Susceptibility of boar spermatozoa to heat stress using in vivo and in vitro experimental models

Induction of heat stress as an experimental procedure in animals is commonly used to examine heat-related impacts on sperm quality. This study aimed to develop potential heat stress models that could be used at any time of the year, to advance the study of seasonal infertility in the pig under controlled conditions. Heat stress was induced by either housing boars (n = 6) at 30 °C inside a hot room for 42 days (55–65% humidity; LD 12:12 h; in vivo), or by heating boar semen (n = 7) for 30 min at various temperatures (35.5, 38.8, 40, 42, 46, 50, 54 and 60 °C; in vitro). Sperm motility was then characterized by computer-assisted sperm analysis (CASA; IVOS version 10: Hamilton Thorne, USA), and DNA integrity was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) and flow cytometry. Our in vivo hot room model induced biologically meaningful levels of DNA damage in boar spermatozoa (10.1 ± 1.9 hot room vs. 6.7 ± 1.7% control; P > 0.05), although not statistically significant from controls. Moreover, sperm concentration and motility parameters did not differ between treatments (P > 0.05). Compared to the 38.8 °C control, our in vitro heat shock model significantly increased sperm DNA damage after incubation at 54 and 60 °C (3.0 ± 1.0, 2.9 ± 1.0, 1.2 ± 0.3, 2.5 ± 0.7, 9.0 ± 3.7, 16.2 ± 7.1, 14.2 ± 5.8 and 41.8 ± 18.6% respectively; P ≤ 0.05). However, these temperatures rendered sperm completely immotile or dead, with most motility parameters declining rapidly to zero above 40 or 42 °C. In conclusion, our results suggest that temperature combined with individual factors may contribute to a boar’s overall susceptibility to heat stress. Refinement of these models particularly of the in vitro heat shock model could be further pursued to overcome environmental variability, reduce whole animal experiments and provide a putative diagnostic fertility screening tool to evaluate heat tolerance in the boar.