Changes in the messenger RNA expressions of the endothelin-1 and angiotensin systems in mature follicles of the superovulated bovine ovary

The aim of the present study was to examine the messenger RNA expressions of the endothelin and angiotensin systems during the periovulatory phase in gonadotrophin releasing hormone (GnRH)-treated cows. Ovaries were collected by transvaginal ovariectomy (n=5 cows/group), and the follicles (n=5, one follicle/cow) were classified into the following groups: before GnRH administration (control, before LH surge), 3-5 h after GnRH (during LH surge), 10 h after GnRH; 20 h after GnRH, 25 h after GnRH (peri-ovulation), and early corpus luteum (CL) (Days 2-3). Expression of mRNA was investigated using quantitative real-time PCR. The expression of angiotensin converting enzyme (ACE) mRNA significantly decreased immediately after onset of the LH surge and remained at low levels. The levels of angiotensin II receptor type 1 (AT1R) and type 2 (AT2R) expression during the periovulatory period significantly decreased compared with other periods. The concentration of angiotensin II in follicular fluid began to increase 10 h after GnRH treatment and further increased as ovulation approached. The level of ET-1 mRNA significantly decreased 10 h after GnRH treatment compared with the levels before GnRH treatment and those of the early CL period. The expression of ETR-A and ETR-B mRNA during the periovulatory period were lower than in other periods. The expression of ECE-1 mRNA began to decrease in the LH surge period and significantly decrease in the periovulatory period compared with other periods. These results suggest that the vasoactive peptides angiotensin and endothelin may be associated with final maturation of follicles.     

Expression of fibroblast growth factor 1 (FGF1) and FGF7 in mature follicles during the periovulatory period after GnRH in the cow

The aim of this study was to evaluate the expression pattern of mRNA for fibroblast growth factor 1 (FGF1), FGF7, and their receptor variants (FGFR2IIIb) in time-defined follicle classes before LH surge, between LH surge and ovulation, and in the early corpus luteum (CL) in the cow. The ovaries were collected by transvaginal ovariectomy (n=5 cows/group), and the follicles (n=5, one follicle/cow) were classified into the following groups: before GnRH administration (before LH surge); 3-5 h after GnRH (during LH surge); 10 h after GnRH; 20 h after GnRH; 25 h after GnRH (periovulation), and early CL (Days 2-3). The mRNA expression was analyzed by quantitative real-time PCR (RotorGene 3000). The mRNA expression of FGF1 showed no significant differences in the follicle groups examined, but increased significantly at the early CL phase. A transient increase in FGF7 mRNA expression was observed 3-5 h after GnRH and again in the early CL phase. In contrast, the expression of FGFR2IIIb was constant throughout the period from the final growth of the follicle to early CL formation. The results of this study suggest that FGF1 and FGF7 may be involved differently in the process of follicle maturation and CL formation, which is strongly dependent on angiogenesis.     

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.     

Hypoxia-inducible factor-1alpha and nitric oxide synthases in bovine follicles close to ovulation and early luteal angiogenesis

The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n = 5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.     

The effects of administering estradiol benzoate plus progesterone during the growth or static phases of the dominant follicle in CIDR-treated lactating dairy cows

We studied the effects of administering estradiol benzoate (EB) plus progesterone (P4) as part of a CIDR-based protocol during the growth or static phases of dominant follicle development on follicular wave emergence, follicular growth, synchrony of ovulation and pregnancy rate following CIDR withdrawal, treatment with PGF(2alpha) and GnRH, and fixed-time artificial insemination (TAI). Forty-one previously synchronized lactating Holstein dairy cows were randomly allocated to three treatment groups. The control group (n=14) received a CIDR on the third day after ovulation only (Day 0). The two treatment groups were administered CIDRs comprising 2 mg EB and 50 mg P4 either on the third (T1, n=14) or eighth day (T2, n=13) after ovulation (Day 0). All cows received PGF(2alpha) after CIDR removal on Day 7, GnRH on Day 9, and TAI 16 h after GnRH treatment. The proportion of cows with follicular wave emergence within 8 days of treatment differed (P<0.01) among the control (14.3%), T1 (85.7%), and T2 groups (92.9%). However, the mean intervals between treatment and wave emergence were not significantly different. There were significant differences in the diameters of the dominant follicles on Day 7 (P<0.01) and in preovulatory follicles on Day 9 (P<0.01), with the largest follicles observed in the control group and the smallest follicles observed in the T2 group. In contrast, the numbers of cows showing synchronous ovulation after GnRH treatment (92.9 to 100.0%) and pregnancy following TAI (46.2 to 50.0%) were similar between the treatment groups. The results showed that, irrespective of the phase (growth or static) of the dominant follicle, administration of 2 mg EB plus 50 mg P4 to CIDR-treated lactating dairy cows induced consistent follicular wave emergence and development, synchronous ovulation after GnRH administration, and similar pregnancy rates following TAI.     

Effects of Full-Length Kisspeptin Administration on Follicular Development in Japanese Black Beef Cows

Kisspeptin is a key molecule that stimulates gonadotropin secretion via release of gonadotropin-releasing hormone (GnRH). In the present study, our aim was to investigate whether kisspeptin has stimulatory effects on follicular development via GnRH/gonadotropin secretion in cows. Japanese Black beef cows were intravenously injected with full-length bovine kisspeptin [Kp-53 (0.2 or 2 nmol/kg)] or vehicle 5 days after they exhibited standing estrus (Day 0). In cows injected with Kp-53 at 2 nmol/kg, the follicular sizes of the first dominant follicles increased on Day 6 and thereafter. Ovulation of the first dominant follicle occurred in 1 out of 4 cows treated with Kp-53 at 2 nmol/kg. Injection of Kp-53 at 2 nmol/kg increased the concentration of plasma luteinizing hormone (LH) but not follicle-stimulating hormone, over a 4-h period following injection in all cows. The present study suggests that administration of full-length kisspeptin causes LH secretion, which is sustained for a few hours, and it is capable of stimulating follicular development and/or ovulation.     

Comparison of oestrus synchronisation programmes in dairy cattle using oestradiol benzoate, short-acting progesterone and cloprostenol, or buserelin and cloprostenol

AIM: To evaluate the efficacy of a programme using oestradiol benzoate, progesterone and the prostaglandin-F2 (PG) analogue, cloprostenol, to synchronise oestrus and ovulation in dairy cows, compared with a programme using a gonadotropinreleasing hormone (GnRH) agonist, buserelin, and cloprostenol. METHODS: Twenty non-lactating dairy cows, at random stages of the oestrus cycle, were randomly assigned to 1 of 2 treatments. In Treatment 1 ( OPPG; n=10), cows were injected with 2 mg oestradiol benzoate intramuscularly (IM) plus 200 mg progesterone subcutaneously (SC) on Day 0, followed by 500 microg cloprostenol IM on Day 9 and 1 mg oestradiol benzoate on Day 10. In Treatment 2 (GPG; n=10), cows were injected with 10 microg buserelin IM on Day 0, 500 microg cloprostenol IM on Day 7 and 10 microg buserelin on Day 9. The ovaries of all cows were examined by ultrasonography, using an 8 MHz probe, from 5 days before the initial treatment until ovulation. Cows were observed for oestrus 3 times daily for 7 days after cloprostenol treatment. Blood samples were collected daily for determination of progesterone, and 6-hourly for 36 h after the second oestradiol or buserelin injection for the determination of follicle stimulating hormone (FSH) and luteinising hormone (LH) concentrations. RESULTS: The percentage of cows observed in oestrus was higher in the OPPG group than in the GPG group (100% vs 55.6%, p=0.018). Treatment with either short-acting progesterone plus oestradiol benzoate or buserelin was followed by atresia or ovulation of the dominant follicle. Emergence of a new follicular wave occurred earlier (p>0.001) in the GPG group (2.2+/-0.2 days) than in the OPPG group (3.6+/-0.2 days). There was no significant difference between treatment groups in the variation of time of follicular wave emergence or size of the largest follicles at either the time of initial treatment (10.8+/-1.4 mm vs 11.1+/-0.8 mm), cloprostenol treatment (13.8+/-0.7 mm vs 14.0+/-1.3 mm) or of ovulation (15.4+/-0.7 mm vs 17.6+/-1.1 mm; p=0.10). The LH surge occurred sooner after the second injection of buserelin (4.0+/-1.0 h) than after the second injection of oestradiol benzoate (22.8+/-1.2 h; p>0.001). The interval between the second injection of oestradiol benzoate or buserelin and ovulation did not differ significantly between treatment groups (1.7+/-0.3 days vs 1.6+/-0.2 days; p=0.69). CONCLUSIONS: The use of short-term progesterone treatment, combined with oestradiol benzoate for follicular wave synchronisation, and cloprostenol to cause lysis of residual luteal tissue, is a promising alternative to established methods of oestrus synchronisation in cows.     

GnRH treatment at CIDR insertion influences ovarian follicular dynamics in Japanese black cows

Ovarian follicular dynamics and estrous synchronization after Gonadotropin-releasing hormone (GnRH) treatment at Controlled Internal Drug Releasing device (CIDR) insertion were investigated in Japanese Black cows. CIDR was inserted for eight cows at 7 days after estrus. Cows were allocated to either Group A: 8-day CIDR insertion with GnRH treatment on d 0 (n=4, d 0=CIDR insertion) or Group B: 8-day CIDR insertion (n=4). Both groups were injected with prostaglandin F2alpha (PGF2alpha) on d 7. Ultrasonography and blood sampling were performed twice daily. Intensive sampling was performed every 15 min for 8 hr to determine the pulsatile release of LH on d -1, d 5 and d 10. Three of four cows showed intermediate ovulation within 2 days after GnRH treatment during CIDR insertion in Group A, whereas no ovulation was found in Group B. Three of four cows in Group A and all four cows in Group B ovulated after CIDR removal. Plasma progesterone concentrations from d 3 to d 7 in three intermediate ovulatory cows in Group A (8.4 +/- 1.6 ng/ml) was significantly higher than those in Group B (4.1 +/- 1.2 ng/ml; 4 cows) during CIDR insertion (P<0.01). Interval to estrus and ovulation after CIDR removal was observed at 60.0 +/- 12.0 hr and 76.0 +/- 6.9 hr in three cows in Group A, and 75.0 +/- 15.1 hr and 93.0 +/- 20.5 hr in Group B, respectively. There was a significant increase in LH pulse frequency on d 10 compared on d -1 or d 5 in both groups (P<0.05), in addition those on d 10 in Group A tended to be higher than in Group B. As a result, GnRH treatment at CIDR insertion at 7 days after estrus induced intermediate ovulation with formation of corpus luteum (CL) and rather synchronized emergence of ovulatory follicle during CIDR insertion. These induced CL increased plasma progesterone concentrations and contributed to precise synchronization.     

Factors Affecting Synchronization and Conception Rate after the Ovsynch Protocol in Lactating Holstein Cows

Objectives were to evaluate risk factors affecting ovulatory responses and conception rate to the Ovsynch protocol. Holstein cows, 466, were submitted to the Ovsynch protocol [day 0, GnRH‐1; day 7, prostaglandin (PG) F_2α; day 9, GnRH‐2] and 103 cows were inseminated 12 h after GnRH‐2. Information on parity, days in milk at GnRH‐1, body condition, milk yield, exposure to heat stress, pre‐synchronization with PGF_2α and the use of progesterone insert from GnRH‐1 to PGF_2α was collected. Ovaries were scanned to determine responses to treatments. Overall, 54.7%, 10.6%, 2.2%, 81.1%, 9.0%, 91.5% and 36.9% of the cows ovulated to GnRH‐1, multiple ovulated to GnRH‐1, ovulated before GnRH‐2, ovulated to GnRH‐2, multiple ovulated to GnRH‐2, experienced corpus luteum (CL) regression and conceived, respectively. Ovulation to GnRH‐1 was greater in cows without a CL at GnRH‐1, cows with follicles >19 mm and cows not pre‐synchronized with PGF_2α 14 days before GnRH‐1. Multiple ovulations to GnRH‐1 increased in cows without CL at GnRH‐1 and cows with follicles ≤19 mm at GnRH‐1. Ovulation before GnRH‐2 was greater in cows without CL at PGF_2α. Ovulation to GnRH‐2 increased in cows that received a progesterone insert, cows with a CL at GnRH‐1, cows with follicles not regressing from the PGF_2α to GnRH‐2, cows with larger follicles at GnRH‐2, cows that ovulated to GnRH‐1 and cows not pre‐synchronized. Multiple ovulations after GnRH‐2 increased in cows with no CL at GnRH‐1, multiparous cows and cows that multiple ovulated to GnRH‐1. Conception rate at 42 days after AI increased in cows with body condition score > 2.75 and cows that ovulated to GnRH‐2. Strategies that optimize ovulation to GnRH‐2, such as increased ovulation to GnRH‐1, should improve response to the Ovsynch protocol.     

Effect of Suppression of FSH with a GnRH Antagonist (Acyline) Before and During Follicle Deviation in the Mare

A GnRH antagonist (Acyline) was used to study the role of FSH in early development of a follicular wave in 61 mares. In Experiment 1, a single dose of 3 mg per mare, compared with 0 and 1 mg, suppressed both the FSH and follicle responses to exogenous GnRH. In Experiment 2, high concentrations of FSH were induced by two successive ablations of all follicles ≥ 6 mm on days 10 and 13 (day 0 = ovulation). A single treatment with Acyline resulted in significantly greater suppression of plasma concentrations of FSH than a single treatment with charcoal-extracted follicular fluid (source of inhibin) or oestradiol. Suppression of FSH was not significantly different between the group treated with Acyline alone and a group treated with a combination of Acyline, inhibin and oestradiol. In Experiment 3, all follicles were ablated on day 10 to induce an FSH surge and a new follicular wave. Acyline treatment on day 10 resulted in an immediate decrease in FSH, without a significant effect on day of emergence of a new wave or growth of follicles from 7 to 11 mm on days 11–13. Treatment on day 15, a day before expected follicle deviation and after the peak of the wave-stimulating FSH surge, resulted in an immediate decrease in FSH and cessation of follicle growth. Results indicated that growth of follicles for about 2 days after wave emergence was independent of FSH. In contrast, during the decline in the wave-stimulating FSH surge and before follicle deviation, growth of follicles was dependent on FSH.     

Timing of preovulatory LH surge and ovulation in superovulated sheep are affected by follicular status at start of the FSH treatment.

The aims of this study were to evaluate the chronology of periovulatory events (oestrus behaviour, LH surge and ovulation) in 16 superovulated Manchega sheep and to determine whether follicular status at start of the FSH supply might affect their occurrence. Mean timing for onset of oestrus behaviour was detected at 28.1 +/- 0.7 h after sponge withdrawal; the preovulatory LH surge and ovulation started at 37.2 +/- 0.7 h and 65.4 +/- 0.7 h after progestagen withdrawal, respectively. The intervals between oestrus, LH surge and ovulation were affected by a high individual variability, which might be the cause for reported decreased efficiency in embryo production. Current results also addressed the role of follicular status at start of the superovulatory treatment on the preovulatory LH surge and the ovulation. The interval LH surge-ovulation was increased in ewes with a growing dominant follicle at starting the FSH treatment (32.3 +/- 0.9 vs 28.6 +/- 0.5 h, p < 0.05). The developmental stage of the largest follicle at starting the superovulatory treatment also affected occurrence of LH surge and ovulation; follicles in growing phase advanced the occurrence of the LH surge and ovulation when compared to decreasing follicles (33.0 +/- 1.0 vs 43.5 +/- 1.1 h, p < 0.05, for LH peak and 60.7 +/- 1.1 vs 72.8 +/- 1.2 h, p < 0.05, for ovulation). Thus, only ewes with growing follicles ovulated prior to 55 h after sponge withdrawal; conversely, no sheep with decreasing follicles ovulated earlier than 67 h, when an 85.7% of the ewes bearing growing follicles has ovulated at 63 h.     

Effect of the preovulatory LH surge on bovine follicular progesterone receptor mRNA expression

A growing body of evidence indicates that intrafollicular progesterone receptor signaling pathways are obligatory for follicle rupture. However, the intrafollicular localization and regulation of progesterone receptor expression during the periovulatory period in cattle are not known. In this study, we determined the effect of the preovulatory gonadotropin surge on localization and expression of progesterone receptor mRNA in bovine periovulatory follicular and luteal tissue. Ovaries containing preovulatory follicles or new corpora lutea (CL) were collected at approximately 0, 6, 12, 18, 24 (preovulatory follicles) and 48 h (CL) after a GnRH-induced LH surge (n=5-8 per timepoint). Expression of progesterone receptor mRNA was detected in periovulatory follicular and luteal tissue at all timepoints examined. Relative levels of progesterone receptor mRNA were dramatically upregulated within 6h after the LH surge compared to all other time points (P<0.0001). In situ hybridization analysis revealed that the significant increase in progesterone receptor mRNA expression was localized to the granulosal layer of preovulatory follicles. Our results indicate that progesterone receptor mRNA expression is upregulated specifically in the granulosal layer of bovine preovulatory follicles following the LH surge. Progesterone receptor signaling pathways may help mediate the effects of the preovulatory LH surge on follicle rupture in cattle.     

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.     

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.     

Expression and Localization of Gap Junctional Connexins 26 and 43 in Bovine Periovulatory Follicles and in Corpus Luteum During Different Functional Stages of Oestrous Cycle and Pregnancy

The aim of this study was to characterize the regulation of connexins (Cx26 and Cx43) in the bovine ovary (experiment 1–3). Experiment 1: ovaries containing preovulatory follicles or corpora lutea (CL) were collected at 0, 4, 10, 20, 25 (follicles) and 60 h (CL) relative to injection of GnRH. Experiment 2: CL were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–16, >18 (after regression) of oestrous cycle and of early and late pregnancy (<4 and >4 months). Experiment 3: induced luteolysis, cows on days 8–12 were injected with PGF2α analogue (Cloprostenol), and CL were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2α injection. Real‐time RT‐PCR was applied to investigate mRNA expression and immunofluorescence was utilized for protein localization. Cx26 mRNA increased rapidly 4 h after GnRH injection (during LH surge) and decreased afterwards during the whole experimental period. Cx43 mRNA expression decreased continuously after GnRH application. Cx26 mRNA in CL increased significantly in the second part of oestrous cycle and after regression. In contrast, the highest mRNA expression for Cx43 in CL was detected during the early luteal phase. After induced luteolysis the mRNA expression of Cx26 increased significantly at 24 h. As shown by immunofluorescence, Cx26 was predominantly localized in the connective tissue and blood vessels of bovine CL, whereas Cx43 was present in the luteal cells and blood vessels. This resulted in a strong increase of Cx26 expression during the late luteal phase and after luteal regression. Subsequently, Cx43 expression was distinctly decreased after luteal regression. These data suggest that Cx26 and Cx43 are involved in the local cellular mechanisms participating in tissue remodelling during the critical time around periovulation as well as during CL formation (angiogenesis), function and regression in the bovine ovary.     

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.     

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography–high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.     

Effects of plasma progesterone concentrations on LH release and ovulation in beef cattle given GnRH

The effects of plasma progesterone concentrations on LH release and ovulation in beef cattle given 100 microg of GnRH im were determined in three experiments. In Experiment 1, heifers were given GnRH 3, 6 or 9 days after ovulation; 8/9, 5/9 and 2/9 ovulated (P<0.02). Mean plasma concentrations of progesterone were lowest (P<0.01) and of LH were highest (P<0.03) in heifers treated 3 days after ovulation. In Experiment 2, heifers received no treatment (Control) or one or two previously used CIDR inserts (Low-P4 and High-P4 groups, respectively) on Day 4 (estrus=Day 0). On Day 5, the Low-P4 group received prostaglandin F(2alpha) (PGF) twice, 12 h apart and on Day 6, all heifers received GnRH. Compared to heifers in the Control and Low-P4 groups, heifers in the High-P4 group had higher (P<0.01) plasma progesterone concentrations on Day 6 (3.0+/-0.3, 3.0+/-0.3 and 5.7+/-0.4 ng/ml, respectively; mean+/-S.E.M.) and a lower (P<0.01) incidence of GnRH-induced ovulation (10/10, 9/10 and 3/10). In Experiment 3, 4-6 days after ovulation, 20 beef heifers and 20 suckled beef cows were given a once-used CIDR, the two largest follicles were ablated, and the cattle were allocated to receive either PGF (repeated 12h later) or no additional treatment (Low-P4 and High-P4, respectively). All cattle received GnRH 6-8 days after follicular ablation. There was no difference between heifers and cows for ovulatory response (77.7 and 78.9%, P<0.9) or the GnRH-induced LH surge (P<0.3). However, the Low-P4 group had a higher (P<0.01) ovulatory response (94.7% versus 61.1%) and a greater LH surge of longer duration (P<0.001). In conclusion, although high plasma progesterone concentrations reduced both GnRH-induced increases in plasma LH concentrations and ovulatory responses in beef cattle, the hypothesis that heifers were more sensitive than cows to the suppressive effects of progesterone was not supported.     

In vivo evidence that local cortisol production increases in the preovulatory follicle of the cow

The aim of the present in vivo study was to monitor real-time fluctuations of cortisol (Cr) in the wall of preovulatory follicles using a microdialysis system (MDS) implanted in the theca layer as well as changes in ovarian venous plasma (OVP) and jugular venous plasma (JVP). Seven cows were superovulated using FSH and prostaglandin F2alpha injections. Dialysis capillary membranes were surgically implanted into the theca layer of mature follicles and connected to a microdialysis system. Fractions of the perfusates were collected from Day -1 (Day 0=LH surge) to Day 3. No difference in the concentrations of Cr between JVP and OVP was detected throughout the experiment. Circulating concentrations of Cr ranged from 20 to 35 ng/ml 8 h after surgery in ovulatory and anovulatory cows. In five ovulatory cows, the Cr concentration decreased to basal levels (<10 ng/ml) between 12 and 24 h after surgery, however, two anovulatory cows retained high Cr levels (>10 ng/ml) up to 42 h after surgery. There was a clear increase in the local concentration of Cr from 13.3+/-2.1 pg/ml at -24 h to 27.5+/-1.7 pg/ml at 0 h (peak of the LH surge) within the wall of ovulatory follicles. This increase was not detected in anovulatory follicles. This transient increase in Cr occurred only in the follicle wall, but not in the OVP or JVP, indicating that the presence of a local regulatory mechanism for Cr production/conversion in ovulatory follicles, and this mechanism may modulate the inflammatory-like reaction induced by LH surge in the follicle wall. The present results demonstrate that the glucocorticoid environment in the follicular wall adjusts at the local level in bovine ovulatory follicles. This mechanism may protect follicles from the adverse effects of glucocorticoid, and it may prevent excess inflammatory reactions associated with ovulation by temporarily increasing local concentrations of glucocorticoid, thus forming an integral part of the regulatory mechanism in ovarian physiology.