported (Kasimanickam et al., 2011; Singh et al.,
2012; Kasimanickam et al., 2012; Singh et al., 2013).
Hinsch et al. (1997) compared Biociphos (soybean
extract based) with Triladyl (egg yolk based) extender by
evaluating viability, acrosomal status, sperm motility and
60–90 days non return rate in Holstein Friesian bulls. There
was no significant difference in these parameters between
extenders. In another experiment conducted in Holstein
bulls (Van Wagtendonk-de Leeuw et al., 2000), Biociphos
produced similar 56 day non return rate with Tris egg yolk
extender. Soy lecithin based extender (Andromed) per-
6 S.S. Layek et al. / Animal Reproduction Science 172 (2016) 1–9
formed better than egg yolk based extender in freezing
semen from Holstein Friesian bulls in the study conducted
by Aires et al. (2003) in. Contrary results were also reported
using both Andromed and Biociphos Plus in comparison
to Biladyl (Tris-egg yolk based extender) where egg yolk
based extender supported greater sperm survival (Muiño
et al., 2007; Arifiantini and Yusuf, 2010). Bousseau et al.
(1998) found that microbiological contamination in Bio-
ciphos plus (lecithin-glycerol extender) was nil whereas
bacteriological contamination was moderate in egg yolk
diluents (Triladyl) and in egg yolk + milk-based extender
(Laciphos). However, there was no difference in fertilizing
capacity between these extenders.
Akhter et al. (2011, 2012) and Singh et al. (2012) evalu-
ated effects of soybean extract–based extenders on Murrah
buffalo bull semen and found that soybean extract can
be a potential substitute for egg yolk for semen cryop-
reservation. Akhter et al. (2012) also reported comparable
fertility with soy lecithin based extender. Singh et al. (2013)
standardized the proportion of soy extract and glycerol
to be used in extender for cryopreservation of Holstein
Friesian crossbred semen and reported comparable post
thaw motility, viability, membrane integrity, acrosome
integrity and cryocapacitation as compared to Tris-egg yolk
semen extender.
In a study conducted by our group (Layek et al. unpub-
lished data), we standardized the extraction method for
soybeans used for preparing extender for bull semen cry-
opreservation. Soybean extract supported sperm functions
during liquid semen storage and post-thaw. Further, soy
based extender maintained in vitro fertilizing ability of cry-
opreserved sperm. However, membrane lipid packing and
fluidity was altered based on merocyanine staining, indi-
cating that further study is needed for making soybean
extract a useful substitute for conventional or soy lecithin
based bovine semen extenders.
Soybean based extenders are also used in human (Reed
et al., 2009; Jeyendran et al., 2008), ram (Gil et al., 2003;
Kasimanickam et al., 2011), dog (Kasimanickam et al.,
2012), and North American bison semen (Krishnakumar
et al., 2011) where sperm functions during liquid semen
preservation or post-thaw are improved or comparable to
existing egg yolk based extenders.
9. Conclusions
With the discovery of glycerol as an effective cry-
oprotective agent for mammalian sperm and the utility
of liquid nitrogen for effective storage and distribution
of frozen semen, the use of artificial insemination has
become the oldest, most useful and widespread repro-
ductive biotechnology for the genetic improvement of
livestock, especially dairy cattle. Equally important, how-
ever, has been the discovery of media components such as
egg yolk, milk products, and most recently soy lecithin that
protect sperm not only during cooling but freezing as well.
Semen quality and fertility of frozen semen have plateaued
somewhat with conventional extenders and semen pro-
cessing methods. However, a concern over the potential
of conventional animal-based extenders that include egg
yolk or milk by-products as a vector for spreading disease
has generated increased interest in developing non-animal
based semen extenders. Studies utilizing commercially
available soy lecithin based extenders demonstrate the
potential of soy by-products in plant-based semen exten-
der. However the precise composition, cost, and validation
of post-thaw semen quality and fertility have thus far lim-
ited the broad acceptance of soy-based extender as the
method of choice in the cattle A.I. industry. Use of soy
milk as a semen extender component shows some poten-
tial as a simple, inexpensive alternative to animal-based
extender by-products, but requires further optimization
during semen cooling and freezing to become an effective,
viable alternative to extenders presently used in commer-
cial semen processing.
Acknowledgement
The authors express their sincere gratitude to Dr. A.
K. Srivastava, Director and Vice Chancellor, National Dairy
Research Institute, Karnal and Dr. John Parks, Cornell Uni-
versity for providing all research facilities and funding
for the successful completion of this study. Thanks to Dr.
Michael Kaproth and his staff at Genex Cooperative, Inc.,
Ithaca, NY for providing fresh bull semen, technical sup-
port, and partial funding for evaluating soybean milk as a
semen extender. The visit of Dr. Layek to Cornell Univer-
sity was funded through a Fulbright Nehru Doctoral and
Professional Fellowship from J. William Fulbright Foreign
scholarship Board. The first author also acknowledges the
support of Department of Science & Technology, Govt. of
India in the form of INSPIRE-DST Fellowship.
Conflict of Interest for submission Transition from egg
yolk to soybean based exextenders. Layek, Siddhartha et al.,
December 2014
Reviewers were selected based on their current interest
or previous work in the subject matter of the manuscript.
We do not believe there is any conflict of interest with the
3 prospective reviewers that we have suggested.
References
Ahmad, K., Foote, R.H., 1985. Post-thaw survival and fertility of frozen
bull spermatozoa treated with antibiotics and detergent. J. Dairy Sci.
69, 535–541.
Aires, V.A., Hinsch, K.D., Mueller-Schloesser, F., Bogner, K.,
Mueller-Schoedder, S., Hinsch, E., 2003. In vitro and in vivo
comparison of egg yolk-based and soybean lecithin-based extenders
for cryopreservation of bovine semen. Theriogenology 60, 269–279.
Aitken, R.J., Baker, M.A., 2004. Oxygene stress and male reproductive
biology. Reprod. Fertil. Dev. 16, 581–588.
Akhter, S., Ansari, M.S., Rakha, B.A., Ullah, N., Andrabi, S.M.H., Khalid, M.,
2011. In vitro evaluation of liquid-stored Buffalo semen at 5 ◦ C
diluted in soya lecithin based extender (Bioxcell® ) tris-citric egg
yolk, skim milk and egg yolk-citrate extenders. Reprod. Domest.
Anim. 46, 45–49.
Akhter, S., Ansari, M.S., Andrabi, S.M., Rakha, B.A., Ullah, N., Khalid, M.,
2012. Soya-lecithin in extender improves the freezability and
fertility of buffalo (Bubalus bubalis) bull spermatozoa. Reprod.
Domest. Anim. 47 (5), 815–819.
Almquist, J.O., Glantz, P.J., Shaffer, H.E., 1949. The effect of a combination
of penicillin and streptomycin upon the livability and bacterial
content of bovine semen. J. Dairy Sci. 32, 183–190.
Amirat, L., Tainturier, D., Jeanneau, L., Thorin, C., Geı́rard, O., Courtens,
J.L., Anton, M., 2004. Bull semen in vitro fertility after
cryopreservation using egg yolk LDL: a comparison with optidyl® , a
commercial egg yolk extender. Theriogenology 61, 895–907.
 S.S. Layek et al. / Animal Reproduction Science 172 (2016) 1–9
Amirat-Briand, L., Bencharif, D., Vera-Munoz, O., Pineau, S., Thorin, C.,
Destrumelle, S., Desherces, S., Anton, M., Jouan, M., Shmitt, E.,
Tainturier, D., 2010. In vivo fertility of bull semen following
cryopreservation with an LDL (low density lipoprotein) extender:
preliminary results of artificial inseminations. Anim. Reprod. Sci.
122, 282–287.
Anderson, R.L., 1992. Effect of steaming on soybean proteins and trypsin
inhibitors. J. Am. Oil Chem. Soc. 69, 1170–1176.
Andrabi, S.M.H., 2007. Fundamental principles of cryopreservation of Bos
taurus and Bos indicus bull spermatozoa: mini review. Int. J. Agric.
Biol. 9, 367–369.
Arifiantini, R.I.I.S., Yusuf, T.L., 2010. Developing of tris soy milk diluent
for frisian holstein bull frozen semen. Hayati J. Biol. 17 (2), 91–94.
Bailey, J.L., Buhr, M.M., 1994. Cryopreservation alters the Ca2+ flux of
bovine spermatozoa. Can. J. Anim. Sci. 74, 45–51.
Bailey, J.L., Robertson, L., Buhr, M.M., 1994. Relationships among in vivo
fertility, computer-analysed motility and in vitro Ca2+ flux in bovine
spermatozoa. Can. J. Anim. Sci. 74, 53–58.
Bailey, J.L., Bilodeau, J.F., Cormier, N., 2000. Semen cryopreservation in
domestic animals: a damaging and capacitating phenomenon. J.
Androl. 21, 1–7.
Bailey, J.L., Morrie, A., Cormier, N., 2003. Semen cryopreservation:
success and persistent in farm species. Canadian J. Anim. Sci. 83,
393–401.
Barbas, J.P., Mascarenhas, R.D., 2009. Cryopreservation of domestic
animal sperm cells. Cell Tissue Bank. 10, 49–62.
Batellier, F., Vidament, M., Fauquant, J., Duchamp, G., Arnaud, G., Yvon,
J.M., Magistrini, M., 2001. Advances in cooled semen technology.
Anim. Reprod. Sci. 68, 181–190.
Becker-Ritt, A.B., Mulinari, F., Vasconcelos, I.M., Carlini, C.R., 2004.
Anti-nutritional and/or toxic factors in soybean (Glycine max (L))
Merril seeds: comparison of different cultivars adapted to the
southern region of Brazil. J. Sci. Food Agric. 84, 263–270.
Bousseau, S., Brillard, J.P., Marquant-Le Guienne, B., Guerin, B., Camus, A.,
Lechat, M., 1998. Comparison of bacteriological qualities of various
egg yolk sources and the in vitro and in vivo fertilizing potential of
Bovine semen frozen in egg yolk or lecithin based diluents. Theriogen
50, 699–706.
Breitbart, H., Naor, Z., 1999. Protein kinases in mammalian sperm
capacitation and the acrosome reaction. Rev. Reprod. 4, 151–159.
Brumm, T.J., Hurburgh, C.R., 2002. Jr. In: Quality of the 2002 soybean crop
from the United States. American Soybean Association, St. Louis, MO.
Breitbart, H., Darshan, R., Rubenstein, S., 1984. Evidence for the presence
of ATP-dependent calcium pump and ATPase activities in bull sperm
head membranes. Biochem. Biophys. Res. Commun. 122, 479–484.
Chatterjee, S., Lamirande, E.D., Gagnon, C., 2001. Cryopreservation alters
membrane sulphydryl status of bull spermatozoa: protection by
oxidized glutathione. Mol. Reprod. Dev. 60, 498–506.
Cheng, F.P., Gadella, B.M., Voorhout, W.F., Fazeli, A., Bevers, M.M.,
Colenbrander, B., 1998. Progesterone-induced acrosome reaction in
stallion spermatozoa is mediated by a plasma membrane
progesterone receptor. Biol. Reprod. 59, 733–742.
Clulow, J.R., Mansfield, L.J., Morris, L.H.A., Evans, G., Maxwell, W.M.C.,
2008. A comparison between freezing methods for the
cryopreservation of stallion spermatozoa. Anim. Reprod. Sci. 108,
298–308.
Cochran, J.D., Amann, R.P., Froman, D.P., Pickett, B.W., 1984. Effects of
centrifugation glycerol level, cooling to 5 ◦ C, freezing rate and
thawing rate on the post thaw motility of equine spermatozoa.
Theriogenology 22, 25–38.
Collin, S., Sirard, M.A., Dufour, M., Bailey, J.L., 2000. Sperm calcium levels
and chlortetracycline fluorescence patterns are related to the in vivo
fertility of cryopreserved bovine semen. J. Androl. 21, 938–943.
Cooter, P.Z., Goolsby, H.A., Prien, S.D., 2005. Preliminary evaluation of a
unique freezingtechnology for bovine spermatozoa
cryopreservation. Reprod. Domest. Anim. 40, 98–99.
de Lamirande, E., Leclerc, P., Gagnon, C., 1997. Capacitation as a
regulatory event that primes spermatozoa for the acrosome reaction
and fertilization. Mol. Hum. Reprod. 3, 175–194.
de Lamirande, E., Harakat, A., Gagnon, C., 1998. Human sperm
capacitation induced by biological fluids and progesterone but not
by NADH or NADPH, is associated with the production of superoxide
anion. J. Androl. 19, 215–225.
Davis, I.S., Bratton, R.W., Foote, R.H., 1963. Livability of bovine
spermatozoa at 5 −25, and −85 ◦ C in tris-buffered and citrate
buffered yolk-glycerol extenders. J. Dairy Sci. 46, 333–336.
De Leeuw, F.E., De Leeuw, A.M., Den Daas, J.H.G., Colenbrander, B.,
Verkleij, A.J., 1993. Effects of various cryoprotective agents and
7
membrane stabilizing compounds on bull sperm membrane
integrity after cooling and freezing. Cryobiology 30, 32–44.
Drobnis, E.Z., Crowe, L.M., Berger, T., Anchordoguy, T.J., Overstreet, J.W.,
Crowe, J.H., 1993. Cold shock damage is due to lipid
phase-transitions in cell-membranes—a demonstration using sperm
as a model. J. Exp. Zool. 265, 432–437.
Eaglesome, M.D., Garcia, M.M., 1995. Comparisons of antibiotic
combinations to control Pseudomonas aeruginosa in Bovine semen.
Can. J. Vet. Res. 59, 73–75.
Foote, R.H., Bratton, R.W., 1949. The fertility of bovine semen cooled
with and without the addition of citrate sulfanilamide yolk extender.
J. Dairy Sci. 32, 856–861.
Foote, R.H., Bratton, R.W., 1950. The fertility of bovine semen in
extenders containing sulfanilamide penicillin, streptomycin, and
polymyxin. J. Dairy Sci. 33, 544–547.
Foote, R.H., Gray, L.C., Young, D.C., Dunn, H.O., 1960. Fertility of bull
semen stored up to four days at 5 ◦ C in 20% egg yolk extenders. J.
Dairy Sci. 43, 1330–1334.
Gebauer, M.R., Pickett, B.W., Komarek, R.J., Gaunya, W.S., 1970. Motility
of bovine spermatozoa extended in “defined” diluents. J. Dairy Sci.
53, 817–823.
Gil, J., Januskauskas, A., Haard, MCh., Haard, M.G.M., Johanisson, A.,
Söderquist, L., Rodrıı́guez-Martıı́nez, H., 2000. Functional sperm
parameters and fertility of bull semen extended in Biociphos-Plus®
and Triladyl® . Reprod. Domest. Anim. 35, 69–77.
Gil, J., Rodriguez-Irazoqui, M., Lundheim, N., Soderquist, L.,
Rodriguez-Martinez, H., 2003. Fertility of ram semen frozen in
Bioxcell and used for cervical artificial insemination. Theriogenology
59, 1157–1170.
Guthrie, H.D., Welch, G.R., 2005. Effects of hypothermic liquid storage
and cryopreservation on basal and induced plasma membrane
phospholipid disorder and acrosome exocytosis in boar
spermatozoa. Reprod. Fertil. Dev. 17, 467–477.
Hammerstedt, R.H., Graham, J.K., 1992. Cryopreservation of poultry
sperm: the enigma of glycerol. Cryobiology 29, 26–38.
Hammerstedt, R.H., Graham, J.K., Nolan, J.P., 1990. Cryopreservation of
mammalian sperm: what we ask them to survive. J. Androl. 11,
73–88.
Harrison, R.A.P., White, I.G., 1972. Glycolytic enzymes in the
spermatozoa and cytoplasmic droplets of bull boar and ram, and
their leakage after shock. J. Reprod. Fertil. 30, 105–115.
Hasan, S., Andrabi, S.M.H., Muneer, R., Anzar, M., Ahmad, N., 2001. Effects
of new antibiotics combination on post-thaw motion characteristics
and membrane integrity of buffalo and Sahiwal bull spermatozoa and
on the bacteriological quality of their semen. Pak. Vet. J. 21 (1), 6–12.
Hinkovska-Galcheva, V., Petkova, D., Koumanov, K., 1989. Changes in the
phospholipid composition and phospholipid asymmetry of ram
sperm plasma membranes after cryopreservation. Cryobiology 26,
70–75.
Hinsch, E., Hinsch, K.D., Boehm, J.G., Schill, W.B., Mueller-Schloesser, F.,
1997. Functional parameters and fertilization success of Bovine
semen cryopreserved in egg-yolk free and egg-yolk containing
extenders. Reprod. Domest. Anim. 32, 143–149.
Holt, W.V., North, R.D., 1994. Effects of temperature and restoration of
osmotic equilibrium during thawing on the induction of plasma
membrane damage in cryopreserved ram spermatozoa. Biol. Reprod.
51, 414–424.
Holt, W.V., 2000. Basic aspects of frozen storage of semen. Anim. Reprod.
Sci. 62, 3–22.
Howard, T.H., Vasquez, L.A., Amann, R.P., 1982. Antibiotic control of
Campylobacter fetus by three extenders of bovine semen. J. Dairy Sci.
65, 1596–1600.
Iritani, A., 1980. Problems of freezing spermatozoa of different species.
Madrid, Spain In: Proc. 9th Int. Congr. Anim. Reprod. Artif. Insemin,
1, pp. 115–131.
Jeyendran, R.S., Acosta, V.C., Land, S., Coulam, C.B., 2008.
Cryopreservation of human sperm in a lecithin-supplemented
freezing medium. Fertil. Steril. 90 (4), 1263–1265.
Johnson, L.A., Hoover, W.J., Deyoe, C.W., 1981. Yield and quality of
soymilk processed by steam infusion cooking. J. Food Sci. 46,
239–248.
Kampshmidt, R.F., Mayer, D.T., Herman, H.A., 1953. Lipid and lipoprotein
constituents of egg yolk in the resistance and storage of bull
spermatozoa. J. Dairy Sci. 36, 733–742.
Karabinus, D.S., Evenson, D.P., Kaproth, M.T., 1991. Effects of egg
yolk-citrate and milk extenders on chromatin structure and viability
of cryopreserved bull sperm. J. Dairy Sci. 74, 3836–3848.
8 S.S. Layek et al. / Animal Reproduction Science 172 (2016) 1–9
Kasimanickam, R., Kasimanickam, V., Tibary, A., Pelzer, K., 2011. Effect of
semen extenders on sperm parameters of ram semen during liquid
storage at 4 ◦ C. Small Rumin. Res. 99, 208–213.
Kasimanickam, V.R., Kasimanickam, R.K., Memon, M.A., Rogers, H.A.,
2012. Effect of extenders on sperm mitochondrial membrane,
plasma membrane and sperm kinetics during liquid storage of
canine semen at 5 ◦ C. Anim. Reprod. Sci. 136 (1–2), 139–145.
Kodama, H., Kuribayashi, Y., Gagnon, C., 1996. Effect of sperm lipid
peroxidation on fertilization. J. Androl. 17, 151–157.
Krishnakumar, S., Whiteside, D.P., Elkin, B.J., Thundathil, C., 2011.
Evaluation of an animal protein-free semen extender for
cryopreservation of epididymal sperm from North American bison
(Bison bison). Theriogenology 76, 252–260.
Krishnan, H.B., 2000. Biochemistry and molecular biology of soybean
seed storage proteins. J. N. Seeds 2, 1–25.
Kumar, V., Rani, A., Tindwani, C., Jain, M., 2003. Lipoxygenase isozymes
and trypsin inhibitor activities in soybean as influenced by growing
location. Food Chem. 83, 79–83.
Kumar, V., Rani, A., Solanki, S., Hussain, S.M., 2006. Influence of growing
environment on the biochemical composition and physical
characteristics of soybean seed. J. Food Compos. Anal. 19, 188–195.
Kumaresan, A., Siqueira, A.P., Hossain, M.S., Bergqvist, A.S., 2011.
Cryopreservation-induced alterations in protein tyrosine
phosphorylation of spermatozoa from different portions of the boar
ejaculate. Cryobiology 63, 137–144.
Kumaresan, A., Johannisson, A., Saraviac, F., Bergqvist, A.S., 2012a. The
effect of oviductal fluid on protein tyrosine phosphorylation in
cryopreserved boar spermatozoa differs with the freezing method.
Theriogenology 77, 588–599.
Kumaresan, A., Johannisson, A., Humblot, P., Bergqvist, A.S., 2012b.
Oviductal fluid modulates the dynamics of tyrosine phosphorylation
in cryopreserved boar spermatozoa during capacitation. Mol.
Reprod. Dev. 9999, 1–16.
Langlais, J., Roberts, K.D., 1985. A molecular model of sperm capacitation
and the acrosome reaction of mammalian spermatozoa. Gamete Res.
12, 183–224.
Leyton, L., Saling, P., 1989. 95 kd sperm proteins bind ZP3 and serve as
tyrosine kinase substrates in response to zona binding. Cell 57,
1123–1130.
Liener, I.E., 1994a. Implications of antinutritional components in
soybean foods. Crit. Rev. Food Sci. Nutr. 34, 31–67.
Liener, I.E., 1994b. Implications of antinutritional components in
soybean foods. Crit. Rev. Food Sci. Nutr. 34, 31–67.
Lin, Y., Kan, F.W.K., 1996. Regionalization and redistribution of
membrane phospholipids and cholesterol in mouse spermatozoa
during in vitro capacitation. Biol. Reprod. 55, 1133–1146.
Liu, Z., Foote, R.H., 1998. Bull sperm motility and membrane integrity in
media varying in osmolality. J. Dairy Sci. 81, 1868–1873.
Lorton, S.P., Sullivan, J.J., Bean, B., Kaproth, M., Kellgren, H., Marshall, C.,
1988. A new antibiotic combination for frozen bovine semen. 2.
Evaluation of seminal quality. Theriogenology 29 (3), 593–607.
Möstl, E., Spendier, H., Kotrschal, H., 2001. Concentration of
immunoreactive progesterone and androgens in the yolk of hen’s
eggs (Gallus domesticus). Vet. Med. Aust. 88, 62–65.
Martin, I.C.A., 1963. Effects of lecithin egg yolk, fructose and period of
storage at 58 ◦ C on bull spermatozoa frozen to −79 ◦ C. J. Reprod.
Fertil. 6, 441–449.
Martin, I.C.A., 1965. Effects of cooling to 5 ◦ C storage at 5 ◦ C, glycerol
concentration, sodium chloride, fructose and glycine on the revival
of deep frozen spermatozoa. J. Agric. Sci. Camb. 64, 425–432.
Mazur, P., Leibo, S.P., Farrant, J., Chu, E.H.Y., Hanna Jr., M.G., Smith, L.H.,
1970. Interactions of cooling rate, warming rate and protective
additive on the survival of frozen mammalian cells. In:
Wolstenholme, G.E.W., O’Connor, M. (Eds.), The Frozen Cell.
Churchill, London, pp. 69–88.
Mazur, P., Katkova, N., Critser, J.K., 2000. The enhancement of the ability
of mouse sperm to survive freezing and thawing by the use of high
concentrations glycerol and the presence of an Escherichia coli
membrane preparation (Oxyrase) to lower the oxygen
concentration. Cryobiology 40, 187–209.
Mazur, P., 1984. Freezing of living cells: mechanisms and implications.
Am. J. Physiol. 247, C125–C142.
Medeiros, C.M.O., Forell, F., Oliveria, A.T.D., Rodrigues, J.L., 2002. Current
status of sperm cryopreservation: why isn’t it better?
Theriogenology 57, 327–344.
Michajilov, N.N., 1950. Sperm dilution in the milk The Czecho- Slovak
Vet. Mag. J. Am. Vet. Med. Assoc., 117, 337.
Milovanov, V.K., Sokolovskaja, I.I., 1980. Long-term storage of ram semen
and new possibilities of large-scale selection in sheep breeding.
Vestn. Skh. Nauki. 12, 122–132.
Moussa, M., Martinet, V., Trimeche, A., Tainturier, D., Anton, M., 2002.
Low density lipoproteins extracted from hen egg yolk by an easy
method: cryoprotective effect on frozen-thawed bull semen.
Theriogenology 57, 1695–1706.
Muiño, R., Fernandez, M., Pena, A.I., 2007. Post-thaw survival and
longevity of bull spermatozoa frozen with an egg yolk-based or two
egg yolk-free extenders after an equilibration period of 18 h. Reprod.
Domest. Anim. 42, 305–311.
Muller, K., Pomorski, T., Muller, P., Herrmann, A., 1999. Stability of
transbilayer phospholipid asymmetry in viable ram sperm cells after
cryotreatment. J. Cell Sci. 112, 11–20.
Nelson, A.I., Steinberg, M.I., Wei, L.S., 1976. Illinois process for
preparation of soymilk. J. Food Sci. 41, 57–61.
Nielsen, N.C., Bassuner, R., Beaman, T., 1997. The biochemistry and cell
biology of embryo storage proteins. In: Larkins, R.A., Vasil, I.K. (Eds.),
Cellular and Molecular Biology of Plant Seed Development. Kluwer
Academic Publishers, Dordrecht, pp. 151–220.
Noiles, E.E., Mazur, P., Watson, P.F., Kleinhans, F.W., Critser, J.K., 1993.
Determination of water permeability coefficient for human
spermatozoa and its activation energy. Biol. Reprod. 48, 99–109.
Noiles, E.E., Bailey, J., Storey, B.T., 1995. Temperature dependence of the
water permeability Lp, of murine sperm shows a discontinuity
between 4◦ and 0 ◦ C. Cryobiology 32, 220–238.
O’Dell, W.T., Almquist, J.O., 1957. Freezing bovine semen. I. Techniques
for freezing bovine spermatozoa in milk diluents. J. Dairy Sci. 40,
1534–1541.
Pace, M.M., Graham, E.F., 1974. Components in egg yolk which protect
bovine spermatozoa during freezing. J. Anim. Sci. 39, 1144–1149.
Pankaj, P.K., 2006. Study of Critical Control Points Associated with
Cryopreservation of Bovine Semen. M.V.Sc. Thesis. NDRI, Karnal.
Parks, J.E., Graham, J.K., 1992. Effects of cryopreservation procedures on
sperm membranes. Theriogen 38, 209–222.
Parks, J.E., Lynch, D.V., 1992. Lipid composition and thermotropic phase
behavior of boar bull, stallion and rooster sperm membrane.
Cryobiology 29, 255–266.
Phillips, P.H., Lardy, H.A., 1940. A yolk-buffer pabulum for the
preservation of bull semen. J. Dairy Sci. 23, 399–404.
Parrish, J.J., Susko-Parrish, J.L., Graham, J.K., 1999. In vitro capacitation of
bovine spermatozoa: role of intracellular calcium. Theriogenology
51, 461–472.
Polge, C., Smith, A.U., Parkes, A.S., 1949. Revival of spermatozoa after
verification and dehydration at low temperatures. Nature (Lond.)
164, 666.
Pugliesi, G., de Carvalho, G.R., Rates, D.M., Ker, P.G., da Matta, M.P., de
Oliveira, R.R., Filho, J.M.D., 2012. Viability and fertility of cooled
equine semen diluted with skimmed milk or glycine egg yolk-based
extenders. R. Bras. Zootecnia 41 (12), 2411–2417.
Rackis, J., Hinig, D.H., Sessa, D.J., Steggerd, F.R., 1979. Flavor problems of
vegetable food protein. J. Am. Oil Chem. Soc. 56, 262–271.
Reed, M.L., Ezeh, P.C., Hamic, A., Thompson, D.J., Caperton, C.L., 2009. Soy
lecithin replaces egg yolk for cryopreservation of human sperm
without adversely affecting postthaw motility, morphology, sperm
DNA integrity, or sperm binding to hyaluronate. Fertil. Steril. 92 (5),
1787–1790.
Rios-Iriarte, B.J., Barnes, R.H., 1996. The effect of overheating on certain
nutritional properties of the proteins of soybean. Food Technol. 32,
836–839.
Robertson, L., Bailey, J.L., Buhr, M.M., 1990. Effects of cold shock and
phospholipase A2 on intact boar spermatozoa and sperm head
plasma membranes. Mol. Reprod. Dev. 26, 143–149.
Rodriguez, O.L., Berndtson, W.E., Ennen, B.D., Pickett, B.W., 1975. Effect
of rates of freezing, thawing and level of glycerol on the survival of
bovine spermatozoa in straws. J. Anim. Sci. 41, 129–136.
Salamon, S., Maxwell, W.M.C., 2000. Storage of ram semen. Anim.
Reprod. Sci. 62, 77–111.
Salisbury, G.W., Fuller, H.K., Willett, E.L., 1941. Preservation of bovine
spermatozoa in yolk-citrate diluent and field results from its use. J.
Dairy Sci. 24, 905–910.
Samper, J.C., Hellander, J.C., Crabo, B.G., 1991. Relationship between the
fertility of fresh and frozen stallion semen and semen quality. J.
Reprod. Fertil. 44 (Suppl), 107–114.
Shannon, P., Curson, B., 1972. Toxic effect and action of dead sperm on
diluted bovine semen. J. Dairy Sci. 55, 614–620.
Shi, Y.G., Ren, L., 1993. Soyfood Technology. Chinese) China Light
Industry Publisher, Beijing, China.
 S.S. Layek et al. / Animal Reproduction Science 172 (2016) 1–9
Shin, S.J., Lein, D.H., Patten, V.H., Ruhnke, H.L., 1988. A new antibiotic
combination for frozen bovine semen. 1. Control of Mycoplasma,
Ureaplasmas, Campylobacter fetus subsp. venerealis and Haemophilus
somnus. Theriogenology 29 (3), 577–591.
Shurtleff, W., Aoyagi, A., 1979. Tofu and Soymilk Production. The Soyfood
Centre, Lafayette, CA.
Singh, R.J., Hymowitz, T., 1999. Soybean genetic resources and crop
improvement. Genome 42, 605–616.
Singh, A.K., Singh, V.K., Narwade, B.M., Mohanty, T.K., Atreja, S.K., 2012.
Comparative quality assessment of buffalo (Bubalus bubalis) semen
chilled (5 ◦ C) in egg yolk- and soya milk—based extenders. Reprod.
Domest. Anim. 47 (4), 596–600.
Singh, V.K., Singh, A.K., Kumar, R., Atreja, S.K., 2013. Development of soya
milk extender for semen cryopreservation of Karan Fries (crossbreed
cattle). Cryo Letters 34 (1), 52–61.
Slaweta, R., laskowska-klita, T., 1985. Glutathione content in the semen
of bulls of the lowland black-white breed. Acta Physiol. Pol. 36,
107–111.
Speroni, F., Añón, M.C., de Lamballerie, M., 2010. Effects of calcium and
high pressure on soybean proteins: a calorimetric study. Food Res.
Int. 43, 1347–1355.
Stradaioli, G., Noro, T., Sylla, L., Monaci, M., 2007. Decrease in glutathione
(GSH) content in bovine sperm afer cryopreservation: comparison
between two extenders. Theriogenology 15, 1249–1255.
Tardif, S., Dubé, C., Chevalier, S., Bailey, J.L., 2001. Capacitation is
associated with tyrosine phosphorylation and tyrosine kinase-like
activity of pig sperm proteins. Biol. Reprod. 65, 784–792.
Thacker, D.L., Almquist, J.O., 1953. Diluters for bovine semen. I. Fertility
and motility of bovine spermatozoa in boiled milk. J. Dairy Sci. 36,
173–180.
Thibier, M., Guerin, B., 2000. Hygienic aspects of storage and use of
semen for artificial insemination. Anim. Reprod. Sci. 62 (1–3),
233–251.
9
Upreti, G.C., Jensen, K., Munday, R., Duganzich, D.M., Vishwanath, R.,
Smith, J.F., 1998. Studies on aromatic amino acid oxidase activity in
ram spermatozoa: role of pyruvate as an antioxidant. Anim. Reprod.
Sci. 51, 275–287.
Van Wagtendonk-de Leeuw, A.M., Haring, R.M., Kaal-Lansbergen,
L.M.T.E., den Dass, J.H.G., 2000. Fertility results using bovine semen
cryopreserved with extenders based on egg yolk and soy bean
extract. Theriogenology 54, 57–67.
Vera Munoz, O., Amirat-Briand, L., Diaz, T., Vasquez, L., Schmidt, E.,
Desherces, S., Anton, M., Bencharif, D., Tainturier, D., 2009. Effect of
semen dilution to low-sperm number per dose on motility and
functionality of cryopreserved bovine spermatozoa using
low-density lipoproteins (LDL) extender: comparison to Triladyl®
and Bioxcell® . Theriogenology 71, 895–900.
Vishwanath, R., Shannon, P., 2000. Storage of bovine semen in liquid and
frozen state. Anim. Reprod. Sci. 62, 23–53.
Wall, R.J., Foote, R.H., 1999. Fertility of bull semen frozen and store in
clarified egg yolk-tris-glycerol extender. J. Dairy Sci. 82, 817–821.
Watson, P.F., Martin, C.A., 1975. The influence of some fractions of egg
yolk on the survival of ram spermatozoa at 5 ◦ C. Aust. J. Biol. Sci. 28,
145–152.
Watson, P.F., 1995. Recent developments and concepts in the
cryopreservation of spermatozoa and the assessment of their
post-thawing function. Reprod. Fertil. Dev. 7, 871–891.
Watson, P.F., 2000. The causes of reduce fertility with cryopreserved
semen. Anim. Reprod. Sci. 60, 481–492.
Wilkens, W.F., Mattick, L.R., Hand, D.B., 1967. Effect of processing method
on oxidative off-flavours of soybean milk. Food Tech. 21, 80–89.
Zarkadas, C.G., Gagnon, C., Gleddie, S., Khanizadeh, S., Cober, E.R.,
Guillemette, R.J.D., 2007. Assessment of the protein quality of
fourteen soybean [Glycine max (L.) Merr.] cultivars using amino acid
analysis and two-dimensional electrophoresis. Food Res. Int. 40,
129–146.