Lactotransferrin in Asian Elephant (Elephas maximus) Seminal Plasma Correlates with Semen Quality

Asian elephants (Elephas maximus) have highly variable ejaculate quality within individuals, greatly reducing the efficacy of artificial insemination and making it difficult to devise a sperm cryopreservation protocol for this endangered species. Because seminal plasma influences sperm function and physiology, including sperm motility, the objectives of this study were to characterize the chemistry and protein profiles of Asian elephant seminal plasma and to determine the relationships between seminal plasma components and semen quality. Ejaculates exhibiting good sperm motility (≥65%) expressed higher percentages of spermatozoa with normal morphology (80.3+-13.0 vs. 44.9+-30.8%) and positive Spermac staining (51.9+-14.5 vs. 7.5+-14.4%), in addition to higher total volume (135.1+-89.6 vs. 88.8+-73.1 ml) and lower sperm concentration (473.0+-511.2 vs. 1313.8+-764.7 x 106 cells ml-1) compared to ejaculates exhibiting poor sperm motility (≤10%; P\u3c0.05). Comparison of seminal plasma from ejaculates with good versus poor sperm motility revealed significant differences in concentrations of creatine phosphokinase, alanine aminotransferase, phosphorus, sodium, chloride, magnesium, and glucose. These observations suggest seminal plasma influences semen quality in elephants. One- and two-dimensional (2D) gel electrophoresis revealed largely similar compositional profiles of seminal plasma proteins between good and poor motility ejaculates. However, a protein of ~80 kDa was abundant in 85% of ejaculates with good motility, and was absent in 90% of poor motility ejaculates (P\u3c0.05). We used mass spectrometry to identify this protein as lactotransferrin, and immunoblot analysis to confirm this identification. Together, these findings lay a functional foundation for understanding the contributions of seminal plasma in the regulation of Asian elephant sperm motility, and for improving semen collection and storage in this endangered species

Insights from the rescue and breeding management of Cuvier’s gazelle (Gazella cuvieri) through whole-genome sequencing

Captive breeding programmes represent the most intensive type of ex situ population management for threatened species. One example is the Cuvier’s gazelle programme that started in 1975 with only four founding individuals, and after more than four decades of management in captivity, a reintroduction effort was undertaken in Tunisia in 2016, to establish a population in an area historically included within its range. Here, we aim to determine the genetic consequences of this reintroduction event by assessing the genetic diversity of the founder stock as well as of their descendants. We present the first whole-genome sequencing dataset of 30 Cuvier’s gazelles including captive-bred animals, animals born in Tunisia after a reintroduction and individuals from a genetically unrelated Moroccan population. Our analyses revealed no difference between the founder and the offspring cohorts in genome-wide heterozygosity and inbreeding levels, and in the amount and length of runs of homozygosity. The captive but unmanaged Moroccan gazelles have the lowest genetic diversity of all genomes analysed. Our findings demonstrate that the Cuvier’s gazelle captive breeding programme can serve as source populations for future reintroductions of this species. We believe that this study can serve as a starting point for global applications of genomics to the conservation plan of this species.K-P. Koepfli and B. Pukazhenthi acknowledge the Sichel Endowment Fund for research support on dama gazelle genomics. M.A.E. is supported by an FPI (Formación de Personal Investigador) PRE2018-083966 from Ministerio de Ciencia, Universidades e Investigación. P.D. was supported as a postdoctoral fellow by the Smithsonian Institution Fellowship Program. K.-P.K. was supported by funding from the Smithsonian Institution's George E. Burch Fellowship in Theoretical Medicine and Affiliated Theoretical Science. T.M.-B. is supported by funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 864203), BFU2017-86471-P (MINECO/FEDER, UE), ‘Unidad de Excelencia María de Maeztu’, funded by the AEI (CEX2018-000792-M), Howard Hughes International Early Career and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880). E.M. received financial support from the project PGC2018-097426-B-C22 (Spanish Ministry of Universities. Spanish State Research Agency. FEDER Program, European Union). E.L. is supported by CGL2017-82654-P (MINECO/FEDER, UE).Peer reviewe

Bilateral Intratesticular Spermatoceles in a Critically Endangered Dama Gazelle (Nanger dama mhorr)

Dama gazelles (Nanger dama mhorr) are considered critically endangered by the IUCN Red List of Threatened Species since the wild population is comprised of fewer than 200 animals. Several zoos in Europe and some private ranches in the US (Texas) maintain this species in captivity as a hedge against extinction. A routine reproductive exam on an adult male revealed bilateral cysts in the testicular parenchyma. The fluid in the cysts contained copious, moderately progressive motile spermatozoa. Following examination, the gazelle was diagnosed with bilateral intratesticular spermatoceles. Electroejaculation yielded 1.5 ml of semen containing 387×106 total sperm with 50% motility and 45% morphologically normal sperm. The spermatoceles did not appear to cause pain or dysfunction, so no treatment was performed at this time. Since fewer than 10 animals are managed in captivity in the US, no intervention (castration/hemicastration) was considered

Linkage between fecal androgen and glucocorticoid metabolites, spermaturia, body weight and onset of puberty in male African lions (Panthera leo).

There is limited physiological information on onset of puberty in male lions. The aim of this study was to use longitudinal non-invasive monitoring to: 1) assess changes in steroid metabolite excretory patterns as a function of age and body weight; 2) determine correlations between fecal androgen (FAM) and glucocorticoid (FGM) metabolite concentrations; and 3) confirm spermiogenesis non-invasively through urinalysis. Specifically, FAM and FGM metabolites were analyzed in samples collected twice weekly from 21 male lions at 17 institutions (0.9-16 years of age) for 3.8 months- 2.5 years to assess longitudinal hormone patterns. In addition, body weights were obtained approximately monthly from 10 individuals at five zoos (0.0-3.0 years), and urine was collected from six males at two facilities (1.2-6.3 years) and evaluated for the presence of spermatozoa. An increase in overall mean FAM occurred at 2.0 years of age, at which point concentrations remained similar throughout adulthood. The onset of puberty occurred earlier in captive-born males (<1.2 years of age) compared to wild-born counterparts (<2.5 years of age). Additionally, males in captivity gained an average of 7.3 kg/month compared to 3.9 kg/month for wild males over the first 2-2.5 years of age. Sperm (spermaturia) was observed in males as young as 1.2 years in captivity compared to 2.5 years in the wild (ejaculates). There was no difference in FAM or FGM concentrations with regards to age or season. Overall, this study demonstrates that: 1) captive male lions attain puberty at an earlier age than wild counterparts; 2) onset of puberty is influenced by body weight (growth rate); and 3) spermiogenesis can be confirmed via urinalysis. Knowledge about the linkage between body weight and onset of puberty could facilitate improved reproductive management of ex situ populations via mitigating the risk of unintended breedings in young animals

Equine Oviductal Organoid Generation and Cryopreservation

Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze&ndash;thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of their normal physiology, including hormonal regulation. These organoids may provide an environment that mimics the in vivo equine oviduct and facilitate improved in vitro embryo production in equids

Progestin Exposure Before Gonadotropin Stimulation Improves Embryo Development after In Vitro Fertilization in the Domestic Cat1

This study investigated the influence of progestin priming and ovarian quiescence on response to exogenous gonadotropin stimulation in the cat. Because a subpopulation of cats routinely ovulated spontaneously, there also was the opportunity to examine the ovary's reaction to the added impact of endogenously secreted progestagen. Queens were given 1) equine chorionic gonadotropin (eCG) plus human chorionic gonadotropin (hCG) only (control; n = 9 cats), 2) GnRH antagonist (antide) injections followed by eCG and hCG (n = 9), and 3) a progestin implant (levonorgestrel) followed by eCG and hCG (n = 9). Laparoscopy was used to assess ovarian activity and aspirate follicular oocytes that were graded on the basis of morphology. In five cats per treatment, half of the high-quality oocytes were assessed for glucose, pyruvate, and lactate metabolism as well as nuclear maturation. Remaining oocytes were inseminated in vitro, cultured, and examined at 72 h after insemination for cleavage. In the remaining four cats per treatment, all oocytes were inseminated in vitro and assessed at 72, 120, and 168 h after insemination for embryo developmental stage. Cats pretreated with progestin had more follicles and produced more embryos per donor (including at the combined morula/blastocyst stage) than controls or females treated with GnRH antagonist (P < 0.05). There were no differences among groups (P > 0.05) in oocyte carbohydrate metabolism, nuclear maturation metrics, or fertilization success, although there was a tendency toward improvements in all three (P < 0.2) in progestin-treated females. Interestingly, cats that spontaneously ovulated within 60 days of treatment onset also produced more embryos per cat than induced-ovulation counterparts (P < 0.05). Results indicate that prior exposure to exogenous progestin (via implant) or endogenous progestagen (via spontaneous ovulation) improves ovarian responsiveness to gonadotropins in the cat through a mechanism that is independent of the induction of ovarian quiescence

Metabolomic profiling implicates mitochondrial and immune dysfunction in disease syndromes of the critically endangered black rhinoceros (Diceros bicornis)

Abstract The critically endangered black rhinoceros (Diceros bicornis; black rhino) experiences extinction threats from poaching in-situ. The ex-situ population, which serves as a genetic reservoir against impending extinction threats, experiences its own threats to survival related to several disease syndromes not typically observed among their wild counterparts. We performed an untargeted metabolomic analysis of serum from 30 ex-situ housed black rhinos (Eastern black rhino, EBR, n = 14 animals; Southern black rhino, SBR, n = 16 animals) and analyzed differences in metabolite profiles between subspecies, sex, and health status (healthy n = 13 vs. diseased n = 14). Of the 636 metabolites detected, several were differentially (fold change > 1.5; p < 0.05) expressed between EBR vs. SBR (40 metabolites), female vs. male (36 metabolites), and healthy vs. diseased (22 metabolites). Results suggest dysregulation of propanoate, amino acid metabolism, and bile acid biosynthesis in the subspecies and sex comparisons. Assessment of healthy versus diseased rhinos indicates involvement of arachidonic acid metabolism, bile acid biosynthesis, and the pentose phosphate pathway in animals exhibiting inflammatory disease syndromes. This study represents the first systematic characterization of the circulating serum metabolome in the black rhinoceros. Findings further implicate mitochondrial and immune dysfunction as key contributors for the diverse disease syndromes reported in ex-situ managed black rhinos