Changes in the ejaculate of the male tammar wallaby (Macropus eugenii) parallel seasonal reproductive activity in the female

The reproductive tract of the male tammar wallaby increases in size and secretory activity when females are in oestrus, perhaps in response to a release of female pheromones (CATLING and SUTHERLAND 1980; INNS 1982; MCCONNELL et al. 1984). This study investigated the effect of changes in male reproductive structures on semen quality throughout the year in this highly promiscuous species. Body size, semen quality and reproductive structures were measured monthly in adult males from January to November. Although there was no significant difference in the weight of males sampled throughout the year, heavier males had heavier testes and epididymides and produced larger ejaculates. The main breeding season in late January, when all females were in synchronous oestrus, coincided with the heaviest testis, epididymis, prostate, Cowper’s glands, crus penis and urethral bulb. Sperm motility, semen volume and coagulation of the ejaculate were greatest at this time. In July, during an almost total absence of mating in the wild, all structures regressed, resulting in significantly reduced sperm number and motility in small volumes of semen that did not coagulate. During the subsidiary breeding season in October/November, when only pubertal females undergo their first oestrus, all male reproductive structures, except the prostate, returned to maximum weights and sperm number and motility were high. In contrast, prostate weight, semen volume and coagulation of the ejaculate reached intermediate levels. Semen volume was positively correlated with prostate weight while the proportion of motile sperm was positively correlated with epididymal weight. Sperm concentration was negatively correlated with semen volume, and sperm numbers remained low when coagulation of ejaculates was greatest. These results show that semen quality is influenced by the change of androgen-dependent reproductive structures in the male tammar wallaby. The male reproductive tract is apparently primed to produce high quality semen when females are in oestrus. Semen quality may therefore improve in response to the number of available oestrous females, which may in turn provide an advantage in sperm competition

Anatomical considerations for the development of artificial insemination by catheter in two marsupials

We recently reported the birth of a tammar wallaby produced by intrauterine AI [Paris DBBP et al., Australian Mammal Society Conference, July 2002]. The use of AI has great potential for the conservation of rare and endangered marsupials, but a non-surgical method of insemination is needed if such a technique is to be widely adopted. In this study we compared the anatomy of the reproductive tract of two monovular marsupials: the tammar wallaby (Macropus eugenii, family Macropodidae) and southern hairy-nosed wombat (Lasiorhinus latifrons, family Vombatidae), in relation to the ability to deposit semen non-surgically by catheter. These species are ideal models for endangered marsupials such as the brush-tailed rock wallaby (Petrogale penicillata ), long-nosed potoroo (Potorous tridactylus) and critically endangered northern hairy-nosed wombat (Lasiorhi-nus krefftii). As in all marsupials studied, tammars and wombats have two completely separate uteri each opening into the anterior vaginal culs desac (AVC) through two distinct cervices. The vaginal canals, unique and highly variable in marsupials, consist of two lateral vaginae (LV) and a third median vagina (MV) connecting the AVC to the urogenital sinus and opening. Adult female reproductive tracts were examined from 13 tammars and 5 wombats. A silicon balloon HSG catheter (5 French 30 cm; Cook, Australia) was introduced at the urogenital opening and navigated through the MV to the AVC, proximal to the cervices. The balloon was inflated to seal off the posterior end of the MV. Two milliliter (tammar) or 1 ml (wombat) of 0.1% Coomassie brilliant blue (Sigma, Australia) was injected into the AVC to stain and simulate the site of insemination. The balloon was defeated, catheter retracted and the extent of dye distribution in the tracts noted. In the tammar, as in all macropodids, the MV is a single open canal, allowing both cervices to draw semen. However, a septum divides the MV in wombats and interestingly, the MV was partially fused at the caudal end. In the tammar, Coomassie blue penetrated the MV, the AVC and cervices. These lie a distance of 7:6 +/-0:3cm from the urogenital opening (see table). In the wombat, the MV was fused caudally and so was difficult to penetrate. The cervices lay 13:3 +/- 0:3 cm from the urogenital opening. Coomassie blue was localized to only one side of the MVand AVC, confirming that the medium septum effectively separates the cervices. These results have important implications for AI using non-surgical catheters for semen deposition. In the tammar wallaby and potentially other macropodids, cervical insemination via the MV is feasible, requiring no ultrasoni cguidan ce. However in the southern hairy-nosed wombat and other vombatids, cervical insemination is unlikely to be successful due to the fused MV. Presence of the septum would require the side of ovulation and thus catheter delivery to be determined by ultrasound

Distribution of spermatozoa and copulatory plug in relation to the time of mating and ovulation in the female tammar wallaby (Macropus eugenii)

In the monovular macropodid marsupial, the tammar wallaby (Macropus eugenii), the cervices are the primary selective barrier to spermatozoa, resulting in differential transport to the non-gravid uterus where a sperm reservoir is established (Tyndale-Biscoe CH and Rodger JC 1978 J. Reprod. Fertil. 52, 37–43). However, due to limited sample size, the dynamics of sperm transport could not be thoroughly examined. In this study, the distribution of spermatozoa, the size of the copulatory plug in the reproductive tract at various times after mating, and the timing of ovulation were characterized in 28 naturally mated female tammars. After the first postpartum (p.p.) mating, adult females were isolated and their reproductive tracts dissected at 0.5, 6, 18, 36, and 40 h post-coitum (p.c.). Each tract was ligated into 13 major anatomical sections, and spermatozoa and eggs were recovered by flushing. Mating occurred 21.7 ± 2.5 h p.p. (mean ± SEM; n = 20) in these animals that were checked frequently and lasted 7.8 ± 0.7 min (n = 15). Within 0.5 h after a single mating (n = 5) the tract contained 2.6 ± 1.0 × 107 spermatozoa and 21.6 ± 8.8 g of copulatory plug, 96 and 70% of which was lost within 6 h p.c., respectively. Spermatozoa reached the uterus, isthmus, and ampulla of the oviduct ipsilateral to the developing follicle within 0.5, 6, and 18 h p.c. respectively, and a uterine population of 2.6 ± 1.2 × 104 spermatozoa (n = 24) was maintained for over 40 h (ANOVA, P > 0.05). Sperm numbers were reduced at the cervix (up to 57-fold) and utero-tubule junction (8-fold), and only 1 in ∼7600 ejaculated spermatozoa (3.4 ± 0.9 × 103; n = 14) reached the oviduct on the side of ovulation. Although sperm numbers were reduced in the gravid uterus (n = 24), differential transport of spermatozoa was not observed (ANOVA, P > 0.05). Ovulation and recovery of sperm-covered eggs from the isthmus of the oviduct occurred 36–41 h p.c. (49–72 h p.p.) (n = 8). Like many eutherian mammals, but in contrast to polyovular dasyurid and didelphid marsupials, the tammar ejaculates large numbers of spermatozoa, but transport is relatively inefficient and sperm storage in the tract before ovulation is limited

Embryos and embryonic stem cells from the white rhinoceros

The northern white rhinoceros (NWR, Ceratotherium simum cottoni) is the most endangered mammal in the world with only two females surviving. Here we adapt existing assisted reproduction techniques (ART) to fertilize Southern White Rhinoceros (SWR) oocytes with NWR spermatozoa. We show that rhinoceros oocytes can be repeatedly recovered from live SWR females by transrectal ovum pick-up, matured, fertilized by intracytoplasmic sperm injection and developed to the blastocyst stage in vitro. Next, we generate hybrid rhinoceros embryos in vitro using gametes of NWR and SWR. We also establish embryonic stem cell lines from the SWR blastocysts. Blastocysts are cryopreserved for later embryo transfer. Our results indicate that ART could be a viable strategy to rescue genes from the iconic, almost extinct, northern white rhinoceros and may also have broader impact if applied with similar success to other endangered large mammalian species

Marsupials: Alternative mammals

Marsupials have come to be regarded as second class mammals, partly because of their unusual mode of reproduction, and partly because they were discovered long after the eutherians (or 'placental' mammals) and were all lumped into group, the Order Marsupialia..

Effect of steroids on thyroid activity and adrenal morphology in tammar wallabies after removal of the corpus luteum

1. 1. Plasma total thyroxine (TT4) levels and plasma free thyroxine (FT4) index were significantly lower in adult tammar wallabies (Macropus eugenii) from which the corpus luteum had been removed than in sham-operated controls. 2. 2. Progesterone injections given for 14 days after corpus lutectomy significantly elevated the plasma free tri-iodothyronine (FT3) index, but had no effect on other thyroid parameters measured. 3. 3. Estrogen and androstenedione given for 14 days after corpus lutectomy had no significant effect on any of the thyroid parameters measured, although in both groups injected with these steroids, the histological appearance of the thyroid was suggestive of an increased activity. 4. 4. The adrenal weight, adrenal somatic index, adrenal cortex area, zona fasciculata width, and zona reticularis width were significantly larger in estrogen-injected corpus lutectomized wallabies than in oil-injected corpus lutectomized control

Uterine and embryonic metabolism after diapause in the tammar wallaby, Macropus eugenii

Pouch young were removed from lactating tammars to terminate embryonic diapause. Uterine metabolism was assessed at periods afterwards by incubating endometrial explants with [3H]leucine, and measuring the incorporation into acid-soluble material. Blastocysts were incubated with [3H]uridine to assess uptake and incorporation into acid-soluble material. Uterine reactivation, shown by an increase in the rate of leucine incorporation into secreted protein, was evident by Day 4 after removal of pouch young and was significantly more in both secreted and tissue protein by Day 6. Both continued to increase in gravid and non-gravid uteri up to Day 12. By the end of pregnancy (Day 26) uterine metabolism in the gravid uterus produced 2-3 times more secreted protein than in the non-gravid uterus, demonstrating a local feto-placental influence on the uterus. Tissue incorporation had declined in endometrium of gravid and non-gravid uteri by Day 26. Day 5 embryos were metabolically more active than in quiescence, although expansion of the embryos was not seen until Day 9. The early reactivation of the uterus and embryo from diapause suggests that it is not triggered by the previously described peaks of progesterone and oestradiol in plasma at Day 5, although there may be an earlier, increased sensitivity to these steroids which allows uterine reactivation to precede changes in peripheral plasma concentration

Effects of progesterone on parturition in the tammar, Macropus eugenii

Summary. Tammar wallabies were treated with progesterone injections or implants during late pregnancy to determine whether progesterone withdrawal was essential for parturition. Neither physiological (implanted group) nor pharmacological (injected group) levels of circulating progesterone prevented parturition occurring at about the expected time in about two-thirds of animals that were pregnant. The neonates of both groups were normal in size and weight, but about a third of treated pregnant animals retained their fetuses or aborted. The retained fetuses were retarded in development. Therefore, progesterone treatment had no influence on the duration of gestation, or parturition, in the tammar wallaby, but high progesterone concentrations may interfere with the normal course of development and birth in a proportion of treated animals

Testicular development and maturation of the hypothalamic-pituitary-testicular axis in the male tammar, Macropus eugenii

Testicular growth and maturation of the hypothalamic–pituitary–testicular axis were assessed in male tammars from 12 to 25 months of age to establish the time of sexual maturity. The testicular dimensions and body weights of 20 male tammars, ∼12 months of age at the beginning of the study, were measured monthly for 1 year. Groups of 3 animals were castrated at 13, 19 and 25 months of age and their testes sectioned for histological examination. Testicular volume increased between 12 and 24 months of age and was highly correlated with body weight (r = 0·91). In the 13-month group the seminiferous tubules were closed with few mitotic figures. Spermatogenesis had begun in 2 of the 19-month animals. All stages of spermatogenesis were present in the other 19-month male, and in all of the 25-month males. Basal FSH concentrations increased with the age of the animal (21·0 ± 32·48, 94·40 ± 55·18 and 193·05 ± 40·21 ng/ml (mean ± s.d.) at 19, 20 and 25 months respectively) while basal LH concentrations were similar at 20 months and 25 months (0·43 ± 0·18 and 0·58 ± 0·25 ng/ml respectively). Basal testosterone concentrations were also similar 0·11 ± 0·04, 0·35 ± 0·16 and 0·22 ± 0·10 ng/ml in 13-, 19- and 25-month-old animals. LHRH injection in tammars at 13, 19 and 25 months of age induced release of both LH and testosterone 10#x2013;30 min after injection. The hormone concentrations increased in both magnitude and duration with increasing age. At 13, 19 and 25 months, peak testosterone values of 1·13 ± 0·27, 1·90 ± 0·45 and 6·58 ± 0·91 ng/ml (mean ± s.d.), respectively, were measured by 90–120 min after injection and peak LH values at 13, 19, 20 and 25 months of age were 2·46 ± 0·82, 8·31 ± 2·02, 9·0 ± 2·18 and 7·86 ± 1·41 ng/ml, respectively, when measured 30 min after injection. By contrast, LHRH injection did not increase FSH concentration at 20 or 25 months of age above the basal concentration. Taken together, the morphological and endocrinological data show that puberty in male tammars begins around 19 months and is complete by 25 months of age