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Anti-Müllerian hormone (AMH) has a conserved role in regulating the reproductive cycle in several species. Its circulating concentration reflects the size of the growing primordial follicle reserve and is a reliable predictor of superovulation response in embryo/oocyte donors. This study investigated the possible application of AMH measurement in dromedary camels (Camelus dromedarius) multiple ovulation embryo transfer programs. In experiment 1, the follicular cycle of synchronized and naturally cycling camels (n = 12) was monitored. Blood was collected at 6 timepoints in 2 consecutive cycles corresponding to emergence, mid-cycle, and dominance in both group and hormonal fluctuations were evaluated for repeatability of measurements within and between cycles. In experiment 2, the correlation between circulating AMH concentrations prior to initiation of superovulation and the outcome of superovulation was evaluated. The results were compared between donors with higher (n = 7) and lower than median (n = 8) AMH values. Mean AMH concentrations in synchronized and non-synchronized camels were 1.46 ± 0.15 and 0.95 ± 0.09, respectively. Intercycle and intracycle values of AMH showed high repeatability in camels of both groups (>96.4% and >92.74%, respectively) with significant correlations between values at different stages of the ovarian cycle (Emergence and mid-cycle: R 2  = 0.82; emergence and. dominance: R 2  = 0.86; Mid-cycle and dominance: R 2  = 0.93, P < 0.05). Total follicles, CLs, and recovered embryos were highly correlated with AMH values prior to superovulation (R 2  = 0.64, R 2  = 0.77, and R 2  = 0.64, respectively, P < 0.05). A greater number of developed follicles prior to mating (17.00 ± 2.09 vs. 7.62 ± 1.06), CLs (12.58 ± 1.36 vs. 5.12 ± 0.93), transferable (10.85 ± 1,31 vs. 3.37 ± 0.82), and spherical embryos (8.14 ± 1.07 vs. 2.62 ± 0.7) were observed in camels with higher than median concentrations of AMH (P < 0.05). Fluctuations in estradiol and progesterone did not affect variations in mean AMH values (r 2 < 0.19 and r 2 < 0.24, respectively, P > 0.05). In conclusion, highly consistent AMH values in dromedary camels are a reliable predictor of superovulation response and outcome in dromedary camels., Competing Interests: Declaration of Competing Interest All authors have declared no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The ability of gonadotropin-releasing hormone (GnRH) to synchronize ovulation and new follicular wave emergence before a "superovulatory Day 0" protocol was assessed in Santa Inês ewes. For estrus synchronization, a 60-mg medroxyprogesterone acetate sponge was inserted for 6 d. One day before sponge removal, 37.5-μg d-cloprostenol and 300 IU equine chorionic gonadotropin were injected intramuscularly (i.m.). After sponge removal, ewes were assigned to the following 3 groups: (1) GC-1 mL saline at 12 h (n = 10); (2) G24h-0.025-mg lecirelin (GnRH agonist) i.m. at 24 h (n = 10); or (3) G36h-0.025-mg lecirelin i.m. at 36 h (n = 9). Ovarian ultrasonography was conducted to assess follicular dynamics. Blood was collected to determine plasma concentrations of progesterone and estradiol. Females from G36h and GC had a greater (P < 0.05) estrous response than those from the G24h group (78.0 and 90.0 vs 0.0%, respectively). Ewes from G24h and G36h had earlier (P < 0.05) ovulation (48.0 ± 10.2 and 56.7 ± 5.7 h) compared with those from Gc (64.1 ± 9.7 h). The mean number of ovulations per ewe was greater (P < 0.05) in Gc (1.9 ± 0.6) and G36h (2.0 ± 1.0) than G24h (1.2 ± 0.4). Plasma concentrations of progesterone and estradiol differed over time. Follicular growth during the postovulatory day was affected (P < 0.05) by day of the estrus cycle as well as by the interaction (P < 0.05) of treatment and day of the estrus cycle. There was a larger (P < 0.05) population of medium follicles during the first 24 h after the ovulation in G24h compared with Gc, and there was an absence of large follicles in G36h between 36 and 72 h after ovulation. In conclusion, the use of GnRH agonist at 36 h more efficiently synchronized ovulation and promoted the absence of dominant follicles during early diestrus and may be used at the start of superovulatory treatment at 80 h in Santa Inês ewes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Follicle-stimulating hormone has been widely used to induce superovulation in buffaloes and cows and usually triggers functional and morphologic alterations in the corpus luteum (CL). Several studies have shown that FSH is involved in regulating vascular development and that adequate angiogenesis is essential for normal luteal development. Angiogenesis is regulated by many growth factors, of which vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) have an established central role. Therefore, we have used a combination of in vitro and in vivo studies to assess the effects of FSH on the expression of VEGF and FGF2 and their receptors in buffalo luteal cells. The in vivo model consisted of 12 buffalo cows, divided into control (n = 6) and superovulated (n = 6) groups, and CL samples were collected on day 6 after ovulation. In this model, we analyzed the gene and protein expression of FGF2 and its receptors and the protein expression of VEGFA systems with the use of real-time PCR, Western blot analysis, and immunohistochemistry. In the in vitro model, granulosa cells were collected from small follicles (diameter, 4-6 mm) of buffaloes and cultured for 4 d in serum-free medium with or without FSH (10 ng/mL). To induce in vitro luteinization, LH (250 ng/mL) and fetal bovine serum (10%) were added to the medium, and granulosa cells were maintained in culture for 4 d more. The progesterone concentration in the medium was measured at days 4, 5, and 8 after the beginning of cell culture. Cells were collected at day 8 and subjected to real-time PCR, Western blot analysis, and immunofluorescence for assessment of the expression of FGF2, VEGF, and their receptors. To address the percentage of steroidogenic and growth factor-expressing cells in the culture, flow cytometry was performed. We observed that in superovulated buffalo CL, the FGF2 system mRNA expression was decreased even as protein expression was increased and that the VEGF protein was increased (P < 0.05). In vitro experiments with granulosa cells showed an increase in the mRNA expression of VEGF and FGF2 and its receptors 1 and 2 and protein expression of VEGF, kinase insert domain receptor, FGF receptor 2, and FGF receptor 3 in cells treated with FSH (P < 0.05), in contrast to the in vivo experiments. Moreover, the progesterone production by FSH-treated cells was elevated compared with untreated cells (P < 0.05). Our findings indicate that VEGF, FGF2, and their receptors were differentially regulated by FSH in vitro and in vivo in buffalo luteal cells, which points toward a role of CL environment in modulating cellular answers to gonadotropins., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Efficiency of superovulatory protocols is affected by the occurrence of reproductive abnormalities, such as the presence of anovulatory follicles. The objective of current study was to assess the incidence and possible causes of anovulatory follicles in superovulated sheep, in order to characterize the endocrine functionality of these follicles in terms of estradiol production and to evaluate their relationship with development of embryos from other follicles. The number and size of all follicles present in the ovaries of 12 sheep treated with a superovulatory FSH step-down treatment was assessed by ultrasonography. On Day 3 after subsequent estrus behaviour, the number of corpora lutea and anovulatory follicles were recorded and the fluid of anovulatory follicles >or=5mm in size was aspirated and assayed for estradiol. At once, embryos were recovered to evaluate their viability. In current study, anovulatory structures averaged 34.6% of the follicles developing to preovulatory sizes. The number of anovulatory follicles was determined by the existence of follicular dominance effects, since they increased with a higher difference in size between the largest and the second largest follicle at the beginning of the superovulatory treatment (P<0.05, r(2)=0.420). Most of the anovulatory follicles showed signs of functionality failures, indicated by a low mean estradiol concentration (9.9+/-1.1 ng/ml). However, a 22.4% of them were highly estrogenic (>200 ng/ml) and their permanence beyond the ovulation was related to a drop in the embryo viability rate (P<0.005), leading to decreased final superovulatory yields.

Ovarian follicular development and plasma concentrations of progesterone (P4), estradiol-17 beta (E2), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were compared during a synchronized estrous cycle between heifers and mature cows displaying contrasting superovulatory responses. Six heifers < 2 years old with a history of good responses to superovulatory (SOV) treatment and six cows 9 to 13 years old with poor responses to SOV treatments were used. Follicular development was monitored by daily ultrasonography. Blood samples were collected two to three times daily for P4 and E2 and thrice daily for LH and FSH analysis. Intensive sampling (samples every 15 min for 6 hr) was performed at critical periods of follicular development to analyze the pulsatile secretion of gonadotropins. In both cattle groups, a transient increase (P = 0.0001) in E2 occurred 4 to 5.7 d after the preovulatory LH surge or 2.3 d before the dominant follicle reached its maximum size. FSH concentrations increased (P = 0.006) before the emergence of the second cohort of follicles and then decreased despite no change in the concentration of E2. Contrary to our expectation and despite differences between groups in terms of age, number of previous SOV treatments, and divergent responses to superovulation, follicular development was similar in both groups. However, during the luteal phase, concentrations of E2 and FSH and LH pulse amplitudes were less (P < or = 0.05) in cows than in heifers. Therefore, follicular development monitored by ultrasonography and endocrine profiles during a synchronized estrous cycle are of limited value to predict quality of embryo donors.

Crossbred heifers (n = 103) were synchronized to estrus with prostaglandin (PGF2 alpha) and superovulated with follicle stimulating hormone (FSH-P). Animals were ovariectomized every 12 hr after the PGF2 alpha injection (n = 7 to 9/time) up to 108 hr to monitor the follicular, hormonal, and oocyte changes associated with follicular development and ovulation. Twenty-eight animals were implanted with Norgestomet implants 12 hr before PGF2 alpha and ovariectomized at 72, 84, 96, and 108 hr post PGF2 alpha injection to monitor effects of progesterone and suppression of the luteinizing hormone (LH) surge on oocyte maturation and quality. Follicular fluid was collected and analyzed for progesterone, estradiol, prolactin, and glycosaminoglycan content in conjunction with cumulus maturation and nuclear stage of oocyte maturation. Analysis of in vivo matured oocytes by in vitro fertilization was carried out at 60, 72, 84, and 96 hr post PGF2 alpha and in vitro matured oocytes at 12 to 108 hr post PGF2 alpha. No developmental changes in cumulus cells surrounding the oocyte of small follicles was noted (< or = 4 mm dia) indicating a static population. Medium (> 4 < or = 8 mm) and large size (> 8 mm) follicles developed to the corona radiata and loose cumulus stages in animals in which an LH surge was detected but cumulus status remained primarily in the tight cumulus stage for animals without an LH surge. The estradiol-to-progesterone ratio for tight cumulus (TC), corona radiata (CR), and loose cumulus (LC) stages was 1.8 +/- .1, 1.0 +/- .1, and .4 +/- .2, respectively (P < .01). Nuclear maturation of oocytes in small follicles from animals without a detectable LH surge seem to indicate early maturation (48 to 72 hr post PGF2 alpha) in conjunction with a high percent of degenerate oocytes not seen in animals exhibiting an LH surge. Oocytes from medium size follicles matured to germinal vesicle breakdown (GVBD) and early meiosis (metaphase I; MI) stages of development in all treatments. Most oocytes were degenerate in Norgestomet-implanted animals. Oocytes from large follicles (> 8 mm dia) from animals exhibiting an LH surge were in MI and metaphase II (MII) stages (48 to 84 hr post PGF2 alpha) in preparation of ovulation whereas oocytes from animals not exhibiting an LH surge had oocytes that early matured to MII (48 to 72 hr post PGF2 alpha), later regressing to degenerate oocytes (84 to 108 hr). Follicular progesterone, estradiol, and prolactin increased with oocyte maturation, particularly in medium and large follicles.(ABSTRACT TRUNCATED AT 400 WORDS)

Estrous cycles of heifers (n = 137) were synchronized with prostaglandin (PGF2 alpha) and follicular development stimulated with follicle stimulating hormone. Twenty-eight animals were administered Norgestomet implants 12 hr prior to the initial PGF2 alpha injection to suppress the LH surge that initiates ovulation. Animals were ovariectomized every 12 hr after the initial PGF2 alpha (7-9/time, 12-108 hr and at 192 and 240 hr post PGF2 alpha) 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 PGF2 alpha 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; < or = 4 mm diameter) and follicular prolactin increased from 12 to 36 hr post PGF2 alpha 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 PGF2 alpha), 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 new wave of growth in small and medium follicles.(ABSTRACT TRUNCATED AT 400 WORDS)