This study aimed to assess the semen quality after the cooling and freezing of the first and second ejaculates of the season, which were collected 1 h apart. After collection (n = 40 ejaculates), the gel-free semen volume, concentration, total number of sperm, and sperm morphology were determined. An aliquot of each ejaculate was extended and cooled for 48 h; a second aliquot was cushion-centrifuged and cooled for 48 h; and a third aliquot was processed and then frozen. The total motility (TM) and progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP) were assessed pre-(0 h), 24 h, and 48 h post-cooling and before and after freezing. The second ejaculate had a lower gel-free semen volume (p = 0.026). The sperm concentration was greater in the first than in the second ejaculate (p < 0.001). The sperm morphology was similar between the ejaculates (p > 0.05). Cushion-centrifugation prevented a reduction in the TM, PM, and PMI over time (p < 0.05). The TM, PM, and PMI decreased after freezing but not between the ejaculates (p > 0.05). The first and second ejaculates of the season, which were collected 1 h apart, varied in quantity but not in quality after cooling and freezing.
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