204THE PRODUCTION OF INTRACYTOPLASMIC SPERM INJECTION LION (PANTHERA LEO) EMBYROS USING SPERMATOZOA COLLECTED BY PERCUTANEOUS EPIDIDYMAL SPERM ASPIRATION FROM VASECTOMIZED MALES

Abstract

Contraception and/or sterilization methods have become an essential component of many captive animal management programs. Sterilization techniques are considered to be the last resort if a viable contraceptive cannot be attained and are generally not considered reversible. The African lion (Panthera leo) is one species in which sterilization techniques have been routinely applied. The objective of this study was to develop and evaluate a method for the collection of spermatozoa from male lions that have been previously rendered sterile by vasectomy. Percutaneous epididymal sperm aspiration (PESA) is a technique in which spermatozoa are aspirated from the epididymis and no surgical incision is required. In the present study, two lions (12 and 19 yrs old) were anesthetized and PESA was attempted. A 21-gauge needle attached to a 10-mL syringe (Norm-Ject) filled with 2–3mL of Tyrodes HEPES medium was gently inserted into the head of the epididymis and aspirated gently until spermatozoa were noted. Spermatozoa were visually assessed for motility (grade 1–5; 1=few motile sperm to 5=all motile), sperm concentrations were determined and then the sperm were cryopreserved. The total sperm concentration collected from the older (19yr) male was lower than that obtained from the younger (12yr) lion (0.08×106sperm/mL v. 65.5×106sperm/mL, respectively). Furthermore, more motile spermatozoa (grade 3) were collected from the younger individual compared to the older male (grade 1). Sperm samples from the 12-yr-old lion were frozen by multi-step addition of TEST yolk buffer+glycerol. Lionesses (n=3) were subjected to laparoscopic oocyte retrieval after gonadotropin treatment. A total of 38 oocytes were retrieved and 74% (28/38) were mature as determined by extrusion of the first polar body. Mature oocytes were subjected to ICSI using frozen-thawed spermatozoa obtained by PESA. More than 60% (17/28) of the injected oocytes cleaved and 100% (17/17) reached the morula stage by Day 5 or 6 of IVC. Embryos were cryopreserved and were subsequently transferred (n=15) into one lioness. We have demonstrated that it is possible to collect viable spermatozoa from sterile male lions using the PESA technique. Spermatozoa collected were motile and could be cryopreserved and functional for assisted reproductive techniques. This technique could be applied to other infertile or sterile males whose genetic background would benefit a current conservation program.