Effects of Intrauterine Devices in Mares: A Histomorphological and Immunohistochemical Evaluation of the Endometrium

Oestrous suppression by intrauterine devices (IUDs) is caused by prolongation of luteal function, but the biological mechanism is unknown. The aim of the study was to investigate mechanisms which could explain the action of IUDs. Thirty mares were age‐matched and either inseminated (AI, n = 15) or fitted with an IUD (IUD, n = 15) and subsequently divided into four groups: AI‐P, pregnant (n = 8); AI‐N, non‐pregnant (n = 7); IUD‐P, prolonged luteal phase (n = 7); and IUD‐N, normal luteal phase (n = 8). The median ages were 5.5 and 7 years in AI‐P and IUD‐P groups and 14 and 11 years in AI‐N and IUD‐N groups, respectively. On Day 15 after ovulation, an endometrial biopsy was obtained to study histomorphological and immunohistochemical expression patterns of uterine proteins (uteroferrin, UF; uterocalin, UC; uteroglobin, UG), oestrogen and progesterone receptors (ER, PR), proliferation marker Ki‐67 and content of inflammatory cells. Expression of UF was higher in IUD mares; the difference between pregnant and IUD‐P mares was significant. Mares exhibiting a prolonged luteal phase (AI‐P, IUD‐P) showed only mild angiosclerosis and lower expression of both ER and PR than mares with a normal luteal phase (AI‐N, IUD‐N). No significant differences were detected in the numbers of inflammatory cells, with the exception of macrophages, which were more numerous in AI‐P than AI‐N mares. Although inflammatory cells were not detected in IUD mares, increased UF levels may indicate chronic inflammation. Young age and normality of the endometrial blood vessels may improve the efficacy of IUDs.     

Equine Endometrial Gland Density and Endometrial Thickness Vary among Sampling Sites in Thoroughbred Mares

The secretions of the equine endometrial glands are essential for the survival, growth, and development of the conceptus in early pregnancy, and endometrial gland density is directly related to successful pregnancy outcome. Endometrial biopsy is routinely used to assess the reproductive potential of broodmares. Some previous studies have shown that equine endometrial glands are uniformly distributed throughout the uterus; however, other work has shown variation of the endometrial architecture between biopsy sites, suggesting that a single biopsy is not representative of the entire endometrium. The aims of this study were to assess and compare the endometrial gland density and thickness at four sampling sites in the uterus (the central segment of each uterine horn, the uterine horn-body junction, and the caudal portion of the uterine body). Endometrial samples from five nulliparous Thoroughbred mares in diestrus were obtained at necropsy and used for subsequent histomorphometric analysis. The caudal uterine body had a significantly lower endometrial gland density and endometrial thickness than the other sites. This may result in nutrient deprivation and reduced survival of embryos or fetuses in this region of the uterus. The endometrial gland density and endometrial thickness did not significantly differ between the other regions sampled, indicating that they are similarly suitable for embryonic implantation and fetal development. Our results suggest that the endometrial structure of the caudal uterine body of the mare is not representative of the endometrial morphology at other sites. Thus, the caudal uterine body is not a suitable site for routine endometrial biopsy.     

Proliferation and Apoptosis in Fetal Membranes and Endometrium During Placental Retention in Heavy Draft Mares

Placental retention (retained placenta [RP]) is a serious and common peripartum disease in mares, but the etiology and pathogenesis of RP still remain unclear. The alteration of cell proliferation and apoptosis is considered to be an important factor in RP. Fetal membranes and endometrial biopsies were collected from mares with RP (n = 8) and from control mares (n = 10). The proliferation and apoptosis levels in the chorionic and the endometrial epithelia were assessed by proliferating cell nuclear antigen immunostaining and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, respectively. The study revealed that there was an insignificant decrease in proliferation and a significant increase in apoptosis in the chorionic epithelium from mares with RP. This result excludes a proliferation imbalance from the possible causes of RP. In the area of the nonpregnant horn of the placenta, proliferation was negatively correlated and apoptosis was positively correlated with the degree of fetomaternal anchorage. It was observed that, in all mares with placental retention, the endometrial epithelium (both luminal and glandular) showed decreased proliferation and increased apoptosis, which may indicate a delay in postpartum uterine regeneration.     

氦氖激光对母兔生殖功能的影响

本试验利用氮氖激光照射8只4月龄母兔的阴蒂及其周围粘膜,用放射免疫测定方法监测15只受试兔外周血浆中孕酮和前列腺素F_(2α)的含量。试验结果表明,除试验组母兔增重速度明显慢于对照组母兔外,生殖首的发育和卵巢内卵泡的数量分别显著(p<0.05)和极显著(p<0.01)优于对照组;并且,试验组母兔的发情次数和产仔数及孕酮含量明显高于对照组。因此,本试验证明,一定能量密度的氦氖激光照射能促进母兔生殖道发育,并且有促进多卵发育、成熟和提高产仔数的作用。关于氦氖激光对母兔增重速度的影响则有待于进一步扩大实验证实。     

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.     

Endometrial Phospholipase A2 Activity During the Oestrous Cycle and Early Pregnancy in Mares

The aim of this study was to determine phospholipase A2 (PLA2) kinetics and activity in the mare’s endometrium during the oestrous cycle and early pregnancy. Phospholipase A2 is responsible for the liberation of arachidonic acid from phospholipids, which is the first limiting step in prostaglandins synthesis. Phospholipase A2 activity was measured using an assay based on the liberation of oleic acid from 1‐palmitoyl‐2‐[¹⁴C] oleoyl phosphatidylcholine. The enzyme was shown to be calcium dependent, to have an optimum pH of 8 and an apparent Michaelis constant of 127 μm . Enzyme activity was low in the endometrium of early luteal phase tissue but increased significantly (p < 0.001) during the late luteal phase (5.39 ± 0.16; 3.48 ± 0.33, 6.85 ± 0.59, and 9.96 thinsp;± thinsp;1.23 thinsp;nmol oleic acid released/mg protein at oestrus, and Days 3, 8 and 14 after ovulation, respectively). The mean PLA2 activity in endometrial tissue from pregnant mares (4.23 ± 0.74) was significantly lower (p < 0.01) than from cyclic animals during late dioestrus (9.96 ± 1.23). The results indicate that PLA2 activity in equine endometrium changes with the stage of the oestrous cycle and thus may be influenced by systemic hormone concentrations. The inhibitory effects of conceptus products on secretion of prostaglandin during early pregnancy were associated with a competitive inhibitor that decreased endometrial PLA2 activity.     

Feasibility and Safety of Endometrial Injection of Autologous Bone Marrow Mesenchymal Stem Cells in Mares

​Chronic degenerative endometritis (CDE) is an important cause of fertility problems in older mares. It is estimated that 30% of mares breeding are over 18 years old and the high value of their progeny encourages the use of these animals in assisted reproduction procedures. Currently, cell-based therapies are broadly used in human and veterinary regenerative medicine and have been showed a good effect on the treatment of liver fibrosis. Thus, the present study aimed to evaluate the feasibility and safety of endometrial injection of autologous bone marrow MSCs in mares. Mares were examined by transrectal ultrasound and the endometrial stem cells injections performed during diestrous, using injection needles coupled to a teflon catheter through the biopsy channel of a flexible endoscope. After treatment clinical evaluations (heart reat, respiratory rate, staining of the mucosa, capillary refill time, body temperature and lameness score) were performed daily during seven days. The intrauterine fluid and endometrial edema was evaluated before, 24 and 48 hours after the procedure by ultrasonographic exams and the inflammatory infiltrate (polymorphonuclear cells and mononuclear cells) and degree of fibrosis by histological evaluations before (D0) and 15 (D15), 30 (D30) and 60 (D60) days after endometrial injections. The results were evaluated by Kolmogorov-Smirnov and Kruskal-Wallis test followed by Dunn test and for fertility rates was used Chi-square, considering a 5% significance level. Neither clinical alteration was observed in mares after treatment, as well as, intrauterine fluid and endometrial edema were not detected in any mare before and after cell therapy. After cell therapy, significantly more PMNs were found in D15 biopsies, however, these cells were not observed in D30 and D60 biopsies. No worsening on the histological architecture after treatment was observed in any mare. In conclusion, the results of this study showed that endometrial hysteroscopic injections of stem cells in mares is a safe procedure.     

Investigations on the Endometrial Response to Intrauterine Administration of N‐Acetylcysteine in Oestrous Mares

In mares, mating‐induced persistent endometritis contributes to low fertility. The condition is in part related to delayed clearance of mucus accumulated within the uterine lumen. The objective of this study was to investigate the endometrial response of healthy mares to intrauterine (i.u.) treatment with N‐acetylcysteine (NAC). Oestrous mares (n = 12) were randomly assigned to a treatment (TM) or control (C) group and received an i.u. infusion of 5% NAC and saline (total volume 140 ml), respectively. Endometrial biopsies were collected in five of the mares 24 h after treatment, in the remaining seven mares 72 h after treatment. Endometrial biopsies were evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki‐67), lectins and periodic acid‐Schiff (PAS). The integrity of endometrial epithelial cells was not affected by treatment (no statistical differences between groups or times). At 24 h after treatment, the mean number of PMN in endometrial biopsies from NAC‐ and C‐mares did not differ, but at 72 h after treatment, number of PMN was significantly higher (p < 0.05) in C (3.9 ± 0.6 PMN/field) compared with NAC‐treated mares (2.3 ± 0.2 PMN/field). At 72 h after treatment, the intensity of staining for COX2 was significantly higher after saline than after NAC treatment (p < 0.05). In the epithelium, no differences in staining for the proliferation marker Ki‐67 were seen with respect to time and treatment. Score for the lectin wheat germ agglutinin (WGA) was slightly higher in NAC‐treated mares than in C‐mares 72 h after treatment (p < 0.05). Score for PAS staining of mucus in deep uterine glands differed significantly between groups at 24 h after treatment (p < 0.05). The present study demonstrates that NAC does not adversely affect the endometrial function. Moreover, an anti‐inflammatory effect on the equine endometrium was observed.     

Cefquinome Concentrations in Endometrium after Intrauterine Treatment of Cobactan 4.5%® in Mares and Inflammatory Response of the Endometrium to this Treatment

This study was conducted to measure the concentration of cefquinome in the endometrium of mares after intrauterine treatment and to evaluate associated inflammation. Mares (n = 14) were randomly assigned to one of the following groups: (i) control (n = 4) were either not treated (n = 2) or received (n = 2) lactated Ringer's intrauterine for 1 or 3 days; (ii) treated mares (n = 10) received intrauterine cefquinome for 1 or 3 days. After at least 10 days had passed following the last treatment and ovulation, mares were given Prostaglandin F2α (PGF2α) and were randomly assigned to an alternate treatment. Endometrial biopsy samples were taken at 2, 8, 24 and 48 h, or at 4, 12 and 36 h, after the last treatment. Biopsy samples were taken at the same time points from control mares (n = 2) and lactated Ringer-treated mares (n = 2). Cefquinome concentrations were quantified using a high-performance liquid chromatography (HPLC) assay and inflammation was assessed using haematoxylin and eosin (H&E)-stained sections. Concentrations of cefquinome [559 (1 day) and 595 μg/g (3 days) at 2 h, and 403 (1 day) and 370 μg/g (3 days) at 4 h] were similar between treatment groups at 2 and 4 h after treatment (p > 0.05). At 8 h, as well as at 24 and 48 h, concentrations were greater in the 3-day group (17 vs 301 μg/g, 3 vs 80 μg/g and 0.1 vs 0.2 μg/g, respectively) (p < 0.05). No significant differences (p > 0.05) in the inflammatory response at 2–48 h after treatment were found between groups.     

Degenerative Endometrial Changes do not Change the Functional Capacity of Immigrating Uterine Neutrophils in Mares

An endometritis model was used to investigate the influence of degenerative endometrial changes (endometrosis) on functional parameters of uterine neutrophils in the horse. Six hours after intrauterine application of recombinant human interleukin‐8 (rhIL‐8), the uteri of 15 mares were flushed with phosphate‐buffered saline. Quantitative and qualitative flow cytometric assays were then made to determine the absolute numbers, viability, phenotype, generation of reactive oxygen species (ROS), and phagocytic activity of immigrated polymorphonuclear neutrophilic granulocytes (PMN). Recombinant hIL‐8 attracted similarly high numbers of similarly viable PMN into the uteri of mares with or without degenerative endometrial changes. Compared with blood PMN, immigrated uterine neutrophils displayed significantly upregulated expression of CD11a/CD18 (LFA‐1) on uterine PMN whereas major histocompatibility complex class I molecules were expressed at lower densities. The ability to phagocytose opsonized streptococci did not differ between uterine and blood PMN. However, uterine PMN displayed a higher capacity to generate ROS. On average, uterine PMN of mares with degenerative endometrial changes showed phenotypical and functional characteristics similar to those of mares with a histologically healthy endometrium. Therefore, degenerative endometrial changes per se did not reduce the functional capacity of equine uterine neutrophils in mares.     

X-ray Microanalysis of the Secretory Epithelium of the Endometrial Glands and Intraluminal Uterine Fluid in Oestrus Mares

X‐ray microanalysis was used to determine the occurrence of transudation as a source of intrauterine fluid in mares during oestrus. The content of some selected elements was studied in secretory vesicles of the epithelium of endometrial glands and in intraluminal fluid of healthy mares of varying age and parity. An in situ cotton tampon was used to absorb intrauterine fluid from 23 gynaecologically healthy mares during oestrus. Immediately after tampon withdrawal, endometrial tissue for biopsy was removed from the uterine body/horn junction while videoendoscopy was performed. The tissue specimens were frozen ultra‐rapidly against a copper surface chilled with liquid N₂. X‐ray microanalysis of frozen fully hydrated endometrial samples and of microdroplets of intraluminal fluid (n = 20) was performed, using a SEM microscope equipped with a cryo‐stage and an X‐ray microanalysis system. The concentrations of sodium (Na), potassium (K), chlorine (Cl), sulphur (S), phosphorus (P) and calcium (Ca) in the epithelium of endometrial glands and in intraluminal fluid were compared with those in serum. There was a significant difference in the concentrations of all analysed elements between the secretory gland epithelium and the intraluminal fluid apart from S. The major elements in glandular epithelium were K and P. Although there was a significant difference in the concentrations of Na, Cl, S and Ca between the uterine fluid and serum, there was still a conspicuous resemblance in the relationship between the different elements in uterine fluid and serum, with Na and Cl being the dominant elements. Sulphur and especially Ca were present only in small amounts throughout. Neither age nor parity affected the elemental concentrations of Na, K, Cl, S, P and Ca in gland epithelium, uterine fluid or serum. It was concluded that serum transudation contributes to a great extent to the composition of uterine fluid, having a diluting effect upon the specific secretion of the uterine glands.     

Effect of Administration of Oxytocin During Diestrus on Corpus Luteum Function and Endometrial Oxytocin Receptor Concentration in Cycling Mares

The objectives of this study were to (1) compare the effect of twice versus once daily administration of oxytocin on days 7-14 after ovulation on the duration of corpus luteum (CL) function and (2) determine the effect of oxytocin treatment on endometrial oxytocin receptor concentration in mares. In experiment 1, mares were randomly assigned to three groups on day 7: (1) untreated control group (n = 7), (2) twice daily oxytocin treatment group (n = 7), and (3) once daily oxytocin treatment group (n = 8). Oxytocin-treated mares received 60 U of oxytocin intramuscularly (IM) the respective number of times each day on days 7 through 14. One of seven control mares (14%), five of seven (71%) twice daily oxytocin-treated mares, and five of eight (63%) once daily oxytocin-treated mares had prolonged CL function. There was no significant difference in the proportion of mares with prolonged CL function between the two oxytocin-treated groups, and collectively, oxytocin treatment increased (P < .05) the proportion of mares with prolonged CL function compared with no treatment. In experiment 2, mares were randomly assigned to two groups (n = 5/group): (1) saline-treated control mares, and (2) oxytocin-treated mares. Beginning on day 7, control mares received 3 mL of sterile saline IM twice daily, and oxytocin-treated mares received 60 U of oxytocin IM twice daily through day 14. On day 15, endometrial oxytocin-binding capacity was determined (as a measure of oxytocin receptor concentration), and there was no difference (P > .1) between control and oxytocin-treated mares (1,465.7 ± 108 and 1,382.8 ± 108 fmol/mg protein [mean ± standard error of mean], respectively).     

Effects of Topical Application of Misoprostol on Cervical Relaxation in Mares

Mares who have not delivered a foal early in life may experience limitations in cervical relaxation, primarily during oestrus. A closed cervix prevents intrauterine deposition of semen during natural breeding, may delay uterine clearance after insemination leading to intrauterine fluid accumulation in, and subsequent infertility. Therefore, a reliable pharmacological method of dilating the equine cervix would have practical application in veterinary medicine. The goal of this study was to investigate the effectiveness of topically applied, synthetic prostaglandin E₁ analogue (PGE₁) for stimulating dilation of the equine cervix. Ten mares in dioestrus were randomly assigned to one of two treatments in a single‐blind crossover study: (treatment) PGE₁ gel (1000 mcg compounded misoprostol cream) applied topically to the external cervical os (n = 5), and (control) a vehicle cream applied topically to the external cervical os (n = 5). Transrectal palpation and ultrasonographic measurements of the cervix were performed prior to, six and 24 h post‐treatment. Digital measurements were taken, per vagina, at six and 24 h post‐treatment. Mares were monitored through the subsequent oestrous cycle for ovulation. Mares were assigned to the opposite treatment group such that each mare served as her own control (crossover). Data were analysed using parametric (split‐plot anova ), as well as nonparametric (Kruskal–Wallis anova , Wilcoxon's rank‐sum test) methods. At six and 24 h there were no significant differences for tone, length, height, degree of relaxation or echotexture between control and PGE₁ treated groups at the measured time points (p > 0.05). Topical cervical application of PGE₁ did not induce a measurable degree of cervical relaxation under the conditions of this experiment.     

Comparison of the Cytology Brush and Cotton Swab in the Cytological Evaluation of the Endometrium in Mares with Regard to Fertility

The aim of this study was to compare the cytology brush (CB) and cotton swab (CS) in the cytological evaluation of the endometrium in mares with regard to fertility. The study was conducted on 26 mares displaying spontaneous estrus. Samples for cytological evaluation were taken from each mare by using commercially available CS and CB. After sample collection, all mares were mated in the same estrus, and pregnancy diagnosis was performed 14-18 days after last mating. No vaginal cells were found in smears, and the CB technique yielded significantly more cells/high-power field (HPF) than the CS technique. Additionally, more cases of endometritis were diagnosed using the CB than the CS. It was also shown that the degree of inflammation is more important in diagnosis of infertility in the mare than the mere presence or absence of inflammation. In conclusion, further studies are needed to establish more precise criteria for the interpretation of inflammation, especially if samples are collected using the CB.     

Effects of Repeated Embryo Collection on Embryo Recovery Rate in Fertile Mares

In mares, repeated embryo collection in successive oestrous cycles is necessary if a greater number of foals should be produced. We investigated effects of repeated embryo collection in fertile donor mares on embryo recovery rates. In addition, an influence of the individual mare and season on embryo recovery rates was studied. In nine mares, a total of 153 embryo collections were performed during 30 months (17 ± 2.2 embryo collections per mare). The overall embryo recovery rate was 64% and did not differ among mares. Between successive embryo collection procedures, recovery rate varied significantly; however, no increase or decrease in the embryo recovery rate with increasing number of successive embryo collections was seen. In three mares, ovulation ceased from November to February. In the remaining six mares, embryo production was successfully continued throughout winter and no influence of the month on embryo recovery rates was detected.     

Induced Lactation with a Dopamine Antagonist in Mares: Different Responses between Ovariectomized and Intact Mares

The aim of this study was to compare the effects of treatment with repeated injections of sulpiride (a dopamine D₂ antagonist) on prolactin secretion and induced lactation in ovariectomized and intact adult mares and to verify if this induction was possible at the beginning and at the end of the birth season. Two experiments were carried out in September [experiment (expt) 1], and in March (expt 2), in France (48°N). In expt 1, three groups of five mares were tested: intact‐control, intact‐treated and ovariectomized‐treated mares. In expt 2, mares previously subjected to artificial photoperiod were assigned in two groups: four intact‐control and five intact‐treated mares. The cyclicity of intact mares was previously synchronized with PGF_2α injections, then all the mares were in the follicular phase at the beginning of treatment. Sulpiride was intramuscularly injected (0.5 mg/kg of BW), twice a day. Mares were milked at 7:30, 11:45, 16:00 and 20:15 hours. Blood samples were collected every day during the treatment for progesterone, total oestrogen and prolactin assays. In the two experiments, only treated intact mares produced milk, with a large inter‐animal variability. Prolactin increase after sulpiride treatment was not so great in the ovariectomized‐treated mares as in the intact‐treated mares. The total correlations between prolactin, progesterone, oestrogen plasma concentrations and daily milk production were significant (0.57, 0.25, 0.17 respectively). This induction of lactation can be performed during the entire birth season in intact mares, but not in ovariectomized mares, indicating that steroids are necessary for this induction in mares treated by dopamine D2 antagonist.     

半导体激光照射对犬创伤愈合的影响

用本地杂种犬１２只分为两组，在背部两侧定边长２ｃｍ的正方形皮肤全层缺损创，对照组左侧作为划体对照，每天对创面愈合情况进行观察记录。结果表明：砷化镓激光照射可促进犬全层皮肤缺损创的愈合，但差异不显著，而对激光照射７天内的愈合速度有显著的促进作用。     

Effects of Epinephrine,Detomidine, and Butorphanol on Assessments of Insulin Sensitivity in Mares

Sympathoadrenal stimulation may perturb results of endocrine tests performed on fractious horses. Sedation may be beneficial; however, perturbation of results may preclude useful information. Four experiments were designed to 1) determine the effects of epinephrine on insulin response to glucose (IR2G), 2) assess the effects of detomidine (DET), alone or combined with butorphanol (DET/BUT), on IR2G and glucose response to insulin (GR2I), and 3) assess the effects of BUT alone on IR2G. In Experiment 1, mares were administered saline or epinephrine (5 μg/kg BW) immediately before infusion of glucose (100 mg/kg BW). Glucose stimulated (P < .05) insulin release in controls at 5 minutes that persisted through 30 minutes; insulin was suppressed (P < .05) by epinephrine from 5 to 15 minutes, rising gradually through 30 minutes. Experiments 2 (IR2G) and 3 (GR2I) were conducted as triplicated 3 × 3 Latin squares with the following treatments: saline (SAL), DET, and DET/BUT (all administered at .01 mg/kg BW). Glucose stimulated (P < .05) insulin release that persisted through 30 minutes in SAL mares; DET and DET/BUT severely suppressed (P < .0001) the IR2G. Sedation did not affect resting glucose and had inconsistent effects on the GR2I when mares were treated with 50 mIU/kg BW recombinant human insulin. Butorphanol had no effect on IR2G. In conclusion, adrenergic agonists severely suppress the IR2G and cannot be used for sedation for this test. The use of DET did not alter the GR2I, and therefore may be useful for conducting this test in fractious horses.