Post-thaw amendment of cryopreserved sperm for use in artificial insemination of a viviparous fish, the green swordtail Xiphophorus helleri

Sperm cryopreservation protocols have been developed for live-bearers such as the green swordtail Xiphophorus helleri and the platyfish Xiphophorus couchianus. Despite the high post-thaw motility (∼ 75%) obtained in both species, the requirements of sperm storage within the female reproductive tract coupled with the process of internal fertilization place functional demands upon cryopreserved sperm samples far beyond those of oviparous species. The purpose of this study was to facilitate the artificial insemination process with cryopreserved sperm of X. helleri through evaluation of parameters related to sperm quality after thawing. Specifically, this study evaluated the effects on motility for fresh and thawed sperm samples of centrifugation (for concentration of sperm and washing for removal of cryoprotectant), ionic composition, and additions of glucose and fetal bovine serum (FBS) in extender solutions. Centrifugation at 1000 ×g for 10 min at 4 °C was found to have no adverse effects on sperm motility of fresh samples, and for cryopreserved samples, the removal of glycerol by washing yielded higher and longer post-thaw motility (e.g., 168 h vs. 48 h for the controls). Suspension of fresh sperm samples in magnesium-free Hanks' balanced salt solution (HBSS) did not affect motility; however, HBSS prepared with the absence of potassium or calcium, and the use of unsupplemented saline (NaCl alone) as extenders significantly reduced sperm motility. The presence of glucose in HBSS yielded higher and longer motility for fresh and thawed samples, but addition of glucose at greater than 2 g/L were unnecessary. Addition of 20% FBS prior to freezing was found to increase the post-thaw motility significantly compared to control treatment with 14% glycerol alone. Also addition of 20% FBS after thawing and centrifugation was found to induce the formation of sperm bundles, which may be beneficial for internal fertilization success. In conclusion, concentration of sperm and the removal of cryoprotectant (through centrifugation), and the addition of 20% FBS in the extender is recommended for future insemination trials with cryopreserved samples.