Large variation in steroid concentrations and insulin-like growth factor binding proteins exists among individual small antral follicles collected from within cows at random stages of the estrous cycle

Variation in the biochemical status of individual small (< or = 5 mm diameter) antral follicles within the ovaries of a cow at any given time likely influences the capacity for undergoing recruitment, selection, and establishing dominance. The objectives of this study were to provide insight into the magnitude of variation in follicular fluid concentrations of steroids and activities of IGFBP that exists among individual small antral follicles within and between cows, and to determine the relationships between follicular fluid IGFBP and steroid concentrations in these follicles. A total of 108 small antral follicles were collected from 6 cows at random stages of the estrous cycle, with 10 to 26 follicles/cow. Concentrations of steroids (ng/mL of follicular fluid) in the overall population of follicles ranged from 0.1 (lowest detectable limit) to 51 for estradiol (E2), 4 to 1,149 for progesterone (P4), and 5 to 504 for androstenedione (A4). Concentrations of E2 and A4 were associated positively (r = 0.2; P < 0.02), but E2 (r = -0.4) and A4 (r = -0.4) were associated negatively, with P4. The proportion of variation in steroid concentrations accounted for by differences among animals (P < 0.05) was small for E2 (12%), moderate for P4 (43%), and greatest for A4 (74%). Least differences between minimum and maximum concentrations of steroids observed in follicles from within a cow were 21-, 5.5-, and 3.5-fold for E2, P4, and A4, respectively, whereas the greatest differences between minimum and maximum concentrations were 505-, 108-, and 26-fold for E2, P4, and A4, respectively. Ranges of IGFBP concentrations (arbitrary densitometer units) detected in fluid from a sub-sample of 43 follicles were 1.18 to 4.50 for IGFBP-3, 0.54 to 4.68 for IGFBP-2, 0.07 to 2.56 for IGFBP-4, and 0.01 to 6.71 for IGFBP-5. Concentrations of E2 were correlated negatively with each IGFBP (r = -0.4 to -0.8; P < 0.05) except IGFBP-3. In contrast, concentrations of A4 were correlated positively with IGFBP-3 (r = 0.4; P < 0.05) but were not correlated with other IGFBP. Concentrations of P4 were correlated positively (r > 0.4; P < 0.05) with IGFBP-4 and -5. The results indicate that steroid concentrations and IGFBP activities vary substantially among small antral follicles collected from within and among individual animals and that increasing production of E2, the hallmark of a developing follicle, was associated with reduced activity of all IGFBP except IGFBP-3, thereby implicating these IGFBP in the regulation of follicular recruitment.     

Ovarian follicular populations and in vitro steroidogenesis on three different days of the bovine estrous cycle

This study was undertaken to determine changes in follicular populations on ovaries of dairy cows during three stages of the estrous cycle and their steroidogenic capacity in vitro. Numbers of small (2.0 to 5.0 mm), intermediate (5.1 to 10 mm) and large (greater than 10 mm) antral follicles on ovaries of multiparous cows and heifers (n = 31) in the early luteal (d 4), mid-luteal (d 12) and follicular phase (d 19) of the estrous cycle were determined (d 0 = estrus), and steroidogenic capacity of intermediate and large follicles was measured in vitro. Total number of follicles and number of small follicles were greatest (P less than .05) on d 19 compared with d 12, with numbers on d 4 not different from either d 12 or 19. Intermediate follicles were fewer (P less than .05) on d 19 compared with d 4 or 12. Numbers of large follicles did not change. The proportion of estrogen active (EA) follicles was greater (P less than .05) on d 19 compared with d 4 or 12. Accumulation of estradiol-17 beta (E) into culture medium by intermediate follicles decreased (P less than .05) with increasing days of the estrous cycle, while accumulation of progesterone (P) was greater on d 19. In large follicles, accumulation of E into culture medium was greatest (P less than .05) on d 19 and the lowest on d 12 (P less than .05). In summary, the proportion of EA follicles increases during the preovulatory period, and E production increases in large EA follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships between ultrasonographic image attributes, histomorphology and proliferating cell nuclear antigen expression of bovine antral follicles and corpora lutea ex situ.

The aim of this study was to examine the relationships between quantitative ultrasonographic image characteristics, histological attributes and cell proliferating ability of bovine antral follicles and corpora lutea (CL) ex situ. Bovine ovaries (n = 30) from animals at various reproductive states (metoestrus-early dioestrus, n = 8; mid-dioestrus, n = 12; oestrous phase of peripubertal heifers, n = 6; and pregnancy, n = 4) were collected at the slaughterhouse. High-resolution ultrasonographic images of the ovaries were obtained in the water bath, digitized and subjected to computerized image analyses. The analyses utilized line and spot techniques designed to determine pixel values of the follicular wall (the largest follicles >2 mm in diameter in each ovary) and CL, respectively. Individual ovarian structures were dissected and processed for histology and immunohistochemical detection of proliferating cell nuclear antigen (PCNA). The mean follicular diameter was negatively correlated with total cell density (r = -0.45, p < 0.05), granulosa layer thickness (r = -0.67, p < 0.001) and the percentage of PCNA-positive cells (r = -0.57, p < 0.001). Numerical pixel values and heterogeneity of the follicular wall were positively correlated with total cell density (r = 0.42, p < 0.05 and r = 0.62, p < 0.05; respectively), granulosa layer thickness (both r = 0.39, p < 0.05), and the percentage of PCNA-positive cells (r = 0.54, p < 0.01 and r = 0.69, p < 0.001, respectively). Estimates of cell density and proliferating cell index were not correlated with the ultrasonographic image attributes of CL. We conclude that follicular size and echotextural variables, as determined by computer-assisted image analysis of ovaries ex situ, are reliable markers of the histophysiological properties of bovine antral follicles, but the ultrasonographic characteristics are not indicative of cell density and proliferation in the bovine CL.     

Association of Fertility with Numbers of Antral Follicles within a Follicular Wave During the Oestrous Cycle in Beef Cattle

The association between conception rate at first service and numbers of follicles developed during a follicular wave was examined in 102 suckled beef cows and 14 heifers. Follicular development was monitored using ultrasonography for either two (trial 1) or three (trial 2) consecutive oestrous cycles (pre-breeding, breeding and post-breeding equivalent). Animals were examined on alternate days from day 6 after first oestrus (day 0) until ovulation and from day 6 after insemination until next ovulation or day 24 of pregnancy and were observed for oestrus twice daily and inseminated artificially at either the second (trial 1) or third oestrus (trial 2). Cows were classified as having two or three waves of follicular development for each oestrous cycle. Numbers of follicles >or=4 mm per wave were determined, and based on the maximum diameter they attained, were classified as small (4-6 mm), medium (7-10 mm) or large (>or=11 mm) follicles. Total numbers of follicles, and primarily numbers of small and medium follicles, were affected by trial and within trial by cow, oestrous cycle and follicular wave. Heifers had more small and total numbers of follicles, but fewer large follicles than cows in trial 1 (p < 0.05). The average number of antral follicles per wave in the breeding cycle or post-breeding period did not affect conception rates, which averaged 84%. Repeatability of the total numbers of antral follicles between and among oestrous cycles and follicular waves ranged from 0.01 to 0.97. In conclusion, fertility was not affected by the numbers of antral follicles >or=4 mm in diameter in a single follicular wave.     

The localization and expression of epidermal growth factor and epidermal growth factor receptor in bovine ovary during oestrous cycle

Epidermal growth factor (EGF) is one of the important regulatory factors of EGF family. EGF has been indicated to effectively inhibit the apoptosis of follicular cells, to promote the proliferation of granulosa cells and the maturation of oocytes, and to induce ovulation process via binding to epidermal growth factor receptor (EGFR). However, little is known about the distribution and expression of EGF and EGFR in cattle ovary especially during oestrous cycle. In this study, the localization and expression rule of EGF and EGFR in cattle ovaries of follicular phase and luteal phase at different time points in oestrous cycle were investigated by using IHC and real-time qPCR. The results showed that EGF and EGFR in cattle ovary were mainly expressed in granulosa cells, cumulus cells, oocytes, zona pellucida, follicular fluid and theca folliculi externa of follicles. The protein and mRNA expression of EGF/EGFR in follicles changed regularly with the follicular growth wave both in follicular and in luteal phase ovaries. In follicular phase ovaries, the protein expression of EGF and EGFR was higher in antral follicles than that of those in other follicles during follicular growth stage, and the mRNA expression of EGFR was also increased in stage of dominant follicle selection. However, in luteal phase ovaries, the growth of follicles was impeded during corpus luteum development under the action of progesterone secreted by granular lutein cell. The mRNA and protein expressions of EGF and EGFR in ovarian follicles during oestrous cycle indicate that they play a role in promoting follicular development in follicular growth waves and mediating the selection process of dominant follicles.     

Ovarian follicular development in cattle selected for twin ovulations and births

Comparisons of numbers of antral ovarian follicles and corpora lutea (CL), of blood hormone concentrations, and of follicular fluid steroid concentrations and IGFBP activity were conducted between cows selected (twinner) and unselected (control) for twin births to elucidate genetic differences in the regulation of ovarian follicular development. Ovarian follicular development was synchronized among cows by a single i.m. injection of PGF2alpha on d 18 of the estrous cycle; six cows per population were slaughtered at 0, 24, 48, and 72 h after PGF2alpha. Jugular vein blood was collected from each animal at PGF2alpha injection and at 24-h intervals until slaughter. Ovaries of twinner cows contained more small (< or = 5 mm in diameter, P < 0.05), medium (5.1 to 9.9 mm, P < 0.05), and large (> or = 10.0 mm, P < 0.01) follicles and more (P < 0.01) CL than ovaries of controls. Follicular fluid concentrations of estradiol, androstenedione, testosterone, and progesterone reflected the stage of follicular development and were similar for twinner and control follicles at the same stage. Earlier initiation of follicular development and/or selection of twin-dominant follicles in some twinner cows resulted in greater concentrations of estradiol in plasma at 0, 24, and 48 h and of estradiol, androstenedione, and testosterone in follicular fluid of large follicles at 0 h after PGF2alpha for twinner vs. control cows (follicular status x time x population, P < 0.01). Binding activities of IGFBP-5 and -4 were absent or reduced (P < 0.01) in follicular fluid of developing medium and large estro-gen-active (estradiol:progesterone ratio > 1) follicles but increased with atresia. Only preovulatory Graafian follicles lacked IGFBP-2 binding, suggesting a possible role for IGFBP-2 in selection of the dominant follicle. Concentrations of IGF-I were twofold greater (P < 0.01), but GH (P = 0.10) and cholesterol (P < 0.05) were less in blood of twinners. Three generations of selection of cattle for twin ovulations and births enhanced ovarian follicular development as manifested by increased numbers of follicles within a follicular wave and subsequent selection of twin dominant follicles. Because gonadotropin secretion and ovarian steroidogenesis were similar for control and twinner cattle, enhanced follicular development in twinners may result from decreased inhibition by the dominant follicle(s), increased ovarian sensitivity to gonadotropins, and/or increased intragonadal stimulation, possibly by increased IGF-I.     

Oxytocin modulates the pulsatile secretion of prostaglandin F2alpha in initiated luteolysis in cattle

Subluteolytic doses of prostaglandin F2alpha analogue (oestrophan) given i.m. and oxytocin (OT) antagonist (CAP) and noradrenaline (NA) infused into the abdominal aorta were used to test the importance of luteal OT in pulsatile secretion of prostaglandin F2alpha (PGF) during luteolysis in heifers (n = 17). In experiment 1, heifers were pre-infused for 30 minutes with saline on either day 17 of the oestrous cycle (group 1; n = 4) or on day 18 of the oestrous cycle (group 2; n = 3), and with CAP (8 mg per animal) on day 17 of the oestrous cycle (group 3; n = 4). Next, heifers were injected with oestrophan (30 microg per animal). Injection of oestrophan in Group 3 increased OT concentrations (P < 0.001) to values similar to those observed during spontaneous luteolysis (50 to 70 pg ml(-1)). PGFM concentrations in this group also increased (P < 0.001), but were lower (P < 0.05) than the values in groups 1 and 2, CAP given prior to oestrophan decreased both PGFM elevation (P < 0.06) and its area under the curve (P < 0.01), compared to the saline pretreated heifers. In experiment 2 NA (4 mg) was infused twice for 30 minutes at five hour intervals to release OT on day 17 of the oestrous cycle (n = 6). However, during hormone analysis it appeared that three of six heifers had elevated PGFM concentrations (group 1) and three others did not (group 2). NA caused the correlated increase of progesterone and OT secretion (r = 0.68; P < 0.05) in both groups but it only influenced PGF secretion in group 1 only (P < 0.05). We postulate that OT can amplify and modulate the course of induced luteolysis as a regulator of the amplitude of pulsatile PGF secretion. PGF analogue stimulates secretion of endogenous PGF from the uterus in cattle and this may be an important component of the luteolytic response to exogenous PGF.     

Follicular progesterone levels decrease during the period of seasonal infertility in sows

Impaired reproductive performance exhibited by the domestic sow during the late summer and early autumn months is referred to as seasonal infertility. This study was carried out to determine whether there are changes in ovarian morphology and follicular steroidogenesis associated with season, which may be associated with seasonal infertility. Ovaries were collected in pairs from sows sourced from two farms and slaughtered 4 days after weaning during winter and summer. The mean progesterone concentration in follicular fluid (FF) collected from small follicles was lower in summer (701.3 ± 115.54 nm) compared with winter (1235.55 ± 164.47 nm; p<0.001). The mean progesterone concentration in the FF of large follicles was also lower in summer (1469.2 ± 156.51 nm) compared with winter (2470.9 ± 169.13 nm; p<0.001). The number of large surface antral follicles (5-8 mm in diameter) on the ovaries recovered from Farm A sows was higher during summer (17.76 ± 0.56) than in winter (15.38 ± 0.54; p<0.05). Similarly, the number of small follicles (3-4 mm in diameter) on Farm A sow ovaries was higher in summer (8.46 ± 0.66) than in winter (4.63 ± 0.53; p<0.001). In contrast, the number of small follicles on the surface of ovaries recovered from Farm B sows was higher during winter (10.17 ± 1.50) than in summer (6.45 ± 1.00; p<0.01). The number of pre-ovulatory follicles (>8 mm in diameter) was also higher in winter (1.23 ± 1.68) when compared to summer (0.51 ± 0.3; p<0.001) on the ovaries of sows from Farm B. The results suggest that there are seasonal differences in follicular steroidogenesis and ovarian dynamics. These findings add support to the theory that altered follicular steroidogenesis and ovarian morphology may possibly be the mechanism behind reduced reproductive performance during the period of seasonal infertility in sows.     

Vascularity and expression of angiogenic factors in bovine dominant follicles of the first follicular wave

To determine the relationships among vascularity, expression of angiogenic factors, and selected intrafollicular factors in dominant and nondominant follicles of the first follicular wave, ovaries were obtained on d 3 of the estrous cycle from mature cross-bred beef heifers (n = 8) after a synchronized estrus. Follicular fluid (FF) was collected from all follicles > or = 3 mm for determination of estradiol-17beta (E), progesterone (P4), vascular endothelial growth factor (VEGF), and IGFBP concentrations. The ovaries were then perfusion-fixed and used for histochemical detection of lectin BS-1 (a marker of endothelial cells and thus vascularization) binding, and immunolocalization of VEGF, endothelial nitric oxide synthase (eNOS), and proliferating cell nuclear antigen, followed by image analysis of selected follicles. Follicles were classified, based on E and P4 concentrations in FF, as dominant, estrogen-active (EA; E:P4 > or = 1) or nondominant, estrogen-inactive (EI; E:P4 <1). Concentrations of E and VEGF in FF, the area of positive staining for lectin BS-1, VEGF, and eNOS, and the labeling index (an index of the percentage of cells proliferating) in granulosa and theca layers were greater (P < 0.05) in the EA than in the EI follicles, but concentrations of P4 and IGFBP in FF were less (P < 0.05) in EA than in EI follicles. In addition, vascularity was positively correlated (P < 0.05) with VEGF and eNOS protein expression, and tended (P < 0.1) to be positively correlated with the E:P4 ratio in FF but tended (P < 0.1) to be negatively correlated with IGFBP and P4 concentrations in FF. These data highlight the importance of vascularity, angiogenic factors, and IGFBP in the health of the dominant follicle in heifers, and indicate that the FF concentrations of E, VEGF, IGFBP, and P4, and the E:P4 ratio can be used as markers of dominant follicles.     

Effect of exogenous administration of oxytocin on postpartum follicular dynamics,oestrous rate and ovulation in Nili-Ravi buffaloes

Based on different surveys, dairy farmers are concerned about extensive use of exogenous oxytocin in buffaloes, which is being held responsible for reproductive problems including irregular oestrous cycle and delayed ovulation. For these concerns, effects of oxytocin injection on postpartum follicular dynamics, postpartum oestrous interval (PEI), oestrous length, the interval from onset of estrus to ovulation and blood progesterone (P4) were studied in Nili-Ravi buffaloes. For this purpose, 23 animals within 1 week after calving were randomly divided into three groups: without oxytocin (CON; n = 7), 10 i.u. oxytocin (LOW; n = 8), 30 i.u. oxytocin – (HIGH; n = 8) and used to record the PEI for the study period of 154 days. At subsequent estrus, three buffaloes from each group (not served) were selected randomly to monitor two cycles for 6 weeks. Transrectal ultrasonography was performed to evaluate follicular and corpus luteum (CL) development, and blood sampling was done for progesterone (P4) analysis. These results revealed that postpartum oestrous interval (PEI) decreased significantly in oxytocin-treated groups. The number of small, medium and total follicles on the left ovary was significantly higher in the HIGH group. However, an overall number of small and total follicles on both right and left ovaries was significantly higher in CON and HIGH groups. On the other hand, there was no difference in the number of follicles on the right ovary among all treatment groups. The same was true for the size of pre-ovulatory follicles, CL, P4 concentrations and oestrous cycle length. The intervals from onset of estrus to ovulation and from standing estrus to ovulation were increased considerably in the HIGH group. It is concluded that exogenous oxytocin administration resulted in the shortening of PEI but triggered a delay in ovulation. Moreover, a higher dose of oxytocin could stimulate the growth of small, medium, and total follicles in postpartum Nili-Ravi buffaloes.     

Ovarian Follicular Dynamics in Buffalo Cows (Bubalus bubalis)

Follicular growth in Egyptian buffalo cows was monitored using genital tracts from 200 buffalo cows collected immediately after slaughter. According to the morphological appearance of the corpus luteum (CL), the corresponding oestrous cycle was divided into four stages: A (days 1–4), B (days 5–10), C (days 11–17) and D (days 18–21). Within these stages the follicular population on the ovaries was evaluated and the dominant follicle (DF) determined in all recovered ovaries. The functional status of the DF and the largest sub‐dominant follicles was examined by histological examination in 31 cases, and Radio Immunoassay (RIA) analyses for estradiol‐17β (E₂) and progesterone (P₄) was performed in the follicular fluid in 23 of the DF. The results showed that DFs changed their endocrine character within the stages of the oestrous cycle. The DFs between days 5 and 10 were functionally active (E₂‐dominant; non‐atretic) in most of the cases. Between days 11 and day 17 half of the DFs became functionally inactive (P₄‐dominant; atretic). At days 18–21 all of the DF became functionally active and non‐atretic. In the specimens that carried two large follicles one of them was regularly atretic and P₄‐dominant whereas the other was non‐atretic and E₂‐dominant. Between days 18 and 21 all ovaries examined showed at least one large follicle. These findings suggest that in most of the cases follicular dynamics occurs in two wave‐like patterns in the Egyptian buffalo cows.     

The effect of steroidal dominance on cAMP, cGMP, progesterone and 17 beta-estradiol levels in the fluid of the largest follicle

Concentrations and mutual correlations of the cyclic adenosin monophosphate (cAMP), quanosin monophosphate (cGMP), progesterone (P4) and 17-beta-estradiol (E2) were studied in the fluid of the largest follicles in cows, in dependence on the steroid dominance: estrogen-dominant (ED), progesterone-dominant (PD) follicles. Mean cAMP and cGMP concentrations in the follicular fluid in the estrogen-dominant follicles were significantly higher than in the progesterone-dominant follicles; in both cases at P less than 0.01. Significant positive correlation between cAMP and cGMP at P less than 0.001 was stated in the evaluation of the correlations. The cAMP and cGMP concentrations were in significantly negative correlations with the P4 concentration at P less than 0.05, or P less than 0.01 and in significantly positive correlations with the E2, at P less than 0.05. The stated correlations suggest a close mutual relation between cyclic nucleotid and E2, or P4 when the follicles' quality changes.     

The Adverse Effect of Phytoestrogens on the Synthesis and Secretion of Ovarian Oxytocin in Cattle

The current investigations were undertaken to study the mechanism of the adverse effect of phytoestrogens on the function of bovine granulosa (follicles >1< cm in diameter) and luteal cells from day 1–5, 6–10, 11–15, 16–19 of the oestrous cycle. The cells were incubated with genistein, daidzein or coumestrol (each at the dose of 1 × 10^?6 m ). The viability and secretion of estradiol (E2), progesterone (P4) and oxytocin (OT) were measured after 72 h of incubation. Moreover, the expression of mRNA for neurophysin‐I/OT (NP‐I/OT; precursor of OT) and peptidyl‐glycine‐α‐amidating monooxygenase (PGA, an enzyme responsible for post‐translational OT synthesis) was determined after 8 h of treatment. None of the phytoestrogens used affected the viability of cells except for coumestrol. The increased secretion of E2 and P4 was only obtained by coumestrol (p < 0.05) from granulosa cells from follicles <1 cm in diameter and decreased from luteal cells on days 11–15 of the oestrous cycle, respectively. All three phytoestrogens stimulated (p < 0.05) OT secretion from granulosa and luteal cells in all stages of the oestrous cycle and the expression of NP‐I/OT mRNA in the both types of cells. The expression of mRNA for PGA was stimulated (p < 0.05) by daidzein and coumestrol in granulosa cells, and by genistein and coumestrol in luteal cells. In conclusion, our results demonstrate that these phytoestrogens can impair the ovary function in cattle by adversely affecting the synthesis of OT in follicles and in corpus luteum. However, their influence on the ovarian steroids secretion was less evident.     

Histological Characteristics and Steroid Concentration of Ovarian Follicles at Different Stages of Development in Pregnant and Non-pregnant Dairy Cows

The aim of the study was to investigate the histological characteristics and steroid concentrations in follicular fluid of different populations of follicles at different stages of development, during pregnancy and the oestrous cycle in cows. Follicles from ovaries collected at a slaughterhouse were allocated into three size categories (small, 2–5.9 mm; medium, 6–13.9 mm; and large, 14–20 mm) in pregnant and non-pregnant cows. Slices were stained with HE and PAS for histological analysis. Follicular fluid was pooled according to size and pregnancy status and estradiol, testosterone and progesterone concentrations in follicular fluid were determined by RIA. Characteristics of healthy follicles did not differ, regardless of follicle size or pregnancy status. Total histological atresia was significantly higher in pregnant cows than in non-pregnant cows (p < 0.05). Estradiol increased and testosterone decreased significantly, while follicles increased in size, in both non-pregnant and pregnant cows (p < 0.05). Nonpregnant cows had the highest estradiol values in follicles of all sizes. Medium and large follicles from pregnant cows showed the lowest testosterone concentration (p < 0.05). Progesterone levels increased with follicle size only in non-pregnant animals. In large follicles, progesterone concentration was significantly higher in non-pregnant cows than in pregnant cows (p < 0.05). Considering steroid concentration and histological findings, most large follicles might be atretic during pregnancy in cattle.     

Impact of Exogenous ACTH During Pro-Oestrus on Endocrine Profile and Oestrous Cycle Characteristics in Sows

Sows housed in freely moving groups have elevated cortisol levels until the rank order is established, which takes place within approximately 48 h. The aim of this investigation was to study the effect of repeated administration of synthetic adrenocorticotropic hormone (ACTH; Synacthen Depot), during the follicular phase (pro-oestrus) on oestrus, ovulation and endocrine parameters. Four multiparous sows were used. Follicular growth and ovulation were recorded by ultrasonography. The first oestrous cycle after weaning was used as control cycle. Onset of oestrus in the sow occurs 3-4 days after the time when plasma progesterone reaches a concentration of 8 nmol/l. The progesterone profile in the control cycle of the individual sow was used for estimation when the ACTH injections should start. In the third pro-oestrus ACTH (2.5 microg/kg) was given via an indwelling catheter every 2 h for 48 h. The sows were euthanased 4-6 days after onset of the third oestrus and the ovaries were examined. Cortisol levels were elevated during the treatment period (p < 0.05). The second cycle, in which the sows were injected with ACTH, was prolonged with 2.5 days compared with the control cycle (p < 0.05). The oestradiol pattern during oestrus was similar in the control and the treatment cycle in ovulating sows. Three sows had ovulated (fresh corpora lutea), but the ovaries contained additionally one or several luteinized follicles/cysts. In conclusion, ACTH administration during pro-oestrus caused a prolongation of the oestrous cycle and a disturbed follicular development.     

The effect of repeated follicular puncture on ovarian function in dairy heifers

Three dairy heifers were examined during three consecutive oestrous cycles (control period, CP). Subsequently, the animals were subjected to 4 and then 5 weeks of twice-weekly ovum pick-up (OPU) (FPP1 and FPP2, respectively) with a recovery period (RP) of two consecutive oestrous cycles between FPP1 and FPP2. After FPP2, the animals were slaughtered and the ovaries were macroscopically examined. Throughout, ovarian activity was monitored by transrectal ultrasonography and concentrations of plasma progesterone. During CP, all the heifers showed normal cyclicity. During FPPs, the heifers occasionally presented oestrous activity. Corpus luteum (CL)-like structures developed from punctured follicles with diameters and life-spans varying from smaller and shorter than those in the CP (P > 0.05) to equal to those in the CP. There was a tendency for a lower number of emerging and punctured follicles in the presence of a CL-like structure. Subsequently to FPP1, all heifers regained normal cyclicity. A thickening of the ovarian tunica albuginea and a slight hardening of the ovaries were found postmortem. In conclusion, dairy heifers can occasionally show cyclic activity and form CL-like structures during twice-weekly OPU. Further, OPU did not seem to cause any major negative effects on ovarian structure and subsequent ovarian function.     

Size Distribution of Dispersed Luteal Cells During Oestrous Cycle in Angora Goats

The present study examines the size distribution of the goat steroidogenic luteal cells throughout the oestrous cycle. Corpora lutea (CL) were collected after laparatomy on days 5, 10 and 16 of the oestrous cycle. Luteal cells were isolated from CL by collagenase digestion. Steriodogenic luteal cells were identified by staining of the cells for 3beta-hydroxysteroid dehydrogenase activity, a marker for steroidogenic cells. Luteal cells having steroidogenic capacity covered a wide spectrum of sizes, ranging from 5 to 37.5 microm in diameter. There was a significant increase in mean cell diameters (p < 0.01) as CL aged. The mean cell diameter on day 5 was 11.55 +/- 0.12 microm, which was significantly increased and reached up to 19.18 +/- 0.24 mum by day 16 of the oestrous cycle. The ratio of large to small luteal cells was 0.06:1.0 on day 5 of the oestrous cycle. This ratio increased to 0.78:1.0 by day 16 of the oestrous cycle. Luteal cell size on days 5, 10 and 16 of the oestrous cycle reached its maximum at 7.5, 10 and 35 microm in diameter, respectively. Development of CL is associated with an increase in luteal cell size in goats. It is likely that small luteal cells could develop into large luteal cells as CL becomes older during oestrous cycle in goats.     

Expression of steroidogenic enzymes and synthesis of steroid hormones during development of ovarian follicles in prepubertal goats

Expression of mRNAs encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 -hydroxylase (P450c17), and cytochrome P450 aromatase (P450arom) were characterized by the RT-PCR technique and concentrations of progesterone (P4), testosterone (T0) and estradiol (E2) were measured by radioimmunoassay during follicular development of prepubertal goats. Synthesis of mRNAs encoding P450scc and P450c17 began in preantral follicles, but mRNA encoding P450arom was not detectable until early antral formation. While mRNA for P450scc was expressed in both theca and granulosa cells, mRNA for P450c17 was expressed only in theca cells while P450arom mRNA only in granulosa cells. In nonatretic follicles from prepubertal ovaries, the relative quantity of mRNA expression of all the three enzymes increased with follicle size; however, while the concentration of P4 and E2 increased, that of T0 decreased with follicle size. While expression of mRNA encoding P450scc was unaffected, that of P450c17 mRNA decreased to the lowest level and mRNA for P450arom became undetectable following atresia; accordingly, while the concentration of P4 increased in the atretic medium follicles, that of T0 and E2 decreased to the lowest level after atresia. While the adult follicular stage follicles showed a similar cytochrome expression as the nonatretic follicles of prepubertal goats, the former contained higher levels of E2 and P4 than the latter. The presence of corpus luteum in an ovary decreased expression of P450scc, significantly in large follicles while it increased concentration of P4. These findings indicated that (1) similar to other species, changes in follicular steroid production in goats were explained in large measure by changes in steroidogenic enzyme expression; (2) while mRNA expression was similar, activities of some of the steroidogenic enzymes may differ between sexually mature and immature goats.