Attempted in vitro maturation and fertilization of postmortem Sumatran rhinoceros (Dicerorhinus sumatrensis) oocytes.

A study was conducted opportunistically to evaluate the potential of rescuing immature oocytes from the ovaries of the Sumatran rhinoceros postmortem. Recovered oocytes (n = 30) were placed in maturation culture for 36 hr and inseminated with frozen-thawed homologous spermatozoa. After culture, evaluation of nuclear maturation status revealed that a large number of oocytes were degenerated (n = 21), but nine oocytes were assessed at the germinal vesicle (n = 3), metaphase I (n = 3), and metaphase II (n = 3) stages. Frozen-thawed Sumatran rhinoceros spermatozoa were capable of binding to the zona pellucida of in vitro matured oocytes, but no fertilization or cleavage resulted. In conclusion, relatively large numbers of oocytes can be obtained by ovarian follicular aspiration postmortem in the Sumatran rhinoceros, and some of these oocytes are capable of achieving nuclear maturation in vitro. However, additional studies are required to improve maturation success and achieve fertilization in culture.

Non-invasive methods to measure inter-renal function in aquatic salamanders-correlating fecal corticosterone to the environmental and physiologic conditions of captive Necturus.

This study sought to develop non-invasive techniques to monitor glucocorticoids in captive Necturus as a means to correlate inter-renal gland function in relation to environmental and physiological changes. Six individually housed breeding pairs of captive Necturus beyeri were subjected to seasonal changes in water temperature (30°F temperature differential) to stimulate natural breeding, specifically spermatophore deposition and oviposition. An enzyme immunoassay was validated for the measurement of N. beyeri faecal corticosterone metabolites (fCMs) by exhibiting parallelism and accuracy to the standard curve. Longitudinal (December 2016-October 2017) assessment of fCM concentrations and pattern of excretion from samples collected from the six breeding pairs revealed a seasonal inter-renal effect with higher concentrations (P < 0.05) excreted during months (December-March) of the year associated with breeding activity and when water temperatures were lowest. Males from each pair produced spermatophores starting on 08 December 8 2016 and ending on 05 April 2017. Females from four of the six pairs went on to successfully oviposit eggs in mid-late April 2017. One clutch was fertile, and three were non-fertile. No differences (P > 0.05) were detected in fCM concentrations between pairs in which oviposition did or did not occur. In addition, a novel waterborne corticosterone metabolite (wCM) assay was validated to overcome challenges associated with faecal collection in a group-housed amphibian. An adrenocorticotropic hormone (ACTH) challenge performed in an adult male Necturus maculosus resulted in a 50-fold increase in wCM at 4 h post-injection and marked the first demonstration of a waterborne inter-renal response to ACTH in Necturus. This study not only provides insight into inter-renal function in an aquatic salamander that exhibits marked reproductive seasonality but also confirms utility of fCM and wCM measurements as non-invasive means of assessment.

Comparison of different sperm cryopreservation procedures on post-thaw quality and heterologous in vitro fertilisation success in the ocelot (Leopardus pardalis).

Cryopreservation of spermatozoa from free-living ocelots (Leopardus pardalis) could benefit their conservation by facilitating gene flow between in situ and ex situ populations without requiring removal of additional cats from the wild. The objective of this study was to investigate three different methods of ocelot sperm cryopreservation to identify the most appropriate technique for use in a field environment. Male ocelots (n = 10), housed in North American zoos, were anaesthetised with tiletamine-zolazepam (7 mg kg(-1) bodyweight; i.m.) and subjected to a regimented electroejaculation procedure. Recovered semen was evaluated for sperm concentration, motility and morphology and processed for cryopreservation by three methods: (1) pelleting on dry ice, (2) freezing in straws over liquid nitrogen vapour; and (3) freezing in straws in a dry shipper. Frozen samples were thawed and assessed for post-thaw acrosome status, viability, motility over time and ability to fertilize viable domestic cat oocytes. Although several post-thaw sperm parameters varied (P < 0.05) among freezing methods, frozen-thawed ocelot spermatozoa from all treatments showed a similar (P > 0.05) capacity to bind, penetrate and fertilize viable domestic cat oocytes. These findings suggest that spermatozoa collected from male ocelots under field conditions may be frozen in straws either using liquid nitrogen alone or in a charged dry shipper to retain adequate functional competence after thawing for use with assisted reproductive procedures.

Enhancing captive Indian rhinoceros genetics via artificial insemination of cryopreserved sperm.

The objective of this study was to design an artificial insemination (AI) protocol using cryopreserved spermatozoa to obtain pregnancies in captive Indian rhinoceroses (Rhinoceros unicornis). Four methods developed varied by timing and approach, as follows; Method 1: females (n=2) were inseminated pre- and post-ovulation under general anesthesia, Method 2: females (n=2) were inseminated pre-ovulation without anesthetic via endoscopy, Method 3: females (n=1) were inseminated pre-ovulation without anesthetic via manual insertion of an insemination catheter, Method 4: females (n=2) were inseminated same as Method 3 with the addition of standing sedation. Semen deposition site varied as a result of changes in AI technology and experience. All females conceived following intrauterine AI using three methods. Four pregnancies (n=3 females) produced via Method 3 and 4 resulted in term births (n=2 male calves, n=2 female calves) at 481.8±12.8days post-AI. Unfortunately, two early pregnancy losses were documented in a fourth female conceiving via Method 2. Pregnancy rates were 0%, 22%, 17%, and 50% for Method 1-4, respectively. Method 3 and 4 rates improved to 29% and 67%, respectively when accounting for AI's conducted only on ovulatory estrous cycles. Spermatozoa (n=5 males) were cryopreserved 0.3-9.3 y prior to successful AI procedures. The lowest dose of frozen-thawed sperm resulting in conception was 500×10(6) motile sperm. Mean time from AI to ovulation in conceptive and non-conceptive cycles was 26±11.8h and 66±80.7h, respectively.

Semen collection in rhinoceroses (Rhinoceros unicornis, Diceros bicornis, Ceratotherium simum) by electroejaculation with a uniquely designed probe.

Electroejaculation in rhinoceroses has historically yielded inconsistent results, with the collection of high-quality, sperm-rich samples rare. The goal of this study was to develop a reliable method of electroejaculation in the rhinoceros by designing a rectal probe that appropriately fits the anatomy of this taxon and refining the procedure. A curved probe handle ending in an oblate, ellipsoid head was built using readily available supplies. A combination of rectal massage, penile massage, and electrical stimulation with a specially designed probe was employed in attempts to collect semen on 14 occasions from greater one-horned rhinoceroses (Rhinoceros unicornis; n = 4), black rhinoceroses (Diceros bicornis; n = 2) and a southern white rhinoceros (Ceratotherium simum; n = 1). During 13 of the 14 attempts, ejaculates were collected in multiple fractions. All but one of the ejaculates contained spermatozoa, and seven ejaculates contained good-quality fractions of semen (-60% sperm motility; > or =20 x 106 spermatozoa/ml) suitable for sperm banking and assisted reproduction procedures. Mean (+/-SEM) values for volume, pH, osmolality, and total sperm number for ejaculates containing good-quality fractions (98.2 +/-21.8 ml, 8.5+/-0.1, 290.4+/-6.7 mOsm, and 37.1+/-12.0 x 10(9), respectively) did not differ (P > 0.05) from those containing only poor-quality samples. Urine and/or erythrocyte contamination was not uncommon in fractions of both ejaculate types. Males producing good-quality samples ranged in age from 7 to 34 yr. None of the samples contained > or =75% morphologically normal spermatozoa. Electroejaculation with a uniquely designed probe consistently produced ejaculates in the rhinoceros. However, the production of high-quality samples continued to be challenging, occurring in only 50% of collection attempts. Regardless, the technology has progressed to a stage at which good-quality semen samples can be produced for sperm banking and assisted reproduction, and thereby can be integrated into intensive rhinoceros management strategies for the ultimate survival of this taxon.