PSX-A-24 Late-Breaking: Testosterone production by theca cells of large preovulatory follicles is affected by growth hormone depending on the hen age and the presence of granulosa cells

Testosterone produced by theca cells may be involved in regulating of the growth and ovulation of hen preovulatory follicles (Rangel, Gutierrez, Gen Comp Endocrinol, 203:250, 2014). In the current research, we studied effects of growth hormone (GH), a known regulator of the hen ovarian function, on in vitro testosterone production by the theca layer (TL) from the two largest yellow follicles in relation to the hen age and the presence of the granulosa layer (GL). Young hens with long clutch (YLC, 32–33 week-old, >10 eggs per clutch) and old hens with short clutch (OSC, 74–76 week-old, 3–6 eggs per clutch) were used. After isolation, TL from F1 and F2 follicles (n = 8–9) was cultured for 18 h in two systems, separately or together with the corresponding GL, in the presence or absence of chicken GH (25 ng/ml). Concentrations of testosterone in the spent media were measured by ELISA. The data were analyzed by RM-ANOVA. In the case of separate TL culture, GH did not change significantly testosterone production in both follicles of YLC hens and reduced it from 338±105 to 152±52 fmol/mg tissue (P < 0.05) in F1 follicles of OSC hens. When TL was cultured in the presence of GL, GH enhanced 1.8-2.6-fold (P < 0.05) the secretion of testosterone in the case of F1 follicles and decreased it 1.8-2.5-fold (P < 0.05) in the case of F2 follicles in both young and old hens. Regardless of the treatment, follicular size or culture system, the production of testosterone in OSC hens was 2–5 times higher than in YLC hens. The results indicate that the interaction between TL and GL changes the steroidogenic response of theca cells from preovulatory follicles to GH in young and old hens. Furthermore, testosterone production is obviously increased with reproductive aging of laying hens. The study was supported by RFBR (19-016-00216).

EFFECT OF THE ADMINISTRATION OF SERUM AND RECOMBINANT EQUINE CHORIONIC GONADOTROPIN ON OVARIAN DYNAMICS AND CONCEPTION RATE IN ANESTRUS PRIMIPAROUS BRANGUS COWS

The objective was to determine the size of the dominant ovulatory follicle, the ovulation time and the conception rate of cows in anestrus treated with serum equine chorionic gonadotropin (eCG), recombinant chorionic gonadotropin and untreated controls. 57 anestrus primiparous Brangus cows were used with 70 ± 26 days of calving and a body condition of 2.50 ± 0.15. They were synchronized with a FTAI protocol based on estrogen and progesterone devices (DI). Upon removal of DI, 3 groups were made according to the type of eCG applied: serum eCG received 400 IU im of equine Chorionic Gonadotropin, recombinant eCG received 140 IU of recombinant Equine Chorionic Gonadotrophin and Control did without treatment. Follicular size and time of ovulation (normal or abnormal) was determined at DI removal, during FTAI and 7 days later. An ANOVA was performed to determine the effect of the treatment on ovarian dynamics and a chi-square test and correspondence analysis to establish associations. In normal ovulation cows at the FTAI, 94.4% of the serum eCG, 55.5% of the recombinant eCG and 71.4% of the Control cows presented a dominant ovulatory follicle (DOF), which in those treated with serum eCG, it was 1.4 mm greater in relation to the Control (P = 0.0073). The ovulation rate, in normal and abnormal ovulation cows, was 94.4% for serum eCG, and for recombinant eCG and 66.6% for Controls. A significant association was detected between ovulation time and the treated groups (P = 0.0042). Normal ovulation was 38.9% higher in cows with serum eCG in relation to those treated with recombinant eCG and 50.8% in comparison with Control. A significant relationship was observed between the conception rate and the groups treated with the different Gonadotropins (P = 0.0574), being 66.7% in serum eCG, 50% in recombinant eCG and 28.6% in the Control. The use of eCG, either serum or recombinant, in anestrus primiparous cows, stimulates the development of DOF at FTAI, increases ovulation rate and improves conception in relation to untreated cows.

Effect of Ubiquinol supplementation on ovulation induction in Clomiphene Citrate resistance

Background Clomiphene Citrate is considered the gold-standard for induction of ovulation and has been used for several years to treat PCOS related infertility. Unfortunately, 15-40% of women with PCOS are resistant to Clomiphene Citrate. The study aimed to evaluate potential benefits of adding the active form of Coenzyme Q10 (Ubiquinol) to Clomiphene Citrate compared with Human Menopausal Gonadotropins (hMG) in Clomiphene Citrate resistant PCOS patients. 148 PCOS Patients with Clomiphene Citrate resistance were randomized into two groups (A and B). In group A, controlled ovarian stimulation was done by Clomiphene Citrate 150 mg daily (from 2nd till 6th day of cycle) together with Ubiquinol starting from 2nd day till day of hCG triggering in a dose of 100 mg orally once daily. In group B, hMG was given from 2nd day of the cycle in a dose ranging from 75 to 225 IU. Serial transvaginal ultrasonography was done starting on cycle day 8 and continued till size of leading follicle reaches 18 mm or more then ovulation triggering was done. Thereafter, patients were advised for a timed intercourse (TI) after 36 hours. A blood sample was withdrawn seven days after hCG triggering, for measurement of serum progesterone. If the Patient presented with a missed period for one week, a serum sample was sent for β-hCG. Results There were no statistically significant differences (P > 0.05) between studied groups regarding; number of cases reaching mature follicular size, number of stimulated cycles, endometrial thickness on the day of hCG triggering, mid-luteal serum progesterone, positive serum pregnancy test and clinical pregnancy rate. Conclusions Addition of Ubiquinol to Clomiphene Citrate improved ovarian responsiveness in Clomiphene Citrate resistant patients with results comparable to conventional hMG stimulation protocol.

The Effect of Estetrol/Drospirenone on Ovarian Function Is Similar to a Well-Established Combined Oral Contraceptive: Results From a Phase 2 Study

Background: Combined oral contraceptives (COCs) often contain ethinylestradiol (EE), an estrogen known to be associated with several side effects including venous thromboembolism. Estetrol (E4) is a native estrogen synthesized by the human fetal liver during pregnancy. Results from a phase 2 dose-finding study showed that E4 15 mg in combination with drospirenone 3 mg (E4/DRSP) resulted in a good bleeding profile and cycle control. Here, we present phase 2 results showing the effect of E4/DRSP on ovarian function. Study Design: A single-center, randomized, open-label, parallel study was conducted in healthy young volunteers with proven ovulatory cycles. Study subjects received either E4 15 mg/DRSP 3 mg (n=41) or EE 20 µg/DRSP 3 mg (n=41) in a 24/4-day regimen for three consecutive cycles. Both in cycle 1 and 3, serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH), estradiol and progesterone levels were determined, and follicular size and endometrial thickness were measured every three days using transvaginal ultrasound. Ovarian function was assessed using the Hoogland score, which considers follicular size and estradiol/progesterone levels. Return of ovulation was determined after treatment cessation. Safety and tolerability were assessed by monitoring adverse events (AEs), vital signs, physical and gynecological examination, clinical laboratory parameters, 12-lead electrocardiogram and echocardiogram. Results: No ovulations were reported during the use of E4/DRSP, while three ovulations occurred in two subjects in the EE/DRSP group. In both groups, most participants had no ovarian activity according to the Hoogland score. In cycle 1, Hoogland scores and follicular diameters were similar in both groups. In cycle 3, these parameters were slightly less suppressed in the E4/DRSP group when compared to EE/DRSP. While mean FSH and LH concentrations were less inhibited by E4/DRSP, mean estradiol and progesterone concentrations and endometrial thickness were similarly suppressed in both groups. Return of ovulation occurred on average 15.5 days after discontinuation of E4/DRSP intake. The number of frequently reported AEs considered to be related to study medication was similar for both treatment groups except for breast pain (11 subjects in the E4/DRSP group versus 4 subjects with EE/DRSP). Most related AEs were of mild or moderate intensity. Three subjects discontinued due to an AE, one with E4/DRSP (severe stress, emotional lability), and two with EE/DRSP (emotional lability, depressed mood). Other safety assessments did not show significant abnormalities. No serious AEs were reported. Conclusions: The combination of E4 15 mg and DRSP 3 mg results in adequate ovulation inhibition and ovarian function suppression, which is similar to a well-established COC containing EE/DRSP. E4/DRSP is considered safe and well-tolerated.

112 Follicular dynamics and oestrus response of Alpine goats with oestrus/ovulation synchronized during the early transitional reproductive phase using gonadotrophin given early or late

Oestrus and ovulation synchronization (E/OS) regimens for fixed-timed breeding are useful when consistently eliciting ovulation. Early synchronization in the reproductive season promotes unreliable oestrus behavioural and physiologic response due to insufficient ovarian priming. In ruminants, equine (eCG) or human chorionic gonadotrophin (hCG) has FSH bioactivity or elicits ovulation, correspondingly. Hence, 120 and 60IU of eCG and hCG, respectively, are included in goat E/OS regimes. This experiment addresses the time when eCG/hCG (CG) is given relative to progestagen (P4) withdrawal and its effect on oestrus and ovulation. Fourteen non-lactating, Alpine breed goats, ranging from 1 to 6 kiddings, averaging±s.d. 3.9±2.0 years of age, and 56.3±5.0kg of bodyweight (BW), and body condition score (BCS) of 2.6±0.2, were evaluated early in the transitional reproductive phase (12h daylight:12h darkness). E/OS was accomplished with 12 day of P4 (200 mg) exposure by intravaginal insert. Three treatments were evaluated: Traditional (T; n=4) did not receive CG and served as the control group; early (E; n=5) received CG 24h before P4 removal; and reverse (R; n=5) received PGF2α 24h before P4 withdrawal as well as CG concurrent with P4 withdrawal. Oestrus response to E/OS was evaluated 24h after breeding using epididymectomized bucks. Ovaries were scanned by transrectal ultrasound (Aloka SSD-500V/7.5-MHz linear array probe) for 4 consecutive days starting with the first i.m. injection of 1.0mL of PGF2α or 1.5mL of CG. Images were digitized (MediCapture™) for later morphometry. The absence of effect (P>0.05) from concomitant variables age, BW, BCS, and parity was ascertained using a logistic regression model (JMP/SAS v15; SAS Institute Inc.). Ovulation, defined as the disappearance of the largest (>7mm) preovulatory follicle on a subsequent observation, was 100, 80, and 100%; and the average±s.e. number of ovulations per goat was 2±0.41, 1±0.32, and 1.3±0.49 (P>0.05). Table 1 summarises follicular size documented of 122 observations of ovulatory areas. Graafian follicle location was not different for left or right ovaries (P>0.05). Oestrus behavioural response to each E/OS treatment (T, E, or L) up to 24h after P4 removal was 50, 80, and 80% (P>0.05), respectively. In summary, oestrus response and ovulation were not affected by the variables studied. In this experience the timing of CG was not relevant. Table 1. Mean diameter (±SE; mm) of nonovulatory and preovulatory follicles on left (L) and right (R) ovaries E/OS protocol Non- and preovulatory Nonovulatory Preovulatory L R L R L R Traditional 6.4±0.6 5.5±0.6 4.8±0.4 4.5±0.4 7.7±0.4 8.9±0.3 Early 5.2±0.3 5.5±0.4 4.9±0.2 4.4±0.3 8.4±1.2 8.1±0.2 Reverse 6.6±0.5 5.3±0.6 5.1±0.4 4.4±0.5 8.7±0.3 8.1±0.2 Overall 5.7±0.19 4.7±0.14 8.3±0.14

Weaned Sows with Small Ovarian Follicles Respond Poorly to the GnRH Agonist Buserelin

The GnRH agonist buserelin (GnRH), used to synchronize ovulation in weaned sows, attains only 70–80% effectivity, owing to several reasons of ovarian origin. This study evaluated in particular whether mean ovarian follicle size at treatment and the season of weaning are among those influencing GnRH responsiveness. The experiment was carried out in a temperate-region farm with 352 sows of 1–6 parities weaned either in winter–spring (WS, 174 sows) or in summer–autumn (SA, 178 sows). The sows were randomized into two groups: GnRH (10 µg of buserelin acetate at 86 h after weaning, 172 sows) and control (180 sows). The ovaries were transrectally scanned from weaning to ovulation and the sows clustered according to their mean follicular size at treatment time: small (<0.5 cm in diameter), medium (0.5 to 0.64 cm) and large (0.65 to 1.09 cm). In total, 88.33% of the GnRH-treated sows ovulated, with 82% of them within the expected time window (120–132 h after weaning). In contrast, 95.45% of the unresponsive sows had small follicles at the time of treatment and were mostly weaned in SA (20.45%) than in WS (4.76%). In conclusion, the conspicuous presence of sows having small ovarian follicles at treatment time compromises the efficiency of the GnRH agonist buserelin to synchronize ovulation in weaned sows, which occurs more frequently in summer–autumn weaning.

Ovarian Follicle Growth during Lactation Determines the Reproductive Performance of Weaned Sows

Factors causing variability in ovarian follicle size among weaned sows are not well known. This field study aimed to disclose influencing factors and evaluate if the differences at weaning were established during lactation. Ovaries were scanned using transrectal ultrasound. The first experiment was conducted over a year with 191 randomly chosen sows that were hierarchically grouped (p < 0.001) according to ovarian follicle diameter reached at weaning: Small (0.20–0.30 cm; n = 37), medium (0.31–0.39 cm; n = 75), and large (0.40-1.00 cm; n = 69). Sows with small follicles showed a higher incidence of post-weaning anestrus (p < 0.01), longer wean-to-estrus/ovulation intervals (p < 0.01) and farrowing smaller litters (p < 0.05). Ovaries with small follicles were more common among sows weaned in summer–autumn than in winter–spring (p < 0.01) and among sows of lower parity (1–3) (p < 0.05). In the second experiment, with 40 sows randomly chosen at farrowing, the ovaries were scanned at 7, 14, and 21 d post-partum. Sows showed great variability in ovarian follicular size during lactation with a consistent relationship between the three measurement times (r = 0.84, p < 0.01). Follicle size was smaller in sows nursing in summer–autumn than in winter–spring (p < 0.05). In conclusion, early lactation dictates the great variability in ovarian follicular diameter at weaning shown by sows. Sows with smaller follicles at weaning had longer intervals for estrus and ovulation and smaller litters at farrowing and they were in greater numbers among sows weaned during the summer and fall and among those with fewer previous farrowing.

Does large (>24mm) follicle yield a competent oocyte/embryo?

Aim: To evaluate the effect of large follicular size ( > 24mm) at day of oocyte retrieval on oocyte/embryo quality. Patients and Methods: A cohort study conducted in a single tertiary medical center between July 2018 and May 2019. Before ultrasound-guided follicular aspiration, follicles were measured and divided into two groups according to their maximal dimensional size: large: ≥24 mm and normal: <24 mm. Microscopic examination of the follicular aspirates was performed by embryologist. Each follicle aspirated was evaluated for oocyte maturation, oocyte fertilization and embryo quality. Results: 428 follicles were measured, including 383 (62.81%) in the normal and 45 (14.06%) in the large follicle groups. Oocytes were achieved during aspiration from 297 (75.5%) and 29 (64.4%) of the normal and large follicle groups, respectively (p=0.05). No in-between group differences were observed in mature oocyte (MII), fertilization and top quality embryo (TQE) rates. Nevertheless, once a zygote (2PN) was achieved, a trend toward a higher TQE rate/2PN was found in the large follicle group [16 /19 (84.2%) vs. 115/171 (67.3%); p=0.062]. Conclusion: While a non-significant decrease in oocyte recovery rate was found in follicles ≥24 mm, the zygote and TQE per follicles were comparable.** Drs. Orvieto and Mohr-Sasson should be considered "similar in author order."