Urinary steroid evaluations to monitor ovarian function in exotic ungulates: VII. Urinary progesterone metabolites in the Equidae assessed by immunoassay

A direct enzyme immunoassay (EIA) for non-specific urinary progesterone (Po) metabolites, utilizing a non-specific monoclonal antibody against pregnanediol-3-glucuronide, was evaluated for the purpose of assessing luteal function in equids. Urinary pregnanediol-3-glucuronide (PdG) and immunoreactive PdG-like conjugate (iPdG) concentrations, indexed by creatinine, were compared to plasma Po concentrations in non-conceptive ovarian cycles through two ovulations in four mares. High-performance liquid chromatography (HPLC) of urine from lutealphase mares and a pregnant zebra revealed an absence of significant concentrations of PdG and the presence of at least three immunoreactive compounds, all of which were more polar than PdG. The concentration of iPdG in the mare ranged from a nadir of approximately 3 ng/mg Cr at the time of ovulation to nearly 400 ng/mg Cr at the mid-luteal-phase peak and paralleled plasma Po concentrations. This non-radiometric assay for iPdG permits the assessment of ovulation, luteal formation and function, and luteolysis in unprocessed urine samples from domestic mares. Data from a single zebra indicate this approach also will permit simplified and non-invasive longitudinal studies of ovarian function among a wide range of Equidae.     

Effects of hormone implants on estrus and ovulation in feral mares

Five groups of 30 captive feral mares each were implanted with silastic rods containing estradiol (E) and/or progesterone (P): E only with 8 g, P only with 24 g, P+HE with 8 g P + 8 g E, HP+E with 12 g P + 4 g E, HP+LE with 12 g P + 2 g E. Arbitrary group designations were differentiated by relative high (H) and low (L) amounts of steroid. Thirty mares received silastic rods containing no hormone (CI). Five mares from each group were bled every 2 wk for 4 mo and monthly for another 5 mo. All mares were tested for estrus by allowing them to stand in an alley between two pens of stallions and visually monitoring her response to the stallion. Serum P levels increased from 0.3 +/- 0.1 to 1.8 +/- 0.1 ng/ml in the P only group during the first 3 wk after implanting. Levels remained stable for the next 2 wk and then began a gradual decline. Serum P levels in the other groups were lower. Serum E levels were slightly increased in the groups receiving 8 g of E (E only and P+HE groups). Significantly fewer animals in the E only and P+HE groups exhibited estrus as compared with control animals (10 of 23 and 13 of 26 versus 22 of 25, respectively, P less than or equal to 0.003). However, animals receiving 24 g of P (P only) showed similar occurrences of estrus as controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation between serum and fecal concentrations of reproductive steroids throughout gestation in goats

Non-invasive techniques such as the measurement of fecal steroids are now widely used to monitor reproductive hormones in captive and free-ranging wild-life. These methods offer great advantages and deserve to be used in domestic animals. The aim of the present study was to determine the endocrine profile of dairy goats throughout pregnancy by the quantification of fecal progestins and estrogens and assess its correlation with serum concentrations. Blood and fecal samples were collected weekly from 11 adult, multiparous goats, from mating through pregnancy and 2 weeks post-partum. The extraction of estradiol and progesterone fecal metabolites was performed by dilution in ethanol. The radioimmunoassay (RIA) in solid phase was used to quantify serum 17beta-estradiol (estradiol) and progesterone, as well as their fecal metabolites. The mean concentrations of both fecal and serum estradiol started to increase between weeks 7 and 11, reached peak values near parturition and then decreased sharply (range: 19.8+/-5.8 ng/g of feces to 608.6+/-472.4 ng/g of feces and 0.007+/-0.005 ng/ml to 0.066+/-0.024 ng/ml). An increase in both fecal and blood progestagens occurred in the second week, mean concentrations remained greater until week 20, and then decreased in the last week of gestation and 2 weeks post-partum (range: 108.8+/-43.6 ng/g of feces to 3119.5+/-2076.9 ng/g of feces and 0.12+/-0.04 ng/ml to 13.10+/-4.29 ng/ml). The changes in blood and fecal hormone concentrations were analyzed and compared throughout gestation for each single goat, for each breed and for the whole group. Results indicated that matched values of serum and fecal hormone concentrations were correlated (r=0.79; p<0.001 for progesterone and r=0.84; p<0.001 for estradiol mean concentrations in the whole group). Regression analysis showed that logarithmic model allows significant prediction of serum from fecal concentrations with an R(2)=0.729 (y=0.013ln x-0.021) for estradiol and R(2)=0.788 (y=3.835ln x-18.543) for progesterone. Neither fecal nor serum concentrations were affected by the breed but a significant effect of the number of fetuses on progestin concentrations was found. Therefore, the profiles of progesterone and estradiol fecal metabolites reflect the serum concentrations of the same hormones in pregnant goats.     

Excretion and measurement of estradiol and progesterone metabolites in the feces and urine of female squirrel monkeys (Saimiri sciureus)

The first objective of the present study was to determine the metabolic form and rate of excretion of ovarian hormone metabolites in the urine and feces of female squirrel monkeys injected with radiolabeled progesterone (Po) and estradiol. The major portion of the urinary metabolites of both hormones was excreted within 16-24 hr post-injection. Estrogen and Po isotopes in feces exhibited an excretion peak at 16 hr post-injection. The majority of recovered radiolabel of both hormones was excreted in feces. Chromatographic separation of fecal extractions indicated that the major estrogen metabolites in feces are in the free as opposed to the conjugated form. The radioactivity and immunoreactivity for estrone and estradiol (E(1) and E(2), respectively) in eluates of fecal samples subjected to celite co-chromatography indicated that both free E(1) and E(2) exist as excretion products in the feces of female squirrel monkeys. The major radioactive peaks for Po metabolites showed peaks in the elution profile at or very near the Po standard, and corresponded with the celite co-chromatography elution profile of Po standard when subjected to enzyme immunoassay (EIA). The second objective was to validate the application of EIA systems to measure fecal metabolites. Reproductive events of one female squirrel monkey across one annual reproductive cycle are described using the endocrine profile generated from fecal steroid assays. Examination of this profile confirmed that longitudinal fecal sampling and steroid hormone metabolite measurement in feces was not only feasible and practical, but accurately detected known reproductive events as well.     

Enzyme immunoassay of progesterone in the feces from beef cattle to monitor the ovarian cycle

The present study was undertaken to measure fecal progesterone concentration of beef cattle using antibody against authentic progesterone and to examine whether this method can monitor the ovarian cycle in beef cattle. Rectal fecal samples collected from 14 beef cattle were mixed with 6 ml of 100% methanol and shaken for 15 min. After centrifugation, supernatant was extracted with petroleum ether followed by an enzyme immunoassay (EIA) for progesterone. Specificity of the assay was examined by HPLC separation of fecal solution followed by the EIA in each fraction. The present assay identified only progesterone but not other metabolites in the feces sample that was extracted with petroleum ether. Sensitivity of the assay was estimated to be 0.0055 ng/ml (0.11 ng/g). Coefficient variations of intra- and inter-assay were 9.6-10.9% and 10.8-16.6%, respectively. Recovery rates ranged between 73 and 84%. Patterns in the fecal progesterone concentrations during the ovarian cycle were almost parallel to the plasma concentrations. A significant positive correlation was established between the fecal and plasma progesterone concentrations in individual animal (r=0.59-0.84, P<0.001, n=10) as well as pooled data (r=0.70, P<0.001, n=65). Fecal progesterone concentrations of day 0 (showing the nadir of concentration) of the ovarian cycle were less than 50 ng/g, which increased significantly toward day 9 (P<0.01). From days 14 to 18, there was significant reduction of fecal progesterone concentration (P<0.01). Ovarian cycles had at least 48 ng/g (mean=74 ng/g) of difference between minimum and maximum fecal progesterone concentrations. All cattle at days 9, 11 and 14 had higher fecal progesterone concentrations by more than 20 ng/g compared with day 0. These results suggest that the present EIA is suitable to measure the progesterone in cattle feces and can monitor ovarian cycle.     

Comparison of three fecal steroid metabolites for pregnancy detection used with single sampling in bighorn sheep (Ovis canadensis)

We conpared three fecal steroid metabolite assays for their usefulness in detecting pregnalcy among free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from Bighorn Canyon National Recreation Area, Wyoming and Montana (USA) and captive bighorn ewes at ZooMontana in Billings, Montana. Fecal samples were collected from 11 free-ranging, radio-collared bighorn ewes in late January-May 2001 and from 20 free-ranging, radio-collared ewes in late March to mid-May 2002. Free-ranging ewes were monitored the following spring to determine whether or not they lambed. In addition, two captive ewes were studied at ZooMontana. With three exceptions, free-ranging bighorn ewes that produced lambs had nonspecific progesterone metabolite (iPdG) levels of >1800 ng/g feces and iPdG levels >7000 ng/gm feces when samples were collected between early March and mid-May. Samples collected earlier in the year were inconclusive. One false negative was suspected to be the result of sample collection error. Of the captive ewes, nonspecific pregnanediol-3alpha-glucuronide (PdG) and iPdG followed a predictable curve over the course of the 180-day pregnancies. We conclude that estrone conjugates are not useful in diagnosing pregnancy; however, fecal steroid analysis of PdG and iPdG can be used to accurately determine pregnancy and reproductive function in bighorn sheep. This holds great potential as a noninvasive technique for understanding the role of reproductive disease in wild bighom sheep.     

Hormonal contraception of feral mares with Silastic rods.

Homogeneous Silastic rods containing ethinylestradiol (EE) (1.5 or 4 g), estradiol-17 beta (E) (4 g) or progesterone (P) (6 g) were implanted into feral mares (Equus caballus) between 4- and 10-yr-old. Six treatment groups (greater than or equal to 10 mares/group) of non-pregnant mares received 36 g P and 12 g E (P+E), 36 g P and 8 g EE (P+HEE), 1.5 g EE (LEE), 3 g EE (MEE, 8 g EE (HEE) or control-implanted mares (CI). CI received implants containing no steroid. Two groups of pregnant mares received P+HEE or HEE. Stallions were placed with the mares 15 to 26 mo after implanting. Blood was collected biweekly for up to 28 mo after implanting and serum analyzed for P by radioimmunoassay. A single P value greater than or equal to 2.5 ng/ml indicated ovulation and 2 consecutive values greater than or equal to 2.5 ng/ml indicated pregnancy. Serum from blood collected before and at 4, 12, 24, 50, 64 and 89 wk after implanting was analyzed for EE concentrations. All animals pregnant at the time of contraceptive placement delivered normal foals. Contraceptive efficacy for groups LEE, MEE, HEE and P+HEE were 75, 75, 100, and 100%, respectively after two breeding seasons. Suppression of ovulation appeared to be inversely related to the concentration of EE used in the implant. The percent of animals ovulating after 2 yr of contraception in each group was 100, 100, 88, 62, 20, and 12 for groups CI, P+E, LEE, MEE, HEE and P+HEE, respectively. The pregnancy rate for the same groups was 100, 78, 25, 25, 0 and 0%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seasonal variation in the endocrine-testicular function of captive jaguars (Panthera onca)

Captive adult male jaguars (Panthera onca) from two locations in southeast Brazil were studied to evaluate the effects of season on endocrine and testicular function. For assessment of testicular steroidogenic activity, androgen metabolite concentrations were measured in fecal samples collected one to three times per week over 14 ( n=14 ), 9 ( n=1 ) or 7 months ( n=1 ). To assess seasonality, data were grouped by season (summer: December-February; autumn: March-May; winter: June-August; spring: September-November). Additionally, samples collected in the dry season (March-August) were compared with those collected in the wet season (September-February). There were no differences ( P>0.05 ) in fecal androgen concentrations in samples collected in spring, summer, autumn, and winter ( 480.8+/-50.4 ng/g, 486.4+/-42.0 ng/g, 335.4+/-37.7 ng/g, and 418.6+/-40.4 ng/g dry feces). However, there were differences ( P<0.05 ) in fecal androgen concentrations between the dry and wet seasons ( 380.5+/-28.0 ng/g versus 483.9+/-32.3 ng/g dry feces). Sperm samples, collected from all males twice (approximately 6 months apart) were similar; mean (+/-S.E.M.) motility, concentration and morphology were 57.0 %4.5%, 6.3+/-2.4 x 10(6) ml(-1), and 60.8+/-3.1 %, respectively. In conclusion, androgen metabolite concentrations in the captive male jaguar were not affected by season, but there was a difference between the wet and dry periods. Further research is needed to verify these results.     

Assessment of pregnancy in Kiang mares (Equus hemionus holdereri ) using estrogen determination in feces

Analysis of fecal estrogens was used to diagnose pregnancy in 6 Kiang mares (Equus hemionus holdereri ) that were kept at Tierpark Berlin. Three extraction methods were compared and were followed by an established RIA for total estrogen. Extraction of desiccated feces with chloroform/n-hexane and KOH, with and without enzyme hydrolysis showed better results than extraction with diethylether without hydrolysis. Pregnancy was confirmed by observation of foaling in 2 mares that showed estrogen concentrations between 800 and 1800 ng/g and in 1 mare that showed widely fluctuating values between 500 and 1300 ng/g of feces. Two mares with estrogen concentrations below 500 ng/g were not seen to foal. The method using chloroform/n-hexane and KOH without enzyme hydrolysis seems practical for non-invasive evaluation of the endocrine status in this endangered Equidae species.     

Use of prostaglandin E2 to ripen the cervix of the mare prior to induction of parturition

Eleven light-breed pregnant mares (335 to 347 d gestaton) were used to evaluate the use of prostaglandin E2 as a cervical ripening agent prior to induction of parturition during the months of April and May. Six hours prior to induction, each mare's cervix was examined per vagina for softness and dilation. Each mare was then assigned to 1 of 2 treatment groups: Group PGE mares (n = 7) received 2.0 to 2.5 mg prostaglandin E2 deposited intracervically; Group SAL mares (n = 4) received 0.5 mL of sterile NaCl deposited intracervically. Six hours later, the mares were readied for parturition by wrapping the tail, scrubbing and rinsing the perineum and udder, and examining the cervix as previously described. Each mare was then administered 15 U, i.v. oxytocin at 15-min intervals until the chorioallantois ruptured. Intervals from initial oxytocin injection until rupture of the chorioallantois, from initial oxytocin injection until delivery of the foal, from delivery of the foal until the foal stood unassisted, and from delivery of the foal until the foal suckled were recorded. Mean cervical dilation immediately prior to induction of parturition tended to be greater in Group PGE mares (3.9 +/- 1.7 cm) than in Group SAL mares (1.9 +/- 1.9 cm; P = 0.10). Mean change in cervical dilation over the 6-h period prior to induction (3.4 +/- 1.9 cm vs 1.5 +/- 2.1 cm), mean number of injections of oxytocin required until the chorioallantois ruptured (1.9 +/- 0.7 vs 2.5 +/- 1.0), and mean intervals from initial injection of oxytocin to rupture of the chorioallantois (20 +/- 10 min vs 28 +/- 19 min) and delivery of the foal (28 +/- 7 min vs 34 +/- 22 min) were not different between Group PGE and SAL mares, respectively (P > 0.10). The proportion of foals that stood within 1 h of birth also did not differ between Group PGE foals (6/7; 86%) and Group SAL foals (3/4; 75%; Chi-square = 0.17; P > 0.10). The proportion of foals that nursed within 2 h of birth was higher in Group PGE foals (6/7; 86%) than in Group SAL foals (1/4; 25%; Chi-square = 4.02; P < 0.05). Premature separation requiring manual rupture of the chorioallantois at the vulvar labia occurred in 1 Group PGE mare (cervical dilation of 1.5 cm at time of induction) and 1 Group SAL mare (cervix closed and firm at time of induction). Foals born from the 2 mares with premature placental separation had the longest intervals from initial oxytocin injection to delivery, delivery to ability to stand unassisted, and delivery to suckling within their respective treatment groups. In summary, it appears that cervical ripening prior to induction of parturition favors shorter deliveries and foal vigor. Intracervical administration of prostaglandin E2 may prove useful for ripening the cervix of the mare prior to induction of parturition. Further studies are indicated to determine optimal dosage and method of administration of prostaglandin E2.     

Evidence for chronic stress in captive but not free-ranging cheetahs (Acinonyx jubatus) based on adrenal morphology and function

The cheetah (Acinonyx jubatus) is highly endangered because of loss of habitat in the wild and failure to thrive in captivity. Cheetahs in zoos reproduce poorly and have high prevalences of unusual diseases that cause morbidity and mortality. These diseases are rarely observed in free-ranging cheetahs but have been documented in cheetahs that have been captured and held in captive settings either temporarily or permanently. Because captivity may be stressful for this species and stress is suspected as contributing to poor health and reproduction, this study aimed to measure chronic stress by comparing baseline concentrations of fecal corticoid metabolites and adrenal gland morphology between captive and free-ranging cheetahs. Additionally, concentrations of estradiol and testosterone metabolites were quantified to determine whether concentrations of gonadal steroids correlated with corticoid concentration and to assure that corticosteroids in the free-ranging samples were not altered by environmental conditions. Concetntrations of fecal corticoids, estradiol, and testosterone were quantified by radioimmunoassay in 20 free-ranging and 20 captive cheetahs from samples collected between 1994 and 1999. Concentrations of baseline fecal corticoids were significantly higher (p = 0.005) in captive cheetahs (196.08 +/- 36.20 ng/g dry feces) than free-ranging cheetahs (71.40 +/- 14.35 ng/g dry feces). Testosterone concentrations were lower in captive male cheetahs (9.09 +/- 2.84 ng/g dry feces) than in free-ranging cheetahs (34.52 +/- 12.11 ng/g dry feces), which suggests suppression by elevated corticoids in the captive males. Evidence for similar sulppression of estradiol concentrations in females was not present. Adrenal corticomedullary ratios were determined on midsagittal sections of adrenal glands from 13 free-ranging and 13 captive cheetahs obtained between 1991 and 2002. The degree of vacuolation of cortical cells in the zona fasciculata was graded for each animal. Corticomedullary ratios were larger (p = 0.05) in captive cheetahs; however, there was no difference (p = 0.31) in the degree of corticocyte vacnolation between the two populations. These data proxile both mnorphologic and functional evidence suggestive of chronic stress in captive cheetahs. Further research into the role of hypercortisolemia in the pathogenesis of the reproductive abnormalities and unusual diseases of captive cheetahs is needed.     

Use of group-specific antibodies to detect fecal progesterone metabolites during the estrous cycle of cows

Progesterone is metabolized to pregnanediones and hydroxylated pregnanes prior to its fecal excretion. Therefore, use of progesterone antibodies underestimates the actual amount of fecal metabolites. To improve the methodology of noninvasive fecal progesterone metabolite analysis, enzymeimmunoassays (EIA) using group-specific antibodies against 5-reduced 20-oxo-pregnane-C3-conjugates were developed. Fecal and milk samples were collected at 1- to 2-d intervals during the morning and evening milking throughout 1 estrous cycle in dairy cows (n = 12). Six immunoreactive metabolites were detected in the feces with high performance liquid chromatography (HPLC), eluting as 5α- and 5β-reduced pregnanes containing a 20-oxo-group (20-oxo-pregnanes). Fecal samples of 3 cows were analyzed by 3 EIAs using antibodies against 4-pregnene-6α-ol-3,20-dione 6HS:BSA (6HS-progesterone), 5α-pregnane-3β-ol-20-one 3HS:BSA and 5β-pregnane-3β-ol-20-one 3HS:BSA, respectively. The follicular and luteal phases were identifiable with each EIA. Luteal phase values and the differences between mean follicular (Days 0 to 2 and 19 to 21) and luteal phase (Days 10 to 16) values obtained with the 5-pregnane EIAs were 3- to 4-fold higher than with the 6HS-progesterone EIA. Since results with the former 2 EIAs were almost identical, the remaining samples were only analyzed by the EIA for 5β-pregnane-3α-ol-20-one. Fecal 20-oxo-pregnane concentrations were parallel to milk progesterone values, but had a lag time of about 0.5 d; the coefficient of correlation (P < 0.001) was 0.76 (y = 155.2 × + 37.2). Fecal 20-oxo-pregnane concentrations during the follicular and luteal phase were 39.5 ± 2.2 and 341 ± 15.2 ng/g feces, respectively. In conclusion, fecal 20-oxo-pregnanes are significantly correlated to milk progesterone concentrations. They consist of several metabolites and compared to a 6HS-progesterone antibody, their evaluation was improved using antibodies against 5-reduced pregnanes.     

Plasma and fecal progestagen evaluations during and after the breeding season of the female vicuna (Vicuna vicuna )

Plasma and fecal progestagen patterns of female (n = 10) vicunas (Vicuna vicuna ) were determined about 1 to 2 mo before and until 4 mo after breeding. The vicunas were caught wild and were penned at the Lauca National Park (Chile, 4470 m above sea level) for 7 mo (December to June). Plasma and fecal samples before and during the mating period (January to March) were collected 4 to 5 times weekly, and once or twice weekly thereafter. The samples were analyzed by enzymeimmunoassays (EIA) using antibodies against progesterone and 20alpha-dihydroprogesterone. High performance liquid chromatography (HPLC) separations confirmed that progesterone and 20alpha-dihydroprogesterone predominated in the plasma, whereas in the feces several unconjugated, immunoreactive progestagen metabolites containing either a 20-oxo- or a 20alpha-OH-group occurred. The coefficients of correlation (n = 409; P < 0.01) between matched plasma and fecal samples were 0.39 and 0.53 for 20-oxo- and 20alpha-progestagens, respectively. Elevated (5 to 6 d) plasma and corresponding fecal progestagens after mating indicated cyclic corpus luteum activity in 5 of the animals. After the mating period (23.2 +/- 3.3 d), corpus luteum function in these 5 animals persisted, as it did in 3 other animals that were not observed to be mating. The persisting corpus luteum function was demonstrated by increased mean plasma and fecal progestagen concentrations (> 1 ng/ml and > 100 ng/g, respectively). Mean plasma 20alpha-dihydroprogesterone concentrations exceeded that of progesterone by about 1 ng/ml (P < 0.01). The results demonstrated that in addition to plasma progesterone, plasma 20alpha-dihydroprogesterone and noninvasive fecal progestagen evaluations are useful, valid tools for determining corpus luteum function in vicunas.     

17α-ethinylestradiol cometabolism by bacteria degrading estrone, 17β-estradiol and estriol

17alpha-ethinylestradiol (EE2), the active compound of the contraceptive pill, is a recalcitrant estrogen, which is encountered at ng/l levels in wastewater treatment plant (WWTP) effluents and rivers and can cause feminization of aquatic organisms. The aim of this study was to isolate micro-organisms that could remove such low EE2 concentrations. In this study, six bacterial strains were isolated from compost that cometabolize EE2 when metabolizing estrone (E1), 17beta-estradiol (E2) and estriol (E3). The strains belong to the alpha, beta and gamma-Proteobacteria. All six strains metabolize E2 over E1, at mug/l to ng/l concentrations. In 4 days, initial concentrations of 0.5 mug E2/l and 0.6 mug EE2/l were degraded to 1.8 +/- 0.4 ng E2/l and 85 +/- 16 ng EE2/l, respectively. No other metabolites besides E1, E2, E3 or EE2 were detected, suggesting that total degradation and cleavage of the aromatic ring occurred. This is the first study describing that bacteria able to metabolize E2, can subsequently cometabolize EE2 at low mug/l levels.     

Comparison of the effects of two GnRH antagonists on LH and FSH secretion,follicular growth and ovulation in the mare

The effects of two GnRH antagonists were tested in order to delay and/or synchronise ovulation in mares. Five mares received Antarelix (0.01 mg.kg(-1)), 5 mares received Cetrorelix (the same dose), 5 mares (control mares) received the vehicle intravenously, twice daily, for 8 days from the day the largest follicle reached 22 mm following prostaglandin administration. Ovulation was postponed in all mares injected with Antarelix (19.4 +/- 1.2 days after the beginning of the treatment) and in 2/5 mares injected with Cetrorelix (20 +/- 1 days) vs. 6.2 +/- 0.4 days in control mares. During the treatment, LH concentrations were strongly depressed in Antarelix and in Cetrorelix mares (1.6 +/- 0.1 and 3.8 +/- 0.5 ng.mL(-1) respectively vs. 21 +/- 2.5 ng.mL(-1) in control mares). In the 3 Cetrorelix mares which ovulated during the treatment. 2 initiated their LH surge at this moment. FSH concentrations were not affected in Antarelix or in Cetrorelix mares during the treatment (11.4 +/- 1.3 and 7.9 +/- 0.8 ng.mL(-1) respectively vs. 10.5 +/- 0.8 ng.mL(-1) in control mares). In conclusion, Antarelix seems more efficient than Cetrorelix for postponing ovulation in mares. The role of LH in antral follicular development before the preovulatory stage is confirmed.     

Effects of reproductive status and management on cortisol secretion and fertility of oestrous horse mares

Stressful events may contribute to low reproductive efficiency due to glucocorticoid-mediated inhibition of hormone secretion in a variety of species. We therefore investigated effects of stress related to management of mares around artificial insemination on secretion of cortisol and fertility parameters. To avoid further disturbance of mares by frequent blood sampling, faecal cortisol metabolites (fCM) were determined instead (sample collection at 8-h intervals). A total of 50 mares (16 maiden, 17 barren, 12 foaling, 5 teaching mares) were included in the study. Mares were brought to the AI centre in vans or trailers (driving time between 30 min and 5 h). Teaching mares were housed in the clinic and had therefore not to be transported. Mares were inseminated either with fresh/cooled-shipped or frozen semen. Rectal palpations and ultrasound examinations were performed at 24- to 48-h intervals, in animals inseminated with frozen semen at 6-h intervals during the last 48 h before ovulation. In maiden, barren and foaling mares, fCM concentrations in faeces tended to be higher than in teaching mares at all times after arrival at the AI centre. At 24 and 48 h after arrival, fCM concentrations in maiden mares were significantly higher than in teaching mares (24h: maiden mares 12.3+/-3.1 ng/g, barren mares 8.5+/-1.2 ng/g, foaling mares 11.0+/-2.4 ng/g, teaching mares 3.8+/-0.6 ng/g, p<0.05). The time from arrival at the AI centre to detection of ovulation did not differ among the different groups of mares and was 4.5+/-0.4, 5.0+/-0.5, 3.8+/-0.5 and 5.6+/-0.9 days in maiden, barren, foaling and teaching mares, respectively (n.s.). Pregnancy rates were 53, 53, 55 and 60%, respectively (n.s.). The time from arrival at the AI centre to detection of ovulation was 4.4+/-0.3 days and 4.9+/-0.3 days in mares inseminated with fresh/shipped (n=39) or frozen semen (n=11; n.s.), respectively. The frequency of follicular checks influenced fCM secretion and was statistically significant at 16 h before ovulation (fresh/shipped semen: fCM 6.9+/-0.7 ng/g faeces, frozen semen: fCM 16.9+/-5.2 ng/g faeces, p<0.01). In the mare, gynaecological examinations seem to act as stressors and may increase cortisol secretion. However, this does not negatively influence fertility and in animals familiar with that procedure fCM concentrations are not elevated.     

Concentrations of altrenogest in plasma of mares and foals and in allantoic and amniotic fluid at parturition

Treatment with the progestin altrenogest is widely used in pregnant mares. The fact that foals born from healthy mares treated with altrenogest until term suffered from neonatal problems raises the question of direct effects of altrenogest on vital functions in the neonate. We have therefore investigated altrenogest concentrations in maternal and neonatal blood plasma and in fetal fluids. Pregnant mares were treated with altrenogest orally once daily (0,088 mg/kg bodyweight, n = 7) or left untreated (n = 8) from 280 d of gestation until foaling. Altrenogest concentration was determined in plasma of the mares, their foals and in amniotic and allantoic fluid. The concentration of altrenogest in plasma from treated mares (2.6 ± 1.0 ng/mL) was significantly lower than in plasma from their foals immediately after birth (5.6 ± 1.9 ng/mL; p < 0.05), but was significantly higher than in their fetal fluids (amniotic fluid: 0.4 ± 0.1 ng/mL; p < 0.05; allantoic fluid: 3.0 ± 1.5 ng/mL). Altrenogest was undetectable in maternal and fetal plasma and fetal fluids of control pregnancies at all times. Altrenogest concentration in plasma of foals from treated mares was strongly correlated to the altrenogest concentration in plasma of their dams (r = 0.938, p < 0.001) and in amniotic (r = 0.886, p < 0.001) and allantoic fluid (r = 0.562, p < 0.05). A significant decrease in altrenogest concentration between the time periods 0-15 min, 30-120 min, and 180-360 min after parturition was seen in the plasma from foals born to altrenogest-treated mares. In conclusion, our data demonstrate that altrenogest reaches the equine fetus at high concentrations.     

Fertility control using intrauterine devices: an alternative for population control in wild horses

The purpose of this study was to develop a contraceptive method for feral horses. The feral horse population has increased significantly in recent years despite attempts to control numbers. As in most wild animal population control programs, contraceptive methods must be easy to apply, cause minimal disruption to the social structure and be fully reversible. In the present study, we tested the effectiveness of an intrauterine device (IUD) for fertility control in mares. Six mares were fitted with a silastic O-ring-shaped IUD on July 1 of Year 1. The IUD-treated mares were turned out with 12 nontreated mares and a fertile stallion in a large pasture until October 20 (112 d). None of the IUD-treated mares and all the nontreated mares became pregnant. The IUD-treated mares were maintained separately from the stallion during the winter. Following removal of the IUD on April 27 of Year 2, the mares were again introduced to the pasture with the stallion together with 6 nontreated mares. For the 6 mares previously treated with an IUD, the mean interval from introduction to the stallion to conception was 17.5 +/- 5 d or 1.3 cycles per pregnancy, and all mares produced a normal foal at term. Subsequently, 19 recorded mare breeding seasons resulted in 18 foals. Uterine cytology and histopathology indicate that the IUD causes mild chronic endometritis without permanent changes in the endometrium. We conclude that based on our observations, the O-ring-shaped IUD is an effective, safe and practical contraceptive method for mares.     

Direct evidence for linkage of roan and extension loci in Belgian horses

A bay roan Brabant Belgian stallion (ERn/ ern) was bred to eight chestnut American Belgian mares (ern/ ern), producing 57 foals. Thirty foals were bay roan, 25 were chestnut, one was bay, and one was chestnut roan. The recombination rate was 0.035 +/- 0.024, indicating fairly close linkage between the roan (Rn) and extension (E) loci.     

Fecal corticosterone assessment in the epaulette shark,Hemiscyllium ocellatum

The present study examined the feasibility of measuring the steroid hormone corticosterone in fecal extracts of epaulette sharks, Hemiscyllium ocellatum. Six immature, captive-raised epaulette sharks (four females and two males) were obtained from two different zoos and were maintained in a closed-system, 530-liter aquarium. After a one-month adaptation, fecal samples were collected daily from each animal for 33 days. Five-day sets of samples were pooled within animals to insure sufficient material for analysis. Fecal hormone extraction was achieved using repeated cycles of dichloromethane and aqueous washes. The levels of corticosterone were measured by reverse-phase high-performance liquid chromatography (HPLC). Corticosterone presence in HPLC eluent peaks from fecal extracts was determined by comparison of the elution pattern of corticosterone standard with the elution patterns of fecal extracts with and without the addition of tritiated corticosterone or exogenous, unlabeled corticosterone. Exclusive presence of corticosterone in HPLC eluent peaks presumed to be corticosterone was determined by nuclear magnetic resonance mass spectrometry. Corticosterone levels, calculated from a 10-point standard curve, ranged from 1.2 to 20.9 ng/g feces across all sharks, with 92.3% of values being < or =13.5 ng/g. Within individuals, the lowest average for corticosterone levels across 33 days was 2.6+/-0.4 ng/g feces, and the highest average was 8.4+/-2.2 ng/g feces. This study demonstrated that corticosterone was extractable from and reliably measurable in fecal extracts of epaulette sharks. This is the first evidence of this hormone in epaulette sharks and the first report of fecal corticosterone in elasmobranchs.