Effect of eFSH on Ovarian Cyclicity and Embryo Production of Mares in Spring Transitional Phase

The use of equine FSH (eFSH) for inducing follicular development and ovulation in transitional mares was evaluated. Twenty-seven mares, from 3 to 15 years of age, were examined during the months of August and September 2004, in Brazil. Ultrasound evaluations were performed during 2 weeks before the start of the experiment to confirm transitional characteristics (no follicles larger than 25 mm and no corpus luteum [CL] present). After this period, as the mares obtained a follicle of at least 25 mm, they were assigned to one of two groups: (1) control group, untreated; (2) treated with 12.5 mg eFSH, 2 times per day, until at least half of all follicles larger than 30 mm had reached 35 mm. Follicular activity of all mares was monitored. When most of the follicles from treated mares and a single follicle from control mares acquired a preovulatory size (≥35 mm), 2,500 IU human chorionic gonadotropin (hCG) was administered IV to induce ovulation. After hCG administration, the mares were inseminated with fresh semen every other day until ovulation. Ultrasound examinations continued until detection of the last ovulation, and embryo recovery was performed 7 to 8 days after ovulation. The mares of the treated group reached the first preovulatory follicle (4.1 ± 1.0 vs 14.9 ± 10.8 days) and ovulated before untreated mares (6.6 ± 1.2 vs 18.0 ± 11.1 days; P < .05). All mares were treated with prostaglandin F_2α (PGF_2α), on the day of embryo flushing. Three superovulated mares did not cycle immediately after PGF_2α treatment, and consequently had a longer interovulatory interval (22.4 vs 10.9 days, P < 0.05). The mean period of treatment was 4.79 ± 1.07 days and 85.71% of mares had multiple ovulations. The number of ovulations (5.6 vs 1.0) and embryos (2.0 vs 0.7) per mare were higher (P < 0.05) for treated mares than control mares. In conclusion, treatment with eFSH was effective in hastening the onset of the breeding season, inducing multiple ovulations, and increasing embryo production in transitional mares. This is the first report showing the use of FSH treatment to recover embryos from the first cycle of the year.     

Gestational Length and First Postpartum Ovulation of Criollo Mares on a Stud Farm in Southern Brazil

The Criollo horse industry requires more efforts toward a better understanding of breed characteristics and physiology; few studies have been conducted in Criollo horses to fulfill this demand. Toward this aim, 70 Criollo mares (between 3 and 28 years of age) underwent physiologic evaluation of the length of gestation, occurrence of foal heat, and interval to postpartum ovulation. Gestation length in the 70 mares was 335.6 ± 10.5 days, varying from 312 to 364 days. The mean (±SD) interval from parturition to first ovulation of 42 mares that foaled between September and December of 2005 and 2006 was 19.9 ± 14.0 days. Eighty-three percent of the mares had an interval to foal heat ovulation shorter than 20 days (35/42). The mean (±SD) parturition to ovulation interval of these mares was 14.2 ± 3.0 days.     

GnRH类似物和孕酮对猪排卵及受胎率的影响

取正常发情青年母猪 16头 ,其中 8头在发情开始后 2 0 h肌注 L RH - A30 .2μg/ kg,结果明显提高了母猪的排卵数和排卵率 ,分别比不作任何处理的 8头对照组母猪增加 6 .12个排卵数和 2 8.5 6个百分点的排卵率。另取 38头母猪 ,随机分成 4组 ,分别在发情配种前 6～ 8h肌注 L RH- A30 .2μg/ kg( 组 )、在发情配种后 2 d肌注孕酮 6 0 mg( 组 )或 L RH- A3与孕酮重复处理 ( 组 ) ,结果表明 ,L RH- A3可提高母猪的受胎率 ,明显提高产仔数 ,而孕酮处理组 ( 组 )与对照组 (不作任何处理 )比较无明显差异。经血清孕酮检测发现 ,L RH- A3可显著提高怀孕早期母猪的血清孕酮水平 ,怀孕后 10、2 0 d,分别比未用 L RH- A3处理的母猪平均高出 7.10 7、8.80 3μg/ L     

Ovulation,Pregnancy Rate and Early Embryonic Development in Vernal Transitional Mares Treated with Equine‐ or Porcine‐FSH

The objective of this study was to compare the efficacy of purified equine‐ and porcine‐FSH treatment regimes in mares in early vernal transition. Mares (n = 22) kept under ambient light were examined ultrasonographically per‐rectum, starting January 30th. They were assigned to one of two treatment groups using a sequential alternating treatment design when a follicle ≥ 25 mm was detected. In the eFSH group, mares were treated twice daily with equine‐FSH, and in the pFSH group mares were treated twice daily with porcine‐FSH; treatments were continued until follicle(s) ≥ 35 mm, and 24 h later hCG was administered. Oestrous mares were inseminated with fresh semen and examined for pregnancy on days 11–20 post‐ovulation. In the eFSH group, 11/11 (100%) mares developed follicle(s) ≥ 35 mm, 8/11 (73%) ovulated and 6/8 (75%) conceived. In the pFSH group, 5/11 (45%) developed follicle(s) ≥ 35 mm, 4/11 (36%) ovulated and 3/4 (75%) conceived. Treatment with eFSH resulted in a greater ovarian stimulation; higher number of pre‐ovulatory‐sized follicles, higher number of ovulations and higher number of embryos (p < 0.05). Following ovulation, serum progesterone concentrations were correlated with the number of CLs and supported early embryonic development; maternal recognition of pregnancy occurred in all pregnant mares. We concluded that eFSH can be used to effectively induce follicular growth and ovulation in vernal transitional mares; however, if bred, diagnosis and management of twins’ pregnancies would be required prior to day 16 because of the increased risk of multiple embryos per pregnancy. Conversely, the current pFSH treatment regime cannot be recommended.     

Stimulation of Ovulation in Seasonally Anovulatory and Vernal Transitional Mares with Estradiol and Domperidone: Dose and Combination Studies

Three experiments were conducted to test the efficacy of different doses of estradiol cypionate (ECP) and domperidone for inducing ovulation in seasonally anovulatory (January; Experiments 1 and 2) and transitional period (March; Experiment 3) mares. In the first two experiments, mares in Kentucky and Louisiana were administered domperidone (3 g in biodegradable particles) alone or after pretreatment with 100 or 150 mg of ECP; another group received ECP, domperidone, and progesterone, and a fifth group received ECP and progesterone only (the latter two in Kentucky). Control mares in both states received no treatment. The proportion of mares ovulating within 35 days (for mares treated in January) was greater (P = .0002) for those receiving any combination of ECP plus domperidone relative to mares not receiving the combined treatment. Addition of progesterone to ECP plus domperidone did not enhance (P = .7) the response relative to the combination alone; domperidone by itself, or ECP plus progesterone, did not alter the response relative to controls (P > .24). Experiment 3 was conducted in Louisiana as a 2 × 3 factorial, with two doses of domperidone (1.5 or 3 g) and three doses of ECP (0, 75, or 150 mg). There was no main effect of domperidone or ECP dose; a greater proportion (P = .055) of mares receiving any combination of ECP plus domperidone ovulated in 21 days compared with those receiving no ECP. In conclusion, pretreatment with ECP before injection of domperidone 10 days later can be used to increase the proportion of mares ovulating early in the year; within the limits of the present experiments, there appears to be no difference in doses of ECP of 75, 100, or 150 mg, and no difference in doses of domperidone of 1.5 or 3 g.     

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.     

长效伊维菌素注射液对免痒螨病的治疗效果及兔场净化效果观察

2007年四川省雅安市某兔场75只兔发生痒螨病,采用长效伊维菌素注射液按0.3 mL/kg体重一次性大腿肌肉注射,治愈率为98.67%.连续观察5个月内复发率为1.52%(1/66).同时,采用长效伊维菌素注射液按0.2 mL/kg体重一次性大腿肌肉注射对该兔场未发病的327只肉兔和216只长毛兔进行了净化处理.处理后5个月内痒螨病发生率为0.37%(2/543).     

hCG‐Induced Ovulation in Thoroughbred Mares Does Not Affect Corpus luteum Development and Function During Early Pregnancy

Our aim was to compare Corpus luteum (CL) development and blood plasma concentration of progesterone ([P₄]) in thoroughbred mares after spontaneous (Control: C) or human chorionic gonadotrophin (hCG)‐induced ovulation. Lactating mares (C = 12; hCG = 21) were daily teased and mated during second oestrus post‐partum. Treated mares received 2500 IU hCG i.v. at first day of behavioural oestrus when dominant follicular size was >35, ≤42 mm and mated 12–24 h after. Control mares in oestrus were mated with dominant follicular size ≥45 mm. Dominant follicle before ovulation, CL and gestational sac were measured by ultrasound and [P₄] by radioimmunoassay (RIA). Blood sampling and ultrasound CL exams were done at days 1, 2, 3, 4, 8, 12, 16, 20, 25, 30, 35, 40, 45, 60 and 90 after ovulation and gestational sac from day 12 after ovulation in pregnant (P) mares; non‐pregnant (NP) were followed until oestrus returned. Data analyses considered four subgroups: hCG‐P, hCG‐NP, C‐P and C‐NP. Preovulatory follicular size was smaller in hCG mares than in C: 39.2 ± 2.7 mm vs 51.0 ± 1.8 mm (p < 0.0001). All hCG mares ovulated 24–48 h after treatment and presented similar oestrus duration as controls. C. luteum size in P mares showed the same pattern of development through days 4–35, presenting erratic differences during initial establishment. Thus, on days 1 and 3, CL was smaller in hCG‐P (p < 0.05); while in hCG‐NP, CL size was greater than in C‐NP on day three (p = 0.03). Corpus luteum size remained stable until day 90 in hCG‐P mares, while in C‐P a transient and apparently not functional increase was detected on days 40 and 45 (p < 0.05) and the decrease from day 60 onwards, made this difference to disappear. No differences were observed in [P₄] pattern between P, or between NP subgroups, respectively. So, hCG‐induced ovulation does not affect CL development, neither [P₄] during early pregnancy. One cycle pregnancy rate tended to be lower in hCG mares while season pregnancy rates were similar to controls.     

The Effect of Cloprostenol on the Incidence of Multiple Ovulation and Anovulatory Hemorrhagic Follicles in Two Mares: A Case Report

Reproductive records of the entire lives of two mares with abnormally high incidence of multiple ovulation and hemorrhagic anovulatory follicles were analyzed retrospectively. Chi-square analysis was used to test statistically the effect of cloprostenol, a prostaglandin F2 analog, on the incidence of multiple ovulations, hemorrhagic anovulatory follicles, and its ultrasonographic appearance. A total of 319 estrous cycles during a 17-year period were analyzed. Cycles induced with cloprostenol were more likely (P < .000) to develop hemorrhagic anovulatory follicles than spontaneous cycles. The incidence of multiple ovulation was higher in induced cycles than in spontaneous cycles in one of the two mares.     

Seasonal Changes in Luteal Progesterone Concentration and mRNA Expressions of Progesterone Synthesis-related Proteins in the Corpus Luteum of Mares

Although circulating progesterone (P(4)) levels tend to change with the season, little is known about the seasonal changes of P(4) synthesis-related proteins in the corpus luteum (CL) of mares. To examine these changes, seventy-four ovaries containing a CL were collected from Anglo-Norman mares at a local abattoir in Kumamoto, Japan (~N32°), five times during one year. The stages of the CLs were classified as early, mid and regressed by macroscopic observation of the CL and follicles. The mid CL, which had the highest P(4) concentration, was used to evaluate the seasonal changes in P(4) synthesis. The luteal P(4) concentration and mRNA expression of luteinizing hormone receptor (LHCGR) were lowest during early winter and highest during late winter. The mRNA expressions of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) were lowest during early winter and increased during late winter. These results suggest that P(4) synthesis in the CL is affected by the seasonal changes in the mRNA expressions of P(4) synthesis-related proteins in mares.     

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.     

Efficacy of Deslorelin Acetate (SucroMate) on Induction of Ovulation in American Quarter Horse Mares

Equine clinicians rely on ovulation induction agents to provide a timed ovulation in mares for optimal breeding management. Numerous studies have been performed on the efficacy of human chorionic gonadotropin (hCG) to induce ovulation in the mare, but limited clinical data are available for the new deslorelin acetate product SucroMate. This study was designed to evaluate the efficacy of SucroMate (deslorelin) in comparison with hCG to induce ovulation. American Quarter horse mares (n = 256) presented to Colorado State University for breeding management were used in this study. Mares received either deslorelin or hCG when a follicle ≥35 mm was detected by transrectal ultrasound in the presence of uterine edema. Ultrasonographic examinations were subsequently performed once daily until ovulation was detected. Deslorelin was administered to 138 mares during168 estrous cycles, and hCG was given to 118 mares during 136 estrous cycles. Mares administered deslorelin had a similar (P < .05) higher ovulation rate (89.9%) within 48 hours following drug administration than mares administered hCG (82.8%). There are no effects of season or age on ovulation rates in either treatment group. Twenty-one mares administered deslorelin and 11 mares administered hCG were monitored by transrectal ultrasound every 6 hours to detect ovulation as part of a frozen semen management program. Average intervals from deslorelin or hCG administration to ovulation were 41.4 ± 9.4 and 44.4 ± 16.5 hours, respectively. Results of this study indicate that SucroMate is effective at inducing a timed ovulation in the mare.     

Faecal Oestrogens and Progesterone Metabolites in Mares of Different Breeds During the Last Trimester of Pregnancy

Non‐invasive pregnancy diagnosis in mares by measuring faecal oestrogens has been performed over years with great accuracy. However, results have indicated breed‐related differences in the amount of excreted steroids during late pregnancy. Therefore faecal samples were collected during the last 4 months of pregnancy of Thoroughbred (n=10), New Forest pony (n=9), Shetland pony (n=10) and Iceland pony mares (n=11). Concentrations of oestrogens, 20α‐hydroxy‐ and 20‐oxopregnanes were measured using enzyme immunoassays. Breed differences concerning both levels (though significant only in case of oestrogens) and time course of measured steroids were observed. There was a highly significant time effect (p < 0.00001) and an interaction between time and breeds (p < 0.02) for all steroids measured, suggesting that the time effect differs for different breeds. Oestrogen concentrations showed a decrease towards parturition, whereas in 20α‐hydroxy‐ and 20‐oxopregnane levels a pronounced increase was found 2 and 1 months, respectively, before parturition. A breed effect was only significant (p=0.001) when comparing oestrogen concentrations and was mainly due to Iceland ponies, which had the lowest concentrations especially during the last 2 months of pregnancy. An almost significant (p=0.06) breed effect was found for 20‐oxopregnanes. In Iceland mares an additional increase in faecal pregnane content was already observed earlier, reaching maximum levels before the 60th day ante‐partum (a.p.), followed by a decrease until the 30th day a.p. The ratio of 20‐oxopregnanes to oestrogens in the samples was significantly higher (p < 0.006) in Iceland ponies in comparison with any other breed throughout all months before parturition. The breed differences observed in the amounts of oestrogens and/or progestagens present during late pregnancy may demonstrate micro‐evolutionary changes in the endocrine system of a species.     

Effect on Perinatal Mortality of a Single Selenium Injection To Sows and Gilts

Selenium treatment of pregnant sows and gilts was conducted as a double-blind placebo-controlled study in 5 commercial herds with a total of 350 sows and gilts (Danish Landrace, Yorkshire and Landrace-Yorkshire crossbreeds). At 3–6 weeks before farrowing the animals were injected once intramuscularly with either Na-selenite (0.05 mg Se per kg) or placebo. Blood samples were taken immediately before treatment and once again at 3–6 weeks after farrowing, to evaluate the Se status. The productivity of the sows and gilts was recorded.Within herds there was no pretreatment difference in Se status between the two treatment groups, while a considerable variation was seen among herds. After farrowing there were only minor differences in Se status between the treatment groups, whereas the variation among herds persisted.Overall, no effect of the Na-selenite injection was seen, in that there was no difference in number of liveborn, stillborn, and weaned piglets between the treatment groups. The number of days from weaning to oestrus and the number of oestrous cycles per pregnancy were not influenced by the Na-selenite treatment.     

Successful Timing of Ovulation Using Deslorelin (Ovuplant®) is Labour-saving in Mares Aimed for Single AI with Frozen Semen

To minimize the number of matings/inseminations, controlled ovulation has been practised since a long time ago. A potent short-term implant, releasing the GnRH analogue deslorelin (Ovuplant((R))) has been used in Australia and North America for several years for hastening the ovulation time in mares, but the product is not registered on the European market. This study was aimed to investigate: (1) ovulation time in mares implanted with Ovuplant when the largest follicle was 42 mm or more in size, (2) repeatability of ovulation time in successive oestruses when treated with Ovuplant, (3) pregnancy rate after single insemination with frozen-thawed semen near ovulation. This study included 11 mares, and altogether 17 timed ovulations. Follicular growth and ovulation were determined by palpation per rectum and by ultrasonography in the morning (at 7:00 hours) every second day until observation of a follicle of at least 42 mm in diameter. Then the mares were re-examined in the afternoon (at 19:00 hours), and an Ovuplant was inserted in the mucosa of the vulva. For detection of ovulation, the mares were palpated and ultrasounded repeatedly from 36-42 h after the insert. The mares were inseminated with frozen-thawed semen once at ovulation. All mares ovulated at 36-48 h after treatment and 94% at 38-42 h after treatment. The six mares that were treated at two oestruses ovulated at 39.9 and 39.7 h, respectively. Five of 11 mares (45.4%), inseminated with frozen-thawed semen at the first oestrous cycle were pregnant day 14-16 after ovulation. Using this protocol, there is no need of palpation/ultrasonography during night hours, and examination at 36 and 41 h after implantation might be enough for estimation of ovulation time.     

米非司酮对早孕猕猴孕酮和雌二醇分泌的研究

为开展生殖生物学研究工作提供理想的动物模型。进行了本项试验,分析了米非司酮(ＲＵ４８６)对猕猴妊娠早期血清中孕酮(Ｐ)和雌二醇(Ｅ２)的影响。结果表明,注射ＲＵ４８６－段时间内孕酮和雌二醇基本不交,但Ｅ２/Ｐ比值有所增加,说明米非司酮干扰内分泌激素之间的平衡,随着出血的发生,孕酮、雌二醇均显著下降。这种降低与出血的发生有着明确的先后关系,从而证实了激素的变化源于蜕膜受损。     

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.     

Effect of Oestrous-cycle Stage on the Response of Mares in a Novel Object Test and Isolation Test

In various species, sex, hormonal treatments and oestrous-cycle stage have been shown to affect the animal's response in behavioural tests. Few such studies have been performed in the horse. The main aim of the present study was to investigate whether oestrous-cycle stage affects mares' response to a novel object test and isolation test and, in part, to study whether mares, assumed to suffer from oestrous-related behavioural problems, respond differently in these tests when compared with controls. Twelve mares were tested twice, in oestrus and dioestrus, in a crossover design. Seven behavioural and two heart rate variables were measured for the novel object test and two heart rate variables for the isolation test. Oestrous-cycle stage and whether a mare was classified as a 'problem' mare did not affect the mare's response. However, test order, i.e. the cycle stage a mare was tested in first, affected its reaction. This effect could partly be explained by significant differences between test occasions 1 and 2 in three behavioural variables and one heart rate variable (p < 0.05) in the novel object test. The mares explored the novel object more and had a higher mean heart rate in the first test. Exploring the novel object more could largely be attributed to those mares tested in dioestrus first, perhaps indicating that the mares in oestrus were less receptive to the novel object. The reason for the differences between test occasions could be an effect of learning or habituation.