Bariushaa Oyuntuya1, MS; William Swanson2, DVM, PhD; Mark Campbell2, DVM; Helen Bateman2, MS; Jason Herrick2, PhD; Colleen Lambo2, DVM; Meredith Brown3, DVM, PhD; Amanda Fine4, VMD, PhD; Steve Ross5, PhD; Bariushaa Munkhtsog6, PhD; Cindy Kreider7; Erika Travis8, DVM, DACZM; Louise Beyea9, DVM; Mike Barrie10, DVM; Ravchig Samiya1, PhD
Collection and cryopreservation of semen from free-ranging wildlife offers a novel means to create gene flow into captive populations without removing animals from the wild. In our previous research in Pallas’ cats, semen was collected from 11 wild males captured on the Mongolian steppes, frozen in 115 semen straws and imported to the U.S.3 Our objectives in the present study were to (1) compare post-thaw motility of wild Pallas’ cat spermatozoa in two culture media, (2) evaluate post-thaw sperm function using heterologous and homologous in vitro fertilization (IVF), and (3) produce offspring via laparoscopic transfer of IVF-derived embryos (ET) or artificial insemination (AI). Frozen semen straws from eight wild males were thawed and diluted in two media (Ham’s F10, FOCM) for motility assays and IVF.2 Oocytes collected via laparoscopy from gonadotropin-treated domestic cats (167 oocytes, 15 females) and Pallas’ cats (73 oocytes, 5 females) were inseminated (5x105 motile sperm/ml) and cultured in vitro for two to seven days before either embryo staining or transfer. Comparing culture medium, percent sperm motility did not differ over time (p>0.05); however, heterologous IVF success and blastocyst formation were higher (p<0.01) in FOCM (64.7% fertilization, 58.5% blastocyst) than in Ham’s F10 (29.3% fertilization, 0% blastocyst). For ET, Pallas’ cat embryos (n=37; 51% fertilization in FOCM) were transferred into the oviducts of seven ovulatory Pallas’ cats synchronized with gonadotropin (eCG/pLH) treatment, but no pregnancies resulted. To assess the feasibility of laparoscopic oviductal AI as an alternative to IVF/ET, one synchronized Pallas’ cat at the Cincinnati Zoo was inseminated with freshly collected semen (5.3x106 motile sperm) from the resident male.1 This female conceived and gave birth to four kittens (three healthy, one stillborn) after a 69-day gestation. Subsequently, oviductal AI was attempted in five Pallas’ cats using frozen-thawed Mongolia semen (mean, 2.9x106 motile sperm/female), but no pregnancies were produced. These findings indicate that Pallas’ cat semen collected and frozen in the field from wild males has adequate post-thaw motility and function to obtain high (50–65%) fertilization success using an optimized feline-specific culture medium. Furthermore, our results show that healthy Pallas’ cat kittens can be produced following oviductal AI of gonadotropin-treated females with non-frozen semen. However, our goal of producing founder offspring from frozen-thawed Mongolian Pallas’ cat semen will require further investigation and continued refinement of assisted reproductive methods for this imperiled wild cat species.
The authors thank Jamsran Gantulga, Galsandorj Naranbaatar, and other field staff for their assistance in Mongolia, and the animal care and veterinary staff at the Cincinnati Zoo and Botanical Garden, Erie Zoo, Utah’s Hogle Zoo, Lake Superior Zoo, and Columbus Zoo and Aquarium for support with domestic cat and Pallas’ cat procedures. This research was supported, in part, by a National Leadership grant (LG-26-08-0155-08) from the Institute of Museum and Library Services.
1. Conforti VA, Bateman HL, Vick MM, Newsom J, Lyons LA, Grahn RA, et al. Improved fertilization success using laparoscopic oviductal artificial insemination with low sperm numbers in domestic cats. In: Proceedings from the Society for the Study of Reproduction. 2011;40(Abstract 173).
2. Herrick JR, Bond JB, Magarey GM, Bateman HL, Krisher RL, Dunford SA, Swanson WF. Development of a feline optimized culture medium: effects of ions, carbohydrates, essential amino acids, vitamins, and serum on the development and metabolism of IVF-derived feline embryos relative to embryos grown in vivo. Biol Reprod. 2007;76:858–870.
3. Oyuntuya B, Swanson W, Munkhtsog B, Ross S, Brown M, Fine A, Samiya R. Assessment of seasonal reproductive traits in a wild Mongolian felid, the Pallas’ cat (Otocolobus manul). In: Proceedings from the Society for Conservation Biology. 2008;123 (Abstract 55.3).