138 Associations of sperm head morphometrics with quality parameters of frozen-thawed ram semen

Abstract

Current methods of mammalian semen evaluation focus on determining spermatozoa motility, concentration, mitochondrial status, and nucleus or chromatin structure integrity, quantifying their ability to bind to ova or measuring seminal plasma content of various biochemical markers. However, there is a paucity of studies that address relationships between sperm head morphometry (the external shape and dimensions of the sperm) and fertilising ability. Sperm head morphometrics are influenced by many molecular and biochemical factors such as genetics, DNA or protein condensation, and cell membrane permeability, all of which can affect semen viability. The objective of this experimental work was to determine quantitative correlations between sperm head dimensions and various indices of sperm quality in frozen-thawed ram semen. Ejaculates were collected from 16 clinically healthy rams (4 Polish Lowland (PON), 4 Olkuska, 5 synthetic line BCP (Berrichon du Cher × Charolais × PON/Polish Merino), and 3 synthetic line SCP (Suffolk × Charolais × PON/Polish Merino) aged 4-12 years) into an artificial vagina in the middle portion of the breeding season. Ejaculates from each ram were divided into two equal portions, diluted with a commercial semen extender prepared in deionised water or nanowater (water declusterised using cold plasma treatment) to a final concentration of 400 × 10⁶ spermatozoa mL⁻¹, and frozen in 0.25-mL plastic straws. After 6 months of being cryogenically preserved, semen samples were thawed and used for the preparation of smears stained with eosin or SpermBlue. Images of the samples containing at least 100 spermatozoa were taken under 200× magnification and used for determination of sperm head morphology with the image analytical software Image Pro Plus (Media Cybernetics Inc.). Sperm progressive motility and survival time, as well as extender concentrations of alkaline phosphatase and aspartate aminotransferase, were measured. Finally, 128 BCP ewes were inseminated laparoscopically with the ram semen and fertility parameters were recorded. The present data were analysed using a multivariate analysis of variance in SAS (SAS Institute Inc.) and Spearman correlation tests. There were no significant effects or interactions of breed, staining method, or extender diluent on sperm head dimensions (head length, width, area, perimeter, and roundness). The mean head length was negatively correlated (P < 0.05) with the percentages of spermatozoa with vacuolated, detached, or amorphous heads or small acrosomes; thick and thin midpiece defects, distal droplet, broken tail plus distal droplet, short tail plus distal droplet, and thick midpiece plus proximal droplet; and sperm progressive motility. In addition, sperm head roundness was negatively correlated with the proportion of spermatozoa with coiled tails. There were no correlations of sperm head dimensions with survival time, alkaline phosphatase and aspartate aminotransferase concentrations, or conception and pregnancy rates of artificially inseminated ewes. Sperm length and roundness (but no other measurements) were significantly correlated with segmental sperm defects and motility that may impinge the fertilising ability of frozen-thawed ram semen.