Mapping the follicle-specific regulation of extracellular vesicle-mediated microRNA transport in the southern white rhinoceros (Ceratotherium simum simum)†.

Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the northern white rhinoceros (NWR; Ceratotherium simum cottoni) to prevent its forthcoming extinction, could be supported by research conducted on the closely related southern white rhinoceros (SWR; Ceratotherium simum simum). Within the follicle, extracellular vesicles (EVs) play a fundamental role in the bidirectional communication facilitating the crucial transport of regulatory molecules such as microRNAs (miRNAs) that control follicular growth and oocyte development. This study aimed to elucidate the dynamics of EV-miRNAs in stage-dependent follicular fluid (FF) during SWR ovarian antral follicle development. Three distinct follicular stages were identified based on diameter: Growing (G; 11-17 mm), Dominant (D; 18-29 mm), and Pre-ovulatory (P; 30-34 mm). Isolated EVs from the aspirated FF of segmented follicle stages were used to identify EV-miRNAs previously known via subsequent annotation to all equine (Equus caballus; eca), bovine (Bos taurus; bta), and human (Homo sapiens; hsa) miRNAs. A total of 417 miRNAs were detected, with 231 being mutually expressed across all three stages, including eca-miR-148a and bta-miR-451 as the top highly expressed miRNAs. Distinct expression dynamics in miRNA abundance were observed across the three follicular stages, including 31 differentially expressed miRNAs that target various pathways related to follicular growth and development, with 13 miRNAs commonly appearing amidst two different comparisons. In conclusion, this pioneering study provides a comprehensive understanding of the stage-specific expression dynamics of FF EV-miRNAs in the SWR. These findings provide insights that may lead to novel approaches in enhancing ARTs to catalyze rhinoceros conservation efforts.

Effect of four eggshell repair materials on weight loss during incubation of white leghorn chicken eggs.

Egg weight loss during incubation is a key indicator used to monitor successful egg development and is closely related to hatchability and chick survival. Artificial incubation is one of the most important captive breeding techniques used in conservation efforts to bolster avian populations. To repair damage to the eggshell and ensure embryonic viability during incubation, a variety of repair coverings can be applied. This study tested the impact of four repair materials (nail polish, synthetic glue, medical dressing, and molten wax film) on egg weight loss during incubation. We found no impact on weight loss for coverings smaller than 35% of the eggshell surface, nor did we find any differences between covering types. The average egg weight loss decreased as the coverage area increased, and the weight loss did not differ when blunt versus sharp-end coverings were compared. Given the relative insensitivity of egg weight loss and survival to the type of patch material used, we concluded that the selection of material for the purpose of weight loss management could be based on practical considerations, such as ease of application and availability.

Sperm cryopreservation in the Burmese python Python bivittatus as a model for endangered snakes.

Burmese pythons Python bivittatus captured in the Florida Everglades as part of an invasive species monitoring program served as a model for the development of sperm cryopreservation protocols for endangered snakes. Spermatozoa were collected from the vas deferens and initial motility, plasma membrane integrity and acrosome integrity were recorded before cryopreservation. Spermatozoa were extended in TES and Tris (TEST) yolk buffer with glycerol (GLY) or dimethyl sulfoxide (DMSO) concentrations of 8%, 12% or 16%, or combinations of GLY and DMSO with final concentrations of 4%:4%, 6%:6% or 8%:8%, and frozen at a rate of 0.3°C min-1 . Sperm frozen in combinations of GLY and DMSO exhibited greater post-thaw motility and plasma membrane integrity than those frozen in GLY or DMSO alone. All DMSO and GLY:DMSO treatments preserved a greater proportion of intact acrosomes than GLY alone. To determine the best overall cryopreservation protocol for this species, a sperm quality index was calculated, giving equal weight to each of the three measured indicators of cryosurvival. This analysis revealed that Burmese python spermatozoa frozen in 6% GLY:6% DMSO or 4% GLY:4% DMSO exhibited the highest post-thaw viability. This study represents the first comparative, comprehensive attempt to develop a sperm cryopreservation protocol for any snake species.

Challenges in the development of sperm cryopreservation protocols for snakes.

Snake populations are declining worldwide, but research devoted to the development of sperm cryopreservation techniques for this taxon is very limited. Spermatozoa were collected postmortem from snakes of four squamate families (Elapidae, Colubridae, Viperidae and Pythonidae). Viability assessment was performed before and after cryopreservation. Spermatozoa were extended in TES and Tris (TEST) yolk buffer with 12% dimethylsulfoxide (DMSO) or 12% glycerol and frozen at a rate of 0.3°Cmin-1. The sperm quality index (SQI), representing three viability parameters (motility, plasma membrane and acrosome integrity), was determined. Despite some species differences, glycerol was a more effective cryoprotectant for Colubridae, whereas DMSO provided greater cryoprotection for spermatozoa of members of the other three families.

Using reproductive technologies to assess the development of secondary sexual characteristics, ovarian senescence and hermaphroditism in the endangered mountain yellow-legged frog Rana muscosa.

Anurans can display a host of intriguing sexual syndromes, including hermaphroditism and sex reversal. Using a multifaceted approach for diagnosing and characterising hermaphroditism in the endangered anuran species Rana mucosa, we tracked changes in female reproductive status using hormone monitoring, ultrasound examinations, individual life history, fertilisation records and post-mortem findings. Seven individuals originally sexed as females developed secondary male sexual characteristics, behaviour and hormone profiles and, in some cases, had testicular tissue despite having previously laid eggs. Our results suggest that reproductive technologies can shed light on life history patterns and reproductive anomalies that may affect endangered anuran survival.

Pregnancies following long luteal phases in southern white rhinoceros (Ceratotherium simum simum).

All extant species in the Rhinocerotidae family are experiencing escalating threats in the wild, making self-sustaining captive populations essential genetic reservoirs for species survival. Assisted reproductive technologies (ARTs) will become increasingly important for achieving and maintaining ex situ population sustainability and genetic diversity. Previous reports have shown that a large proportion of captive southern white rhinoceros (SWR) females are irregularly cyclic or acyclic, and that cycling females display two different estrous cycle lengths of approximately 30 or 70 days. It has been suggested that the longer estrous cycle length is infertile or subfertile, as no term pregnancies have been observed following long cycles. Here we report the achievement of two pregnancies following long luteal phases, using ovulation induction and artificial insemination with either fresh or frozen-thawed semen. One female SWR conceived on the first insemination attempt and gave birth to a live offspring. A second female conceived twice in consecutive long cycles although the first embryo was resorbed by 33 days post-insemination. A pregnancy from this female's second insemination is ongoing with expected parturition in November 2019. Whether prolonged estrous cycles in SWR are subfertile or infertile in natural breeding situations remains unclear. However, our findings demonstrate that the application of ARTs following prolonged cycles can result the successful establishment of pregnancies in SWR. Therefore, with ARTs, female SWR otherwise considered nonreproductive due to long estrous cycles may still have the potential for representation and contribution to the ex situ population.

Serum prolactin and testosterone levels in captive and wild brown kiwi (Apteryx mantelli) during the prebreeding, breeding, and incubation periods.

In brown kiwi (Apteryx mantelli), the male is the primary incubator, a trait that is relatively rare among birds. The maintenance of avian incubation behavior is controlled by the protein hormone prolactin (PRL). Although steroid hormone concentrations in both wild and captive kiwi have previously been reported, this study is the first to report levels of PRL in captive and wild male and female kiwi through the prebreeding and breeding seasons, and to directly compare testosterone (T) concentrations between captive and wild males during the breeding and incubation periods. Female PRL concentrations increased at the time of oviposition, whereas male PRL concentrations rose gradually between the prebreeding and incubation periods. Although males are considered the main incubator, an increase in PRL levels could help females maintain behaviors such as nest guarding, or to take over incubation the event of mate loss. A gradual increase in PRL allows the male to be ready for incubation during the long breeding season. Interestingly, T concentrations in captive males did not decrease during incubation and was significantly higher than in wild males. Continual elevated T could have an impact on sperm production through negative feedback, thereby contributing to the low egg fertility seen in captive kiwi. Therefore, determining the underlying reason for the differences in hormone levels could be significant, if not vital, for improving the success of captive kiwi breeding programs.

VAGINOSCOPIC IDENTIFICATION OF A VERTICAL VAGINAL SEPTUM IN ONE PRIMIPAROUS AND THREE NULLIPAROUS SOUTHERN WHITE RHINOCEROS (CERATOTHERIUM SIMUM SIMUM).

Vaginoscopy using a 10-mm, 30° forward viewing rigid endoscope was used to evaluate the caudal reproductive tract of four subadult southern white rhinoceros (Ceratotherium simum simum). A vertical vaginal septum was documented in all four animals, including a primiparous cow that gave birth to a stillborn calf 14 months before vaginoscopy. Vaginoscopy using a 57-cm-long, 10-mm, 30° forward viewing endoscope provides adequate visualization of the caudal reproductive track in the southern white rhinoceros, and a detailed description of the vertical vaginal septum is presented. Additionally, the presence of a vertical vaginal septum in a primiparous southern white rhinoceros suggests the presence of this anatomic structure cannot be used as a proxy of nulliparity for captive southern white rhinoceros.

Approaches to studying behavior in captive sloth bears through animal keeper feedback.

Animal keepers at zoos and wildlife rescue centers often possess in-depth knowledge of the health and behavior of the individuals under their care. While it is often not feasible for keepers to regularly collect behavior data through formal scientific methods, efforts should be made to find alternative means to capture this knowledge. We investigated the use of keeper feedback to study the behavior of sloth bears at the Agra Bear Rescue Facility (ABRF; Agra, India). We prepared a survey with 5 questions focused on behaviors indicative of playfulness, boldness, aggressiveness, and the tendency to express self-directed behaviors (SDB). We asked keepers to rate bears on a Likert scale from 1 (least likely to exhibit a behavior) to 5 (most likely) for 44 adult female bears (5-21 years of age). We validated this method by comparing keeper ratings of SDB with formal behavior observations, finding that time of day had an influence on the accuracy of keeper assessments. We found a significant negative correlation between housing bears in larger groups (>15) and SDB. In addition, we correlated ratings given by keepers for all study behaviors. Social play had significant negative correlation with aggression toward people. There was no correlation between social play and aggression toward other bears, possibly due to the existence of cohesive social groups in group housing or high dimensionality of the data. We found that keeper feedback is an efficient tool to gather behavior data on captive sloth bears and recommend its use in future studies.

Estrogenicity of captive southern white rhinoceros diets and their association with fertility.

The captive southern white rhinoceros (SWR) population is not currently self-sustaining, primarily due to poor or absent reproduction of captive-born (F1+) females. In this study, we investigate the role of dietary phytoestrogens in this reproductive phenomenon by characterizing activation of SWR estrogen receptors (ESRs) 1 and 2 by diet items from nine North American institutions and comparing female SWR fertility to total diet estrogenicity. Of the diet items tested, alfalfa hay and soy and alfalfa-based commercial pellets were found to be the most potent activators of SWR ESRs. In contrast, most grass hays tested were not estrogenic. The estrogenicity of total diets varied across the institutions surveyed and the degree of diet estrogenicity was positively associated with the percentage of the total diet comprised by pellets. Comparisons of fertility records of the institutions surveyed showed no significant relationship between diet estrogenicity and fertility for female SWR conceived or born in the wild (F0). However, for F1+ females, there was a significant negative relationship between institutional diet estrogenicity and fertility. Taken together, these data suggest that developmental exposure to phytoestrogens may be the cause of poor fertility in captive-born female SWR. Whether the low fertility of the current population of captive-born female SWR is permanent or can be reversed by removing phytoestrogens from the diet remains unclear. However, our findings suggest that in order for the SWR population to become self-sustaining, the development and feeding of low phytoestrogen diets should be strongly considered.