Endometrial nitric oxide production and nitric oxide synthases in the equine endometrium: Relationship with microvascular density during the estrous cycle

Nitric oxide (NO) plays an important role in angiogenesis and in the regulation of the blood flow. This study was carried out to investigate (i) the effects of endogenous estrogens and progestins and exogenous progesterone (P₄) (5 ng/ml or 1 μg/ml) or estradiol 17β (E₂β) (50 pg/ml or 1 μg/ml) on in vitro endometrial NO synthesis; (ii) the presence of different isoforms of NO synthase; (iii) and their relationship to microvascular density in the equine endometrium during the estrous cycle. NOS expression was also evaluated in the myometrium. Expression of endothelial and inducible forms of NOS in the uterus was assessed by Western blot and immunocytochemistry. Vascular density in endometrial tissue was determined on histologic sections. In the luteal phase, compared to the follicular phase, endometrial NO production increased without exogenous hormones and with exogenous E₂β (1 μg/ml). Although immunocytochemistry revealed iNOS and eNOS expression in the endometrium, no positive signal for iNOS was detected by Western blot. Endothelial NOS was observed in endometrial glands, endothelial cells, fibroblasts, blood and lymphatic vessels. Endometrial eNOS expression was the highest in the follicular and mid-luteal phases while it was found to be the lowest in the early luteal phase. In the follicular phase, hyperplasia of endometrial tissue with respect to myometrium was detected. No difference in vascular density was present between phases. All together, NO may play some roles in both proliferative and secretory phases of endometrial development in the mare.     

Follicular fluid concentration of transforming growth factor-beta1 is negatively correlated with estradiol and follicle size at the early stage of development of the first-wave cohort of bovine ovarian follicles

The objectives of this study were to characterize the relationship between estradiol and transforming growth factor-β1(TGF-β1) concentrations in follicular fluid of growing bovine ovarian follicles, and to examine the effect of TGF-β1 on FSH-stimulated estradiol secretion in cultured bovine granulosa cells. Follicular fluid was collected from individual follicles >5 mm in diameter by ultrasound-guided transvaginal puncture (n = 12 heifers). Follicles were sampled at four different stages of development of the first post-ovulatory wave during selection of the single dominant follicle. Estradiol, progesterone and total TGF-β1 were measured in follicular fluid of the three or four largest follicles sampled when the largest follicle (F1) had reached either 6.5, 7.5, 8.5 or 9.5 ± 0.5 mm stage of development. There was a significant negative relationship between follicular fluid TGF-β1 and estradiol concentrations (R² = 0.44; p < 0.002), and between TGF-β1 concentrations and follicle diameter (R² = 0.23; p < 0.01) in cohort follicles at the 6.5 mm stage, but not at any later stage of development of the follicle wave. There was no correlation between progesterone and TGF-β1 concentrations at any stage. To assess the causal relationship between TGF-β1 and estradiol, granulosa cells from follicles measuring 2–5 mm at dissection were placed in serum-free culture. TGF-β1 caused a dose-dependent decrease in FSH-stimulated estradiol secretion (P < 0.001). These findings suggest that TGF-β1 has an inhibitory effect on estradiol secretion in FSH-stimulated follicles and that a reduction in TGF-β1 inhibition may be part of the mechanism of selection of a single dominant follicle.     

Concentration effect of prostaglandin E2 on the growth factor expression and cell proliferation in bovine endometrial explants and their kinetic characteristics

Bovine endometrium undergoes various physiological and histological changes that are necessary for blastocyst implantation during oestrous cycle. From pro‐oestrus to late‐oestrus, endometrium thickens gradually for implantation preparation and exhibits remarkable capacity for self‐repair after uterine lining shedding while implantation does not occur. The prostaglandin E₂ (PGE ₂) secretion pattern is synchronized with endometrial growth during oestrous cycles in bovine endometrium; however, limited information is available regarding the association between PGE ₂ secretion and endometrial growth. In this study, the concentration (10^?9 to 10^?5 M) and time effect (2–36 hr) of PGE ₂ treatment on a series of growth factors are essential for endometrial growth including connective tissue growth factor (CTGF ), fibroblast growth factor‐2 (FGF ‐2), interleukin‐8 (IL ‐8), transforming growth factor‐β1 (TGF ‐β1), matrix metalloproteinase‐2 (MMP ‐2), and vascular endothelial growth factor A (VEGFA ) mRNA and protein expression, and proliferation of epithelial and fibroblast cells was investigated in bovine endometrial explants in vitro. The results indicated that PGE ₂ at concentration about 10^?7 to 10^?5 M could up‐regulate CTGF , FGF ‐2, IL ‐8, MMP ‐2, TGF ‐β1, VEGFA mRNA and protein expression, and could induce the proliferation of epithelial and fibroblast cells and reduce the proapoptotic factor (caspase‐3) expression in bovine endometrial explants in vitro. These results collectively improved the possibility of PGE ₂ functions in endometrial growth during oestrous cycles.     

Hormonal Stimulation in a Gonadal Dysgenesis Mare

The present case report aimed to determine the responsiveness of the endometrium and the ovaries of an X0 mare after hormonal treatment. On transrectal palpation, the uterus was flaccid and smaller than normal, and the ovaries were small and smooth. The endometrium had normal histological architecture, with an atrophic glandular epithelium. A karyotype evaluation was performed, and 70 cells presented 63 chromosomes, lacking one sex chromosome. Circulating hormonal levels of total estrogens were 43.93 pg/mL; progesterone 0.01 ng/mL; testosterone 48 pg/mL; FSH 30.3 ng/mL; and LH 1.71 ng/mL. Immunohistochemistry tests showed the presence of estrogens and progesterone receptors in the endometrial samples of the X0 mare. 17β estradiol was administrated on three consecutive days and long-action progesterone on the fourth day. After hormonal stimulation, the mare showed changes in endometrial ultrasonography and histology. After treatment with estradiol, uterine edema was noted, and after progesterone, a reduction in edema was observed. At the request of the owner, no further treatment or follow-up occurred. This report showed that the endometrium is functional, but the ovaries did not change macroscopically under hormonal therapy.     

Direct effects of linoleic and linolenic acids on bovine uterine function using in vivo and in vitro studies

The aim of the present study was to evaluate the effects of continuous administration of linoleic acid or linolenic acid into the intra-uterine horn, ipsilateral to the corpus luteum, on the duration of the estrous cycle and plasma progesterone (P4) concentration. The effects of linoleic and linolenic acids on bovine uterine and luteal functions were also studied using a tissue culture system. Intra-uterine administration of linoleic or linolenic acid (5 mg/10 ml of each per day) in cows, between days 12 and 21, resulted in a prolonged estrous cycle compared to the average duration of the last one to three estrous cycles before administration in each group (P < 0.05). Moreover, plasma P4 concentration in cows treated with linoleic or linolenic acid was high between days 19 and 21 (linoleic acid), or on day 20 (linolenic acid), compared to that of the control cows (saline administration; P < 0.05 or lower). Both linoleic (500 µg/ml) and linolenic (5 and 500 µg/ml) acids stimulated prostaglandin (PG) E2 but inhibited PGF2α production by cultured endometrial tissue (P < 0.01), while P4 production by cultured luteal tissue was not affected. These findings suggest that both linoleic and linolenic acids support luteal P4 production by regulating endometrial PG production and, subsequently, prolonging the duration of the estrous cycle in cows.     

Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14 ± 0.4 postpartum (n = 12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling.     

Production of calcium maintenance factor Stanniocalcin-1 (STC1) by the equine endometrium during the early pregnant period

A factor responsible for progression to pregnancy establishment in the mare has not been definitively characterized. To identify factors possibly involved in the establishment of equine pregnancy, the endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19 and 25 pregnant animals. From initial subtractive hybridization studies, a calcium regulating factor, Stanniocalcin-1 (STC1) mRNA, was found as a candidate molecule expressed uniquely in the pregnant endometrium. Endometrial expression of STC1 mRNA was noted on day 19 and was markedly increased in the day 25 gravid endometrium. STC1 protein was found in the extracts of day 25 gravid endometrium and immunochemically localized in the uterine glands. In addition, STC1 protein was detected in uterine flushing media collected from day 25 pregnant mares. High concentrations of estradiol-17 β (E(2)) were detected in day 25 conceptuses. E(2) levels were much higher in the gravid endometrium than in other regions, whereas progesterone levels did not differ among the samples from different endometrial regions. Expression of STC1 mRNA, however, was not significantly upregulated in cultured endometrial explants treated with various concentrations of E(2) (0.01-100 ng/ml) with or without 10 ng/ml progesterone. These results indicate that an increase in STC1 expression appears to coincide with capsule disappearance in the conceptus, and suggest that STC1 from the uterine glands likely plays a role in conceptus development during the pregnancy establishment period in the mare.     

Comparative profiles of different lipoprotein cholesterol parameters and Growth Hormone during hot humid and winter season in Murrah Buffaloes

Now, it is well known that in bovines HDL-C act as substrate for ovarian steroidogenesis. The restriction of entry of other lipoprotein parameters is due to ovarian blood barrier, which restricts the entry of other lipoproteins, which are bigger in size. In vitro studies have indicated their role in mediating proliferation of cells. Besides gonadotropin, Growth Hormone (GH) is also gaining importance in the field of reproduction. GH receptors have been localized on the ovaries and follicle of bovines. Further it has been suggested that it can be the direct role of the hormone or it can be mediated through IGF-I. It has been assigned various roles in mediating follicle development, ovulation and corpus luteum maintenance. The river buffaloes are capable of breeding throughout the year, but during certain period of time were found to be more favourable than the others. It has been found that, winter season and hot humid season exhibit two extreme conditions of temperature variation; Hot humid season being unfavourable than the winter season because of high humidity and reasonably high temperature. It has been observed that during June to August months, conception rate and exhibition of estrus behaviour is low. This study was undertaken to analyze different lipoprotein cholesterol parameters namely Total cholesterol (TC), High-density lipoprotein cholesterol (HDL-C), Triglycerides (TG) and Low density lipoprotein cholesterol (LDL-C) in the plasma of cyclic Murrah buffaloes throughout the estrous cycle. The mean plasma concentration (μg/ml) of different lipoproteins during hot humid season were TC 592 ± 40, HDL-C 229 ± 42, TG 345 ± 95, LDL-C 285 ± 94.Where as in winter season their concentrations were 1381.0 ± 31.0, 1793.0 ± 110.0, 511.0 ± 21.0, 608.0 ± 94.0 respectively.

The mean ± SEM circulatory level of GH was low during HH season than during winter (6 ± 2 ng/ml vs. 17 ± 2 ng/ml). During estrous cycle only one peak of GH was exhibited during hot humid season where as three peaks were exhibited during winter season. It can be concluded that winter season is favourable for maintaining physiological levels of hormones and metabolic parameters, which in turn may increase the reproductive efficiency of bovines. During winter season average temperature was 15 ±5 °C. During hot humid season average temperature was 34 ± 3 °C. THI was more than 75%.     

Characteristics of protein-protease inhibitors, 17-beta estradiol and progesterone in the blood of heifers returning to estrus following LH/FSH-RH treatment]

Tests were performed to study the inhibitive capacity of protein-protease inhibitors, 17-beta estradiol, and progesterone in the blood serum of 21 heifers returning to oestrus which had been treated intramuscularly with 400 microgram LH/FSH-RH. Four selected heifers were examined as to the relationships between the concentrations of 17-beta estradiol, progesterone and the inhibitive capacity of the serum. Heifer no. 1 had low concentrations of 17-beta estradiol and higher concentrations of progesterone (average levels 7.8 and 657.4 pg ml-1 of serum). On the other hand, heifer no. 4 was found to have higher concentration of 17-beta estradiol and lower values of progesterone (average levels 28.3 and 325 pg ml-1 of serum). Having shown heat, the heifers were fertilized and remained in calf. Heifer no. 2 with a low concentration of progesterone (292 pg ml-1 of serum) and 17-beta estradiol (12.4 pg ml-1 of serum), as well as heifer no. 3 with a high concentration of both (830 pg ml-1 of serum and 22.7 pg ml-1 of serum, respectively) showed no heat and they were not mated. Heifers with significant differences in the concentrations of progesterone and 17-beta estradiol and with low values of protein-protease inhibitory capacities remained in-calf, whereas heifers with lower progesterone and 17-beta estradiol values, or adversely, with higher inhibition values, showed no heat. Synthetic LH/FSH-RH was found not to increase the amount of protein-protease inhibitors in heifer serum, as distinct from the cervical mucus of breeding cows treated with gestagens in heat synchronization.     

奶牛子宫内膜组织体外培养方法的建立及前列腺素E_2和F_（2α）受体分布的研究

[目的]建立体外培养荷斯坦奶牛子宫内膜组织的方法及检测子宫内膜组织中前列腺素E_2和前列腺素F_（2α）受体的分布情况。[方法]挑选新鲜、健康的发情前期奶牛子宫内膜组织,采集双侧子宫角部位内膜组织,体外悬浮培养组织小块并通过组织学形态鉴定培养效果。通过RT-qPCR方法检测PGE_2受体EP1、EP2、EP3（EP3A、EP3B、EP3C、EP3D）、EP4和PGF_（2α）受体FP的表达情况。[结果]HE染色表明,体外培养的奶牛子宫内膜组织结构完整,细胞和腺体形态完好,细胞核完好;发情前期奶牛子宫角部位内膜组织中PGE_2和PGF_（2α）受体均有表达,且表达量存在差异,其中PGE_2受体EP4相对表达量最高,而EP1、EP3B及PGF_（2α）受体FP的相对表达量较低。[结论]成功地建立了奶牛子宫内膜组织的体外培养方法;PGE_2和PGF_（2α）的受体在奶牛子宫内膜组织中均有表达,但不同受体的表达量有所不同。     

Truncation of the mRNA cap-binding protein eIF4E is specific for the non-invasive implantation in pigs

The mechanism of implantation is species specific (pig: epitheliochorial, bovine: synepitheliochorial, mouse: hemochorial). Recently, we have shown that proteolytical cleavage of the prototypical 25 kDa mRNA cap-binding protein eIF4E (eukaryotic initiation factor 4E) produces a stable variant with a molecular mass of approximately 23 kDa in porcine endometrium at the time of implantation. Here, we investigate if an eIF4E truncation also takes place in the endometrium of species with other implantation forms. Thus, eIF4E and its repressor protein 4E-BP1 were investigated in porcine, murine and bovine endometrium during the time of implantation. Our results show that eIF4E truncation is specific for the porcine implantation. In bovine and mouse uterine tissue, no cleavage of eIF4E was observed. Whereas no difference of bovine 4E-BP1 was found, in murine samples, increased phosphorylation during implantation was observed. However, porcine samples exhibit an opposite behaviour, the abundance and mainly the phosphorylation of 4E-BP1 decrease. We propose that the translation initiation in the endometrium is differently regulated by the two eIF4E forms with regard to different 4E-BP1 abundance and phosphorylation as well as different eIF4E/4E-BP1 binding dynamic depending on the type of implantation.     

Nutritional secondary hyperparathyroidism in rabbits

The present study was designed to document the effect of a low (0.6%) calcium-high (1.2%) phosphorus (LCaHP) diet on the development of parathyroid gland hyperplasia in rabbits and to describe the dynamics of parathyroid function (PTH–Ca²⁺ curves) in rabbits with nutritional secondary hyperparathyroidism (N2HPT). Parathyroid gland weight, parathyroid cell proliferation (measured as percentage of cells in S-phase), and parathyroid calcium (CaRmRNA) and Vitamin D (VDRmRNA) receptor expression were measured in normal rabbits and in rabbits with N2HPT. The PTH–Ca²⁺ curve was studied in normal rabbits (Group I) and in rabbits with N2HPT at two stages: 2–3 weeks (Group IIA) and 5–6 weeks (Group IIB) after being fed LCaHP diet. An increase in parathyroid gland weight and percentage of cells in S-phase was detected in the course of N2HPT. After receiving a LCaHP diet for 6 weeks rabbits had decreased levels of CaRmRNA but VDRmRNA remained unchanged. A progressive increase in the concentrations of plasma PTH (Group IIA = 167 ± 14 pg/ml and Group IIB = 377 ± 54 pg/ml, P < 0.05 versus Group I = 27 ± 3 pg/ml) was detected in the rabbits fed a LCaHP diet. This was accompanied by an increase in maximal and minimal PTH, reductions in plasma Ca²⁺ and calcitriol and elevations in plasma phosphate and creatinine. In conclusion, feeding a LCaHPD results in a rapid induction of N2HPT in rabbits. After 6 weeks on the LCaHPD rabbits develop parathyroid hyperplasia characterized by increases in PTH secretion, glandular weight and proliferation and by a decrease in CaRmRNA.     

The concentration of bovine placental lactogen and the incidence of different forms in fetal cotyledons and in fetal serum

The concentration of bovine placental lactogen (bPL) was determined in fetal placentomes, allantoic fluid, amniotic fluid, maternal and fetal plasma throughout pregnancy. In addition, chromatofocusing chromatography was used to separate the different forms of bPL found both in fetal serum and in placental homogenates in order to determine whether the different forms that have been reported to exist in the cotyledon are also found in the fetal circulation. Reproductive tracts were collected from cows between 109 and 247 days of pregnancy. The concentration of bPL in the fetal cotyledonary tissue was measured by both radioreceptor assay and radioimmunoassay, both assays showed that the concentration of bPL in the fetal portion of the placentomes remained constant throughout the period of pregnancy tested. The mass of the placenta increased approximately 10-fold during the period of study but the concentration of bPL in the maternal plasma was low (0.9±0.1 ng/ml) at all stages of pregnancy tested. The mean concentration of bPL (Mean ± S.E.M.) in amniotic and allantoic fluid was 0.4±0.1 and 1.2±0.2 ng/ml respectively. Fetal blood contained the highest concentrations of bPL, from 11.6 to 18.4 ng/ml, and the concentration tended to decrease with advancing gestation (slope = 0.07, P = 0.001). Several forms of bPL were found in the fetal circulation; however, a higher percentage of forms with more acidic isoelectric points were found in the fetal serum than in placental homogenates. These results suggest that either some forms of bPL are more stable or that the hormone isolated from placental tissue is not representative of the final secreted product.     

Endotoxin challenge increases xanthine oxidase activity in cattle: effect of growth hormone and vitamin E treatment

In addition to its basic role in the metabolism of purine nucleotides, xanthine oxidoreductase (XOR) is involved in the generation of oxygen-derived free radicals and production and metabolic fate of nitric oxide (NO). Growth hormone (GH) and Vitamin E (E) have been shown previously to modify immune response to infection. Our objective was to determine in heifers the effect of endotoxin challenge (LPS; 3.0 μg/kg BW, i.v. bolus, Escherichia coli 055:B5) on xanthine oxidase (XO) activity in plasma and liver and the modification of this response by daily treatment with recombinant GH (0.1 mg/kg BW, i.m., for 12 days) or GH+E (E: mixed tocopherol, 1000 IU/heifer, i.m., for 5 days). In experiment 1, 16 heifers (348.7±6.1 kg) were assigned to control (C, daily placebo injections), GH, or GH+E treatments and were challenged with two consecutive LPS injections (LPS1 and LPS2, 48 h apart). After LPS1, plasma XO activity increased 290% (P<0.001) at 3 h, reached peak (430%) at 24 h and returned to basal level by 48 h after LPS2. XO responses (area under the time×activity curve, AUC) were greater after LPS1 than LPS2 (P<0.001). Total plasma XO responses to LPS (AUC, LPS1+LPS2) were augmented 55% (P<0.05) over C with GH treatment but diminished to C responses in GH+E. There was a linear relationship (r²=0.605, P<0.001) between total response in plasma XO activity and plasma nitrate+nitrite concentration. In experiment 2, 24 heifers (346±6 kg) were assigned to C or GH treatments and liver biopsy samples were obtained at 0, 3, 6, and 24 h after a single LPS challenge. Hepatic XO activities increased 63.3% (P<0.05) 6 h after single LPS challenge and remained elevated at 24 h (100.1%, P<0.01) but were not affected by GH treatment. Results indicate that LPS-induced increases in plasma XO activity could be amplified by previous GH treatment but attenuated by E administration. The data also suggest that E may be effective in controlling some mediators of immune response associated with increased production of NO via the effect on XO activity and its production of superoxide anion as well as uric acid.     

Reproductive tract lesions in heifers after intrauterine inoculation with infectious bovine rhinotracheitis virus

Cross-breed heifers given infectious bovine rhinotracheitis virus by intrauterine inoculation 1 day after natural mating with a noninfected bull were killed on postinoculation days 4 to 14. When reproductive organs were examined for gross and microscopic lesions and for virus infection, the most severe uterine lesions were found in the body and caudal portions of the uterine horns of heifers killed between postinoculation days 4 and 9. Primary pathologic features were necrosis, edema, hemorrhage, and a diffuse accumulation of mononuclear cells, mostly lymphocytes; numerous lymphocytes were in mitosis. In cranial parts of uterine horns, the only lesions observed were a few small lymphocytic foci in the endometrial lamina propria. Lesions were not seen in the oviducts. In many heifers, the ovarian corpus luteum (CL) was cystic. In a few of these heifers, the cyst had a necrotic wall that was bordered by a zone of proliferating mononuclear cells. Focal necrosis and lymphoid proliferation were common in the parenchyma of cystic and noncystic CL. Similar necrotizing lesions were sometimes present in non-CL ovarian tissue. Infectious bovine rhinotracheitis virus was most frequently isolated from the uterine body, the internal os of the cervix, and the CL. Isolations were not made from blood samples taken at the time of necropsy. Isolation of virus from the CL correlated with the detection of luteal inflammation by light microscopy, but did not correlate with the presence of cysts. There also was no correlation between cystic CL and the severity of uterine lesions.     

Sex differences in oestrogen receptor levels in adrenal glands of sheep during the breeding season

The concentrations of the oestrogen receptor (ER), and the mRNA levels of ER, progesterone receptor (PR) and insulin-like growth factor I (IGF-I) were characterised in adrenal glands and uterine tissue of adult Corriedale sheep during the breeding season. The sheep were of different sex and gonadal status. Ewes had higher levels of cytosolic ER in the adrenals than the rams (mean±S.E.M.: 7.3±2.0 fmol/mg protein and 2.5±1.0 fmol/mg protein, respectively; P=0.0091) and gonadectomy increased ER (mean±S.E.M.: 2.9±1.2 fmol/mg protein and 8.6±2.3 fmol/mg protein, intact and gonadectomised sheep, respectively; P=0.0071). No differences could be observed in mRNA levels for ER and IGF-I in the adrenal glands of all of the sheep. PR mRNA levels were reduced in ovariectomised ewes and enhanced in castrated rams (sex×gonadal status: P=0.009). PR mRNA levels tended to be higher in ewes in the follicular phase than in ovariectomised ewes and intact rams (P<0.1). All of the animals had positive nuclear staining for ER in the adrenal cortex, but no differences were observed between the groups. In this study, we demonstrated the existence of ER in the adrenal gland of sheep and found varying sensitivity to oestrogens as the ER levels differed among sex and gonadal status. These findings indicate that oestrogens most likely affect steroidogenesis directly at the adrenal cortex and suggest that oestrogens are partly responsible for the sex differences in cortisol secretion in sheep.     

Progesterone and pregnancy status of buffaloes treated with a GNRH agonist

The primary aim of the present study was to establish whether the treatment with a GnRH agonist on Day 5 after AI may result in the formation of an accessory corpus luteum, greater progesterone secretion, and the increased likelihood of pregnancy success in buffaloes. The study was conducted during a period of increasing daylight length when progesterone secretion is suppressed and embryonic mortality is relatively high in buffaloes. In Experiment 1, treatment with a GnRH agonist (buserelin, 12 μg) on Day 5 after AI induced acute increases in circulating concentrations of LH, FSH and oestradiol-17β. Pregnant buffaloes (n = 14) at Day 40 following AI showed an increase (P < 0.01) in milk whey progesterone concentration between Day 5 (310 ± 55 pg/ml) and Day 15 (424 ± 50 pg/ml). The non-pregnant buffaloes (n = 7) showed a decrease (P < 0.01) in progesterone level from Day 5 (410 ± 87 pg/ml) to Day 15 (188 ± 30 pg/ml) following AI. In Experiment 2, the treatment with buserelin (12 μg) on Day 5 after AI induced ovulation in 62% of the buffaloes (31/50) and these buffaloes showed a progressive increase in milk whey progesterone concentration on Day 10, 15 and 20 of pregnancy. Buffaloes that did not ovulate, recorded a relatively constant milk whey progesterone level from Day 10 to Day 20 following AI. Milk whey progesterone concentrations increased after the administration of the GnRH agonist in 97% of the pregnant buffaloes and 68% of the non-pregnant buffaloes. The diameter of the largest follicle in buffaloes that ovulated (ovulated n = 31) (8.9 ± 0.04 mm; range 4.2 – 13.0 mm) did not differ significantly from the diameter of the largest follicle in buffaloes that did not ovulate (not ovulate n = 19) (8.7 ± 0.04 mm; range: 4.0 – 12.0 mm). The latter observation suggested that notional ovulatory size follicles in buffaloes are heterogeneous with respect to stage of follicle maturation and capacity to respond to plasma LH. The present study showed that treatment with a GnRH agonist on Day 5 following AI provides a strategy to increase progesterone secretion and the likelihood of pregnancy in buffaloes mated during periods of increasing daylight length.     

Quantitative unbiased estimates of endometrial gland surface area and volume in cycling cows and heifers

Entire reproductive tracts were removed from seven normal healthy heifers and qualitative unbiased estimates made of endometrial gland volume density and glandular surface density. After examining approximately 55 microscopic fields of endometrium from each tract, a mean glandular surface density value of 10.2 mm(2)/mm(3) (CE 3.1%) was obtained. The stereological method was then employed in optimising the design of the main study. The endometrial height was measured for 17 healthy cycling heifers and 19 similar cows. Subsequently, unbiased estimates were made of intercaruncular endometrial gland volume per unit surface for all cattle were investigated; differences between heifers and cows generally, and the possible effect of the follicular and luteal phases of the oestrous cycle were compared. The mean surface area of glands per unit area of endometrium at the intercaruncular site in heifers and cows was approximately 18 mm(2)/mm(2) in the follicular phase and 26 mm(2)/mm(2) in the luteal phase, figures similar to the gland area found in women. The intercaruncular gland volume increased significantly, by about 30% during the luteal phase of the bovine oestrous cycle in heifers, from 0.01 to 0.13 per mm(3). The differences in endometrial anatomy between site of sampling and either follicular or luteal phases of the oestrous cycle were always more significant in heifers than cows. The endometrial thickness in cows was always greater than for heifers, irrespective of the site of sampling. It was concluded that the intercaruncular endometrium of cattle was far more active physiologically than recognised previously.     

Establishment and characterization of a coculture system of equine endometrial epithelial and stromal cells

To investigate the equine endometrium as close to the in vivo situation as possible, we established a coculture system for epithelial and stromal cells (ECs/SCs). ECs and SCs were isolated from nine endometrial tissue specimens. ECs obtained as glandular formations were cultivated on one side of the semipermeable membrane of a Millicell^® insert. After 2 days, SCs (2 × 10⁴ cells/membrane) were seeded onto the other side of the same membrane. During cocultivation, the low serum containing culture medium (Theuß et al., 2010) was supplemented with different concentrations and combinations of 17β‐estradiol (2.0–3.0 pg/ml medium) and progesterone (0.5–15.0 ng/ml medium). Once the cocultures formed continuous cell layers as determined by phase‐contrast microscopy, the membranes were fixed and processed for light microscopical examination. Cytokeratin 19, steroid hormone receptors and the uterine proteins uteroglobin and calbindin_D9k were detected using immunocytochemistry to determine the degree of culture purity and functional cellular differentiation. The culture purity of the EC layer averaged ≥95%. Uteroglobin and calbindin_D9k were consistently expressed in ECs, while hormone receptors were predominantly absent in both cell populations. An explicit cytomorphological epithelial differentiation with formation of round‐oval to polygonal cell forms was encountered in ≤50% of all ECs and independent of supplemented steroids. Based on the findings altogether, and despite the partly absent congruence to the in situ prerequisites, we established a standardized and reproducible coculture system, which offers a basic approach for studies of physiologic and pathophysiologic issues in the mare.