Sperm cryopreservation in guppies and black mollies--a generalized freezing protocol for livebearers in Poeciliidae

In this study, we evaluated various parameters of sperm cryopreservation in two livebearers, guppies (Poecilia reticulata) and black mollies (P. latipinna). Our results suggested a common freezing protocol for the guppies and mollies: suspend sperm in Hanks' balanced salt solution (HBSS) at 300 mOsm/kg, use 14% glycerol as cryoprotectant, cool at 25 degrees C/min, and thaw at 40 degrees C in a water bath for 7s. Live young were produced from females inseminated with frozen-thawed sperm in both species. In guppies, percent fertilization (F) and the number of embryos (N) produced with cryopreserved sperm (F=50%, N=74, from 26 females) were similar to those of fresh controls (F=54%, N=61, from 22 females). Interestingly, this same freezing protocol has been used successfully for sperm cryopreservation in green swordtails Xiphophorus helleri, and platyfish of X. couchianus with post-thaw motility as high as 80%. All these species belong to the family of Poeciliidae, and their sperm are similar in morphology exhibiting the absence of acrosome, elongate sperm head, and the long mitochondrial sheaths. Besides their internal fertilization reproduction mode, these fish are also small in size (2-4 cm) and live in a freshwater environment. Sperm cryopreservation in fish has been generally recognized as species specific, and new protocols are required for new species. However, results presented in this study suggested otherwise. Thus, sperm cryopreservation methods optimized for one species may be applicable to others if they are taxonomical closely related species with similar sperm morphology and reproduction mode. Considering the enormous number of fish species on the planet, development of generalized sperm freezing protocols for species in groups could have additional advantages for genetic conservation.