Reproductive anatomy of the female grey short-tailed opossum (Monodelphis domestica): considerations for developing artificial insemination by catheter

Due to the variability in vaginal anatomy among marsupials, the female reproductive tract must be examined for each species prior to developing artificial insemination using catheters. The grey short-tailed opossum (Monodelphis domestica) is a nonseasonal, polyovular didelphid and, as in all marsupials studied, has two completely separate uteri each opening into an anterior vaginal expansion through two distinct cervices. In the opossum, however, a septum divides the anterior vaginal expansion into two vaginal culs-de-sac, two lateral vaginae connect the anterior vaginal culs-de-sac to the urogenital sinus and opening, and the median vagina is a transient pseudo-vaginal birth canal. In this study we examined the site for insertion of artificial insemination catheters and the distribution of fluids inseminated within the vaginal complex of the opossum. Reproductive tracts were examined from four similarly sized unpaired adult one- to two-year-old females. A 2.3 mm diameter silicon balloon HSG catheter or a 0.8 mm diameter malleable obturator (Cook Australia, Brisbane, Australia) was introduced at the urogenital opening and navigated through the urogenital sinus, lateral vaginae, and cervices and the depth of insertion noted. Second, 0.15 mL of 1% Methylene blue dye (Sigma, Poole, UK) was injected by HSG catheter into the anterior urogenital sinus and the extent of dye distribution in the tract noted. In all tracts, the HSG catheter easily reached the anterior urogenital sinus (1.5 ± 0.3 cm; mean ± SEM), but would not pass into the lateral vaginae or bladder. The malleable obturator easily passed into the bladder or through each lateral vagina into the anterior vaginal cul-de-sac (2.9 ± 0.2 cm). The obturator also passed through the each cervix into the opening of the uterus (3.1 ± 0.3 cm), but the tract required manipulation to navigate it through the convoluted lateral vaginae. Dye injected into the anterior urogenital sinus was distributed throughout the lateral vaginae and anterior vaginal culs-de-sac. No dye was detected in the bladder or had passed through the cervices into the uteri. In two of three tracts studied, dye was observed to migrate between each anterior vaginal cul-de-sac across the posterior end of the median septum that divides them. In conclusion, the use of larger diameter catheters (∼2.3 mm) is recommended for unguided insemination of the anterior urogenital sinus in this species. Catheters less than 1 mm diameter require ultrasonic or endoscopic guidance to reach the uteri in order to avoid insemination into the bladder. Dye studies indicate that the median septum may not completely separate the cervices in the opossum, suggesting that inseminates delivered to one side of the tract may be able to fertilize oocytes on the contralateral side. In addition, the cervices appear to restrict inseminates from entering the uteri. Spermatozoa may thus be required to pass through the cervices under their own motility

In vitro cell culture from the Black Rhinoceros (Diceros bicornis minor): a novel approach to genetic conservation

The Black Rhinoceros (Diceros bicornis) is critically endangered primarily due to poaching for horns. While access to animals and success in gamete collection and assisted breeding is lim­ited at this stage, opportunities for gene banking in cases of routine veterinary procedures, fieldwork and animal death are being lost. In this study, we investigated the feasibility of in vitro cell culture as an alternative strategy to genetically preserve the gene pool of this species in light of emerging reproductive technologies such as somatic cell cloning and cross species nuclear transfer. Three as­pects were assessed: 1) time of initiation and rate of growth of primary cell cultures, 2) quantitative harvest at early passages (P), and 3) maintenance of health and vigor in vitro. Three l .5cm3 skin biopsies were taken from a 6½-year-old male Southern Black'Rhinoceros (Diceros bicornis minor), divided into epidermis (skin) and dermis (under layer), then mechanically minced. Subsequently, selected groups were enzymatically digested with 200U/ml crude collagenase type IA (Sigma, Aus­tralia) or 0.25% trypsin/lmM EDTA (Life Technologies, Australia) over 4h, 16h or 24h, to produce primary cultures of fibroblast cells. Fibroblasts were grown until confluency whereafter, most cells were cryopreserved for future use in 90% Foetal Bovine Serum (FBS) + 10% DMSO at -I °C/min until -80°C and then stored. Remaining cells were seeded at 104cells/ml and growth rates (% con­fluence, % viability, viable cell concentration and total viable cell yield) were monitored for up to 15 passages. Common Wombat (Vombatus ursinus) fibroblasts, routinely grown in our laboratory (Wolvekamp MCJ et al., Theriogenology 2000;53:345 abst), were cultured in parallel as control. Data were tested for significance using the appropriate t-Test with Microsoft Excel 97 software. Time of initiation and rate of growth of primary rhino cell cultures varied with tissue type, digestion time and type of digestion enzyme used. No difference in tissue type was observed after 4h collage­nase digestion. However, epidermis was more sensitive than dermis after 16h collagenase digestion, having a significantly later initiation and near-significantly slower growth rate. Digestion time had no effect on dermis, but 16h collagenase digestion was harsher than 4h on epidermis having a signif­icantly later initiation. Trypsin was harsher than collagenase after 24h digestion on dermis having significantly slower growth rate and later initiation. Early passage rhino cells (PO and Pl) had an average (±SEM) of 89.1 ± 1.5% confluence, 94.1±0.4% viability, 103.9±4.2x 104 viable cells/ml and 2.1±0.lxl06 total viable cell yield. Rhino fibroblasts maintained their % confluence and % viability for the whole 15 passages up to the termination of the experiment. However, viable cell concentration and total viable cell yield decreased significantly from Pl (142.0±46.0xl04cells/ml and 2.8±0.9xl06cells resp.) to P3 (22.8±7.4xl04cells/ml and 0.5±0.2xl06cells resp.), and never recovered, remaining only 10-20 fold higher than seeded amounts. In summary, digestion using collagenase (using short times for epidermis) is the best method to establish rhinoceros primary cell cultures. Also, rapidly increasing fibroblast cell numbers should be cryopreserved at early pas­sage number before they significantly decline. These results demonstrate a successful protocol in the black rhinoceros for the establishment of primary fibroblast cell cultures that may be applicable to other rhinoceros species

Developmental changes in expression of pluripotent genes in early equine embryos.

Genes involved in maintaining pluripotency have potential use in establishing cell lines for regenerative medicine. However, the genes differ subtly between species, and are poorly described in the horse. In this study we examined changes in expression of pluripotency-associated genes in horse embryos during blastocyst formation. Twenty-one grade 1–2 embryos where recovered from mares by uterine lavage on Day 6–7 after ovulation. Embryos were classified by developmental stage (morula, early or expanded blastocyst: n = 5, 7 and 9, respectively) and their diameter measured by micrometer, before being snap frozen. Subsequently, mRNA from individual embryos was extracted, DNAse-treated and synthesized into cDNA using an AllPrep Mini Kit and Superscript III Reverse Transcriptase (Qiagen, Venlo, and Invitrogen, Breda respectively, the Netherlands). Equine-specific intron-spanning/overlapping primers were designed using PerlPrimer v1.1.14 by BLAST searching the NCBI horse genome for 5 genes associated with pluripotency in other species (octamer binding protein OCT4, transcription factor NANOG, developmental pluripotency-associated DPPA4, growth and differentiation factor GDF3 and telomerase reverse transcriptase TERT) and 2 reference genes (signal recognition particle SRP14 and phosphoglycerate kinase PGK1). Relative gene expression was then examined by quantitative PCR using an iQ5 RT PCR Detection System (BioRad, Veenendaal, the Netherlands). Relationships were tested by Pearson correlations and differences between developmental stages were tested by ANCOVA. Embryos ranged in diameter from 126 to 680 μm. As expected, absolute expression of all pluripotency markers increased with increasing embryo diameter (P = 0.000; R = 0.93, 0.92, 0.88, 0.86 and 0.76 for NANOG, DPPA4, GDF3, OCT4 and TERT, respectively). After normalization with SRP14 and PGK1, significant negative correlations with embryo diameter were apparent for OCT4, NANOG and DPPA4 (P < 0.001; R = –0.73, –0.69 and –0.53, respectively). Moreover, all 5 pluripotency genes were down-regulated as embryonic development progressed (P < 0.05), although the time-course differed between genes. The DPPA4 and OCT4 expression decreased significantly at both the morula-early blastocyst and early-expanded blastocyst transitions, whereas NANOG expression only decreased significantly between the early-expanded blastocyst stages and GDF3 and TERT expression only between the morula-early blastocyst stages. Down-regulation of pluripotent gene expression during early development is consistent with increased cohorts of cells differentiating into trophectoderm and primitive endoderm, leaving an ever decreasing proportion of pluripotent cells in the inner cell mass. Furthermore, the different time courses of down-regulation may reflect different roles of the examined genes in developmental processes. For example, early and continued down-regulation of OCT4 is consistent with a role in differentiation between ICM and trophectoderm, whereas the slightly later onset of NANOG down-regulation better fits a role in determining between pluripotent epiblast and primitive endoderm, as described in other species

Tropical summer induces sperm DNA damage in boars which can be mitigated by antioxidant therapy

Despite pork being the most widely consumed meat in the world, summer infertility due to heat stress significantly reduces pig production and can potentially limit sustainable pork supply particularly in tropical regions. The boar's characteristic non-pendulous scrotum, inefficient capacity to sweat and the high susceptibility of boar sperm to temperature shock makes this species particularly vulnerable to heat stress. While traditionally considered a sow problem, recent studies demonstrate that DNA damage in sperm caused by heat stress can result in early embryo loss in mice. The aim of this study was to determine whether sperm DNA damage is higher during summer in boars and evaluate antioxidant supplementation to alleviate the problem

Antioxidant supplementation alleviates DNA damage in boar sperm induced by tropical heat stress

Seasonal heat stress is known to significantly diminish reproductive performance in pigs, particularly in the tropics, costing the industry millions in annual loses. The boar’s reduced capacity to sweat, non-pendulous scrotum, and widespread use of European breeds in the tropics, makes this species particularly vulnerable to heat stress. While traditionally considered a sow problem, recent mouse studies demonstrate that heat stress-induced sperm DNA damage can result in arrested development and loss of early embryos. Our study investigated the impact of tropical summer heat stress on the quality and DNA integrity of boar sperm, and trialled antioxidant supplementation to alleviate the problem. Data, expressed as mean ± SEM, were analysed by one-way repeated measures ANOVA with pairwise Bonferroni tests. Motility of sperm obtained from n = 5 Large White boars housed in the dry tropics of Townsville, North Queensland, Australia was characterized by Computer-Assisted Sperm Analysis but did not differ between summer, winter or spring (total motility: 71.3 ± 8.1 vs. 90.2 ± 4.2 vs. 70.8 ± 5.5% respectively, P > 0.05; progressive motility: 35.4 ± 7.0 vs. 46.6 ± 4.0 vs. 41.7 ± 2.8% respectively, P > 0.05). Sperm DNA integrity in 20,000 sperm/boar/season, evaluated using TUNEL and flow cytometry, revealed 16-fold more DNA damaged sperm in summer than winter, and nearly 9-fold more than spring (16.1 ± 4.8 vs. 1.0 ± 0.2 vs. 1.9 ± 0.5% respectively, P ≤ 0.05). However, boar feed supplemented with 100g/boar/day of proprietary custom-made antioxidants during summer significantly reduced sperm DNA damage to 9.9 ± 4.5% and 7.2 ± 1.6% (P ≤ 0.05) after 42 and 84 days treatment respectively. Total and progressive motility were not altered by the supplement. In summary, sperm DNA integrity is compromised in boars during summer, suggesting boar factors may contribute to seasonal embryo loss in sows. Moreover, such damage appears undetectable using traditional measures of sperm motility. Antioxidant supplementation during summer appears to mitigate the negative impact of heat stress on sperm DNA integrity

Successful cryopreservation of African wild dog (Lycaon pictus) spermatozoa: towards developing the frozen zoo

Sperm freezing and artificial insemination can aid species management and conservation of the African wild dog (Lycaon pictus). Freezing attempts have previously been unsuccessful with sperm motility dropping to nearly 0% within 2 h of thawing. We examined the quality of wild dog spermatozoa subjected to 2 routine canine cryopreservation protocols: 1) 1-step dilution in TRIS-egg yolk extender containing 8% glycerol and 20% egg yolk; and 2) 2-step dilution in TRIS-egg yolk extender to a final concentration of 5% glycerol, 20% egg yolk and 0.5% Equex STM. Protocol 2 showed a significantly higher post-thaw viability, acrosome integrity and longevity of spermatozoa with motility present for up to 8 h after thawing; making it suitable for sperm banking and downstream use in species management by artificial insemination for the first time

Migration efficiency of paired sperm in the tract of the peri-ovulatory female grey short-tailed opossum (Monodelphis domestica)

American marsupials are the only mammals known to ejaculate paired spermatozoa, which confer a motility advantage in vitro over single spermatozoa in viscous environments. In the only American marsupial examined, the Virginian opossum (Didelphidae), relatively low numbers of spermatozoa are ejaculated (106), but transport is extremely efficient with ~1 in 20 spermatozoa reaching the site of fertilization compared to ~1 in 10,000 in the rabbit. This study examined the post-copulatory distribution and state (paired or single) of spermatozoa at various times in the female reproductive tract of another didelphid, the polyovular grey short-tailed opossum (Monodelphis domestica). After a single mating, the reproductive tracts of 19 females were dissected at 0.5 (n=4), 6 (n=4), 12 (n=3), 18 (n=3) and 24h (n=5) post coitum (p.c.). Each tract was dissected into 8 major anatomical sections and spermatozoa were recovered by flushing. Mating occurred 5.4 ± 0.4d (mean ± SEM; n=19) after pairing, copulation lasted 4.4 ± 0.2 min (n=18) and ovulation occurred 18.0 - 24.1h p.c. (n=5). Shortly after mating (0.5h p.c.) the tract contained 1.2 ± 0.2g of seminal gel (n=2) and 2.0 ± 1.3 x106 spermatozoa (n=3; 38% of which were paired) found predominantly in the anterior vaginal culs de sac. A uterine sperm reservoir was never observed, but spermatozoa reached the isthmus and ampulla within 6 and 18h p.c. respectively. Paired spermatozoa localized almost exclusively in the isthmus from 6h p.c., and pairing decreased to only 4% of the total sperm population in the tract by the start of ovulation. In total ~1 in 300 ejaculated spermatozoa (~6.5 x103; n=9) reached the oviduct. In conclusion, sperm pairing appears to confer effective colonization of the isthmus in M. domestica and, like the Virginian opossum, transport of spermatozoa is relatively efficient

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

Social dominance does not affect semen quality in African wild dogs (Lycaon pictus)

Sperm banking and artificial insemination could benefit conservation of endangered African wild dogs (AWD). However, it is not clear whether their strict dominance hierarchy causes subfertility in subdominant males that typically do not breed. Our study investigated the effect of dominance on male reproductive parameters including: faecal glucocorticoids (fGCMs) and androgens (fAMs), testis and prostate volume, preputial gland size, semen collection success, and the number, motility, morphology, viability, acrosome integrity (PSA-FITC), and DNA integrity (TUNEL) of spermatozoa collected by electroejaculation. Samples were obtained from n=12 captive AWDs (4 US packs) in the pre-breeding season and n=28 captive AWDs (n=11 from 4 US packs; n=17 from 3 Namibian packs) in the breeding season. Male hierarchy was clearly determined by behavioural observations in all but 1 Namibian pack. Data were grouped by dominance status and means compared by ANOVA or t-test. P≤0.05 was significant. In the pre-breeding season, there was no significant difference in body weight, fGCMs, fAMs, or prostate and testis volume between dominance groups. Semen was successfully collected from all alphas but only half the subdominants; with urine contamination negatively associated with dominance. Sperm quality was low (17.3 ± 10.2% total motility, 12.8 ± 8.5% progressive motility, 27.4 ± 11.5 x 106 ejaculated spermatozoa, 40.6 ± 9.8% normal morphology, 63.1 ± 5.1% viability, 72.6 ± 5.2% acrosome integrity) with no difference observed in any parameter except progressive motility and normal sperm morphology; which were significantly lower in subdominants (27.7 ± 16.8% vs. 0.0 ± 0.0% and 59.8 ± 13.0% vs. 21.4 ± 5.7%). From pre-breeding to breeding season, testis and prostate volume increased significantly; particularly in beta and gamma males respectively. Prostate volume was higher in alpha than beta males (16.0 ± 6.4 cm3 vs. 5.7 ± 1.4 cm3), but testis volume, body weight, fAMs and fGCMs did not differ between dominance groups (12.0 ± 0.9 cm3, 28.5 ± 0.8 kg, 0.51 ± 0.07 µg/g dry weight - DW, 30.6 ± 2.3 ng/g DW). Semen was successfully collected from 75% of males; with reduced urine contamination. Collection success, urine contamination and preputial gland size were not associated with dominance. Sperm quality improved with significantly greater number, viability, and total motility. However, sperm quality did not differ between dominance groups (47.4 ± 6.7% total motility, 30.5 ± 5.8% progressive motility, 32.3 ± 9.2 x 106 ejaculated spermatozoa, 50.9 ± 5.2% normal morphology, 74.4 ± 4.2% viability, 85.6 ± 3.0% acrosome integrity, 99.7 ± 0.1% DNA integrity). In conclusion, subdominant males are at higher risk of urine contamination and have lower sperm motility and normal morphology when semen is collected in the pre-breeding season. However, their semen is of similar quality to dominant males in the breeding season, indicating that reproductive suppression of subdominant males is only behavioural. Thus, AWD males of all social ranks in the breeding season are suitable candidates for sperm banking

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