120 Effect of bull exposure to high temperature-humidity index levels on the quality of sperm selected through density gradient centrifugation for in vitro fertilisation

Bulls exposed to hot and humid conditions experience adverse effects on sperm quality and in vitro embryo production outcomes. Commonly, a sperm subpopulation with favourable functional characteristics is selected by means of a sperm separation method and thereafter, used for IVF. Therefore, to study the effect of sperm modifications on in vitro embryo production, it is essential to focus on the detailed functional analysis of the sperm fraction selected for IVF. This study aimed to evaluate the functional status of frozen-thawed bovine sperm produced under high or low temperature-humidity index (THI) selected through a density gradient centrifugation (DGC), using computer-assisted sperm analysis (CASA) and flow cytometry. In this regard, we examined 12 ejaculates collected from six mature Simmental bulls (aged 6 ± 1 years), housed in an AI centre in southern Germany and exposed to high (n = 6) and low (n = 6) THI during the epididymal maturation phase (7 days before sperm collection). For each bull, two 0.25-mL straws per ejaculate were pooled post-thaw and motile sperm were separated using the BoviPure® density gradient system (Nidacon International AB). Samples of the selected sperm population were examined after 0 and 3 h of incubation at 38°C in HEPES-TALP medium. The percentage (%) of total and rapidly motile sperm was evaluated using CASA. Flow cytometry was used to determine the percentage (%) of sperm with intact plasma membrane and acrosome (PMAI), with high DNA fragmentation index (%DFI) as well as the size of the sperm subpopulation with high esterase activity, intact plasma membrane and acrosome, low Ca²⁺  levels, and high mitochondrial membrane potential (C_posPI_negPNA_negF_negM_pos sperm). Differences in sperm parameters were explored using the least-squares means procedure and Tukey’s honestly significant difference pairwise comparisons were established. Sperm of the high-THI group showed a profound deterioration of their % DFI values between 0 and 3h (5.9 ± 6 and 11.5 ± 9, respectively; P < 0.05), whereas such deterioration was not observed for the low-THI group (5.2 ± 6 and 9.2 ± 8, respectively at 0 and 3 h; P > 0.05). Selected sperm of both THI groups showed similar values of CASA traits at both 0 and 3 h (P > 0.05), while PMAI and C_posPI_negPNA_negF_negM_pos values decreased after 3 h of incubation in both high- and low-THI groups (P < 0.05). In conclusion, our results suggest a relationship between bulls with high-THI exposure and the chromatin integrity of frozen-thawed sperm selected for IVF; however, the observed chromatin instability appears to be subtle and only evident after incubation-induced stress.