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 Spermatology. SRF Vol. 65. ERS Roldan and M Gomendio (eds) Nottingham University Press, Nottingham

Seminal plasma proteins 217

Seminal plasma proteins: functions and interaction

with protective agents during semen preservation
Puttaswamy Manjunath1,2,3, Annick Bergeron3, Jasmine Lefebvre2,3
and Jinjiang Fan3
Departments of 1Medicine and 2Biochemistry, University of Montreal and 3Guy-Bernier Research
Center, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada, H1T 2M4

Seminal plasma contains factors that are beneficial and/or detrimental to

sperm function and/or storage. However, the nature and characteristics
of these factors are not well understood. The major protein fraction (50-
70%) of bovine seminal plasma is represented by a family of phospholipid-
binding proteins collectively called BSP proteins. The BSP protein
signature is characterised by two tandemly repeated fibronectin type 2
(Fn2) domains. It is now well established that BSP proteins and their
relatives represent a new emerging superfamily of proteins in mammals.
They bind to sperm membrane choline phospholipids at ejaculation. They
also bind to capacitation factors, namely, high-density lipoproteins and
glycosaminoglycans and promote sperm capacitation induced by these
molecules, indicating their beneficial role in sperm function and fertility.
In contrast, BSP proteins also induce changes in the sperm plasma
membrane by stimulating cholesterol and phospholipid efflux. Thus, the
continuous exposure of sperm to seminal plasma that contains BSP proteins
is detrimental to the sperm membrane, which may render the membrane
very sensitive to sperm storage in the liquid or frozen states. Interestingly,
BSP proteins specifically bind low-density lipoproteins present in egg
yolk, a compound commonly used in semen extenders. This interaction
appears to abolish the detrimental effect of BSP proteins on the sperm
membrane. Therefore, BSP proteins in seminal plasma act like a double-
edged sword, being both beneficial and detrimental to sperm.

Introduction
Seminal plasma is a complex mixture of secretions from the testes, epididymides and accessory
sex glands (seminal vesicles, ampulla, prostate, bulbourethral glands). Despite its physiologi-
cal significance as the carrier of spermatozoa to the female reproductive tract, the biochemical
characteristics and physiological roles of the various seminal plasma proteins are poorly under-
stood. While some seminal plasma proteins appear to be beneficial to sperm functions such as
motility, capacitation, acrosome reaction, viability and/or sperm-egg interaction, others seem
to be detrimental, particularly to sperm preservation including cryopreservation (for a review
see Manjunath and Therien, 2002; Bergeron and Manjunath, 2006). In this review, we describe

Corresponding author E-mail: puttaswamy.manjunath@umontreal.ca

15-Manjunath.p65 217 3/12/2007, 11:37 AM

 218 P. Manjunath et al.

the characteristics and functions of a family of phospholipid-binding proteins widely distributed

in mammalian seminal plasma. In addition, we describe the interaction of these proteins with
egg yolk extender components and discuss the implication of this interaction in the context of
sperm storage in the liquid and frozen states. We also explain why these lipid-binding seminal
plasma proteins are beneficial to sperm function, yet detrimental to sperm preservation.

BSP proteins
In bovine, the major protein fraction of seminal plasma (30-50 mg/ml) constitutes a family of
closely related proteins named BSP-A1, BSP-A2, BSP-A3 and BSP-30-kDa (BSP proteins)
(Manjunath, 1984; Manjunath and Sairam, 1987. These proteins are the secretory products of
the seminal vesicles. The biochemical properties and structure of these proteins have been
studied in detail (Manjunath, 1984; Manjunath and Sairam, 1987; Manjunath et al., 1987;
Seidah, Manjunath, Rochemont, Sairam and Chretien, 1987; Manjunath, Baillargeon, Marcel,
Seidah, Chretien and Chapdelaine, 1988; Leblond, Desnoyers and Manjunath, 1993; Desnoyers,
Therien and Manjunath, 1994). They are small acidic proteins with apparent molecular weights
of 15-16 kDa (BSP-A1, -A2 and -A3) and 28-30 kDa (BSP-30-kDa), which are all glycoproteins
with the exception of BSP-A3. BSP-A1 and BSP-A2 (also called PDC-109) originate from the
same gene but differ in the degree of glycosylation (glycoforms). All of these proteins contain
two homologous type II domains similar to those found in the gelatin-binding domains of
fibronectin (called Fn2 domains) and an amino-terminal extension that is variable between
each protein (Fig. 1) (reviewed in Manjunath and Therien, 2002; Fan, Lefebvre and Manjunath,
2006). The crystal structure of BSP-A1/-A2 (PDC-109) has been determined (Wah, Fernandez-
Tornero, Sanz, Romero and Calvete, 2002), allowing us to create homology models of BSP-A3
and BSP-30kDa (Fig. 2). Homologues of BSP proteins have been isolated and characterised in
seminal plasma or seminal vesicle secretions from numerous other mammals, namely boar and
stallion (Menard, Nauc, Lazure, Vaillancourt and Manjunath, 2003; Calvete, Mann, Schafer,
Sanz, Reinert, Nessau, Raida and Topfer-Petersen, 1995; Calvete, Raida, Gentzel, Urbanke,
Sanz and Topfer-Petersen, 1997; Sanz, Calvete, Mann, Gabius and Topfer-Petersen, 1993),
goat (Villemure, Lazure and Manjunath, 2003), bison (Boisvert, Bergeron, Lazure and Manjunath,
2004), ram (Bergeron, Villemure, Lazure and Manjunath, 2005) and water buffalo (unpub-
lished). All of these species contain 3-4 molecular forms, except boar, which contains one form
(Table 1). BSP-like antigens are also present in rat, mouse and hamster seminal vesicle fluid as
well as in human seminal plasma (Leblond et al., 1993). The concentration of BSP homologues
in seminal plasma varies from species to species (Table 2). At the genetic level, the cloning
and sequencing of the cDNA corresponding to BSP proteins has been reported (Salois, Menard,
Paquette and Manjunath, 1999; Kemme and Scheit, 1988). In a recent study, three new BSP
related genes (BSPH4, BSPH5 and BSPH6) were discovered in bovine (Fan et al., 2006). In
addition, an exhaustive genomic search indicated the presence of BSP-related DNA sequences
in human, rat, mouse, chimpanzee, dog, horse and rabbit (Fan et al., 2006). Molecular evolu-
tionary analysis revealed that all BSP-related sequences could be grouped into three sub-fami-
lies: BSPH4, which includes BSP-A1/-A2, BSP-A3 and BSP-30 kDa and is expressed in seminal
vesicles, BSPH5 and BSPH6, which are expressed in the epididymis and testis (Fig. 3). It is
becoming increasingly evident that BSP proteins and their homologous proteins represent a
new emerging superfamily in mammals.

15-Manjunath.p65 218 3/12/2007, 11:37 AM

 Seminal plasma proteins 219

BSP-A1/-A2 proteins
Q DE
D G
1 V R N R K H
S V Y
F F I Y G G K
P P F
T F K
D F Y 80
E V F L 40 T C C V
C P S M W M E
P W S G H V K W
E C A A C S T 109
T E C 60 Y Q Y G C C
Q R D S
D S K L I K T Y
D E L W K
G 20 D R S
P A D Y V G P N A W
P Y D K D R
A EL 100

BSP-A3 protein
E D
S N
L
Q V
I
Q L G N K K
D 1 P F I Y Y 40 80
P F P F I Y E G K
K F F S
V D V Y
E F L S C C N M F
K C L T W Y D
K W G H L K T T
K C C T A C
N S Y K N S 100 S I C C 115
D D Y
D K L G I K Y
P L W K
A K D R S S W
A D Y T G
T N Y D E D G V
20 S
G A E 60

BSP-30-kDa protein
80
G K K
Y K
F T Y 120
Y I Y R K K S
F
P
M F P F
A V W F Y
L L V S C C R
C T
W R 140 F E
A W N K R E
S
C C T P C S
P F E E S H C C 158
S K R D V R T Y
G L
E L W K
D N T W
F T E Q G S A
100 Y N Y D R D K
V
A
N 60 40
L D
N A K S L L S P F P K D D Y P M E E Q P Y I T R
T P P G M A D P
K P E L P T E T Y D L P P E I Y T T T F L
S 20
G
P
D P I D G
1

Figure 1. Primary structure of BSP proteins. The carbohydrate moieties are indicated by
the symbol (•). Modified from Manjunath and Therien (2002).

BSP proteins play a key role in sperm capacitation

Studies aimed at elucidating the biological role of BSP proteins revealed that they are secreted
by seminal vesicles (Manjunath et al., 1987) and bind to the sperm surface at ejaculation
(Manjunath, Chandonnet, Leblond and Desnoyers, 1994). Using various biochemical techniques,
it was established that the binding sites for BSP proteins on the sperm membrane are choline
phospholipids (Desnoyers and Manjunath, 1992), specifically phosphatidylcholine, phosphati-
dylcholine plasmalogen and sphingomyelin. Such choline phospholipids account for >60% of

15-Manjunath.p65 219 3/12/2007, 11:37 AM

 220 P. Manjunath et al.

Figure 2. Ribbon representation of homology models of BSP proteins. The potential ligand,
phosphorylcholine, is represented in ball and stick structure. The figure was prepared with
Swiss-PbdViewer 3.7b2 (Guex and Peitsch, 1997) and MOLMOL 2K.2 (Koradi, Billeter
and Wuthrich, 1996). The BSP-A1/-A2 (PDC-109) template is from the Research
Collaboratory for Structural Bioinformatics Protein Data Bank (1H8P), in which chains A
and B correspond to 2 monomers, because this protein forms dimers when associated
with phosphorylcholine.

Table 1. BSP protein homologues isolated from seminal plasma of various species.

Species BSP protein homologs

Bull BSP-A1, BSP-A2, BSP-A3, BSP-30kDa
Boar pB1
Stallion HSP-1, HSP-2, HSP-12kDa
Goat GSP-14kDa, GSP-15kDa, GSP-20kDa, GSP-22kDa
Ram RSP-15kDa, RSP-16kDa, RSP-22kDa, RSP-24kDa
Bison* BiSV-16kDa, BiSV-17kDa, BiSV-18kDa, BiSV-28kDa

*Isolated from seminal vesicle secretions.

From Bergeron and Manjunath (2006).

Table 2. Concentration of BSP protein homologues in the seminal plasma of

different mammals.

BSP proteins/homologues in seminal plasma

(mg/ml) (% of total proteins)
Bull 39.1 65
Bison* 3.0 25
Ram 15.0 30
Goat 5.6 20
Stallion 2.0 20
Boar 0.3 1.1

*BSP proteins in seminal vesicle secretions.

From Bergeron and Manjunath (2006).

15-Manjunath.p65 220 3/12/2007, 11:37 AM

 Seminal plasma proteins 221

98/ 90/ 86 SP1

SP2
pB1
CDK105
BSPH4
EP52c1
BSP -A1/ -A2
83/62/61
65/-/ - BSP -A3
BSPH4
BSP -30kDa

100/100/99 pBSPH1
hBSPH1
99/84/89
81/ 73/ 100 mBSPH1 BSPH5
rBSPH1
64/ 53/-
BSPH5

56/ 55/- mBSPH2

95/ 53/ 71 rBSPH2
99/94/97 mBSPH3 BSPH6

BSPH6
0.2

Figure 3. Neighbour-joining trees of two Fn2 domains of BSP-related proteins. BSP pro-
teins and their relatives are separated into three subfamilies: BSPH4, BSPH5 and BSPH6.
Bootstrap values from 1000 replicates for neighbour-joining (1st number), 1000 replicates
for maximum parsimony (2nd number) and 100 replicates for maximum likelihood (3rd
number) are indicated at the nodes and were used to assess the robustness of the trees.
Only bootstrap values larger than 50% are shown and the bold numbers indicate the three
main branches. Genetic distance is indicated as the number of substitutions per amino
acid site. BSP protein, bovine seminal plasma protein; BSPH, BSP protein homologue;
SP1/2, equine seminal plasma protein 1/2; pB1, boar seminal plasma protein pB1; CDK105,
canine gene; EP52c1, epididymal protein 52 from rabbit. The small letter preceding each
abbreviation represents the name of the species (h, Homo sapiens; p, Pan troglodytes; m,
Mus musculus; r, Rattus norvegicus) from which the orthologous protein originates. Modi-
fied from Fan et al. (2006).

the total phospholipids of the sperm membrane (Parks, Arion and Foote, 1987). Further studies
indicated that BSP proteins bind to high-density lipoproteins (HDL) (Manjunath et al., 1988;
Manjunath, Marcel, Uma, Seidah, Chretien and Chapdelaine, 1989; Therien, Soubeyrand and
Manjunath, 1997; Therien, Bousquet and Manjunath, 2001), and glycosaminoglycans (GAGs)
such as heparin (Chandonnet, Roberts, Chapdelaine and Manjunath, 1990), heparan sulfate and
chondroitin sulfate (Therien, Bergeron, Bousquet and Manjunath, 2005). HDL and GAGs are
components of follicular and oviductal fluids and are physiological inducers of sperm capacita-
tion. Furthermore, BSP proteins potentiate capacitation induced by follicular fluid-HDL and -
GAGs (Therien et al., 1997, Therien et al., 2001). It was shown that epididymal sperm exposed
to BSP proteins followed by incubation with GAGs or HDL exhibit a higher incidence of

15-Manjunath.p65 221 3/12/2007, 11:37 AM

 222 P. Manjunath et al.

capacitation compared to GAGs or HDL alone. In addition, BSP proteins mediate cholesterol
efflux from sperm, which is also accompanied by choline phospholipid efflux (Therien, Moreau
and Manjunath, 1998; Therien, Moreau and Manjunath, 1999). The removal of cholesterol from
the sperm membrane is deemed to be essential during capacitation.
The studies described above led us to propose that BSP proteins participate in the sperm
membrane lipid modification events that occur during capacitation in the female reproductive
tract (Manjunath and Therien, 2002; Therien et al., 2001, Therien et al., 2005). Two distinct
mechanisms, both mediated by BSP proteins but one induced by HDL and the other by GAGs,
were deduced (Fig. 4). In brief, at ejaculation, sperm are mixed with accessory gland secretions
containing BSP proteins, contributed by the seminal vesicles. During this brief exposure, BSP
proteins remove some cholesterol (first cholesterol efflux) accompanied by the release of some
phospholipids. This lipid efflux may slightly destabilise the sperm membrane (priming). At the
same time, BSP proteins coat the sperm surface via their interaction with the membrane cho-
line phospholipids. Within the next 10-20 min, sperm travel through the cervical mucus into
the uterus, leaving behind most of the seminal plasma. During this time, bound BSP proteins
may prevent the free movement of phospholipids, thereby stabilising the sperm membrane
(arrested state). BSP protein-coated sperm then travel through the female genital tract and reach
the oviduct, the site of fertilisation, where they encounter HDL and/or GAGs. With HDL-
induced capacitation, oviductal- or follicular fluid-HDL remove BSP proteins from the sperm
membrane. As a result, sperm membrane lipids are free to move. In addition, HDL may induce
a second efflux of cholesterol. Since cholesterol is recognized to have a stabilising effect on
membranes (Yeagle, 1985), its efflux would be expected to provoke further reorganization or
destabilization of the membrane and trigger some unknown signal transduction pathways. Such
events could regulate the surface expression of sperm zona pellucida receptors, and adhesion
to the zona pellucida could trigger the acrosome reaction. With GAG-induced capacitation,
sperm-bound BSP proteins may play a role, via their interaction with GAGs, in the uptake of
Ca2+, intracellular alkalinization and protein tyrosine phosphorylation (Therien et al., 2001;
Therien et al., 2005). The role of tyrosine phosphorylation in capacitation in various species
(human, bovine, murine, porcine) has been investigated in detail (Galantino-Homer, Visconti
and Kopf, 1997; Leclerc, de Lamirande and Gagnon, 1996; Aitken, Harkiss, Knox, Paterson and
Irvine, 1998; Tardif, Dube, Chevalier and Bailey, 2001; Osheroff, Visconti, Valenzuela, Travis,
Alvarez and Kopf, 1999; de Lamirande, Leclerc and Gagnon, 1997; Visconti, Westbrook,
Chertihin, Demarco, Sleight and Diekman, 2002; Naz and Rajesh, 2004; Baldi, Luconi,
Bonaccorsi, Muratori and Forti, 2000). HDL-induced capacitation, in contrast to GAG-induced
capacitation, is not associated with an increase in tyrosine phosphorylation (Lane, Therien,
Moreau and Manjunath, 1999).
The first functional role identified for BSP proteins is that they promote the capacitation of
bull sperm, which involves a complex series of events and is schematised in Figure 4 (Therien,
Bleau and Manjunath, 1995; Therien et al., 1997, 1998a, 2005; Manjunath and Therien, 2002).
Recently, two additional functions were proposed for BSP proteins. The group of Yu et al. (Yu,
Zhao, Zhao, Chen, Liu, Zhang, Fu, Zong, Yu and Guan, 2003) demonstrated that, in vitro, the
activity of protein kinase C (PKC) and of tyrosine protein kinase (TPK) are inhibited by PDC-
109 (BSP-A1/-A2) and proposed that the inhibition of PKC may serve to prevent the premature
acrosome reaction of sperm in the female reproductive tract. The Suarez group demonstrated
that BSP proteins mediate the binding of sperm to the oviductal epithelium and proposed that
they are involved in prolonging sperm survival during storage and in the maintaining sperm
motility in the oviduct (Ignotz, Lo, Perez, Gwathmey and Suarez, 2001; Gwathmey, Ignotz
and Suarez, 2003; Gwathmey, Ignotz, Mueller, Manjunath and Suarez, 2006). Therefore, BSP
proteins play multiple roles in sperm functions and have a beneficial effect on sperm fertility.

15-Manjunath.p65 222 3/12/2007, 11:37 AM

 Seminal plasma proteins 223

Epididymal spermatozoa Accessory gland secretions

Ejaculation
First cholesterol efflux

BSP proteins
Heparin-binding protein
Sperm membrane

cholesterol
Ejaculated sperma tozoa phospholipid

Female genital tract

Arrested state

GAG -induced capacitation HDL-induced capacitation

(Second cholesterol efflux)
Heparin/GAGs Oviductal and Follicular fluids HDL

AC

pH Ca2+ cAMP ATP

A
PK-A

Protein Tyrosine Protein Tyrosine

kinase Phosphatase ?
(No Protein Tyrosine Phosphorylation)

Protein Tyrosine Phosphorylation

CAPACITATION

Figure 4. Mechanism of BSP-mediated sperm capacitation. Modified from Therien et al.

(2001) and Therien et al. (2005).

15-Manjunath.p65 223 3/12/2007, 11:37 AM

 224 P. Manjunath et al.

BSP proteins are detrimental to sperm storage

In contrast to their beneficial role in sperm function, BSP proteins may be detrimental to sperm
in the context of sperm storage. This is because BSP proteins stimulate cholesterol and phos-
pholipid efflux from the sperm membrane in a time and concentration dependent manner
(Therien et al., 1998, 1999). Therefore, a continuous exposure of sperm to seminal plasma (as
in the case of semen collected for in vitro storage), which contains BSP proteins, causes con-
tinuous cholesterol and phospholipid removal from the sperm membrane, which can render
sperm very sensitive to storage in the liquid or the frozen states (for a review, see (Bergeron
and Manjunath, 2006)). Thus, BSP proteins are beneficial or detrimental to sperm depending on
the concentration and duration of exposure.

Egg yolk low-density lipoproteins interact with BSP proteins

Since BSP proteins are choline phospholipid-binding proteins and that low-density lipoproteins
(LDL) contain these lipids, we investigated whether or not BSP proteins interact with LDL, the
component responsible for sperm protection by egg yolk (Manjunath, Nauc, Bergeron and
Menard, 2002). Interestingly, BSP proteins bind to LDL in a rapid and saturable manner. More-
over, the binding capacity of LDL is very high. In fact, one molecule of LDL (Mr 0.6-1.4 x 106
Da) can bind 240-555 molecules of BSP proteins. After freeze-thawing semen, BSP proteins are
still associated with egg yolk-LDL. This interaction appears to have significant effects on the
preservation of sperm functions (Bergeron, Crete, Brindle and Manjunath, 2004).

Novel mechanism of sperm protection by egg yolk-LDL

In view of the above study, a novel mechanism of sperm protection by LDL has been proposed
(Fig. 5). Upon ejaculation, BSP proteins secreted by the seminal vesicles are added to sperm
(Manjunath et al., 1994). BSP proteins then bind to the sperm membrane (Desnoyers and
Manjunath, 1992; Manjunath et al., 1994) and induce cholesterol and phospholipid efflux
(Therien et al., 1998, 1999). If semen is not diluted, sperm are continuously exposed to a high
concentration of BSP proteins and the lipid removal continues, resulting in decreased sperm
resistance to cold shock and freezing. Since ejaculates are diluted with egg yolk extenders
within minutes after collection, LDL sequestrates most of the BSP proteins present in semen.
This could result in minimal modification of the sperm plasma membrane and allow better
sperm storage. Thus, LDL may offer protection to sperm by reducing the deleterious effect of
BSP proteins on the sperm membrane. In support of this novel mechanism, we observed that
when semen is diluted with egg yolk- or LDL-containing extenders, 50-80% less BSP proteins
associate with sperm and concomitantly, lipid (cholesterol and phospholipid) efflux from the
sperm membrane is prevented during storage (Bergeron et al., 2004). Consequently, sperm
functions such as motility, acrosomal integrity and viability are protected. Since BSP proteins
are involved in sperm capacitation and sperm binding to oviductal cells (Manjunath and Therien,
2002; Gwathmey et al., 2003, 2006), the decrease in sperm-bound BSP proteins in the pres-
ence of egg yolk extender may be one of the reasons why cryopreserved sperm have impaired
fertility compared to fresh semen.
It is remarkable that extender-containing egg yolk is also used to store semen from other
mammals such as boar, ram, goat, stallion, buffalo, dog, monkey, gazelle and human (for a
review, see Bergeron and Manjunath, 2006). Since BSP proteins are ubiquitous among mam-
mals and the BSP homologues found in the seminal plasma of stallion, boar, goat, bison and

15-Manjunath.p65 224 3/12/2007, 11:37 AM

 Seminal plasma proteins 225

Figure 5. Mechanism of sperm protection by egg yolk (Manjunath et al., 2002).

ram also bind to LDL (Menard et al., 2003; Villemure et al., 2003; Boisvert et al., 2004;
Bergeron et al., 2005), the mechanism of sperm protection by egg yolk may be the same for all
mammals.

Conclusions
Elucidating the mechanisms occurring during fertilisation is fundamental to resolve causes of
infertility, develop contraceptives and sperm storage media, and optimise in vitro fertilisation.
One of the key events regulating fertility is sperm capacitation, which is a complex and multi-
step phenomenon. It occurs in the female reproductive tract and requires factors present in the
oviduct. Despite more than 50 years of research and efforts by hundreds of scientists, the
molecular events involved in sperm capacitation are still being investigated. Our laboratory
has identified a family of lipid-binding proteins of seminal plasma (BSP) proteins that play a
key role in this complex process. Based on our studies aimed at investigating the effects of
purified BSP proteins, isolated follicular fluid-HDL and -GAGs on epididymal sperm, we have
proposed the sequence of events shown in Figure 4. This investigation also unravelled another
important mechanism that is sperm protection by egg yolk during storage (Fig. 5). While a brief

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 226 P. Manjunath et al.

exposure of sperm to BSP proteins (as in natural mating) is beneficial to sperm (promotes
capacitation), a continuous exposure to BSP proteins (as in the case of semen collected for
artificial insemination) is detrimental. However, the detrimental effect of seminal plasma BSP
proteins is abolished or minimised when they associate with LDL, the protective agent present
in egg yolk extender. This discovery may lead to improvements in the existing protocols for
semen preservation and in developing novel protocols for the preservation of semen from
domestic, farm, zoo and endangered animals as well as from human. The existence of benefi-
cial and detrimental factors for sperm in seminal plasma has been known for many years.
However, the fact that the same family of proteins acts like a double edged-sword that is both
beneficial and detrimental to sperm depending on the context (capacitation or preservation) is
surprising. We hope that this review will create renewed interest in the significance of seminal
plasma proteins as well as their effect on sperm storage.

Acknowledgements
This review, which is based on the plenary talk by PM to the 10th International Symposium on
Spermatology, was possible due to a “IHDCYH Lectureship in Child and Reproductive Health”
awarded by the Institute of Human Development, Child and Youth Health of the Canadian
Institutes of Health Research. This work was supported by grants from the Canadian Institutes of
Health Research, Natural Sciences and Engineering Research Council of Canada, Lalor Founda-
tion, World Health Organization, Semex Alliance, Cattle Breeding Research Council and
L’Alliance Boviteq (Canada). We appreciate the collaboration of Mr Yves Brindle, Centre
d’Insémination Artificielle du Québec. We thank all the graduate students, postdoctoral fel-
lows and research assistants who contributed to the progress of this research since the project
was initiated in 1984.

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