Salivary luteinizing hormone: An open window to detect oestrous period in buffalo

Silent oestrus is an unsurmountable problem in the management of buffalo reproduction. In addressing this issue, we have earlier reported variation in the levels of urinary luteinizing hormone (LH) through the different phases of oestrous cycle with an extended window during the mid-oestrous phase. Based on this report, the present study is designed to assess the salivary LH levels in buffalo during the different phases of oestrous cycle. Bovine LH ELISA kit was used to determine the level of salivary LH. We observed a notable variation in salivary LH levels during the different phases of oestrous cycle. The maximum LH level, 39.07 mIU/ml, observed during oestrus, which was significantly (p < .05) higher than other consecutive phases. Altogether, the results showed a significant (p < .05) fold variation during oestrus compared with other phases. Therefore, the study convincingly shows that salivary LH has the potential of application in development of a modality for non-invasive oestrous detection in buffalo.     

Ion,Protein, Phospholipid and Energy Substrate Content of Oviduct Fluid During the Oestrous Cycle of Buffalo (Bubalus bubalis)

The aim of this research was to analyse the composition of oviduct fluid (ODF) in buffalo cows at different oestrous cycle phases to fulfil the requirements of buffalo embryos in vitro. ODF was collected by chronic cannulation from three cows that were synchronized by administering a synthetic prostaglandin. Based on hormonal profiles, the pre‐ovulatory, ovulatory, post‐ovulatory and luteal phases of the oestrous cycle were defined. The volume of ODF produced (ml/24 h) was influenced by the oestrous cycle, with values (mean ± SE) around ovulation (1.0 ± 0.2) greater (p < 0.05) than in both the luteal (0.4 ± 0.1) and the post‐ovulatory phases (0.5 ± 0.1), but not different from the intermediate values in the pre‐ovulatory phase (0.8 ± 0.2). Among cycle phases, no differences were found in sodium, potassium, calcium and magnesium concentrations (130.0 ± 1.1, 5.1 ± 0.3, 2.8 ± 0.1 and 0.59 ± 0.04 mmol/l respectively). Interestingly, the chloride secretion (μm /24 h) was higher (p < 0.05) at ovulation (150.2 ± 16.5) than during both the luteal (73.7 ± 22.0) and the post‐ovulatory phases (63.7 ± 11.2), with intermediate values in the pre‐ovulatory phase (113.4 ± 23.5). Glucose concentration (mmol/l) was higher (p = 0.056) in the pre‐ovulatory phase (0.06 ± 0.02) than in the luteal (0.02 ± 0.01) and post‐ovulatory (0.02 ± 0.01) phases but not different from values in the ovulatory phase (0.04 ± 0.02). Concentrations of pyruvate and lactate among oestrous cycle phases were similar (0.08 ± 0.01 and 1.0 ± 0.1 mmol/l respectively). The total quantity of phospholipids (μmol/24 h) was greater (p < 0.05) at ovulation (0.21 ± 0.02) compared with the luteal, pre‐ovulatory and post‐ovulatory phases of the cycle (0.09 ± 0.02, 0.13 ± 0.02 and 0.09 ± 0.01 respectively). No differences were found in either the protein concentration (1.8 ± 0.3 mg/ml) or the quantity of proteins secreted in 24 h (1.8 ± 0.4 mg) among oestrous cycle phases. In conclusion, this study provides the first characterization of buffalo ODF during the oestrous cycle, showing species‐specific differences that may be useful for developing suitable media for buffalo in vitro embryo production.     

Histological Study of Canine Mammary Gland During the Oestrous Cycle

The development of the mammary gland is a puzzling phenomenon and the research on this field has been focussed mostly on the carcinogenesis, with a less goal‐oriented concern in basic histology. In order to determine the histological features of normal mammary gland in the different oestrous phases we used 39 non‐pregnant female dogs of various breeds and ages. The animals were grouped in: pre‐pubertal, pro‐oestrous, oestrous, early and late dioestrous, early and late anoestrous phases. Major changes of the canine mammary histology throughout the oestrous cycle were identified in this study. A rudimentary gland with few ducts in the base of the nipple was observed in pre‐pubertal female individuals and pubertal pro‐oestrous female ones. In the oestrus, small inactive lobules associated with ductal branching and inconspicuous regressive changes were observed, while in early dioestrus, a ductal arborization was present. In late dioestrus, a complete lobuloalveolar differentiation and secretory capacity was achieved. The regressive histological features were abundant on early anoestrus, and markedly generalized on late anoestrus. The regressive process was longer in the more caudal gland pairs. This work provides baseline knowledge of canine mammary gland that may be relevant for interspecies comparative purposes and for pathologists dealing with mammary gland tumours.     

Relationship between Ultrasonographic Assessment of the Corpus Luteum and Plasma Progesterone Concentration during the Oestrous Cycle in Monovular Ewes

Ultrasonographic observations of the corpus luteum (CL) and collection of blood samples for progesterone radioimmunoassay were performed daily during 15 oestrous cycles in Spanish Merino ewes, a consistently monovular breed. Ultrasonographic image of the CL changed during the oestrous cycle, increasing its echogenic pattern from ovulation to luteolysis. The size of the CL and mean progesterone levels were significantly affected by day of cycle (p < 0.05 and p < 0.001, respectively). Both increased their values from day 1 to day 12 (from 49.6 ± 7.4 to 154.6 ± 11.8 mm² and from 0.2 ± 0.0 to 2.8 ± 0.5 ng/ml, respectively) and then declined sharply until day 0 (28.2 ± 5.3 mm² and 0.1 ± 0.0 ng/ml, respectively). There was a significant correlation between CL area and plasma progesterone concentrations during the entire oestrous cycle, taking the developing and regressing phases of the CL separately (p < 0.05). A central cavity was observed in 33.3% of the CL studied. The presence of this cavity had no effect in total luteal‐tissue area of the CL nor on oestrous cycle length or on progesterone concentrations. Likewise, the cavity did not affect the correlations observed between CL size and progesterone levels, CL size and day of cycle and progesterone levels and day of cycle. It is concluded that ultrasonographic assessment of CL area is a reliable method for estimating peripheral plasma progesterone levels, regardless to the presence or absence of a cavity in the CL.     

Are oestrogen receptors and protein tyrosine kinases involved in phytoestrogen-modulated steroid secretion by porcine adrenocortical cells?

The phytoestrogens genistein and daidzein had been found to affect the function of some tissues via oestrogen receptors (ER). In addition, genistein, but not daidzein, is considered to be a protein tyrosine kinase (PTK) inhibitor. Thus, the involvement of oestrogen receptors and PTK in phytoestrogen action on adrenocortical porcine steroidogenesis was examined in this study. The aims of the experiment were to test the effects of (i) ICI 182, 780 (ICI), an ER antagonist, on genistein- and daidzein-modulated cortisol and androstenedione (A4) secretion by adrenocortical cells isolated during the luteal and follicular phases of the porcine oestrous cycle; (ii) tyrphostin AG 957 (TAG), a nonsteroidal PTK inhibitor, on cortisol and A4 secretion by the cells and (iii) the phase of the porcine oestrous cycle on the mechanism of phytoestrogen action. Adrenals were harvested during the luteal (n = 5 animals) and follicular (n = 5 animals) phases of the oestrous cycle from locally slaughtered crossbred gilts. The isolated adrenocortical cells were incubated for 8 h (37 °C, 95% air, 5% CO2) with genistein (5 or 10 μM) or daidzein (5 or 10 μM) in the presence or absence of ICI (0.5 μM) or TAG (5 or 10 μM). Genistein and daidzein inhibited cortisol secretion and stimulated A4 secretion by porcine adrenocortical cells harvested during both the luteal and follicular phases of the oestrous cycle. The ER antagonist ICI did not eliminate phytoestrogen-induced changes in steroidogenesis. In contrast to genistein, TAG reduced the secretion of A4 and did not affect cortisol secretion. There was no observable effect due to the phase of the cycle. It is suggested that the mechanism of genistein and daidzein action in the adrenocortical cells of pigs is independent of ER and PTK. It is possible that PTK are involved in A4 secretion by porcine adrenocortical cells.     

Expression Pattern of Markers in the Canine Ovarian Cycle

Although hormonal changes during different phases of the oestrous cycle of bitches are well-described, knowledge about the luteal phase and anoestrus is incomplete. Furthermore, which paracrine and autocrine critical factors that differentiate between follicles destined for atresia and those that continue to develop are unknown. In this study, ovarian tissue was collected from 39 healthy bitches that were subject to ovariectomy or ovariohysterectomy for surgical neutering or medical purposes such as unwanted pregnancy. Bitches were allocated to different groups depending on the stage of the oestrous cycle. Serum progesterone, LH, FSH and 17β-estradiol (E₂) -levels were determined and immunhistochemistry was performed for a variety of receptor antigens; Ki-67, vimentin, pan cytokeratin antibody, p53 and oestrogen receptor (ER) α antigens. Marked differences were found in progesterone concentration between pregnant and non-pregnant animals. Oestrogen concentration was significantly lower in pro-oestrus and ovulation than during the luteal phase. Although progesterone could be detected in cytoplasm of ovarian cells at each stage, its presence was restricted to follicular cells during anoestrus. A strong presence of AE1/AE3, vimentin and p53 was found in each oestrous stage, in contrast with Ki67. The localization of ERα appeared to vary during the oestrous cycle, a phenomenon that suggests a switch between target cells of oestrogen; while as a proliferation marker, the mild reaction of p53 during parturition suggests an apoptotic process at this stage of the cycle.     

Evaluation of von Willebrand Factor During Pregnancy,Lactation and Oestrous Cycle in Bitches Affected and Unaffected by von Willebrand Disease

Plasmatic concentrations of von Willebrand Factor (vWF) increase during pregnancy in humans and dogs; however the mechanism of such increase is still not well defined. The aims of this study were: (i) to evaluate changes in vWF concentration during pregnancy and during the subsequent oestrous cycle in bitches affected and unaffected by von Willebrand Disease (vWD); (ii) to correlate the vWF levels and cortisol levels in both groups. Seven vWD affected (GI) and nine unaffected (GII) bitches were used. The animals were assessed during pregnancy, parturition, lactation and non‐gestational oestrous cycle in 11 moments (Pregnancy 1, Pregnancy 2, Parturition, Lactation 1, Lactation 2, Lactation 3, Anestrus, Proestrus, Oestrus, Diestrus 1, and Diestrus 2). The following tests were performed; measurement of von Willebrand factor antigen (vWF:Ag), albumin and cortisol. In both groups, vWF concentration remained stable during the non‐gestational oestrous cycle, but increased during pregnancy, with the highest value observed at parturition. Increases of 70% and 124% in vWF were seen in GI and GII, respectively, compared to anestrus. No correlation was found between vWF and cortisol. Values of vWF:Ag changed during pregnancy, with a peak at parturition, both in vWD affected and unaffected animals. Values of vWF were not altered in the different phases of the oestrous cycle following pregnancy in both groups. Evaluation of vWF during pregnancy can cause false negative results for vWD, but assessment can be performed at any point in the oestrous cycle of non‐pregnant bitches.     

Endometrial glycogen, protein, nucleic acids and phosphatases during oestrous cycle in buffaloes (Bubalus bubalis).

Distinct cyclic variations were observed in the concentrations of endometrial glycogen, protein, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and activities of acid phosphatase (ACP) and alkaline phosphatase (AKP) during the follicular and luteal phases of the oestrous cycle in buffaloes. Glycogen, protein, DNA and RNA concentrations were significantly (P less than 0.01) higher in buffalo endometrium during the follicular phase than the luteal phase of the oestrous cycle whereas ACP and AKP activities were significantly (P less than 0.01) elevated during the luteal phase compared with the follicular phase. These results are discussed in relation to the nourishment of the blastocyst during the pre-implantation period.     

Peripheral Blood Neutrophil Function and Lymphocyte Subpopulations in Cycling Mares

The purpose of this study was to evaluate different parameters of the immune status in the mare, during the follicular and the luteal phases of the oestrous cycle, in two consecutive years. Functional competence of peripheral blood neutrophils, such as chemotaxis, phagocytosis and oxidative burst was assessed under physiological cyclic conditions (Exp. I). In the second year of this study (Exp. II), besides peripheral blood neutrophil phagocytosis and oxidative burst analysis, circulating lymphocyte subsets were also characterized. The reproductive status in a total of 17 adult cycling mares was evaluated by ultrasonography and further confirmed by plasma progesterone levels. Chemotaxis tests were performed using porous membranes inserted in transwell chambers. Lipopolysaccharide (LPS) from Escherichia coli and N‐formyl‐Met‐Leu‐Phe (fMLP) were used as chemoattractants. Measurement of phagocytosis and oxidative burst in blood neutrophils were assessed by flow cytometry using commercially available kits. Quantification of T‐lymphocyte subsets was assessed by indirect immunofluorescence staining after incubation with monoclonal antibodies specific for CD2, CD3, CD4 and CD8 cell markers by flow cytometry. Natural killer cells and B cells were estimated mathematically. No significant difference was found in migration, phagocytosis and oxidative burst at either phase of the oestrous cycle. Statistical analysis of total white blood cell counts also showed no significant difference between either phase of the oestrous cycle, although there was a tendency for blood neutrophils to increase in number under the progesterone influence (p = 0.09). Lymphocytic subpopulations did not differ throughout the oestrous cycle. Overall, our results suggest that luteal and follicular phases in cycling mares may not influence the immune status of the mare.     

Tumour Necrosis factor-α Receptors are Present in the Corpus Luteum Throughout the Oestrous Cycle and During the Early Gestation Period in Pigs

To determine the physiological significance of tumour necrosis factor‐α (TNFα) in the regulation of luteal functions in pig, this study was conducted to identify the presence of functional TNFα receptors in porcine corpora lutea (CL) throughout the oestrous cycle and the early gestation. The CL were isolated from pigs on days 4, 6, 8, 12 or 15 of the oestrous cycle (n=3; day 0 = oestrus) and days 15, 20 or 25 of gestation (n=3; day 0 = mating). A Scatchard analysis revealed the presence of a high‐affinity binding site for TNFα in all samples (dissociation constant; 2.7 ± 0.51 to 5.8 ± 0.50 nM ). The concentration of TNFα receptors was higher on day 15 of the oestrous cycle than on days 4 and 8 of the oestrous cycle (p < 0.05). Furthermore, TNFα receptor concentrations in the CL on days 15, 20 and 25 of gestation were significantly lower than on day 15 of the oestrous cycle (p < 0.05). On day 9 of the oestrous cycle, exposure of cultured luteal cells to 0.06–60 nM TNFα stimulated prostaglandin (PG) F_2α and PGE₂ secretion in a dose‐dependent manner (p < 0.05). These results indicate that functional TNFα receptors are present in the porcine CL throughout the oestrous cycle and early gestation, and suggest that TNFα plays one or more physiological roles in regulating CL function throughout the oestrous cycle and the early gestation period. In addition, TNFα receptor concentration in the CL of the late luteal stage (day 15) of the oestrous cycle was higher than on the respective day in the early pregnant pig, suggesting that TNFα plays a role in accomplishing luteolysis in the porcine CL.     

Absorption of neomycin from the equine uterus: effect of bacterial and chemical endometritis

Plasma concentrations of neomycin were measured after intrauterine infusion of 3.3 mg/kg neomycin sulphate. Mares infected two hours previously with an intra-uterine infusion of beta-haemolytic streptococci absorbed approximately 12 per cent of the neomycin in both the oestrous and the luteal phases of the cycle. Normal mares in oestrus absorbed 6 per cent of the neomycin infused and luteal mares absorbed 56 per cent. In infected mares the peak plasma concentrations occurred two hours after neomycin infusion, earlier than in healthy mares. Cervical flushings after neomycin infusion in infected luteal mares revealed an increased reflux of neomycin when compared with healthy mares. Prior infusion of 30 ml of 10 per cent Lugol's iodine into the uterus resulted in 31 per cent of neomycin being absorbed by oestrous mares and 64 per cent by mares in the luteal phase. Peak plasma concentrations occurred 30 minutes after infusion in both phases. In the luteal phase the mares' absorption of neomycin may have been maximal.     

The Endometrium of Cycling Cows Contains Populations of Putative Mesenchymal Progenitor Cells

Endometrial stem cells have been identified in humans, mice and pigs. This study was designed to determine whether the uterine endometrium of cycling cows contains such cells, to identify markers of stemness and ultimately to isolate putative stem/progenitor cell and evaluate their capability to differentiate into mesodermal derivatives. Uteri from healthy cows in the early (days 1–5) and late luteal phases (days 13–18) of the oestrous cycle were collected. Total RNA and proteins were isolated and searched for gene markers of embryonic (OCT4, NANOG, SOX2) and mesenchymal (CD44, STAT3, CD‐117) stem cells and for protein markers (Oct4, Sox2, Cd44) in Western blots or immunostaining of paraffin‐embedded tissue. Primary cell cultures were isolated; characterized in terms of morphology, colony formation and gene/protein expression; and induced osteogenic and chondrogenic differentiation. We identified expression of embryonic (OCT4 and SOX2, but not NANOG) and mesenchymal (STAT3, CD44 and c‐KIT) gene markers in the endometrium of cycling cows and the encoded proteins (Oct4, Sox2 and Cd44) in both stages of the oestrous cycle. Derived cell lines displayed essentially the same gene expression pattern; however, at the protein level, Oct4 was not detected. No clear influence of the stage of the oestrous cycle was found. Cell lines from late luteal phase displayed osteogenic and chondrogenic differentiation potential upon chemical stimulation. In this research, we demonstrated the presence of mesenchymal progenitor cell populations of apparently mesenchymal origin in the endometrium of cycling cows, in both the early and late phases of the oestrous cycle. The cells isolated from the late luteal phase were more acquiescent to differentiate into mesodermal derivatives than cells in the early luteal phase. Our findings might have implications for the understanding of uterine stem cell biology in cows and other farm animal species.     

奶牛发情周期中生殖激素浓度与卵泡发育对比分析

选择从澳大利亚购进的2岁左右发情周期为21d育成母牛10头,用放射免疫测定法（RIA）测定发情周期血清中促卵泡素、促黄体素、孕酮、雌二醇17β的浓度,用B-型超声波诊断仪检测发情周期卵巢卵泡发育变化。结果显示：优势卵泡发育体积和FSH、LH、与E2浓度在奶牛发情周期中主要表现出两个波峰．对比分析显示奶牛在发情周期第12天（优势卵泡峰时）不发情与不排卵主要原因可能是体内前列腺素等激素量不足所致。     

Variation in Ano‐Genital Distance in Spontaneously Cycling Female Mice

A recently observed developmental instability of the ano‐genital distance (AGD) in female mice indicates that natural prenatal androgens do not have such a robust effect on female genital morphology as has been generally assumed. Part of this instability might be caused by oestrous cyclicity. To check this assumption, we examined the effect of the stage of the oestrous cycle on the AGD in adult (61–75 days old) female mice. Consistent with our assumption, the female AGD (1) varied during the oestrous cycle (p < 0.05), indicating thus rapid changes in morphology of female external genitalia, and (2) showed good repeatability (>0.66) in each stage of the oestrous cycle, suggesting that female genital morphology systematically varied within the oestrous cycle. Therefore, the stage of the oestrous cycle should be considered when assessing prenatal masculinization in adult female mice.     

Involvement of beta-adrenoceptors in the regulation of luteal function in cattle.

The infusion of beta-adrenomimetic drug (bamethan sulphate--BS) on day 11 of the oestrous cycle markedly increased peripheral progesterone concentration whereas infusion of beta-antagonist (propranolol) decreased plasma progesterone levels in heifers. Oxytocin injected on days 2-6 of the oestrous cycle shortened the length of the cycle (to 7-9 days) whereas the length of the cycle in heifers injected with oxytocin followed by injections of either BS or xanthinol (theophylline nicotinate--a potent vasodilatory drug) was normal (21.8 +/- 1.7). Heifers injected with BS on days 13-21 of the oestrous cycle had increased levels of progesterone on days 13-19 of the cycle and the length of the cycle was normal (22.3 +/- 0.6 compared with 21.3 +/- 1.5 for the control). The data indicate that: (1) beta-adrenergic stimulation overcomes the action of oxytocin given on days 2-6 of the cycle, whereas the stimulation of beta-receptors on days 13-21 of the oestrous cycle raised progesterone plasma concentration but did not prevent spontaneous luteolysis. (2) Basal secretion of progesterone in cattle is partly dependent on constant beta-adrenergic stimulation.     

Size Distribution of Dispersed Luteal Cells During Oestrous Cycle in Angora Goats

The present study examines the size distribution of the goat steroidogenic luteal cells throughout the oestrous cycle. Corpora lutea (CL) were collected after laparatomy on days 5, 10 and 16 of the oestrous cycle. Luteal cells were isolated from CL by collagenase digestion. Steriodogenic luteal cells were identified by staining of the cells for 3beta-hydroxysteroid dehydrogenase activity, a marker for steroidogenic cells. Luteal cells having steroidogenic capacity covered a wide spectrum of sizes, ranging from 5 to 37.5 microm in diameter. There was a significant increase in mean cell diameters (p < 0.01) as CL aged. The mean cell diameter on day 5 was 11.55 +/- 0.12 microm, which was significantly increased and reached up to 19.18 +/- 0.24 mum by day 16 of the oestrous cycle. The ratio of large to small luteal cells was 0.06:1.0 on day 5 of the oestrous cycle. This ratio increased to 0.78:1.0 by day 16 of the oestrous cycle. Luteal cell size on days 5, 10 and 16 of the oestrous cycle reached its maximum at 7.5, 10 and 35 microm in diameter, respectively. Development of CL is associated with an increase in luteal cell size in goats. It is likely that small luteal cells could develop into large luteal cells as CL becomes older during oestrous cycle in goats.     

Immunohistochemical Localization of the Progesterone and Oestrogen α Receptors in the Uterine Horns of the African Giant Rat (Cricetomys gambianus)

The present study investigated the immunolocalization of the progesterone and oestrogen α receptors in the uterine horns of the African giant rat during the oestrous cycle. The progesterone and oestrogen α receptors were demonstrated in various cellular constituents of the endometrium, myometrium and perimetrium. The intensity of progesterone and oestrogen α receptor immunostaining in the endometrial and myometrial layers of the uterine horns varied during the oestrous cycle. The intensity of oestrogen α receptor immunoreactivity in the luminal epithelium was high during pro‐oestrus, oestrus and dioestrus. Progesterone and oestrogen α receptor immunoreactivity in the endometrial epithelia was absent during metoestrus. Moderate to strong immunostaining for the progesterone and oestrogen α receptors was demonstrated in the myometrial smooth muscle cells during pro‐oestrus, oestrus and dioestrus. The intensity of progesterone and oestrogen α receptor immunostaining in the myometrial smooth muscle cells was low during metoestrus. Stromal cells in the perimetrium consistently expressed progesterone and oestrogen α receptor immunoreactivity throughout the oestrous cycle. The findings of the study indicate that in the giant rat the immunolocalization of the progesterone and oestrogen α receptors, in endometrial and myometrial regions of the uterine horns, varies during the oestrous cycle.     

Progesterone profiles of fat-tailed tuj ewes following tactile and visual separation of rams at the beginning of the breeding season

The aim of this study was to determine the level and duration of progesterone secretion during the sequential oestrous cycles in fat-tailed Tuj ewes following tactile and visual separation of rams during the breeding season. For these purposes, rams were separated from the main flock for 50 days starting from the beginning of the breeding season and 21 ewes were randomly selected for the current experiment. In order to assess luteal activity and the length of oestrous cycles, the ewes were blood-sampled thrice or twice weekly for the measurement of progesterone in plasma. The data showed that 3 +/- 0.2 oestrous cycles were observed in this time window, and the first oestrous cycle observed was shorter (P < 0.05) than the following cycles (mean +/- SEM, 14.6 +/- 0.82, 16.5 +/- 0.48 and 17.0 +/- 0.54 days, respectively, for cycles 1, 2 and 3). Progesterone production was significantly lower in the first oestrous cycle compared with the second and third cycles on days 9, 12 and 14 of the cycles. The follicular periods of ewes showed four accumulations (maximum values using a 3-day moving average technique) throughout the study and the percentages of ewes at each accumulation (peak day +/- 1 day) were 50, 35, 65 and 80% for the first, second, third and fourth accumulations, respectively (P < 0.05). It was concluded that progesterone production was lower and the oestrous cycles were shorter during the first oestrous cycle and that tactile and visual separation of rams at the beginning of the breeding season might enhance the synchronizing effect towards the end of the breeding season in the fat-tailed Tuj ewes.