Reproductive considerations: mare and stallion.

Functional alterations within the reproductive system and in other supporting systems may limit the reproductive capacity of geriatric patients; however, the age of onset and degree of compromise show wide individual variation. Aging of the hypothalamopituitary-ovarian axis in the mare manifests as delayed entry to the breeding season, prolonged follicular phases, reduced response to ovulation induction, irregular cycles, oocyte defects, increased early embryonic death, and, eventually, persistent anestrus. Aging of the reproductive tract may increase her susceptibility to endometritis, compromise placental formation with long-term effects on the fetus and viability of the resulting foal, increase her risk of ascending placentitis, alter gestation length, and make her more prone to catastrophic rupture of the uterine arteries at the time of parturition. Effects on the stallion are less well documented but include a reduction in sperm output associated with progressive testicular degeneration and potential compromise of libido and mating ability that is often associated with degenerative conditions of the musculoskeletal system.     

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (?Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P‐tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30‐ and 120‐min incubation. Membrane fluidity (MC540) increased in both media at 30‐ and 120‐min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2‐ and 4‐hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation‐related parameters in stallion sperm.     

Inhibin activity in the mare and stallion

An overnight double antibody RIA, employing a rabbit antiserum raised to bovine 31 kDa inhibin (rAs-#1989, NICHD) and purified bovine 31 kDa inhibin (bINH-I-90/1, NICHD) as trace and standard, was validated to measure immunoreactive inhibin (iINH) concentrations in equine peripheral plasma, follicular fluid (FF), ovarian vein (OV) plasma, testicular tissue extracts (TTE) and testicular vein (TV) plasma. The dynamic relationship of iINH and follicle stimulating hormone (FSH) was investigated during the estrous cycle of the mare and the annual reproductive cycle of the stallion.In the RIA, parallel dose-response curves were observed between the bovine inhibin standard and serial dilutions of equine FF, OV, TTE, TV and plasma. The average recovery of a known amount of purified bovine inhibin added to gelding plasma was approximately 100%. In the inhibin bioassay, serial dilution of equine FF and TTE were observed to be parallel to the bovine inhibin standard. A five-fold difference (p<0.05) between jugular and gonadal vein plasma iINH concentrations was observed in the mare and an eight-fold difference (p<0.05) was observed in the stallion. Plasma levels of iINH in ovariectomized mares or geldings were undetectable in the RIA.Concentrations of FSH, estradiol and iINH changed significantly in the mare during the estrous cycle (p<0.05). Immunoreactive inhibin levels were highest (0.54 ± 0.06 ng/ml) on the day of ovulation, declined rapidly following ovulation and reached a nadir (0.21 ± 0.03 ng/ml) on day 7 post-ovulation. Plasma iINH and estradiol concentrations followed a similar profile and were found to be positively correlated (r=0.7064; p<0.01), whereas iINH and FSH levels demonstrated an inverse relationship (r=−0.7359, p<0.01) throughout the estrous cycle. Concentrations of FSH were also inversely related (−0.8498, p<0.01) with estradiol during the cycle. In the stallion, plasma iINH and FSH levels changed significantly during the year (p<0.05). The iINH profile reflected seasonal changes in testicular activity, with highest concentrations in late spring (3.37 ± 0.44 ng/ml) and lowest concentrations in the fall (2.21 ± 0.33 ng/ml). Plasma concentrations of iINH were positively correlated (r=0.7691, p<0.01) with FSH concentrations throughout the year.In conclusion, a specific and sensitive RIA for iINH has been validated for plasma and biological fluids in the horse. Furthermore, the gonads appear to be the source of bioactive and immunoreactive inhibin as observed in other species. The dynamic relationship between iINH and FSH that is present in both the mare and stallion suggests that iINH may be a useful marker of gonadal activity in this species.     

Certain physiochemical properties of uterine tubal fluid, follicular fluid, and blood plasma in the mare.

Uterine tubal fluids were collected twice a day from mares for 5 consecutive estrous cycles between March 15 and September 1. Follicular fluids were aspirated from the follicles of exteriorized ovaries of 3 mares between days 2 and 5 of estrus. Uterine tubal fluid and follicular fluid were analyzed for osmolarity, dry matter, total lipids, total free fatty acids, glucose, fructose, and lactic acid. Blood samples were collected (jugular venipuncture) throughout the estrous cycle, and the same physical and biochemical analyses were made on blood plasma. A difference (P less than 0.01) was found for osmolarity between uterine tubal fluids collected during estrus and those collected during anestrus. The osmolarity of uterine tubal fluid during anestrus was greater than that of blood plasma; follicular fluid was similar in osmolarity to blood plasma. The dry matter in blood plasma was greater (P less than 0.01) than that in either uterine tubal fluid or follicular fluid. Cyclic variations in dry matter content were not observed in uterine tubal fluid. Total lipids in blood plasma and follicular were greater (P less than 0.01) than those in uterine tubal bluid. The concentration of total lipid in uterine tubal fluid was similar during estrus and anestrus. Myristic acid (C14:0) in blood plasma and myristoleic acid (C14:1) in uterine tubal fluid were the only free fatty acids that had cyclic variation. The fatty acids in the greatest concentration in uterine tubal fluid and blood plasma were palmitic acid (C16:0) and linoleic acid (C18:2). Concentrations of linoleic acid and stearic acid (C18:0) were greater (P less 0.01) in follicular fluid than in uterine tubal fluid or blood plasma. Only trace amounts of glucose were detected in uterine tubal fluid, whereas a considerable amount of glucose was found in follicular fluid. Fructose was not detected in any of the fluids. Lactic acid concentrations did not differ between estrus and anestrus. Lactic acid concentration was significantly greater (P less than 0.01) in uterine tubal fluids and follicular fluids than in blood plasma.     

The relation of infection to infertility in the mare and stallion.

Many normally fertile stallions harbour bacteria in and on the genital organs. Many mares served by such stallions are unaffected by the bacteria to which they are thus exposed; however, some mares so exposed will become infected and diseased. Presumably, the genital defenses of such mares had been compromised. Strain differences in pathogenicity of bacteria do exist. Some mares affected with pyometra had irregular ovarian activity and some had normal ovarian cycles. In the former group, destruction of the endometrium many have prevented the production of endogenous luteolysin. The leukopenia which occurs in both groups is due to neurtropenia.     

Uterine torsion and uterine tear in a mare.

A 15-year-old Standard-bred mare was examined because of signs of abdominal discomfort in late gestation. Palpation per rectum revealed tight broad ligaments above and below the uterus, with the right broad ligament running across the top of the uterine body down toward the left, ventral side of the abdomen. A diagnosis of counterclockwise uterine torsion was made and surgical correction was approached via a left, flank laparotomy with the horse standing. The uterus was repositioned and a uterine tear encompassing 180 degrees of the uterine surface was found in the lateral, uterine body just cranial to the cervix. A live colt was delivered vaginally after uterine repositioning and the laparotomy incision was closed. The uterine tear was then repaired via a blind, vaginal approach. The mare was discharged 10 days after surgery. Repair per vaginum of a uterine tear is presented as an alternative treatment in cases for which the tear is recent, abdominal contamination is minimal, and the tear is easily accessible from the vaginal approach.     

Factors affecting the composition of mare uterine fluid

The influx of protein and polymorphonuclear leucocytes (PMN) into the uterine lumen was examined at different intervals after intrauterine infusion of fluids. The intrauterine infusion of both phosphate buffered saline (PBS) and a solution of lipopolysaccharide (LPS) derived from Escherichia coli resulted in a biphasic influx of protein in the uterine flushings peaking three and six hours after infusion. LPS infusion caused an additional influx of protein at 24 hours. The initial influx of protein preceded a biphasic influx of PMN which peaked six and 24 hours after both infusions. Uterine flushings obtained following PBS and LPS infusion contained both serum-derived and uterine-specific proteins. To investigate whether the influxes were a general response to reproductive mucosal stimulation, several regions of the reproductive tract were subjected to physical manipulation. Results suggested that these influxes were initiated chiefly by stimulation of the cervical and, or, uterine region.