Functional Significance of the Sperm Head Morphometric Size and Shape for Determining Freezability in Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm Samples

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Abstract
In the present study, computer‐automated sperm head morphometry of epididymal samples was used to determine if sperm head area and shape are useful measurements for separating “good” and “bad” Iberian red deer freezers. A microscope slide was prepared from single diluted sperm fresh samples collected from 38 mature stags. Slides were air‐dried and stained with Hemacolor. The sperm head area and shape (length/width) for a minimum of 145 sperm heads were determined for each male by means of the Sperm‐Class Analyser. The remainder of each sample was frozen. After thawing, sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. All sperm parameters evaluated at thawing were placed in a statistical database and a multivariate cluster analysis performed. Mean sperm parameters of the 2 clusters generated (“bad” and “good” freezers) were compared by ANOVA. Our results show that sperm quality at thawing for all sperm parameters evaluated was significantly higher (P < .01) for “good” freezers than for the “bad” ones (sperm motility index: 67.4±2.0 vs 57.1±2.8; NAR: 67.1±2.5 vs 54.5±3.5; viability: 68.8±2.0 vs 60.1±2.8; HOST: 71.3±2.2 vs 63.1±3.1). Additionally, differences (P < .01) in epididymal sperm head area and shape were found between “good” and “bad” freezers before freezing, with the smallest overall sperm head dimensions found in the “good” freezers group (area: 32.04 μm2vs 34.42 μm2). Thus, the lower the sperm head area in the fresh samples, the greater the sperm cryoresistance. Our results show that the 2 groups of males also differ in sperm head shape in fresh samples (good: 1.96 vs poor: 1.72;P< .01). It is possible that sperm head area and shape influence total sperm volume, thus causing differences in heat exchange as well as in movements of water, ions, and cryoprotectants and, in turn, on sperm freezability.

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