Intra-uterine artificial insemination in the tammar wallaby (Macropus eugenii)
Paris, D.B.B.P., Taggart, D.A., Temple-Smith, P.D., Shaw, G., and Renfree, M.B. (2002) Intra-uterine artificial insemination in the tammar wallaby (Macropus eugenii). In: Proceedings of the 2nd International Symposium on Assisted Reproductive Technology for the Conservation and Genetic Management of Wildlife. pp. 80-84. From: The 2nd International Symposium on Assisted Reproductive Technology for the Conservation and Genetic Management of Wildlife, 28-29 Spetember 2002, Omaha, NE, USA.
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Abstract
One of the most successful examples of artificial insemination as a conservation management tool has been the production and genetic management of over 87 black-footed ferrets and selected re-introduction into their previous home range (Howard et al., 2001). While AI has the capability to supplement inbred populations, prevent disease transmission associated with the movement of whole animals, and overcome mate incompatibility, little effort has been devoted to this area in Australia. AI into the urogenital sinus has been successful in the koala (Phascolarctos cinereus) with 6 live births from 11 inseminations reported (Johnson et al., 1999 abst.). Super-ovulated brush-tailed possums (Trichosurus vulpecula) and tammar wallabies (Macropus eugenii) inseminated into the uterus resulted in 1-4 cell embryos (Molinia et al., 1998a).
The tammar wallaby (M. eugenii) is one of 56 species of the family Macropodidae (Kirsch and Calaby, 1977), comprising small rat kangaroos, wallabies and large kangaroos. As in all marsupials studied, females have two separate uteri each with a separate cervix that open into the anterior vaginal culs de sac. The vaginal apparatus is made up of two lateral vaginae and a third, in macropodids permanent, median vagina (birth canal) that connects the anterior vaginal culs de sac to a urogenital sinus and opening.
Female reproduction is extremely well characterized in M. eugenii (Tyndale-Biscoe and Renfree, 1987) making it an ideal model to develop AI for application in endangered counterparts including the brush-tailed rock wallaby (Petrogale penicillata) and long-nosed potoroo (Potorous tridactylus). M. eugenii is monovular and polyoestrus, with a breeding season from mid January to mid May. They have a post-partum oestrus, and a multiple male mating strategy. Embryonic diapause is a phenomenon common to many macropodids, and occurs when a developing embryo (conceived post-partum) arrests at the unilaminar blastocyst stage in the uterus for the duration of lactation and suckling by the pouch young on the teat (Sharman, 1959; Tyndale-Biscoe et al., 1974; Renfree, 1981). If the sucking stimulus stops, the embryo reactivates and birth occurs 26.4±0.5 days later (Merchant, 1979; Renfree et al., 1989). Oestrus and mating occur 1-6h post-partum (Rudd, 1994) and ovulation from 37-43h post-partum (Renfree and Lewis, 1996). Sperm are found in the uterus 6-24h post-coitum (Tyndale-Biscoe and Rodger, 1978). Thus by removing the sucking stimulus we can naturally synchronize the birth, oestrus and ovulation in M. eugenii and introduce sperm relative to the time it is found naturally in the tract. The aim of this study was to develop artificial insemination in M. eugenii. Twenty-four adult females were isolated from males and their reproductive cycles naturally synchronized as described above. Semen was collected from adult males in Ham’s F10 +5% fetal calf serum and 50IU/mL penicillin-50±g/mL streptomycin (Life Technologies, Australia) using a 10mm diameter, 3-strip electrode rectal probe with a 20Hz sine wave electro-ejaculation unit (CGS, Australia)(Taggart et al., 1998). Sperm was assessed for motility and concentration, and inseminated within 12h of birth into 4 different regions of the female reproductive tract: (i) urogenital sinus via modified syringe (n=7) (ii) anterior vaginal culs de sac via silicon balloon HSG catheter (Cook, Australia)(n=7) (iii) uterus via a trans-cervical catheter during laparotomy (n=5) (iv) uterus directly via needle during laparotomy (n=5) Animals were checked for births from 26 days after AI.
Sperm inseminated had a mean (±SEM) sperm motility index of 245.7±14.9 (0-400 scale), motility of 93.1±0.9%, and concentration of 16.3±2.0x106 sperm/mL across all animals treated. One of the five females (♀0651), inseminated by direct intra-uterine artificial insemination, gave birth (4.2% success across all animals inseminated). This is the first macropodid offspring produced by AI. ANOVA analysis (SYSTAT 9.01) indicated that ♀0651 was an outlier with respect to a later insemination time (~16.5h post-partum), than other animals (10.6±0.7h post-partum). This may be a critical factor that resulted in success in this animal. Sperm used in this female was also of a considerably higher quality with a sperm motility index of 318.2, 96.3% motility and concentration of 10.1x106 sperm/mL.
Based on our findings a second group of females (n=7) was inseminated by intra-uterine AI using insemination times further from oestrus but closer to ovulation in the female. These ranged from approximately 15.5 to 27.5h post-partum. Inseminates had a higher mean concentration of 10.3 ± 4.4x107 sperm/mL, and a sperm motility index of 237.5±7.3 and 83.9±2.6% motility. To date, one of these animals has not been successful but the remaining 6 animals may have undergone seasonal embryonic diapause. Investigations to confirm pregnancies are currently underway.
The ultimate goal of this work is to improve the efficiency of AI in M. eugenii and work toward a non-surgical technique that can be readily adopted in endangered macropodids. We know that sperm capacitation is important in fertilization in marsupials (Mate and Rodger, 1996), and anaesthetic can negatively affect sperm transport (Wildt et al., 1987) and ovulation (Howard et al., 1992). These and other factors need to be taken into consideration to further optimize this technique.
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