After copulation, insect females store sperm in a spermatheca for some duration until fertilization. At the beginning of their adult lives, ant queens can preserve numerous viable sperm cells from copulation for over ten years. However, the key factors influencing long-term sperm storage have not been identified. Here we show that the spermathecal environment is nearly anoxic, which induces sperm immobilization. Furthermore, mitochondrial respiratory inhibitors suppress sperm motility, suggesting that sperm immobilization may be caused by a shortage of ATP generated from only glycolysis under near-anoxic conditions. Sperm immobilization is not induced by acidification via glycolytic metabolism because the spermathecal fluid is not acidic. Finally, we show that artificial anoxic conditions rather than aerobic conditions sustain viable sperm cells. Therefore, near-anoxia is a key factor influencing long-term sperm storage in ant queens. The viability of sperm cells under artificial anoxia, however, is lower than that of those dissected immediately from queens. Moreover, the immotile sperm cells under more than 4 h of anoxia do not begin swimming after aerobic exposure, unlike those under anoxic conditions for less than 2 h. This finding indicates that factors other than anoxia are also necessary for long-term sperm preservation.
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