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Reproductive steroids testosterone (T) and estrone sulfate (E1S) are used as diagnostic markers for cryptorchidism in horses. The human chorionic gonadotropin (hCG) stimulation test is used as a diagnostic aid because administration of this hormone results in greater incremental differences in circulating steroid concentrations. Thoughts regarding optimal sampling times following hCG administration, however, are inconsistent. Additionally, determination of half-life of these steroids is important in postsurgical samples to confirm complete removal of testicular tissue. Objectives of this study, therefore, were to determine optimal sampling periods for peak T and E1S after hCG administration and half-life of these steroids after castration. Eight pony stallions were randomly assigned to control or treatment groups (5000 IU hCG). Blood samples were collected following hCG administration. Subsequently, stallions were castrated and blood samples were collected post-castration. The T concentrations were greatest at 72 h after hCG and were greater (P < 0.02) in samples from hCG-treated than control animals: 9,903.4 ± 384 and 784.0 ± 192 pg/mL, respectively (Mean ± SEM). The T concentrations were also greater at 1, 12, 24, 48 and 96 h. The E1S concentrations did not change after administration of hCG. The T response to hCG administration was biphasic with a maximal response between 48-96 h after administration. Half-lives of T and E1S were 1.1 and 0.7 h, respectively, and concentration of T and E1S was similar to that of geldings at 24 h post-castration, which, therefore, should be considered an optimal time to ensure complete castration has occurred., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Endometritis in horses caused by Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) may be underdiagnosed due to traditional diagnostic methods lacking sensitivity and specificity. We serendipitously identified a bacterial growth medium (bActivate) that appeared capable of inducing growth of dormant S. zooepidemicus, which subsequently allowed detection by standard diagnostics. To assess the effect of bActivate we compared its ability to activate dormant S. zooepidemicus in a group of potentially infected subfertile mares with phosphate-buffered saline (PBS). All mares had to test negative for S. zooepidemicus on a low-volume uterine lavage, be negative on endometrial cytology and without clinical signs of endometritis to be included in the investigation. The mares were instilled with bActivate or PBS in the uterus. Growth of S. zooepidemicus was induced by bActivate in 64% (16/25) and PBS in 8% (1/12) of the mares, respectively (p<0.002). In vitro studies supported that some strains of S. zooepidemicus were able to form persister cells tolerating 32-times of the minimal inhibitory concentration of penicillin compared to normal growing cells. Persister cells had not acquired penicillin resistance, but seemed to tolerate the antimicrobial due to dormancy. This is, to our knowledge, the first description of controlled growth induction of dormant bacteria from a subclinical infection. Moreover we demonstrated how endometritis can origin from a reservoir of dormant bacteria residing within the endometrium, and not only as an ascending infection. Further studies should aim at determining the prevalence of dormant S. zooepidemicus, impact of activation on diagnostic and treatment efficacy, uterine health and mare fertility., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The objective of this study was to compare expression of estrogen receptor alpha (ER-α), β (ER-β), progesterone receptor (PR), as well as prostaglandin E2 type 2 (EP2) and 4 (EP4) receptors in the equine myometrium and endometrium during estrus, diestrus and early pregnancy. Tissues were collected during estrus, diestrus, and early pregnancy. Transcripts for ER-α (ESR1), ER-β (ESR2), PR (PGR), EP2 (PTGER2) and EP4 (PTGER4) were quantified by qPCR. Immunohistochemistry was used to localize ER-α, ER-β, PR, EP2 and EP4. Differences in transcript in endometrium and myometrium were compared by the ΔΔCT method. Expression for ESR1 (P<0.05) tended to be higher during estrus than diestrus in the endometrium (P=0.1) and myometrium (P=0.06). In addition, ESR1 expression was greater during estrus than pregnancy (P<0.05) in the endometrium and tended to be higher in estrus compared to pregnancy in the myometrium (P=0.1). Expression for PGR was greater (P<0.05) in the endometrium during estrus and diestrus than during pregnancy. In the myometrium, PGR expression was greater in estrus than pregnancy (P=0.05) and tended to be higher during diestrus in relation to pregnancy (P=0.07). There were no differences among reproductive stages in ESR2, PTGER2 and PTGER4 mRNA expression (P>0.05). Immunolabeling in the endometrium appeared to be more intense for ER-α during estrus than diestrus and pregnancy. In addition, immunostaining for PR during pregnancy appeared to be more intense in the stroma and less intense in glands and epithelium compared to estrus and diestrus. EP2 immunoreactivity appeared to be more intense during early pregnancy in both endometrium and myometrium, whereas weak immunolabeling for EP4 was noted across reproductive stages. This study demonstrates differential regulation of estrogen receptor (ER) and PR in the myometrium and endometrium during the reproductive cycle and pregnancy as well as abundant protein expression of EP2 in the endometrium and myometrium during early pregnancy in mares., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Bacteria (environmental contaminants and occasionally potential pathogens) are found in most stallion ejaculates and may negatively affect sperm quality during storage. Since the use of antibiotics can lead to the development of resistance, an alternative means of microbial control is desirable. The removal of bacteria from stallion semen using Single Layer Centrifugation through Androcoll-E was investigated. Known doses of cultured bacteria were added to freshly collected ejaculates (15mL aliquots) before processing by Single Layer Centrifugation. The resulting sperm pellets and controls (not processed by Single Layer Centrifugation) were cultured and the bacteria identified. In experiment 1, doses of E. coli from 2×10(2) to 2×10(7) colony forming units were added to aliquots of semen. In experiment 2, Taylorella equigenitalis or a mix of E. coli, Klebsiella pneumoniae and Streptococcus equi subsp. zooepidemicus (approximately 7×10(6), 5×10(6), and 6×10(6)cfu, respectively) were added to 15mL aliquots of semen. In experiment 1, more than 90% of the bacteria were removed where loading doses were >×10(4)cfu/mL. In experiment 2, varying proportions of different bacteria were removed, ranging from 68% for naturally occurring Corynebacterium spp. to >97% for added cultured E. coli. Thus, Single Layer Centrifugation can separate spermatozoa from many, but not all bacteria in stallion ejaculates and could be a useful alternative to adding antibiotics to semen extenders to control bacterial contamination. However, further research is needed to determine the effect of small numbers of bacteria on sperm quality., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Biological effects of prostaglandin E2 are mediated via one of four receptors designated EP1, EP2, EP3 and EP4 which are encoded by separate genes. In general, EP1 and EP3 induce smooth muscle contraction whereas EP2 and EP4 induce smooth muscle relaxation. The objective of the current study was to characterize the expression of the genes for PGE2 receptors (EP2 and EP4) in the horse oviduct based upon immunohistochemistry (IHC) and quantitative PCR (qPCR). Oviductal tissue was collected from mares at estrus (n=5), at 5 days post-ovulation (n=4), and from prepubertal mares (n=5). Isthmic and ampullar regions of the oviduct were fixed for IHC or preserved for RNA isolation. Prostaglandin E2 receptors EP2 and EP4 were strongly expressed by the luminal epithelium of both the isthmic and ampullar regions of the horse oviduct with a lesser immuno-expression noted within the smooth muscle in both regions of the oviduct. Based upon qPCR, relative amounts of EP2 or EP4 mRNA did not differ across estrous cycle stage or from prepubertal mares. However, across region and estrous cycle stage, relative amount of EP2 was greater (P<0.05) than EP4, and relative amount of EP2 mRNA was greater (P<0.001) in the ampullar compared with the isthmic oviduct. The relative roles of these receptors in regulating oviduct function in the mare remains to be determined., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Ultrasonic-guided transvaginal follicle aspiration was performed in 58 crossbreed mares in order to determine whether aspiration of various dominant follicle diameters resulted in luteal tissue capable of producing progesterone (P(4)). The mares were randomly assigned to three groups according to follicular diameter (25-29 mm; 30-35 mm and >35 mm). Mares that had ovulations naturally served as controls. The serum progesterone (P(4)) concentrations in the aspirated mares were greater (P < 0.0001; r(2) = 0.6687; CV = 21.52) in mares with natural ovulation compared to mares with aspirated follicles regardless of groups. Serum P(4) concentration in aspired mares with follicular diameter of 25-29 mm declined 0.365 ng/ml/day (P = 0.0065) from the day of aspiration (D0) up to D8. In mares with follicle diameter of 30-35 mm, serum P(4) concentration increased (0.258 ng/ml/day; P = 0.001), as well as in the mares with follicles >35 mm diameter (0.481 ng/ml/day; P < 0.0001), and in mares with natural ovulation (1.236 ng/ml/day; P < 0.0001). Out of the 25 mares with follicular aspirations that formed Corpora hemorragica (P(4) >1 ng/ml), 23 (92%) had greater (>2 ng/ml) serum P(4) concentrations on Day 8 after aspiration. Of these 23 mares, 75% were in the 25-29 mm group, 9/10 (90%) in the 30-35 mm group, and 11/11 (100%) of the mares in the >35 mm follicular diameter group had luteinization (P(4) >2 ng/ml). These results suggest that a functional Corpus luteum can be induced in mares using follicular aspiration and that a minimum 35 mm follicular diameter is needed to reach a progesterone serum concentration compatible with that of a Corpus luteum produced by natural ovulation., (Copyright 2009 Elsevier B.V. All rights reserved.)

Seminal plasma has been suggested to be involved in sperm transport, and as a modulator of sperm-induced inflammation, which is thought to be an important part of sperm elimination from the female reproductive tract. This article reports on recent experiments on the importance of seminal plasma components in sperm transport and elimination. In Experiment 1, hysteroscopic insemination in the presence (n = 3) or absence (n = 3) of 2 ng/mL PGE showed an increased portion of spermatozoa crossing the utero-tubal junction in the presence of PGE in two mares, while no difference was observed between treatments in a third mare. In Experiment 2, whole seminal plasma, heat-treated seminal plasma (90 degrees C for 45 min), and charcoal-treated seminal plasma were added to: (1) sperm samples during opsonization prior to polymorphonuclear neutrophil(s) (PMN)-phagocytosis assays (n = 5); or to (2) phagocytosis assays (n = 5). Opsonization of spermatozoa was suppressed in the presence of whole seminal plasma, compared with samples without seminal plasma (p < 0.05). Charcoal treatment did not remove the suppressive effect of seminal plasma on opsonization, but heat treatment of seminal plasma reduced its suppressive properties (p < 0.05). The addition of whole seminal plasma to opsonized spermatozoa almost completely blocked phagocytosis (p < 0.05). Charcoal treatment did not remove the suppressive effect of seminal plasma. However, heat-treated fractions of seminal plasma removed the suppressive effect of seminal plasma on phagocytosis (p < 0.05). In Experiment 3, viable and non-viable (snap-frozen/thawed) spermatozoa were subjected to in vitro assays for PMN binding and phagocytosis with the following treatments (n = 3): (1) seminal plasma (SP), (2) extender; (3) ammonium sulfate precipitated seminal plasma proteins with protease inhibitor (SPP+); or (4) ammonium sulfate precipitated seminal plasma proteins without protease inhibitor (SPP-). Treatment was observed to impact binding and phagocytosis of viable and non-viable spermatozoa (p < 0.05). SP and SPP+ suppressed PMN-binding and phagocytosis of viable sperm. This effect was also seen, but to a lesser degree, in SPP- treated samples. Non-viable spermatozoa showed less PMN-binding and phagocytosis than live sperm in the absence of SP. The addition of SP promoted PMN-binding and phagocytosis of non-viable spermatozoa. SPP- treated samples also restored PMN-binding of non-viable spermatozoa. The addition of protease inhibitors removed this effect. In Experiment 4, seminal plasma proteins were fractionated based on MW by Sephacryl S200 HR columns (range 5000-250,000 kDa). Fractionated proteins were submitted to sperm-PMN binding assays. A protein fraction <35 kDa suppressed PMN-binding to live and snap-frozen spermatozoa. A greater MW protein fraction appeared to promote binding between PMNs and snap-frozen spermatozoa. While the addition of protease inhibitors was necessary to maintain the protective effect of seminal plasma proteins on viable spermatozoa, the promotive effect of seminal plasma on non-viable spermatozoa appeared to require some protease activity. It was concluded from these experiments that components of seminal plasma play active roles in transportation and survival of viable spermatozoa in the female reproductive tract and in the elimination of non-viable spermatozoa from the uterus.

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.