Growth hormone receptors in the porcine testis during prepuberty.

Supplementation of exogenous growth hormone (GH) during prepuberty advances onset of spermatogenesis in boars, but the mechanism of action is unknown. The present study is an investigation of the presence and characteristics of testicular growth hormone receptors (GHR). A total of 36 boars were castrated, three boars every 10 days, between the ages of 10 and 120 days. Testicular membrane preparations of 10, 20, 30, 50, 70, 100 and 120-day-old boars were used to determine (125)I-bGH binding and Scatchard analysis. Liver from a 60-kg barrow was used for comparison. Specific (125)I-bGH binding to testicular membrane preparations occurred in all age groups with the exception of 20-day-old boars at levels of 30-40% of liver binding. At 30 days of age the unlabelled bGH at 1.1 ng/tube achieved half maximal inhibition (ID(50)). Results of Scatchard analysis indicated a single class of binding sites. Binding affinity was 2.89 x 10(9) m with a binding capacity of 12 fmole/mg membrane protein. The results from this study suggest that GH may act directly on the cells of the prepubertal boar testis.

Interaction between equine semen and the endometrium: the inflammatory response to semen.

Insemination of mares with bacteria-free equine spermatozoa results in an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen. In vitro studies have demonstrated that equine spermatozoa activate complement, resulting in cleavage of factors C5a and C3b. Since uterine secretion is rich in complement, it is likely that an interaction between spermatozoa and uterine secretion results in C5a-mediated chemotaxis and migration of PMNs into the uterine lumen. Once in the uterine lumen, the PMNs phagocytize bacteria and spermatozoa, which is an important part of sperm elimination from the reproductive tract. It is not clear how the spermatozoa are opsonized, or if phagocytosis of equine spermatozoa is a selective or non-selective process. Breeding-induced endometritis appears to be both up and down regulated by seminal components. A modulatory role on the inflammation has been suggested for equine seminal plasma. Seminal plasma suppressed complement activation, PMN-chemotaxis and phagocytosis in vitro. Preliminary in vivo experiments also support a suppressive role of seminal plasma in breeding-induced endometritis. The duration but not the magnitude of the PMN-influx into the uterine lumen was shortened when seminal plasma was included in an insemination dose. The presence of PMNs in the uterus affects the motion characteristics of spermatozoa in vitro. Both progressive motility and mean path velocity were impaired when spermatozoa were incubated in uterine secretion from mares with ongoing breeding-induced endometritis. The binding of spermatozoa to PMNs was prominent in all samples collected from mares with an ongoing endometritis. The motility remained impaired, but the binding of the spermatozoa to PMNs was reduced when the spermatozoa were incubated in uterine secretion in the presence of seminal plasma. Preliminary characterization of the immune-suppressive component in seminal plasma suggests that it is one or more molecule(s) with a molecular weight between 50 and 100 kDa, partially inactivated by charcoal stripping and partially heat-inactivated at 95 degrees C for 45 min.

Effect of scrotal insulation on clusterin-positive cells in ram semen and their relationship to semen quality.

Scrotal contents of 2 rams were insulated for 96 hours and the fraction (as a percentage) of clusterin-positive cells (CPCs) and its relationship to semen quality was investigated. Semen collection was started 18 days before insulation and was terminated on day 78 and day 63 after insulation in animals 1 and 2, respectively. Sperm clusterin was localized by immunostaining with anti-bovine clusterin antibody (anti-bCAb) and fluorescein isothiocyanate-conjugated immunoglobulin G. Scrotal insulation led to deterioration of semen quality and increased the percentage of CPCs in both rams. Two types of sperm reactivity were observed: an extensive, intensive staining pattern (ESP); and a localized, less-intensive staining pattern (LSP). The percentage of ESP-CPCs began to increase from day 6 and reached 88.8% and 100% on day 15 after insulation in animals 1 and 2, respectively. The increase in CPCs coincided with the presence of a high percentage of teratoid forms (88.3%) in semen from animal 1, and detached heads (81.4%) in semen from animal 2. After normal semen production was restored on day 60 in animal 1, the percentages of ESP-CPCs and LSP-CPCs returned to preinsulation rates, whereas only the ESP-CPCs returned to normal in animal 2. A negative relationship was observed between ESP-CPCs and total sperm/ejaculate (r = -.62), motility (r = -.78), viability (r = -.68), and filtration rate (r = -.71) in semen from animal 1. Conversely, a positive relationship was seen between ESP-CPCs and total abnormal spermatozoa (r = .82). Similar results were obtained in semen from animal 2. CPCs were nearly completely absent in glass wool-Sephadex (GWS)-filtered semen, suggesting a role for clusterin in the process of trapping abnormal spermatozoa in the GWS filters. We conclude that aberrant spermatogenesis induced by scrotal insulation increases the percentage of CPCs in ram semen. We suggest that the percentage of CPCs in ram semen could be a useful marker in poor-quality ejaculates.

Localization of clusterin on freeze-preserved bull spermatozoa before and after glass wool-sephadex filtration.

Clusterin is a major protein in bull reproductive tract secretions and sperm membrane extract. A polyclonal antibody was produced against clusterin from bull cauda epididymal fluid (CEF) and used for the localization of the protein on bull spermatozoa. Immunoblotting of unreduced bovine samples showed that the anticlusterin antibody reacted with a protein of approximately 94- to 100-kd in rete testis fluid (RTF), a approximately 57- to 76-kd protein in CEF, and with a approximately 57- to 60-kd protein from cauda epididymal sperm membrane extract. The antibody also reacted with stallion RTF and both ram CEF and RTF at relative molecular weights (Mr) that were consistent with the anticipated size of clusterin in these species. Less intense immunostaining was observed for a protein of about 2 times the predicted size of clusterin in unreduced ovine RTF, suggesting the presence of multimers of clusterin in ovine RTF. Also, a dimeric clusterin-sized protein was detected in reduced bovine CEF, suggesting the presence of unprocessed clusterin in bovine CEF. By immunofluorescence, clusterin was detected on only a small fraction of bull spermatozoa, which were morphologically abnormal. Neither permeabilization nor the method of dilution affected the reactivity of the antibody with spermatozoa (P > .05). Average clusterin-positive spermatozoa (CPS) in unpermeabilized, permeabilized, abruptly diluted, and gradually diluted semen were 10.1%, 11.3%, 15.0%, and 14.4%, respectively. CPS were eliminated from semen after filtration through glass wool-Sephadex (GWS) columns. Average CPS in unfiltered and filtered semen were 14.3% and 1.1%, respectively. We conclude that sperm clusterin in bull semen is associated with morphologically abnormal spermatozoa and that clusterin is implicated in the process of abnormal spermatozoa trapping in GWS columns. We suggest that the fraction of CPS in bull semen is a potential marker for poor semen quality.

The chemotactic properties of porcine seminal components toward neutrophils in vitro.

Our objectives were to investigate the mechanisms of postbreeding inflammation in swine by examining the chemotactic properties of polymorphonuclear neutrophilic granulocytes (PMN) and of various populations of spermatozoa and seminal plasma. Epididymal spermatozoa from two boars obtained under sterile conditions, washed ejaculated spermatozoa from two boars, and pooled seminal plasma from eight boars of known fertility were examined for chemotaxis to PMN. The chemotaxis of blood-derived PMN in response to sperm and seminal plasma was evaluated and expressed as a percentage of a positive control (lipopolysaccharide-activated blood plasma). The mean chemotactic effect of washed sperm alone (4.4+/-0.04) and of epididymal sperm alone (3.4+/-0.06) was not different from that of the negative controls (3.1+/-0.05) of McCoy's medium with 10% heat-inactivated fetal calf serum. A marked chemotactic effect was detected when washed ejaculated and epididymal sperm were incubated with blood plasma, compared with blood plasma without spermatozoa (P < 0.001). Washed sperm in blood plasma (86.2+/-5.6) and epididymal sperm in blood plasma (83.9+/-7.7) were different from blood plasma alone (11.2+/-1.5), but no differences were detected between the two populations of sperm. This effect, however, was not completely inhibited by heat inactivation of the blood plasma. The chemotactic response of washed ejaculated and epididymal spermatozoa incubated in lipopolysaccharide-treated, heat-inactivated blood plasma were greater than that of the negative control (P < 0.05). Polymorphonuclear neutrophilic granulocyte migration toward seminal plasma was similar to the negative control (4.0+/-0.04 vs 3.1+/-0.05). It seems that porcine epididymal sperm and ejaculated sperm activate chemotactic components in porcine blood plasma and heat-inactivated blood plasma, suggesting that, at least partially, a heat-stable (noncomplement) blood plasma component may be involved in sperm-induced PMN chemotaxis. In contrast, porcine seminal plasma was not chemotactic to PMN. These results support the hypothesis that spermatozoa play an active role in initiating postbreeding endometritis.

Correlation between clusterin-positive spermatozoa determined by flow cytometry in bull semen and fertility.

The objectives were to 1) develop a rapid and accurate method for detection of clusterin-positive spermatozoa (CPS) in bull semen and 2) determine the utility of incidence of CPS for prediction of fertility of bull semen in comparison to routine semen quality traits. Semen from 3 bulls was immunostained with anti-bovine clusterin antibody and with FITC-conjugated anti-rabbit IgG for method development. Clusterin-positive spermatozoa were determined by flow cytometry (FCM) and fluorescence microscopy, and results were compared by paired t test. There was no difference between FCM and microscopic techniques (P = .81). Flow cytometry was then used for determination of CPS in semen of 48 bulls with known fertility. Significant inverse relationships were found between the percentage of CPS and raw nonreturn rate (r = -.30), adjusted nonreturn rate (r = -.58), and estimated relative conception rate (ERCR; r = -.60). Estimated relative conception rate is potentially a very accurate method for determining fertility, and it resulted in highest correlation with CPS. An inverse relationship was observed between the percentage of CPS and prefreeze and postfreeze motility (r = -.51), whereas a direct relationship was found between CPS and primary, secondary, tertiary, and total sperm abnormalities (r = .52, .77, .32, and .58, respectively). The fractions of motile and abnormal spermatozoa, with the exception of tertiary abnormalities, were inversely correlated with 2 or more of the fertility estimates, but none of them showed the characteristic increase in correlation with improvement of accuracy of fertility estimate as demonstrated by CPS. We conclude that FCM is useful for objective and efficient detection of CPS in bull semen. The results suggest that the percentage of CPS in bull semen is potentially a better predictor of fertility than sperm motility or abnormal morphology.

Effect of sterile service on estrus duration, fertility and prolificacy in artificially inseminated dairy goats.

The effect of sterile service on estrus duration, fertility and prolificacy in artificially inseminated dairy goats during breeding season was studied. Nubian does (n=126) were divided into 2 equal groups: service and control. Estrus was synchronized with intravaginal sponges containing either fluorgestone acetate (FGA; 40 mg) or medroxiprogesterone acetate (MAP; 60 mg) for 12 or 14 d, respectively. Two vasectomized teaser bucks were used to detect estrus at 6-h intervals for 5 d after sponge removal (0600, 1200, 1800 and 2400 h). The teasers were fitted with aprons and permitted to mount all does in both groups, but to penetrate only the service does within the first 12 h of estrus. Does in both groups were inseminated twice at 12 and 24 h after estrus was first detected, using 1 straw per insemination containing 200 million of cooled spermatozoa from 1 buck. The semen was placed in mid-cervix. Estrus duration for the service and control does was (mean +/- SD) 29.4 +/- 6.5 and 41.8 +/- 9.6 h, respectively. Fertility for the service does was 73.7% (46/63); for control does it was 58.7% (37/63). Prolificacy was 2.1 (96/46) and 2.0 (74/37) for service and control does, respectively. Estrus duration (P<0.001) and fertility (P<0.05) differed between the service and control group, but prolificacy was similar (P>0.05). It is concluded that sterile service reduces the duration of estrus and increases fertility in artificially inseminated dairy goats.

The importance of seminal plasma on the fertility of subsequent artificial inseminations in swine.

Yorkshire x Landrace sows and gilts were used in a 3x2 factorial arrangement of treatments to determine the effect of uterine inflammation induced by either killed spermatozoa (KS) or bacterial lipopolysaccharide (LPS) on the fertility of a subsequent, optimally timed AI. Estrus was detected with a mature boar twice daily. Twelve hours after the first detection of estrus, females received intrauterine infusions of an inflammatory stimulus consisting of a 100-mL dose of extender containing 3x10(9) KS (n = 40), 20 microg of LPS (n = 40; positive control) or extender alone (n = 40; negative control). An insemination was performed 12 to 18 h later with 3x10(9) motile spermatozoa (i.e., fertile AI) suspended in either 100 mL of seminal plasma (SP; n = 60) or extender replenished with of estrogens (5 microg of estradiol-17beta, 4.5 microg of estrone sulfate, and 2 microg of estrone; n= 60). Transcutaneous ultrasound was performed at the time of fertile AI and again 24 h later to detect the presence or absence of preovulatory follicles. A fertile AI performed within 24 h before ovulation was considered optimal. Conception (CR) and farrowing rates (FR) were greater in females that received a fertile AI diluted with SP compared with extender (P<.01), and there was a significant (P<.05) treatment x fertile AI dilution medium interaction for both CR and FR. Females that received a fertile AI 12 h after infusion of extender had similar CR and FR regardless of fertile AI dilution medium. After inducing an inflammatory response with either KS or LPS, CR and FR were higher in females that received a fertile AI diluted with SP compared with fertile AI dilution with extender (P<.05). The effects of treatment and AI dilution media and their interactions were not significant for litter size in females that farrowed. These results show that the fertility of a subsequent AI can be impaired when semen is deposited into an inflamed environment created by an earlier AI, and this impairment was offset by inclusion of SP in the subsequent insemination.

The role of seminal plasma in post-breeding uterine inflammation.

The effect of seminal plasma on polymorphonuclear neutrophil (PMN) chemotaxis, PMN phagocytosis and complement-induced cytolysis was determined using blood plasma pooled from four horses and seminal plasma pooled from two stallions. To investigate chemotaxis, complement in blood plasma was activated with E. coli lipopolysaccharide in the presence of 0-50% seminal plasma diluted with a standardized volume of McCoy's medium and placed in a chemotactic chamber. Chemotaxis of blood derived equine PMNs toward the chemoattractants was determined after incubation at 37 degrees C for 45 min. To investigate phagocytosis, equine spermatozoa (5 x 10(7) ml(-1); n=5) from fertile stallions (n=2) were mixed with blood plasma and increasing concentrations of seminal plasma (50-90%). The seminal plasma was removed after opsonization at 37 degrees C for 30 min and 4 x 10(6) PMNs were added to the sperm cells and incubated at 37 degrees C for 30 min. The number of phagocytosed spermatozoa per 100 PMNs and the proportion of PMNs that had ingested at least one spermatozoon were counted under a microscope. Antibody-sensitized sheep red blood cells were used to investigate complement-induced cytolysis. The 50% haemolytic complement activity (CH50) was determined in blood plasma and in the presence of increasing concentrations of seminal plasma (3-40%). Haemolysis was quantified by spectrophotometry at 540 nm. A dose-dependent inhibition of chemotaxis (P < 0.001), phagocytosis (P < 0.001) and complement-induced cytolysis (P < 0.001) was observed when seminal plasma was included in the assays. These results indicate that seminal plasma may have a downregulatory role in post-breeding endometritis.

The effect of spermatozoa and seminal plasma on leukocyte migration into the uterus of gilts.

Yorkshire x Landrace gilts were used to determine the effect of spermatozoa and seminal plasma on postbreeding uterine leukocyte influx. Estrus detection was performed with a boar at 12-h intervals following synchronization with 400 IU eCG and 200 IU of hCG. All gilts were AI once, 24 h after the detection of estrus following random assignment to a 2x2x3 factorial arrangement of treatments (sperm or sperm-free AI doses), AI dose medium (seminal plasma or PBS), and lavage time following AI. Gilts were treated with sperm (5x10(9) spermatozoa; SPZ; n = 30) or sperm-free (SF; n = 30) doses containing either 100 mL of seminal plasma (SP; n = 15/treatment) or PBS (n = 15/treatment). Uterine lavage was performed once on each gilt (n = 20/time) at one of three times after AI (6, 12, or 36 h) to determine the total number of uterine leukocytes. The leukocytes consisted predominately (92 to 99%) of polymorphonuclear neutrophilic granulocytes (PMN). There was an AI x medium interaction on uterine PMN numbers. The number of uterine PMN recovered from gilts inseminated with sperm suspended in PBS was greater than the number of PMN recovered from the uterine lumen of gilts inseminated with sperm in SP, SP alone, or PBS alone (P<.05). Furthermore, SP accelerated the rate of uterine clearance when suspended with sperm cells during the first 36 h following AI (P<.05). These results indicate that seminal plasma suppresses PMN migration into the uterus following breeding and enhances the rate of disappearance of uterine inflammation.

Reproductive tract secretions and bull spermatozoa contain different clusterin isoforms that cluster cells and inhibit complement-induced cytolysis.

Clusterin from bull rete testis fluid (RTF), cauda epididymal fluid (CEF), and octyl-beta-D-glucopyranoside extract of cauda epididymal sperm (CES) was identified and characterized using monoclonal and polyclonal antibodies (Abs) developed against ram clusterin and a beta-subunit-specific oligopeptide of porcine clusterin. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting showed that bovine RTF clusterin had dimeric and monomeric molecular weights (M(r)s) of approximately 94 kDa and of 42 and 43 kDa, respectively. Clusterin in CEF and CES had similar dimeric M(r)s (74 kDa). Reduced CEF clusterin appeared as three monomers (M(r)=40, 39, and 38 kDa), whereas reduced CES clusterin appeared only at M(r)40 kDa. Enzymatic deglycosylation resulted in similar M(r)s of clusterin from RTF, CEF, and CES. The M(r) of RTF clusterin decreased from 94 kDa to 51 kDa, indicating a carbohydrate content of 45%. After deglycosylation, the M(r) of the CEF clusterin decreased from 74 kDa to two distinct bands at 51 and 50 kDa (with carbohydrate contents of 31 and 32%, respectively), suggesting that two isoforms of the heterodimeric protein are present because of the two isoforms of the alpha-subunit. Under nonreduced conditions, a beta-subunit-specific Ab reacted with M(r) of 36-38 kDa, indicating the existence of free clusterin beta-subunits in CES. RTF, CEF, and CES extracts all caused mouse fibroblastic L-cell aggregation. CEF cell aggregation was inhibited by Hyb-17 Ab but not by other Abs. Both RTF and CEF caused a dose-dependent inhibition of complement-induced cytolysis, although RTF clusterin was more potent than CEF clusterin. We conclude that several isoforms of clusterin occur in the bull reproductive tract and that the variation in carbohydrate content among these isoforms may affect the biological or functional activity of the protein.

Transient expression of clusterin (sulfated glycoprotein-2) during development of rat pancreas.

Clusterin has been known to play important roles not only in remodeling damaged tissues, but also in tissue reorganization during embryonic development. In the present study, we have investigated the expression of clusterin in the endocrine pancreas during embryonic development. Although a weak immunoreaction was detected in some pancreatic primordial cells at day 14 of gestation, distinct clusterin expression was identified by immunocytochemistry and Northern blot analysis at the 16th day of gestation. Clusterin-producing cells, which corresponded to insulin-containing cells, accounted for the major portion of the developing islet of Langerhans up to 18 days of gestation. Thereafter, clusterin-producing cells display similar distribution and morphological features to glucagon-producing cells. Clusterin expressed in the pancreas was shown by Western blot analysis to be a disulfide-linked heterodimer of 70 kDa with an alpha-subunit of 32 kDa. During early developmental stages, however, we found that proteolytic internal cleavage of the clusterin molecule occurred from the 18th day of gestation. Only one 70 kDa band on the 16th day and two bands (32 kDa and 70 kDa) on the 18th day of gestation were detected by Western blot analysis even in reducing conditions, while only a single 32 kDa band was detected on the second day after birth. The levels of clusterin mRNA in the pancreas transiently increased from the 16th day of gestation to the second day after birth, during the period when active cellular reorganization takes place to form the classic cellular features of the islet. Among various tissue (kidney, brain, liver, heart, lung and pancreas) the levels of clusterin mRNA were the highest in the pancreas from the 18th day of gestation to the second day after birth. In contrast, the lowest expression was observed in adult pancreatic tissue. The higher expression of clusterin in developing pancreas must indicate its involvement in tissue organization during development.

Characterization of uterine leukocyte infiltration in gilts after artificial insemination.

The objective of this study was to characterize the uterine leukocyte influx after artificial insemination (AI). After detection of oestrus with a boar at intervals of 1.5 h, seventy-two gilts were randomly assigned to a 2 x 3 x 4 factorial arrangement. AI was performed with 100 ml extended semen containing 5 x 10(9) spermatozoa (semen; n = 36) or 100 ml VSP semen extender (extender; n = 36) at one of three times after detection of oestrus: 12, 24 or 36 h (n = 24/time). The uterus was lavaged at 6, 12, 18 or 24 h (n = 18/time) after AI to determine the total number of uterine leukocytes. In addition, uterine lavage was performed on nine untreated gilts immediately after the detection of oestrus to establish a baseline number of leukocytes. The leukocyte response in all samples consisted predominately (92-99%) of polymorphonuclear neutrophilic granulocytes (PMNs). The mean number of PMNs recovered from the uteri of gilts treated with semen was greater than in gilts treated with extender and in untreated gilts (P < 0.01). The greatest number of PMNs in semen-treated gilts was found 12 h after AI (P < 0.01), and this number was sustained for 24 h. In contrast, the number of uterine PMNs recovered from extender-treated gilts reached a peak at 6 h and had declined by 12 h after AI (P < 0.05). It was concluded that an extensive influx of PMNs into the uterus is a normal sequence to AI. The consequences and importance of semen-induced uterine leukocytosis needs further investigation.

Sperm transport and survival in the mare.

Following the deposition of semen in the mares uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mares reproductive tract. Fertilizable sperm are present in the oviduct within 4 hours after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa trigger an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen via activation of complement. Furthermore, seminal plasma appears to have a modulatory effect on the post-mating inflammation through its suppressive effect on PMN chemotaxis and migration. Spermatozoa that safely have reached the oviduct can be stored in a functional state for several days, but prolonged sperm storage in the female tract is not required for capacitation and fertilization in the horse. The caudal isthmus has been proposed as a sperm reservoir in the mare. The pattern of sperm transport and survival of spermatozoa in the mares reproductive tract are different between fertile and subfertile stallions, between fertile and some infertile mares, and between fresh and frozen-thawed semen. Possible explanations for these differences include a selective phagocytosis of damaged or dead spermatozoa, impaired myometrial activity in subfertile mares, bio-physiological changes of spermatozoa during cryopreservation, and the removal of seminal plasma during cryopreservation of equine semen.

Effect of eCG on the pregnancy rate of ewes transcervically inseminated with frozen-thawed semen outside the breeding season.

An experiment was conducted to determine whether factors affecting pregnancy rate out-of-season are associated more with transcervical artificial insemination (T-AI) procedures or with the reproductive state of the ewe. Twenty Finncross ewes were treated with progesterone sponges, and at sponge removal (0 h) 10 ewes were treated with eCG. Blood samples were collected for LH and progesterone analyses, and follicular development was monitored using ultrasonography. Ewes were inseminated from 48 to 52 h with 200 million motile frozen-thawed spermatozoa. The incidence of estrus, LH surges and ovulation was greater (P < 0.01) and intervals to these responses were shorter (P < 0.01) in the eCG-treated ewes. The number of follicles > 5 mm was higher (P < 0.05) in eCG-treated than control ewes. Progesterone concentrations increased and remained elevated through Day 19 in 7 eCG-treated and in 1 control ewe, and these ewes were pregnant based upon ultrasonographic examination. The results demonstrate that the T-AI technique using frozen-thawed semen produces a relatively high (70%) pregnancy rate out-of-season. The pregnancy rate was found to reflect primarily the reproductive condition of the ewe.

Sperm transport and survival in the mare: a review.

After the deposition of semen in the mare's uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mare's reproductive tract. Fertilizable sperm are present in the oviduct within 4 h after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa trigger an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen via activation of complement. Furthermore, semen plasma appears to have a modulatory effect on the post-mating inflammation through its suppressive effect on PMN chemotaxis and migration. Spermatozoa that safely have reached the oviduct can be stored in a functional state for several days, but prolonged sperm storage in the female tract is not required for capacitation and fertilization in the horse. The caudal isthmus has been proposed as a sperm reservoir in the mare. The pattern of sperm transport and survival of spermatozoa in the mare's reproductive tract are different between fertile and subfertile stallions, between fertile and some infertile mares, and between fresh and frozen/thawed semen. Possible explanations for these differences include a selective phagocytosis of damaged or dead spermatozoa, impaired myometrial activity in subfertile mares, bio-physiological changes in spermatozoa during cryopreservation, and the removal of semen plasma during cryopreservation of equine semen.

Late estrus or metestrus insemination after estrual inseminations decreases farrowing rate and litter size in swine.

A study was conducted with 360 gilts and sows from four herds to determine whether fertility was affected when the final of multiple inseminations was performed in late estrus or metestrus (late insemination). Sows and gilts were inseminated at 24-h intervals immediately after the detection of estrus. After receiving a first insemination, females were paired according to parity and estrus status on d 2 of estrus. Each pair set was inseminated with extended semen from the same semen collection(s). Control females were inseminated once on d 1 if they were not in estrus on d 2 (n = 31) or on both days if they were in estrus on d 2 (n = 149). Late inseminated females in each pair were managed and inseminated in the same manner as control females and then inseminated again 24 h later regardless of estrus status. Overall reproductive performance was similar among the four herds. Late insemination caused a drop in farrowing rate in parity 1 and 2 females (23 and 22%; P < .05 and P < .01, respectively) and average litter size decreased by 1.1 pigs per litter (P < .05) regardless of parity. There were no differences in either litter size or farrowing rate between late inseminated females in estrus and those that were in metestrus at the time of their last insemination.

Monitoring of porcine reproductive and respiratory syndrome virus infection in boars.

A major concern exists on transmission of porcine reproductive and respiratory syndrome virus (PRRSV) via semen and effect of vaccination on PRRSV shedding in semen. Recent reports suggest that the virus can be transmitted by semen from boars infected experimentally or from natural sources. Seminal shedding, viremia, and changes in semen quality in boars with or without vaccination were examined. Nine boars were divided into three groups (three boars/group). Group I boars were vaccinated with 2 ml of RespPRRS vaccine (NOBL Laboratory) intramusculary and groups II and III were non-vaccinated. At 28 post-vaccination study days, group I and group II boars were challenged with virulent PRRSV VR-2332 at 2 ml of 10(4.0) TCID50 per boar intranasally. Group III served as non-vaccinated and non-challenged control. Semen and serum samples were collected from -9 pre-vaccination study days to 85 post-challenge study days and tested for the presence of PRRSV by virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR). Prior to detection of PRRSV RNA from samples, conditions for RT-nPCR were optimized. Two primer sets, an external and an internal, were selected for RT-nPCR. The first round of PCR using an external primer set could detect 10 TCID50 of PRRSV/reaction. However, nested PCR could detect as little as 0.01 TCID50 of PRRSV/reaction. PRRS vaccine virus was not isolated from vaccinated pigs, but the vaccine virus RNA was detected from three boars, at day 6 to 15, 9 to 12, and 15 to 21 post-vaccination by RT-nPCR. Following challenge, two of non-vaccinated/challenged boars shed virus into semen up to 50 and 57 days post-challenge, respectively. The group I vaccinated boars did not shed virus into semen after challenge. The non-vaccinated/challenged group featured sperm abnormalities in the form of significantly increased incidence of proximal droplets and abnormal tails at 36-50 days post-challenge. The latter defect was observed to increase similarly in vaccinated/challenged boars as well.

Diverse testicular responses to exogenous growth hormone and follicle-stimulating hormone in prepubertal boars.

The effects of exogenous growth hormone (GH) and FSH on development of the testes in intact prepubertal boars was investigated. Twenty-four boars received one of four daily treatments from 8 through 40 days of age: 1) 90 micrograms porcine (p) GH/kg body weight (BW), 2) 100 micrograms pFSH/kg BW, 3) GH + FSH, or 4) vehicle only (control). Plasma testosterone levels, measured at 10-day intervals, were similar among groups of boars throughout the study. Body weights among groups were similar during treatment, and testicular weight between treatment groups did not differ at castration (100 days of age). However, total length of the seminiferous tubule per testis in FSH-treated boars was 59% greater than in GH-treated animals (2705 vs. 1704 m; p < 0.05). Diameter of the tubule in GH-treated boars was 58% greater than in FSH-treated boars (p = 0.03). Relative mass of spermatocytes and spermatids in GH-treated animals exceeded that in controls by 2.5-fold and that in FSH boars by 75-fold (p = 0.05). There were no differences in effects of GH + FSH treatment as compared to control treatment; none of the treatments affected any interstitial tissue parameter measured. These results suggest that exogenous FSH had a mitogenic effect on Sertoli cells while delaying tubular maturity, whereas exogenous GH promoted tubular and Sertoli cell maturation, defined as increased Sertoli cell size, lumen formation, and onset of spermatogenesis.

Artificial insemination in swine.

This article analyses the advantages and disadvantages of artificial insemination with semen purchased from a center as well as from the herd boars on the farm. Intensive swine production could benefit greatly by adapting artificial insemination with herd boars, particularly from savings in labor and boar numbers. The techniques for semen collection, extension, and insemination are described, and sources for equipment given. Expected results of artificial insemination are quoted from experiments and international field experience.