Patterns of follicular growth during the four-day estrous cycle of the rat

Female Sprague-Dawley rats underwent laporatomy during metestrus at 70 to 75 days of age or remained untreated to study the effects of surgical stress on follicular growth. Groups of rats were killed on each day of a 4-day estrous cycle, serial sections of the ovaries were prepared histologically and the number and size of follicles with one or more complete layers of cuboidal granulosa cells were determined. Since no differences due to surgery were found, the data were pooled by day of the estrous cycle (17 or 18 rats/day of cycle) for characterization and comparison of size distribution of follicles on different days of the estrous cycle. Follicles were classified as atretic or healthy and divided into groups by increments of 20 micron of diameter for graphing. Data were analyzed by analysis of variance and least squares means. Significant differences were found in the distribution of both healthy and atretic follicles among days of the estrous cycle. At least 21 follicles/ovary were recruited from less than 260 micron into greater than 260 micron in diameter between proestrus and estrus, and the follicles for ovulation were selected by diestrus. A greater number of growing follicles of 70 to 100 micron in diameter were present at diestrus. From the disappearance of follicles greater than 260 micron between estrus and proestrus, it appears that atresia is a very rapid process.     

Quantitative determination of follicle size distribution in the guinea pig ovary after hemicastration and PMSG treatment

In order to investigate the action point of intraphysiological or supraphysiological elevation of FSH during the preovulatory period on follicular development, adult guinea pigs underwent unilateral ovariectomy on days 10, 12 and 14 of the estrous cycle (N = 6 each group). Thereafter, guinea pigs were injected twice daily with either vehicle or pregnant mare's serum gonadotropin (PMS). After 2 days, the remaining ovaries were removed. The resected ovaries were fixed, embedded in paraffin, serially sectioned (7 microns) and stained with Azan. All follicles greater than 70 microns were classified by size and atretic stage. The follicular size distribution was not affected by hemicastration at day 10, although the ratio of atretic follicles (greater than 400 microns) decreased from 51% to 32% (P less than 0.01). Hemicastration at day 12 increased the largest nonatretic population (70-99 microns group) from 17% to 26%, and the ratio of atretic follicles (greater than 400 microns) decreased from 35% to 23%. The peak size distribution of follicles was shifted from 70-99 microns to 200-299 microns by PMS, and follicles 600-899 microns in size contained an increased percentage of atresia, which resulted in the bimodal distribution of viable follicles greater than 400 microns. These data suggest that 2 day hemicastration promotes an influx of primordial follicles into growing follicles and suppresses the atretic process by a different mechanism depending on the date of hemicastration in the estrous cycle. Conversely, hemicastration + PMS accelerated viable follicle growth to increase the percentage of atresia.     

Age-related alterations in follicular development and hormonal profiles in rats with 4-day estrous cycles

Two experiments were conducted to examine the hypothesis that an alteration in follicular development is associated with advancing maternal age in the absence of prolonged estrous cycles. In Experiment 1, serum and four follicles (from one ovary per rat) were collected from young and middle-aged, 4-day cycling rats on estrus or metestrus. Number and diameter of nonatretic antral follicles greater than 200 microns in diameter were determined from serial sections of the other ovary from each rat. In Experiment 2, serum and follicles (12 +/- 2) from both ovaries were collected from young and middle-aged rats on each day of a 4-day estrous cycle. All microdissected follicles were assayed for estradiol-17 beta (E2) and all sera were assayed for E2, follicle-stimulating hormone, and luteinizing hormone by radioimmunoassay. Numbers of follicles greater than 400 microns in diameter did not differ, while numbers of follicles 200-400 microns in diameter were reduced in middle-aged rats compared to young rats (Experiment 1). The mean diameter of follicles greater than 400 microns in diameter and the follicular content of E2 was greater in middle-aged than in young rats. In Experiment 2, a greater proportion of large follicles were observed in middle-aged rats than in young rats on all days, and a greater proportion of follicles with high concentrations of E2 were observed on diestrus. We interpreted these data as indicative of an early age-related change in the control of follicular recruitment, growth, and maturation.     

Development during single IVP of bovine oocytes from dissected follicles: Interactive effects of estrous cycle stage,follicle size and atresia

Previous work suggests that a number of factors such as follicle size, day of estrous cycle, and level of atresia influence the developmental potential of bovine oocytes in vitro. To understand better the interactions of these factors, 1299 follicles ≥3 mm in diameter were dissected from ovaries of synchronized dairy cows on four days (d2, d7, d10, or d15) during the estrous cycle. The oocyte from each follicle was collected and matured, fertilized, and cultured singly to d8 (d0 of culture = IVF). Control follicles (302) were similarly dissected and processed from an ovary pair randomly collected from the abattoir on each slaughter day. Results showed that development to blastocyst was greater in oocytes collected during phases of follicular growth (d2 and d10) than those collected during phases of follicular dominance (d7 and d15; 44.8% vs. 36.0%, respectively: P < 0.001) over all follicle size categories (3–5 mm, 6–8 mm, 9–12 mm and ≥13 mm). Oocyte competence tended to increase with increasing follicle size (P < 0.1). Follicular cells from follicles containing an oocyte that developed to morula or greater by d8 (484 samples) were analyzed by flow cytometry to measure the level of apoptosis. Results showed an increase in mean percent apoptotic cells in subordinate follicles (18.65 ± 0.86 over all size categories), particularly those of medium size (25.55 ± 2.2 for 6–8 mm size follicles; P < 0.001), during the dominance phase compared to growth phase (9.25 ± 0.95 over all sizes; P < 0.05). These results show a significant affect of the stage of estrous cycle on both oocyte competence and levels of follicular atresia. Mol. Reprod. Dev. 53:451–458, 1999. © 1999 Wiley‐Liss, Inc.     

A light- and electron-microscope study of the oocyte nucleus during development of the antral follicle in the prepubertal mouse.

The ordered changes which occur in the structural organization of the mouse oocyte nucelus during the preparatory, the maturative and the preovulatory stages of antral follicle development, have been studied under both light and electron microscopy. All observations have been made on those antral follicles whose development is initiated on postnatal day 14 and completed by postnatal day 28 in prepubertal animals of the ICR albino mouse strain. The formed entities that can be recognized within the oocyte nucleus during that period are the condensing bivalents, the heterochromatic knobs, the nucleolus and the extranucleolar bodies. At the onset of antral follicle development, the highly unravelled dictyate bivalents are seen to take on a lampbrush-type configuration. Subsequent condensation of these lampbursh bivalents appears to be a very gradual and lengthy process that extends over almost the entire period of antral follicle development. The shortening and thickening of the lampbrush bivalents are best interpreted as resulting from the withdrawal of their lateral loop-like projections into the chromosome axes and from the focal aggregation of these axes into compact chromatin masses. Electron-opaque granules, which appear within the oocyte nucleus during the preparatory and maturative follicle stages, are seen to be intimately associated with these condensing bivalents. A number of Feulgen-positive heterochromatic knobs make their appearance in contact with certain bivalents during the preparatory follicle stage. These knobs are not reincorporated as such into the condensing chromatin masses and undergo disintegration and dissolution during the preovulatory follicle stage. The size, shape and ultrastructural features of the nucleolus remain unchanged thoughout the period of antral follicle development. Breakdown and dissolution of the nucleolar mass is a swift process that takes place only in the fully mature preovulatory follicle and more or less concomitantly with the dismantling of the nuclear envelope. The extranucleolar bodies increase noticeably in size during the preparatory and the maturative follicle stages; they shrink in size and undergo dissolution during the preovulatory stage of antral follicle development. An attempt is made to interpret these morphological changes in the light of current knowledge concerning the architectural and functional organization of the oocyte nucleus in general during meiotic prophase. The relevant observational evidence would be consistent with the view that, during antral follicle deveopment, the mouse oocyte nucleus is not, as too often assumed, in a period of arrested evolution; its formed components undergo structural, maturational and functional changes which are of significance not only for the resumption of the first meiotic prophase but also for the early development of the embryo.     

Functional anatomy of the female genital organs of the wild black agouti (Dasyprocta fuliginosa) female in the Peruvian Amazon

This study examined anatomical and histological characteristics of genital organs of 38 black agouti females in the wild in different reproductive stages, collected by rural hunters in the North-eastern Peruvian Amazon. Females in the follicular phase of the estrous cycle had greater antral follicle sizes than other females, the largest antral follicle measuring 2.34mm. Antral follicles in pregnant females and females in luteal phase of the estrous cycle had an average maximum diameter smaller than 1mm. In black agouti females in follicular phase, some antral follicles are selected to continue to growth, reaching a pre-ovulatory diameter of 2mm. Mean ovulation rate was 2.5 follicles and litter size was 2.1 embryos or fetuses per pregnant female, resulting in a rate of ovum mortality of 20.8%. Many follicles from which ovulation did not occur of 1-mm maximum diameter luteinize forming accessory CL. The constituent active luteal tissues of the ovary are functional and accessory CL. Although all females had accessory CL, transformation of follicles into accessory CL occurred especially in pregnant females, resulting in a contribution from 9% to 23% of the total luteal volume as pregnancy advances. The persistence of functional CL throughout pregnancy might reflect the importance for the maintenance of gestation and may be essential for the continuous hormonal production. The duplex uterus of the agouti female is composed by two completely independent uterine horns with correspondent separate cervices opening into the vagina. In pregnant females, most remarkable observed uterine adaptations were induced by the progressive enlargement caused by the normal pregnancy evolution. The wild black agouti showed different vaginal epithelium features in accordance with the reproductive state of the female.     

Influence of the dominant follicle on oocytes from subordinate follicles

As the oocyte grows within the follicle, a number of factors influence its health and developmental competence. These factors include follicle size, day of estrous cycle, level of atresia and influence of other follicles such as the dominant follicle. Follicles were dissected from ovaries of synchronized dairy cows on four days during the estrous cycle, and the oocyte from each follicle collected, matured, fertilized and cultured singly until Day 8. Development to blastocyst was greater in oocytes collected during phases of follicular growth than those collected during phases of follicular dominance (P<0.001) over all follicle size categories. Oocyte competence tended to increase with increasing follicle size (P<0.1). Follicular cells analyzed by flow cytometry showed an increase in proportion of apoptotic cells in subordinate follicles during the dominant phase compared to growth phase (P<0.05). Thus, the dominant follicle on both oocyte competence and levels of atresia. Further studies on the effect of dominance has shown that lactate production in cumulus-oocyte-complexes (COCs) from medium-sized follicles collected during a dominance phase and small follicles collected during a growth phase are no different from other follicles, despite having significantly lower uptake of glucose (P<0.1). Thus, COCs from different follicle subclasses differ in their nutrient requirements, and current IVM technology needs further improvement to better assist those oocytes that are developmentally challenged.     

Use of high-resolution transrectal ultrasonography to assess changes in numbers of small ovarian antral follicles and their relationships to the emergence of follicular waves in cyclic ewes

Transrectal ovarian ultrasonographic studies have shown that, in cattle, follicular wave emergence is associated with a large increase in the number of small antral follicles (4-6mm in diameter); an analogous association has not been found for small follicles (2-3mm in diameter) in the ewe. In previous studies in ewes, accurate assessment of the number of follicles has been limited to follicles > or =2 or 3mm in size. Newer, high-resolution equipment allowed us to identify follicles > or =0.4mm and to quantify all antral follicles > or =1mm in diameter in seven cyclic Western White Face ewes. This allowed us to expand the small follicle pool examined, from 1 to 4 follicles/day (2-3.5mm in diameter) in earlier studies, to 8-18 follicles/day (1-3mm in diameter). Total number of small follicles (> or =1 and < or =3mm in diameter) increased between Days -1 and 0 (Day 0=day of ovulation), and declined between Days 1 and 3 (P<0.05). There were no significant changes in the number of small or medium (4mm in diameter) follicles around days of follicle wave emergence (+/-2 days). The 1-3 follicles in the 2-3mm size range, which constituted a follicle wave (i.e. grew to > or =5mm in size before regression or ovulation), were the only small follicles to emerge in an orderly succession during the estrous cycle, approximately every 3-5 days. Thus, unlike in cattle, there is no apparent increase in numbers of small follicles at follicle wave emergence in cyclic sheep, and little evidence for selection of recruited follicles and follicular dominance.     

Relationship among the status of the human oocyte, the 17 beta-estradiol concentration in the antral fluid and the follicular size

We studied the relationship among the status of the human oocytes, the E2 concentration in the antral fluid and the follicular size in the different phases of the menstrual cycle, in order to determine the microenvironment of the follicles with healthy or degenerative oocytes in the human ovary. In the follicular phase of the menstrual cycle, follicles which contained a healthy but not degenerative oocyte had a significantly higher level of 17 beta-estradiol (E2). In the late follicular phase, the larger follicles (greater than or equal to 13 mm, in diameter) had only health oocytes. It seems that the follicle containing a degenerative oocyte does not develop physiologically until maturation of the preovulatory follicle. In the luteal phase, there were no relationships among the status of the oocyte, E2 concentration in the antral fluid and the follicular size. However, the E2 levels of the antral follicles with healthy oocytes in an ovary with corpus luteum were significantly lower than those in the contralateral ovary. The results suggest that the corpus luteum may exert an influence on the adjacent follicles.     

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.     

In vitro DNA synthesis by isolated preantral to preovulatory follicles from the cyclic mouse

In recent studies, we have shown that the smallest preantral follicles in the cyclic hamster increase DNA synthesis in the periovulatory period in response to surge levels of FSH. The current investigation was designed to determine whether the same phenomenon occurs in the cyclic mouse. Intact mouse follicles were isolated with watchmaker forceps (stages 4-6) or by enzymatic digestion (stages 1-4) at 0900 h and 1500 h on each day of the 5-day estrous cycle. The isolated follicles were classified into 6 stages: stages 1 and 2: follicles with 1 and 2 layers of granulosa cells; stage 3: follicles with 3 or more layers of granulosa cells and formation of theca; stages 4-6: incipient, small, and preovulatory antral follicles. The follicles at each stage were incubated for 3 h with [3H]thymidine. DNA content in stages 1-4 of follicles remained unchanged during the estrous cycle; for stages 5 and 6, DNA content was higher on the afternoon of proestrus than on other days of the cycle. Incorporation of [3H]thymidine for stages 1-3 (preantral follicles) started to increase at 1500 h of proestrus and peaked at 0900 h on estrus, whereas for stages 4-6, DNA synthesis peaked on proestrus (1500 h) and then fell by the morning of estrus. Thus, the rate of DNA replication in preantral and antral mouse follicles were different. Similarities and differences in folliculogenesis between mouse and hamster are discussed. These results suggest that DNA synthesis and the growth of all stages of follicles in the cyclic mouse may be associated with changing levels of periovulatory gonadotropins.     

Immunocytochemical localization of albumin in ovarian follicles of fertile rats

Summary The purpose of this study was to investigate whether albumin (Alb) can be detected in ovarian rat granulosa cells. Using immunocytochemistry and morphometrics, the percentages of Alb-positive follicles (follicle-index), of Alb-positive granulosa cells (granulosa-index), and of strongly reacting follicles (intensity-index) were evaluated in intact and regressing follicles of different diameter groups during different stages of the estrous cycle. In intact follicles, the follicle- and the granulosa-index increased from small-sized to large-sized follicles. Although the follicle-index did not change in any group during the stages of the estrous cycle, the granulosa-index was higher during proestrus than during the other stages. Intact follicles showed a stronger immunoreactivity than regressing follicles throughout the stages of the estrous cycle. Thus, Alb may be a requirement for the control of follicle growth in fertile rats. This Alb function may be attributable to Alb binding to specific cell-membrane components followed by the intracellular uptake of Alb-bound substances.Supported by a DFG grant no. Sp 232/2-2     

Synchronization rate, size of the ovulatory follicle, and pregnancy rate after synchronization of ovulation beginning on different days of the estrous cycle in lactating dairy cows

Recently a protocol was developed that precisely synchronizes the time of ovulation in lactating dairy cows (Ovsynch; GnRH-7d-PGF2 alpha-2d-GnRH). We evaluated whether initiation of Ovsynch on different days of the estrous cycle altered the effectiveness of this protocol. The percentage of cows (n = 156) ovulating to the first GnRH was 64% and varied (P < 0.01) by stage of estrous cycle. Treatment with PGF2 alpha was effective, with 93% of cows having low progesterone at second GnRH. The overall percentage of cows that ovulated after second GnRH (synchronization rate) was 87% and varied by response to first GnRH (92% if ovulation to first GnRH vs 79% if no ovulation; P < 0.05). There were 6% of cows that ovulated before the second injection of GnRH and 7% with no detectable ovulation by 48 h after second GnRH. Maximal diameter of the ovulatory follicle varied by stage of estrous cycle, with cows in which Ovsynch was initiated at midcycle having the smallest follicles. In addition, milk production and serum progesterone concentration on the day of PGF2 alpha affected (P < 0.05) size of the ovulatory follicle. Using these results we analyzed pregnancy rate at Days 28 and 98 after AI for cows (n = 404) in which Ovsynch was initiated on known days of the estrous cycle. Pregnancy rate was lower for cows expected to ovulate larger follicles than those expected to ovulate smaller follicles (P < 0.05; 32 vs 42%). Thus, although overall synchronization rate with Ovsynch was above 85%, there were clear differences in response according to day of protocol initiation. Cows in which Ovsynch was initiated near midcycle had smaller ovulatory follicles and greater pregnancy rates.     

Ovarian folliculogenesis in collared peccary, Pecari tajacu (Artiodactyla: Tayassuidae)

The sustainability and production of collared peccary (Pecari tajacu) has been studied in the last few years; however, further information on its reproduction is necessary for breeding systems success. Understanding folliculogenesis aspects will contribute to effective reproductive biotechniques, which are useful in the preservation and production of wildlife. The aim of this study was-to evaluate the ovarian folliculogenesis in collared peccary. Ovaries from six adult females of collared peccary were obtained through ovariectomy and analyzed. These were fixed in aqueous Bouin's solution and sectioned into 7 microm slices, stained with hematoxilin-eosin and analyzed by light microscopy. The number of pre-antral and antral follicles per ovary was estimated using the Fractionator Method. The follicles, oocytes and oocyte nuclei were measured using an ocular micrometer. Results showed that the length, width, thickness, weight, and the gross anatomy of the right and left ovaries were not significantly different. However, the mean number of corpora lutea was different between the phases of the estrous cycle (p<0.05), with the highest mean in the luteal phase. Primordial follicles were found in the cortex; the oocytes were enveloped by a single layer of flattened follicular cells. In the primary follicles, proliferation of the follicular cells gave rise to cuboidal cells (granulosa cells). The secondary follicle was characterized by two or more concentric layers of cuboidal cells (granulosa), beginning of antrum formation, and the presence of pellucid zone and theca cells. Antral follicles were characterized by a central cavity (antrum), the presence of cumulus oophorus and theca layers (interna and externa). In the right ovary, the values of the primordial and primary follicles were similar, but significantly different from the secondary ones (p<0.05). In the left ovary, significant differences were observed between all follicles in the follicular phase (p<0.05); the mean number of primordial and primary follicles was similar in the luteal phase. The mean number of pre-antral follicles and antral follicles in the follicular phase was higher in the left ovary (p<0.05). The mean number of antral follicles in the luteal phase was similar in both ovaries. We also found significant differences in mean diameter of preantral follicles, oocyte, granulosa layer and oocyte nucleus during the estrous cycle. In the antral follicles a significant difference was observed only in follicular diameter (p<0.05). The predominance of active primordial and primary follicles was found in both phases; otherwise the secondary follicles and antral follicles showed a high degree of degeneration. The results obtained in the present work will strengthen the development of biotechnology programs to improve the productive potential and conservation of the collared peccary.     

Ovarian prostaglandin endoperoxide synthase: cellular localization during the rat estrous cycle

The specific cellular localization of prostaglandin endoperoxide (PGH) synthase was studied throughout the rat estrous cycle. Animals were necropsied at 1300 h on each day of the 4-day cycle, and an additional group was necropsied at 2300 h on proestrus. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. At all stages of the cycle, intense immunostaining was observed in newly formed corpora lutea. Luteal cells were immunoreactive, but the connective tissue centrum was unstained. Interstitial tissue contained heavily labeled cells, whereas the germinal epithelium exhibited faint staining. During estrus, metestrus, and diestrus, thecal cells from preantral and antral follicles contained PGH synthase immunoreactivity, but granulosa cells were unstained. Faint staining of mural granulosa cells was observed first in 78% of preovulatory follicles (less than 400-microns diameter) in ovaries collected on the afternoon of proestrus. After the luteinizing hormone surge, 95% of the preovulatory follicles exhibited PGH synthase staining. The percentage of immunoreactive granulosa cells in these preovulatory follicles increased 4-fold in ovaries collected at 2300 h on proestrus. The presence of ovarian PGH synthase throughout the rat estrous cycle and the changes in cellular localization may reflect the potential role of PGs in follicular and luteal function.     

Intraovarian relationships among dominant and subordinate follicles and the corpus luteum in heifers

Previous studies demonstrated that waves of follicular activity develop approximately every 9 d in cattle during the estrous cycle and early pregnancy. A dominant follicle develops from each wave and the remaining follicles (subordinates) begin to regress after a few days. In this study, intraovarian luteal and follicular interrelationships were examined during the follicular waves of the estrous cycle and pregnancy using data obtained by ultrasonography. During the estrous cycle, no intraovarian relationships were found between the ovary containing the corpus luteum and the ovary containing the dominant follicle (n = 165), or between the location of the corpus luteum and the characteristics of the dominant follicle. During pregnancy, however, the frequency distribution for the number of follicular waves with the dominant follicle and corpus luteum on the same or opposite ovaries differed (P<0.05) among Waves 1 to 10. The two structures (dominant follicle and corpus luteum) were more often in opposite ovaries during Waves 3 to 10 (combined frequency, 75%) than during Waves 1 and 2. During pregnancy, dominant follicles of consecutive waves differed (P<0.05) among Waves 1 to 8 in the frequency with which they appeared in the same versus the opposite ovary. The difference seemed primarily due to an increased frequency of consecutive follicles on the same ovary for Waves 4 to 8 (combined frequency, 80%). During both the estrous cycle and pregnancy, there was no significant intraovarian effect of the dominant follicle on the day of detection of the next dominant follicle, on the growth rate of the largest subordinate follicle, or on the length of the interval from wave origin to cessation of growth of the largest subordinate; these results indicate that previously postulated suppressive effects between follicles are exerted through systemic channels.     

Confocal laser scanning microscopy of rat follicle development.

This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pro, LysoTracker Red, hydroethidine, ethidium bromide, and 7-aminoactinomycin-d) were applied either to fresh tissue or to tissue that had been fixed with glutaraldehyde or paraformaldehyde. After fixation and staining, the tissue was dehydrated with MEOH and cleared with benzyl alcohol/benzyl aldehyde. CLSM was then used with the appropriate laser excitation, dichroics, and bandpass filters to acquire images of oocytes contained in follicles. Analysis of the data revealed three principal findings. First, a rapid increase in oocyte size occurred in the preantral stages of follicle development. In the antral stage of follicle development, there was a rapid increase in follicle size without any substantial increase in oocyte size. Second, accompanying these changes in oocyte and follicle growth was a differential staining pattern in which the nucleus stained more than the cytoplasm in a young follicle, but stained less than the cytoplasm as the follicle enlarged into the late antral stage. Lastly, using CLSM, atretic follicles showed increased LysoTracker Red staining in the granulosa region of the antral follicle, suggestive of cell death.     

Waves of follicle development during the estrous cycle in sheep

The pattern of ovarian follicle development in maiden cyclic lambs was characterized using the definition of a follicle wave as the changes in the number of follicles among the days of the estrous cycle, as originally defined in cattle by Rajakoski in 1960. We also examined the steroid content relationships among follicles on Days 5 (Wave 1) and 14 (Waves 2 and 3) of the estrous cycle. In Experiment 1, the ovaries of 20 cyclic lambs (40 to 45 kg) were examined daily using transrectal ultrasonography for 1 or 2 estrous cycles (n = 31 cycles). The number of small (2 and 3 mm in diameter), medium (4 and 5 mm) and large (> or = 6 mm) follicles were aligned with the beginning and end of the average length estrous cycle and then compared among days. Identified follicles were defined as those that grew to > or = 4 mm and remained at > or = 3 mm for > or = 3 d. The number of identified follicles emerging (retrospectively identified at 2 or 3 mm) per ewe per day was also aligned with the average length estrous cycle. In Experiment 2, ewe lambs were ovariectomized on Day 5 (n = 6) or 14 (n = 5) of the estrous cycle, then follicle diameters and follicular fluid concentrations of estradiol and progesterone were compared among follicles. Data were analyzed by repeated measures ANOVA and compared among days using Fisher's LSD. In Experiment 1, either 2 (n = 10 cycles), 3 (n = 20 cycles) or 4 (n = 1 cycle) periods of emergence of identified follicles occurred during individual cycles, with estrous cycle lengths of 15.6 +/- 1.6, 16.1 +/- 1.1 and 17 d respectively. In animals with 2 or 3 periods of emergence of identified follicles, the total number of small, medium and large follicles differed (P < 0.05) among days of the estrous cycle showing a wave-like pattern. In Experiment 2, a single follicle collected on each of Days 5 and 14 of the cycle (6.2 +/- 0.2 and 3.9 +/- 0.2 mm in diameter) had a higher (P < 0.05) concentration of follicular fluid estradiol (36.2 +/- 4.4 and 50.9 +/- 21.6 ng/mL) than other follicles collected on the same day (next largest follicle: 4.3 +/- 0.3 and 3.5 +/- 0.4 mm; 4.3 +/- 0.9 and 18.2 +/- 6.7 ng/mL estradiol). The results showed that 1) there was a synchronous emergence of follicles associated with fluctuations in the number and size of follicles during the estrous cycle; 2) within a wave there was a hierarchy among follicles for diameter and steroid content; 3) ovarian follicle growth in ewe lambs occurred in 2 or 3 organized waves during the estrous cycle.     

Effects of Calf Removal at Parturition on Postpartum Ovarian Activity in Zebu (Bos indicus) Cows in the Humid Tropics

To assess endocrine and morphological responses of ovaries to total weaning at parturition, 6 Zebu (Bos indicus) cows 5 years or older were investigated. Following parturition, blood samples were collected daily during the first month and twice weekly thereafter until day 60 to determine concentrations of progesterone (P4) and prostaglandin F2α metabolite. It took between 25 to 32 days to complete uterine involution.The prostaglandin metabolite remained elevated for a mean period of 14.2 days (range, 4-21) postpartum. Five of the animals resumed cyclicity with a short estrous cycle starting between days 7 to 34 and lasting between 7 and 14 days. No estrous behavior was recorded prior to the short estrous cycles, but subsequent normal-length estrous cycles were all preceded by signs of estrus. In the 1 animal that resumed cyclicity with an estrous cycle of normal length on day 37 (length 20 days), the cycle was preceded by estrous behavior. Progesterone concentrations reached a mean maximum of 4.8 nmol liter−1 during the short estrous cycles, and prostaglandin metabolite concentrations peaked while P4 concentrations were decreasing. P4 concentrations reached a mean maximum of 12.2 nmol liter−1 during the estrous cycles of normal length. The interval from parturition to the first estrous cycle of normal length varied between 16 and 48 days, and the length of the cycle was 18 to 22 days. Starting 2 days postpartum, ovaries from 5 of the cows were scanned by ultrasonography every second day until day 30 postpartum. Medium-sized follicles were detected between days 4 to 7 postpartum in 4 of the scanned cows that later had short estrous cycles. The time between parturition and the appearance of the first dominant follicle was 7.6 days (range 6-10 days). The interval between parturition and the appearance of the first ovulatory-sized follicle was 10.2 days (range 8-13 days). In 3 of the scanned cows this ovulatory-sized follicle ovulated. We conclude that cyclic ovarian activity in Zebu cows can start early in the postpartum period in the absence of offspring, and that short luteal phases, not preceded by estrous behavior, may play an important role in establishing normal postpartum ovarian activity.