The robustness of faecal steroid determination for pregnancy testing Kaimanawa feral mares under field conditions

Aims: To investigate the utility of faecal oestrone sulphate (OS) concentrations for detecting pregnancy in mares during behavioural studies of feral horses, in which the collection and preservation of samples is not immediate.

Methods: Oestrone sulphate concentrations were measured in fresh dung samples collected from 153 free-roaming Kaimanawa mares throughout the year. In addition, multiple samples were taken from the same pile to investigate the reliability of diagnosis from a single sample, as well as the influence of time until preservation on OS concentrations. Samples were also taken before and after a 10mm simulated rainfall event to test for dilution of OS concentrations by rain. Oestrone sulphate concentrations in all samples were measured using an enzymeimmunoassay.

Results: From approximately 150 to 250 days of gestation, OS concentrations were consistently >80 ng/g in mares which subsequently foaled. Mares which did not foal and had low faecal OS concentrations in multiple samples throughout the year had faecal OS concentrations of 31 ±13 ng/g (mean ± s.d.) with an upper 95% confidence limit of 57 ng/g. Mares sampled from 1 week before to 1 month after behavioural oestrus, and that did not foal in the previous and subsequent seasons, had OS concentrations of 37 ± 32 ng/g (mean ± s.d.) with an upper 95% confidence limit of 100 ng/g.The standard error of oestrone sulphate concentrations in multiple samples from the same dung pile ranged from 1 to 37% of the mean. This large within-pile variation, however, did not result in incorrect diagnoses from single samples unless mares were within 18 days of parturition. Keeping samples at ambient temperatures for up to 16 hours did not affect OS concentrations. Simulated rainfall caused a 17% mean reduction in OS concentrations, but did not change pregnancy diagnoses.

Conclusions: Faecal OS concentrations > 100 ng/g were indicative of pregnancy in Kaimanawa mares. For mares more than 150 days post-mating, OS concentrations <57 ng/g were indicative of non-pregnancy, while concentrations between 57 and 100 ng/g provided an inconclusive diagnosis. A single sample from each dung pile collected within 16 hours of defecation was sufficient to accurately diagnose pregnancy in mares 150-250 days post conception.

Clinical Relevance: Measurement of OS concentrations in dung samples was a reliable and robust indicator of pregnancy status in feral mares 150-250 days post mating. This corresponds approximately to the period from May to August, given the seasonal breeding pattern in this population. This method of determining pregnancy status is suitable for field use in behavioural and demographic studies of wild horse populations.     

Enzymeimmunoassay of oestrone sulphate concentrations in faeces for non-invasive pregnancy determination in mares

AIM: To determine the suitability of measuring faecal oestrone sulphate (OS) by enzymeimmunoassay as a means of determining pregnancy status in mares bred under New Zealand conditions. METHODS: An antibody-coated microtitre plate-based enzymeimmunoassay was used to determine the concentration of OS in faecal and plasma samples obtained from pregnant and non-pregnant mares. RESULTS: In non-pregnant mares, the mean faecal OS concentration was 34 ng/g, and the value three standard deviations above this was 80 ng/g. None of 427 faecal samples collected from 116 non-pregnant mares over a l-year period had an OS concentration >80 ng/g. Only five samples from three mares had an OS concentration >65 ng/g, the value two standard deviations above the mean non-pregnant value. Analysis of faecal OS concentrations in 532 faecal samples collected from 39 pregnant mares showed that as pregnancy progressed, an increasing proportion of faecal samples had OS concentrations >80 ng/g. None of the mares 150 days or more pregnant had faecal OS concentrations <50 ng/g, and 204/220 samples obtained from these mares had faecal OS concentrations >80 ng/g. Following foaling or foetal death, elevated faecal OS concentrations returned quickly to non-pregnant levels. The mean +/- s.e.m. plasma level of OS in five mares bled daily throughout one oestrous cycle was 1.7 +/- 0.2 ng/ml. Sixty-eight blood samples from pregnant mares bled up to five times between 92 days after mating and foaling all had plasma OS concentrations >30 ng/ml, with 64/68 being >50 ng/ml. CONCLUSIONS: This study shows that measuring faecal OS concentrations by enzymeimmunoassay offers a convenient, accurate, non-invasive means of determining pregnancy status in mares from 150 days after mating onwards. Mares with faecal OS concentrations <50 ng/g can be considered not pregnant, while mares with faecal OS concentrations >80 ng/g can be considered pregnant. Those few mares returning a faecal OS concentration between 50 and 80 ng/g should be retested to obtain a conclusive result. Measuring plasma OS concentrations allows pregnancy status to be determined earlier (from 100 days after mating). Moreover, the discrimination between non-pregnant and pregnant levels is greater for OS in plasma than in faeces. CLINICAL RELEVANCE: Measurement of OS concentrations in faeces provides an alternative and non-invasive means of determining pregnancy status in mares from 150 days after mating.     

Concentrations of oestrone sulphate during pregnancy in milk from Jersey and Friesian dairy cows differing in milk yields and composition

Oestrone sulphate concentrations were measured by radioimmunoassay in milk samples obtained weekly during pregnancy from Jersey and Friesian cows, with each breed grazed at two different stocking rates. Mean milk yields differed significantly (P<0.05) between the four herds, while mean percentage milk fat and protein values differed significantly (P<0.05) between the two breeds. In all four herds, oestrone sulphate concentrations in milk rose progressively during pregnancy from a mean value of approximately 80-100 pg/ml at 60-80 days of pregnancy to a plateau value of approximately 1 ng/ml at 181-200 days. In non-pregnant cows, oestrone sulphate concentrations in milk ranged from non-detectable to 110 pg/ml, with a mean +/- s.e.m. value of 59 +/- 4 pg/ml. There was considerable variation in milk oestrone sulphate concentrations between cows in each herd, and oestrone sulphate concentrations could also fluctuate markedly within cows from week to week. Despite this variation, the concentration of oestrone sulphate in 98% of milk samples obtained after 120 days of pregnancy was greater than the highest concentration found in milk from non-pregnant cows. Measurement of oestrone sulphate concentrations in milk samples taken at least 120 days after mating or insemination may provide an alternative, non-invasive means of determining or confirming pregnancy in New Zealand dairy cows.     

Effects of short-term early gestational exposure to endophyte-infected tall fescue diets on plasma 3,4-dihydroxyphenyl acetic acid and fetal development in mares

Consumption of wild-type (toxic) endophyte-infected tall fescue (E+) by horses during late gestation is known to adversely affect pregnancy outcome; however, little is known of the potential disruptive consequences of E+ consumption by mares during the critical phases of placentation and fetal development in early pregnancy. The objective of this study was to evaluate the detrimental effects of feeding E+ to mares during early gestation. Mares (n = 12) paired by stage of gestation (d 65 to 100) were assigned to diets (six per diet) consisting of endophyte-free (E-) or E+ tall fescue seed (50% E- or E+ tall fescue seed, 45% sweet feed, and 10% molasses fed at 1.0% of BW/d). Mares also had ad libitum access to E+ or E- annual ryegrass hay, and were fed diets for 10 d. Following removal from the tall fescue diet on d 11, mares were placed on common bermudagrass pasture and monitored until d 21. Morning and evening rectal temperatures were recorded and daily blood samples were collected for progesterone and prolactin (PRL) analyses, whereas samples for 3,4-dihydroxyphenyl acetic acid (a catecholamine metabolite) analysis were collected on alternate days. For clinical chemistry analysis, blood samples were collected on d 0, 5, 10 and 21. Daily urine samples were collected for ergot alkaloid analysis, and ultrasonography was performed for presence of echogenic material in fetal fluids. Rectal temperatures (E+ 37.76+/-0.03; E- 37.84+/-0.03 degrees C) and serum PRL concentrations (E+ 14.06< or =0.76; E- 12.11+/-0.76 ng/mL) did not differ (P = 0.96) between treatments. Measuring the change in basal serum concentration from d 0 over time, progesterone concentrations did not differ (-0.64 +/-1.49 and -0.55+/-1.47 ng/mL for E+ and E- mares, respectively). There was no negative pregnancy outcome, and ultrasonography indicated no increase in echogenic material in fetal fluids. Plasma 3,4-dihydroxyphenyl acetic acid concentrations decreased (P < 0.05) in E+ compared with E- mares (2.1+/-0.14 and 4.4+/0.43 ng/mL, respectively). Urinary ergot alkaloid concentration was greater (P < 0.01) in mares consuming E+ compared with E- (532.12+/- 52.51 and 13.36+/-2.67 ng/mg of creatinine, respectively). Although no fetal loss was observed during the current study, elevated concentrations of urinary ergot alkaloid were consistent with depressed endogenous catecholamine activity, suggestive of an endocrine disruptive effect of hypothalamic origin.     

Pregnancy status determination in mares using a rapid lateral flow test for measuring serum oestrone sulphate

AIMS: To develop a means of determining pregnancy status in horses based on measuring serum oestrone sulphate (OS) concentrations using a rapid lateral flow immunoassay, and to determine the assay's effectiveness using a visual end-point. METHODS: Serum samples from mares >100 days post-mating (n=701) were assayed using a nitrocellulose membrane-based lateral flow immunoassay device. The device was developed using membrane-bound 1,3,5 (10)-estratrien-3-ol-17-one conjugated to bovine serum albumin as the capture antigen, and an OS-detection monoclonal antibody coupled to colloidal gold as the visible detection reagent. Concentrations of the coating antigen and OS monoclonal antibody were optimised so that the working range would allow pregnancy status to be determined from a visual end-point. The test was run by adding 0.1 ml serum to the sample well of a plastic cassette encasing the test membrane. As the serum migrated along the membrane, a test dot and control line were generated on it within 5-10 min. The intensity of the test dot was inversely proportional to the concentration of OS in the serum sample being tested. Results were compared with those from a validated OS enzyme immunoassay (EIA) and subsequent foaling or return to oestrus of the mares. RESULTS: Serum samples with OS concentrations <10 ng/ml, indicative of non-pregnancy in mares >100 days post-mating, generated a test end-point consisting of a highly visible test dot and control line, whereas serum OS concentrations >50 ng/ml, indicative of pregnancy, generated a control line only. The test correctly identified 384/389 (98.7%) non-pregnant mares tested, and 303/312 (97.1%) pregnant mares tested that were >100 days post-mating. The lateral flow test devices were stable for at least 12 months when stored at 4 degrees C, sealed in aluminium pouches with desiccant. CONCLUSION: This novel, rapid, easy-to-use, lateral flow immunoassay offers a practical alternative to traditional laboratory- based immunoassays for measuring serum OS concentrations in mares for determining their pregnancy status.     

Pregnancy status determination in mares using a rapid lateral flow test for measuring serum oestrone sulphate

AIMS: To develop a means of determining pregnancy status in horses based on measuring serum oestrone sulphate (OS) concentrations using a rapid lateral flow immunoassay, and to determine the assay's effectiveness using a visual end-point.

METHODS: Serum samples from mares >100 days post-mating (n=701) were assayed using a nitrocellulose membrane-based lateral flow immunoassay device. The device was developed using membrane-bound 1,3,5 (10)-estratrien-3-ol-17-one conjugated to bovine serum albumin as the capture antigen, and an OS-detection monoclonal antibody coupled to colloidal gold as the visible detection reagent. Concentrations of the coating antigen and OS monoclonal antibody were optimised so that the working range would allow pregnancy status to be determined from a visual end-point. The test was run by adding 0.1 ml serum to the sample well of a plastic cassette encasing the test membrane. As the serum migrated along the membrane, a test dot and control line were generated on it within 5–10 min. The intensity of the test dot was inversely proportional to the concentration of OS in the serum sample being tested. Results were compared with those from a validated OS enzyme immunoassay (EIA) and subsequent foaling or return to oestrus of the mares.

RESULTS: Serum samples with OS concentrations <10 ng/ml, indicative of non-pregnancy in mares >100 days post-mating, generated a test end-point consisting of a highly visible test dot and control line, whereas serum OS concentrations >50 ng/ml, indicative of pregnancy, generated a control line only. The test correctly identified 384/389 (98.7%) non-pregnant mares tested, and 303/312 (97.1%) pregnant mares tested that were >100 days post-mating. The lateral flow test devices were stable for at least 12 months when stored at 4°C, sealed in aluminium pouches with desiccant.

CONCLUSION: This novel, rapid, easy-to-use, lateral flow immunoassay offers a practical alternative to traditional laboratory-based immunoassays for measuring serum OS concentra- tions in mares for determining their pregnancy status.     

Review of seven cases of granulosa cell tumour of the equine ovary

This paper reviews the equine granulosa cell tumour (GCT) and describes the clinicopathological features, treatment and outcome in seven cases of GCT in mares. Mares were presented with unilateral ovarian enlargement during the 2007 to 2010 breeding seasons. The mean (sd) age of the mares was 11.7 (5.96) years. Three mares were multiparous barren, three were nulliparous and one was primigravida. Behaviour at presentation was 57 per cent anoestrus, 28 per cent with stallion-like behaviour and 14 per cent with persistent oestrus. All mares had unilateral ovarian enlargement. Six non-pregnant mares had a small and inactive contralateral ovary; the pregnant mare had a single small corpus luteum on the contralateral ovary and was at three-and-a-half months' gestation. Enlarged ovaries measured 7 cm to an estimated 30 cm in diameter. 28 per cent had a multicystic ultrasound appearance, 57 per cent were dense structures and 14 per cent were of mixed appearance. Mean concentrations of progesterone were <1 ng/ml, oestrone sulphate 3.06 (2.32) ng/ml and testosterone 0.58 (0.64) nmol/l in non-pregnant mares. Inhibin was elevated in all non-pregnant cases at 7.6 (12.45) ng/ml. The pregnant mare had concentrations of progesterone 2.5 ng/ml, oestrone sulphate 81.0 ng/ml, testosterone 1.9 nmol/l and inhibin 1.31 ng/ml. Mares demonstrating stallion-like behaviour had a significantly higher (P<0.001) testosterone concentration (1.85 [0.07] nmol/l) than those that did not (0.34 [0.26] nmol/l). Three mares underwent unilateral ovariectomy and resumed cyclic ovarian activity within nine months of surgery.     

Seasonal serum concentrations of melatonin in cycling and noncycling mares

To determine whether secretory patterns of melatonin change throughout the seasons in mares, blood samples were drawn byvenipuncture from nine mares at noon and midnight for five successive days at monthly intervals from August through July at the University of Missouri in Columbia, MO. In addition, during September, December, March, and June, blood samples were drawn from indwelling catheters at 2-h intervals for 48 or 72 h. Mares were predominantly Quarter Horses weighing approximately 450 kg and ranged from 3 to 12 yr of age. Mares were housed in outdoor paddocks with three-sided run-in sheds for shelter. During the noon and midnight bleeding period, mares were placed in a larger open-sided barn with outside runs. Mares remained outdoors with the barn being used as a shelter in the event of inclement weather. All lights in the shed were converted to red light. Often, moonlight provided enough illumination to collect blood samples. Mares were returned to their normal paddock after each sampling period. For analysis of data, a mare was considered to be cycling if serum concentrations of progesterone were greater than 1 ng/ mL. For a mare to be classified as exhibiting a nocturnal rise of melatonin, serum concentrations of melatonin had to be at least two times greater at midnight than at noon. By month, a relationship did not exist (chi2; P > 0.05) among mares that were exhibiting estrous cycles and exhibiting nocturnal rises of melatonin. Likewise, examination of serum profiles of melatonin taken at 2-h intervals for 48 h revealed considerable variation among mares throughout the seasons. A nocturnal rise in serum melatonin was observed only in June (P < 0.02). In March and December, serum melatonin was greater in cycling mares than noncycling mares, but the elevation was not associated with light-dark periods (P < 0.01). Two of the mares exhibited estrous cycles throughout the seasons but melatonin secretion in these two mares were similar to that observed in the seven mares that demonstrated seasonal anestrous. From these results, it does not appear that changes in serum concentrations of melatonin are used as a cue to regulate cyclic activity in the mare throughout the seasons.     

Stress Response of Three-year-old Horse Mares to Changes in Husbandry System During Initial Equestrian Training

For initial training, horses are often transferred from group housing to individual boxes, which is a potential stressor. In this study, salivary cortisol concentrations, locomotion activity, and heart rate (HR) were analyzed and the HR variability (HRV) variables standard deviation of beat-to-beat interval (SDRR) and root mean square of successive RR differences (RMSSD) were calculated in 3-year-old mares (n = 8). Mares were transferred abruptly from a group stable with access to a paddock to individual boxes without a paddock and were studied from 4 days before to 5 days after changing the stable. Mares underwent routine equestrian training for young horses. On the days before mares were moved to individual boxes, cortisol concentrations showed a diurnal rhythm with values approximately 0.6 ng/ml in the morning and a decrease throughout the day. When horses were moved to individual boxes, cortisol concentrations increased to 1.8 ± 0.2 ng/ml within 30 minutes and did not return to baseline values within 6 hours (0.7 ± 0.1 ng/ml, P < .05 over time). On the following days, a diurnal rhythm was re-established but at a higher level than before the change of stable. Locomotion activity was higher when mares had access to a paddock than when kept in individual boxes. Heart rate increased for approximately 60 minutes when mares were separated from their group. In conclusion, separating young horses from their group and individual stabling are perceived as stressful.     

Effect of a Nonsurgical Embryo Transfer Procedure and/or Altrenogest Therapy on Endogenous Progesterone Concentration in Mares

Endogenous progesterone levels may decline after transcervical embryo transfer in some mares. Progestogen therapy is commonly used to support endogenous progesterone levels in embryo transfer recipient mares or those carrying their own pregnancy. The goal of this study was to determine the effects of the transcervical transfer procedure and/or altrenogest therapy on luteal function in mares. Mares were assigned to one of six treatment groups: group 1 (untreated control; n = 7 cycles), group 2 (sham transfer, no altrenogest; n = 8 cycles), group 3 (sham transfer plus altrenogest; n = 8 cycles), group 4 (pregnant, no altrenogest; n = 9 mares), group 5 (pregnant plus altrenogest; n = 9 mares), and group 6 (nonpregnant plus altrenogest; n = 10 cycles). Mares in groups 4-6 were bred and allowed an opportunity to carry their own pregnancy. Blood samples were collected for 22 days beginning on the day of ovulation. Sham embryo transfer (groups 2 and 3, combined) did not result in a decline in endogenous progesterone levels compared with control mares (group 6). However, sham embryo transfer did result in luteolysis and an abrupt decline in endogenous progesterone levels in one of the 16 (6.2%) sham-transferred mares. Altrenogest therapy in sham-transferred mares (group 3) was associated with lower endogenous progesterone levels on days 10, 12, and 13 postovulation when compared with sham-transferred mares that did not receive altrenogest (group 2). Administration of altrenogest to pregnant mares (group 5) was associated with lower concentrations of endogenous progesterone from days 14 to 18 and on day 21 compared with endogenous progesterone levels in pregnant mares not administered altrenogest (group 4). In conclusion, a transcervical embryo transfer procedure can cause luteolysis in a low percentage of mares. Altrenogest therapy may be associated with a reduction in endogenous progesterone secretion, presumably mediated by a reduction in pituitary luteinizing hormone (LH) release and a decrease in luteotropic support.     

Measurement and clinical significance of equine fetal protein in pregnant mares serum

An assay for the determination of the concentration of an equine fetal protein (EQFP) in pregnant mares serum was developed and used to quantitate more than 2000 serum samples from Thoroughbred mares during pregnancy. The concentration of EQFP increased throughout pregnancy. Mares with unproblematic (normal) pregnancies were used to establish the reference values at given gestational ages (GA). Mares with pregnancy failures (n=58) were found to have EQFP concentrations outside the reference values established at given GA's; 10 were high and 48 were low. Twin pregnancies (n=21), placentitis and impending abortion (n=71) were associated with elevated concentration. Twin pregnancies were detected by elevated EQFP concentration, in one case as early as 12 days after breeding.     

Use of progesterone in microspheres for maintenance of pregnancy in mares.

Administration of progesterone in poly(d-,l-lactide) microspheres was used to maintain pregnancy in mares after luteolysis was induced by treatment with prostaglandin F2 alpha at day 14 of pregnancy. Mares were given vehicle only (control, n = 6) or 0.75 g (n = 7), 1.5 g (n = 8), or 2.25 g (n = 5) of microencapsulated progesterone at days 12 and 22 of pregnancy. Serum progesterone concentrations were determined daily, and pregnancy was evaluated by transrectal ultrasonography on alternate days. Significantly (P less than 0.05) more mares given 1.5 or 2.25 g of progesterone (6 of 8 and 4 of 5 mares, respectively), but not those given 0.75 g (3 of 7 mares), maintained pregnancy through day 32, compared with control mares (0 of 6). Progesterone concentrations decreased significantly (P less than 0.025) in all groups after administration of prostaglandin F2 alpha at day 14, and significant (P less than 0.05) effects of time and treatment on progesterone concentrations were found between days 12 and 22, and 22 and 32. Although treatment with 1.5-g and 2.25-g doses of microencapsulated progesterone improved maintenance of pregnancy, compared with that of vehicle-treated controls, administration of 2.25 g of microencapsulated progesterone appeared to be most efficacious in maintenance of pregnancy during the study interval.     

Serum thyroxine concentrations and pregnancy rates 15 to 16 days after ovulation in broodmares

OBJECTIVE: To determine whether serum thyroxine (T4) concentration was associated with pregnancy rates 15 to 16 days after ovulation in mares and to determine whether thyroid hormone supplementation would enhance fertility in mares. DESIGN: Cohort study. ANIMALS: 329 clinically normal broodmares. PROCEDURE: Mares were examined 15 to 16 days after ovulation to determine whether they were pregnant; blood samples for determination of serum T4 concentration were collected at the same time. Sixty mares were receiving thyroid hormone supplementation prior to the study because of low serum T4 concentration (< 16 microg/dl) prior to breeding. RESULTS: Serum T4 concentration ranged from 4.5 to 53.9 mg/dl. Forty (12%) mares had low (< 16 microg/dl) concentrations, 283 (86%) had normal concentrations, and 6 (2%) had high (> 45 microg/dl) concentrations. Two hundred thirty-one mares were pregnant 15 to 16 days after ovulation. A significant association between serum T4 concentration (low, normal, or high) and pregnancy (yes or no) was not detected, and logistic regression analysis indicated that serum T4 concentration was not significantly related to pregnancy. Of the 269 mares not receiving thyroid hormone supplementation, 187 were pregnant, and of the 60 mares receiving thyroid supplementation, 44 were pregnant. There was no significant relationship between thyroid hormone supplementation and pregnancy status. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that serum T4 concentration in mares is not significantly associated with pregnancy 15 to 16 days after ovulation. Results also suggest that supplementation of mares that only have low T4 concentrations is not indicated or likely to be beneficial.     

Quantitative and qualitative assessment of milk production after pharmaceutical induction of lactation in the mare

The induction of lactation is performed in ruminants by steroidogenic impregnation, followed by drugs intended to increase prolactin secretion. The aim of this study was to induce lactation in barren mares and to evaluate milk production. Five treated and 5 control mares were used in June and September in year 1, and 12 mares were used in year 2. Mares were administered a vaginal pessary (500 mg altrenogest and 50 mg estradiol benzoate) for 1 week. The 2nd week, another sponge with 100 mg estradiol benzoate was administered, together with 50 mg/100 kg body weight (BW) sulpiride in oil (IM q12h). All mares were milked by hand. Drug treatment was stopped after I L was obtained. Milk production and composition and plasma prolactin concentration were measured. In year 2, the same steroid treatment was applied, but mares received sulpiride (n = 6) or domperidone (1.1 mg/kg PO q12h) (n = 6). A milking machine and oxytocin injections 1 minute before the start of milking were used. In year 1, all treated mares started milking within 1-5 days after sulpiride treatment. Mean daily milk production was 0.88 +/- 0.52 L/500 kg BW. Milk immunoglobulin G (IgG) contents increased in all mares (IgG concentration range, 14-92 g/L). Plasma prolactin increased during sulpiride treatment (range. 27.7 +/- 2.9 to 43.7 +/- 6.7 ng/mL [before] to 289.0 +/- 7.8 ng/mL during treatment, P < .001). In year 2, results were similar to those in year 1, with peak IgG concentrations ranging from 4.2 to 106.7 g/L and a larger daily milk production (3.13 +/- 0.75 with sulpiride and 3.45 +/- 0.51 L/500 kg BW with domperidone). In conclusion, lactation can be induced in mares within 2 weeks, and some mares produce good-quality colostrum.     

Melengestrol acetate as a tool for inducing early ovulation in transitional mares

The efficacy of melengestrol acetate (MGA) to shorten the vernal transition of mares by synchronising and accelerating the first ovulation of the year after 60 days of phototherapy was determined by ultrasonographic monitoring. Sixteen mares in late transition were fed two doses of MGA (150 mg/mare/day and 100 mg/mare/day, respectively) for 10 days. A luteolytic dose of prostaglandin was administered to each mare one day after the end of MGA treatment. The presence and duration of oestrus, follicular growth, uterine oedema and presence of ovulation were monitored by ultrasonography and the cervical tone was evaluated by rectal palpation. Ovulation was detected in 87.5% of the mares treated with 150 mg MGA/mare/day for 10 days, and in 62.5% of the mares receiving 100 mg MGA/mare/day for 10 days. This was statistically different (P = 0.03) from the untreated control mares having an ovulation rate of 20%. Mares that received 150 mg MGA/day for 10 days had a mean treatment to ovulation interval of 13.1 +/- 5.97 days after the end of treatment, while mares that received 100 mg MGA/day for 10 days had a mean of 25.6 +/- 10.50 days (P = 0.01) to ovulation. These results suggest that MGA can be used for synchronising and hastening the first ovulation of the year in mares.     

Prolactin and progesterone concentrations during early pregnancy and relationship to pregnancy loss prior to day 45

Eleven multiparous Quarter Horse and Thoroughbred mares were used to determine the plasma concentrations of progesterone and prolactin during early pregnancy and to examine the relationship of plasma progesterone and prolactin to pregnancy loss prior to d 45 of gestation. Plasma samples were collected at two day intervals beginning on d 14 of pregnancy (d 0 = ovulation) and countinued to d 80. Ovulation and pregnancy status were determined by ultrasonography. Four mares experienced pregnancy loss between d 28 and 44 and plasma samples were collected for 10 days beyond the detected loss. Seven mares had successful pregnancies (Term group).Plasma progesterone concentrations peaked by d 28 in the Term group, with individual peak values ranging from 14.9 to 31.9 ng/ml. Values then declined until d 36, followed by a rise until d 80. Prior to d 45 of gestation 5 of the 7 mares had a peak in excess of 15 ng/ml and 3 of these had brief periods, ranging fron 1 to 8 days when progesterone dropped to less than 2 ng/ml. The other 2 mares had peak plasma progesterone concentrations of less than 7 ng/ml, but maintained concentrations in excess of 2 ng/ml during this period.Within the mares experiencing pregnancy loss, 2 mares had a decline in plasma progesterone concentrations prior to pregnancy loss, while the other 2 had typical progesterone patterns beyond the detected loss. Differences in individual patterns of plasma progesterone concentration were observed in both groups (P<.01).Plasma prolactin concentrations ranged from less than .32 to 4.58 ng/ml in the Term group and from .31 to 1.9 ng/ml in the mares experiencing pregnancy loss. Differences in the individual patterns of prolactin secretion were observed in both groups (P<.01).A correlation between progesterone and prolactin was observed in the Term group between day 14 and 37 (P<.01, r²=.88).     

Dimeric inhibin concentrations in mares with granulosa-theca cell tumors

OBJECTIVE: To determine whether concentrations of dimeric inhibin (CaCA) are greater in plasma and tumor fluid from mares with granulosa-theca cell tumors (GTCT), compared with concentrations in plasma and equine follicular fluid (eFF) from control mares. ANIMALS: 6 mares with GTCT and 12 clinically normal mares. PROCEDURE: The alphabetaA immunoradiometric assay used 2 antibodies, one against each subunit of inhibin (alpha and betaA subunits). Tumor tissue, tumor fluid, and a single blood sample were collected at the time of surgical removal of the GTCT. A single blood sample was collected from 7 control mares during various stages of the estrous cycle. Five other control mares were ovariectomized when their ovaries contained growing follicles of 25 to 35 mm in diameter. A blood sample and eFF from the largest follicle were collected at the time of ovariectomy. RESULTS: Mares with GTCT had significantly greater plasma concentrations of betabetaA (mean +/- SEM, 0.86 +/- 0.53 ng of recombinant human-alphabetaA/ml), compared with control mares (0.14+/-0.02 ng/ml). Concentrations of alphabetaA in tumor fluid and eFF were similar. Concentrations of alphabetaA were significantly lower after ovariectomy. CONCLUSIONS AND CLINICAL RELEVANCE: Dimeric inhibin concentration was higher in plasma from mares with GTCT than in plasma from control mares. Increased granulosa cell mass and loss of mechanisms regulating alphabetaA release in mares with GTCT likely accounted for the increase in plasma concentrations. Measurement of alphabetaA concentrations may be useful for identifying mares with GTCT.     

Mid‐gestation pregnancy is not disrupted by a 5‐day gastrointestinal mucosal cytoprotectant oral regimen of misoprostol

Reasons for performing study: To investigate effects of a 5‐day oral misoprostol regimen recommended for use in horses as a gastrointestinal mucosal cytoprotectant during colic on mid‐gestation pregnancies. Objectives: To monitor cervical tone, ultrasonographic characteristics of the uterus, cervix and conceptus, as well as serum progesterone and oestrone sulphate concentrations, and observations of general health, behaviour and comfort of mid‐gestation mares given a 5‐day course of misoprostol or control treatment. Methods: Eleven light horse and pony mares with known breeding dates were administered 5 µg/kg bwt misoprostol orally, twice daily for 5 days. General health and pregnancy status were monitored daily during treatment via general physical examination, as well as palpation and ultrasonography per rectum of the uterus, cervix and conceptus. Jugular serum was obtained during and for 5 days following treatment for assay of progesterone and oestrone sulphate concentrations. Additionally, daily 12 h video samples of the mares were obtained to evaluate behaviour and comfort. Results: All findings, including cervical tone, ultrasonographic characteristics of the uterus, cervix and conceptus, as well as progesterone and oestrone sulphate concentrations, and observations of general health, behaviour and comfort, were similar during misoprostol and control treatment. Conclusions: Treatment of pregnant mares with a gastrointestinal mucosal cytoprotectant regimen of oral misoprostol for 5 days did not disrupt pregnancy, nor adversely affect the general health and comfort of these mares. Additional investigation of treatment at earlier and later stages of gestation, for longer‐term treatment, as well as evaluating neonates for developmental disturbances, would add further information on safety of misoprostol during gestation. Potential relevance: These results provide some assurance of safety of a 5‐day gastrointestinal mucosal cytopretectant regimen of oral misprostol in mid‐gestation pregnant mares.     

Effect of repeated administration of oxytocin during diestrus on duration of function of corpora lutea in mares

OBJECTIVE: To determine whether IM administration of exogenous oxytocin twice daily on days 7 to 14 after ovulation blocks luteolysis and causes prolonged function of corpora lutea (CL) in mares. DESIGN: Prospective study. ANIMALS: 12 mares. PROCEDURES: Beginning on the day of ovulation (day 0), jugular blood samples were collected every other day until day 40 for determination of progesterone concentration. On day 7, mares (n = 6/group) were treated with saline (0.9% NaCl) solution (control group) or oxytocin. Beginning on day 7, control mares received 3 mL of sterile saline solution every 12 hours, IM, and oxytocin-treated mares received 60 units of oxytocin every 12 hours, IM, through day 14. Mares were considered to have prolonged CL function if progesterone concentration remained > 1.0 ng/mL continuously through day 30. RESULTS: The proportion of mares with prolonged CL function was significantly higher in the oxytocin-treated group (6/6), compared with the control group (0/6). All control mares underwent luteolysis by day 16, at which time their progesterone concentrations were < 1.0 ng/mL. In contrast, all 6 oxytocin-treated mares maintained progesterone concentrations > 1.0 ng/mL continuously through day 30. CONCLUSIONS AND CLINICAL RELEVANCE: IM administration of 60 units of oxytocin twice daily on days 7 to 14 after ovulation was an efficacious method of inhibiting luteolysis and extending CL function in mares. Disrupting luteolysis by administering exogenous oxytocin during diestrus appears to be a plausible and practical method of long-term suppression of estrus in mares.     

Plasma prolactin concentrations in mares and their neonates after oxytocin induction of parturition

Studies were undertaken to investigate the effects of oxytocin induction on prolactin release in term (Group II) and preterm (Group III) mares and to compare these effects to spontaneously foaling mares (Group I). Since physiological concentrations of prolactin in blood have not been measured in the neonatal foal, experiments were designed to monitor prolactin in the cord artery and jugular blood of the foals from all groups of mares. Although prolactin levels varied in term mares (Group I and II) during the last 11 days of pregnancy, an increase was observed between Day -6 and Day 0 (2.7 and 11.9 ng/ml respectively; P less than 0.1). The average concentration of prolactin over the last 4 days (Days -3 to 0) had increased by 40% when compared to the average concentration on Days -6, -5, and -4. These findings indicate a rising trend which appears to occur concomitantly with changes in concentrations of 2 mammary components tested, sodium and potassium. Prolactin concentrations did not significantly increase in term mares after oxytocin treatment or in spontaneously foaling mares. However, the preterm induced mares had higher prolactin concentrations during the first stage of labor (19.3 +/- 7.2 ng/ml) than prior to treatment with oxytocin (4.7 +/- 2.0 ng/ml; P less than 0.01). Levels of prolactin in all groups significantly declined by 20-min post-placental expulsion. For the first 30 min after birth, prolactin concentrations in foals from oxytocin-induced mares appeared to be 2-fold higher than those from spontaneously foaling mares. Thereafter, prolactin values declined to baseline values by 48 hrs. When comparing cord arterial plasma with cord venous plasma in each group, prolactin concentrations were similar. However, the average prolactin levels in both the cord artery and vein appeared higher (ave: 1.1 ng/ml) in Group II and III than in Group I (less than 0.5 ng/ml). From these results, the authors suggest that 1) prolactin may have a role in regulating mammary secretory products in mares just prior to parturition; 2) oxytocin may increase prolactin secretion in preterm induced mares; 3) oxytocin induction may have a short term effect to increase circulatory prolactin concentrations in neonates in utero regardless whether their dams were treated preterm or term.