Seminal plasma is generally viewed as a transport and survival medium for sperm. However, its role is now recognized to extend beyond this to targeting the female tissues after insemination. Studies in rodents, livestock species, and humans show that introduction of semen into the female reproductive tract elicits molecular and cellular changes, with the physiological consequences bearing on conception and pregnancy. Factors in seminal plasma and associated with sperm that have potential biological effects in the female include cytokines, sex hormones, and prostaglandins (Mann, 1964; Aumuller and Riva, 1992; Maegawa et al., 2002). These molecules bind to target cells in the cervix, uterus, and oviduct, activating changes in gene expression that impact the function of the tract and the likelihood of successful establishment of pregnancy (Robertson and Sharkey, 2001; Robertson, 2005).
In this short review, we summarize our current understanding of the physiological significance of seminal fluid signaling in rodent and porcine species, which share the attribute of in utero insemination and the capacity for seminal fluid targeting of the upper region of the female reproductive tract. A better knowledge of this relatively unexplored aspect of reproductive biology might bring new insight to the events of early pregnancy and have implications for farm practice in the pig breeding industry.
THE INFLAMMATORY RESPONSE TO INSEMINATION
The immediate response to insemination in mammals is a rapid and dramatic influx of inflammatory cells into the site of semen deposition. This inflammatory reaction to semen was first observed in rabbits (Taylor, 1982) but has since been described in several other species (Robertson, 2005). Studies in rodents provide a comprehensive understanding of the molecular and cellular regulation of the inflammatory cascade and have informed experiments in pigs and other animals, as well as in humans. In mice, the response is initiated when seminal proteins interact with estrogen-primed cervical and uterine epithelial cells to activate synthesis of proinflammatory cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, macrophage chemotactic protein-1, IL-8, and an array of other chemokines (Sanford et al., 1992; Robertson et al., 1996; Robertson et al., 1998). Within hours of insemination, the surge in proinflammatory cytokine release causes inflammatory leukocytes, including macrophages, dendritic cells, and granulocytes, to traffic from the blood and accumulate in the subepithelial stromal tissue. During the initial acute phase, large populations of neutrophils migrate through the epithelial surface and efflux into the uterine luminal cavity (De et al., 1991; McMaster et al., 1992; Robertson et al., 1996). The response is short-lived and is resolved before embryo implantation, in parallel with the decline in inflammatory cytokine release as rising progesterone levels inhibit synthesis of GM-CSF and proinflammatory chemokines (Robertson et al., 1996, 1998).
An inflammatory cascade regulated by similar processes occurs after insemination in the pig uterus. Neutrophils are recruited into the uterine lumen within hours of insemination (Lovell and Getty, 1968; Claus, 1990; Rozeboom et al., 1998), and this is accompanied by accumulation of macrophages and dendritic cells, granulocytes, and lymphocytes in the endometrial stroma (Bischof et al., 1994, 1995; Engelhardt et al., 1997). Sperm and seminal plasma contribute to this response; experiments with vasectomized boars show that seminal plasma constituents can mediate an increase in endometrial macrophage numbers (Bischof et al., 1994; O’Leary et al., 2004), whereas insemination with washed sperm caused a greater influx of neutrophils into the luminal cavity than seminal plasma or whole semen (Rozeboom et al., 1999).
Studies with gilts administered pooled seminal plasma by transcervical catheter showed that specific factors present in seminal plasma interact with uterine cells to induce expression of GM-CSF, IL-6, and monocyte chemoattractant protein-1, and that neuroendocrine stimulation resulting from the physical act of mating is not necessary for this response (O’Leary et al., 2004). These inflammatory factors have chemotactic properties and are implicated in regulating macrophages and dendritic cell recruitment into the endometrial stroma. Although the luminal neutrophil response is resolved within 24 h (Rozeboom et al., 1998), the inflammatory cells infiltrating the endometrium appear to persist and undergo differentiation in the tissue for several days, expanding local activated macrophage and dendritic cell populations for the duration of the preimplantation period (O’Leary et al., 2004). This transition in leukocyte phenotype is likely to be driven by the more rapid termination of inflammatory cytokine synthesis that occurs in gilts exposed to seminal plasma (O’Leary et al., 2004).
INFLUENCE OF SEMINAL PLASMA ON FEMALE REPRODUCTIVE EVENTS
The inflammatory response to seminal fluids has a central role in female tract processing of seminal material and recovery of tissue homeostasis after mating. In addition, there is mounting evidence to suggest that exposure to seminal fluids can proactively influence subsequent events in the female tract to promote conception and progression of pregnancy. Although the success of AI shows that seminal constituents other than sperm are not mandatory for pregnancy, there are data showing that reproductive success and quality of the outcome can be compromised if females are not exposed to seminal plasma. Depending on the species, effects ranging from reduced fertilization and embryo implantation to altered growth of the placenta and fetus are observed when seminal signaling is perturbed.
The most compelling information comes from rodents and shows that seminal fluid can influence sperm survival and competence, development of the preimplantation embryo, and receptivity of the uterine endometrium. Experiments in which the seminal vesicle, prostate, or coagulating glands are surgically removed from mice, rats, or hamsters before mating each show that seminal vesicle fluid is the most vital nonsperm component of the ejaculate (Pang et al., 1979; Queen et al., 1981). In the absence of seminal plasma, impaired fertility appears to result from a reduction in the forward motility and survival of sperm, resulting in reduced fertilization (Peitz and Olds Clarke, 1986); in hamsters, this is accompanied by a slower cleavage rate in preimplantation embryos and greater fetal loss after implantation (O et al., 1988). In mice, embryo transfer protocols generally employ recipients exposed to seminal plasma by mating to vasectomized males, but fetal loss and abnormality is considerably greater when embryos are transferred after pseudopregnancy achieved without exposure to male fluids (Watson et al., 1983). Confirmation that the effects of seminal plasma are mediated, at least partly, through promoting receptivity in the female tract is provided by studies showing that when recipient females are mated with seminal vesicle-deficient males, transferred embryos implant normally but give rise to fetuses with retarded growth trajectories and placental development (Bromfield et al., 2004). In rats, implantation rates and fetal growth are similarly impaired unless females are inseminated before embryo transfer (Carp et al., 1984).
Well-designed studies to evaluate seminal fluid exposure in pigs are scarce. Reports on fertility rates using AI compared with natural mating are difficult to reconcile across studies, with considerable variation in reproductive performance reflecting different breeding practices including the inseminator’s experience, the method of estrous detection, and the insemination protocol. Comparisons made when AI was first implemented suggested that conception rates and litter sizes were compromised by AI compared with natural mating (Baker et al., 1968; Skjervold, 1975; Claus, 1990). However, with current AI practice in modern, large-scale facilities, farrowing rates and litter sizes are comparable to natural service. Pregnancy rates of >90%, farrowing rates of >85%, and litter size of >9 are routinely achieved after AI in pigs using a single dose of the sperm-rich fraction of semen diluted 20 to 30 times (Vazquez et al., 2005). This suggests that seminal fluid stimulation of the female tract is not essential, particularly when insemination occurs close to the time of ovulation, and when sperm are deposited high in the reproductive tract to minimize physical loss (Vazquez et al., 2005).
In herds with poorer reproductive performance or a less than optimal breeding practice, seminal plasma can improve reproductive outcomes even after natural mating with an intact boar. Increases in farrowing rates from 70 to 81% were reported when seminal plasma was administered before natural service, and seminal plasma treatment increased farrowing rate and litter size when given with AI plus natural service (Flowers and Esbenshade, 1993). Similar results were reported when mating with an intact boar was supplemented by seminal plasma provided by a vasectomized boar (Mah et al., 1985). Uterine infusion with heat-killed semen during the previous estrus can also increase litter size and improve farrowing rate (Murray et al., 1983, 1986), and similar effects result from mating with vasectomized boars in previous estrous periods (Flowers and Esbenshade, 1993), suggesting that some benefit persists into subsequent cycles. Similarly, when AI is impaired by a less than optimal female condition, seminal plasma may promote pregnancy success. When tracts were previously exposed to an adverse inflammatory treatment, improvements in conception rate and farrowing rate resulted from addition of seminal fluid to sperm at AI (Rozeboom et al., 2000).
TRANSFORMING GROWTH FACTOR-β AND OTHER ACTIVE FACTORS IN SEMEN
Depending on the animal species, the proinflammatory signals in seminal fluid have been associated with both sperm and seminal plasma. Experiments with mice from which the accessory glands were surgically removed showed that in this species the active inflammation-inducing moieties are derived from the seminal vesicle, where the majority of seminal fluid is produced (Robertson et al., 1996). Using protein chromatographic techniques and neutralizing antibodies, transforming growth factor-β (TGF-β) was identified as the principal trigger for induction of uterine inflammatory responses (Tremellen et al., 1998; Robertson et al., 2002). Seminal vesicle TGF-β synthesis is testosterone-dependent, with a severe reduction evident after castration, and partial recovery after administration of exogenous testosterone (Robertson et al., 2002). The majority of the TGF-β present in male seminal fluids is synthesized in the latent form and appears to be activated in the female reproductive tract by plasmin and other enzymes after insemination (Robertson et al., 2002).
In pigs, it appears likely that different active fractions associated with sperm and seminal plasma can interact with each other and with female tract cells to attenuate the quality and duration of the uterine inflammatory response. Seminal plasma attenuation of the inflammatory infiltrate (Rozeboom et al., 1999, 2001a) is mediated by seminal plasma inhibition of boar sperm-induced activation of chemotactic components in plasma (Rozeboom et al., 2001b). However, the precise nature of the active constituents remains to be identified. Although TGF-β cytokines have been detected in high concentrations in boar seminal fluid (O’Leary et al., 2002), and immunosuppressive activity characteristic of TGF-β is associated with protein fractions of the appropriate size in boar seminal fluid (Claus, 1990), whether this cytokine family contributes to regulation of the female inflammatory response in gilts is unproven.
A recent study failed to find any beneficial effect of recombinant TGF-β1 administered at AI on total or live implantation rate at d 80 of pregnancy (Rhodes et al., 2006); however, with only 9 to 11 gilts per treatment, this study was not powered to detect the small changes in litter size expected on the basis of previous studies with seminal plasma. Estrogens are another candidate mediator because boar seminal plasma is a rich source of estrogens, and transcervical administration of estrogen can elicit some improvement in farrowing rate (Flowers and Esbenshade, 1993) and mimic some aspects of the physiological response to insemination (Claus, 1990). Prostaglandins and oxytocin have also shown promise in eliciting farrowing rate and litter size increases (Flowers and Esbenshade, 1993).
FEMALE TRACT RESPONSES ACTIVATED BY SEMINAL FLUID
The inflammatory response stimulated by seminal fluid affects several reproductive processes by virtue of the wide range of biological downstream effects of the leukocytes recruited into the endometrial and cervical tissues (Figure 1). These inflammatory changes are likely to act in concert with the direct effects of seminal fluid to coordinate insemination with the events of ovulation and maximize the chances of conception and initiation of pregnancy. Four categories of effector function are likely: (1) clearance of superfluous sperm and microorganisms introduced into the uterus at mating; (2) activation of female immune responses specific to paternal transplantation proteins and other antigens present in semen; (3) tissue remodeling associated with preparation of endometrial receptivity; and (4) activation of expression of cytokines and growth factors implicated in preimplantation embryo development. Together, these seminal plasma-induced changes in the female tissues act to assist in delivery of the most competent male gametes to the oocyte and maximize the opportunity for fertilization, embryo development, and successful implantation.
IMPACT ON SPERM SURVIVAL AND TRANSPORT
Seminal plasma is a diluent and vehicle for sperm that supports sperm viability through nutritive and protective activities (Mann, 1964). In pigs, seminal plasma facilitates delivery of sperm to the upper reproductive tract through direct effects on uterine contractility mediated by estrogen, which acts through inducing PGF2α release immediately after insemination (Claus, 1990). Inflammatory cells recruited in response to seminal fluid further influence the capacity of sperm to reach the oocyte, particularly the abundant populations of neutrophils that infiltrate the uterine lumen to remove superfluous sperm, microorganisms, and seminal debris. The upper reproductive tract is normally sterile, with intromission providing the opportunity for access by commensal microorganisms originating from male and female tissues, or sexually transmitted pathogens. A male contribution to maintenance of a sterile female tract would assist sperm survival and increase the likelihood of a mating event resulting in pregnancy. At least in humans, there is evidence of selective sperm phagocytosis acting to filter morphologically abnormal spermatozoa (Tomlinson et al., 1992). Through differential resistance to phagocytosis, individual sperm could thus be selected for fertilization competence. There is evidence that apparently viable and morphologically normal spermatozoa are also targeted, suggesting selection may occur on the basis of morphological or antigenic parameters other than the ability to fertilize (Taylor, 1982; Roldan et al., 1992). This raises the prospect of the inflammatory response providing a mechanism for active female tract selection of male gametes permitted to advance to fertilization of oocytes, although such a mechanism remains hypothetical until a molecular basis for leukocyte discrimination between sperm is identified.
The physiological significance of seminal plasma in uterine clearance and immune regulation might be particularly important in species where rapid resolution of the uterine inflammatory response is linked with reproductive success. In swine, AI into an inflamed uterus impairs conception rate and successful progression to farrowing, but coadministration of seminal plasma alleviates the effect of a hostile environment (Rozeboom et al., 2000). This study concluded that seminal plasma acts largely through protecting sperm from neutrophil attack by reducing the numbers of neutrophils accessing the luminal compartment (Rozeboom et al., 1999). Together with the direct effects of seminal plasma on neutrophils (Rozeboom et al., 2001a), seminal plasma regulation of uterine chemotactic factors is likely to contribute. Exposure to seminal plasma initially activates endometrial expression of cytokines and chemokines, but this is followed by more rapid termination of GM-CSF, monocyte chemoattractant protein-1, and cyclooxygenase-2 (COX-2) mRNA synthesis than in tracts exposed to carrier alone (O’Leary et al., 2004). Furthermore, it is important to note that the activation phenotypes of inflammatory cells are highly variable and responsive to microenvironmental signals (Hunt and Robertson 1996), so that the immune regulatory cytokines induced by seminal plasma are likely to attenuate the functional properties of the inflammatory leukocytes recruited into the female tract.
ACTIVATION OF MATERNAL IMMUNE TOLERANCE OF PREGNANCY
In rodents and pigs, macrophages and dendritic cells comprise the major populations of inflammatory cells retained in the endometrial stromal tissue after exposure to semen (Robertson et al., 1996, O’Leary et al., 2004) because the neutrophil response is short-lived, with the majority of these cells eliminated via the uterine lumen within 24 h (McMaster et al., 1992; Rozeboom et al., 1998). Macrophages and dendritic cells are professional antigen processing and presenting cells that take up and transport seminal antigens to draining lymph nodes, resulting in activation of immune responses to paternal major histocompatibility complex (MHC) and other antigens in semen. The female immune response to seminal antigens is characterized in mice by hypertrophy of lymph nodes draining the uterus and evidence of lymphocyte activation (Beer and Billingham, 1974; Johansson et al., 2004).
Hostile immune responses to seminal antigens would be incompatible with fertility by preventing the female tract from tolerating future semen exposures. There would also be consequences for pregnancy because the conceptus shares paternal antigens with those in semen (Thaler, 1989). However the immune activation elicited by semen does not cause rejection of male antigens, due to the presence in seminal plasma of several powerful immunoregulatory molecules, such as PGE and TGF-β, that dampen potentially destructive type-1 (cell-mediated) immune responses and instead skew the immune response toward regulatory lymphocyte activation (Letterio and Roberts, 1998; Weiner, 2001). Consistent with the expected actions of PGE and TGF-β, the female tract immune response to semen results in a state of functional immune tolerance to male antigens (Robertson et al., 1997). Because there are similarities between the MHC and other antigens carried by semen and the conceptus, the female immune response to ejaculate antigens might also confer immune protection to the conceptus (Robertson et al., 1997).
There are few studies to evaluate the existence of homologous immune events in large animal species; however, their occurrence is likely given the presence of the necessary antigenic elements and immune-deviating compounds in semen. The endometrial inflammatory response after insemination in gilts is accompanied by expression of activation markers (MHC class II and IL-2 receptor) expression in draining lymph nodes (Bischof et al., 1994; Waberski et al., 2006), reflecting lymphocyte activation in response to seminal antigens and the possibility of immunological consequences for an ensuing pregnancy. Observations that administration of pooled leukocytes to the AI dose can improve embryo viability (Almlid, 1981) are also consistent with semen providing an antigenic stimulus that activates the female immune response in readiness for pregnancy. A mechanism involving immune priming to seminal antigens would also explain the beneficial effects of uterine treatments with seminal or leukocyte antigens at previous estrous events (Murray et al., 1983, 1986; Flowers and Esbenshade, 1993).
REGULATION OF ENDOMETRIAL TISSUE REMODELING AND UTERINE RECEPTIVITY
Leukocytes can exert effects in their local milieu by secreting an array of potent enzymes and signaling molecules that affect extracellular matrix turnover and the behavior of endothelial cells in the endometrium. The leukocytes recruited in response to semen, particularly macrophages, are implicated in restructuring the endometrial environment to facilitate implantation and placental development (Hunt and Robertson, 1996).
Regulation of angiogenesis is the major potential avenue for macrophage effects on implantation. Activated macrophages have the capacity to influence each phase of the angiogenic process, including alterations of the local extracellular matrix, induction of endothelial cells to migrate and proliferate, and formation of capillaries (Sunderkotter et al., 1994). Consistent with a proangiogenic role for semen-induced inflammatory changes, vascular endothelial growth factor mRNA abundance in hamsters is reduced after mating with accessory gland-deficient males (Chow et al., 2003). An additional target for macrophage-secreted products is the extracellular matrix of the endometrial stroma, which is remodeled before and during the decidual transformation, with breakdown of the existing matrix and the deposition of new components (Aplin, 2002). Matrix metalloproteinases (MMP) regulate this process after coordinated increases in their transcription, secretion, and proteolytic activation, along with their regulatory proteins, the tissue inhibitors of metalloproteinases. Macrophages are a major source of a broad range of MMP under the influence of cytokines, extracellular matrix, and PG (Goetzl et al., 1996). Consistent with a role for semen-induced inflammatory cells in MMP regulation is the finding in golden hamsters that the absence of male accessory gland fluids is associated with reduced expression of MMP-2 in the implantation site (Chow et al., 2003).
Macrophage mediators potentially also target the luminal epithelial cells involved in embryo attachment during the initial phases of implantation (Kosaka et al., 2003). Macrophages interdigitate between epithelial cells in the endometrium (Hunt and Robertson, 1996), where they would be well-positioned to influence integrin expression at the paracrine level. In pigs, an increase in the number of endometrial glands is one of the most striking changes induced by exposure to a vasectomized boar (Bischof et al., 1994), indicating that macrophages recruited after seminal exposure might release cytokines that promote epithelial cell proliferation. Evidence of increased endometrial vascularity and edema was observed during the acute phase of the inflammatory response to seminal plasma in gilts (O’Leary et al., 2004), but whether any seminal fluid-induced changes in vascular or matrix structure persist through the pre-implantation period remains to be evaluated.
SYNTHESIS OF EMBRYOTROPHIC CYTOKINES
Cytokines synthesized by uterine and oviductal epithelial cells are secreted into the uterine luminal fluid, where they interact with the developing embryo before implantation. Several of these embryotrophic cytokines are regulated by semen exposure, providing a further nexus through which seminal fluid signaling can promote embryo development and implantation success. A principal cytokine in the postmating inflammatory response, GM-CSF, targets the preimplantation embryo to increase the number of viable blastomeres through inhibiting apoptosis and regulating glucose uptake (Robertson et al., 2001). Other cytokines targeting the developing blastocyst, including IL-6 and leukemia inhibitory factor, are induced after exposure to semen (Robertson et al., 1992). Perturbations in the growth factor environment experienced by the preimplantation embryo impair normal development of the placenta and fetus, with long-term consequences for postnatal health and metabolic programming in the progeny (Sjoblom et al., 2005).
The quality of the uterine environment is a well-recognized factor in embryonic viability and development in pigs. Treatment of gilts with seminal plasma has been shown to increase the numbers of viable embryos and to alter the embryo growth trajectory during the preimplantation period (O’Leary et al., 2004). Surprisingly, this study showed the increased number of viable blastocysts retrieved from the tract on d 9 of pregnancy was associated with a significant reduction in their average size, indicating seminal conditioning of the tract may act to support embryo survival, while constraining their pace of development. This increase in embryo survival presumably reflects indirect effects of seminal constituents on the reproductive tract environment, acting to reduce early embryo mortality, most likely through regulating epithelial cell secretion of embryotrophic factors or nutritional support. Greater developmental synchrony within the cohort of embryos, mediated through the effects of cytokines or reflecting the timing of ovulation (see below), could also contribute to improved embryo viability in a manner similar to that observed in Meishan pigs (Rivera et al., 1996).
REGULATION OF OVARIAN FUNCTION
The actions of seminal fluid can reach beyond the immediate site of deposition and exert effects in organs and systems elsewhere in the female body. In mice, macrophage populations in corpora lutea are augmented by exposure of the female tract to seminal plasma constituents (Gangnuss et al., 2004). In estrous pigs, there is a reduction in the interval between the LH surge and ovulation in response to natural mating (Signoret et al., 1972) or seminal plasma administered transcervically (Waberski et al., 1997). The effects appear to be mediated by local transport of seminal constituents or semen-induced effector molecules originating in the upper region of the tract because the effect is seen only in ovaries ipsilateral to uterine horns receiving the seminal stimulus. In recently published work, we have shown an effect of seminal plasma on development of the corpus luteum and its steroidogenic capacity (O’Leary et al., 2006), with infiltrating macrophages implicated as the mediating cells in this process.
The mechanism and route of signal transfer from the uterus to the ovary is unknown, but an attractive possibility is the vascular counter-current mechanism linking the uterus and ovary, with uterine prostaglandins targeting the ovary to induce local inflammatory changes. Studies in which uterine horns are unilaterally ligated implicate a local communication pathway between the uterine horn and the adjacent ovary (Waberski et al., 1995). The prostaglandins PGF2α and PGE, known to be induced in the endometrium by seminal plasma estrogens (Claus, 1990) and facilitated by upregulated COX-2 expression (O’Leary et al., 2004) are likely candidates in influencing follicular development and rupture. These endometrial prostaglandins have been shown to reach the ovary by countercurrent exchange between the closely apposed uterine venous network and the ovarian artery (Claus, 1990). In nonmated gilts, PGF2α is the major eicosanoid, but in mated gilts peak prostaglandin secretion occurs earlier and PGE2 predominates, antagonizing the luteolytic effects of PGF2α (Christenson et al., 1994). The increase in COX-2 mRNA after exposure to seminal plasma thus raises the tantalizing possibility of a new role for seminal plasma in amplifying uterine-derived prostaglandin signals that act to precipitate ovulation, exert antiluteolytic effects, and increase progesterone synthesis (Figure 2).
SUMMARY AND CONCLUSIONS
Exposure to seminal fluid elicits inflammatory changes in the reproductive tract of all mammalian species so far examined. Studies in rodents have provided the greatest insight into the molecular and cellular basis of this response and its role in reproductive physiology, underpinning the view that seminal fluid must be considered not just a survival medium for sperm, but as a means for communication between the male and female reproductive tissues and a key factor in conditioning the female tract for pregnancy. In rodents, seminal fluid exposure contributes to optimal pregnancy outcome, through activating inflammatory and immunological changes in the female reproductive tract that have beneficial effects for embryonic development and implantation. Many aspects of the mechanisms explaining these effects are now elucidated, with uterine cytokines and leukocytes playing central roles.
New studies informed by knowledge gained in the mouse are beginning to identify cytokines and other components of seminal fluid signaling pathways in pigs. However, there is still much to learn in this species regarding (1) the nature of and interaction between active moieties present in sperm and seminal plasma, (2) the identity of the cytokine and leukocyte responses elicited in the female tissues, and (3) the downstream consequences for the immune response to male gametes, embryonic development, and endometrial receptivity. Whether seminal fluid signaling has any beneficial role in establishing pregnancy in pigs, or alternatively is concerned more with maintaining female tract homeostasis, requires further evaluation. To address this, well-designed experiments with adequate power are required to quantify the effects of exposure to seminal fluid and its constituents in early pregnancy. In addition to pregnancy and farrowing rates, characteristics including fetal development and postnatal growth trajectory, and the viability, health, and body composition of offspring all warrant investigation. These studies need to take into account the significance of collection and storage conditions on seminal fluid signaling potency and the possible importance of matching between seminal plasma and sperm donors, as well as confounding factors such as the timing of treatment relative to estrus and AI, and variation in breeding performance of different herds.
A better understanding of seminal plasma signaling in pigs could lead to improved breeding management protocols to reduce embryo mortality in early pregnancy and ensure maximal reproductive efficiency. Options to explore might include more widespread utilization of vasectomized males, less extensive dilution of seminal fluid, or utilization of proteins present in seminal fluid to improve the function of commercial semen extenders.