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-miRNA 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.

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte.

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) exist within the reconstructed embryo: one within the recipient ooplasm and one within the donor somatic cell. This mitochondrial heteroplasmy can lead to developmental issues in the embryo and the fetus. Handmade cloning protocols include oocyte bisection, which can be used to decrease the mtDNA copy number, reducing the degree of mitochondrial heteroplasmy in a reconstructed embryo. Centrifugation of denuded, mature bovine oocytes produced a visible mitochondria-dense fraction at one pole of the oocyte. Oocytes' zonae pellucidae were removed by exposure to a pronase solution. Bisection was performed using a microblade to remove the visible mitochondria fraction. qPCR was used to quantify the mtDNA present in DNA samples extracted from whole oocytes and bisected ooplasts, providing a comparison of mtDNA copy numbers before and after bisection. Copy numbers were calculated using cycle threshold values, a standard curve's regression line formula, and a ratio that included the respective sizes of mtDNA PCR products and genomic PCR products. One bovine oocyte had an average mtDNA copy number (± standard deviation) of 137,904 ± 94,768 (n = 38). One mitochondria-depleted ooplast had an average mtDNA copy number of 8,442 ± 13,806 (n = 33). Average mtDNA copies present in a mitochondria-rich ooplast were 79,390 ± 58,526 mtDNA copies (n = 28). The differences between these calculated averages indicate that the centrifugation and subsequent bisection can significantly decrease the mtDNA copy numbers present in the mitochondria-depleted ooplast when compared to the original oocyte (P < 0.0001, determined by one-way ANOVA). The reduction in mtDNA should decrease the degree of mitochondrial heteroplasmy in a reconstructed embryo, possibly fostering standard embryonic and fetal development. Supplementation with mitochondrial extract from the somatic donor cell may also be essential to achieve successful embryonic development.

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.

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.

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.

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.

Ovulation induction in anovulatory southern white rhinoceros (Ceratotherium simum simum) without altrenogest.

All species in the extant Rhinocerotidae family are experiencing increased threats in the wild, making captive populations essential genetic reservoirs for species survival. However, managed species face distinct challenges in captivity, resulting in populations that are not self-sustaining. Captive southern white rhinoceros (Ceratotherium simum simum) have low reproductive rates and presumed acyclicity is common among females. Although many females fail to ovulate, follicle growth may occur and ovulation can be hormonally induced. Female southern white rhino (n = 6), housed as a bachelorette group, were determined to be ovulatory (n = 1) or anovulatory (n = 5) by serial ultrasound and fecal progestagen analysis. When follicles reached pre-ovulatory size (~35 mm), females (n = 4) were induced to ovulate in 11 trials with a GnRH analog (4.5 mg, SucroMate™) via single intramuscular injection. Nine trials resulted in ovulation (81.8%), all between 36 and 48 hours post-treatment. Ovulations were confirmed by progestagen elevation above baseline coincident with visualization of a corpus luteum (CL). Luteal phases were characterized as short (<50 days) or long (≥50 days). Between short and long cycles, only the number of days of progestagen above baseline was significantly different (P < 0.05), while days with visible luteal structures was not significant (P = 0.11). Both cycle types were observed following both spontaneous and induced ovulations. Furthermore, we showed that longer cycle lengths do not necessarily indicate early pregnancy loss as none of the females were bred or inseminated during the study. While anovulation is common in the southern white rhino captive population, ovulation induction can be achieved efficiently and predictably for use in conjunction with artificial insemination or to facilitate natural breeding. This information will lead to more efficient use of assisted reproductive technologies to overcome reproductive challenges in this species and to generate genetically healthy captive populations as a hedge against extinction.

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.

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.

Gut Microbiota and Phytoestrogen-Associated Infertility in Southern White Rhinoceros.

With recent poaching of southern white rhinoceros (SWR [Ceratotherium simum simum]) reaching record levels, the need for a robust assurance population is urgent. However, the global captive SWR population is not currently self-sustaining due to the reproductive failure of captive-born females. Dietary phytoestrogens have been proposed to play a role in this phenomenon, and recent work has demonstrated a negative relationship between diet estrogenicity and fertility of captive-born female SWR. To further examine this relationship, we compared gut microbial communities, fecal phytoestrogens, and fertility of SWR to those of another rhinoceros species-the greater one-horned rhinoceros (GOHR [Rhinoceros unicornis]), which consumes a similar diet but exhibits high levels of fertility in captivity. Using 16S rRNA amplicon sequencing and mass spectrometry, we identified a species-specific fecal microbiota and three dominant fecal phytoestrogen profiles. These profiles exhibited various levels of estrogenicity when tested in an in vitro estrogen receptor activation assay for both rhinoceros species, with profiles dominated by the microbial metabolite equol stimulating the highest levels of receptor activation. Finally, we found that SWR fertility varies significantly not only with respect to phytoestrogen profile, but also with respect to the abundance of several bacterial taxa and microbially derived phytoestrogen metabolites. Taken together, these data suggest that in addition to species differences in estrogen receptor sensitivity to phytoestrogens, reproductive outcomes may be driven by the gut microbiota's transformation of dietary phytoestrogens in captive SWR females.IMPORTANCE Southern white rhinoceros (SWR) poaching has reached record levels, and captive infertility has rendered SWR assurance populations no longer self-sustaining. Previous work has identified dietary phytoestrogens as a likely cause of this problem. Here, we investigate the role of gut microbiota in this phenomenon by comparing two rhinoceros species to provide the first characterizations of gut microbiomes for any rhinoceros species. To our knowledge, our approach, combining parallel sequencing, mass spectrometry, and estrogen receptor activation assays, provides insight into the relationship between microbially mediated phytoestrogen metabolism and fertility that is novel for any vertebrate species. With this information, we plan to direct future work aimed at developing strategies to improve captive reproduction in the hope of alleviating their threat of extinction.

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.

Use of urinary 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) concentrations to diagnose pregnancy and predict parturition in the giant panda (Ailuropoda melanolecua).

Pregnancy determination is difficult in the giant panda (Ailuropoda melanolecua), representing a challenge for ex situ conservation efforts. Research in other species experiencing pseudopregnancy indicates that urinary/fecal concentrations of 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) can accurately determine pregnancy status. Our objective was to determine if urinary PGFM concentrations are associated with pregnancy status in the giant panda. Urinary PGFM concentrations were measured in female giant pandas (n = 4) throughout gestation (n = 6) and pseudopregnancy (n = 4) using a commercial enzyme immunoassay. Regardless of pregnancy status, PGFM excretion followed a predictable pattern: 1) baseline concentrations for 11-19 weeks following ovulation; 2) a modest, initial peak 14-36 days after the start of the secondary urinary progestagen rise; 3) a subsequent period of relatively low concentrations; and 4) a large, terminal peak at the end of the luteal phase. Pregnant profiles were distinguished by an earlier initial peak (P = 0.024), higher inter-peak concentrations (P < 0.001), and a larger terminal peak (P = 0.003) compared to pseudopregnancy profiles. Parturition occurred 23 to 25 days from the initial PGFM surge and within 24 hours of the start of the terminal increase. These pattern differences indicate that urinary PGFM monitoring can be used to predict pregnancy status and time parturition in the giant panda. Furthermore, this is the only species known to exhibit a significant PGFM increase during pseudopregnancy, suggesting a unique physiological mechanism for regulating the end of the luteal phase in the giant panda.

Reproductive performance parameters in a large population of game-ranched white rhinoceroses (Ceratotherium simum simum).

The population of free-roaming white rhinoceroses (Ceratotherium simum) is under serious threat. Captive breeding of this species is therefore becoming more important, but this is challenging and often not successful. Obtaining reproductive reference values is a crucial aspect of improving these breeding results. In this study performed between 2008 and 2016, reproductive performance was analysed in 1,354 animals kept in a 8000 hectares game-ranched environment. Descriptive statistics of this captive population showed an average annual herd growth (%) of 7 .0±0.1 (min -9 -max 15). Average calving rates were calculated as an annual calving rate of 20% and biennial calving rate of 37% adult females calving per year. Females had a median age of 83.2 months at first calving (IQR 72.9-110.7) and inter-calving intervals of 29.2 (IQR 24.6-34.8) months. Furthermore, translocations of animals did not interfere with reproductive success in terms of inter-calving periods or age at first calving. Multivariate models showed a clear seasonal calving pattern with a significant increase of the number of calvings during December-April when compared to April-December. Our results did not show any significant skewed progeny sex ratios. Weather observations showed no significant influence of rain or season on sex ratios of the calves.

Red light improves spermatozoa motility and does not induce oxidative DNA damage.

The ability to successfully fertilize ova relies upon the swimming ability of spermatozoa. Both in humans and in animals, sperm motility has been used as a metric for the viability of semen samples. Recently, several studies have examined the efficacy of low dosage red light exposure for cellular repair and increasing sperm motility. Of prime importance to the practical application of this technique is the absence of DNA damage caused by radiation exposure. In this study, we examine the effect of 633 nm coherent, red laser light on sperm motility using a novel wavelet-based algorithm that allows for direct measurement of curvilinear velocity under red light illumination. This new algorithm gives results comparable to the standard computer-assisted sperm analysis (CASA) system. We then assess the safety of red light treatment of sperm by analyzing, (1) the levels of double-strand breaks in the DNA, and (2) oxidative damage in the sperm DNA. The results demonstrate that for the parameters used there are insignificant differences in oxidative DNA damage as a result of irradiation.

Development of a sperm cryopreservation protocol for the Argentine black and white tegu (Tupinambis merianae).

Of the 934 lizard species evaluated by the International Union for the Conservation of Nature (IUCN), at least one-third is threatened with extinction. However, there are no reports of semen cryopreservation efforts for lizards. Invasive Argentine black and white tegus were captured in the Florida Everglades, and sperm was collected postmortem. Initial motility score (IMS; % motile × speed of progression2 × 100), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded. Sperm was diluted in TEST-yolk buffer with a final glycerol or dimethylsulfoxide (DMSO)concentration of 8%, 12%, or 16%, and frozen at 0.3 °C, 1.0 °C, or 6.3 °C/min. At thaw, all variables were expressed as the percentage of initial (%IMS, %IPL, and %IAC). The 0.3 °C freeze rate was more successful than 1.0 °C and 6.3 °C/min in preserving %IMS and %IPL. DMSO preserved %IMS, %IPL, and %IAC better than glycerol. To determine the best overall cryopreservation protocol, a sperm quality index was calculated, giving equal weight to each of the three indicators of cryosurvival. Because there were significant interactions between freeze rate and cryoprotectant concentration, each treatment was compared with all others. The sperm quality index analysis revealed that tegu sperm frozen at 0.3 °C/min with 12% DMSO exhibited the highest postthaw viability compared with all other treatments.

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.

Rewinding the process of mammalian extinction.

With only three living individuals left on this planet, the northern white rhinoceros (Ceratotherium simum cottoni) could be considered doomed for extinction. It might still be possible, however, to rescue the (sub)species by combining novel stem cell and assisted reproductive technologies. To discuss the various practical options available to us, we convened a multidisciplinary meeting under the name "Conservation by Cellular Technologies." The outcome of this meeting and the proposed road map that, if successfully implemented, would ultimately lead to a self-sustaining population of an extremely endangered species are outlined here. The ideas discussed here, while centered on the northern white rhinoceros, are equally applicable, after proper adjustments, to other mammals on the brink of extinction. Through implementation of these ideas we hope to establish the foundation for reversal of some of the effects of what has been termed the sixth mass extinction event in the history of Earth, and the first anthropogenic one. Zoo Biol. 35:280-292, 2016. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc.

Chelonian perivitelline membrane-bound sperm detection: A new breeding management tool.

Perivitelline membrane (PVM)-bound sperm detection has recently been incorporated into avian breeding programs to assess egg fertility, confirm successful copulation, and to evaluate male reproductive status and pair compatibility. Due to the similarities between avian and chelonian egg structure and development, and because fertility determination in chelonian eggs lacking embryonic growth is equally challenging, PVM-bound sperm detection may also be a promising tool for the reproductive management of turtles and tortoises. This study is the first to successfully demonstrate the use of PVM-bound sperm detection in chelonian eggs. Recovered membranes were stained with Hoechst 33342 and examined for sperm presence using fluorescence microscopy. Sperm were positively identified for up to 206 days post-oviposition, following storage, diapause, and/or incubation, in 52 opportunistically collected eggs representing 12 species. However, advanced microbial infection frequently hindered the ability to detect membrane-bound sperm. Fertile Centrochelys sulcata, Manouria emys, and Stigmochelys pardalis eggs were used to evaluate the impact of incubation and storage on the ability to detect sperm. Storage at -20°C or in formalin were found to be the best methods for egg preservation prior to sperm detection. Additionally, sperm-derived mtDNA was isolated and PCR amplified from Astrochelys radiata, C. sulcata, and S. pardalis eggs. PVM-bound sperm detection has the potential to substantially improve studies of artificial incubation and sperm storage, and could be used to evaluate the success of artificial insemination in chelonian species. Mitochondrial DNA from PVM-bound sperm has applications for parentage analysis, the study of sperm competition, and potentially species identification.

Identification of California Condor Estrogen Receptors 1 and 2 and Their Activation by Endocrine Disrupting Chemicals.

Recently, California condors (Gymnogyps californianus) have been reintroduced to coastal regions of California where they feed on marine mammal carcasses. There is evidence that coastal-dwelling condors experience reproductive issues, such as eggshell thinning, likely resulting from exposure to endocrine-disrupting chemicals (EDCs). To address this problem, we have identified and cloned condor estrogen receptors (ESRs) 1 and 2 and characterized their activation by EDCs present in the coastal habitats where condors reside. Dichlorodiphenyltrichloroethane (DDT) and its metabolites all activated ESR1 and ESR2, although their relative potency differed between the receptors. Bisphenol A, dieldrin, trans-nonachlor, and polychlorinated biphenyl 52 (PCB52) moderately activated both ESRs, whereas PCB138 and PCB153 stimulated little to no activation. Overall, EDC activation of condor ESR2, which is the first ESR2 cloned from a raptor species, was greater than that of ESR1. Significant activation of both condor ESRs by EDCs occurred at high concentrations (≥1µM), which are within the range of plasma levels of certain EDCs (eg, dichlorodiphenyldichloroethylene [p'p-DDE]) in coastal-dwelling condors. Finally, phylogenetic analyses of ESRs of 41 avian species identified a single amino acid position in ESR2 under positive selection. Mutation of this amino acid affected receptor activation by EDCs, suggesting the identity of this amino acid may influence EDC sensitivity of avian species. Together, these findings broaden our understanding of EDC interactions with ESRs in avian species. For condors specifically, these data could be used to evaluate EDC exposure risk at future release sites to identify those least likely to compromise the continued recovery of this species.