Changes in follicular vascularity during the first follicular wave in lactating cows

Increase in the blood supply to individual follicles appears to be associated with follicular growth rates and the ability to become the dominant follicle, while reduced thecal vascularity appears to be closely associated with follicular atresia. Therefore, this study aimed to determine the real-time changes in the vascularity of the follicle wall during the first follicular wave in cycling Holstein cows. Normally cycling and lactating cows (n=5) were examined by transrectal color Doppler ultrasonography (the sensitivity for velocity: > 2 mm/sec) to determine the changes in the vasculature of the follicle wall (presence or absence of blood flow) and the diameter of follicles. A new follicular wave and ovulation were induced by GnRH injection at 48 h after an injection of PGF2alpha analogue. The ovaries were scanned daily for 7 days after GnRH injection. Follicles >2.5 mm were classified into 3 groups by the changes in diameter as follows: 1) largest follicle, 2) second largest follicle, and 3) small follicles, which included all other follicles >2.5 mm. Before the follicle selection, there was no significant difference in the percentage of follicles with detectable blood flow between the subsequently determined largest and second largest follicles. After the follicle selection, the percentage of follicles with detectable blood flow significantly decreased among the second largest follicles. In addition, small follicles with detectable blood flow kept larger diameters than those without detectable blood flow from one day before the occurrence of follicle selection. It is likely that maintenance of follicle vasculature and appropriate blood supply to the larger follicles is essential for follicle dominance. In small follicles, the presence of blood flow within the wall also appears to be required for recruitment. Consequently, the data suggest that the change of the blood supply to an individual follicle closely relates to the dynamics of follicular growth in the first follicular wave in the cow.     

Dynamics of ovarian follicular development in cattle during the estrous cycle, early pregnancy and in response to PMSG

Ovarian follicular dynamics of cattle were examined during the estrous cycle, early pregnancy and in response to PMSG. Number and size of follicles were monitored by ultrasonographic examinations. During the estrous cycle, distinct periods of follicular dominance (measured by the increase in difference in size between the largest and second largest follicle) occurred in both the luteal (Days 6-8) and proestrus (18-22) phases of the estrous cycle (two follicular waves). Associated with the well timed development of the first dominant follicle was a change in distribution of follicle numbers in small (less than 5 mm; increased on Days 2-4), medium (6-8 mm; increased on Days 3-5) and large (greater than or equal to 9 mm; increased on Days 6-9) follicular size classes. Follicular development was greater on the ovary bearing the CL for the period that the CL was present. The dominant follicle formed during the first follicular wave was capable of ovulating (6 of 8 heifers) following an injection of a synthetic analogue of prostaglandin F-2 alpha on Day 9 of the estrous cycle. During early pregnancy (Days 6-34), follicular development (size of largest follicle, number of follicles and total accumulated size of all follicles) on the ovary bearing the CL was suppressed between Days 24 and 34 of pregnancy. This was a local effect in that follicular development was sustained on the contralateral ovary. Therefore, the CL or conceptus may be regulating follicular development in a manner to help prevent luteolysis. Associated with the injection of PMSG was an initial increase in the number of small follicles followed by their recruitment into medium and large size classes leading to ovulation. Number of follicles greater than 5 mm on the Day of estrus was related (r = .97) to the number of subsequent embryos and oocytes collected. Ultrasonography is a valuable technique to monitor ovarian follicular dynamics in cattle, and can thereby be used to infer changes in physiological and endocrine states.     

Dynamics of ovarian follicular development in cattle following hysterectomy and during early pregnancy

Three experiments were conducted to evaluate ovarian follicular dynamics and functional activity during pregnancy in cattle. In 11 pregnant Charolais cows of Experiment I, size of largest follicle, number of follicles and accumulated follicle size were reduced by day 27 of pregnancy on the ovary bearing the corpus luteum (CL) but not on the non-CL bearing ovary. In experiment II, local attenuation of ovarian follicular development on the CL bearing ovary of seven pregnant heifers was evident compared to the contralateral ovary without the CL. However, in four hysterectomized heifers, follicular development was sustained on both the CL- and non-CL bearing ovaries when CL maintenance was achieved without presence of the uterus or conceptus. In Experiment III, steroidogenic characteristics of the largest and second largest follicles at 17 d postestrus were evaluated for seven pregnant and six cyclic cattle. Follicle by physiological status interactions were detected for both aromatase activity of the follicle and follicular fluid concentrations of estradiol and progesterone. In cyclic cows, the largest follicle had appreciably more aromatase activity than did the second largest follicle; whereas, aromatase activity of the largest follicle from pregnant cows was less than that of cyclic cows. However, in pregnant cows the second largest follicle became the estrogen-active follicle, and this follicle occurred with a higher frequency on the ovary contralateral to the CL-bearing ovary. These changes in aromatase activity were reflected by parallel changes in estrogen concentrations of follicular fluid. The higher progesterone concentration in follicular fluid of the largest follicle in pregnant cows provided further confirmation of their atretic status. In conclusion, during early pregnancy the conceptus and/or uterus ipsilateral to the conceptus appear to secrete compounds which alter local follicular steroidogenic activity and attenuate subsequent follicular growth between 17 to 34 d of pregnancy on the CL-bearing ovary. This local mechanism acting within the ovary may contribute to the antiluteolytic effects of early pregnancy in cattle.     

Human chorionic gonadotropin influences ovarian function and concentrations of progesterone in prepubertal Angus heifers

The effects of administering hCG on subsequent ovarian structure dynamics and concentrations of progesterone in prepubertal heifers were evaluated. Forty-seven purebred Angus heifers were assigned randomly to 1 of 3 treatments: 1) 100 μg of injection of GnRH (GnRH; n = 16); 2) 1,000 IU injection of hCG (H1000; n = 16); and 3) 500-IU injection of hCG (H500; n = 15). From d -1 to 9 relative to treatment (d 0), daily blood samples were collected to determine concentrations of progesterone and ovaries of each heifer were examined daily by transrectal ultrasonography. Diameter of all follicles ≥ 4 mm and all luteal structures were mapped. Disappearance of the largest follicle occurred within 2 d in a greater percentage (P < 0.05) of all heifers in the H1000 treatment (87.5%) compared with GnRH heifers (43.8%), whereas H500 heifers (73.7%) were intermediate. A new luteal structure formed after follicle disappearance in a greater (P < 0.05) percentage of all heifers treated with H1000 (87.5%) and H500 (73.7%) heifers compared with that in GnRH-treated heifers (18.8%). The largest follicle present in ovaries of H1000 and H500 heifers was smaller (P < 0.05) than that of GnRH heifers from d 2 to 5. Heifers treated with H1000 (1.72 ± 0.19 ng/mL) had peak concentrations of progesterone on d 6 that were greater (P < 0.05) than H500 heifers (1.34 ± 0.20 ng/mL), which were greater than heifers treated with GnRH (0.31 ± 0.19 ng/mL). The mean volume of luteal tissue was greater (P < 0.05) in H1000 heifers (1.54 ± 0.15 cm(3)) than in H500 heifers (1.15 ± 0.15 cm(3)), which was greater (P < 0.05) than in heifers treated with GnRH (0.23 ± 0.15 cm(3)). We conclude that hCG was more effective than GnRH in its ability to cause disappearance of the largest follicle, increase volume of luteal tissue in the subsequent developing luteal structures, and increase concentrations of progesterone in prepubertal heifers. In addition, hCG seems to be more effective when administered at 1,000 IU than at 500 IU.     

Effect of the Type and Number of Prostaglandin Treatments on Corpus Luteum, the Largest Follicle and Progesterone Concentration in Dairy Cows

Lactating dairy cows (n = 72) with a mature corpus luteum (CL) (diameter of > or = 17 mm) determined by ultrasonography and having a follicle with a diameter of > or = 10 mm were randomly assigned to four groups. Cows were treated with cloprostenol i.m. once or twice, or with dinoprost i.m. once or twice with an 8-h interval. The ovaries of each cow were scanned daily by transrectal ultrasonography to measure the changes in the areas of CL and the largest follicle and to determine the occurrence of ovulation. Oestrus was verified twice daily. In addition, blood sample was withdrawn from each cow daily for measuring progesterone (P4) concentrations. Significant decreases in the percentage changes relative to areas of CL and P4 concentrations or increases in the percentage changes in the area of the largest follicle on day 0 were detected in each group during the experiment. However, the type of the drug and the number of the treatments had no significant effect on those parameters. Cows ovulated with or without showing oestrus (group A) and cows exhibiting no oestrus and ovulation (group B) were also evaluated. In contrast to the mean area of the CL and the mean concentration of P4 on day 0, the mean area of the largest follicles between the two groups on day 0 differed significantly. Significant decreases in the percentage changes relative to the area of the CL and P4 concentration or increases in the percentage changes relative to the area of the largest follicle during the experiment were detected in both groups; however, there were no group differences. Treatment of dairy cows with two injections of prostaglandins (cloprostenol or dinoprost) at an 8-h interval resulted in more cows being observed in oestrus within 5 days after treatment and having significantly higher pregnancy rate than those treated with a single prostaglandin injection.     

Studies of follicular vascularity associated with follicle selection and ovulation in cattle

We reviewed recent in vivo studies of the real-time changes in the vasculature of the follicle wall during selection of the dominant follicle as well as during ovulation in cows. Changes in follicle diameter and vascularity were determined by transrectal ultrasonography. Blood flow within the walls of the two largest follicles was detected at the time of wave emergence (largest follicle=5 mm in diameter). Before selection of a follicle (largest follicle <8.5 mm in diameter), the degrees of vascularity of the two largest follicles were not significantly different. After the largest follicle reached a diameter of 10 mm, the vascularity of the largest (dominant) follicle was higher than that of the second largest (subordinate) follicle. In the preovulatory follicle, follicular vascularity gradually increased, and as ovulation approached, the LH-surge induced an increase in blood flow within the follicle wall. The above results suggest that maintenance of follicular vasculature and appropriate blood supplies to follicles are essential for establishment of follicular dominance. Consequently, only a dominant follicle with high vascularity may have a chance to reach final maturation and acquire ovulatory capacity.     

Ovarian and hormonal responses of cows to treatment with an analogue of gonadotrophin releasing hormone and prostaglandin F2 alpha

Blood samples were taken from 11 cows and their ovaries were scanned by ultrasound at least daily. Around day 5 of an induced cycle, they were injected with 10 micrograms buserelin, an analogue of gonadotrophin releasing hormone, and on day 12 they received 0.5 mg cloprostenol, an analogue of prostaglandin F2 alpha (PGF2 alpha). Two days later six of the cows (the treated group) received a second injection of 10 micrograms buserelin, but the remaining five received no further treatment (control group). The dominant, that is, the largest follicle in each cow disappeared after the first buserelin injection and was replaced by a new one which grew synchronously in all the cows until after the treatment with PGF2 alpha. Ovulation occurred significantly earlier after PGF2 alpha in the treated group than in the control group (72 to 96 hours v 96 to 120 hours; P < 0.05). Plasma progesterone concentrations then increased more rapidly in the treated group than in the control group and were significantly higher on days 3 and 4 after ovulation (P < 0.05).     

Dynamics and histological observation of first follicular wave in Japanese black cows

AIMS: To determine the turnover of the first follicular wave in Japanese black cows and quantitative immunohistological characteristics of the previously in vivo identified dominant follicle (DF) and largest subordinate follicle (SF) derived from ovariectomy on Day 7 (3 cows) and Day 10 (3 cows) (Day 0=estrus). Six cases of first follicular wave in cows were observed twice daily by ultrasound scanning. The number of follicles, diameter of DF and SF, and prevalence of apoptotic granulosa cells (GC) and theca cells (TC) were studied by TUNEL methods. At follicular wave emergence, 13.5 +/- 9.5 Class I (2-5 mm in diameter follicles) were found 12 hr after ovulation, and increased its number until Day 1 pm. Future DF and SF observed retrospectively were 4.9 +/- 0.8 mm and 4.9 +/- 0.9 mm at wave emergence. Deviation of DF and SF occurred on Day 3 pm with mean diameters of 8.9 +/- 1.3 mm and 6.8 +/- 0.9 mm, respectively. DF developed until Day 8 am with a maximum diameter of 14.4 +/- 1.8 mm (n=3) and then regressed. The follicular wall of the DF had a characteristic image of a healthy follicle on Day 7 and slightly atretic DF on Day 10, whereas SF showed heavy atresia on both Day 7 and Day 10 under HE staining. In the prevalence of apoptotic cells, DF were 4.4 +/- 1.0% and 17.9 +/- 4.9% on Day 7 and on Day 10 in GC, respectively, and 2.4 +/- 0.7% and 8.0 +/- 1.4% on Day 7 and on Day 10 in TC, respectively. These results showed that, 1) the first follicular wave in cows is characterized by 24 hr recruitment of small follicles and a gradual divergence of growth rates in future DF and SF, and 2) early regression of DF on Day 10 was preceded by severe apoptosis.     

Inhibition of intrafollicular PGE2 synthesis and ovulation following ultrasound-mediated intrafollicular injection of the selective cyclooxygenase-2 inhibitor NS-398 in cattle

Ultrasound-mediated intrafollicular injection and aspiration procedures were used to investigate the ability of the selective cyclooxygenase-2 inhibitor, NS-398, to inhibit intrafollicular PGE2 synthesis and suppress ovulation in dairy cattle. Follicular growth and timing of the preovulatory gonadotropin surge were synchronized in 55 Holstein cows and the position of the ovulatory follicle was determined by daily ultrasound scanning. Preovulatory follicular fluid was aspirated from the largest follicle in four animals at 0, 6, 12, 18, and 24 h after GnRH injection (n = 20). The remaining 35 animals were subjected to ultrasound-mediated intrafollicular injection of NS-398 (10 microM final concentration; n = 19) or diluent (n = 16; controls). At 24 h after GnRH injection, follicular fluid was harvested from a subset of NS-398- (n = 9) and diluent-treated animals (n = 6). The remaining NS-398- and diluent-treated animals were subjected to ultrasonography every 6 h for 36 h after intrafollicular injection, and then daily through d 7 of the subsequent luteal phase to monitor ovulation and corpus luteum development. Follicular fluid PGE2 concentrations were increased following GnRH injection and reached a maximum at 24 h (P < 0.05). Follicular fluid PGE2 concentrations were decreased in NS-398- vs. diluent-treated follicles (7.2 vs. 52.2 ng/mL respectively; P < 0.05), but progesterone concentrations did not differ. Intrafollicular injection of NS-398 also inhibited follicle rupture (P < 0.001). All 10 control animals ovulated within 30 h of GnRH injection. Nine out of the ten NS-398-injected animals failed to ovulate. The NS-398-injected follicles developed morphological and endocrine characteristics resembling luteinized, unruptured follicles. Thus, intrafollicular PGE2 synthesis and follicle rupture, but not luteinization, were inhibited in cattle following ultrasound-mediated intrafollicular injection of NS-398. Ultrasound-mediated intrafollicular injection of NS-398 is a useful tool for mechanistic studies of intrafollicular regulation of the ovulatory process in cattle.     

Uterine Infection Influences Size and Follicular Fluid Composition of the Largest Follicle in Buffalo (Bubalus bubalis)

The effect of uterine infection on size and follicular fluid composition of the largest follicle was studied in buffalo. Reproductive tracts were collected from 102 graded Murrah buffaloes at an abattoir. Uterine infection was diagnosed by physical examination of uterine mucus, white side test and uterine cytology. Samples with pus‐containing mucus, positive reaction on white side test and/or >5% neutrophils were considered to be positive for uterine infection. Diameter of the largest follicle was measured, and follicular fluid was aspirated and assayed for nitric oxide (NO), ascorbic acid (AA), cholesterol, oestradiol (E₂) and progesterone (P₄). Infected buffaloes had smaller‐sized (p < 0.0001) largest follicles than non‐infected buffaloes. Follicular fluid collected from the largest follicle in infected buffaloes had greater (p < 0.0001) NO and P₄ concentrations coincident with lesser AA (p < 0.001), cholesterol (p < 0.0001) and E₂ (p < 0.0001) concentrations. Results indicated that uterine infection has an inhibitory effect on growth of the largest follicle in buffalo. The changes in follicular fluid composition in infected buffaloes suggest that the direct effect of uterine infection on ovarian function may be mediated through an alteration in the follicular microenvironment. Greater NO and lesser AA concentrations in the follicular fluid of infected animals are novel findings.     

Application of timed artificial insemination protocols to grazing Japanese black cattle with long open period

To investigate the viability of timed artificial insemination (TAI) protocols in grazing Japanese Black cattle with long open period, ovarian status and progesterone and estradiol-17 beta profiles of the animals during the protocol were monitored. In 1998, prosta-glandin F(2a) (PG) was administered to 36 animals seven days after GnRH injection. Three out of the 36 animals were inseminated after detection of estrus and did not receive further treatment. The second GnRH was injected to the remaining 33 animals 48 hr after the PG injection and TAI was performed 24 hr later. In 1999, PG was injected to 25 animals six days after GnRH, the second GnRH was injected to 22 animals 48 hr after PG, and TAI was performed 16 hr later (The other three animals were inseminated before the time of TAI). The percentage of the animals with at least one functional corpus luteum and one follicle equal to or greater than 10 mm in diameter at PG injection was similar between the groups in 1998 and 1999. Likewise, the hormonal profiles were similar between the two groups. Pregnancy rates (PR) after the TAI protocols and natural mating in 1998 and 1999 were 75.0% and 88.0%, respectively. These figures were comparable to the PR obtained by conventional estrus synchronization protocols using PG (in 1995; 69.4%) or CIDR (in 1996; 59.1%). In conclusion, the TAI protocol can be applicable into grazing Japanese Black cattle with long open period.     

Follicular development,oocyte viability and recovery in relation to follicular steroids,prolactin and glycosaminoglycans throughout the estrous period in superovulated heifers with a normal LH surge,no detectable LH surge,and progestin inhibition of LH surge

Estrous cycles of heifers (n = 137) were synchronized with prostaglandin (PGF_2α) and follicular development stimulated with follicle stimulating hormone. Twenty-eight animals were administered Norgestomet implants 12 hr prior to the initial PGF2α injection to suppress the LH surge that initiates ovulation. Animals were ovariectomized every 12 hr after the initial PGF2α (7–9/time, 12–108 hr and at 192 and 240 hr post PGF2α) and divided into three treatment groups to consist of: 1) animals exhibiting a normal luteinizing hormone (LH) surge (n = 86), 2) animals in which no LH surge was detected (n = 23), and 3) suppression of the LH surge via Norgestomet implants (72–108 hr, n = 28). Follicular diameter was measured and follicular fluid was collected for analysis of prolactin, estradiol, progesterone and glycosaminoglycan concentrations. Progesterone concentrations were increased in animals exhibiting an LH surge as compared to animals in which no LH surge was detected; primarily in large follicles (> 8 mm diameter) after the LH surge. Animals not exhibiting an LH surge also had increased follicular progesterone concentrations compared to Norgestomet-implanted animals (242.3 ± 36.3 vs 86.7 ± 6.4 ng/ml, respectively, P < .01), indicating some LH stimulation. Follicular estradiol in animals exhibiting an LH surge increased up to the time of LH surge detection and then declined whereas animals with no LH surge detected had follicular estradiol concentrations that declined after the PGF_2α injection. No differences were noted between those that did not exhibit an LH surge or in which the LH surge was suppressed with Norgestomet in relation to follicular estradiol concentrations. Follicular estradiol concentrations increased with follicular size in all treatment groups (P < .01). Follicular concentrations of prolactin were increased in small follicles (P < .05; ≤ 4 mm diameter) and follicular prolactin increased from 12 to 36 hr post PGF2α injection, then declined after the LH surge. Follicular glycosaminoglycan concentrations decreased with increases in follicular size (P < .01) and were higher in animals that did not exhibit an LH surge (P < .01). No differences in follicular glycosaminoglycans were noted between Norgestomet-implanted animals and those not exhibiting an LH surge. In the animals representing days 4 and 6 of the subsequent estrous cycle (192 and 240 hr post PGF2α), numbers of small-sized follicles were increased. Follicular progesterone and estradiol concentrations were related to atretic large follicles unovulated from the prior estrus and a wave of growth in small and medium follicles. Follicular prolactin and glycosaminoglycans increased with time of the new estrous cycle and were increased in smaller follicles (P < .01). Suppression of LH with progestin implants (Norgestomet) may relate to early effects of progesterone, which may not be totally eliminated at target tissues and subsequently alters the LH surge, steroidogenesis of the follicle, and ovulation. Oocytes were predominantly found in the follicular fluid from animals in which an LH surge was detected and in the buffer wash of follicles in which no LH surge was detected. Oocyte viability was higher in animals exhibiting an LH surge (75% viable) whereas the oocytes of Norgestomet-implanted animals were 75% degenerate.     

Role of prostaglandin F2 alpha in follicular development and subsequent luteal life span in early postpartum beef cows.

In postpartum cows expected to have corpora lutea (CL) of normal (norgestomet-treated) compared to short (control) life spans, function of the largest follicle increases after an increase in concentrations of prostaglandin F2 alpha (PGF). To determine whether PGF alters follicular growth and subsequent life span of the CL, 43 crossbred beef cows (19 to 22 d postpartum) were assigned to one of four treatments: 1) control (C; n = 10), 2) control+PGF (CPGF; n = 10), 3) norgestomet (N; n = 13), 4) norgestomet+flunixin meglumine (NFM; n = 10). Flunixin meglumine inhibits prostaglandin endoperoxide synthase. On day 0, N and NFM cows received a 6 mg implant of norgestomet. From days 3 through 8, CPGF and NFM cows were injected every 8 hr with 10 mg PGF im or 1 g FM iv, respectively. Implants were removed on day 9. On day 11, each cow received 1000 IU of hCG im to induce formation of CL. Follicular growth was monitored by daily ultrasonography from days 6 through 11. In a majority of the cases (25/32), the largest follicle present on day 6 was still the largest on day 11; frequency of persistence did not differ with treatment. Rate of growth of the largest follicle was greater in CPGF than in N cows (.6 +/- .1 vs .3 +/- .1 mm/d, respectively; P less than .05) but did not differ between C and NFM cows (.4 +/- .1 and .5 +/- .1 mm/d, respectively). Concentrations of estradiol in NFM cows were higher (P less than .05) on day 3 and declined to concentrations similar to those of the other treatments on day 9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood LH level and induced ovulation after hCG and PMSG treatment in ovarian quiescent cattle

Ovarian quiescent cattle bearing follicle with palpable size were treated with single intramuscular injection of 750-6,000 IU of human chorionic gonadotrophin (hCG) in 13 cases and 1,000-2,000 IU of pregnant mare serum gonadotrophin (PMSG) in 5 cases. Changes of blood luteinizing hormone (LH) level, estrus and ovulation after the treatments were examined. After the hCG treatment LH level became slightly high from 0.2-0.6 ng/ml of pre-treatment to 0.3-1.9 ng/ml of post-treatment and maintained the level up to ovulation without the ovulatory LH surge. Ovulation was induced about 36 hr after the treatment in 12 cases. The ovulations were all silent ovulations. After the PMSG treatment LH level became slightly high from 0.6 ng/ml of pre-treatment to 1.3 ng/ml of post-treatment and the level lasted until the ovulatory LH surge. The ovulatory LH surge occurred about 39 hr after the PMSG treatment in 4 cases with a peak of about 32 ng/ml. Ovulation was induced about 74 hr after the treatment in all 5 cases. Four cases showed estrus but one in which the LH surge could not be confirmed did silent estrus preceding the induced ovulations. It was demonstrated that hCG induced ovulation without the LH surge but PMSG induced the ovulatory LH surge and the subsequent ovulation in ovarian quiescent cattle.     

In situ hybridization analysis of ovarian prostaglandin endoperoxide synthase mRNA throughout the periovulatory period of the ewe.

Cellular alterations in level of expression of mRNA encoding for prostaglandin endoperoxide synthase were quantified within ovarian tissues of sheep obtained before, during and after induction of the preovulatory surge of LH and ovulation with LHRH. This was accomplished by isotopic in situ hybridization using a selective cRNA probe to ovine prostaglandin endoperoxide synthase mRNA. A significant elevation in mRNA was detected within the theca interna of the preovulatory follicle at 8, 16 and 24 hr following administration of LHRH. Very close to the time of ovulation (ie., at 24 hr post-LHRH) a marked rise in mRNA was observed in association with epithelial cells covering the apical surface of the follicle. Ovarian cyclooxygenase metabolites of arachidonic acid produced during the ovulatory process in the ewe originate within the thecal layer and germinal epithelium of the follicle destined to ovulate.     

Effect of a nonsteroidal aromatase inhibitor on in vitro and in vivo secretion of estradiol and on the estrous cycle in ewes.

Three experiments were performed to study effects of decreased concentrations of estradiol-17β (E₂) on lifespan and function of ensuing ovine corpora lutea (CL). In experiment 1, 52 follicles were collected from 10 ewes and placed into individual culture with 0 or .01 μCi ³H-androstenedione (10 ng; ³H-A) and 0, 10⁻¹¹, 10⁻⁹, 10⁻⁷, or 10⁻⁵ M of a nonsteroidal aromatase inhibitor, CGS16949A (CGS). Concentrations of E₂ secreted into the medium, and synthesis of estrogens as estimated by formation of ³H-water from ³H-A were decreased by 10⁻⁵ and 10⁻⁷ (P<.01), but not 10⁻⁹ or 10⁻¹¹ M CGS. In experiment 2, luteolysis was induced in 24 ewes by injection of PGF₂ on days 5 to 10 of the estrous cycle (0 hr). Ewes received 0, 0.5, 1.0, 2.0 or 4.0 mg CGS per kg BW i.v. at −12, 0, 12 and 24 hr, and an ovulatory dose of hCG at 36 hr. Jugular (P<.001) and vena caval (P<.001) concentrations of E₂ were decreased by CGS at all doses tested for 8 to 10 hr, but had returned to levels similar to control ewes by the time of the next injection. Concentrations of E₂ around the time of the LH surge were similar in control and treated ewes. During the subsequent luteal phase, concentrations of progesterone (P₄) were similar in control and treated ewes. Thus, transient decreases in E₂ during the follicular phase were not deleterious to the subsequent luteal phase. In experiment 3, luteolysis was induced in 18 ewes by injection of PGF₂ on days 6 or 7 (0 hr) of the estrous cycle. Ewes received 0 or 1 mg CGS per kg BW i.v. every 8 hr from 0 to 40 hr. Ovulation was induced with hCG at 36 hr. CGS reduced jugular (P<.001) and vena caval (P<.001) concentrations of E₂, prevented an endogenous surge of LH (P<.05) and increased (P<.001) concentrations of FSH. All ewes had ovulated a marked follicle by 72 hr, but onset of the luteal phase, as assessed by concentrations of P₄, was delayed (P<.01) in ewes receiving CGS. Delayed luteal phases were not solely attributable to the presence of new CL or to luteinization of follicular cysts. When data were aligned according to the day ewes were observed in estrus, profiles of P₄ did not differ with treatment. Therefore, normal luteal function ensued following estrus whether or not ewes re-ovulated. In conclusion, decreased secretion of E₂ by the preovulatory follicle was not involved in the ontogeny of CL of short lifespan or subnormal function. Instead, adequate production of E₂ or precisely timed E₂ secretion may be required during follicular development for subsequent functional luteinization.     

Follicular development in cattle; changes in their count and size during the ovarian cycle

Follicular development was examined by transrectal ultrasound scanning in 12 heifers during 51 oestrous cycles. Internal diameters of largest and second largest follicles and the number of smaller ovarian vesicles were determined. Diameters of dominant follicles showed inverse growth pattern to the second largest follicles and numbers of smaller follicles (greater than or equal to 5 mm). There was an increase in diameters of the largest follicles from beginning of dioestrous to day 9 and from time of luteolysis to ovulation, which was coincident which a decrease in diameters of the second largest follicles and numbers of smaller ovarian vesicles. Smaller follicles increased in count and diameter, when the dominant follicle achieved its largest dimension and started to regress. The cyclic corpus luteum had no local influence on diameters of the largest and second largest follicles in the ovary bearing the corpus luteum versus the contralateral ovary. Internal diameters of oestrous follicles measured 14.7 +/- 2.6 mm in heifers and 15.3 +/- 2.9 mm in cows at the day of oestrous (p greater than 0.05; t-test). Dioestrous follicles with similar size were detected during various stages of the oestrous cycle. The diameter of the dominant follicle is not an accurate criterion for determining the stage of the oestrous cycle.     

Supplementation of heterologous complement induces anti-Thy-1.1 nephritis in the Mongolian gerbil (Meriones unguiculatus)

Anti-Thy-1.1 nephritis in the rat is a popular experimental model for mesangial proliferative glomerulonephritis (GN). This model is characterized by direct binding of anti-Thy-1.1 antibody with Thy-1.1 antigen expressed on mesangial cells (MCs) of glomeruli in the rat. A single injection of anti-rat thymocyte serum (ARTS) results in GN with proteinuria and extensive mesangiolysis. Development of mesangiolysis and proteinuria are complement-dependent. We previously demonstrated Thy-1.1 antigen, similar to the rat, in thymocytes, brain cells and MCs of the kidney in the Mongolian gerbil (MG). In this study, we attempted to develop a MG nephritis model, but an injection of ARTS did not induce GN. An additional injection of guinea pig serum as a complement after ARTS injection resulted in anti-Thy-1.1 nephritis in MG. Degeneration of MCs and neutrophil infiltration were observed 1 hr after GP serum injection. Mesangiolysis and fibrin exudation occurred 12 hr after the injection and MC proliferation was apparent 7 days after the injection. In the complement-dependent hemolytic test, MG serum could not hemolyze sheep erythrocytes. These results suggested low activity, or depletion of some factors, in complements of MG serum.     

Effect of the dominant follicle aspiration before or after luteinizing hormone surge on the corpus luteum formation in the cow

Luteinizing hormone (LH) surge and follicle rupture act as trigger to start corpus luteum (CL) formation. Thus, we aimed to investigate whether a dominant follicle that has not been exposed to an LH surge can become a functional CL. For this purpose, follicular fluid from the dominant follicles (DF) of cows was aspirated before or after a GnRH-induced LH surge, and subsequent CL formation was observed. Holstein cows were divided into four groups as follows: Luteal phase, a DF was aspirated 7 days after GnRH injection; Pre-LH surge, a DF was aspirated 42 h after PGF(2alpha) injection during the mid luteal phase; Post-LH surge, a DF was aspirated 24 h after GnRH injection following PGF(2alpha); and Intact follicle, ovulation was induced by GnRH injection after PGF(2alpha). Observation of morphological changes in the aspirated follicle using color Doppler ultrasonography and blood sampling was performed on Days 0, 3, 6, and 9 (Day 0 = follicle aspiration). CL formation following DF aspiration was observed only in the Post-LH surge group. In both the Luteal phase and Pre-LH surge groups, however, none of the cows showed local blood flow at the aspirated site or CL formation. Luteal blood flow area, CL volume, and plasma progesterone concentration in the Post-LH surge group were no different from those in the Intact follicle group. The present results clearly demonstrate that rather than follicle rupture, it is the LH surge that is essential for CL formation in cows.     

Expression pattern of HIF1alpha and vasohibins during follicle maturation and corpus luteum function in the bovine ovary

The aim of this study was to characterize expression patterns of hypoxia‐inducible factor‐1alpha (HIF1A) and vasohibin family members (VASH1 and VASH2) during different stages of ovarian function in cow. Experiment 1: Antral follicle classification occurred by follicle size and estradiol‐17beta (E2) concentration in the follicular fluid into 5 groups (<0.5, 0.5–5, 5–40, 40–180 and >180 E2 ng/ml). Experiment 2: Corpora lutea (CL) were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–16 and >18 (after regression) of oestrous cycle and of pregnancy (months 1–2, 3–4, 6–7, >8). Experiment 3: Cows on days 8–12 were injected with a prostaglandin F2alpha (PGF) analogue and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64 hr after PGF injection. Expression of mRNA was measured by qPCR, steroid hormone concentration by EIA and localization by immunohistochemistry. HIF1A mRNA expression in our study increases significantly in follicles during final maturation. The highest HIF1A mRNA expression was detected during the early luteal phase, followed by a significant decrease afterwards. In contrast, the mRNA of vasohibins in small follicle was high, followed by a continuous and significant downregulation in preovulatory follicles. The obtained results show a remarkable inverse expression and localization pattern of HIF1A and vasohibins during different stages of ovarian function in cow. These results lead to the assumption that the examined factors are involved in the local mechanisms regulating angiogenesis and that the interactions between proangiogenic (HIF1A) and antiangiogenic (vasohibins) factors impact all stages of bovine ovary function.