78 CRYOPRESERVATION OF FROG (RANA PIPIENS) SPERM CELLS COLLECTED BY NON-LETHAL METHODS

Abstract

There are few reported studies of cryopreservation of frog spermatoazoa. None have used non-lethal methods for collection, which involves a simple IP injection, followed by collection of sperm from the urine. Our study compared post-thaw survival of spermatozoa frozen using three different diluents: Salamon's ram semen diluent (SSD) (Evans and Maxwell 1987 Salamon's Artificial Insemination of Sheep and Goats, Sydney, Australia: Butterworths, pp. 122–141); our modification of Salamon's diluent (MSSD), containing an additional 5.9 mL glycerol and 6.6 g of sucrose per 100 mL; and a diluent reported to be currently the most successful for freezing frog sperm: Browne's frog diluent (BFD) (Browne et al. 2002 Cryobiology 44, 251–257). The 1 × SSD was composed of 2.9 g Tris, 0.4 g glucose, 3.2 g citric acid monohydrate, 12 mL egg yolk, and 4.5 mL glycerol per 100 mL. The composition of 1 × BFD was 10 g sucrose and 15 mL DMSO per 100 mL. Ten male frogs of unknown age were injected with 200 IU hCG (Chorulon; Intravet, Millsboro, DE, USA) in 500 µL PBS. Urine specimens were collected by cloacal lavage 30, 60, and 90 min post-injection. Specimenswere stored in a cold room at 8°C until all collections were finished. Diluents requiring addition of egg yolk were completed with that from a freshly laid chicken egg. Sperm cells were pooled and concentrations were measured using microscopy. Sperm cells were divided and combined with diluents in 1.5-mL microcentrifuge tubes. Tubes were suspended in a 500-mL beaker, which was placed in an ice bath to allow slow cooling to 2°C over 45 min. Aliquots of 100 µL were pipetted into indentations on dry ice and left to freeze for 3 min. Frozen pellets were plunged into liquid N₂, and while under LN₂, pellets were placed in microcentrifuge tubes, which were then stuffed with cotton wool. Upon thawing, pellets were removed from LN₂ and dropped into 200 µL of thawing solution (0.9085 g Tris, 0.25 g fructose, and 0.45 g citric acid per 15 mL final volume) at 21°C while gently vortexing. Spermatozoa were assessed using live/dead cell stain (Molecular Probes, Eugene, OR, USA) and fluorescent microscopy. Percentages of live spermatozoa from the three treatments were 58.5%, 70.5%, and 44.1% in SSD, MSSD, and BFD, respectively. When considering the number of sperm cells lysed along with live/dead cell stain data, 40%, 55.2%, and 34.4% in SSD, MSSD, and BFD, respectively, survived post-thaw. All values were significantly different (P < 0.001). Superior results were observed with MSSD; use of SSD also resulted in better results than BFD. The most prevalent method for collection of frog sperm is to pith the frog and then macerate the testis. With frog populations dwindling worldwide, use of a technique that is virtually harmless such as used here in conjunction with cryopreservation of specimens could be useful for preserving genetic variation in endangered frog species.