Time of ovarian follicle selection during the porcine estrous cycle

Two experiments were conducted to: 1) determine the time during the procine estrous cycle when compensation in ovulation rate after unilateral ovariectomy (ULO) ceases to be complete, 2) compare the follicle selection process in gilts selected for high ovulation rate with unselected control gilts and 3) determine the number of follicles on the right ovary at various stages of the estrous cycle. Experiment I included 25 crossbred gilts, while Experiment II included 17 gilts selected for high ovulation rate and 16 unselected control gilts. The right ovary was removed via a mid-ventral laparotomy on either day 13, 15, 17 or 19 of the cycle. In Experiment I, compensation in ovulation rate ceased between days 13 and 15; whereas, in Experiment II, cessation occurred between days 15 and 17. Selected and control gilts responded alike to ULO, indicating similarity in the follicle selection process. Follicle numbers in the right ovary showed a general decline, especially between days 17 and 19, indicating that atresia was occurring during the follicular phase. The results indicate that the selection of ovarian follicles for ovulation at the ensuing estrus occurs before day 17 of the porcine estrous cyle.     

Effects of injection of hcg during the estrous cycle on follicle development and the inter-estrous interval

Pseudopregnancy in pigs can be induced by the administration of a single dose of hCG at Day 12 of the estrous cycle. However, the resulting length of pseudopregnancy can be extremely variable. In this study, it was investigated whether time of hCG administration (day of the cycle) and degree of follicle growth after hCG administration were related to the length of inter-estrous interval (pseudopregnancy). In the first experiment, groups of cyclic gilts were given 1500 IU hCG at either Day 11 (D 11; n=14) or Day 12 (D12; n=14) after onset of estrus, or not treated (Control; n=13). Follicle development was assessed daily using transcutaneous ultrasonography. Follicle size in the Control gilts remained relatively constant between Days 11 and 17, whereas in the treated gilts, follicle size increased (P < 0.001) within 4 days (D11) and 2 days (D12) after treatment. The inter-estrous interval was increased (P < 0.01) in the hCG-treated gilts (34.7+/-6.3 and 37.6+/-11.1 days in the D11 and D12 gilts, respectively), compared to Controls (22.3+/-5.2 d). About two-thirds of the treated gilts returned to estrus between Days 32 and 39 after onset of first estrus. No relationships were found between follicle development after treatment and length of the inter-estrous interval. In a second experiment, 16 cyclic gilts were treated with 1500 IU hCG at Day 12 and Day 15 of the estrous cycle. Follicle development was assessed at Days 12, 15 and 18. At Day 18, average follicle size was 8.4+/-2.0 mm. The inter-estrous interval was 39.7+/-5.4 days and 14 of 16 gilts returned to estrus between Days 34 and 44 after onset of first estrus. Again, no relationships were found between follicle development after treatment and the duration of the inter-estrous interval. We conclude that, based on the duration of the inter-estrous interval, administration of hCG during the luteal phase induced a short pseudopregnancy. However, the induction of accessory corpora lutea or follicular luteinization cannot be discounted.     

Involvement of apoptosis in the atresia of nonovulatory dominant follicle during the bovine estrous cycle

The present study was designed to 1) investigate whether apoptosis is responsible for the atresia of nonovulatory dominant follicle (DF), 2) to determine if atresia of a nonovulatory DF is associated with alterations in Bcl-2 and Bax expression, 3) to test whether progesterone P(4) has a direct effect on apoptosis in bovine follicles, and 4) to study the pattern of expression of Bcl-2 and Bax in follicles at different developmental stages (small, medium, and large). In experiment 1, 16 cycling cows received a norgestomet ear implant at proestrus (Day 1) for 9 days to mimic the subluteal phase. The cows were assigned either to a control (n = 4) or P(4)-treated groups (n = 12). Injections of P(4) (150 mg, i.m.) were given on Day 3 (n = 4); on Days 3 and 4 (n = 4), and on Days 3, 4, and 5 (n = 4) of the implant period. Controls received injections of corn oil on Days 3, 4, and 5. Unilateral ovariectomy was performed on Days 4, 5, and 6 to recover DFs from cows that had been treated with P(4) for 24, 48, and 72 h, respectively. DFs in the control group were collected on Day 6. The onset of atresia of DFs was assessed morphologically by ultrasound to determine DF diameters, histologically by light microscopic inspection of tissue sections, and functionally by quantification of follicular fluid steroid hormone levels. Apoptosis was detected by DNA analysis and in situ TUNEL labeling. Expression of Bcl-2 and Bax proteins was examined by Western blot analysis. The earliest signs of atresia were detected 24 h after P(4) injection as evidenced by decreased diameter, degeneration and detachment of granulosa cells (GCs) from the basal lamina, and a dramatically reduced ratio of estrogen to P(4). Electrophoretic analysis of DNA extracted from DFs of cows treated with P(4) for 24 h revealed a distinct ladder pattern of DNA fragments. In contrast, this pattern was not obvious in DFs from control cows. Similar results were also obtained from TUNEL analysis of DFs. Furthermore, both Bcl-2 and Bax were found to be present in all DFs; however, the ratio of Bcl-2 and Bax protein levels was significantly reduced by 24 h of P(4) treatment compared with DFs from the control group (P < 0.05). Experiment 2 investigated the direct effect of P(4) (4 ng/ml) on apoptosis of cultured GCs using ovaries obtained from a local slaughterhouse. In addition, the pattern of expressions of Bcl-2 and Bax in follicles at different developmental stages (small, medium, and large) was studied. No increase in apoptotic DNA fragments was detected in GCs treated with P(4). The ratio of Bcl-2 and Bax protein levels was variable in small follicles; however, Bax protein level was always relatively higher than that of Bcl-2 in medium and large follicles. In conclusion, our study suggests that apoptosis is the mechanism that underlies the atresia of nonovulatory DFs that develops during the luteal phase of bovine estrous cycle.     

Circulating concentrations of oxytocin during the estrous cycle and early pregnancy in sheep

Plasma concentrations of oxytocin and progesterone have been measured by radioimmunoassay in jugular venous blood obtained daily from 5 sheep during 2 estrous cycles and in early pregnancy.Concentrations of oxytocin were relatively high (15–30 pg/ml) during the luteal phase of the cycle, but fell at estrus (to 1–17 pg/ml). A fall in oxytocin was also observed on day 15 of pregnancy, when, as expected, progesterone levels remained high. It is suggested that raised basal levels of oxytocin are unlikely to cause the increasing uterine release of prostaglandin F_2α which occurs at the end of the estrous cycle.     

Ovulation synchronization following commercial application of ultrasound-guided follicle ablation during the estrous cycle in mares

A regimen of progesterone plus estradiol (P&E) was used as a standard for ovarian synchronization to test the efficacy and evaluate the commercial application of ultrasound-guided follicle ablation as a non-steroidal alternative for ovulation synchronization in mares. Recipient mares at a private embryo transfer facility were at unknown stages of the estrous cycle at the start of the experiment on Day 1 when they were randomly assigned to an ablation group (n=18-21 mares) or to a P&E group (n=20-21 mares). In the ablation group, mares were lightly sedated and all follicles > or = 10 mm were removed by transvaginal ultrasound-guided follicle aspiration. In the P&E group, a combination of progesterone (150 mg) plus estradiol (10mg) prepared in safflower oil was given daily (im) for 10 d. Two doses of prostaglandin F(2alpha) (PGF, 10mg/dose, im) were given 12 h apart on Day 5 in the ablation group, or a single dose on Day 10 in the P&E group. Human chorionic gonadotropin (hCG, 2500 IU/mare, im) was given at a fixed time, 6 and 10 d after PGF treatment in the ablation and P&E groups, respectively, with the expectation of a follicle > or = 30 mm at the time of treatment. In both the ablation and P&E groups, transrectal ultrasonography was done at the start of the study (Day 1) and again on the day of hCG treatment and daily thereafter to determine the presence of a CL, measure diameter of the largest follicle and detect ovulation. The mean interval from the start of the study and from PGF treatment to ovulation was shorter (P<0.0001) in the ablation group (13.7 and 9.7 d, respectively) compared to the P&E group (22.3 and 13.2 d, respectively). Following fixed-day treatment with hCG after PGF treatment, the degree of ovulation synchronization was not different (P>0.05) between the ablation and P&E groups within a 2-d (56 and 70%) or 4-d (83% and 90%) period. Although ultrasound-guided follicle ablation may not be practical in all circumstances, it excluded the conventional 10-d regimen of progesterone and estradiol and was considered an efficacious and feasible, non-steroidal alternative for ovulation synchronization in mares during the estrous cycle.     

Plasma follicle stimulating hormone and luteinizing hormone patterns during the estrous cycle of ewes

Fifteen Suffolk ewes were used in three experiments to compare plasma follicle stimulating hormone (FSH) and luteinizing hormone (LH) patterns during the estrous cycle and to determine whether FSH levels undergo changes in pulse frequency. Luteinizing hormone changed inversely with progesterone levels whereas FSH and progesterone concentrations revealed no obvious relationship. Unlike LH, FSH levels did not pulsate during the follicular phase. Higher FSH levels were detected on days 1, 6 and 12 and lower levels on days 0, 4 and 16. Coincident preovulatory LH and FSH surges were observed and this was the only time FSH and LH levels appeared to be jointly controlled.     

A simple mathematical model of the bovine estrous cycle: follicle development and endocrine interactions

Bovine fertility is the subject of extensive research in animal sciences, especially because fertility of dairy cows has declined during the last decades. The regulation of estrus is controlled by the complex interplay of various organs and hormones. Mathematical modeling of the bovine estrous cycle could help in understanding the dynamics of this complex biological system. In this paper we present a mechanistic mathematical model of the bovine estrous cycle that includes the processes of follicle and corpus luteum development and the key hormones that interact to control these processes. The model generates successive estrous cycles of 21 days, with three waves of follicle growth per cycle. The model contains 12 differential equations and 54 parameters. Focus in this paper is on development of the model, but also some simulation results are presented, showing that a set of equations and parameters is obtained that describes the system consistent with empirical knowledge. Even though the majority of the mechanisms that are included in the model are based on relations that in the literature have only been described qualitatively (i.e. stimulation and inhibition), the output of the model is surprisingly well in line with empirical data. This model of the bovine estrous cycle could be used as a basis for more elaborate models with the ability to study effects of external manipulations and genetic differences.     

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.     

Follicular development during early pregnancy and the estrous cycle of the sow

The objective of this study was to monitor and compare follicle populations and follicular development in pregnant and nonpregnant sows from Day 3 to Day 20 after breeding. Twenty-four sows were paired within parity on the day of artificial insemination and were randomly allocated within pair for insemination with either killed (n=12) or live spermatozoa (n=12). All the sows were artificially inseminated with the pooled ejaculate of the same boar. From Day 3 through Day 20 post estrus, ovarian follicles were scanned daily by ultrasonography. Ultrasound images were recorded on videotape and were retrospectively analyzed. Follicles were mapped to indentify the existence of follicular waves. The follicles were then classified as small (< 3 mm), medium (3-5 mm), or large (>/=5 mm). Pregnancy diagnosis was performed on Day 21 by ultrasonography. Pregnant sows maintained a constant proportion of the follicle population in the small, medium and large follicle categories. However, in the nonpregnant sows, the proportion of follicles in the various size categories remained constant until Day 15. Thereafter, the proportion of small follicles decreased (P < 0.05) from Day 15 to 20, and the proportions of medium and large follicles increased (P < 0.05). The predictability of pregnancy status on Day 20 based on follicle populations in any of the 3 follicle categories was low. Moreover, there was no evidence of follicular waves during the estrous cycle or early pregnancy. In conclusion, the proportion of small follicles decreased while medium and large follicle increased from Day 15 through Day 20 of the estrous cycle, but not during a similar stage of pregnancy. This latter finding concurs with follicle recruitment from the pool of small follicles for ovulation following PGF2alpha secretion to induce luteolysis, which reduces progesterone concentrations and thereby allows for the stimulation of the pool of small follicles by gonadotropins.     

Preantral follicle development during the menstrual cycle in the Macaca mulatta ovary

Ovaries obtained from 18 adult, regularly cycling rhesus monkeys were evaluated to determine the status of preantral follicle development at various stages throughout the menstrual cycle. The ovaries were serially sectioned, and all preantral follicles on every 20th section were classified as developing or atretic, counted and/or measured, and grouped according to size. Results from this study revealed (1) that a significant increase (P less than 0.05) occurred in the mean percentage of developing preantral follicles greater than 100 microns in diameter during the periovulatory period, suggesting that follicles in this size range had developed a sensitivity to the unique hormonal milieu present during that stage of the cycle; (2) that similar numbers of primordial and developing preantral follicles were present in the right and left ovaries of a pair, showing that neither ovary had a predominance over the other; (3) that the mean number of developing preantral follicles varied directly with the size of the primordial follicle pool; (4) that atresia was minimal with no significant differences between the various stages of the cycle in any size group; and (5) that polyovular follicles were abundant in certain pairs of ovaries, but could not be related to age or stage of cycle.     

Pattern of follicular development in high fecundity Olkuska ewes during the estrous cycle

Folliculogenesis was studied daily in the 18 oestrous cycles in six prolific Olkuska ewes from October to December using transrectal ultrasonography to record the number and size of all ovarian follicles > or =2 mm in diameter. Blood samples were taken once a day and were analyzed for concentrations of FSH, LH, estradiol and progesterone. Follicular and hormonal data were analyzed for associations between different stages of development of the follicular waves and concentrations of FSH and estradiol. The first wave during which at least one follicle reached maximum diameter of > or =4 mm after ovulation, was defined as a wave 1, and the following waves were numbered sequentially. Waves 1, 2, 3, 4 and the ovulatory one emerged on days: -2 to 4, 4 to 8, 6 to 11, 10 to 12 and 11 to 15, respectively. The mean number of follicles per wave that reached diameter of > or =4 mm was 4.15 +/- 1.1 and 16.62 +/- 8.6 follicles per estrous cycle of a total 299 follicles were observed. Significantly more follicles (p> or =0.05) emerged on days 2, 8 and 13 than in other days. Serum FSH concentrations fluctuated from 0.11 ngml(-1) on day 2 to preovulatory maximum 1.81 ngml(-1) on day 17 of the estrous cycle. The emergence of follicular waves was associated with elevations of FSH concentrations in blood serum. The mean increase in FSH concentration was followed by the recruitment of follicles of the next wave. The mean daily FSH concentration and the mean number of follicles emerging each day were negatively correlated. The length of the interwave interval (4.4 +/- 1.6 days) did not differ significantly from the interval between pulses of FSH (4.8 +/- 0.3 days). The mean serum estradiol concentrations showed fluctuations until day 14 and then gradually increased from 5.47 +/- 0.3 pgml(-1) to reach a peak 13.14 +/- 0.2 pgml(-1) on the day before ovulation. To summarize, the growth of ovarian follicles during the estrous cycle in high fecundity Olkuska sheep exhibited a distinct wave-like pattern. Ovarian follicles emerged from the pool of 2 mm follicles. The preovulatory follicles originated from the large follicle population were present in the ovary at the time of luteal regression. The initial stages of the growth of the largest follicles appears to be controlled primarily by increases in FSH secretion.     

Expression profile analysis of microRNAs during hair follicle development in the sheep foetus

MicroRNAs (miRNAs) regulate the development and growth cycle of hair follicles (HFs). The molecular mechanism by which miRNAs determine the development of HFs in the sheep foetus remains elusive. In this study, the expression profiles of miRNAs at 11 development periods (45, 55, 65, 75, 85, 95, 105, 115, 125, 135 and 145 d) in sheep foetus skin were analysed by high-throughput sequencing and bioinformatics analysis. A total of 72 conserved miRNAs, 44 novel miRNAs and 32 known miRNAs were significantly differentially expressed. qRT-PCR results for 18 miRNAs were consistent with the sequencing data. 85 d of foetal development was the starting point for secondary hair follicle (SF) development according to tissue morphology and cluster analysis. In SF development, the prolactin signalling pathway and platelet activation played important roles, and 10 miRNAs were potential candidate miRNAs in SF initiation.     

Divergent changes in the concentrations of gonadotropin beta-subunit messenger ribonucleic acid during the estrous cycle of sheep

Cyclic changes in the production of the pituitary gonadotrophic hormones, LH and FSH are essential events in the maintenance of the reproductive system of female mammals. While studies have examined changes in the secretion of LH and FSH during the estrous cycle and demonstrated the importance of these hormones in regulation of ovarian development and gametogenesis, considerably less is known concerning the regulation of the biosynthesis of these hormones. Although initial studies have examined changes in LH subunit mRNA concentrations during the rat and ovine estrous cycles, no information concerning the physiological regulation of FSH beta mRNA concentrations has been available. In the present study we have examined the relationship between pituitary concentrations of LH and FSH subunit mRNAs and the serum concentrations of these gonadotropins. The results demonstrate a very different pattern of change for FSH beta subunit mRNA than that observed for alpha and LH beta subunit mRNAs. In fact, FSH beta mRNA concentration decline substantially during the preovulatory period, reaching minimal values at a time when alpha and LH beta mRNA levels are near maximal. Furthermore, this decline in FSH beta mRNA amounts occurs when serum FSH concentrations are maximal. Thus, FSH beta mRNA concentrations follow a very different pattern than that of serum FSH. In contrast, LH beta mRNA and serum LH concentrations tend to increase at the same time. These findings provide evidence that concentrations of LH beta and FSH beta mRNAs are likely regulated by different mechanisms.     

Effect of season on estrous cycle of Yankasa sheep

An investigation was conducted to establish the effects of harmattan and hot-dry season on estrous cycle length, onset, and duration of estrus in Yankasa sheep indigenous to the Nigerian guinea savanna zone. Mean cycle lengths were 16.8 +/- 0.58 and 16.4 +/- 0.53 days during harmattan and hot-dry seasons, respectively; short cycles, 5-13 days, and long cycles, 21 to 30 days, were observed during both seasons. During the harmattan season, 57.1% of estrus began at night while 70% started at night during the hot-dry season. The duration of normal estrus observed during the harmattan, 33.6 +/- 5.87h, significantly decreased (P0.05) during the hot-dry season (24.0 +/- 5.45h). It is suggested that twice daily observation at 12-hour intervals will suffice to detect estrus in this breed of sheep.     

Pituitary alpha subunit mRNA amounts during the sheep estrous cycle. Assessment by cDNA hybridizations

Pituitary glycoprotein hormones exhibit a dimeric structure consisting of a common alpha subunit and similar beta subunits. In this study, alpha subunit mRNA amounts have been examined in sheep pituitaries during defined times of the normal estrous cycle. These times were designed to include events prior to, and including the beginning of, the preovulatory luteinizing hormone surge. Criteria such as serum and pituitary luteinizing hormone, serum progesterone, and ovarian morphology were used to classify the groups as: 1) Day 12 of the cycle; 2) 24 h before behavioral estrus (E-24); and 3) 5 h after estrus (E + 5). RNA was extracted from the pituitaries and amounts of alpha subunit mRNA quantitated using cell-free translations and cDNA hybridizations. Both Northern transfers and RNA dot blots were used. The amount of alpha subunit mRNA in the Day 12 group was the lowest of the three groups and was similar to that seen in the pituitary from an anestrous ewe. The amount observed in the E-24 animals was only slightly increased over the Day 12 (approximately 2-fold); however, a greater increase was observed when the E + 5 group was examined (approximately 4-8-fold). These results suggest that the amount of alpha subunit mRNA increases during the time of the preovulatory luteinizing hormone surge in the normal estrous cycle of the sheep and thus probably plays a role in the important physiological event.     

Midcycle and luteal elevations of follicle stimulating hormone in squirrel monkeys (Saimiri boliviensis) during the estrous cycle

Follicle stimulating hormone (FSH) has fundamental importance in reproductive function, but its cyclic pattern has not previously been described in the squirrel monkey, due primarily to the lack of a suitable assay. An homologous radioimmunoassay (RIA) based on recombinant cynomolgus FSH measured changes in serum FSH relative to patterns of bioactive luteinizing hormone (LH), estradiol, and progesterone during the estrous cycle. FSH was observed to have a sharp peak during the late follicular phase coincident with the LH surge and then rose again during the luteal phase. Estradiol was low except for the midcycle rise, suggesting an inhibitory relationship. The rat granulosa cell in vitro FSH bioassay confirmed high levels of this hormone. Measurement of FSH in the squirrel monkey has found a pattern different from Old World primates in the luteal phase, which may provide insight into the reproductive mechanisms of this species.     

Apoptosis in the canine endometrium during the estrous cycle

Apoptotic cell death in the endometria of 58 female dogs in different stages of the estrous cycle was assessed (in formalin-fixed, paraffin-embedded sections) with both the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and immunohistochemical detection of caspase-3 activity. For both techniques, the apoptotic index was determined in the surface epithelium, stroma, crypts, and basal glands by counting the percentage of stained cells in a total of 500 cells in each category. In the surface epithelium and stroma, TUNEL- and caspase-3-positive cells were rare (apoptotic index<1) throughout the estrous cycle. However, caspase-3 detection showed a significant increase in the apoptotic index in the stroma during anestrus as well as an increase in the index in both the stroma and surface epithelium in late metestrus. The apoptotic index increased during late metestrus and anestrus in the crypts and basal glands; in the crypts, this increase was significant only when caspase-3 detection was used, whereas in basal glands, significant differences were found for both techniques. In conclusion, apoptosis was present in canine endometrial cells during the estrous cycle, but caspase-3 detection showed more significant differences than the TUNEL assay. Furthermore, a high apoptotic index (suggestive of endometrial desquamation) was not detected in the surface epithelium and there was no significant correlation between the apoptotic index in any cell group and serum progesterone concentrations.