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Summary.

Two trials were conducted to examine the effect of three progestagens, chlormadinone acetate (CAP), melengestrol acetate (MGA) and megestrol acetate (MA) on the sexual activity of ranch cows.

In the first trial, injection of CAP (6 mg/day) inhibited manifestation of oestrus in ten out of ten cows, while six of these cows showed full heat 4 to 6 days after cessation of treatment. When CAP was administered by intravaginal tampon (200 mg CAP/tampon), eight out of ten cows showed full heat during the 18 days of treatment, while two out of ten cows showed full heat in the 7 days immediately after cessation of treatment.

In the second trial, CAP was injected at 6 mg/day for 16 days and at 12 mg every alternate day for 16 days. MA or MGA was administered by intramuscular injection at 4 mg/day or 0·5 mg/day respectively or by intravaginal tampon at 100 mg/tampon or 60 mg/tampon respectively. With the exception of one cow (MA), all progestagens completely inhibited manifestation of oestrus when injected. When the progestagen was administered by tampon, two out of ten cows receiving MGA and two out of ten cows receiving MA showed oestrus during treatment. During the period 5 to 10 days after cessation of treatment, heat was shown by 100%, 80%, 70% and 70% of cows which received injections of CAP daily, CAP on alternate days, MGA or MA, respectively. The high incidence of anovulatory heats (20 to 80%) indicated that the first heat after cessation of progestagen treatment would be associated with low fertility.

Luteinized unruptured follicle (LUF) syndrome is a recurrent anovulatory dysfunction that affects up to 23% of women with normal menstrual cycles and up to 73% with endometriosis. Mechanisms underlying the development of LUF syndrome in mares were studied to provide a potential model for human anovulation. The effect of extended increase in circulating LH achieved by administration of recombinant equine LH (reLH) or a short surge of LH and decrease in progesterone induced by prostaglandin F₂α (PGF₂α) on LUF formation (Experiment 1), identification of an optimal dose of COX-2 inhibitor (flunixin meglumine, FM; to block the effect of prostaglandins) for inducing LUFs (Experiment 2), and evaluation of intrafollicular endocrine milieu in LUFs (Experiment 3) were investigated. In Experiment 1, mares were treated with reLH from Day 7 to Day 15 (Day 0=ovulation), PGF₂α on Day 7, or in combination. In Experiment 2, FM at doses of 2.0 or 3.0 mg/kg every 12 h and human chorionic gonadotropin (hCG) (1500 IU) were administered after a follicle ≥32 mm was detected. In Experiment 3, FM at a dose of 2.0 mg/kg every 12 h plus hCG was used to induce LUFs and investigate the intrafollicular endocrine milieu. No LUFs were induced by reLH or PGF₂α treatment; however, LUFs were induced in 100% of mares using FM. Intrafollicular PGF₂α metabolite, PGF₂α, and PGE₂ were lower and the ratio of PGE₂:PGF₂α was higher in the induced LUF group. Higher levels of intrafollicular E2 and total primary sex steroids were observed in the induced LUF group along with a tendency for higher levels of GH, cortisol, and T; however, LH, PRL, VEGF-A, and NO did not differ between groups. In conclusion, this study reveals part of the intrafollicular endocrine milieu and the association of prostaglandins in LUF formation, and indicates that the mare might be an appropriate model for studying the poorly understood LUF syndrome.

Epigenetic modifications are key mediators of the interactions between the environment and an organism's genome. DNA methylation represents the best-studied epigenetic modification to date and is known to play key roles in regulating transcriptional activity and promoting chromosome stability. Our laboratory has previously demonstrated the utility of the American alligator (Alligator mississippiensis) as a sentinel species to investigate the persistent effects of environmental contaminant exposure on reproductive health. Here, we incorporate a liquid chromatography–tandem mass spectrometry method to directly measure the total (global) proportion of 5-methyl-2′-deoxycytidine (5mdC) in ovarian and whole blood DNA from alligators. Global DNA methylation in ovaries was significantly elevated in comparison with that of whole blood. However, DNA methylation appeared similar in juvenile alligators reared under controlled laboratory conditions but originating from three sites with dissimilar environmental qualities, indicating an absence of detectable site-of-origin effects on persistent levels of global 5mdC content. Analyses of tissues across individuals revealed a surprising lack of correlation between global methylation levels in blood and ovary. In addition, global DNA methylation in blood samples from juvenile alligators was elevated compared with those from adults, suggesting that age, as observed in mammals, may negatively influence global DNA methylation levels in alligators. To our knowledge, this is the first study examining global levels of DNA methylation in the American alligator and provides a reference point for future studies examining the interplay of epigenetics and environmental factors in a long-lived sentinel species.