Identification of fertility associated sperm sub-populations and development of quality control assays for cryopreserved semen in buffaloes (Bubalus bubalis).

The fertility of buffalo bull semen is crucial to the success of buffalo breeding programs. However, traditional methods for determining semen fertility frequently fall short, resulting in inefficiencies in artificial insemination (AI). Recent advances have highlighted the importance of sperm sub-populations as critical indicators of semen fertility. Frozen semen from 30 Mehsana buffalo bulls with known field fertility was analysed for 25 sperm traits using CASA and flow cytometry. The data was utilized to phenotypically characterize the two groups of bulls and create a model to predict their fertility. Sperm progressive motility, viability, acrosome intactness, intracellular calcium status, mitochondrial membrane potential (MMP), tyrosine phosphorylation status (PTP) and DNA fragmentation index (DFI%) differed (p < 0.05) between high and low fertility bulls. The relationship of sperm progressive motility, viability, acrosomal integrity, low intracellular calcium status, high MMP and PTP positivity were significantly positive (p < 0.05), while the relationship of acrosome reacted population, high intracellular calcium status and DFI% were significantly negative (p < 0.05) with semen fertility. Principal component analysis revealed that three components explained a total of 72.66% variation in the dataset. A stepwise regression analysis revealed that the model comprising of three variables, which include DFI%, live intracellular calcium negative and live acrosome reacted population explained 70% variation (Adjusted R² value: 0.70; p < 0.001) in the bull fertility. In conclusion, the study identified sperm quality traits explaining the variability in bull fertility, and the fertility prediction model developed in the study have the potential to be used for quality control of cryopreserved buffalo semen for obtaining high fertility in AI.