Large Preovulatory Follicles Research Articles

The metabolomic composition of the oviductal fluid is controlled by the periovulatory hormonal context in Bos indicus cows.

In cattle, oviductal function is controlled by the ovarian sex-steroids estradiol and progesterone. Here, we tested the hypothesis that the exposure to contrasting sex-steroid milieus differentially impacts the oviductal fluid composition. Estrous cycles of non-lactating, multiparous Nelore cows were pre-synchronized and then synchronized with a protocol designed two induce ovulation of large (LF group) or small (SF group) follciles. Larger preovulatory follicle (day 0) and corpora lutea (day 4) and greater estradiol (day 0) and progesterone (day 4) concentrations were observed in the LF group. Four days after induced ovulation, oviductal fluid was collected post-mortem. Quantitative mass spectrometry was used to determine the concentration of amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, lysophosphatidylcholines, sphingomyelins, hexoses, prostaglandins and related compounds. Multivariate analyses (OPLS-DA) were conducted to compare the metabolomic signatures of oviductal fluids. Correlation network analysis was conducted to measure the strength and hierarchy of associations among metabolites. Of the 205 metabolites quantified, 171 were detected in at least 50% of the samples and were included in further data analysis. After OPLS-DA analysis, samples of the LF and SF were divided clearly into two non-overlapping clusters. Twenty metabolites had different or tended to have different concentrations in the oviductal fluid when comparing groups. Seven of these 20 analytes had greater concentrations in LF cows. Moreover, total sum of biogenic amines, phosphatidylcholines, and prostaglandins were higher in the SF group. The correlation network showed that the LF group metabolites' concentrations were highly intercorrelated, which was not observed in the SF group. We concluded that the periovulatory endocrine milieu regulates the composition of the oviductal fluid.

Optimisation of Early Pregnancy Diagnosis andResynchronisation in Beef Cows.

The objectives of this study were (1) to investigate the effects of the preovulatory follicle (POF) size on the accuracy of Doppler-based early pregnancy detection, and (2) to determine whether the removal of PGF2α (PGF) treatment during the resynchronisation protocol would affect fertility in beef cows. In Experiment 1, Nelore suckling cows (n = 224) were enrolled in an estradiol-progesterone-based timed artificial insemination (TAI) protocol. At TAI, cows were separated based on the range of POF diameters, as follows: ≤11.0 mm (n = 50), 11.1-12.9 mm (n = 64), 13.0-14.4 mm (n = 62) and ≥14.5 mm (n = 48). On day 22 after TAI, the corpus luteum (CL) blood flow (CLBF) of all cows was examined by colour Doppler ultrasonography to diagnose nonpregnant cows. The cows with the largest POF had the greatest positive predictive value (88.6%; 31 of 35) and diagnostic accuracy (91.7%; 44 of 48). In Experiment 2, Nelore cows (n = 233) were subjected to the same TAI protocol. Fourteen days after TAI, all cows were started on a resynchronisation protocol. Cows diagnosed as nonpregnant based on CLBF, on day 22, received 0.5 mg estradiol cypionate intramuscular (im) and were assigned to receive either 150 μg of PGF (PGF; n = 50) or 2 mL of saline (control; n = 47). Cows treated with PGF had a P/AI of 30.0% compared with a 48.9% P/AI in controls (p = 0.06). Our findings demonstrate that the POF size affects the accuracy of a CLBF-based early pregnancy diagnosis and that the removal of PGF treatment from the resynchronisation protocol tended to increase P/AI of the second TAI.

Associations among the largest follicle, preovulatory estradiol concentrations, and predominant vaginal epithelial cells at the completion of hormonal ovarian stimulation for fixed-time artificial insemination in goats.

The objective of the present study was to investigate the association among the largest follicle (LF), preovulatory estradiol (E2), and predominant vaginal epithelial cell at the completion of hormonal ovarian stimulation for fixed-time artificial insemination (FTAI) in goats. Thirty-seven crossbred Boer does received gonadotropin-releasing hormone (GnRH) and intravaginal progesterone (P4)-releasing devices (day 0). On day 5, P4 devices were removed and does received prostaglandin F2α and equine chorionic gonadotrophin. On day 7, does received GnRH, and FTAI was undertaken. On day 7, does were divided into three groups, i.e. small-sized (3-3.9 mm; n = 5), medium-sized (4-4.9 mm; n = 8), and large-sized (≥5 mm; n = 24) according to the diameter of the ovarian LF; follicular characteristics (number and diameter) were identified, and blood samples and vaginal smears were collected. The average diameters of total antral follicles and LF and the percentage of superficial cell were greatest in large-sized LF does (p < .01). The average diameters of total antral follicle (r = .68) and LF (r = .71), number of preovulatory follicle (r = .58), and plasma E2 concentrations (r = .61) were positively correlated with the percentage of superficial cells (p < .01). The likelihood of a pregnancy outcome after the FTAI increased by 13.71 times in does with a greater average diameter of antral follicle, 14.18 times with emergence of a large preovulatory follicle, and 36.83 times with a higher percentage of vaginal superficial cells (p < .01). It was concluded that there is a relationship between the cell types of the vaginal epithelium, the diameters of the largest ovarian follicles, and the concentration of E2 in goats subjected to FTAI protocols.

The Presence of TGFβ3 in Human Ovarian Intrafollicular Fluid and Its Involvement in Thromboxane Generation in Follicular Granulosa Cells through a Canonical TGFβRI, Smad2/3 Signaling Pathway and COX-2 Induction.

Ovarian follicular fluid (FF) has a direct impact on oocyte quality, playing key roles in fertilization, implantation, and early embryo development. In our recent study, we found FF thromboxane (TX) to be a novel factor inversely correlated with oocyte maturation and identified thrombin, transforming growth factor β (TGFβ), TNF-α, and follicular granulosa cells (GCs) as possible contributors to FF TX production. Therefore, this study sought to investigate the role of TGFβ3 in regulating TX generation in human ovarian follicular GCs. TGFβ3 was differentially and significantly present in the FF of large and small follicles obtained from IVF patients with average concentrations of 68.58 ± 12.38 and 112.55 ± 14.82 pg/mL, respectively, and its levels were correlated with oocyte maturity. In an in vitro study, TGFβ3 induced TX generation/secretion and the converting enzyme-COX-2 protein/mRNA expression both in human HO23 and primary cultured ovarian follicular GCs. While TGFβRI and Smad2/3 signaling was mainly required for COX-2 induction, ERK1/2 appeared to regulate TX secretion. The participation of Smad2/3 and COX-2 in TGFβ3-induced TX generation/secretion could be further supported by the observations that Smad2/3 phosphorylation and nuclear translocation and siRNA knockdown of COX-2 expression compromised TX secretion in GCs challenged with TGFβ3. Taken together, the results presented here first demonstrated that FF TGFβ3 levels differ significantly in IVF patients' large preovulatory and small mid-antral follicles and are positively associated with oocyte maturation. TGFβ3 can provoke TX generation by induction of COX-2 mRNA/protein via a TGFβR-related canonical Smad2/3 signaling pathway, and TX secretion possibly by ERK1/2. These imply that TGFβ3 is one of the inducers for yielding FF TX in vivo, which may play a role in folliculogenesis and oocyte maturation.

Nitrophenols disrupt the expression and activity of biotransformation enzymes (CYP3A and COMT) in chicken ovarian follicles in vivo and in vitro.

Nitrophenols are environmental pollutants and xenobiotics, the main sources of which are diesel exhaust fumes and pesticides. The biotransformation processes that take place in the liver are defence mechanisms against xenobiotics, such as nitrophenols. Our previous study showed that the chicken ovary is an additional xenobiotic detoxification place and that nitrophenols disrupt steroidogenesis in chicken ovarian follicles. Therefore, the present study aimed to determine the in vivo and in vitro effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on the expression and activity of phase I (CYP3A) and phase II (COMT) biotransformation enzymes in chicken ovary. In an in vivo study, hens were treated with a vehicle or 10mg PNP or PNMC/kg b.wt. per day for 6 days. In an in vitro study, prehierarchical white and yellowish follicles, as well as the granulosa and theca layers of the three largest preovulatory follicles (F3, F2 and F1), were isolated and then incubated in a control medium or medium supplemented with PNP (10-6M) or PNMC (10-6M) for 24 or 48 h. Both in vivo and in vitro studies showed that nitrophenols exert tissue- and compound-dependent (PNP or PNMC) effects on CYP3A and COMT gene (real-time PCR) protein (Western blot) expression and their activity (colorimetric methods). The inhibitory effect of nitrophenols in vivo on the activity of biotransformation enzymes suggest that the ovary has the capacity to metabolise PNP and PNMC.

Clutch Size, Clutch Frequency, and Egg Characteristics of Diamond-Backed Terrapins (Malaclemys terrapin) in Southwestern Louisiana

The reproductive output of Malaclemys terrapin (Diamond-backed Terrapin) is highly variable across its geographic range. While much of the literature is based on populations from the Atlantic coast of the United States, little reproductive-output data exists for the Gulf of Mexico populations. We captured female Diamond-backed Terrapins in southwestern Louisiana and assessed their reproductive output using radiographic- and ultrasound-imaging techniques. X-rays identified that average clutch size was 7.2 and average egg width was 21.9 mm. We revealed a positive relationship between female size (plastron length) and both clutch size and egg width, but no relationship between egg size and clutch size. Ultrasound imaging in a subset of individuals revealed large pre-ovulatory follicles and fully shelled eggs concurrently in females, indicating the potential for at least 2 clutches annually in this region. Our results suggest Diamond-backed Terrapins in southwestern Louisiana show variation in reproductive output compared to other populations at similar latitudes, indicating the need for additional study within and between populations in the region.

Anogenital distance is associated with postpartum estrous activity, intensity of estrous expression, ovulation, and progesterone concentrations in lactating Holstein cows.

The objectives of this retrospective observational study were to determine the associations of anogenital distance (AGD) with (a) postpartum estrous activity, (b) diameter of the preovulatory follicle, (c) intensity of estrous expression, (d) postestrus ovulation, (e) corpus luteum (CL) size, and (f) concentrations of progesterone at estrus and on d 7 after estrus. Lactating Holstein cows (n = 178; 55 primiparous, 123 multiparous) were enrolled into the study during the first postpartum week. All cows were continuously monitored by a pedometer-based automated activity monitoring (AAM) system for estrus. Postpartum estrous activity was assessed using the AAM estrus alerts, in which cows with at least one true estrus alert (i.e., a relative increase in steps from each cow's baseline detected by the AAM and the presence of at least one follicle >15 mm, a CL <20 mm, or no CL detected by ultrasound) by the first 50 d in milk (DIM) were considered to have commenced estrous activity. At the estrus alert >60 DIM, ovulation was determined by ultrasound at 24 h, 48 h, and 7 d after estrus, and blood samples were collected at estrus alert and on d 7 after estrus for progesterone analysis. The AGD was measured from the center of the anus to the base of the clitoris and classified as either short- or long-AGD using 2 cut-points of 148 mm (predictive of the probability of pregnancy to first insemination; short-AGD, n = 115; long-AGD, n = 63) and 142 mm (the median AGD; short-AGD, n = 90; long-AGD, n = 88). Regardless of the cut-point used, early postpartum estrous activity by 50 DIM (67 vs. 54%), duration of estrus (11.6 vs. 9.7 h), and preovulatory follicle diameter (20 vs. 19 mm) were greater in short-AGD than in long-AGD cows. Increased peak of activity at estrus in short-AGD cows (354 vs. 258% mean relative increase) was affected by an interaction between AGD and parity in which multiparous long-AGD cows had lesser relative increase in activity than primiparous cows (217 vs. 386%, respectively). Mean progesterone concentration at estrus was lesser in short-AGD (0.47 vs. 0.61 ng/mL) than in long-AGD cows. The ovulatory response at 24 h did not differ, but at 48 h (91 vs. 78%) and on d 7 after estrus (97 vs. 84%) it was greater in short-AGD cows. Although CL diameter on d 7 after estrus did not differ, short-AGD cows had greater progesterone concentration 7 d after estrus than long-AGD cows (4.1 vs. 3.2 ng/mL, respectively). In conclusion, greater proportions of short-AGD cows commenced estrous activity by 50 DIM, had larger preovulatory follicles, exhibited greater duration of estrus, had reduced progesterone concentration at estrus, had greater ovulation rates and progesterone concentration 7 d after estrus compared with long-AGD cows, with no difference in CL size between AGD groups. Because all the differences in physiological characteristics of short-AGD cows reported herein favor improved reproductive outcomes, we infer that these are factors contributing to improved fertility reported in short-AGD cows compared with long-AGD cows.

Type of CIDR but not eCG administration affected pregnancy per AI in Holstein heifers subjected to a 5-day estrus synchronization protocol

ABSTRACT This study compared the effects of CIDR type and eCG treatment on ovarian follicular dynamics, estrus response and pregnancy per AI (P/AI) in Holstein heifers subjected to a 5-d estrus synchronization protocol. On Day 0, heifers (n = 133) received either a new or a once-used CIDR. On Day 5, CIDR was removed, 500 μg cloprostenol administered and heifers received or not 300 IU eCG (2 × 2 design). Heifers in estrus were inseminated 12 h after first observation. All heifers without estrus by 84 h after CIDR removal received 100 μg GnRH concurrently with AI. Ultrasonography was done in a subset of 77 heifers at CIDR insertion and removal, at AI and every 12 h after AI to determine follicular dynamics. Diameter of largest follicle at CIDR removal and AI was larger in heifers given a used CIDR (P < .05). The overall estrus response and ovulation (85.0 and 95.5%) did not differ between CIDR types, heifers given a new CIDR had greater P/AI (67.2 vs. 42.6%; P = .004). In summary, P/AI was reduced in dairy heifers treated with a used CIDR, despite having larger preovulatory follicles at AI. In addition, eCG treatment did not affect follicular dynamics, estrus response nor P/AI.

Postponing TAI in beef cows with small preovulatory follicles

The aims of this study were (1) to establish an optimal cut-off point for evaluating the effect of the preovulatory follicle (POF) diameter in timed AI (TAI) programs, and (2) to evaluate the effect of postponing TAI in cows with smaller follicles on pregnancy per AI (P/AI). In Study 1, Nelore cows (n = 426) were subjected to an estradiol-progesterone (P4) based TAI protocol. The diameter of POF was measured at TAI, 48 h after P4 insert removal. From the ROC curves for determining the relationship between the POF diameter and pregnancy, the cut-off was 11 mm (P < 0.001). Cows with larger POF had greater (P < 0.05) P/AI (62.5%) than cows with smaller POF (34.8%). In Study 2, Nelore cows (n = 1257) were subjected to the same TAI protocol as in Study 1. Before TAI, cows were separated according to POF diameter in three groups: (1) Larger POF (LP, n = 873; POF ≥11 mm), (2) Smaller POF 0 h (SP0, n = 195; POF <11 mm), and (3) Smaller POF 24 h (SP24, n = 189; POF <11 mm). Cows in the LP and SP0 were TAI at 48 h and cows in the SP24 group were TAI at 72 h after P4 insert removal. Cows in the LP group had the greatest (P < 0.05) P/AI (60.25%), followed by cows in the SP24 group (41.8%), while cows in the SP0 group had the lowest P/AI (31.8%). In conclusion, cows with smaller follicles have lower P/AI, but postponing the TAI by 24 h in these cows increases P/AI.

Dynamic transcriptome and chromatin architecture in granulosa cells during chicken folliculogenesis

Folliculogenesis is a complex biological process involving a central oocyte and its surrounding somatic cells. Three-dimensional chromatin architecture is an important transcription regulator; however, little is known about its dynamics and role in transcriptional regulation of granulosa cells during chicken folliculogenesis. We investigate the transcriptomic dynamics of chicken granulosa cells over ten follicular stages and assess the chromatin architecture dynamics and how it influences gene expression in granulosa cells at three key stages: the prehierarchical small white follicles, the first largest preovulatory follicles, and the postovulatory follicles. Our results demonstrate the consistency between the global reprogramming of chromatin architecture and the transcriptomic divergence during folliculogenesis, providing ample evidence for compartmentalization rearrangement, variable organization of topologically associating domains, and rewiring of the long-range interaction between promoter and enhancers. These results provide key insights into avian reproductive biology and provide a foundational dataset for the future in-depth functional characterization of granulosa cells.

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

Abstract 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, &amp;gt;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 &amp;lt; 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 &amp;lt; 0.05) the secretion of testosterone in the case of F1 follicles and decreased it 1.8-2.5-fold (P &amp;lt; 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).

The Involvement of Lumican in Human Ovulatory Processes.

Based on a previous global transcriptome sequencing project, we hypothesized that Lumican (LUM) might play a role in ovulatory processes. We sought to determine LUM gene expression under various conditions in human preovulatory follicles. The in vitro expression of LUM mRNA in mural (MGCs) and cumulus (CGCs) granulosa cells was characterized using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical staining was used to identify human LUM expression in follicles at different developmental stages. Cell signaling studies were performed by treating human MGCs with human chorionic gonadotropin (hCG) and both, different stimulators and inhibitors to determine their effect on LUM expression by using qRT-PCR. Cell confluence studies were carried out to study the correlation between LUM expression and follicle cell proliferation. Follicular MGCs and CGCs of women undergoing in vitro fertilization (IVF) procedures due to endometriosis were analyzed for differences in LUM expression patterns by qRT-PCR. LUM mRNA expression was significantly higher in MGCs as compared to CGCs. In CGCs, LUM mRNA was higher in mature metaphase II (MII) oocytes than in germinal vesicle (GV) and metaphase I (MI) oocytes. LUM expression was significantly upregulated in response to hCG in cultured MGCs. Immunohistochemistry of human ovaries revealed LUM was mostly present in MGCs of large preovulatory and postovulatory follicles and absent from primordial follicles. Using pharmacological activators and inhibitors, we demonstrated that LUM induction by luteinizing hormone (LH)/hCG is carried through the mitogen-activated protein kinase (MEK) pathway. LUM expression was induced in high-density cell cultures in a confluence-dependent manner. MGCs from follicles of subjects with endometriosis exhibited reduced mRNA transcription levels compared to control subjects. Our study confirms that LUM is a newly discovered ovulatory gene. LUM might play an important role during the preovulatory period up until ovulation as well as in endometriosis infertility. A better understanding of LUM's role might provide potential new treatment paradigms for some types of female infertility.

239 Body Weight and Growth in Normal Cyclic and Acyclic Gilts

Abstract Anestrus, or failure to express estrus during boar exposure, is commonly observed in replacement gilts, and results primarily from either delayed onset of puberty (prepubertal; PP) or cyclic ovulations without behavioral estrus (behavioral anestrus; BA). Gilts born between 2007 and 2018 at USMARC were observed for age at puberty between 160 and 240 days of age. Mature boars were placed in an alleyway pen while a herdsman observed gilts for standing lordosis in response to the back pressure test. Gilts failing to be observed in estrus by 240 days of age were slaughtered (250.4 ± 0.3 days of age) and reproductive tracts recovered to determine if gilts had ovulated. Gilts were defined as PP (n = 606), BA (n = 649), or Peripubertal (n = 118; PP with large preovulatory follicles on the ovary). There were 96 age-matched, cyclic contemporary gilts included as cyclic control gilts. Body weights were recorded at birth, weaning, 8 weeks, and 21 weeks of age with hot carcass weight (HCW) recorded at slaughter. The objective was to retrospectively determine if growth and HCW differed between these groups. Data were analyzed as a mixed ANOVA using group as a fixed effect with sire and farrowing group to which the gilt was born as random effects. Birth weight, weaning weight, ADG at weaning, and weight at 8 weeks did not differ between groups (P &amp;gt; 0.16). The BA gilts had greater growth rate (weight per day of pig age at 21 weeks; P &amp;lt; 0.02) and HCW (P &amp;lt; 0.0001) than PP and Peripubertal gilts, which also had smaller HCW than control gilts. Some cyclic control gilts (7.3%) that displayed estrous behavior had a prepubertal reproductive tract with no ovulatory activity at slaughter. Results indicated that gilts exhibiting delayed puberty grow slower late in development and during boar exposure. Estrus without ovulation in replacement gilts may be more prevalent than assumed. USDA is an equal opportunity provider and employer.

Effect of age on the secretory activity of different layers of preovulatory follicles in laying hens

Age-related deterioration in female fertility is mainly associated with a decrease in the number and quality of ovarian follicles, which, in turn, leads to various endocrine disorders. In the present work, the steroidogenic activity of theca and granulosa layers from preovulatory follicles of laying hens of different ages was studied in vitro. Young hens aged 32-33 weeks with a long egg clutch and hens aged 74-76 weeks with a short egg clutch were used in the experiments. The granulosa and theca layers were isolated from the two largest preovulatory follicles F1 and F2 and cultured separately for 18 h. After culture, the concentration of sex steroid hormones in the media was determined by ELISA. The production of progesterone by the granulosa layer was 1.5-2.0 times higher in reproductively aged layers than in young layers. Concurrently, the secretory activity of granulosa cells increased with the development of follicles from the F2 stage to the F1 stage only in aged birds (from 74.0±7.5 to 97.0±10.9 pmol/mg tissue, P&lt;0.05). Furthermore, testosterone production by the theca layer in aged hens was 2.0-2.8 times higher than that in young hens, but did not change significantly with the growth of follicles in birds of both groups. At the same time the ability of theca cells to secrete estradiol-17β was 1.6-2.3 times lower (P&lt;0.001) in F1 than in F2 follicles, regardless of the age of the birds. The findings indicate that the processes associated with ovarian aging modulate the steroidogenic activity of follicular cells in laying hens, with the age-related reduction in the egg clutch being related to an increase in the basal production of progesterone and testosterone in the two largest preovulatory follicles.

PSII-32 In vitro effect of growth hormone on progesterone production by large preovulatory follicles depends on the hen age and follicular layers interaction

Abstract Growth hormone (GH) is an endocrine and paracrine/autocrine regulator of the hen ovarian function, with the GH receptor concentration in the granulosa layer (GL) being maximum in the largest preovulatory follicle (Lebedeva et al. 2004, Biol.Reprod. 71:1174–1181). In the present study, GH effects on in vitro production of progesterone by GL from the two largest yellow follicles (F1 and F2) were investigated due to the hen age and the presence of the theca layer (TL). Young hens with long clutch (YLC, 32–33 week-old, &amp;gt;10 eggs per clutch) and old hens with short clutch (OSC, 74–76 week-old, 3–6 eggs per clutch) were used. After isolation, GL from F1 and F2 follicles (n = 8–9) was cultured separately or jointly with the respective TL for 18 h in the presence or absence of chicken GH (25 ng/ml). Concentrations of progesterone in culture media were measured by ELISA. The data were analyzed by repeated measures ANOVA. When GL from F1 follicle cultured alone, GH did not affect progesterone production in YLC hens and decreased it from 30,5±3,4 to 20,5±2,9 ng/mg tissue (P &amp;lt; 0.01) in OSC hens. Conversely, when tested GL from F2 follicle, GH increased progesterone output from 15,8±2,4 to 20,4±2,5 ng/mg tissue (P &amp;lt; 0.05) in YLC birds and had no effect on the output in OSC birds. During co-culture of GL and TL, GH raised 1.4–1.5 times the production of progesterone in the case of F1 follicle and did not change it in the case of F2 follicle in hens of both ages. The findings indicate that the steroidogenic response of GL from the two largest preovulatory follicles to GH differs in young and old hens. However, the interaction with TL modifies the GL response and makes it similar in birds regardless the age and reproductive status. The study was supported by RFBR (19-016-00216).

Human chorionic gonadotrophin does not induce ovulation in the tammar wallaby

We investigated whether human chorionic gonadotrophin (hCG), rather than porcine luteinising hormone (pLH), could induce ovulation in the tammar wallaby (Notamacropus eugenii) after priming with pregnant mare serum gonadotrophin (PMSG). pLH is effective, but is more expensive and supply is problematic. On Day 0 pouch young were removed (RPY) and 5mgkg−1 of bromocriptine mesylate was administered to all non-pregnant wallabies (n=12) to reactivate an oestrous cycle. On Day 20 RPY each female received 20IU PMSG i.m. and three days later (Day 23 RPY), the Single-Dose group (n=6) received 500IU of hCG suspended in oil at 0900 hours, and the Multi-Dose group (n=6) received 500IU of hCG at 0900, 1230 and 1630 hours. From Day 25 to Day 28 RPY copulatory plugs were observed in the Single-Dose (n=4 of 6; Day 25.3 to 28.3 RPY) and Multi-Dose (n=5 of 6; Day 25 to 28 RPY) groups. Autopsy on Day 31 RPY revealed that all females had reactivated, and despite all having large preovulatory follicles (4–6mm) in both ovaries, no ovulations or embryos were observed. We conclude that hCG is not an effective source of exogenous LH for the induction of ovulation in the tammar wallaby.

Inhibin A regulates follicular development via hormone secretion and granulosa cell behaviors in laying hens.

Inhibin A regulates follicular development, and its expression level is related to physiological activities, such as the recruitment, selection, and predominance during follicular development. Therefore, examining inhibin A and its regulatory effects on the reproductive performance of poultry is crucial. In this study, we measured the mRNA and protein abundances of INHA and INHBA in the chicken reproductive system and determined the hormone secretion and apoptosis of follicular granulosa cells (GCs) after being treated with inhibin A protein, and flow cytometry was performed to analyze GC apoptosis in INHA-specific small RNA interference (siRNA). We detected that INHA and INHBA were mainly expressed in chicken follicles. The highest INHA mRNA abundance was found in the fifth largest preovulatory follicle (F5) (P < 0.05). INHBA mRNA expression in the largest preovulatory follicle (F1) was significantly higher than those in other follicles (P < 0.05). Similar results were found for INHA and INHBA protein expression in those follicles (P < 0.05). Treatment with inhibin A protein increased the activity of GCs in a dose-dependent manner (P < 0.05), which was characterized by decreased gene expression of pro-apoptotic factors Bax and Caspase-3 (P < 0.05) and increased expression of proliferation genes Bcl-2 and PCNA (P < 0.05). Additionally, inhibin A significantly increased the secretion of progesterone and estradiol (P < 0.05). RNAi-mediated knockdown of INHA increased apoptosis in GCs via a Caspase-3-dependent mitochondrial pathway.

Role of the superior ovarian nerve in the regulation of follicular development and steroidogenesis in the morning of diestrus 1.

Little is known about the role of the superior ovarian nerve (SON) in follicular development during the estrus cycle. The aim of the present study was to analyze the role of neural signals arriving through the SON at the ovaries in the regulation of follicular development and ovarian steroid secretion in diestrus 1 of cyclic rats. Cyclic rats were subjected to left, right, or bilateral SON sectioning or to unilateral or bilateral laparotomy at diestrus 1 at 11:00h. Animals were sacrificed 24h after surgery. Compared to laparotomized animals, unilateral SON sectioning decreased the number of preovulatory follicles, while bilateral SON sectioning resulted in a decreased number of atretic preantral follicles. An important observation was the presence of invaginations in the follicular wall of large antral and preovulatory follicles in animals with denervation. Furthermore, left SON sectioning increased progesterone levels but decreased testosterone levels, which are effects that were not observed in animals that were subjected to right denervation. At 11:00h of diestrus 1, the SON was found to stimulate follicle development, possibly via neural signals, such as noradrenaline and/or vasoactive intestinal peptide, and this stimulation induced the formation of follicle-stimulating hormone receptors. The role of the SON in the regulation of ovarian steroid secretion is asymmetric: the left SON inhibits the regulation of progesterone and stimulates testosterone secretion, and the right nerve does not participate in these processes.

Randomized, Controlled Pilot Study of Low-Dose Human Chorionic Gonadotropin Administration Beginning From the Early Follicular Phase for Women With Polycystic Ovarian Syndrome Undergoing Ovarian Stimulation Using the Progesterone Protocol.

Purpose: To illustrate whether low-dose human chorionic gonadotropin (hCG) administration during the early follicular phase could reduce the number of large preovulatory follicles in women with polycystic ovarian syndrome (PCOS) undergoing ovarian stimulation using the progesterone protocol.Methods: We performed a randomized, controlled pilot trial at a university-affiliated tertiary hospital. A total of 40 infertile women undergoing their first in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment with the freeze-all strategy were included. Human menopausal gonadotropin (hMG) and progesterone soft capsule 100 mg/d were added simultaneously beginning from menstrual cycle day 3 for all participants. Low-dose hCG (200 IU) was injected every 3 days in the study group from the first day of ovarian stimulation until trigger. The primary outcome was the number of large preovulatory follicles. Secondary outcomes included the incidence of ovarian hyperstimulation syndrome (OHSS); the number of oocytes retrieved, mature oocytes, and good-quality embryos; and clinical results after frozen-thawed embryo transfer (FET) cycles.Results: The study group had slightly more large preovulatory follicles than the control group (17.75 ± 10 vs. 13.2 ± 5.34; P > 0.05). None of the participants experienced severe OHSS. There were no statistically significant differences in the number of oocytes retrieved (15.9 ± 8.46 vs. 15.75 ± 6.96), mature oocytes (13.55 ± 6.56 vs. 13.4 ± 6.34), and good-quality embryos (5.5 ± 3.41 vs. 4.9 ± 2.99) between the two groups (P > 0.05). Clinical pregnancy rates (65.52 vs. 41.94%; P = 0.067) and live birth rates (48.28 vs. 35.48%; P = 0.315) per transfer following FET of the study group were higher than those of the control group, but without statistical significance.Conclusions: Administration of low-dose hCG from the early follicular phase for PCOS patients undergoing ovarian stimulation with progesterone protocol may lead to slightly more early preovulatory follicles and marginally, but not significantly, higher clinical pregnancy rates. A continuous trial should be performed to explore the effects of supplementation with different doses of hCG from the start of ovarian stimulation in PCOS patients using the progesterone protocol.Clinical Trial Registration: Chictr.org.cn, identifier: ChiCTR-IOR-15007165

171 Follicular and luteal characteristics and pregnancy rates in Bos indicus suckled cows treated with two oestradiol/progesterone-based protocols with a prolonged pro-oestrus

An experiment was designed to evaluate the effect of the length of insertion of a progesterone (P4)-releasing device on follicular and luteal characteristics and pregnancy rates to fixed-time AI (P/FTAI) in Bos indicus cows treated with oestradiol/P4-based treatments with a prolonged pro-oestrus. Multiparous Bos indicus suckled beef cows (n=659), 60-90 days postpartum, with a corpus luteum (CL) or at least one follicle ≥8mm in diameter and with a body condition score between 2 and 3.5 (1-to-5 scale) were synchronised using three treatments. On Day 0, all cows received 2mg of oestradiol benzoate (Sincrodiol, Ourofino) and an intravaginal device with 1g of P4 (Sincrogest, Ourofino). The P4 device was removed on Day 6 in cows in the J-Synch 6 group (n=195) and on Day 7 in cows in the J-Synch 7 group (n=262) and conventional group (n=202). All cows received 500μg of cloprostenol (Sincrocio, Ourofino) and 400IU of equine chorionic gonadotrophin (SincroeCG 6000UI, Ourofino) at the time of P4 device removal. Cows in the conventional treatment group also received 0.5mg of oestradiol cypionate (SincroCP, Ourofino) at the same time. In addition, all cows were tail-painted for oestrous detection (CeloTest, Biotay). Cows that had lost ≥50% of the tail paint 62-66h (J-Synch groups) or 48-52h (conventional group) after device removal were FTAI at that time. Cows not showing oestrus by 62-66h (J-Synch groups) or 48-52h (conventional group) received 10μg of GnRH (Sincroforte, Ourofino) at the same time and were FTAI 8h later. A subset of 20 cows per group was also examined using ultrasonography (Mindray DP50 Vet) every 12h from the time of device removal to determine the time of ovulation, 6 days after ovulation to determine CL diameter, and 30 days after FTAI to determine P/AI. Data were analysed using the GLM Mixed procedure (InfoStat). The results are shown in Table 1. The diameter of the dominant preovulatory follicle was larger and the interval from device removal to ovulation was longer in cows in the J-Synch groups compared with those in the conventional group (P&amp;lt;0.05). Furthermore, the diameter of the CL was larger and P/FTAI was higher in cows in the J-Synch 7 group compared with those in the J-Synch 6 and conventional groups. In conclusion, delaying the removal of the P4 device by 1 day (i.e. Day 7 vs. Day 6) in the J-Synch groups and prolonging the pro-oestrus period by not giving oestradiol cypionate at P4 device removal (i.e. J-Synch 7 vs. conventional) resulted in a larger preovulatory follicle and CL and higher P/FTAI in Bos indicus suckled cows. Table 1.Mean (±s.e.m.) diameter of the preovulatory follicle (P/Foll) and corpus luteum (CL), interval from progesterone (P4) device removal to ovulation, and pregnancy rates to fixed-time AI (P/FTAI) in Bos indicus suckled cows Treatment P/Foll diameter, mm CL diameter, mm Interval to ovulation, h P/FTAI,% (n) J-Synch 6 15.5±0.3a 18.7±0.8a 83.0±4.0b 41.0 (80/195)a J-Synch 7 16.4±0.2b 21.9±0.8b 86.0±3.0b 52.0 (136/262)b Conventional 13.8±0.2a 16.8±0.9a 64.0±3.0a 37.1 (75/202)a a,bDifferent superscripts denote differences between means or P/FTAI.