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In this study, we tried to optimize the porcine semen extender conditions to maximize the differences between live X chromosome‐bearing (X) spermatozoa and to Y chromosome‐bearing (Y) spermatozoa without a decline in the fertility rate at different pH conditions during storage. We observed the viability of X and Y boar spermatozoa in acidic (pH 6.2), original (pH 7.2), and alkaline condition (pH 8.2) for 5 days to investigate the effect of storage conditions on the X to Y spermatozoa ratio. The functional parameters of spermatozoa were also examined to evaluate sperm quality. Sperm motility was preserved at pH 7.2 and pH 6.2 for 3 days, while sperm motility at pH 8.2 decreased significantly after 2 days. Non‐capacitated spermatozoa increased while capacitated spermatozoa decreased during storage. Sperm viability decreased significantly duration‐dependent under all pH conditions, but there was no significant difference during storage at pH 6.2 and 7.2. The X: Y ratio of live spermatozoa in acidic condition was maximized (1.2:1) without affecting the sperm function and fertility‐related protein expression after 2 days compared to original conditions. Moreover, insemination of sows using acidic extender increased the number of female pups on days 1 and 2 of preservation. These results indicate that the production of female offspring may increase when acidic BTS is used for 2 days without affecting the success rate of AI. Above all, this method is simple and economical compared to other methods. [ABSTRACT FROM AUTHOR]

Background: Cryoprotective agent (CPA) addition during sperm cryopreservation causes detrimental effects on sperm function and quality. We previously reported that CPA addition adversely affects bull sperm physiological functions and shows differentially expressed proteins. Objectives: To study functional and proteomic alterations between high CPA‐tolerant spermatozoa (HCS) and low CPA‐tolerant spermatozoa (LCS) in bull. Materials and methods: Bull semen was collected from the cauda epididymides of Korean bull (Hanwoo) and suspended in Tris‐egg yolk buffer (TYB). The collected fresh semen was diluted down to a final concentration of 6% glycerol TYB solution. After CPA exposure to the sperm cells from individual bulls, the percentage of sperm motility was examined by utilizing a computer‐assisted sperm analysis system. According to sperm motility value, the HCS (motility above 80%) and LCS (motility below 60%) groups were evaluated for sperm function parameters (swimming speed, capacitation, viability, and mitochondrial function) and protein expression. Results: The HCS group had good sperm function parameters following CPA addition, whereas sperm functions in the LCS group were significantly reduced. There were differentially expressed proteins between the HCS and LCS groups. Cytosolic 5‐nucleotidase 1B and fumarate hydratase were abundantly expressed in the HCS group, while F‐actin‐capping protein subunit beta, voltage‐dependent anion‐selective channel protein 2, and cytochrome b‐c1 complex subunit 1 had a lower expression in the HCS group than in the LCS group. Discussion and conclusion: Identified proteins implicate potential markers to predict CPA‐tolerable spermatozoa, which could provide a method of selecting animals and breeds with cryoprotectant resistance. [ABSTRACT FROM AUTHOR]