Effect of hypothermal storage on motility, viability, acrosome integrity and intracellular zinc of boar sperm

Boar semen is commonly stored at 17°C to preserve sperm quality to be used for insemination within 3 d. However, the first stages of decline of sperm quality occurs as sublethal damage, but eventually result in sperm death and loss of fertility potential. Alternatively, hypothermal (5°C) storage is useful to control bacterial growth but limiting due to the high sensitivity of boar sperm to chilling injuries. Therefore, we aimed to evaluate the effect of hypothermal storage on sperm quality and intracellular Zinc2+ as indicative of capacitation status. Semen from commercial boars (n=15) was collected, extended in Androstar®Plus (Minitube) at 3 ×109 sperm/75 ml and shipped (17°C) overnight. Paired-samples were stored at 17 and 5°C. Motility (IVOS II, Hamilton Thorne) and flow cytometric assessment of viability, acrosome integrity [100 nM SYBR®14, 10 µm propidium iodide (PI), 5.6 µg/ml PNA Alexa- FluorTM647; Molecular Probes] and intracellular Zinc2+ [8 µg/mL Hoecsht-33342, 1 µM FluoZin™-3-AM (FluoZin), 10 µM PI] were performed at Days -1, -4 and -7 after collection. Results were analyzed by 2-factor mixed model for repeated measure and Tukey-adjusted pairwise comparisons and expressed in percentages (mean±SEM). Total motility was higher (P<0.05) at Day -1 (85.9±3.1) compared to Day -4 (17°C: 78.9±3.1; 5°C: 73.7±3.1) or Day -7 (17°C: 71.8±3.1; 5°C: 73.5±3.1), with no differences between and within temperatures. Progressive motility remained similar (P>0.05) during 17°C-storage (Day -1: 47.5±4.1; Day - 4: 48.1±4.1; Day 7: 53.8±4.1), but it was reduced (P<0.05) at Day -4 (36.9±4.1) compared to Day -7 (47.4±4.1) at 5°C and to samples held at 17°C. For live sperm with intact acrosome (SYBR+/PI−/PNA−), percentages of 17°C (Day -4: 85.3±2.1; Day -7: 82.8±2.1) or 5°C-storage (Day -4: 78.9±2.1; Day -7: 81.3±2.1) did not differ compared to Day -1 (84.3±2). However, live sperm with intact acrosome at Day -4 was higher (P<0.05) at 17°C than at 5°C. Samples stored at 17°C had reduced (P<0.05) intracellular Zinc2+ and plasma membrane integrity (FluoZin+/PI–) at Day -4 and Day -7 (62.2±4.3; 67.4±4.3) than Day -1 (77.6±4.3), but they did not differ from samples kept at 5°C (Day -4: 61.5±4.3; Day -7:67.9±4.3). We concluded that storing boar sperm either at 5 or 17°C reduced motility and plasma membrane integrity, but live sperm subpopulation maintain high intracellular Zinc2+, indicative of non-capacitated status, further than 4 d of storage.