The use of steroid hormones in superovulation of Nelore donors at different stages of estrous cycle

The objective of the present study was to evaluate the superovulatory response and ova/embryo recovery from Nelore donors following treatment with a controlled internal drug releasing device and estradiol benzoate (CIDR-B program) at different stages of the estrous cycle. The control group (TI; n=40) received a standard superovulation protocol with females of this group being between days 9 and 12 of the estrous cycle (estrus = day 0). The donors that received a CIDR-B program containing 1.9 g progesterone and an intramuscular injection of estradiol benzoate (2 mg) were at day 0 (TII; n=30), between days 2 and 6 (TIII; n=30), days 7 and 12 (TIV; n=30), days 13 and 16 (TV; n=30) and days 17 and 20 (TVI; n=30) of the estrous cycle. Superovulation was induced with 400 IU of p-FSH, divided into eight decreasing doses (80/80; 60/60; 40/40; 20/20) at intervals of 12h. The donors received PGF2alpha (Cloprostenol) 48 h after beginning the treatment and CIDRs were removed 12h later. Artificial inseminations (AI) were performed 12 and 22 h after the initiation of estrus and embryos were collected 7 days after AI. The mean numbers (+/-S.E.M.) of total ova and embryos, viable (transferable) and degenerated embryos were 14.2+/-11.3, 7.4+/-6.9 and 3.2+/-3.5 (TI), 13.3+/-10.4, 7.1+/-6.2 and 3.3+/-4.3 (TII), 13.5+/-7.0, 8.1+/-6.7 and 2.3+/-3.0 (TIII), 17.4+/-9.9, 9.4+/-6.9 and 4.0+/-4.4 (TIV), 16.9+/-8.8, 9.8+/-8.1 and 2.7+/-2.5 (TV) and 13.0+/-7.8, 7.2+/-6.9 and 2.3+/-2.5 (TVI), with no significant differences (P>/=0.05) among groups. Pregnancy rates of 67.1% (TI; n=86/128), 60.8% (TII; n=59/97), 62.5% (TIII; n=73/115), 64.1% (TIV; n=84/131), 72.3% (TV; n=81/112) and 60.6% (TVI; n=63/104) were obtained with embryos transferred from these collections and did not differ significantly (P>/=0.05) among groups. The results of the present study allow us to conclude that a combination of steroid hormones may be used prior to superovulation in Nelore donors, at any stage of the estrous cycle without affecting the efficiency of embryo transfer programs.     

Changes in the pituitary and hypothalamic content of methionine-enkephalin during the estrous cycle of rats

Met-Enkephalin content of the anterior hypothalamic-preoptic area, medial basal hypothalamus, anterior pituitary, intermediate and posterior pituitary was measured using a specific radioimmunoassay. Met-Enkephalin content of the anterior hypothalamic-preoptic area, medial basal hypothalamus and anterior pituitary was very high on the morning of proestrus but decreased on the afternoon of proestrus and on estrus. The content of met-Enkephalin was more variable in the anterior pituitary than in the anterior hypothalamic-preoptic area and medial basal hypothalamus during the estrous cycle. The results suggest that the met-Enkephalin may be involved in regulating the hypothalamo-hypophyseal function during estrous cycle in the rat.     

Superovulatory and endocrine responses in heifers treated with FSH-P at different stages of the estrous cycle

Forty-two Holstein heifers were superovulated with FSH-P (total dose, 30 mg) and cloprostenol. Treatment was initiated on Day 3 (Group D3, n = 11), Day 6 (Group D6, n = 11), Day 9 (Group D9, n = 10) or Day 12 (Group D12, n = 10) of the estrous cycle. Heifers were bled daily for serum progesterone and estradiol-17beta determinations and every 6 h for a 48-h duration at the expected time of estrus for luteinizing hormone (LH) assay. Ova and embryos were flushed from the reproductive tracts and the number of corpora lutea (CL) were recorded after slaughter on Day 7 post-estrus. Mean (+/- SEM) numbers of observed CL were higher (P < 0.05) in Group D9 (33.3 +/- 4.8) than in Group D3 (15.3 +/- 3.8), with Group D6 (17.0 +/- 2.9) and Group D12 (23.9 +/- 7.3) being intermediate. Similarly, mean (+/- SEM) numbers of fertilized embryos were highest (P < 0.05) in Group D9 (13.3 +/- 2.2). There was also a nonsignificant trend for the number of transferable embryos to be greatest in Group D9. Neither serum progesterone concentrations 3 d after the LH peak nor peak serum estradiol 17beta concentrations differed among groups, but both were significantly correlated with numbers of observed CL and total ova and embryos.     

Sperm migration into and through the oviduct following artificial insemination at different stages of the estrous cycle in the rat

In order to examine whether sperm migration into and through the oviduct follows an invariable pattern or is subject to regulation, rats in proestrus, estrus, metestrus, or diestrus were inseminated in the upper third of each uterine horn with 10-20 million epididymal spermatozoa. Three or eight hours later, the numbers of spermatozoa free and adhering to the epithelium in the ampullary and isthmic segments were determined. A significantly higher number of spermatozoa were recovered in estrus than in other stages, at 3 h than at 8 h, and at all stages from the isthmus than from the ampulla. Spermatozoa adhering to the epithelium were observed only in proestrus and estrus and in the isthmus. The effect of exogenous estradiol-17beta (E2) and progesterone (P4) on sperm migration was investigated in rats in which the estrous cycle was inhibited pharmacologically. E2 facilitated sperm migration into the oviduct and P4 antagonized this effect, whereas P4 alone had no effect. Concomitant treatment with E2+P4 induced adhesion of spermatozoa to the oviductal epithelium. In conclusion, the pattern of sperm migration into and through the rat oviduct varies with the stage of the cycle, being dependent on E2 and P4. The adhesion of spermatozoa to the rat oviductal epithelium is stage- and segment-specific and requires the combined action of both hormones.     

Ovarian superstimulation after follicular wave synchronization with GnRH at two different stages of the estrous cycle in cattle

The objective of this study was to evaluate superovulatory programs based on synchronization of follicular waves with GnRH at 2 different stages of the estrous cycle. Sixteen Holstein cows were randomly assigned to 1 of 3 groups and administered GnRH (Cystorelin, 4 ml i.m.) between Days 4 and 7 (Groups 1 and 3) or between Days 15 and 18 (Group 2) of the estrous cycle (estrus = Day 0). Four days after GnRH treatment, > or = 7-mm follicles were punctured in Groups 1 (n = 6) and 2 (n = 6) or were left intact in Group 3 (n = 4). All cows were superstimulated 2 d later (i.e., from Days 6 to 10 after GnRH treatment) with a total of 400 mg NIH-FSH (Folltropin-V) given twice daily in decreasing doses. The GnRH treatment caused a rapid disappearance of large follicles (P < 0.005), rapid decrease in estradiol concentrations (P < 0.003), and increase in the number of recruitable follicles (4 to 6 mm; P < 0.04), indicative of the emergence of a new follicular wave within 3 to 4 d of treatment. Between 4 and 6 d after GnRH treatment, the mean number of 4- to 6-mm follicles decreased (4.7 +/- 1.8 to 1.5 +/- 3.3) in the nonpunctured group but increased (3.9 +/- 1.0 to 7.3 +/- 1.9) in the punctured group of cows (P < 0.05). In response to FSH treatment, the increase in the number of > or = 7-mm follicles was delayed by approximately 2 d in the nonpunctured group (P < 0.006). Moreover, the mean number of > or = 7-mm follicles at estrus was higher (16.9 +/- 1.7 vs 11.5 +/- 3.0; P < 0.1) in the punctured than the nonpunctured group. The increase in progesterone concentration after estrus was delayed in the nonpunctured group (P < 0.1) compared with the punctured follicles. Mean numbers of CL as well as freezable (Grade 1 and 2) and transferable (Grade 1, 2 and 3) embryos were similar (P > 0.1) in punctured and nonpunctured groups. Spontaneous estrus did not occur prior to cloprostenol-induced luteolysis in any group, and stage of the estrous cycle during which GnRH was given did not affect (P > 0.1) hormonal and follicular responses in the punctured groups. In conclusion, GnRH given at different stages of the estrous cycle promotes the emergence of a follicular wave at a predictable time. Puncture of the newly formed dominant follicle increases the number of recruitable follicles (4 to 6 mm) 2 d later and, in response to superstimulation with FSH, causes a greater number and faster entry of recruitable follicles into larger classes (> or = 7 mm) and a faster postovulatory increase in progesterone concentrations.     

The day of the estrous cycle that FSH is started and superovulation in cattle

A total of 993 commercial donor cows were superovulated with 28 mg FSH-P. The first injection of FSH-P was administered on one of days 9 through 13 of the donor's estrous cycle. The day that FSH was started did not affect total embryos collected, the number of transferable embryos or the percent transferable. These results did not support the current hypothesis that because the population of antral and preantral follicles in the ovaries on days 9 and 10 is higher, best embryo production should be achieved by starting donor cows on day 9 or 10 of the estrous cycle.     

Oxytocin secretion induced by osmotic stimulation in rats during the estrous cycle and after ovariectomy and hormone replacement therapy

Hyperosmolality is a potent stimulus for the secretion of oxytocin. Oxytocinergic neurons are modulated by estrogen and oxytocin secretion in rats varies according to the phase of the estrous cycle, with higher activity during proestrus. We investigated the oxytocin secretion induced by an osmotic stimulus (0.5 M NaCl) in female rats. Plasma oxytocin and the oxytocin contents in the neurohypophysis and the paraventricular and supraoptic nuclei were determined during the morning (8-9 h) and afternoon (17-18 h) of the estrous cycle and after ovariectomy followed or not by hormone replacement. Plasma oxytocin peaked in control animals during proestrus. Oxytocin content decreased in the paraventricular and supraoptic nuclei during proestrus and estrus compared to diestrus and increased in the neurohypophysis during proestrus morning. No significant difference was observed in the oxytocin content of the neurohypophysis, nuclei or plasma between ovariectomized animals and ovariectomized animals treated with estrogen or estrogen plus progesterone. Therefore, any ovarian factor other than estrogen or progesterone seems to play a direct or indirect role in the increase in oxytocin secretion. The osmotic stimulus caused an increase in plasma oxytocin throughout the estrous cycle. A reduction in oxytocin content during diestrus and an increase during proestrus were observed in the paraventricular nuclei. In ovariectomized animals, the treatment with estrogen potentiated the response of oxytocin to the osmotic stimulus, with the response being even stronger in the case of estrogen plus progesterone. In conclusion, the ovarian steroids estrogen plus progesterone could modulate the osmoreceptor mechanisms related to oxytocin secretion.     

Uterine epithelial and eosinophil estrogen receptors in rats during the estrous cycle

Summary In the uterus of the adult female rats, the luminal epithelial cells and the eosinophil leukocytes are rich in cytoplasmic estrogen receptors. During the estrous cycle, the epithelial estrogen receptor concentration reaches its peak level, in proestrus, drops precipitously in estrus, and hits the trough, at metestrus. Repopulation of the cytoplasm with estrogen binding sites occurs during diestrus. This pattern of cyclic change is indicative of a rapid turnover of estrogen receptors in the epithelial cells and its regulation by endogenous estrogens. The concentration of estrogen receptors in the cytoplasm of the eosinophils does not appear to fluctuate during the cycle. But the intrauterine, distribution of these leukocytes is clearly cyclic in pattern, ostensibly influenced by estrogens. While progesterone binding activity is consistently demonstrated in tandem with estrogen receptors in the cytoplasm of the epithelial cells, it has not been observed in the eosinophil leukocytes. These findings support the claim that there are two estrogen receptor systems in the rat uterus, one mediating the intracellular events of the genomic response to estrogens, and the other being concerned with non-genomic responses.Abbreviations BSA Bovine serum albumin - FITC fluorescein isothiocyanate - TMRITC tetramethylrhodamine isothiocyanate - CMO O-carboxymethyl oxime     

Changes of progesterone level in the plasma of sows during the estrous cycle

Changes of progesterone level in the plasma of sows during the estrous cycle. Acta Physiol. Pol., 1978, 29 (2): 177--183. The investigations were carried out on 12 sows of the Polish Large White and Polish White Longeared breeds. Blood samples were taken daily from auricular veins between 8 and 11 a.m. The progesterone level was determined in the blood plasma by the competitive protein--binding technique. The results obtained were within the limits of below 1 to 25 ng/ml plasma. During ovulation the plasma progesterone level was lowest. It increased markedly beginning with the 4th to 6th day of the estrous cycle, reaching its peak on the 14th and 15th day and then fell significantly to a minimum value in the estrous period. Individual differences in the plasma progesterone level between the sows were also noted.     

Pattern of follicular development during the estrous cycle of aged rats

Summary The pattern of follicular development during the estrous cycles of aged rats was examined and compared with that of mature rats. In both, preovulatory follicles are derived from a select group of small pre-Graafian follicles which begin to develop at estrus and reach the preovulatory size by the morning of proestrus, but the rate of growth, as judged by an increase in the percentage of granulosa cells incorporating ³H-thymidine, is accelerated in the follicles of aged rats. A second mechanism, which accounts for preovulatory follicles in aged rats, involves the rescue from atresia of pre-Graafian and preovulatory follicles. The existence of this mechanism is supported by the observation that at metestrus in aged rats virtually all follicles, regardless of their state of atresia, possess a high percentage of granulosa cells incorporating ³H-thymidine, indicating that the follicles are growing rapidly. However, some of these rapidly growing follicles show signs of atresia such as pyknotic nuclei within their granulosa cell layers. Since follicles in the initial stage of atresia contain defective oocytes (Peluso et al. 1979b), their rescue and development into preovulatory follicles would result in the ovulation of defective oocytes, a fact which accounts in part of the lower fertility in these older animals.     

The antiepileptic effect of sodium valproate during different phases of the estrous cycle in PTZ-induced seizures in rats

Catamenial epilepsy is a form of epilepsy which is related to the menstrual cycle. Cyclic variation in the levels of ovarian hormones plays a pivotal role in its pathogenesis. Sodium valproate (VPA) is one of the oldest antiepileptic drugs (AEDs) which inhibits hepatic metabolizing enzymes. The aim of this study was to evaluate the antiepileptic effects of VPA during different phases of the estrous cycle in rats. 72 adult female Wistar rats in three groups (control, 75 and 100 mg/kg VPA), each with four subgroups (proestrous, estrous, metestrous and diestrous) were used (n = 6). Initially, puberty was assessed using vaginal smears and rats with two regular cycles were selected. VPA with doses 75 and 100 mg/kg was administered intraperitoneally (i.p) in the treatment groups followed by i.p. injection of 80 mg/kg pentylentetrazol (PTZ) in the treatment and control groups. After induction of seizure by PTZ, initiation time of myoclonic seizures (ITMS), initiation time of tonic–clonic seizures (ITTS), seizures duration (SD) and mortality rate (MR) were recorded for 30 min. Data were presented as mean±SD, one-way ANOVA followed by Tukey–Kramer multiple comparison post hoc test were used for analysis of data (P < 0.05). The results of this study showed that VPA significantly improved antiepileptic parameters including ITMS, ITTS, SD, and MR, in which they were significantly more prominent during the luteal phase than the follicular phase (P < 0.05). In addition, there was no significant difference neither between proestrous and estrous nor between metestrous and diestrous in each separately group of rats (P > 0.05).     

Changes in the distribution of sizes of ovine luteal cells during the estrous cycle

The ovine corpus luteum is composed of two types of steroidogenic cells, which are referred to as small and large luteal cells. In this study, the size and number of steroidogenic cells were determined in corpora lutea collected on Days 4, 8, 12, and 16 of the estrous cycle. Corpora lutea were dissociated into single-cell suspensions that were stained for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, a marker for steroidogenic cells. The size of 3 beta-HSD-positive cells was measured with a Zeiss Videoplan Image Analyzer. On Day 4, most of the 3 beta-HSD-positive cells were less than 18 microns in diameter, the median being 11.2 microns. By Day 8, the number of 3 beta-HSD-positive cells increased 3-fold, and the median diameter increased to 12.8 microns. Although the number of 3 beta-HSD-positive cells was reduced by approximately 50% on Day 16, the median size on Days 12 and 16 was 14.6 and 16.8 microns, respectively. The ratio of large (greater than 18 microns) to small (less than 18 microns) luteal cells was 0.11 +/- 0.03 on Day 4; the ratio increased linearly to 0.67 +/- 0.09 by Day 16. This increase between Days 4 and 12 was attributable to an overall increase in the size of the cells; the increase between Days 12 and 16, however, was due to a loss of small luteal cells. When the experiment was conducted near the end of the breeding season, before animals became anestrous, the median size of the luteal cells did not change at different times of the estrous cycle but remained constant throughout. These data suggest that development of the corpus luteum is associated with an increase in the size and number of steroidogenic luteal cells, and that luteolysis is associated with a preferential loss of small luteal cells.