Improving Pregnancy Success with Laparoscopic Artificial Insemination in Brazilian Ocelots (Leopardus pardalis mitis)
Many of the world’s 36 wild felid species are threatened with extinction, including the ocelot (Leopardus pardalis), most notably the two distinct subspecies endemic to southern Texas (L. p. albescens) and southern Brazil (L. p. mitis). To help conserve this species, the Association of Zoos and Aquariums (AZA) established a Species Survival Plan (SSP) to coordinate a cooperative breeding program among AZA institutions housing Brazilian ocelots. However, limited founder numbers and poor breeding success are impairing the SSP’s ability to achieve its genetic management goals. Assisted reproductive techniques, such as artificial insemination (AI), could be invaluable for improving reproduction of behaviorally or physically incompatible pairings or for creating genetic exchange between disjunct populations without requiring the transport of living animals. However, for AI to be an effective and reliable genetic management tool, the success of this technique must be improved for use with either freshly-collected or frozen-thawed spermatozoa.
The standard AI protocol for felids requires treatment of females with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) to induce ovarian follicular growth and ovulation, respectively, followed by laparoscopic intra-uterine sperm deposition.3 Although this approach has resulted in multiple pregnancies in domestic cats and cheetahs, AI success in other felid species, including the ocelot, has been less consistent, possibly related to the gonadotropin regimen used and/or the site of insemination. Because hCG persists in circulation for up to 5 days post-injection, the formation of ancillary follicles and secondary corpora lutea (CLs) may disrupt the post-ovulatory endocrine environment and impair oviductal embryo transport. Furthermore, intra-uterine AI techniques often require substantial sperm numbers (>10 million motile sperm/AI) for fertilization, which limits the number of potential AI procedures per ejaculate collected. This is especially true in small-sized cats which often have low total sperm numbers per ejaculate or when using frozen-thawed spermatozoa that has been compromised by cryopreservation-related damage.
In our earlier domestic cat studies, we obtained promising pregnancy results with a novel laparoscopic AI approach using porcine luteinizing hormone (pLH) as an alternative to hCG for ovulation induction and depositing low numbers of spermatozoa directly into the oviduct.1 In the current study, our objectives were to: 1) evaluate the ovarian and hormonal response of Brazilian ocelots following eCG/pLH treatment; 2) assess pregnancy success after intra-uterine vs. intra-oviductal AI in eCG/pLH-treated females; and 3) investigate the reliability of a commercially available canine relaxin kit to detect urinary relaxin for pregnancy diagnosis in ocelots. For ovarian stimulation, Brazilian ocelot females (n=5 cats; 7 AI procedures) received 400 IU eCG followed 84 hr later with 3000 IU pLH. At 39–42 hr after pLH injection, females were anesthetized for laparoscopic insemination bilaterally into the uterine horns (n=5 AIs) or oviducts (n=2 AIs) with freshly-collected semen from resident males. Fecal samples were collected for 50–80 days post-AI and assessed for fecal progesterone metabolite levels using a validated enzyme immunoassay. Fresh urine samples collected at Day 50–61 post-AI were evaluated for urinary relaxin using a commercially available canine test kit.2
All ocelot females ovulated in response to eCG/pLH treatment, averaging (±SEM) 2.7±0.5 CL and 4.4±1.7 unovulated follicles per female. Five females were inseminated using the intra-uterine technique, receiving an average of 16.6±6.2 million motile spermatozoa per AI. One female conceived and gave birth to a single, healthy female kitten after a 79 day gestation. Two females were inseminated using the intra-oviductal technique (10.2±3.0 million motile spermatozoa per AI). One female conceived and gave birth to a single, healthy female kitten after an 80 day gestation. Fecal progesterone monitoring in non-pregnant females revealed luteal phases lasting 44.2±3.8 days (range, 31–55 days) post-AI. Thus, fecal progesterone levels that remained elevated past 55 days were indicative of pregnancy. Relaxin kit testing of concentrated urine samples collected from pregnant and non-pregnant ocelots were all negative for the presence of relaxin.
Our findings indicate that the eCG/pLH regimen is effective in ocelots for inducing ovarian stimulation and ovulation for AI procedures and that both intra-uterine and oviductal laparoscopic insemination may be used to produce pregnancies in eCG/pLH-treated females. These results also suggest that oviductal AI offers the potential of improved pregnancy success in ocelots while using fewer spermatozoa or spermatozoa of poorer quality such as occurs following cryopreservation. Fecal progesterone monitoring was found to be a reliable method of pregnancy diagnosis in ocelots beginning at 55 days post-AI, but use of a commercial canine relaxin kit for urinary relaxin detection was not accurate and is not recommended for pregnancy diagnosis in ocelots.
1. Conforti VA, Bateman HL, Vick MM, Newsom J, Lyons LA, Grahn RA, Deddens JA, Swanson WF. Improved fertilization success using laparoscopic oviductal artificial insemination with low sperm numbers in domestic cats. Ann Meet Soc Study Reprod. In press.
2. Harris LA, Steinetz BG, Bond JB, Lasano S, Swanson WF. Refinement of a commercial bench-top relaxin assay for pregnancy diagnosis using urine from domestic and non-domestic felids. J Zoo Wildl Med. 2008;39:170–179.
3. Howard JG, Barone MA, Donoghue AM, Wildt DE. The effect of pre-ovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cats. J Reprod Fert. 1992;96:175–186.