Effects of Mixed Pasture Legume Phytoestrogens on Superovulatory Response and Embryo Quality in Angus Cows.

Ovulation and artificial insemination rates have been observed to decrease in sheep and cows when exposed to dietary phytoestrogens at concentrations greater than 25 mg/kg DM. A grazing trial was undertaken to investigate the effects of coumestrol and other key phytoestrogens on the superovulatory response, embryo numbers and quality in beef cows grazing legume pastures. A 7-week controlled grazing trial was conducted with legume and ryegrass pasture treatments, with cows exposed to legumes at two timed treatments, 4 and 7 weeks. Twenty Angus cows were subjected to a conventional estrus synchronization and superovulation protocol. Embryos were recovered via conventional uterine body flushing 7 days post artificial insemination (AI). Numerous phytoestrogens were identified in both pasture and plasma samples, including coumestrol and formononetin. Concentrations of phytoestrogens in the pasture ranged from 0.001 to 47.5 mg/kg DM and 0 to 2.6 ng/mL in plasma. Approximately 50% of cows produced viable embryos 7 days post AI. A significant interaction between the effect of treatment groups on the embryo stage was observed (p < 0.05). The results suggest that concentrations of >25 mg/kg DM of phytoestrogens less than 20 days preceding AI may negatively affect oocyte developmental competence, reduce progesterone production and thus contribute to early embryonic loss.

3-Methoxytyrosine as an indicator of dopaminergic manipulation in equine plasma.

The use of catechol-O-methyltransferase inhibitors may mask doping agents, primarily levodopa, administered to racehorses and prolong the stimulating effects of dopaminergic compounds such as dopamine. It is known that 3-methoxytyramine is a metabolite of dopamine and 3-methoxytyrosine is a metabolite of levodopa thus these compounds are proposed to be potential biomarkers of interest. Previous research established a urinary threshold of 4,000 ng/mL for 3-methoxytyramine to monitor misuse of dopaminergic agents. However, there is no equivalent biomarker in plasma. To address this deficiency a rapid protein precipitation method was developed and validated to isolate target compounds from 100 µL equine plasma. A liquid chromatography-high resolution accurate mass (LC-HRAM) method using an IMTAKT Intrada amino acid column provided quantitative analysis of 3-methoxytyrosine (3-MTyr) with lower limit of quantification of 5 ng/mL. Reference population profiling (n = 1129) investigated the expected basal concentrations for raceday samples from equine athletes and showed a right-skewed distribution (skewness = 2.39, kurtosis = 10.65) which resulted from large variation (RSD = 71%) within the data. Logarithmic transformation of the data provided a normal distribution (skewness = 0.26, kurtosis = 3.23) resulting in the proposal of a conservative threshold for plasma 3-MTyr of 1,000 ng/mL at a 99.995% confidence level. A 12-horse administration study of Stalevo® (800 mg L-DOPA, 200 mg carbidopa, 1600 mg entacapone) revealed elevated 3-MTyr concentrations for 24-hours post-administration.

Kisspeptin stimulates LH secretion but not ovulation in mares during vernal transition.

Managing the return to regular cyclicity after the winter anestrous period in the mare has been a challenge for the equine breeding industry. Specifically, efforts have been made to shift or shorten the vernal transition period and to have it followed by a predictable first ovulation at the commencement of the breeding season. Intravenous administration of kisspeptin is known to stimulate an LH response in both reproductively active and inactive mares. This study examined the effects of a constant rate infusion (CRI) of kisspeptin on mares during vernal transition. Mares were given a 30 hours infusion of kisspeptin at a low and high rate (66 nmol [88 µg] and 100 nmol [130 µg] per hour, respectively) or saline, and the LH and follicular response tracked. Plasma samples were collected every 15 minutes for the first 6 hours to determine if there is an acute effect of kisspeptin infusion on LH secretion. Plasma samples were then collected every 3 hours for a total of 72 hours to examine the ability of kisspeptin to stimulate an LH surge. A CRI of kisspeptin increased LH secretion in these mares but was not able to stimulate an LH surge. To examine the effect of kisspeptin on ovarian activity, follicular measurements were collected ultrasonographically until ovulation occurred or the follicles regressed. CRI of kisspeptin at these rates was unable to induce ovulation earlier than controls.