Effects of Bisphenol A and Bisphenol F on Porcine Uterus Contractility.

Introduction: Bisphenols, as endocrine disruptors, may cause a wide range of health problems in humans, but so far, not all of them have been confirmed in animals, including pigs. Since animals are also exposed to bisphenols, we hypothesised that these substances may have an effect on uterine contractility in pigs. Therefore, the aim of the study was to investigate the effect of the most-used bisphenol, bisphenol A (BPA), and a selected analogue, bisphenol F (BPF), on the contractile activity of the pig uterus. Material and Methods: The investigation utilised smooth muscles from immature pigs (n = 6), cyclic pigs on days 12-14 of the oestrous cycle (n = 6) or early pregnant pigs on days 12-16 of pregnancy (n = 6). Strips of the myometrium were exposed to BPA and BPF at concentrations of 10-13-10-1 M. Smooth muscle contractility was determined with equipment for measuring isometric contractions. Results: BPA caused a significant decrease in contraction amplitude, and frequency and in myometrial tension in all groups examined. BPF caused a decrease in the amplitude and frequency of contractions in all groups and in myometrial tension in the early pregnant group. Conclusion: The obtained results indicate that both BPA and BPF relaxed the porcine myometrium, but these changes, especially in the amplitude and frequency of contractions, were more evident after BPF treatment. The extent of relaxation is dependent on the physiological status of the animals.

Quercetin affects uterine smooth muscle contractile activity in gilts.

Quercetin is a polyphenolic flavonoid occurring in leaves, stems, flowers and fruits of many plants. In traditional Chinese medicine, it is used as a natural therapeutic agent with a broad spectrum of activities (antioxidant, neuroprotective, anti-inflammatory, anticancer, antibacterial and antiviral). Moreover, quercetin affects function of the reproductive tract, however the knowledge of this activity is still fragmentary. Therefore, this study aimed to determine the influence of quercetin on the contractile activity of the porcine myometrium collected from immature (n = 6), cyclic (n = 6) and early pregnant (n = 6) gilts. Strips of the myometrium (comprising longitudinal and circular layer) were resected from the middle part of the uterine horns and the isometric contractions were recorded. After 60-90 min of preincubation, the strips were stimulated with quercetin in increasing (10-13-10-1 M) concentrations and the changes in the tension amplitude and frequency of contractions were measured. Quercetin decreased (P<0.01-0.001) the amplitude of contractions at concentrations 10-11-10-1 M and 10-10-10-1 M in cyclic and early pregnant groups, respectively. The frequency of contractions decreased in all groups but was the highest (at concentrations 10-11-10-1 M; P<0.05-0.001) in the cyclic group and the lowest (at concentrations 10-5-10-1 M; P<0.01) in the immature group. The tension decreased only in the cyclic group after quercetin administration in high concentrations (10-6-10-1 M; P<0.05-0.01). The results indicate that quercetin causes relaxation of the porcine uterine smooth muscle but this activity is strongly related to the physiological status of the gilts.

The Influence of an Adrenergic Antagonist Guanethidine on the Distribution Pattern and Chemical Coding of Caudal Mesenteric Ganglion Perikarya and Their Axons Supplying the Porcine Bladder.

This study was aimed at disclosing the influence of intravesically instilled guanethidine (GUA) on the distribution, relative frequency and chemical coding of both the urinary bladder intramural sympathetic nerve fibers and their parent cell bodies in the caudal mesenteric ganglion (CaMG) in juvenile female pigs. GUA instillation led to a profound decrease in the number of perivascular nerve terminals. Furthermore, the chemical profile of the perivascular innervation within the treated bladder also distinctly changed, as most of axons became somatostatin-immunoreactive (SOM-IR), while in the control animals they were found to be neuropeptide Y (NPY)-positive. Intravesical treatment with GUA led not only to a significant decrease in the number of bladder-projecting tyrosine hydroxylase (TH) CaMG somata (94.3 ± 1.8% vs. 73.3 ± 1.4%; control vs. GUA-treated pigs), but simultaneously resulted in the rearrangement of their co-transmitters repertoire, causing a distinct decrease in the number of TH+/NPY+ (89.6 ± 0.7% vs. 27.8 ± 0.9%) cell bodies and an increase in the number of SOM-(3.6 ± 0.4% vs. 68.7 ± 1.9%), calbindin-(CB; 2.06 ± 0.2% vs. 9.1 ± 1.2%) or galanin-containing (GAL; 1.6 ± 0.3% vs. 28.2 ± 1.3%) somata. The present study provides evidence that GUA significantly modifies the sympathetic innervation of the porcine urinary bladder wall, and thus may be considered a potential tool for studying the plasticity of this subdivision of the bladder innervation.

Selected Biological Medicinal Products and Their Veterinary Use.

Definitions of biological medicinal products (BMPs) vary depending on the source. BMPs are manufactured using complex biological/biotechnological processes involving living cell lines, tissues and organisms such as microorganisms, plants, humans and even animals. Advances in modern biotechnological methods and genetic engineering have made it possible to search for new drugs with a targeted effect and simultaneous reduction of adverse effects, which has resulted in BMPs dynamically increasing their share in the pharmaceutical market. Currently, these drugs are widely used in the treatment of many human diseases, but an increasing number of drugs of this group are also being used in the treatment of animals, mainly in dermatology, rheumatology and oncology. This article presents the current state of knowledge in the field of biological medicinal products used in animal therapy.

Prostaglandin E2 exerts the proapoptotic and antiproliferative effects on bovine NK cells.

The aim of this research was to determine whether prostaglandin E2 (PGE2) affects bovine NK cells in respect of their counts, apoptosis and proliferation, and if it does, then which of the PGE2 receptor (EP) subtype(s) mediate(s) these effects. We here report that long-term, but not short-term, exposure of bovine peripheral blood mononuclear cells to PGE2 at 10(-5)M, 10(-6)M and 10(-7)M, but not at 10(-8)M, caused a significant increase in the percentage of early apoptotic cells among NK cell subset. Moreover, PGE2 at 10(-5)M and 10(-6)M, but not at 10(-7)M and 10(-8)M, induced a considerable decrease in the absolute count of NK cells. The magnitude of these effects increased with an increasing concentration of PGE2. The blockade of EP1, EP2, EP3 and EP4 receptors did not prevent the PGE2-induced apoptosis and depletion of NK cells. The results suggest that the proapoptotic effect of PGE2 is secondary in character and the induction of this effect is not mediated through EP receptors. Furthermore, the studies demonstrated that PGE2 at 10(-5)M and 10(-6)M, but not at 10(-7)M and 10(-8)M, highly significantly reduced the percentage of proliferating NK cells. The EP1, EP1/2 and EP3 receptor antagonists were unable to block this effect significantly, whereas the selective blockade of EP4 receptors prevented the PGE2-induced inhibition of NK cells proliferation. These results indicate that PGE2 at certain concentrations may impair the proliferation of NK cells and this effect is mediated via the EP4 receptor.

Comparison of the porcine uterine smooth muscle contractility on days 12-14 of the estrous cycle and pregnancy.

BACKGROUND: Uterine contractile activity is very important for many reproductive functions including embryo transport, implantation, gestation and parturition. Abnormal contractility leads to implantation failure, spontaneous miscarriage, preterm birth and many other disorders. The objective of the present study was to assess the effects of acetylcholine (ACh), noradrenaline (NA), oxytocin (OT) and prostaglandins F2α (PGF2α) and E2 (PGE2) on the contraction of uterine strips collected from the horns of cyclic gilts (12-14 days of the estrous cycle-group I) and from pregnant (12-14 days after first insemination gilts in which one of the uterine horn was gravid (group IIa) and the second one was non-gravid (group IIb). Uterine strips consisting of the endometrium with the myometrium and myometrium alone were examined. RESULTS: ACh increased the tension in all groups as compared to the pretreatment period, and the increase was the highest in group IIb; the amplitude decreased in all groups, and the frequency increased mainly in groups I and IIa. NA did not affect the tension in any group, but decreased the amplitude and frequency in group IIb as compared to groups I and IIa. OT caused the highest increase in the tension in group IIb, a decrease in the amplitude and an increase in the frequency of contractions as compared to the pretreatment period. PGF2α induced the highest increase in the tension and amplitude in group IIb, with a decline in the frequency in this group. PGE2 increased the tension and frequency only in group IIb, and caused the greatest eduction in the amplitude in this group. CONCLUSIONS: These results indicate that contractility of the porcine smooth muscle collected from uterine horns with embryos was different from those obtained from the uterine horns without embryos and the horns of cyclic gilts.

Botulinum toxin type A induces changes in the chemical coding of substance P-immunoreactive dorsal root ganglia sensory neurons supplying the porcine urinary bladder.

Botulinum toxin (BTX) is a potent neurotoxin which blocks acetylcholine release from nerve terminals, and therefore leads to cessation of somatic motor and/or parasympathetic transmission. Recently it has been found that BTX also interferes with sensory transmission, thus, the present study was aimed at investigating the neurochemical characterization of substance P-immunoreactive (SP-IR) bladder-projecting sensory neurons (BPSN) after the toxin treatment. Investigated neurons were visualized with retrograde tracing method and their chemical profile was disclosed with double-labelling immunohistochemistry using antibodies against SP, calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase activating polypeptide (PACAP), neuronal nitric oxide synthase (nNOS), galanin (GAL), calbindin (CB), and somatostatin (SOM). In the control group (n = 6), 45% of the total population of BPSN were SP-IR. Nearly half of these neurons co-expressed PACAP or CGRP (45% and 35%, respectively), while co-localization of SP with GAL, nNOS, SOM or CB was found less frequently (3.7%, 1.8%, 1.2%, and 0.7%, respectively). In BTX-treated pigs (n = 6), toxin-injections caused a decrease in the number of SP-IR cells containing CGRP, SOM or CB (16.2%, 0.5%, and 0%, respectively) and a distinct increase in these nerve cells immunopositive to GAL (27.2%). The present study demonstrates that BTX significantly modifies the chemical phenotypes of SP-IR BPSN.

Prostaglandin E2 down-regulates the expression of CD25 on bovine T cells, and this effect is mediated through the EP4 receptor.

A crucial event in the initiation of an immune response is the activation of T cells, which requires IL-2 binding to its high-affinity IL-2 receptor for optimal signaling. The IL-2 receptor α-chain (CD25) is needed for the high affinity binding of IL-2 to effector cells and is potently induced after T cell activation. The aim of this research has been to determine whether prostaglandin E2 (PGE2) affects the CD25 expression on bovine T cells, and if it does, then which of the PGE2 receptor (EP) subtype(s) mediate(s) this effect. Herein, we report that exposure of peripheral blood mononuclear cells (PBMC) to PGE2 considerably reduces the percentage and absolute counts of CD25(+)CD4(+), CD25(+)CD8(+) and CD25(+)WC1(+) T cells, significantly increases the value of these parameters with respect of CD25(-)CD4(+), CD25(-)CD8(+) and CD25(-)WC1(+) T cells, and does not affect counts of the total populations of CD4(+), CD8(+) and WC1(+) T cells. These results indicate that PGE2 down-regulates the CD25 expression on bovine T cells. Moreover, we show that the selective blockade of EP4 receptor, but not EP1 and EP3 receptors, prevents this effect. Interestingly, the exposure of PBMC to a selective EP2 receptor agonist leads to a substantial increase in the percentage and absolute number of CD25(+)CD4(+), CD25(+)CD8(+) and CD25(+)WC1(+) T cells. In conclusions, the PGE2-induced down-regulation of CD25 expression on bovine CD4(+), CD8(+) and WC1(+) T cells should be considered as immunosuppressive and anti-inflammatory action, because these lymphocytes primarily represent effector cells and adequate CD25 expression is essential for their correct functioning. The PGE2-mediated down-regulation of the CD25 expression on bovine T cells is mediated via the EP4 receptor, although selective activation of the EP2 receptor up-regulates the CD25 expression on these cells. Thus, with respect to the effect of PGE2 on the CD25 expression on bovine T cells, EP4 receptor serves as an inhibitory receptor, whereas EP2 receptor functions as a stimulatory receptor. The fact that non-selective stimulation of EP receptors, i.e. triggered by PGE2, leads to weaker CD25 expression proves that inhibitory actions prevail over stimulatory ones. These results indicate the possibility of pharmacological manipulation of the CD25 expression on T cells via selective antagonists and agonists of EP2 and EP4 receptors.

Influence of oral co-administration of a preparation containing calcium and magnesium and food on enrofloxacin pharmacokinetics.

The objective of this study has been to determine the influence of food and ions on the pharmacokinetics of enrofloxacin (ENRO) in turkeys, administered per os at a dose of 10 mg/kg of body weight (b.w.). Co-administration of ENRO with ions or with food significantly retarded its absorption, and the interaction was more pronounced when the drug was given together with food. The bioavailability of ENRO was 65.78 ± 7.81% and 47.99 ± 9.48% with ions and food, respectively. The maximum concentration (Cmax) in plasma of animals exposed to ions reached 0.87 ± 0.26 µg/ml in a tmax of 2.07 ± 0.76 h; in animals which were fed while medicated, the analogous parameters were 0.36 ± 0.13 µg/ml and 8.06 ± 3.08 h. The PK/PD analysis demonstrated that a decrease in the concentration of ENRO in turkeys' blood due to the interaction with ions or food might impair the drug's clinical efficacy toward some pathogenic microorganisms in turkeys if a routine dose of 10 mg ENRO/kg b.w. is administered.

Neoadjuvant treatment for unresectable rectal cancer: an interim analysis of a multicentre randomized study.

PURPOSE: To present an interim analysis of the trial comparing two neoadjuvant therapies for unresectable rectal cancer. METHODS: Patients with fixed cT3 or cT4 or locally recurrent rectal cancer without distant metastases were randomized to either 5 × 5 Gy and 3 courses of FOLFOX4 (schedule I) or 50.4 Gy delivered in 28 fractions given simultaneously with 5-Fu, leucovorin and oxaliplatin (schedule II). Surgery in both groups was performed 12 weeks after the beginning of radiation and 6 weeks after neoadjuvant treatment. RESULTS: 49 patients were treated according to schedule I and 48 according to schedule II. Grade III+ acute toxicity was observed in 26% of patients in group I and in 25% in group II. There were two toxic deaths, both in group II. The microscopically radical resection (primary endpoint) rate was 73% in group I and 71% in group II. Overall and severe postoperative complications were recorded in 27% and 9% of patients vs. 16% and 7%, respectively. Pathological complete response was observed in 21% of the patients in group I and in 9% in group II. CONCLUSIONS: The interim analysis revealed no major differences in acute toxicity and local efficacy between the two evaluated strategies.

Effects of dexamethasone and meloxicam on bovine CD25+ CD8+ and CD25- CD8+ T cells--in vitro study.

The aim of undertaken research was an in vitro evaluation of the effects of dexamethasone and meloxicam on selected bovine CD8(+) T lymphocyte subpopulations. Dexamethasone induced a fast-occurring and lasting depletion of CD25(-)CD8(+) cells. This was primarily the result of the proapoptotic effect of dexamethasone, but the antiproliferative effect of the drug was clearly responsible for the deepening of this disturbance. Dexamethasone transiently increased the relative and absolute CD25(high)CD8(+) and CD25(low)CD8(+) cell numbers. This effect was not a consequence of increased proliferation, but at least partly resulted from the antiapoptotic effect of the drug on these cells. The obtained results indicate that induction of CD8(+) lymphocyte depletion and impairment of IFN-γ production by these cells participate in the production of the anti-inflammatory and immunosuppressive effect of dexamethasone in cattle. An increase in Foxp3, IL-10 and TGF-ß production by CD8(+) lymphocytes is not involved in the production of these effects because the drug did not affect the percentage of TGF-ß(+)CD8(+) cells, while paradoxically reducing the percentage of cells with the suppressive phenotype, i.e. IL-10(+)CD25(low)CD8(+) and Foxp3(+)CD25(low)CD8(+) cells. Meloxicam did not substantially affect CD8(+) lymphocytes as to their percentage, absolute number, apoptosis, proliferation, Foxp3 expression and IFN-γ, IL-10 and TGF-ß production. Thus, in the context of the parameters being estimated, meloxicam seems a relatively safe anti-inflammatory drug to be used in infectious diseases in cattle.

Application of ultra-performance columns in high-performance liquid chromatography for determination of albendazole and its metabolites in turkeys.

Methods for determination of albendazole (ALB), albendazole sulfoxide (SOX) and albendazole sulfone (SON) in turkey blood plasma, using high-performance liquid chromatography (HPLC) with fluorescence detection, were developed. Moreover, comparison of HPLC columns with ultra-performance liquid chromatography (UPLC) columns was performed. Albendazol was administered orally in 5-week-old birds (n = 18) at a dose of 25 mg/kg b.w. Accuracy and precision of the developed method were satisfactory and stability studies showed acceptable variation (below 15%) in ALB, SOX and SON concentrations when the samples were stored at -75°C for 15 days. UPLC(®) columns gave higher peaks from typical HPLC columns retaining high quality of analysis. Pharmacokinetic analysis indicated quick elimination of ALB from turkey blood plasma. The mean residence time of SON was at least two times longer than that of SOX and four times longer than that of ALB. The elimination half-lives for ALB, SOX and SON were 0.7 ± 0.27, 5.37 ± 6.03, 9.17 ± 5.12 h, respectively. The obtained results indicate that the described method allows for precise determination of albendazole and its metabolites in turkey plasma. Moreover, using UPLC columns in HPLC apparatus results in higher sensitivity as compared with the classical HPLC columns.

Acute changes in circulating concentrations of progesterone and nitric oxide and partial pressure of oxygen during prostaglandin F2alpha-induced luteolysis in cattle.

To examine whether oxygen (O(2)) and nitric oxide (NO) are temporally associated with the acute changes in luteal function during luteolysis, we determined the real-time changes in the circulating concentrations of progesterone (P4) and nitrite/nitrate (the stable metabolites of NO) and the partial pressure of oxygen (pO(2)) during prostaglandin F(2alpha) (PGF(2alpha))-induced luteolysis in cattle. Catheters for frequent blood sample collection were inserted into the ovarian vein (OV), jugular vein (JV) and aorta abdominalis (AA) in 12 cows on Day 9 of the oestrous cycle (oestrus=Day 0). On Day 10, the cows were randomly divided into two groups and treated with a luteolytic dose of a PGF(2alpha) analogue or saline solution (control). Blood samples were collected at -2, -1, 0, 0.25, 0.5, 0.75, 1 and 2 h and then at 2-h intervals until 12 h after treatment (0 h). Injection of a PGF(2alpha) induced a significant decrease in the concentrations of P4 in OV plasma within 2 h. The decrease in P4 concentrations was preceded by an increase in the NO concentrations in the blood collected from OV, JV and AA. Basal pO(2) was significantly higher in OV blood than in JV blood (P<0.05). PGF(2alpha) injection increased pO(2) in OV blood between 0.5 and 2 h. These results demonstrate that PGF(2alpha) induced an acute increase in pO(2) and NO in the ovarian circulation and suggest that O(2) and NO are involved in the early events of CL regression, including inhibition of P4 secretion and output, in cattle.

Activity of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in the sera of patients with breast cancer.

Numerous experiments have shown that alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) play a significant role in the metabolism of many biological substances. Some metabolic disorders that can lead to breast carcinogenesis may be the cause of changes in ADH and ALDH activity. In previous experiments we found a changed level of class I ADH activity in breast cancer tissues. The changed level of this enzyme in cancer cells may be reflected by the enzyme's activity in the serum. Therefore, in this study we measured the activity of ADH isoenzymes and ALDH in the sera of patients with breast cancer. Serum samples were taken for routine biochemical investigation from 45 women with breast cancer before treatment. Among all tested classes of ADH isoenzymes, only class I had higher activity in the serum of patients with breast cancer in stage IV. The total ADH and ALDH activities were not significantly higher in patients with breast cancer than in healthy controls. The changes in activity, especially in class I ADH, appear to be caused by isoenzymes being released from the organ damaged by metastatic disease.

The distribution and influence of calcitonin gene-related peptide (CGRP) on the perfusion pressure in the isolated ovarian artery in the pig.

A moderate number of delicate CGRP-immunoreactive (CGRP-IR) varicose nerve terminals were found at the adventitia-media border of the sexually mature porcine ovarian artery by means of a routine single immunolabelling technique. Additionally, a pharmacological analysis was performed of the function fulfilled by alpha-CGRP and its C-terminal fragment (Tyr27) alpha-CGRP(27-37) in the porcine isolated ovarian artery, collected on days 8-13 of the oestrous cycle. It was shown that alpha-CGRP (10(-8) M) caused a decrease in the perfusion pressure of the porcine isolated ovarian artery of 16% (p < 0.05), while (Tyr27) alpha-CGRP(27-37), added at the same concentration, reduced perfusion pressure by 13% (p > 0.05). Thus, we concluded that alpha-CGRP released from perivascular terminals may cause relaxation of the ovarian artery and, furthermore, that the potency of this action is dependent on the length of the chain of this peptide produced during the deactivation of the molecule by tissue proteases.

NPY: its occurrence and relevance in the female reproductive system.

Neuropeptide Y (NPY), an amidated peptide composed of 36 amino acid residues, is the most widely distributed neuropeptide that performs a broad spectrum of physiological functions in both the central and peripheral nervous systems. Among numerous other actions, this peptide is involved, at the periphery, in the neural regulation of blood pressure and blood flow through the organs, and also, acting via Y2 and/or Y5 receptors, in the regulation of angiogenesis. NPY influences blood vessels via its own Y receptors, predominantly of the Y1 subtype. As a sympathetic co-transmitter NPY causes vasoconstriction, stimulates vascular growth and potentiates the contractile activity of noradrenaline (NA), and as a parasympathetic neurotransmitter it is involved in the regulation of vasodilatation within e.g. the uterine artery. In the female reproductive system, NPY not only regulates the blood flow, but also the contractile activity of non-vascular smooth muscle cells of the uterus and oviduct, as well as the secretory function of the ovary. Both the concentration of NPY and its influence on the blood flow through the female reproductive organs are finely tuned by fluctuations in the concentration of ovarian steroid hormones. Thus, the present review was aimed at summarizing the current knowledge dealing with the physiological relevance of NPY in the regulation of female gonad and genital tract function, with a special regard to the pig as a model animal.

Localization of neuropeptide Y and norepinephrine in the porcine ovarian artery and their influence on the local blood pressure.

The aim of the present study was to determine: (i) the presence of dopamine-beta-hydroxylase (DbetaH)- and neuropeptide Y (NPY)-immunoreactive (IR) nerve fibres in the wall of the porcine ovarian artery, (ii) the influence of NPY and norepinephrine (NE) on the contractile activity of the pig ovarian arteries, and (iii) the pharmacological analysis of the interaction between NPY and NE in the isolated porcine ovarian arteries collected from immature pigs and from animals in different days of the estrous cycle. Ovarian arteries for immunohistochemistry and isolated arteries for pharmacological studies were excised from immature pigs and mature animals on days 1-5, 8-13 and 17-20 of the estrous cycle. The study showed that both DbetaH- and NPY-IR nerve fibres were present in the pig ovarian arteries in all periods examined, and, that in some fibres DbetaH and NPY were co-localized. Both NE (10(-6) M) and NPY (10(-7) M) increased blood pressure of examined preparations, however, NE caused stronger changes in the vessel wall tension (P<0.001), than did NPY. NE significantly increased (P<0.001) blood pressure of all isolated arteries, however, this response was stronger in vessels from days 1-5 of the cycle, when compared to days 8-13 (P<0.01), 17-20 and immature pigs (P<0.001). NPY increased significantly blood pressure in isolated arteries from days 8-13 and 17-20 of the cycle (P<0.001), while in preparations taken from immature pigs and animals on days 1-5 of the estrous cycle this response was somewhat weaker (P<0.01). A higher elevation (P<0.001) of blood pressure after NPY administration was observed in isolated arteries from days 17-20 of the cycle, when compared to vessels from days 1-5 and 8-13 and those from immature pigs. Moreover, NE significantly intensified (P<0.001) an increase in the blood pressure in isolated arteries pre-treated with NPY in all periods examined. NPY insignificantly (P>0.05) potentiated increase of blood pressure in NE pre-treated vessels of immature pigs and in isolated arteries from days 17-20 of the cycle and significantly (P<0.05) in vessels from days 1-5 and 8-13 of the estrous cycle. Our results indicate that DbetaH- and NPY-IR nerve fibres are present in the pig ovarian arteries. NE and NPY administered alone increased blood pressure in the pig isolated ovarian artery and simultaneous administration of both substances caused each other potentiation of vasocontractile effect, however, the strength of observed changes was dependent on the stage of the estrous cycle.