Expression of Aquaporin Water Channels in Equine Endometrium is Differentially Regulated During the Oestrous Cycle and Early Pregnancy

The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non‐pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time‐dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non‐pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14 days after ovulation from non‐pregnant animals, in particular luminal epithelial staining. Samples collected 8 days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up‐regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine‐specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare.     

Luteal vascularity and embryo dynamics in mares during early gestation: Effect of age and endometrial degeneration

The present study characterized the luteal status and the dynamic of the conceptus during the first 20 days of gestation in mares with different ages and degrees of endometrial degeneration. Total area of the corpus luteum (CL), luteal vascularity, CL area with blood signals, progesterone concentrations (P4), embryonic vesicle diameter, number of embryonic location changes, embryonic fixation position and uterine contractility were evaluated. In Experiment 1, mares ≤6 years of age (Young group, 5.6 ± 0.2 years, n = 7 mares) and mares ≥15 years of age (Old group, 17.2 ± 0.9 years, n = 6 mares) were used to investigate the effect of age. In Experiment 2, the luteal and embryonic parameters were compared between mares with minimal (Mild group, endometrial category I, n = 9 mares) and severe (Severe group, endometrial category III, n = 7 mares) endometrial degeneration. The Old and Severe groups had greater (p ≤ 0.04) total CL area and reduced luteal vascularity (p ≤ 0.04) than the Young and Mild groups, respectively. However, P4 levels and CL area with blood signals were similar (p ≥ 0.8) between the groups. A negative effect of age (p < 0.01), but not of endometrial degeneration (p = 0.6), was found for the embryonic vesicle diameter. The conceptus mobility was high (p > 0.1) until day 14 of gestation in the Severe group, while a reduced number of changes of the embryo location was detected earlier (p < 0.05) in the Old group. In conclusion, the newly formed CL of aged mares and mares with severe endometrial degeneration suffered a structural remodelling to safeguard the local blood supply and the continuous P4 output during early gestation. Moreover, an earlier reduction of the embryonic mobility and a delayed development of the conceptus were associated with advanced age, regardless of the degree of endometrial degeneration.     

Follicle Diameter and Systemic Hormone Interrelationships during Induction of Follicle Collapse with Intrafollicular Prostaglandin E2 and F2α in Mares

The objectives were to determine: (i) whether intrafollicular administration of PGE2 and PGF2α to mares would hasten follicle collapse and (ii) the differences in reproductive hormone characteristics in mares with spontaneous and prostaglandin‐induced follicle collapses. Six mares were followed for two oestrous cycles each: when the mares reached a follicle diameter of 30–35 mm and showed mild‐to‐moderate endometrial oedema, mares were administered a single 0.5 ml dose containing 500 μg PGE2 and 125 μg PGF2α (treatment cycle) or a placebo (0.5 ml of water for injection; control cycle) into the preovulatory follicle (Hour 0). Blood samples were collected, and serial ultrasound examinations were performed until follicle collapse. Treated mares showed follicle collapse significantly earlier (20.0 ± 5.9 h) than the control mares (72.0 ± 10.7 h). The LH, progesterone, total oestrogens and oestradiol concentrations did not differ between groups; however, the progesterone concentration increased more between 48 and 72 h after follicle injection in the treatment compared to the control cycles (P < 0.05). In conclusion, intrafollicular treatment with PGE2 and PGF2α hastened follicle collapse in mares without the simultaneous use of an inductor of ovulation; despite the early induction of follicle collapse, the profiles of LH and oestradiol were not altered. This study provides information on the role of prostaglandins (PGs) in the process of follicle wall rupture and collapse and suggests that this may happen even before the beginning of the sharp rise in circulating LH at the final stage of the ovulatory surge.     

Oestradiol enhances plasma growth hormone and insulin-like growth factor-I concentrations and increased the expression of their receptors mRNAs in the liver of ovariectomized cows

Many metabolic hormones, growth hormone (GH), insulin-like growth factor-I (IGF-I) and insulin affect ovarian functions. However, whether ovarian steroid hormones affect metabolic hormones in cattle remains unknown. This study aimed to determine the effect of sex steroids on the plasma profiles of GH, IGF-I and insulin and their receptors in the liver and adipose tissues of dairy cows. Ovariectomized cows (n = 14) were randomly divided into four groups: control group (n = 3) was treated with saline on Day 0; oestradiol (E2) group (n = 3), with saline and 1 mg oestradiol benzoate (EB) on Day 0 and 5, respectively; progesterone (P4) group (n = 4) with two CIDRs (Pfizer Inc., Tokyo, Japan) from Day 0; and E2 + P4 group (n = 4) with two CIDRs on Day 0 that were removed on Day 6 and were immediately injected with 1 mg EB. The animals were euthanized after the experiment, and liver and adipose tissues samples were quantitatively analysed using real-time PCR for the expression of mRNA for the GH (GHR), IGF-I (IGFR-I) and insulin (IR) receptor mRNAs. Oestradiol benzoate significantly increased the number of peaks (p < 0.05), pulse amplitude (p < 0.05) and area under the curve (AUC; p < 0.01) for plasma GH; moreover, it increased plasma IGF-I concentration (p < 0.05), but it had no effect on the plasma insulin profile. P4 significantly decreased the AUC (p < 0.01), compared with the control group, whereas it did not affect the number of peaks and the amplitude of GH pulses. P4 + E2 did not affect the GH pulse profile. E2 increased the mRNA expression of GHR, IGFR-I and IR in the liver (p < 0.05), whereas both P4 and E2 + P4 did not change their expressions. Our results provide evidence that the metabolic and reproductive endocrine axes may regulate each other to ensure optimal reproductive and metabolic function.     

Development of an in vitro model for the analysis of bovine endometrium using simple techniques

This study aimed to develop an in vitro model for the analysis of the bovine endometrium. Immunofluorescent staining revealed that the hetero‐spheroids and the cultured explants showed almost similar structure in the localization of bovine endometrial epithelial cells and endometrial stromal cells, except the glandular‐like structure of the epithelial cells inside the explants. Gelatin zymography revealed that the hetero‐spheroids did not express matrix metalloproteinases (MMPs) after 4 days of culture, but strong MMP expressions were observed in the cultured explants until 7 days of culture. Additionally, expression of progesterone receptor (PR), estrogen receptor alpha (ERα), type I interferon receptor 1 (IFNAR1) and 2 (IFNAR2) messenger RNA was observed both in the homo‐ and hetero‐spheroids. The expression of oxytocin receptor (OTR) mRNA in E2 and E2+P4 (1,3,5(10)‐Estratrien‐3, 17β‐diol + 4‐Pregnen‐3, 20‐dinone) treated groups were significantly (P < 0.05) higher than that of the control group of spheroids. In case of cultured explants, the expression of PR and OTR mRNA were significantly (P < 0.05) higher in E2 treated groups compared to the control groups. Hepatocyte growth factor (HGF) mRNA expression was also higher in P4 treated groups at 10 days in culture (P < 0.05). In a nutshell, the in vitro model developed in this study for the analysis of the endometrium may provide a new platform for extensive research on bovine endometrial function.     

Effect of altrenogest‐treatment of mares in late gestation on adrenocortical function,blood count and plasma electrolytes in their foals

Reasons for performing study: Mares with compromised pregnancies are often treated with altrenogest to prevent abortion. However, there is only limited information about effects on the foal when altrenogest treatment is continued during final maturation of the fetus. Objectives: To determine effects of altrenogest treatment during late gestation in mares on maturity, haematology changes, adrenocortical function and serum electrolytes in their newborn foals. Methods: Six mares were treated with altrenogest (0.088 mg/kg bwt) once daily from Day 280 of pregnancy until foaling and 7 mares served as controls. Results: Foals born to altrenogest‐treated mares had a significantly lower neutrophil/lymphocyte ratio on the first day after birth than control foals (P<0.05). Basal plasma cortisol concentrations immediately after birth were higher in foals of altrenogest‐treated mares than in control foals (P<0.05). Cortisol release in response to exogenous adrenocorticotropic hormone (ACTH) ‐ except for higher values 15 min after ACTH injection in foals of altrenogest‐treated mares on Day 1 ‐ revealed no differences in adrenocortical function between the groups of foals. Plasma potassium concentration in foals from altrenogest‐treated mares compared to control foals was significantly lower immediately after birth (P<0.05) and plasma ionised calcium concentration was significantly lower 3 h after birth (P = 0.01). Conclusions and potential relevance: Altrenogest treatment of pregnant mares prolonged labour had no major effects on adrenocortical function in foals. A reduced neutrophil/ lymphocyte ratio in these foals may suggest either immunomodulatory effects of altrenogest or dysmaturity of the foals.     

The effect of pulsatile gonadotropin-releasing hormone and estradiol administration on luteinizing hormone and follicle-stimulating hormone concentrations in pituitary stalk-sectioned ovariectomized pony mares

Hourly pulses of gonadotropin-releasing hormone (GnRH) or bi-daily injections of estradiol (E2) can increase luteinizing hormone (LH) secretion in ovariectomized, anestrous pony mares. However, the site (pituitary versus hypothalamus) of positive feedback of estradiol on gonadotropin secretion has not been described in mares. Thus, one of our objectives involved investigating the feedback of estradiol on the pituitary. The second objective consisted of determining if hourly pulses of GnRH could re-establish physiological LH and FSH concentrations after pituitary stalk-section (PSS), and the third objective was to describe the declining time trends of LH and FSH secretion after PSS. During summer months, ovariectomized pony mares were divided into three groups: Group 1 (control, n = 2), Group 2 (pulsatile GnRH (25 μg/hr), n = 3), and Group 3 (estradiol (5 mg/12 hr), n = 3). All mares were stalk-sectioned and treatment begun immediately after stalk-section. Blood samples were collected every 30 min for 8 h on the day before surgery (DO) and 5 d post surgery (D5) to facilitate the comparison of gonadotropin levels before and after pituitary stalk-section. Additionally, jugular blood samples were collected every 12 hr beginning the evening of surgery, allowing for evaluation of the gonadotropin secretory time trends over the 10 d of treatment. On Day 10, animals were euthanized to confirm pituitary stalk-section and to submit tissue for messenger RNA analysis (parallel study). Plasma samples were assayed for LH and FSH by RIA. Mean LH secretion decreased from Day 0 to Day 5 in Groups 1 and 3, whereas LH secretion tended (P < 0.08) to decrease in Group 2 mares. On Day 5, LH was higher (P < 0.01) in Group 2 (17.26 ± 3.68 ng/ml; LSMEANS ± SEM), than either Group 1 (2.65 ± 4.64 ng/ml) or group 3 (4.28 ± 3.68 ng/ml). Group 1 did not differ from Group 3 on Day 5 (P < 0.40). Similarly, mean FSH levels decreased in all groups after surgery, yet Group 2 mares had significantly (P < 0.001) higher FSH concentrations (17.66 ± 1.53 ng/ml) than Group 1 or Group 3 (8.34 ± 1.84 and 7.69 ± 1. 63 ng/ml, respectively). Regression analysis of bi-daily LH and FSH levels indicated that the time trends were not parallel. These findings indicate: 1) Pituitary stalk-section lowered LH and FSH to undetectable levels within 5 d after surgery, 2) pulsatile administration of GnRH (25 μg/hr) maintained LH and FSH secretion, although concentrations tended to be lower than on Day 0, and 3) E2 did not stimulate LH or FSH secretion.     

Exercising the Pregnant Mare from Day 16 to Day 80 of Gestation

Our previous research has demonstrated moderate exercise can be detrimental to early pregnancy in the mare, but little work has examined exercise after pregnancy has been detected. We exercised mares (n = 8) 6 days a week for 45 min from Day 16 until Day 80 of gestation. Color Doppler ultrasonography was used to evaluate embryonic vesicle size, fetal length, and uterine blood flow. Blood was sampled every other day to analyze cortisol and progesterone concentrations. Results indicated that exercising pregnant mares (n = 4) led to greater (P < .01) cortisol concentrations 30 min after the exercise period. No overall treatment effect could be detected in progesterone concentrations; however, following Day 60 of gestation, progesterone concentrations were lower (P < .05) in exercised mares. Additionally, progesterone concentrations peaked earlier in exercised mares at Day 52 of gestation compared to peak levels at Day 68 of gestation for control mares. No significant effects were detected in embryonic vesicle size. Fetal length tended (P = .06) to be longer in the conceptus of exercised mares. Uterine blood flow did not differ between groups but increased as pregnancy progressed (P < .001) in both groups. All mares in this study went on to deliver healthy foals and suffered no difficulties during parturition. These results indicated that moderate exercise was not detrimental to mare pregnancy.     

Proteins and Enzymes in Uterine Lavage Fluid of Postpartum and Nonparturient Mares

Uterine lavage fluids from postpartum and nonparturient mares were compared to determine when the normal secretory capacity of the postpartum uterus is restored. Lavage fluids were obtained from cyclic nonparturient mares on the second, fourth or fifth day of oestrus, and 3, 8, or 14 days after ovulation (seven mares/sampling day). Twelve intact postpartum mares were sampled 1 to 28 days postpartum (group A: 1, 6, 12 and 20; group B: 2, 8, 14 and 24; group C: 4, 10, 16 and 28 days postpartum; four mares/group). Three ovariectomized (OVX) postpartum mares were sampled as mares in group C. Samples were analysed for neutrophils, bacteria, total protein concentration, proteolytic and antiproteolytic activities and for various lysosomal enzyme activities. In nonparturient mares, activities of acid phosphatase, β‐glucuronidase (B‐Gase), and N‐acetyl‐β‐D‐glucosaminidase (NAGase) in uterine lavage fluids were significantly higher in mid‐ and late‐dioestrus than in mid‐ to late‐oestrus (p < 0.05). Lysozyme concentration, trypsin‐inhibitor capacity (TIC), and plasmin activity were below the detection limit in nonparturient mares. One to four days postpartum, total protein, acid phosphatase, B‐Gase, and NAGase were high but declined rapidly thereafter. Lysozyme and plasmin activities were high 1 to 6 days postpartum. TIC peaked around day 6 postpartum. On day 16 postpartum, acid phosphatase, B‐Gase, and NAGase, being progesterone‐dependent, tended to be higher in intact mares than in OVX ones (p < 0.1). Total protein and lysozyme concentrations, TIC, and B‐Gase (p < 0.01) and acid phosphatase (p < 0.05) activities were significantly higher in parturient mares during postpartum oestrus than in oestrous nonparturient mares. High total protein concentration and TIC, and detectable lysozyme and plasmin activities during postpartum oestrus were associated with uterine inflammation. During dioestrus, differences between postpartum and nonparturient mares were not statistically significant and suggested that the endometrium of postpartum mares had resumed its normal secretory capacity by this time.     

Melatonin and CIDR improved the follicular and luteal haemodynamics,uterine and ovarian arteries vascular perfusion,ovarian hormones and nitric oxide in cyclic cows

This study hypothesizes that melatonin with exogenous progesterone (CIDR) can improve follicular, luteal, ovarian and uterine haemodynamic of heat-stressed cows. Holstein cows (N = 12) studied for two spontaneous oestrous cycles during winter then divided equally during summer into the CIDR group received CIDR for 7 days and the melatonin group (Mel) received three injections of melatonin (75 mg/head) at the CIDR insertion, removal and ovulation days. Blood samples were collected to assay oestradiol (E2), progesterone (P4) and nitric oxide (NO). On day 0 (Ovulation), Mel had more small follicles (p < .05), higher ipsilateral and contralateral ovarian arteries (Ov.A.) peak systolic velocity (PSV), higher ipsilateral uterine artery (Ut.A.) PSV (p = .031) and blood flow volume (BFV), also Mel elevated contralateral Ut.A. PSV and BFV (p < .0001) but lowered contra Ut.A. pulsatility index (PI, p < .0001), E2 (p < .01) and NO (p < .0001). Mel increased the corpus luteum diameter (CL, p < .001), coloured area (p < .007) and P4 (p < .0001) on day 5 and reduced them (p < .05; p < .01) on Day 14. On day 10, Mel obtained CL diameter (p < .03) and coloured area (p < .002) of spontaneous that was higher than CIDR and decreased P4 (p < .003). Mel increased CL diameter, area and coloured area and decreased them thereafter. Mel increased the ipsilateral ovarian and uterine arteries PSV and BFV before ovulation and until day 8. Mel increased P4 and decreased NO until days 6 and 14. In conclusion, the improvement in follicular, luteal, ovarian and uterine haemodynamic and the decrease of NO production proved our hypothesis Melatonin doses higher than 75 mg/head is recommended to improve the heat-stressed cow's fertility.     

The Effect of Mycobacterium Cell Wall Fraction on Histologic,Immunologic, and Clinical Parameters of Postpartum Involution in the Mare

Maintaining yearly foal production is important for the economic success of the broodmare, and this requires breeding to occur as quickly postpartum as possible. The initial postpartum estrus occurs within 5–20 days postpartum, whereas the uterus is still undergoing repair from tissue alterations during pregnancy and parturition, a process known as involution. Attempts have been made to hasten this process, but with minimal success. Mycobacterium cell wall fraction (MCWF) is an immunomodulator that has been shown to reduce bacterial growth and alter aspects of the immune response to breeding, but it is unknown if MCWF hastens the process of involution. Therefore, the objectives of this study were to (1) investigate the effect of MCWF on tissue remodeling, (2) assess the effect of MCWF on the local immune system of the uterus, and (3) determine the optimal treatment interval needed for these processes to occur. We hypothesize that repeated treatments of MCWF postpartum will hasten the process of involution. To study this, 16 pregnant mares of mixed breeds were evaluated postpartum. Control mares (n = 4) received 1.5 mL lactated Ringer’s solution intravenously on Day 1 (Day 0 = day of parturition) postpartum and again on Day 7, whereas treated mares either received 1.5 mL Settle intravenously on Day 1 and Day 7 (TX1; n = 6) or 1.5 mL Settle intravenously on Day 1 and then every 3 days until ovulation was detected (TX2; n = 6) and then evaluated until 15 days postpartum. Mares were assessed every 3 days for clinical, immunologic, and histologic parameters. Clinical parameters were assessed with transrectal ultrasonography and included ovarian activity, uterine fluid retention, and measurement of the uterine diameter, in addition to endometrial culture. Immunologic parameters included endometrial biopsies for quantitative polymerase chain reaction for expression of various cytokines (interleukin [IL]-1β, IL-1RN, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor [TNF], interferon [IFN]-γ, and granulocyte-macrophage colony-stimulating factor) in addition to endometrial cytology. Formalin-fixed endometrial biopsies were histologically assessed for the retention of microcaruncles, dilation of endometrial glands, and inflammation of the mucosa, stratum compactum, and spongiosum. Statistics were performed using SAS 9.4, using a mixed model for repeated measures with mare and treatment as a random effect. All post-hoc analysis was done using a Tukey’s honestly significant difference test. Involution was considered complete by Day 15 postpartum in all mares, and the day postpartum had a significant effect on almost all parameters investigated, indicating the immunologic process of involution. Treatment with MCWF decreased the magnitude of bacterial growth in addition to time to negative culture. In addition, MCWF increased the expression of IL-1β, IFNγ, and TNF. Although minimal treatment effect was noted histologically, a decrease in mucosal inflammation was seen in MCWF-treated mares. In conclusion, involution appears to be influenced by the immune system. In addition, MCWF appears to have a bactericidal effect on the postpartum mare, and this may be because of an increase in proinflammatory cytokines. It is unknown if this bactericidal property will improve fertility on the first estrous cycle postpartum, and future studies are needed to determine this.     

Ruminal epithelial insulin‐like growth factor‐binding proteins 2, 3, and 6 are associated with epithelial cell proliferation

The aim of this study was to identify factors that regulate ruminal epithelial insulin‐like growth factor‐binding protein (IGFBP) expression and determine its role in rumen epithelial cell proliferation. Primary bovine rumen epithelial cells (BREC) were incubated with short‐chain fatty acids (SCFAs) at pH 7.4 or 5.6, lactate, lipopolysaccharide (LPS), insulin‐like growth factor‐I (IGF‐I), ‐II (IGF‐II), or recombinant bovine IGFBP2 (rbIGFBP2). The mRNA expression levels of IGFBP in BREC were analyzed using quantitative real‐time polymerase chain reaction (qRT‐PCR). The proliferation rate of BREC was analyzed using a WST‐1 assay. IGFBP2 gene expression tended to be lower with SCFA treatment (p < .1), and IGFBP6 gene expression was significantly lower with SCFA treatment (p < .05). IGFBP3 and IGFBP6 gene expression tended to be higher with d ‐Lactate treatment (p < .1). IGFBP3 gene expression was significantly higher (p < .05) with LPS treatment. BREC treated with IGF‐I grew more rapidly than vehicle control‐treated cells (p < .01); however, recombinant bovine rbIGFBP2 inhibited IGF‐I‐induced proliferation. IGF‐II and/or rbIGFBP2 did not affect BREC proliferation. Taken together, SCFA treatment decreased IGFBP2 and IGFBP6 expression in rumen epithelial cells, and lower expression of these IGFBP might promote rumen epithelial cell proliferation by facilitating IGF‐I.     

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.     

Early luteal development in Santa Inês ewes superovulated with reduced doses of porcine follicle‐stimulating hormone

The aim was to compare the early luteal development in ewes superovulated with different doses of pFSH. Twenty‐nine Santa Inês ewes received a progesterone device (CIDR®) for 8 days. Gonadotrophic treatment started on Day 6: G200 (control, n = 9, 200 mg); G133 (n = 10, 133 mg); and G100 (n = 10, 100 mg of pFSH). On Day 6, all females received eCG (300 IU). B‐mode and spectral Doppler ultrasonography were performed daily during the early luteal phase (Days 11–15) to monitor the development of corpora lutea (CLs; dimensions) and ovarian arteries indices. CLs were also classified as normal or prematurely regressed (PRCL) on Day 15 by videolaparoscopy. Ewes from G100 and G133 showed gradual increase in luteal diameter during the early luteal phase (p < 0.001), whereas G200 animals presented increase from Day 11 to Day 13, and then decrease on Days 14 and 15 (p < 0.001). The G200 females showed greater percentage of PRCL (45.20%) than those of the other groups (p < 0.001). The normal CLs number was greater in G100 than in G133 (p = 0.04), while the PRCL number was greater in G200 than in the other groups (p = 0.03). Resistive index (RI) was greater in G200 than in G100 (p = 0.02). RI was lower in Day 12 than Day 15 (p = 0.02). Pulsatility index (PI) was greater on Days 14 and 15 (p < 0.01). In conclusion, the lowest dose of pFSH (100 mg) can be considered sufficient for an efficient superovulatory response in sheep, producing better CLs development dynamic in early luteal phase and ovarian blood perfusion and smaller number of PRCL than the traditional (200 mg) pFSH dose.     

Markers for Predicting Overweight or Obesity of Broodmares

Obesity has become of great concern to all equine community from both veterinary and welfare points of view. For estimating obesity markers of brood mares, 17 mares with body conditions were subjected to blood sampling and ultrasound examination to measure rump fat for 6 consecutive weeks. Body length (L), girth (G), and height (H) were measured to estimate body weight (BW), body fat %, body fat mass (BFM) and body mass index (BMI). Mares were classified into three groups according to body condition score (BCS) and rump fat thickness (RF). Overweight mares (O) had BCS >7 and RF >7 mm, moderate (M) had BCS and RF >3 to ≤7, and emaciated (E) had BCS and RF ≤ 3 mm. Glucose, triglycerides, nitric oxide (NO), insulin, insulin-like growth factor-I (IGF-1), leptin, ovarian hormones, and thyroid hormones were measured. Results revealed that BCS, G, L, L × G × H, BW, RF, fat %, and BFM correlated significantly (P < .0001) with body condition. Tetraiodothyronine concentrations of E mares were significantly high (P = .04), but triiodothyronine concentrations tended (P = .07) to be low. Insulin (P = .06) and IGF-1 (P = .07) concentrations tended to be high in O mares. Moderate mares had the highest leptin concentrations (P = .007), but E mares had the lowest P4 concentrations (P = .01). Overweight mares had nonsignificantly high glucose, NO, and triglycerides. In conclusion, back fat and morphometric measurements are the easiest and simple assessment of overweight and obesity. Obese and overweight mares showed slight hyperinsulinemia, hypertriglyceridemia, and hyperglycemia. Hyperleptinemia alone is not indicative of obesity.     

Post natal oestrogen administration stimulates precocious endometrial gland development in the horse

Reasons for performing study: Fillies completely devoid of endometrial glands (uterine gland knockout; UGKO) would make ideal experimental models in which to study the role of endometrial histotroph in embryogenesis and early fetal development in the mare. Hypothesis: Administration of a synthetic progestagen plus oestrogen to newborn filly foals and, thereafter, at regular intervals to age 6 months, would permanently suppress endometrial gland development. Methods: Nine half‐sister Thoroughbred filly foals were treated, in 3 groups, with: A) the weakly active progestagen, norgestomet, administered from birth to age 6 months, in subcutaneous implant form plus oestradiol valerate and norgestomet i.m. at fortnightly intervals; B) the strongly active oral progestagen, altrenogest, administered daily from birth to age 6 months plus fortnightly injections of oestradiol valerate and norgestomet; C) nothing (untreated controls). Endometrial biopsies were recovered from all fillies at ages 6 months and 2 years to assess the degree of endometrial gland morphogenesis and to determine immunohistochemically the presence or absence of oestrogen and progesterone receptors in the endometrial tissues. Results: Groups B and C showed no endometrial gland development, whereas Group A fillies showed a high degree of endometrial gland development, plus strong staining for both oestrogen and progesterone receptors at age 6 months. All 9 fillies showed full normal endometrial gland morphogenesis, development and function at age 2 years. Conclusions and relevance: While the administration of a strongly active progestagen over‐rode the actions of the concomitantly administered oestrogen and suppressed endometrial gland development during the period of administration, treatment with oestradiol valerate together with a weakly active progestagen, stimulated precocious endometrial gland development. Neither steroid was able to create the desired UGKO experimental model and all fillies showed normal endometrial gland development and fertility after puberty. Hence, ovarian oestrogen, not progesterone, appears to be the basic stimulus for endometrial gland morphogenesis in the horse.     

Escherichia coli Lipopolysaccharide-Induced Immunomodulation Along With Oxytocin Administration After Mating as a Treatment Protocol for Persistent Endometritis in Mares

Endometritis is accepted as a major hindrance to achieve optimal reproductive efficiency in mares. The objective of the present study was to evaluate the therapeutic efficacy of combined therapy of immunomodulator and ecbolic as an alternative stand-alone therapy for mares with persistent endometritis. On the basis of history, culture, endometrial cytology, and per rectal and/or ultrasonographic genital examinations, 76 subfertile mares were selected and assigned to three age groups and four treatment (G-1, 2, 3) and control (G-4) groups. At estrus, all the mares were bred once naturally. Thereafter, the mares of G-1 (n = 28) were aseptically treated at 6-hours after natural service with intrauterine infusion (in 50 mL normal saline solution) of 100 μg of Escherichia coli lipopolysaccharide (LPS). Additionally, these mares received two injections of 20 IU of oxytocin (IV) at 12 and 24-hours after infusion. Mares in group G-2 (n = 11) were treated with LPS as mares of G-1, whereas mares in G-3 (n = 12) received oxytocin injections only. The mares of G-4 (n = 23) did not receive any treatment. Pregnancy rates at day 21 and foaling rates were higher (P < .001) in group G-1 than in G-4. In G-1, higher percentage of mares at ages 6–10 years conceived and foaled than mares aged ≥16 years. On re-swabbing of mares that remained nonpregnant, the majority of G-1 and G-2 mares demonstrated sterile cultures and negative cytology, whereas uterine inflammation persisted in mares of G-3 and G-4. In conclusion, the combined therapy was effective for the elimination of persistent endometritis and improved reproductive performance of subfertile mares.     

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.