Assessment of ovarian cycles in the African elephant (Loxodonta africana) by measurement of salivary progesterone metabolites.

Monitoring ovarian cycles through hormonal analysis is important in order to improve breeding management of captive elephants, and non-invasive collection techniques are particularly interesting for this purpose. However, there are some practical difficulties in collecting proper samples, and easier and more practical methods may be an advantage for some institutions and/or some animals. This study describes the development and validation of an enzymeimmunoassay (EIA) for progestins in salivary samples of African elephants, Loxodonta africana. Weekly urinary and salivary samples from five non-pregnant elephant cows aged 7-12 years were obtained for 28 weeks and analyzed using EIA. Both techniques correlated positively (r = 0.799; P < 0.001), and the cycle characteristics obtained were identical. The results clearly show that ovarian cycles can be monitored by measuring progestins from salivary samples in the African elephant. This is a simple and non-invasive method that may be a practical alternative to other sampling methods used in the species.

Development of a versatile enzyme immunoassay for non-invasive assessment of glucocorticoid metabolites in a diversity of taxonomic species.

Endocrinology is a useful tool for conservation biologists and animal managers, and measuring glucocorticoids can help understand biological mechanisms associated with species decline and animal welfare. The current study describes the development and optimization of a glucocorticoid enzyme immunoassay (EIA) to non-invasively assess adrenal activity in a variety of taxa. The antiserum (CJM006) was raised in rabbits to a corticosterone-3-CMO-BSA immunogen and used in a standard competitive EIA system. However, the EIA initially produced results with unacceptably high inter-assay variation, attributed to consistent patterns observed within the optical density of developing plates. To determine the cause of this variability, a number of factors were examined using synthetic corticosterone standard and endogenous faecal extract, including: plate type (Nunc MaxiSorp® II versus Immulon IB plates); the use of non-specific secondary antibody; type (artificial versus natural) and presence (light versus dark) of light during incubation; plate loading temperature (4°C versus room temperature); and substrate reagent temperature (4°C versus room temperature). Results indicated that variability was associated with plate location effects, which were not initially detected because control samples were always run in the same positions across plates. Light and temperature were the two major factors that affected EIA reliability. For this assay, the standard protocol required slight modification, with the optimal protocol using Nunc MaxiSorp® plates, room temperature substrate reagents and dark incubation conditions. Following optimization, this EIA was then validated biochemically for 38 species, through parallel displacement curves and interference assessment tests of faecal and urine samples. Additionally, biological validation was performed opportunistically in a subset of species, with use of this EIA demonstrating significant elevations in faecal glucocorticoid metabolites following potentially challenging events. In summary, this glucocorticoid EIA cross-reacts with excreted glucocorticoid metabolites across a wide range of taxa, including ungulates, primates, felids, birds, rodents and amphibians. We conclude that when used with optimal reagent and incubation conditions, this EIA will be useful for non-invasive monitoring of adrenal activity in a wide range of wildlife species.

Serum 17alpha-hydroxyprogesterone concentrations during the reproductive cycle in healthy dogs and dogs with hyperadrenocorticism.

OBJECTIVE: To determine concentrations of 17alpha-hydroxyprogesterone (17OHP) in serum of healthy bitches during various stages of the reproductive cycle and in bitches with hyperadrenocorticism and to compare the dynamics of 17OHP with those of progesterone. DESIGN: Prospective evaluation study. ANIMALS: 15 healthy sexually intact bitches and 28 spayed bitches with hyperadrenocorticism. PROCEDURES: 11 healthy bitches were evaluated during estrus, nonpregnant diestrus, and anestrus (group 1); 4 other healthy bitches were evaluated during pregnancy and after ovariohysterectomy (group 2). Cycle stages were determined via physical examination, vaginal cytologic evaluation, and serum progesterone concentration. Bitches with hyperadrenocorticism were evaluated once at the time of diagnosis (group 3). Serum hormone concentrations were determined with immunoassays. RESULTS: In group 1, the serum 17OHP concentration was significantly higher in diestrus (median, 1.8 ng/mL) than in estrus (median, 1.1 ng/mL) and anestrus (median, 0.2 ng/mL) and higher in estrus than in anestrus. Changes in serum progesterone concentrations accounted for 22% (estrus) or 23% (diestrus) of the variation in serum 17OHP concentrations. In group 2, 17OHP and progesterone concentrations were significantly higher during pregnancy than after ovariohysterectomy. The serum 17OHP concentration in group 3 was significantly lower (median, 0.2 ng/mL) than in group 1 in estrus and diestrus and in group 2 during pregnancy (median, 0.7 ng/mL) but was not different from 17OHP concentrations in anestrus or after ovariohysterectomy (median, 0.2 ng/mL). CONCLUSIONS AND CLINICAL RELEVANCE: Serum 17OHP concentrations in healthy bitches increased during estrus, diestrus, and pregnancy and at those times were higher than in spayed bitches with hyperadrenocorticism.

Granulosa cell tumors of the equine ovary.

The granulosa cell tumor is the most common ovarian tumor in mares. A clinical diagnosis can be made based on the presence ofa unilaterally enlarged ovary and a small inactive contralateral ovary. Endocrine testing may be beneficial to confirm a diagnosis. Surgical removal of the tumor eliminates the adverse effect on pituitary function and results in resumption of follicular development and ovulation in the opposite ovary over time.

Gonadotropin secretion and pituitary responsiveness to GnRH in mares with granulosa-theca cell tumor.

Granulosa-theca cell tumors (GTCTs) are able to secrete variable amounts of sex steroids and immunoreactive inhibin (ir-INH). Although the pituitary appears to be affected by the presence of a GTCT, pituitary responsiveness to exogenous GnRH has not been examined. The aims of the present study were to: (i) assess the plasma hormone concentrations of ir-INH, gonadotropins and sex steroids in eight mares with GTCT and (ii) assess the responsiveness of pituitary gonadotroph cells to exogenous GnRH stimulus both before and after tumor removal. In seven mares, the contralateral ovary was firm, small and inactive. Histopathological observations of the tumors confirmed the presumptive diagnosis of a GTCT. Four mares, judged to be in vernal transition period (n=2) and in the breeding season (n=2), were used as controls. A single intravenous injection of 40 microg of GnRH agonist was given to each mare and blood samples were collected every 15 min from 2 h before to 4 h after injection. In four GTCT mares, this procedure was repeated 20 (n=2) and 90 (n=2) days after tumors removal. All plasma samples were analyzed for concentrations of ir-INH, LH, FSH, estradiol-17beta (E2), testosterone (T) by RIA and progesterone (P) by EIA. Results showed that E2 levels were significantly higher (P<0.001) in control animals compared to E2 levels in GTCT mares before and after surgery. P and T concentrations were not statistically different between the groups. Baseline levels of ir-INH were greater (P<0.05) in GTCT mares before surgery than in control mares, and decreased to undetectable levels after neoplasia ablation. Baseline FSH did not differ between control and GTCT animals either before or after the ovaries were removed. LH baseline values appeared to be higher for affected mares, but the difference was not statistically significant. Maximum release (MR) and area under the gonadotrophin release curve (AUC) after the GnRH challenge for both the gonadotrophins were similar between the groups.

Urinary estrone conjugate and pregnanediol 3-glucuronide enzyme immunoassays for population research.

BACKGROUND: Monitoring of reproductive steroid hormones at the population level requires frequent measurements, hormones or metabolites that remain stable under less than ideal collection and storage conditions, a long-term supply of antibodies, and assays useful for a range of populations. We developed enzyme immunoassays for urinary pregnanediol 3-glucuronide (PDG) and estrone conjugates (E1Cs) that meet these criteria. METHODS: Enzyme immunoassays based on monoclonal antibodies were evaluated for specificity, detection limit, parallelism, recovery, and imprecision. Paired urine and serum specimens were analyzed throughout menstrual cycles of 30 US women. Assay application in different populations was examined with 23 US and 42 Bangladeshi specimens. Metabolite stability in urine was evaluated for 0-8 days at room temperature and for 0-10 freeze-thaw cycles. RESULTS: Recoveries were 108% for the PDG assay and 105% for the E1C assay. Serially diluted specimens exhibited parallelism with calibration curves in both assays. Inter- and intraassay CVs were <11%. Urinary and serum concentrations were highly correlated: r = 0.93 for E1C-estradiol; r = 0.98 for PDG-progesterone. All Bangladeshi and US specimens were above detection limits (PDG, 21 nmol/L; E1C, 0.27 nmol/L). Bangladeshi women had lower follicular phase PDG and lower luteal phase PDG and E1Cs than US women. Stability experiments showed a maximum decrease in concentration for each metabolite of <4% per day at room temperature and no significant decrease associated with number of freeze-thaw cycles. CONCLUSIONS: These enzyme immunoassays can be used for the field conditions and population variation in hormone metabolite concentrations encountered in cross-cultural research.

Brain natriuretic peptide concentration in dogs with heart disease and congestive heart failure.

Plasma brain natriuretic peptide concentration ([BNP]) is high in humans with cardiac disease and is further increased with congestive heart failure (CHF). The hypotheses of this study were that dogs with moderate to severe mitral regurgitation due to myxomatous mitral valve disease (MVD) would have increased plasma [BNP] compared to normal dogs, that plasma [BNP] would be higher in dogs with CHP, and that plasma [BNP] would predict premature death from cardiovascular disease. The study population consisted of 34 dogs: 9 normal dogs and 25 dogs with MVD. Patients were divided into 4 groups: group 1-10 dogs with moderate to severe MVD and no radiographic evidence of CHF; group II--6 dogs with severe MVD and mild CHF; group III--7 dogs with severe MVD and moderate CHF; and group IV--2 dogs with severe MVD and severe CHF. Diagnostic tests included thoracic radiographs, an echocardiogram, a serum chemistry profile, and the measurement of plasma [BNP] by a canine-specific radioimmunoassay. There was a significant positive correlation between the plasma [BNP] and heart disease/failure groups (P = .0036). Plasma [BNP] increased with progressively increasing severity of MVD and CHE Group I dogs had higher plasma [BNP] than did control dogs (P < .0001), and plasma [BNP] was higher in dogs with CHF (groups II-IV versus group I; P = .012). Plasma [BNP] was also weakly positively correlated with left atrial size (r = 0.43, P = .04). For every 10-pg/mL increase in plasma [BNP], the mortality rate over 4 months' time increased approximately 44%.