The expression of interferon-stimulated gene 15 in equine endometrium

Establishment of pregnancy is critically dependent upon a precisely orchestrated embryo-maternal interaction leading to a receptive uterine environment. The up-regulation of the interferon-stimulated protein 15 kDa (ISG15) during pregnancy has been described in various species and has been hypothesized to be part of the molecular repertoire that makes the uterus receptive to conceptus development. In the current study, the expression of ISG15 and enzymes involved in ISG15ylation was examined at the mRNA and protein level in equine endometrium at Day 14 of the luteal phase and at Day 14 and 50 of pregnancy. ISG15 mRNA showed a 2.63-fold higher expression at Day 14 of pregnancy when compared to Day 14 of the cycle, while mRNA abundance at Day 50 of pregnancy was unchanged compared to Day 14 of the cycle. Upon Western blot analysis using anti-ISG15 antibody, several higher molecular weight bands could be observed, representing proteins conjugated to ISG15. No free ISG15 could be detected. The pattern of ISG15 reactive proteins differed from those observed in non-uterine samples. Upon immunohistochemistry, ISG15 reactive proteins located primarily to luminal and glandular epithelial cells, while stromal cells showed weaker staining. In conclusion, the expression of ISG15-conjugated proteins in equine endometrium did not differ between cyclic and pregnant 14 days after ovulation and Day 50 of pregnancy. It is hypothesized that the unique subset of ISG15ylated proteins expressed in endometrial tissue contributes to normal cellular function and that, unlike other species, the modification of ISG15-conjugated proteins is not an active contributor to conceptus-maternal interaction in the mare.     

Effect of seminal plasma concentration and various extenders on postthaw motility and glass wool-Sephadex filtration of cryopreserved stallion semen

OBJECTIVE: To compare the effect of semen extender and seminal plasma on postthaw motility and filtration through a glass wool-Sephadex (GWS) filter for frozen stallion semen. SAMPLE POPULATION: 7 stallions from which we collected > or = 3 ejaculates/stallion. PROCEDURES: 4 experiments were conducted to evaluate postthaw quality of frozen stallion semen. Kenney extender was compared with glucose-EDTA extender by use of various dilution rates that resulted in differing concentrations of seminal plasma. Stallions known to produce semen with poor postthaw quality were used to investigate whether a particular extender or dilution rate could improve ability of such semen to survive freeze-thaw procedures. RESULTS: Use of Kenney extender as the centrifugation extender significantly improved postthaw motility and GWS filtration, compared with glucose-EDTA. Extending semen at a dilution of 1:3 was significantly better than 1:1 for both motility and GWS filtration. In addition, including seminal plasma at a concentration of 5% in the cryopreserved semen resulted in significantly higher yield of spermatozoa after GWS filtration, compared with complete removal of SP or use of seminal plasma at 25%. Lastly, semen with poor postthaw quality had significantly improved postthaw quality in regard to motility and GWS filtration when semen was frozen with seminal plasma at a concentration of 5%, compared with semen frozen with seminal plasma at a concentration of 25%. CONCLUSIONS AND CLINICAL RELEVANCE: Use of Kenney extender at a high dilution (> or = 1:3) immediately after collection of semen can improve postthaw quality of frozen stallion semen.     

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.     

Endometrial Phospholipase A2 Activity During the Oestrous Cycle and Early Pregnancy in Mares

The aim of this study was to determine phospholipase A2 (PLA2) kinetics and activity in the mare’s endometrium during the oestrous cycle and early pregnancy. Phospholipase A2 is responsible for the liberation of arachidonic acid from phospholipids, which is the first limiting step in prostaglandins synthesis. Phospholipase A2 activity was measured using an assay based on the liberation of oleic acid from 1‐palmitoyl‐2‐[¹⁴C] oleoyl phosphatidylcholine. The enzyme was shown to be calcium dependent, to have an optimum pH of 8 and an apparent Michaelis constant of 127 μm . Enzyme activity was low in the endometrium of early luteal phase tissue but increased significantly (p < 0.001) during the late luteal phase (5.39 ± 0.16; 3.48 ± 0.33, 6.85 ± 0.59, and 9.96 thinsp;± thinsp;1.23 thinsp;nmol oleic acid released/mg protein at oestrus, and Days 3, 8 and 14 after ovulation, respectively). The mean PLA2 activity in endometrial tissue from pregnant mares (4.23 ± 0.74) was significantly lower (p < 0.01) than from cyclic animals during late dioestrus (9.96 ± 1.23). The results indicate that PLA2 activity in equine endometrium changes with the stage of the oestrous cycle and thus may be influenced by systemic hormone concentrations. The inhibitory effects of conceptus products on secretion of prostaglandin during early pregnancy were associated with a competitive inhibitor that decreased endometrial PLA2 activity.     

The Use of Equine Follicle Stimulating Hormone to Increase Equine Chorionic Gonadotropin in the Pregnant Mare

Equine chorionic gonadotropin (eCG), obtained from pregnant mares, is used for assisted reproductive technologies in laboratory rodents and livestock. The objective of the present study was to use equine follicle-stimulating hormone (eFSH) to increase the incidence of twin pregnancies, through multiple ovulations, and increase eCG. Nineteen light horse–type mares were enrolled in the study. The control group (n = 9) was bred with fresh or cooled semen and given human chorionic gonadotropin (hCG) at the time of breeding. The second group (n = 10) was given 12.5 mg of eFSH intramuscularly twice a day beginning 5–7 days after ovulation. Prostaglandin F2α was administered intramuscularly the second day of eFSH treatment. Treatment with eFSH continued until follicles were >35 mm in diameter, and mares were then given no treatment for 36 hours. The mares were then bred with fresh or cooled semen from the same stallion as the control group and given hCG. Blood samples were taken weekly from day 35 to day 105 after ovulation. Serum concentration of eCG was obtained, and data were analyzed with multivariate analysis using the mixed procedure. Significance was set at P < .05. Data were combined for all mares carrying twins and compared with those carrying singletons. The group of mares carrying twins had higher peak concentrations of eCG and higher values for area under the curve compared with mares carrying singletons (P < .05). These results suggest inducing twins could be a method used to increase eCG production.