Different Stages Of Estrous Cycle Research Articles

Detection and quantification of TIMP1 and miR-141 through RT-LAMP and TT-LAMP in serum samples during estrous cycle in buffalo

Estrus identification is a common problem in the reproductive management of farm animals. Hence, several studies have been conducted to explore biomarkers for estrus detection. One of our previous studies identified the abundance of RNA biomarkers such as TIMP1 and miR-141 in buffalo saliva during the estrus stage. However, the level of these RNA biomarkers in buffalo serum during estrous cycle is undetected. Therefore, the present study was designed to quantify TIMP1 and miR-141 in serum during buffalo estrous cycle. Blood samples were collected in different stages of estrous cycle from four healthy cyclic buffaloes. The quantification of TIMP1 and miR-141 was performed with direct serum using RT-LAMP and TT-LAMP technologies, respectively. The LAMP amplification was confirmed by agarose gel electrophoresis and the color change was quantified in comparison to a non-template control using ImageJ software. A decreased abundance of TIMP1 at the diestrus stage and a decreasing trend of miR-141 from proestrus to diestrus stages were observed, which was further reinforced by simulated random populations generated with R programming. Specifically, TIMP1 was found significantly (P < 0.0001) abundant at estrus and metestrus stages as compared to the diestrus stage, whereas miR-141 was significantly (P < 0.001) higher during the proestrus stage as compared to the other stages of estrous cycle. The ROC curve analysis showed miR-141 to be a better biomarker than TIMP1 as it distinguished the proestrus stage from diestrus with a sensitivity and specificity of 83 % and 98 %. This study also marked the first use of TT-LAMP technology for rapid miRNA detection in livestock.

P-227 Exposure to triclosan modifies oocyte development and embryo metabolism

Abstract Study question Does the endocrine disruptor, triclosan, affect the function of cumulus-oocyte-complexes (COCs), and if so, do these effects persist in early embryo developmental competence and metabolism? Summary answer Triclosan caused COCs to i) increase progesterone secretion, ii) switch from releasing to consuming pyruvate, and iii) produce fewer blastocysts that were iv) metabolically compromised. What is known already Triclosan (TCS) is used widely as an antimicrobial agent in a range of consumer healthcare products, including hand sanitiser, toothpastes, soaps, and shampoos. Its prevalent use has led to accumulation in the environment and human exposure. Indeed, TCS has been identified in a range of bodily fluids. It is classed as an endocrine-disrupting chemical and there are reports of its detrimental impact on the vertebrate reproductive system. However, the direct effects of TCS on mammalian Cumulus-Oocyte-Complexes’ (COCs) function and subsequent development of the embryo remains unknown. Study design, size, duration COCs, isolated from abattoir-derived bovine ovaries on 5 independent occasions, were exposed of two environmentally-relevant doses of TCS (10nM and 1nM) during in vitro maturation. The TCS-exposed COCs were subsequently fertilised in vitro without further TCS addition, and the cleavage and blastocyst rate were recorded on Day 2 and Day 7/Day 8, respectively. The resulting blastocysts were collected and further cultured individually for 24 hours, and depletion/release of key-metabolites quantified. Participants/materials, setting, methods In vitro production was performed for the maturation of the bovine oocytes and the embryo culture. The production of steroid hormones (17b-estradiol and progesterone) during IVM was determined by ELISA. The concentrations of glucose, pyruvate, lactate during IVM and blastocyst formation were quantified in the spent media using microfluorometric assays. Main results and the role of chance TCS at 1nM had a significant impact on steroidogenesis during COCs in vitro maturation; progesterone production was significantly higher compared to control group (4.784 ±1.69 vs 2.85 ±0.83 pg/hour/COCs, p = 0.035; n = 5), although oestrogen release was not affected. In addition, there was a dramatic shift in pyruvate metabolism. Over the course of in vitro maturation, COCs exposed to TCS depleted pyruvate from the culture media regardless of the administrated dose whilst net pyruvate concentration increased in the medium of COCs in the control groups. The cleavage rate of bovine embryos produced from TCS-exposed COCs was not significantly different to control group. However, COCs-exposed to 1nM TCS produced significantly fewer blastocysts (12.22% vs 29.11% n = 5; p = 0.02). Those blastocysts that did originate from COCs treated with TCS depleted significantly more pyruvate from the culture media compared to controls, regardless of TCS dose (19.86 ±10.54 vs 10.12 ±12.5 pmol/hour/embryo, p = 0.011 and 21.41 ±10.08 vs 10.12 ±12.5 pmol/hour/embryo, p = 0.002, TCS-10nM and TCS-1nM compared to Control, respectively). Additionally, while glucose depletion did not differ, the lactate release from blastocysts derived from COCs exposed to 10nM TCS was approximately halved compared to controls (15.20 ±14.59 vs 31.83 ±22.73 pmol/hour/embryo p = 0.0054). Limitations, reasons for caution The origin of the ovaries may be a limitation since they came from animals at different stages of estrous cycle. Furthermore, pre-exposure to TCS prior to animal slaughter cannot be discounted. Finally, these observations were made in an animal model in a laboratory setting which may differ from natural exposure. Wider implications of the findings TCS modifies oocyte maturation, developmental competence, and metabolic activity of resulting embryos. The differences in pyruvate metabolism and steroidogenesis point to altered mitochondrial function. These data indicate that TCS directly modulates oocyte and embryo physiology which may relate to fertility in domestic livestock species and may be extrapolated to humans. Trial registration number not applicable

Repeatability of anogenital distance measurements from birth to maturity and at different physiological states in female Holstein cattle

The inverse association between anogenital distance (AGD; the distance from the center of the anus to the base of the clitoris) and fertility, its moderate heritability, and high variability reported in dairy cattle make AGD a promising candidate for further exploration as a reproductive phenotype. In addition to heritability, repeatability (i.e., consistency in measurements taken at different time points) is important for a reproductive phenotype to be considered useful in genetic selection. Therefore, our primary objective was to determine the repeatability of AGD from birth to breeding age (≈16 mo) in Holstein heifer calves, and during different stages of the estrous cycle, gestation, and lactation in Holstein cows. We also determined the associations among AGD, height (at the hip), and body weight (BW) at birth. In calves (n = 48), we recorded BW (kg) and height (cm) at birth and measured AGD (mm) at approximately 0, 2, 6, 9, 12, and 16 mo of age. In cows, AGD was measured at different stages of the estrous cycle (proestrus, estrus, metestrus and diestrus; n = 20), gestation (30, 90, 180, and 270 d; n = 78), and lactation (30-300 d in milk in 30-d increments; n = 30). Calf height and BW at birth had a weak positive association with AGD at birth. The AGD increased linearly from birth to breeding age, but there was no association between the AGD at birth and at breeding age in heifers. Although any 2 consecutive AGD measurements were correlated, 6 mo was the earliest age at which AGD was moderately correlated (r = 0.41) with that of breeding-age heifers. The AGD was neither influenced by the different stages of estrous cycle nor lactation and remained highly repeatable (r ≥ 0.95). Although AGD measurements at 30, 90, and 180 d of gestation (126.9, 126.7, and 127.7 mm, respectively) were strongly correlated (r ≥ 0.97) with each other, AGD at 270 d of gestation (142.8 mm) differed from AGD at all earlier stages of gestation. In summary, AGD measured at birth did not reflect AGD at breeding age in heifers, but AGD measurements in cows had high repeatability at all stages of the estrous cycle, gestation, and lactation, except at 270 d of gestation. Therefore, AGD could be measured reliably at any of the aforesaid physiological states in cows due to its high repeatability, except during late gestation. The earliest gestational stage when pregnancy-associated increase in AGD occurred, however, could not be definitively established in the present study.

Influence of genistein and diadizine on regularity of estrous cycle in cyclic female Wistar rat: interaction with estradiol receptors and vascular endothelial growth factor.

Isoflavones are estrogenic compounds that exist in soy, clover, and peanuts. They are selective estrogen receptor modulators. The study was planned to explain the interactions of isoflavones with estrogen receptors alpha (ERα), beta (ERβ), and vascular endothelial growth factor (VEGF) expressions in ovarian and uterine tissues during different stages of the estrous cycle of regular cyclic female Wistar rats. Thirty-two regular cyclic females were divided equally into control group: fed casein-based diet and isoflavones group: fed casein-based diet and gavaged 50 mg/kg/day soy isoflavones extract 40%. The regularity of estrus cycles was monitored. Final body weight (FBW), weight gain (BWG), and ovarian and uterine weights were estimated. Histopathology and immunohistochemistry for ERα, Erβ, and VEGF in ovarian and uterine tissues were performed. All females (100%, n = 16) in control group showed regularity in estrous cycle compared to 62.5% (n = 10) in isoflavones group. Estrus and diestrus phases revealed prolongation and shortening in isoflavones rats than control, respectively. Nonsignificant variation was noted in the duration of the whole cycle of both groups. FBW and BWG significantly decreased however, ovarian and uterine weights increased significantly in all estrous phases of isoflavones group than control. Histopathology demonstrated an increase in number of follicles/ovaries besides, hyperplasia and proliferation of luminal epithelium with hydropic degeneration in the isoflavones group. Also, uterine connective tissue stroma showed edema in the isoflavones group during all estrous phases. Immunostaining percentages of ERα, Erβ, and VEGF protein expression were significantly elevated in the isoflavones group during all estrous phases. Isoflavones induced irregularity of the estrous cycle that was encountered by increased and altered ERα, Erβ, and VEGF expressions in ovarian and uterine tissues.

Faecal microbiome analysis reveals Clostridiales and Bacteroidales as signature gut microbes during estrus of buffalo

The non-pathogenic intestinal microbes that conquer our intestines are not an accidental jumble of organisms, but rather a disparate community of microbes that coexist, and sustain a mutualistic and symbiotic relationship with the host. The gut microbiome has been shown to be influenced by animal physiology and vice versa. However, information is still scanty. The present study aimed to analyse the variation between faecal bacteria of three different stages (proestrus, estrus and postestrus) of the estrous cycle of Murrah buffalos. A phylogenetic study of buffalo faeces derived from three different stages of estrous cycle was conducted in order to compare the bacterial diversity among these three stages. We performed an exploratory microbiome analysis of buffalo faeces using 16S rRNA sequencing during these stages of the buffalo estrous cycle. A total of three bacterial phyla with six different bacterial orders and twenty-three different genera were identified among all the three comparative phases of the estrous cycle. Among them, the Clostridiales were found to be the most abundant, and Bacteroidales were present exclusive during the estrus phase. As faeces is a source of gut microbes and a non-invasive representative of the metabolic steroids and perceptible pheromones, the profiling of gut microbes during estrous cycle would provide clues towards the major microbes contributing to the perceptible pheromones during estrus stage. To the best of our knowledge, this is the first ever report describing the faecal bacterial diversity during estrous cycle of any ruminant species. Although future studies are required to understand the role of Clostridiales and Bacteroidales in faecal pheromone metabolism.

Production of IL-1β, IL-6 and CXCL8 by endometrium of crossbred heifers stimulated with various pathogen-associated molecular patterns

Uterine bacterial infections are common during the post-partum period of dairy herds and, apparently, incidences in crossbred cattle are less than in Holsteins. The aims of this study were (I) to evaluate production of interleukin 1-β (IL-1β), interleukin-6 (IL-6) and chemokine CXCL8 using endometrial explants from Bos indicus crossbred heifers at diestrous, stimulated by various pathogen-associated molecular patterns (PAMP), and (II) assess production of these cytokines by lipopolysaccharide-stimulated endometrial explants from heifers when samples were collected at different stages of estrous cycle. In the first experiment, endometrial explants from heifers at diestrous were stimulated by ten-fold serial dilutions of lipopolysaccharide (LPS), triacylated lipopeptide (PAM3) or peptidoglycan (PGN). In the second experiment, endometrial explants collected at different stages of the estrous cycle were treated with LPS. Concentrations of IL-1β, IL-6 and CXCL8 were quantified in supernatant. There was a marked (P < 0.05) production of IL-1β, IL-6, and CXCL8 in response to LPS treatment. There was also production of IL-1β (P < 0.05) in response to PGN treatment. Explant samples collected at different stages of the estrous cycle responded to LPS treatment with production of IL-1β and IL-6, but with no differences (P > 0.05) between stages of estrous cycle. In conclusion, endometrial samples of crossbred Zebu-based heifers collected during diestrous produced IL-1β, IL-6 and CXCL8 in response to LPS and IL-1β in response to PGN. The cytokine production in response to LPS, however, was not affected by the stage of the estrous cycle in Bos indicus crossbred heifers.

Influence of estrous stages on electrocardiography, clinical pathology and ovarian weight of experimental beagle dogs: a retrospective analysis.

Estrous cycle is a repetitive phenomenon occurring during the reproductive life of a female dog. The duration of the canine estrous cycle is considerably longer than one in the most of the other animals and is broadly grouped into follicular phase (proestrus and estrus), luteal phase (diestrus) and non-seasonal anestrus. Dogs in the same stage of cycle can be inadvertently assigned to same group during routine safety and metabolic studies leading to possible erroneous interpretation of test-item related effects. This retrospective analysis was conducted by analyzing data of 86 female beagle dogs from control/placebo treated groups to correlate any possible effect of estrous stages with electrocardiography, clinical pathology and ovarian weight. Different estrous cycle stages of beagles were confirmed histologically by evaluating ovary, uterus, vagina and mammary glands. The incidence of beagles in diestrus was the highest, followed by anestrus, proestrus and estrus. No significant effect was noticed on heart rate, P–A, P–D, RR, QRS and QT intervals across different stages of estrous cycle. However, significantly higher PQ (PR) interval in dogs in proestrus stage was observed compared to dogs in anestrus and estrus. Marginally higher WBCs, neutrophils, lymphocytes, RBCs, hemoglobin, AST and lower hematocrit, lipid profile (total cholesterol, HDL, LDL, triglycerides), ALP level was evident in estrous period. Relative ovary weight was significantly higher in dogs in diestrus stage. Considering these results, one may need to exercise caution while interpreting experimental data from female beagle dogs.

Morphological characterization of vaginal epithelial cells of santa inês ewes subjected to estrus synchronization

Vaginal cytology analysis has been used to evaluate the different stages of estrous cycle of several species; it presents a direct correlation with the animal’s hormonal state and provides essential information about the female reproductive tract conditions. Two staining methods were tested to evaluate the vaginal epithelial cell morphology of nulliparous and multiparous ewes during the estrus period. An intravaginal device impregnated with medroxyprogesterone acetate was kept into 10 nulliparous and 10 multiparous ewes for 14 days for estrus synchronization. Then, the progesterone device was withdrawn, and 300 IU of eCG was administered intramuscularly. Vaginal smears were prepared for posterior staining with Panotico or Giemsa stains when estrus was detected. The cells were classified into nucleated superficial, anucleate superficial, intermediate, parabasal, and basal. The Panotico and Giemsa staining of the different cell types studied were satisfactory. A predominance of intermediate epithelial cells (p&lt;0.05) was found after staining. No difference in percentages of the different types of vaginal epithelial cells between nulliparous and multiparous ewes were found. Therefore, both staining methods were efficient, and a predominance of intermediate cells is found in nulliparous and multiparous ewes during the estrus period.

Plasma Progesterone and Vaginal Bacterial Flora at Different Stages of Estrous Cycle in Breeding Bitches

This study determined plasma progesterone and vaginal bacterial flora, using Enzyme Linked Immunosorbent Assay (ELISA) and bacterial culture and isolation respectively in 41 exotic breeding bitches at different stages of estrous cycle. Proestrual, estrual, diestrual and anestrual bitches had mean progesterone of 0.92 ± 1.05 ng/ml, 14 ± 17.1ng/ml, 35.7 ± 12.6ng/ml and 0.5 ± 0.4ng/ml respectively. There was a significant difference (P

Functional Characterization of Corpus Luteum and its Association with Peripheral Progesterone Profile at Different Stages of Estrous Cycle in the Buffalo

Functional Characterization of Corpus Luteum and its Association with Peripheral Progesterone Profile at Different Stages of Estrous Cycle in the Buffalo

Global proteomic analysis of water buffalo (Bubalus bubalis) saliva at different stages of estrous cycle using high throughput mass spectrometry

Accurate and efficient detection of estrus is one of the major constraints for exploitation of the production potential of buffalo owing to its poor manifestation of estrus signs, seasonal differences in expression and higher incidences of silent estrus (29%). The current study focused on identification of estrus specific candidate proteins in saliva of buffaloes. Estrus was detected based on behavioral signs in response to the teaser and changes in reproductive organs and confirmed by per-rectal examination, trans-rectal USG of reproductive organs, cervico-vaginal mucus characteristics and blood serum progesterone estimation. Day of onset of estrus was considered as day 0 and day −3, +3, +10 were considered as proestrus, metestrus and diestrus stage of the estrous cycle respectively. A total of 19 animals and their 38 estrous cycles (two from each) were included in this study. Saliva was collected from these animals during different stages of estrous cycle. Out of these, 08 animals were selected for global proteome analysis of saliva using in-solution digestion and nano-LC-MS/MS. A total of 275, 371, 304 and 565 proteins were identified with ≥2 peptides during proestrus, estrus, metestrus and diestrus stages of estrous cycle. Among the identified proteins 31, 62, 32 and 104 proteins were found specific to proestrus, estrus, metestrus and diestrus stage of the estrous cycle. Few salivary proteins such as Cullin-associated NEDD8-dissociated protein 1, Heat shock 70 kDa protein 1A, 17-beta-hydroxysteroid dehydrogenase type 1, Inhibin beta A chain, testin were identified as estrus specific and are important for estrus physiology. Taken together, these estrus specific proteins could be considered as the candidate biomarker for detection and confirmation of estrus in buffalo after thorough validation.

Effect of Heat Stress on Reproductive and Productive Traits in Baladi and Crossbred Goat Does under Subtropical Conditions

This experiment was conducted to study the effect of heat stress on some reproductive and productive performance in purebred and crossbred female goats under both of hot (summer season) and mild climatic (winter season) conditions. Forty mature female goats (20 Baladi and 20 crossbred (50% Zaraibi x 50% Baladi) used in this research. Results- cundity were higher in experimental does during mild climate conditionsthan hot climate conditions. Birth weight recorded higher values inmild climate conditions in comparison with hot climate conditions. Concerningto breed, reproductive traits were higher in crossbred goat doescompared with purebred goat does. Crossbred goat does showed higher(P<0.01 or P<0.0001) in all productive traits than Baladi goat does. Cortisolhormone concentration showed lower (P<0.0001) concentration undermild climate conditions than hot climate conditions during differentstages of estrous cycle, pregnancy and postpartum periods. On the otherin crossbred goat does compared with purebred does during differentstages of estrous cycle, except diestrous period. Similar trend was foundduring pregnancy and postpartum periods (P<0.01 or P<0.0001). It could-productive and productive traits of goats and in the same time, crossbredwas better than purebred goats.

Co-relation of estrous cycle phases with uterine bacterial and fungal flora in non-pregnant female laboratory rabbits.

This study was designed to investigate the relationship between the estrous cycle phases with uterine bacterial and fungal flora in non-pregnant female rabbits. Thirty laboratory mature multiparous rabbits were used for this purpose. Samples from uterine lavage for culture of bacteria and fungi were collected at different stages of estrous cycle (based on vaginal cytology), and histopathological observations were evaluated based on the scoring system used for defining the infection of the uterus. Various types of bacteria and fungi were isolated from rabbits at all stages of estrous cycle. The widest variety of bacteria and fungi was isolated at Di-estrous stage and the lowest variety was detected at estrous stage. Klebsiella oxytoca as well as yeast have been isolated at all stages of estrous cycle. This study showed that infection with K. oxytoca and yeast had no relationship with different stages of estrous cycle but other bacteria and fungus were associated with one or more stages of the estrous cycle in rabbits.

Expression and localization of fibroblast growth factor (FGF) family in corpus luteum during different stages of estrous cycle and synergistic role of FGF2 and vascular endothelial growth factor (VEGF) on steroidogenesis, angiogenesis and survivability of cultured buffalo luteal cells

The aim of this study was to document the expression and localization of fibroblast growth factor (FGF) family members comprising of fibroblast growth factor (FGF1, FGF2, FGF7, FGF10), and their receptors (FGFR1, FGFR2, FGFR3, FGFR4, FGFR2IIIB, FGFR2IIIC) in buffalo corpus luteum (CL) obtained at different stages of the estrous cycle. In addition, the synergistic role of FGF2 and/or vascular endothelial growth factor (VEGF) on P4 secretion and mRNA expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 (CYP11A1), 3-beta-hydroxysteroid dehydrogenase (3βHSD), proliferating cell nuclear antigen (PCNA), BCL-2 associated X protein (BAX) and von willebrand factor (vWF) were studied in luteal cell culture obtained from mid-luteal phase (MLP) of estrous cycle in buffalo. Real-time PCR (qPCR), western blot, and immunohistochemistry were used to investigate mRNA and protein expressions, and the localization of examined factors whereas P4 secretion was assessed by RIA. The mRNA and protein expression of FGF1 and FGFR1 were maximum (P<0.05) during MLP whereas FGF2 was maximum (P<0.05) during early luteal phase (ELP). FGF7, FGF10, FGFR2, FGFR3, FGFR4, FGFR2IIIb, and FGFR2IIIc mRNA and protein expression did not change among luteal phases. FGF family members were localized in cytoplasm of luteal cells as well as in endothelial cells. P4 secretion in luteal cells treated with FGF2 or VEGF alone showed the maximum values (P<0.05) with the highest dose at 72h. P4 secretion was found to be greater (P<0.05) in luteal cells treated with FGF2+VEGF compared to FGF2 or at 72h of incubation. The mRNA expression of all factors were maximum (P<0.05) whereas BAX was minimum (P<0.05) at highest dose cultured for 72h of luteal cells subjected with either protein alone or in combination. Summarizing, the present findings explore the synergistic role of FGF2 and VEGF on steroidogenesis, angiogenesis, cell viability through an autocrine and paracrine actions in buffalo CL.

Alleviation of thermoregulatory responses of Baladi Does by chromium and Selenium-E supplementation in subtropical areas.

This experiment was conducted to study the effect of the treating Baladi female goats with chromium and selenium-E during estrous cycle, pregnancy and postpartum periods to alleviate thermoregulatory responses of animals under hot conditions in Egypt to improve their performance throughout these stages. It has been used in this research 72 mature Baladi does (36 animal / season) with an average age of 2-3 years and an average body weight of 25 Kg, the experiment lasted for two seasons (mild and hot). Animals were divided randomly into three similar groups, the 1st group was kept without any treatments (control), the 2nd group was supplemented by chromium (chromium chloride, Cr). The 3rd group was injected intramuscularly twice a week with 2ml viteselen®, contained 0.5 mg selenium and 10.7 IU vitamin E (Se-E). Animals were kept in semi-open pens during the experimental period and allowed to graze for five hours daily at least. Thermoregulatory responses of the animal were estimated in terms of rectal temperature (RT), skin temperature (ST) and respiratory rate (RR). Results showed significant decrease in RT during the estrous cycle due to Cr and Se-E treatments. Also, Selenium-E injection showed lower ST during most of the different stages of estrous cycle. Under hot season, each of chromium and selenium-E reduced ST during estrous cycle while this effect was only due to Se-E for RR without chromium .Both Cr and Se-E treatments decreased RT and ST throughout different stages of pregnancy, except at late pregnancy of Se-E group. Under hot season conditions, does injected with Se-E were the lowest in RR during the mid and late pregnancy compared with other groups. While Cr group did not reveal significant effect on RR. During postpartum period Cr and Se-E supplementation under hot condition showed significant decrease in RT and ST. Both treatments decreased (P<0.05) RR during the postpartum period, only Se-E exhibited this decline under hot season.

Streptozotocin-Induced Diabetic that Histochemical and Immunohistochemical Examination of Mast Cells Distribution in Ovary and Uterus During Different Stages of Estrous Cycle in Rats

Streptozotocin-Induced Diabetic that Histochemical and Immunohistochemical Examination of Mast Cells Distribution in Ovary and Uterus During Different Stages of Estrous Cycle in Rats

Stimulatory effect of luteinizing hormone, insulin-like growth factor-1, and epidermal growth factor on vascular endothelial growth factor production in cultured bubaline luteal cells

The purpose of this study was to evaluate the temporal (24, 48, and 72 hours) and dose-dependent (0, 5, 10, and 100 ng/mL of LH, insulin-like growth factor 1 [IGF-1], and EGF) in vitro expression and secretion patterns of vascular endothelial growth factor (VEGF) in luteal cell culture during different stages of estrous cycle in water buffaloes. Corpus luteum samples from ovaries of early luteal phase (ELP; Days 1–4), midluteal phase (Days 5–10), and late luteal phase (Days 11–16) were collected from a local slaughterhouse. The samples were then processed and cultured in (serum containing) appropriate cell culture medium and incubated separately with three factors (LH, IGF-1, or EGF) at the previously mentioned three dose–duration combinations. At the end of the respective incubation periods, VEGF was assayed in the spent culture medium by ELISA, whereas the cultured cells were used for VEGF mRNA expression by quantitative real-time polymerase chain reaction. The results of the present study disclosed dose- and time-dependent stimulatory effects of LH, IGF-1, and EGF on VEGF production in bubaline luteal cells. The VEGF expression and secretion from the cultured luteal cells were highest during the ELP, intermediate in the midluteal phase, and lowest in the late luteal phase of the estrous cycle for all the three tested factors. Comparison of the results of the three treatments depicted EGF as the most potent stimulating factor followed by IGF-1 and LH. Immunocytochemistry findings in luteal cell culture of ELP agreed with the VEGF expression and secretion. In conclusion, mRNA expression, protein secretion, and immunolocalization of VEGF data clearly indicated for the first time that LH, IGF-1, and EGF play an important role in stimulating luteal angiogenesis in buffalo CL. The highest expression and secretion of VEGF in the ELP might be associated with the development of blood vessels in early growth of CL, which in turn gets augmented by the aforementioned factors emphasizing their regulatory role in luteal angiogenesis. Further studies are however necessary to divulge more information on other factors which regulate VEGF secretion in bubaline CL and the synergistic effects existing among such growth factors.

Respiratory Control in Female Adult Rats

Sex hormones may influence many physiological processes. It is still unclear whether sex hormones and hormonal fluctuations that occur during the estrous cycle can affect respiratory control. Our study aimed to evaluate the ventilatory responses to hypoxia in female adult rats. To do so, we measured ventilation (VE), tidal volume (VT), respiratory frequency (fR), oxygen consumption (VO2) and body temperature (Tc) of female rats at different stages of estrous cycle (proestrus, estrus, metaestrus and diestrous) and ovariectomized (OVX) female rats during normoxia (21%O2) and hypoxia (7% O2). VE, VT, fR, VO2 and VE/VO2 were not different among animals in different stages of the estrous cycle in normoxia and hypoxia. However, Tc was higher in female rats in estrus. Hormone replacement in females did not change the ventilatory and metabolic parameters. However, females OVX, OVX + E2 and OVX + EP showed a lower ventilatory response to hypoxia compared with intact females in estrus. Thus, our data suggest that the different phases of the estrous cycle did not alter ventilation in normoxia and hypoxia. OVX animals presented an attenuated response to hypoxia. Hormone replacement with E2 or P4 did not cause significant difference in hypoxic ventilatory response. Our data suggest that other gonadal factors might be involved in the process. Financial Support: FAPESP

Influence of Estrous Cycle ond Sexual Hormones in Ventilatory Control during Hypercapnia

Our study aimed to evaluate the ventilation (VE), tidal volume (VT), respiratory frequency (fR), oxygen consumption (VO2) ventilatory equivalent (VE/VO2) and body temperature (Tb) of rats at different stages of estrous cycle (proestrus, estrus, metaestrus and diestrus) and ovariectomized rats (OVX) with hormone replacement to estrogen (OVX + E2) and estrogen + progesterone (OVX + EP). Additionally, we compared the ventilatory and metabolic responses during normocapnia and hypercapnia in intact rats and orchidectomized (ORX) with intact estrous (phase that plasma concentrations of E2 and P are lower) and ovariectomized (OVX) rats during normocapnia and hypercapnia (7% CO2). VE, VT, fR, VO2 and VE/VO2 were not different among animals in different stages of the estrous cycle in normocapnia and hypercapnia, Tb was higher in female rats in estrus. Hormone replacement in females did not change the ventilatory and metabolic parameters. However, females OVX, OVX+E2 and OVX+EP showed lower ventilatory responses to hypercapnia compared to intact females in estrus phase. Intact animals (both males and females in estrus) showed a significantly higher VE and VE/VO2 in hypercapnia than castrated animals. We also observed that females in estrus had a higher ventilatory response to CO2 compared to intact male rats. Thus, our data demonstrated that the different phases of the estrous cycle did not change ventilation in normocapnia and hypercapnia. In addition, gonadectomy reduces the ventilatory response to CO2 and that rats in estrus phase have a higher ventilatory responses to hypercapnia than males. Supported by FAPESP and CNPq.

Expression of Pathogen Recognition Receptors and Pro-Inflammatory Cytokine Transcripts in Clinical and Sub-Clinical Endometritis Cows

The present study was carried out to examine the expression profile of pathogen recognition receptors (CD14 and toll-like receptor 4) and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNFα) in endometrial tissue of cows with endometritis at different stages of estrous cycle. Genital tracts were collected from 60 cows at slaughter from the killing village. The genitalia were examined for clinical endometritis (CE) and subclinical endometritis (SCE) through physical examination, white side test of cervico-vaginal mucus, endometrial cytology and histopathology. The stage of estrous cycle for each genitalia was determined by visual examination of both the ovaries and classified as either follicular (F) or luteal (L). Depending on the degree of inflammation and stage of estrous cycle, the genitalia were categorized in four groups i.e., FCE, FSCE, LCE, and LSCE with six genitalia in each group. Furthermore, 12 healthy genitalia comprise of six each of follicular (FN) and luteal (LN) were included as control. Endometrial tissue scrapings were collected ex vivo from all the genitalia. Total RNA was extracted and cDNA was transcribed for each sample and relative quantification of mRNA of target genes was done by real-time PCR. The results revealed a significant up-regulation of CD14 (11 fold) and IL-8 (13 fold) in follicular stage and IL-6 (8 fold) and TNFα (29 fold) in luteal stages in SCE cows. However, the majority of pro-inflammatory cytokine and pathogen recognition receptors expressed at significant higher level in both follicular and luteal stages in cows with CE. Thus, it is concluded that the endometrial transcripts of pathogen recognition receptors and pro-inflammatory cytokines expressed differentially in cows with endometritis, whereas the fold change is dependent on the severity of inflammation and the stage of cyclicity. Therefore, endometrial transcript profile with a defined threshold level could be used as a possible diagnostic marker in cows with SCE.