Luteal expression of factors involved in the metabolism and sensitivity to oestrogens in the dog during pregnancy and in non-pregnant cycle.

The canine corpus luteum (CL) is the main source of reproductive steroids during dioestrus in the dog and remains active even in the absence of pregnancy (non-pregnant dioestrus, physiological pseudopregnancy). Whereas the biological effects of 17ß-oestradiol (E2) in the canine CL remain unclear, the transcriptional availability of oestrogen receptors, ESR1 and ESR2, as well as other modulators of local availability of E2, for example, HSD17B7 (converts oestrone into oestradiol), SULT1E1 (inactivates E2 binding capacity to its own receptors through sulphonation) and STS (reverts E2 sulphonation), were previously detected in the CL of non-pregnant bitches. The aim of the present work was to evaluate the mRNA amounts of these factors involved in luteal sensitivity and metabolism of E2 in the canine CL during the course of non-pregnant dioestrus (days 10, 20, 30, 40, 50 and 60 post-ovulation, n = 5/group) and at different stages of pregnancy (n = 4-6/group): pre-implantation (days 8-12), post-implantation (days 18-25), mid-gestation (days 35-40) and prepartum luteolysis. During pregnancy, the availability of ESR1, HSD17B7, SULT1E1 and STS decreased from mid-pregnancy to prepartum luteolysis. The main findings during non-pregnant dioestrus were as follows: increased ESR2:ESR1 ratio on days 40 and 50 after ovulation, decreasing during luteal regression (day 60); increased STS at day 30 when SULT1E1 levels decreased; increased availability of SULT1E1 transcripts during luteal regression; and decreased amounts of HSD17B7 mRNA in early dioestrus, increasing towards later stages. These results suggest that E2 signalling and biologically active local concentrations could diverge in response to time and pregnancy status of the bitch.

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment.

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine® , 10 mg/kg) was injected subcutaneously 5 days before ovariohysterectomy in the treatment group (n = 6); bitches without treatment served as a control group (n = 6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p < .05) and ERα mRNA increased (p < .01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p < .05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p < .01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.

A comparison of the uterine proteome of mares in oestrus and dioestrus.

Proteomic analysis of mare uterine flush fluid provides a minimally invasive technique for studying protein changes associated with the oestrous cycle. The aim of this study was to identify differentially abundant proteins in the uterine flush fluid of mares in oestrus and dioestrus. In this study, uterine flush fluid samples were collected from eight reproductively healthy mares in either oestrus (n = 5) or dioestrus (n = 3). Proteomic analysis was performed using liquid chromatography-tandem mass spectrometry. Of 172 proteins identified, six proteins (immunoglobulin lambda-like polypeptide 1, haemoglobin subunit alpha, alpha-1B-glycoprotein, serotransferrin, apolipoprotein A-1, and haemoglobin subunit beta) were significantly more abundant in oestrus. These proteins may contribute to the endometrial defence system through roles in inflammation, immunity or antimicrobial activity. In other species, some of these proteins have been described as immunoglobulins, negative acute phase proteins or defence agents against micro-organisms. During dioestrus, immunoglobulin alpha-1 chain C region-related, complement factor I, CD 109 antigen and uterocalin, were significantly more abundant. Research in other species suggests that these four proteins contribute to the immune response through proposed immunoregulatory characteristics, complement system involvement or roles in B cell-T cell interactions. In conclusion, ten differentially abundant proteins were identified in the uterine flush fluid of mares in oestrus and dioestrus. Targeted studies on these proteins could elucidate their role in uterine defence mechanisms during the oestrous cycle in the mare.

Oxytocin receptors in dioestrous and anoestrous canine uteri.

The aim of the study was to localize oxytocin receptors (OTR) and measure mRNA expression of OTR in the canine uterus with and without the influence of progesterone. Uterine samples were taken from nine anoestrous and eight dioestrous bitches during ovariohysterectomy. Histological changes were evaluated in haematoxylin and eosin (HE)-stained samples. Purified polyclonal antibody for OTR was used in immunohistochemistry to localize receptors in uterine layers. Relative mRNA concentration of OTR was evaluated with real-time PCR from full-thickness uterine samples taken from the middle horn and the body. Myometrial smooth muscle cells, endometrial luminal epithelium (LE) and deep and superficial glandular epithelium were positively stained for oxytocin receptors in non-pregnant animals. No significant difference in staining intensity was detected between uterine middle horn and body. However, the staining intensity of LE was significantly higher in dioestrous than in anoestrous uteri (p < .05). Leucocytes and endothelium of blood vessels were also positively stained for OTR. Real-time PCR showed no significant differences in OTR mRNA expression between the middle horn and the body of the uterus, or between anoestrous and dioestrous uterus. No correlation was noted between OTR mRNA expression and blood progesterone concentration. In conclusion, despite the apparent inactivity, the uterus of the non-pregnant bitch expresses OTR. The distribution or relative expression of OTR does not differ between uterine horn and body in dioestrus or anoestrus except in LE. LE may have more oxytocin-dependent activity during dioestrus than anoestrus.

Variation of Physiological and Behavioural Parameters during the Oestrous Cycle in Mares.

The behaviour of oestrous mares is well-known in horse breeding. However, alterations in their physiological and behavioural indices during the whole oestrous cycle are scarcely known. The objective of the study was to analyse changes in cardiac activity variables, rectal and superficial temperatures, behaviour towards humans and conspecifics, and the time of standing and locomotor activity in mares during their oestrous cycle. Fifteen adult mares in oestrus were examined in the morning and evening (six successive days) and in dioestrus (five days-once every third day). The oscillation of physiological and behavioural variables accompanies changes in mares' sexual behaviour. Most physiological variables studied in oestrus indicate the elevated activity of the adrenergic nervous system and, opposite to that, both behaviour towards humans and conspecifics and the time of standing relate to a relaxed state. The end of oestrus, manifested by a rapid decrease in most of the physiological variables studied, is followed by changes of behavioural variables at the beginning of dioestrus. The time of locomotor activity arises at the end of oestrus. The outcomes may contribute to the knowledge of, among others, mare owners who evaluate the oestrus by mares' sexual behaviours without regarding other rhythmically changing variables.

Studying the heterogeneous pathogenesis of canine diabetes: Observational characterization of an island population.

BACKGROUND: Canine diabetes mellitus has mostly been studied in northern European, Australian and American populations, whereas other regions have received less attention. OBJECTIVES: We evaluated the epidemiological, clinical and histopathological features of diabetic dogs in Gran Canaria, Spain. METHODS: Prevalence and incidence were estimated. Clinical features were analysed, and serum and genomic DNA were obtained. Dogs with presumed idiopathic or immune-mediated diabetes, were DLA-typed and antibodies against GAD65 and IA-2 were assessed. Pancreases from ten diabetic dogs were examined and compared with pancreases from non-diabetic dogs. RESULTS AND CONCLUSIONS: Twenty-nine diabetic dogs were identified in a population of 5,213 (prevalence: 0.56%; incidence: 0.37%). Most were female (79%) and sexually intact (87% of females, 83% of males). Diabetes secondary to dioestrus (55.2%) and insulin-deficient diabetes (20.7%) were the most frequent types. Antibodies against GAD65 and IA-2 were identified in two out of five cases and DLA-genotyping revealed novel haplotypes. Breed distribution differed between diabetic and non-diabetic dogs. Reduced number of pancreatic islets and ß-cell mass were observed, with vacuolation of islet cells and ductal epithelium. In this population, where neutering is not standard practice, diabetes secondary to dioestrus is the most frequent diabetes subtype. Genetic susceptibility also differed from previous studies. These results support the heterogeneous pathogenesis of canine diabetes.

Expression Profiles of the Progesterone Receptor, Cyclooxygenase-2, Growth Differentiation Factor 9, and Bone Morphogenetic Protein 15 Transcripts in the Canine Oviducts during the Oestrous Cycle.

The gene expression in the canine oviduct, where oocyte maturation, fertilization, and early embryonic development occur, is still elusive. This study determined the oviductal expression of (PR), cyclooxygenase-2 (COX-2), growth differentiation factor 9 (GDF-9), and bone morphogenetic protein 15 (BMP-15) during the canine oestrous cycle. Samples were collected from bitches at anoestrus (9), proestrus (7), oestrus (8), and dioestrus (11), after routine ovariohysterectomy and the ovarian surface structures and plasma progesterone concentration evaluated the physiological status of each donor. The oviductal cells were isolated and pooled. Total RNA was isolated, and gene expression was assessed by qPCR followed by analysis using the t-test and ANOVA. The PR mRNA increased (P < 0.05) from the anoestrus to dioestrus with the plasma progesterone concentration (r = 0.8). COX-2 mRNA expression was low in the anoestrus and proestrus, and negligible in the oestrus, while it was around 10-fold higher (P < 0.05) in the dioestrus. The GDF-9 mRNA was expressed during all phases of the oestrous cycle and was most abundant (P < 0.05) during oestrus phase. The BMP-15 mRNA decreased (P < 0.05) in the anoestrus and proestrus phases. Thus, the transcripts were differentially expressed in a stage-dependent manner, suggesting the importance of oestrous cycle regulation for successful reproduction in dogs.

Oestrous phase cyclicity influences judgment biasing in rats.

The identification of cognitive bias has become an important measure of animal welfare. Negative cognitive biases develop from a tendency for animals to process novel information pessimistically. Judgment-bias testing is the commonplace methodology to detect cognitive biases. However, concerns with these methods have been frequently-reported; one of which being the discrepancy between male and female cognitive expression. The current study assessed the factors of social status and oestrus, to investigate whether oestrous cycle rotation, or subordination stress encouraged an increase in pessimistic responses. Female Sprague-Dawley rats (n = 24) were trained on an active-choice judgment bias paradigm. Responses to the ambiguous probe were recorded as optimistic or pessimistic. Oestrous phase was determined by assessing vaginal cytology in stained vaginal cell smears. Rats in the dioestrous phase and those rats considered to be subordinate demonstrated an increased percentage of pessimistic responses. However, no interaction between these factors was observed. This suggests that oestrous cyclicity can influence the judgment biases of female animals; a previously unreported finding. On this basis, researchers should be encouraged to account for both oestrous phase cyclicity and social status as an additional fixed effect in study design.

The precision of peri-oestrous predictors of the date of onset of parturition in the bitch.

Precise prediction of the date of onset of parturition in the bitch is clinically important. The study compared the precision with which four peri-oestrous predictors predict the date of onset of parturition. The predictors evaluated in 24 bitches were: the date of the first or only day of the LH surge, the date on which the concentration of progesterone in the blood plasma first exceeded 6 nmol/L, the date on which the concentration of progesterone in the blood plasma first exceeded 16 nmol/L and the date of onset of cytological dioestrus. Among the 24 bitches, the date of onset of cytological dioestrus predicted the date of onset of parturition with greater precision than the other three predictors. Following the evaluation of another 218 intervals between the onset of cytological dioestrus and the date of onset of parturition, it was shown that the onset of cytological dioestrus predicted the date of onset parturition with a precision of ±1 d, ± 2 d and ±3 d in 88%, 99% and 100% of the 242 pregnancies. This study concludes that the first day of cytological dioestrus is a useful predictor of the date of onset of parturition.

Canine diabetes mellitus risk factors: A matched case-control study.

Different subtypes of canine diabetes mellitus (CDM) have been described based on their aetiopathogenesis. Therefore, manifold risk factors may be involved in CDM development. This study aims to investigate canine diabetes mellitus risk factors. Owners of 110 diabetic dogs and 136 healthy controls matched by breed, sex, and age were interviewed concerning aspects related to diet, weight, physical activity, oral health, reproductive history, pancreatitis, and exposure to exogenous glucocorticoids. Two multivariate multivariable statistical models were created: The UMod included males and females without variables related to oestrous cycle, while the FMod included only females with all analysed variables. In the UMod, "Not exclusively commercial diet" (OR 4.86, 95%CI 2.2-10.7, P<0.001) and "Overweight" (OR 3.51, 95%CI 1.6-7.5, P=0.001) were statistically significant, while in the FMod, "Not exclusively commercial diet" (OR 4.14, 95%CI 1.3-12.7, P=0.01), "Table scraps abuse" (OR 3.62, 95%CI 1.1-12.2, P=0.03), "Overweight" (OR 3.91, 95%CI 1.2-12.6, P=0.02), and "Dioestrus" (OR 5.53, 95%CI 1.9-16.3, P=0.002) were statistically significant. The findings in this study support feeding not exclusively balanced commercial dog food, overweight, treats abuse, and diestrus, as main CDM risk factors. Moreover, those results give subside for preventive care studies against CDM development.

Vaginal cytology after induction of ovulation in the queen: comparison of postoestrus and dioestrus.

OBJECTIVE: To compare the vaginal cytology of ovulating and non-ovulating queens. PROCEDURE: The study group comprised 15 queens showing behavioural oestrus. Ovulation was induced in 7 (dioestrus group) and 8 were left untreated (postoestrus group). Vaginal smears were collected from all animals prior to ovariohysterectomy on day 7. Epithelial cells were classified as basal-parabasal, intermediate, superficial, or anucleated superficial cells and counted using computer-assisted image analysis. From each smear, 50 representative vaginal epithelial cells were chosen. Digital images of cells were taken and cell area, cytoplasm area, nucleus area, cell diameter, cell perimeter, nucleus/cytoplasm ratio and red-green-blue (RGB) values were measured using image analysis software. Measurement data were compared between groups. RESULTS: Ovulation induction was successful in all animals. The swabbing procedure in oestrus did not induce ovulation in any postoestrus queens. Mean duration of oestrus was 6.65 ± 0.44 and 4.71 ± 0.32 days (P > 0.05) in the postoestrus and dioestrus queens, respectively. Intermediate cell count averaged 21.43% in dioestrus cats and 10.76% in postoestrus cats (P < 0.05). Epithelial cells in the postoestrus group had higher cell area, cytoplasm area, cell diameter and cell perimeter measurements (P < 0.01). Red (90.9 ± 1.6), green (76.1 ± 1.3) and blue (83.6 ± 1.4) channel values in postoestrus were higher than the values (81.3 ± 0.8, 65.8 ± 0.9 and 74.0 ± 0.7, respectively) in dioestrus (P < 0.01). CONCLUSION: Induction of ovulation in oestrus queens results in a significant increase in the number of intermediate cells and a significant decrease in both the dimensions and RGB values of vaginal epithelial cells on day 7.

The effect of dioestrus on the racing performance of Greyhounds.

The degree of performance change during the dioestrus of racing Greyhounds has long been the subject of debate. Assessments have previously been on a qualitative basis. The analyses in this paper are unique and produce a quantitative assessment of the change in performance during dioestrus. By accessing a large dataset, race form and oestrous data were analysed using a longitudinal observational study design. The performance changes in dioestrus were modelled with a series of multilevel linear regressions revealing a pattern such that the performance loss varied from 0.031 to 0.733 s (90% confidence interval) between 41 and 56 days since oestrus over 450 m, returning to the baseline (anoestral) performance level after about 80-100 days. The changes in performance formed a temporal match with the changes in serum progesterone concentration noted by other workers. Whilst serum hormone concentrations were not measured, it is suggested that the performance changes are linked with the changes in serum progesterone concentrations rather than prolactin. It is therefore proposed that a minimum time off of 70 days could be set, which would capture the majority of females with a genuine change in performance, and with a caution regarding the use of entire female Greyhounds for performance studies.

The expression difference between estrus and non-estrus of ERβmRNA of beagles by real-time quantitative PCR / 中国比较医学杂志

Objective To quantitative the changing information of estrogen receptor βgene which was in tissue and organ of sex gland during oestrus and dioestrus of Beagles, and to show the different expression situation of hypothalamus-pituitary-gonad axis during oestrus and dioestrus, and providing the basic of theory to research deeply the mechanism of heat of Beagles. Methods As the key gene in regulation reproduction, ERβgene is located in hypothalamus-pituitary-gonad axis, so using Beagles which was in oestrus and dioestrus, and extract the RNA from hypothalamus、pituitary、ovary and uterus respectively,after reverse transcription we detected the expression of ERβgene by real-time quantitative PCR.Results The expression of ERβgene mRNA from ovary、uterus、pituitary、hypothalamus of Beagles which was in dioestrus was 0.35 times, 0.17 times, 0.44 times and 0.43 times than the expression of ERβgene mRNA from ovary, uterus, pituitary, hypothalamus of Beagles which was in oestrus.Conclusion The expression of ERβgene was up-regulation in hypothalamus-pituitary-ovary axis of Beagles which was in oestrus.