226 DAY LENGTH DOES NOT AFFECT LIVE SPERM NUCLEAR SHAPE IN THE STALLION

Abstract

Fourier harmonic analysis (FHA) of sperm nuclei is a precise and objective method to evaluate shape of the sperm head, with the calculated harmonic amplitudes highly related to male fertility. The FHA approach has been developed for use in the bull and the boar but has not yet been applied to the stallion. Direct utilization of the previous fluorescent approaches to identify and image live sperm nuclei in the bull cannot be used in the stallion due to the increased thickness of the post-nuclear region and thin anterior region of the sperm head. An alternative approach was developed in which live and motile sperm were isolated after filtration of an ejaculate through a Sephadex G-15 column. The resulting live sperm were sonicated briefly to separate tails and heads. The heads were isolated on a 45–90 discontinous Percoll gradient, fixed with paraformaldehyde (0.2%), centrifuged onto glass slides, and dried. The slides were then stained with eosin (1%), cleared with water, and dried again; Permount was added, followed by a coverslip. Slides were imaged with phase contrast microscopy; digital images were acquired and evaluated with custom software to identify perimeter coordinates of sperm nuclei. The perimeter coordinates were next converted to Fourier harmonic amplitudes 0–5 (HA0–HA5) using trigonomic regression at 1 degree equally spaced angles. Fertility of bulls were previously reported to be most related to changes in HA0 and HA2 but no information is available on stallions. As fertility data on stallions is limited, to evaluate FHA in the equine, the day length (period of light) was increased in January from the ambient 9–10 h to 16. Semen samples from 5 light horse stallions were collected at weekly intervals for 8 weeks following the increase in light. It was hypothesized that fertility would increase for each stallion over the course of the experiment, as testosterone increases and spermatogenesis improves with increasing day length, as previously shown. Each week semen samples were evaluated for FHA live sperm nuclei, as described above. All parameter means and variations were recorded on 100 randomly selected sperm nuclei per semen sample evaluated. There was no difference relative to week 0 in the least squares mean or SD of HA0, HA1, HA2, HA3, HA4, or HA5 over the 8 weeks (P > 0.05). However stallions were consistently different for all HAs (P < 0.05). The overall mean HA0–HA5 ± SEM were 1.973 ± 0.38, 0.087 ± 0.002, 0.721 ± 0.021, 0.051 ± 0.004, 0.208 ± 0.014, and 0.031 ± 0.002, respectively. Even though libido increased during the experiment, confirming the effect of light on the stallions, no affect on sperm nuclear shape or its variation was detected using FHA. Based on work in other species involving numbers of sperm inseminated, if large numbers of sperm from these stallions were inseminated in mares, we would predict no change in fertility due to season. Further research is needed to confirm this prediction.