Determination of serum anti‐Müllerian hormone concentrations for the diagnosis of granulosa‐cell tumours in mares

Reasons for performing study: Endocrinological assays are important for evaluation of mares with granulosa‐cell tumours (GCTs), and our research in mares indicates that anti‐Müllerian hormone (AMH) may be a good biomarker for this type of ovarian tumour. Objectives: To evaluate the use of serum AMH concentrations for endocrine diagnosis of GCTs in mares. Methods: Archived serum samples (n = 403) previously assayed for determination of serum inhibin, testosterone and progesterone concentrations (GCT panel) were assayed for serum AMH concentrations using a heterologous enzyme‐linked immunosorbent assay previously validated by our laboratory. For a subset (n = 44) of these samples, a clinical diagnosis of GCT was confirmed by histopathology. Results: Overall, the sensitivity of AMH (98%) for detection of histologically confirmed GCTs was significantly (P<0.05) greater than that of either inhibin (80%) or testosterone (48%) or the combination of inhibin and testosterone (84%). Conclusions: Determination of serum AMH concentrations is a useful biomarker for detection of GCTs in the mare. Potential relevance: Measurement of serum AMH concentrations can be used for diagnosis of GCTs in the mare. As serum AMH concentrations do not vary significantly during the oestrous cycle or pregnancy, interpretation of these results is not confounded by these physiological states.     

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.     

Granulosa cell tumours in the mare: A review of 52 cases

Granulosa cell tumour (GCT) is reported to be the most common ovarian neoplasm in the mare. A multicentre retrospective study evaluating the medical records of horses undergoing ovariectomy for treatment of a GCT was undertaken, documenting the presenting clinicopathological features, treatment and outcome. Follow‐up information was obtained by telephone questionnaire. Fifty‐two mares were included. Most presented with behavioural/reproductive abnormalities, including stallion‐like behaviour (50%), aggression (31%), prolonged oestrus or nymphomania (19%), and persistent anoestrus (8%). Most (93%) affected ovaries had a multilocular appearance when examined ultrasonographically. Only 60% of horses with histopathologically confirmed GCTs demonstrated elevations in serum inhibin concentrations. Twenty‐five percent of horses had histopathologically confirmed bilateral GCTs. Ninety‐four percent of horses that underwent ovariectomy were discharged from the hospital. Standing laparoscopic ovariectomies had a lower complication rate (34%) than other surgical methods. Long‐term complications were uncommon (11%). In conclusion, histopathologically confirmed GCTs can occur in the presence of normal serum inhibin concentrations. The high prevalence of bilateral GCTs indicates that thorough evaluation of the contralateral ovary is necessary. Surgical ovariectomy for treatment of GCTs has favourable short‐ and long‐term outcomes. Standing laparoscopic ovariectomy may be associated with a lower complication rate than other surgical methods.     

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.     

Elevated Serum Anti-Müllerian Hormone in an Arabian Mare with Granulosa Cell Tumor

Anti-Müllerian hormone (AMH), produced by granulosa cells, has been reported to be elevated in mares with granulosa cell tumor. A 13-year-old Arabian mare was referred after 3 years of infertility. Rectal and ultrasonographic examination revealed enlargement and multicystic appearance of the left ovary. The mare had substantially higher concentration of AMH (21.6 ng/mL) compared with normal cyclic mares (n = 5; 0.36 ± 0.02 ng/mL). Testosterone (50 pg/mL) concentration was also elevated, but progesterone concentration (0.4 ng/mL) was lowered. Accordingly, the mare was presumptively diagnosed with granulosa-theca cell tumor and a left ovariectomy implemented using ventral midline approach under general anesthesia. Histopathological examination confirmed the diagnosis of granulosa-theca cell tumor. Serum concentrations of AMH decreased to 2 ng/mL and 0.5 ng/mL 2 and 4 weeks after tumor removal, respectively. In conclusion, the current report presents confirmatory evidence that evaluation of serum AMH could be useful for diagnosis of granulosa cell tumor in mare.     

Effect of Chronic Administration of Oxytocin on Corpus Luteum Function in Cycling Mares

The objective of this study was to determine if intramuscular administration of 60 units of oxytocin once daily for 29 days, regardless of when treatment was initiated during the estrous cycle (i.e., without monitoring estrous behavior and/or detecting ovulation), would induce prolonged corpus luteum (CL) function in cycling mares. Mares were randomly assigned to two groups: (1) saline-treated control (n = 7) and (2) oxytocin-treated (n = 9) subjects. Control mares received 3 cc of saline, and oxytocin-treated mares received 60 units (3 cc) of oxytocin intramuscularly for 29 consecutive days. Treatment was initiated in all mares on the same day (day 1), independent of the day of the cycle. Jugular blood samples for determination of progesterone concentration were collected three times weekly (M, W, and F) for 21 days before treatment was initiated to confirm that all mares had a luteal phase of normal duration immediately before treatment. Beginning on the first day of treatment, blood samples were collected daily for eight days and then three times weekly through day 80. Mares were considered to have prolonged CL function if serum progesterone remained >1.0 ng/mL continuously for at least 25 days after the end of the treatment period. The proportion of mares with prolonged CL function was higher in the oxytocin-treated group than in the saline-treated group (7/9 vs. 1/7, respectively; P < .05). Three of the seven oxytocin-treated mares that developed prolonged CL function initially underwent luteolysis within 4–7 days of the start of oxytocin treatment and then developed prolonged CL function after the subsequent ovulation during the treatment period. In the other four oxytocin-treated mares that developed prolonged CL function, progesterone remained >1.0 ng/mL throughout the treatment period and into the post-treatment period. All mares with prolonged CL function maintained elevated progesterone concentrations through at least day 55 of the study. In conclusion, intramuscular administration of 60 units of oxytocin for 29 consecutive days effectively prolonged CL function in mares, regardless of when treatment was initiated during the estrous cycle. Importantly, this represents a protocol for using oxytocin treatment to prolong CL function that does not require detection of estrous behavior or day of ovulation.     

Ovarian activity of the mare during winter and spring at a latitude of 19° 21′ north

The ovulatory activity of mares located at 19° 21′N was monitored from November to June. Twenty-one mares were used (11 quarter-mile mares and 10 hybrids). Every week the mares were subjected to ultrasonographic observations of the ovaries, and blood samples for progesterone determinations were collected. Estrus activity was evaluated by teasing with a stallion. A mare was considered to be in an anovulatory state if progesterone concentrations during 5 or more consecutive samples were below 1 ng/mL. Cyclic ovulatory activity during the entire duration of the study was recorded in 62% of the mares. Only 38% of the mares (5 quarter-mile and 3 hybrid mares) had anovulatory periods, which lasted 128.5 ± 14.4 days (mean ± SEM) and occurred at some point between November and May. The duration of the anovulatory period varied from 70 to 196 days. Estrous behavior was shown during the anovulatory period in 75% (6/8) of the mares that had such a period. Most mares maintain regular ovulatory activity during the winter and spring at 19° 21′ north, and that a wide variation occurs in the duration of the anovulatory period of those mares that stop cycling at this latitude.     

Hyperleptinemia in Mares: Prevalence in Lactating Mares and Effect on Rebreeding Success

Previous research from our laboratory showed that approximately one third of obese, nonfoaling mares displayed a condition of hyperleptinemia coupled with hyperinsulinemia that resembled type 2 diabetes in humans. The current study was performed to evaluate the prevalence of the hyperleptinemic syndrome in lactating mares and its possible impact on their rebreeding success. Additionally, we investigated possible relationships between leptin levels in lactating versus nonlactating mares. In experiment 1, jugular blood samples were collected from 198 lactating mares on two occasions approximately 2 weeks apart. The mares resided on eight farms in Louisiana; breeds included Thoroughbred (n = 86), Quarter Horse (n = 71), Warmblood (n = 24), and draft-type (n = 17). Body condition scores (BCS) were measured at the time of blood sampling; plasma samples were assessed for leptin and progesterone concentrations. Reproductive and medical histories, as well as feeding regimens, were compiled on each mare. Based on our previous reports and examination of the current data, a mare was considered hyperleptinemic if her plasma samples contained greater than10 ng/mL leptin; normal was considered 6.0 ng/mL or less; mares with levels above 6.0 and 10 ng/mL or greater were classified as intermediate. Overall mean leptin concentration was 4.7 ng/mL, and average BCS was 5.5. After analysis, 24 mares were classified as hyperleptinemic (12%), 138 were classified as normal (70%), and 36 were classified as intermediate (18%). Leptin concentrations were affected by BCS (P = .08), with higher concentrations in mares with higher body condition; however, there were hyperleptinemic mares with BCS of 4 to 5.5. Feeding regimen affected leptin concentrations (P < .01), with mares on pasture full-time having the highest concentrations. There was no effect of breed, mare age, number of years the mare had been bred, number of live foals, progesterone concentrations, or last foaling date on leptin concentrations. Rebreeding success averaged 81% overall and was not affected by leptin classification. In experiment 2, nonfoaling mares kept on pasture had mean leptin concentrations of 7.0 ng/mL; 8 of 31 mares (26%) displayed hyperleptinemia. Mean leptin concentration was correlated with BCS (R² = 0.65; P < .02) but was not affected by age of the mare. It was concluded that the hyperleptinemic condition occurs in lactating broodmares, even at BCS as low as 4. The overall incidence appears to be lower in broodmares than in nonfoaling mares, likely because of their lower BCS in general and the energy demands of lactation. Hyperleptinemia did not affect rebreeding success at the end of the breeding season.     

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.     

Endocrine Profile Comparisons of Fat Versus Moderately Conditioned Mares Following Parturition

The influence of progesterone, luteinizing hormone (LH), and estrogen on the mare's estrous cycle has been well researched and documented, but other endocrine profiles have not received as much attention. To evaluate endocrine concentrations in fat-conditioned (body condition score [BCS] of 7–8) versus moderately conditioned mares (BCS of 5–6), 24 mares were allotted to and maintained in respective groups from late gestation until pregnancy was confirmed after breeding on the second postpartum estrus. Serum concentrations of thyroxine (T₄), insulin-like growth factor-1 (IGF-1), and leptin were assayed to characterize circulating blood concentrations. Additionally, LH and progesterone serum concentrations were assayed to evaluate the estrous cycle status of the mares. Leptin and progesterone concentrations were not different (P > .05) between the groups. Nevertheless, serum concentrations of T₄ were higher (P < .01) and IGF-1 concentrations lower (P < .01) in moderately conditioned as compared with fat-conditioned mares during times of ovulation and the interovulatory period. Furthermore, serum concentrations of LH were found to be different between the groups only when the estrous cycle approached the second ovulation (P < .0001). Results of this study suggest that mares maintained in a BCS of 5 or greater are similar in terms of reproductive efficiency. Although the circulating serum concentrations of T₄ and IGF-1 are different after parturition, their influence does not affect reproductive capabilities of mares with a BCS of 5 or greater.     

Sexual maturity,seasonal estrus,and gestation in female Indo-Pacific bottlenose dolphins Tursiops aduncus inferred from serum reproductive hormones

Reproductive hormones in serum concentrations of progesterone, estradiol, and testosterone in female Indo-Pacific bottlenose dolphins (Tursiops aduncus, n = 12) housed in Ocean Park Hong Kong were investigated in the present study. Results showed that, onset of puberty of captive Indo-Pacific bottlenose dolphins was at 5 years while sexual maturity was at 6. Average serum progesterone concentrations in non-pregnant sexually mature individuals was 0.33 (0.25–0.97) ng/mL (interquartile), significantly higher than in immature ones 0.26 (0.25–0.38) ng/mL. This study found significant difference in serum estradiol concentrations between individuals at the onset of puberty (9.5 ± 1.7 pg/mL, ±SD) and not (below detection limit 9 pg/mL). A slightly seasonal breeding pattern, with progesterone values tend to be higher from February to October (0.38 [0.25–1.07] ng/mL) was inferred. During pregnancy, serum progesterone concentrations range from 10.54 ± 8.74 ng/mL (indexed month post-conception [IMPC] 0) to 25.49 ± 12.06 ng/mL (IMPC 2), and display a bimodal pattern with 2 peaks in early- (25.49 ± 12.06 ng/mL, IMPC 2) and late-pregnancy (21.71 ± 10.25 ng/mL, IMPC 12), respectively. Serum estradiol concentrations can seldom be detected in early-pregnancy and increase constantly in mid- (9.45 ± 1.83 pg/mL) and late-pregnancy (11.88 ± 3.81 pg/mL), with a spike (15.45 ± 6.78 pg/mL) 1 month prior to delivery. Serum testosterone concentrations elevate significantly in IMPC 7 (0.36 ± 0.10 ng/mL) compared to other months (0.16 ± 0.10 ng/mL) of the year. The present study provides normal concentration profiles for some reproductive hormones in female Indo-Pacific bottlenose dolphins and can contribute to the breeding monitoring of this species. Also, our study would shed further light on the reproductive physiology of small cetaceans.     

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).     

Changes of serum anti‐Müllerian hormone in a mare with granulosa cell tumour following surgery and reinitiation of follicular activity

Anti‐Müllerian hormone (AMH) has been reported to be elevated in mares with granulosa cell tumour (GCT). An 8‐year‐old Thoroughbred mare was presented for not being observed in oestrus after the beginning of the breeding season. Rectal palpation and ultrasonography revealed enlargement and cystic appearance of the left ovary while the right ovary was small with an anoestrous‐like appearance. The AMH concentration was 694.9 ng/ml. Presumptively diagnosed with GCT, the mare was subjected to tumour removal. Histopathology confirmed GCT. To evaluate changes of AMH concentration following surgery, blood samples were collected immediately prior to surgery, and on Days 1, 2, 4, 8, 16 and 32 after surgery. Thereafter, blood samples were collected monthly and also at the time the mare was observed in oestrus (148 days after tumour removal). The AMH concentrations decreased over the first 2 months after surgery (from 721.2 ng/ml to 0.1 ng/ml). Subsequently, AMH concentration increased and peaked at the time of oestrus expression (0.7 ng/ml). The mare then became anoestrous, and AMH concentration decreased and reached 0.2 ng/ml, which was not significantly different from the mean concentration of AMH in normal anoestrous mares (n = 5; 0.26 ± 0.07 ng/ml). In conclusion, the present report implies the potential use of AMH for determination of the time of follicular resumption in mares after GCT removal.     

Evaluation of the PATHFAST Chemiluminescent Enzyme Immunoassay for Measuring Progesterone in Whole Blood and Serum of Mares

Evaluation of a new chemiluminescent enzyme immunoassay, the PATHFAST assay system (PATHFAST), for measurement of circulating progesterone in mares was performed. Five mares at the mid-luteal stage were administrated a single i.m. injection of prostaglandin F2α analog (PGF2α; cloprostenol 250 μg/ml), and then blood samples were collected from the jugular vein at 0, 15, 30 and 45 min, at one-hour intervals until 24 and at 48 hr via a catheter in the jugular vein. To monitor the physiological changes in circulating progesterone in mares after induced luteolysis, concentrations of progesterone in whole blood and serum samples were measured by PATHFAST. In addition, concentrations of progesterone in serum samples measured by PATHFAST were compared with those measured by radioimmunoassay (RIA) and enzyme immunoassay (EIA). Using PATHFAST, the serum concentrations of progesterone in mares correlated highly with those of whole blood samples (r=0.9672, n=88). The serum concentrations of progesterone as measured by PATHFAST correlated well with RIA (r=0.9654, n=88) and EIA (r=0.9323, n=112). An abrupt decline in circulating progesterone in whole blood samples was observed within 2 hr (50%), followed by a gradual decline until 48 hr later. The results for progesterone in whole blood samples correlated highly with those in serum samples, and the declining pattern paralleled that of the serum samples. These results demonstrated that PATHFAST is useful in the equine clinic as an accurate diagnostic tool for rapid assay of progesterone within 26 min, using unextracted whole blood.     

Testosterone administration to mares during estrus: duration of estrus and diestrus and concentrations of LH and FSH in plasma

To study the possible role of ovarian androgens in regulation of follicle stimulating hormone (FSH) secretion in the cycling mare, five mature, intact mares were treated with testosterone (20 micrograms/kg of body weight) daily during estrus; five control mares received safflower oil on the same schedule. Mares were teased for estrus and samples of jugular blood were drawn daily through one full estrous cycle. Concentrations of FSH in plasma were measured by a newly developed radioimmunoassay based on anti-ovine FSH serum and radioiodinated equine FSH. Testosterone treatment during estrus had no effect on duration of estrus, diestrus or the total cycle. Concentrations of FSH in plasma during estrus were unaffected by testosterone treatment. However, FSH concentrations in testosterone-treated mares were elevated (P less than .05) compared with controls during mid-diestrus (d 6 through 11). The magnitude and timing of the LH peaks were unaffected by treatment, as was the day on which the first elevated progesterone concentration occurred. These data are consistent with a model of FSH secretion in which ovarian androgens cause an accumulation of FSH in the pituitary during estrus in preparation for the surges that occur in FSH secretion during diestrus.     

Effect of a Single Injection of Long-acting Progesterone on the First Ovulation in Early and Late Spring Transitional Mares

Since 1966, exogenous progestins have been used in equine practice for pregnancy maintenance, estrous suppression, and control of erratic sexual behavior. This study was designed to investigate the use of a new compounded controlled-release progesterone preparation (BioRelease P4 LA 300) in early and late spring transitional mares. In the first experiment, the pharmacodynamic properties of the preparation were studied in five geldings. In the second experiment, the use of a single intramuscular injection (600 mg) was tested in 68 embryo-recipient mares maintained under natural photoperiod in the Southern Hemisphere. Experiment 1 demonstrated elevated serum concentrations of progesterone (>1 ng/mL) for 7.6 ± 2.2 days. In experiment 2, there was no effect of treatment in mares that were treated on September 18, independent of their follicular status at day of treatment (10 to 15 mm; 20 to 25 mm, respectively). When mares with a follicular size of 20 to 25 mm were treated on October 14, significantly more progestin-treated mares (10/12; 83%) ovulated between 10 and 24 days after treatment than untreated controls (3/12; 25%) (P < .05). Additionally, there was a trend in mares treated on October 14 for a shorter treatment to ovulation interval (mean ± SD, 18.6 ± 8.7 days) compared with untreated controls (mean ± SD, 26.7 ± 14.7 days) (P = .07). Administration of one single injection of long-acting progesterone is a simple and effective method of controlling the first ovulation of the season in late transitional mares.     

Effects of vitamin E and selenium administration on pregnant,heavy draft mares on placental retention time and reproductive performance and on white muscle disease in their foals

This study investigated the effects of administering vitamin E and selenium to pregnant heavy draft horsemares on the incidence of retained placenta and postpartum reproductive performance and on the prevention of the white muscle disease in their foals. In study A, 1,000 mg of vitamin E and 50 mg of selenium (E-SE 20 mL) were given to 22 mares 3 weeks before expected parturition (335 days counted from last mating), whereas 28 mares were used as controls. In study B, E-SE were administered 2 weeks before expected parturition at 2 dose levels, with 25 mares receiving 20 mL E-SE, 19 mares receiving 10 mL, and 29 mares kept as controls. Vitamin E and selenium were assayed in serum collected from some of the mares before administration of E-SE and again postpartum and from the foals immediately after birth. Serum selenium concentrations before E-SE administration were deficient (<65 ng/mL) in all mares (n = 48) but were increased in the postpartum sample from treated mares regardless of the dose or timing of administration (n = 31) (P = .05). Only study B mares were deficient in vitamin E prepartum, and both dose levels of E-SE had corrected this in the postpartum sample (P = .01). All foals were selenium deficient regardless of whether their dams had received E-SE or not, although concentrations were higher in foals from treated study A mares than from controls (P = .05). Mares with the highest selenium concentrations prepartum (40 ng/mL and over) had shorter placental retention times than mares with lower selenium concentrations (P = .05) and did not respond to E-SE with a further reduction in retention time. By contrast, mares with prepartum selenium concentrations between 20 and 40 ng/mL tended to respond to E-SE with a shortened placental retention time (P = .07). E-SE administration reduced the mean number of days from parturition to last mating (nonpregnant term) in study B mares (P = .05) and in mares with adequate prepartum vitamin E concentrations (>300 g/mL, P = .05). We conclude that maintaining high level serum vitamin E and selenium concentrations of prepartum mares is expected to increase fertility of selenium-deficient mares. Therefore, the regimen of vitamin E and selenium administrations to selenium deficient mares should be developed.     

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.     

Effect of hypothyroidism on the blood lipid response to higher dietary fat intake in mares

Blood lipid and lipoprotein concentrations were measured and compared between euthyroid and thyroidectomized mares on low-fat or high-fat diets to test the hypothesis that hypothyroidism alters the blood lipid response to higher dietary fat intake. Four healthy adult mares and four adult mares that had been thyroidectomized 3 to 6 mo earlier were placed on low-fat or high-fat diets according to a replicated 2 x 2 Latin square design consisting of two 5-wk feeding periods separated by a 2-wk washout interval. Plasma lipid concentrations were measured at 0, 3, 4, and 5 wk, and plasma lipase activities were measured at the end of each 5-wk feeding period. Compared with euthyroid mares (0.46 ng/mL [range 0.34 to 0.68 ng/mL T3], and 21.5 ng/mL [range 18.1 to 25.1 ng/mL T4], respectively), median serum concentrations of T3 and T4 were lower (P = 0.029 and P = 0.021, respectively) in thyroid-ectomized mares (0.26 ng/mL [range 0.23 to 0.26 ng/ mL T3], and undetectable T4). Serum T4 concentrations were below the limits of detection in thyroidectomized horses. Alterations in body weight over 5 wk did not differ between groups. Mean plasma very low density lipoprotein (VLDL) and triglyceride (TG) concentrations were higher (P = 0.045 and 0.034, respectively) in hypothyroid mares (55.42 +/- 35.05 mg/dL and 52.83 +/- 34.46 mg/dL, respectively) compared with euthyroid mares (28.28 +/- 13.76 mg/dL and 23.53 +/- 9.84 mg/dL, respectively). Mean plasma total cholesterol (TC) concentrations increased from 88.73 +/- 25.49 mg/dL at baseline to 103.93 +/- 24.42 mg/dL after 5 wk on the low-fat diet, but increased by a greater magnitude (P = 0.006 diet +/- time interaction) in mares that were on the high-fat diet (81.05 +/- 17.24 mg/dL and 123.84 +/- 32.27 mg/ dL, respectively). Mean plasma TC concentrations were higher (P = 0.099) in hypothyroid mares (116.16 +/- 32.89 mg/dL) than in euthyroid mares (89.56 +/- 14.45 mg/ dL). Higher post-heparin plasma lipoprotein lipase and hepatic lipase activities (P = 0.012 andP = 0.017, respectively) were detected in mares that were on the high-fat diet (2.66 +/- 0.91 micromol FA x mL(-1) x h(-1) and 2.95 +/- 0.49 micromol FA x mL(-1) x h(-1), respectively) vs. a low-fat diet (1.75 +/- 0.55 micromol FA x mL(-1) x h(-1) and 2.27 +/- 0.59 micromol FA x mL(-1) x h(-1), respectively). We conclude that plasma VLDL and TG concentrations are elevated in hypothyroid mares, but the blood lipid response to higher dietary fat intake is not influenced by hypothyroidism.     

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.