247 SUCCESSFUL ARTIFICIAL INSEMINATION IN THE CORN SNAKE (ELAPHE GUTATTA), USING FRESH AND COOLED SEMEN

Abstract

The purpose of this investigation was to develop a non-invasive technique to artificially inseminate snakes using the corn snake, Elaphe gutatta, as the model representative for this taxon. Semen was collected by first applying pressure to the lower abdomen in a continuous distal motion toward the cloaca to remove any feces or urates. The cloaca was then gently washed using phosphate-buffered saline, and a more localized pressure was applied to each side of the vent to evert the hemipenes and, subsequently, the ejaculate. The semen was collected using a sterile transfer pipette and placed into 70 to 90 µL of medium (TL-HEPES solution; Cambrex Bio Science, Inc., Baltimore, MD, USA04–616F) in a sterile microcentrifuge tube, and then analyzed for overall motility, rate of forward progression (RFP, 0–5), and concentration. Based on a previously reported procedure, 10 females were inseminated with either fresh (n = 5) or cooled semen (n = 5; refrigerated for 3 days) one week after recovering from a hibernation period required to stimulate reproduction in this species. The overall sperm motility and concentration for females inseminated with fresh or cooled semen was 92%, 9.6 million sperm mL^-1; and 85%, 6.1 million sperm mL^-1, respectively. Immediately prior to insemination, the same method for expressing feces and urates in the males was applied to the females. The insemination dose (50 µL semen per oviduct) was drawn into a 1-mL latex- and silicone-free tuberculin syringe (Norm-Ject; VWR, Batavia, IL, USA) that was connected to a feeding/dosing needle (EJAY International, Issaquah, WA, USA) with a ball tip to prevent any potential damage during the insemination. The tip of the needle was then moved around the inner tissue of the vent to relax the cloaca, and the insertion continued until resistance was found indicating the vicinity of the oviducts. The extended semen was carefully deposited on both sides, and then the needle was slowly withdrawn. The offspring were tested for parentage to verify the success of the insemination. Blood was collected from the dorsal aorta posterior to the cloaca and stored in 10 mM Tris at 4°C. The DNA was extracted using a phenyl : chloroform : isoamyl alcohol (PCI) extraction method. Eight microsatellite loci were used for the paternity exclusion analysis: Eobµ1, Eobµ3, Eobµ10, Eobµ13, Eobµ16, Eobµ34, Eobµ366, and Eobµ373 (IDT, Coraville, IA, USA). All males and females in the collection were tested, and parental candidates were excluded if 2 or more allele mismatches occurred. From the total number of females inseminated, 3 females laid 51 eggs. Two females inseminated with cooled semen laid 36 eggs, of which 5 eggs were hatched, and the remaining were either unfertilized (n = 25) or non-viable (n = 6). All 5 hatched eggs were laid by one of the females. The third female inseminated with fresh semen laid 15 eggs, resulting in 5 hatching and 10 unfertilized eggs. The parentage test validated the AI a success as the alleles correlated between the adults and the offspring. In conclusion, artificial insemination was successful using both freshly collected and cooled (3 days) semen. Further studies are underway to improve the success rate in order to maximize the efficiency of this technology, and thus assist in the genetic preservation of endangered snake species.