Effect of intrafollicular administration of PGE2 or PGF2α in early estrus on ovulation, hemorrhagic anovulatory follicles formation, progesterone secretion and pregnancy outcome in the mare.

This experiment was performed to evaluate whether intrafollicular treatment of PGE2 or PGF2α administered in early estrus would induce normal ovulation, progesterone production (Experiment 1) and pregnancy (Experiment 2). In Experiment 1, mares in estrus after 2 days of endometrial edema were injected in all largest dominant follicles (28-35 mm in diameter) with 0.5 mL of sterile water containing 500 µg PGE2 (n = 6), 125 µg PGF2α (n = 6) or placebo (n = 7) (Hour 0). Ultrasound examinations were performed daily, until ovulation or anovulation was detected, and daily blood samples were taken for 8 days. In Experiment 2, mares with a dominant follicle ≥35 mm after at least three days of slight-to-moderate endometrial edema, were injected with 500 µg PGE2 diluted in 0.5 mL of sterile water for injection in the follicle (PGE2 group; n = 9 mares and 11 dominant follicles). No puncture was performed in the control group (n = 9 mares and 11 dominant follicles). Mares from both groups were inseminated. In Experiment 1, all mares (6/6) in the PGE2 group ovulated within 24 h of treatment. The mean interval from intrafollicular injection to ovulation was shorter (P < 0.001) in PGE2 mares (24 ± 0 h) than in control mares (77 ± 9 h). Mares from the PGF2α group developed hemorrhagic anovulatory follicles (HAF) more often (7/7) than control mares (2/7); P < 0.05). The progesterone concentration in mares from the PGF2α group was lower (P < 0.004) than control mares in the early post-ovulatory period. The first significant increase in post-ovulatory progesterone concentration occurred earlier (P < 0.05) in mares from the control group than in mares from the PGF2α and PGE2 groups. In Experiment 2, more mares from the control group (7/9, 78 %) became pregnant than from the PGE2 group (2/9, 22 %) (P = 0.015). In conclusion, PGE2 alone induced follicle collapse in all treated mares within 24 h of administrations, while PGF2α blocked ovulation and induced formation of HAFs. However, the post-ovulatory rise in progesterone production was delayed and the fertility reduced in mares with ovulation induced by PGE2 compared to control mares.

Epigallocatechin-3-Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm-Zona Pellucida Heterologous Binding.

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin-3-gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2-h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm, 500 nm and 5 µm) and EGCG (10, 20 and 60 µm), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 µm (but not of 60 µm) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm, the presence of EGCG at all the concentrations tested (10, 20 and 60 µm) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone.

Caspase activation, hydrogen peroxide production and Akt dephosphorylation occur during stallion sperm senescence.

To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.

Sex sorting increases the permeability of the membrane of stallion spermatozoa.

At present, the only repeatable means of selecting the sex of offspring is the Beltsville semen sorting technology using flow cytometry (FC). This technology has reached commercial status in the bovine industry and substantial advances have occurred recently in swine and ovine species. In the equine species, however, the technology is not as well developed. To better understand the changes induced in stallion spermatozoa during the sorting procedure, pooled sperm samples were sorted: sperm motility and kinematics were assessed using computer assisted sperm analysis, sperm membrane integrity was assessed using the YoPro-1 assay, while plasmalemmal stability and lipid architecture were assessed using Merocyanine 540/SYTOX green and Annexin-V, respectively. Lipid peroxidation was also investigated with the probe Bodipy(581/591)-C11. All assays were performed shortly after collection, after incubation and after sex sorting using FC. In order to characterize potential molecular mechanisms implicated in sperm damage, an apoptosis protein antibody dot plot array analysis was performed before and after sorting. While the percentage of total motile sperm remained unchanged, sex sorting reduced the percentages of progressive motile spermatozoa and of rapid spermatozoa as well as curvilinear velocity (VCL). Sperm membranes responded to sorting with an increase in the percentage of YoPro-1 positive cells, suggesting the sorted spermatozoa had a reduced energy status that was confirmed by measuring intracellular ATP content.