Search

Increased poaching in northern South Africa has necessitated relocation of large numbers of southern white rhinoceros (Ceratotherium simum simum) to the Eastern Cape Province. The climate and grassland ecology of this province differ from that of northern South Africa which may impact the health of this species. This assessment of fecal steroid levels and microbiome in 10 free-ranging southern white rhinoceros in the Eastern Cape will provide insights into white rhinoceros physiology in this biome. Fecal steroid metabolites were analyzed using enzyme immunoassay (EIA) and ultra-performance convergence chromatography tandem mass spectrometry (UPC 2 -MS/MS). Fecal microbial composition was assessed via next generation sequencing. EIAs with antibodies raised against progesterone (P4; mouse monoclonal - CL425 clone), testosterone (T; rabbit polyclonal), corticosterone (B; sheep polyclonal) were utilized. Pregnant females had large quantities of fecal progesterone metabolites (FPMs) detected by CL425 EIA. Pregnant females also had native P4 and 11α-hydroxydihydroprogesterone (11αOHDHP4; 4-pregnen-11α-ol-3,20-dione) detected by UPC 2 -MS/MS but these concentrations were 1000-fold less than the concentrations of FPMs detected by the CL425 EIA. By contrast, non-pregnant females had FPM concentrations detected by CL425 EIA which were similar to native P4 and 11αOHDHP4 concentrations detected by UPC 2 -MS/MS. Mean fecal androgen metabolite (FAM) concentrations detected by the T EIA were similar between males and females. 11-ketoandrostenedione (11KA4) detected by UPC 2 -MS/MS was higher in females than males. However, there was no difference between males and females in the concentration of fecal glucocorticoid metabolites (FGMs) detected by the B EIA. Bacteroidia, followed by Clostridia, was the most abundant classes of fecal microbes. The unfiltered microbiome of females was more diverse than that of males. The core fecal microbiome of young rhinoceros had a higher observed species richness (Shannon diversity index, and Simpson diversity index) than that of old rhinoceros. In the alpha male, immobilization was associated with an increase in FGMs detected by 11-deoxycortisol (S) detected by UPC 2 -MS/MS coupled with decreased abundance of Spirochaetia. We detected substantially different FAM and FPM concentrations from those previously reported for both captive and wild white rhinoceros. Comparison of our UPC 2 -MS/MS and EIA results underscores the fact that most EIAs are highly cross reactive for many steroid metabolites. Our data also demonstrates a distinct effect of stress not only on FGMs but also on the fecal microbiome. This is the first non-invasive assessment of fecal steroid metabolites by UPC 2 -MS/MS and the fecal microbiome in wild white rhinoceros., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

The measurement of progesterone (P4) and estradiol (E2) is essential for monitoring reproductive cycles and can aid in diagnosing the cause of poor reproductive performance in dairy cattle. Readily available, reproducible, accurate, non-radioactive assays are needed for the assessment of P4 and E2 in bovine serum. The gold standard for hormone assessment, radioimmunoassay (RIA), was compared with enzyme-linked immunoassay (EIA). Serum collected from various points in the estrous cycle was extracted with radiolabeled P4 (ie, 3 H-P4; HE) and without 3 H-P4 (CE) before being used in the assay. For the assessment of P4, there is a great degree of correlation between the RIA and EIA (adjusted R-square = 0.95; Pearson correlation coefficient (PCC) = 0.98, P < 0.001). A difference between the RIA and EIA methods was not detected for E2 concentrations (P = 0.16), but the correlation between techniques was poor (adjusted R-squared = 0.73; PCC = 0.87, P = 0.002). There was no difference in the serum extraction efficiency as measured with 3 H-P4 as opposed to without (P = 0.94). The two methods for the measurement of serum extraction efficiency were highly correlated (adjusted R-square = 0.83; PCC = 0.92, P < 0.001). The concordance correlation coefficient (CCC) showed an excellent agreement between RIA and EIA for P4 determination (0.89) and between HE and CE methods (0.90). Although the 95% limits of agreement of the Bland-Altman plots encompassed 89% (8/9) and 92% (12/13) of the differences between methods for P4 quantification and extraction respectively, the CCC indicated an excellent agreement among them. The CCC between RIA and EIA for E2 quantification was 0.68 which corresponds with a fair agreement; however, the 95% limits of agreement of the Bland-Altman plot encompassed 100% (9/9) of differences between methods. The EIA and CE methods are comparable alternatives to the RIA and HE methods, respectively and can be used to quantify P4 and E2 for bovine serum., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The discrete effects of obesity on infertility in females remain undefined to date. To investigate obesity-induced ovarian dysfunction, we characterized metabolic parameters, steroidogenesis, and folliculogenesis in obese and lean female Ossabaw mini-pigs. Nineteen nulliparous, sexually mature female Ossabaw pigs were fed a high fat/cholesterol/fructose diet (n=10) or a control diet (n=9) for eight months. After a three-month diet-induction period, pigs remained on their respective diets and had ovarian ultrasound and blood collection conducted during a five-month study period after which ovaries were collected for histology, cell culture, and gene transcript level analysis. Blood was assayed for steroid and protein hormones. Obese pigs developed abdominal obesity and metabolic syndrome, including hyperglycemia, hypertension, insulin resistance and dyslipidemia. Obese pigs had elongated estrous cycles and hyperandrogenemia with decreased LH, increased FSH and luteal phase progesterone, and increased numbers of medium, ovulatory, and cystic follicles. Theca cells of obese, compared to control, pigs displayed androstenedione hypersecretion in response to in vitro treatment with LH, and up-regulated 3-beta-hydroxysteroid dehydrogenase 1 and 17-beta-hydroxysteroid dehydrogenase 4 transcript levels in response to in vitro treatment with LH or LH + insulin. Granulosa cells of obese pigs had increased 3-beta-hydroxysteroid dehydrogenase 1 transcript levels. In summary, obese Ossabaw pigs have increased transcript levels and function of ovarian enzymes in the delta 4 steroidogenic pathway. Alterations in LH, FSH, and progesterone, coupled with theca cell dysfunction, contribute to the hyperandrogenemia and disrupted folliculogenesis patterns observed in obese pigs. The obese Ossabaw mini-pig is a useful animal model in which to study the effects of obesity and metabolic syndrome on ovarian function and steroidogenesis. Ultimately, this animal model may be useful toward the development of therapies to improve fertility in obese and/or hyperandrogenemic females or in which to examine the effects of obesity on the maternal-fetal environment and offspring health.