Bo G. Crabo

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.

Some factors affecting loss of intracellular enzymes from spermatozoa.

Abstract Diluted semen from the bull, boar, and turkey was plunged into liquid nitrogen. After separation of the cells from the extracellular medium, the total releasable amounts of glutamic oxaloacetic transaminase (GOT), lactic dehydrogenase (LDH), cholinesterase, acid phosphatase, and alkaline phosphatase were determined. Turkey semen was low in enzyme content when compared to the bull and boar. Correlations between sperm cell concentration and enzyme concentration along with the amounts of enzyme in the extracellular medium after minimum and maximum damage indicated that GOT was present primarily in the cell. Therefore, of the five enzymes studied, GOT release into the extracellular medium was the best indicator of cell damage. Comparison of filtering semen through cellulose powder with discontinuous centrifugation to separate the cells from the extracellular medium was studied. Discontinuous gradient centrifugation resulted in significantly lower release of GOT from the cells in the boar and turkey than filtration. Thus discontinuous gradient centrifugation was the superior method for separating spermatozoa from the extracellular medium.

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.

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.

Concentration of viable spermatozoa for artificial insemination

Glass wool microfiber code 112 (approximately 30 mg) was gently packed to a depth of 3 mm in a 3-ml disposable syringe barrel. The column was rinsed repeatedly with Tyrode solution to remove loose glass wool fibers. Approximately 0.3 to 0.5 ml of concentrated spermatozoa (after centrifugation of semen at 500 X g for 5 minutes) was layered over the wet column and allowed to filter by gravity. The filtered spermatozoa demonstrated a significantly higher (P less than 0.001) mean percentage of motility, progressive motility, sperm unstained by eosin Y dye, and sperm with functionally intact membranes. Although there was a considerable loss of sperm, the filtered specimen contained all viable spermatozoa present before filtration. Therefore, it appears that this procedure yields a high percentage of viable spermatozoa, potentially capable of fertilization, for use in in vitro fertilization or, possibly, artificial insemination.

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.

In vitro capacitation of stallion spermatozoa in calcium-free Tyrode's medium and penetration of zona-free hamster eggs

Capacitation of stallion spermatozoa in Tyrodes calcium-free (TCF) medium was assessed. Twelve gel-free ejaculates were collected. After removal from the seminal plasma, cells were washed three times with 0.85% saline containing 0.1% bovine serum albumin (BSA) and resuspended in TCF. Both washing and incubation media were adjusted to pH 8 and 300 to 310 mOsm. Final sperm concentration during incubation was 2 x 10(6) cells/ml. The diluted ejaculates were incubated for 2, 4, 6, 8 and 10 h at 37 degrees C in an atmosphere containing 5% CO(2). Acrosomes were stained with naphthol yellow and erythrocin B initially and after each incubation period and evaluated microscopically. Transmission electron microscopy was used to verify whether normal acrosome reaction was occurring or if cells were degenerating. Penetration of zona-free hamster oocytes was evaluated using 10(3) to 10(4) sperm/ml suspension and coincubating eggs for 3.5 to 4 h with sperm. Penetration tests were done for wash and incubation treatments and recorded positive when swollen sperm heads or male pronuclei were present. Incubation time affected acrosome integrity (P<0.001). Incubation for 8 to 10 h significantly improved acrosome reaction (P<0.001) and the percentage of reacted acrosomes increased sharply after 6 h of incubation (P<0.001). None of the washed sperm penetrated zona-free eggs at zero time, but sperm from all incubation treatments penetrated eggs. A peak penetration rate of 29.9% was observed at 8 h (P<0.001). Results indicate that under the conditions used, the requirement for Ca(++) in the medium for the process of capacitation and acrosome reaction can be substituted for by elevated pH.

Assay of capacitated, freeze-damaged and extended stallion spermatozoa by filtration

Abstract Results of stallion semen assay with glass wool/Sephadex filters are highly correlated with fertility. A hypothesis was tested that mechanically damaged spermatozoa are trapped in glass wool while capacitated spermatozoa are trapped in Sephadex. Another objective was to assay stallion semen in extenders known to vary in their ability to maintain sperm fertility with these filters, and for sperm motility. In Experiment 1, ejaculates were split so that a portion was used for fresh semen filtration and another portion plunged into liquid nitrogen repeatedly to maximize the number of broken acrosomes. The remainder of the ejaculate was inseminated into the uterus of an estrous mare which was flushed 6–8 hours later. Semen from each treatment was filtered through cotton with and without Sephadex, and through glass wool with and without Sephadex. Ejaculates were also washed and incubated in a capacitation medium. Aliquotes were filtered every 2 hours for 10 hours through glass wool and Sephadex. In Experiment 2, stallion semen diluted in egg yolk-lactose, skim milk-glucose and Triscitrate-yolk was evaluated for motility and with glass wool and Sephadex filters daily for 4 days. In Experiment 1, more frozen-thawed spermatozoa were trapped in glass wool than in Sephadex filters. Filters containing Sephadex trapped all uterine-incubated cells, while cotton did not affect the trapping of spermatozoa. In the capacitation medium, the passage of cells through all filters decreased with time, and glass wool and Sephadex had additive effects. In Experiment 2, fewer spermatozoa passed Sephadex filters than glass wool filters after storage in the Tris-citrate extender, which is known to be detrimental to the fertility of stallion semen although it did maintain sperm motility. We suggest that Sephadex traps spermatozoa with capacitation-like changes.

Continuous presence of rams hastens the onset of estrus in ewes synchronized during the breeding season

Abstract The objective of the study was to investigate whether ram exposure would affect the onset of estrus in synchronized ewes, during the breeding season. Ninety pluriparous ewes (30 White Face, 30 Suffolk and 30 Hampshire) were divided by breed and estrous synchronized with intravaginal sponges. These sponges were impregnated with medroxyprogesterone acetate and inserted for 12 days at unknown stages of the estrous cycle. Half of the females in each breed were exposed to a ram immediately following sponge removal and throughout an 120-h period (group IMM) while the other half was subjected to ram exposure at 48 h after sponge removal (group LAT). The same ram was used for both groups. In the LAT group the male was housed in a pen adjacent to the female pen. Estrus onset for group IMM occurred (mean±S.D.) 32.9±12.3 h after sponge removal while in group LAT estrus occurred at 53.1±19.6 h, respectively ( P P P P >0.05). These results show that immediate exposure to a ram at sponge removal hastens estrus onset in ewes estrous synchronized during the breeding season.