Effects of bone morphogenetic protein 4, gremlin, and connective tissue growth factor on estradiol and progesterone production by bovine granulosa cells

Bone morphogenetic proteins (BMP) are members of the transforming growth factor β (TGFβ) family of proteins that have been implicated in the paracrine regulation of granulosa cell (GC) function, but whether responses to BMPs change with follicular size or interact with connective tissue growth factor (CTGF) or BMP antagonists (e.g., gremlin) to directly affect GC function of cattle is unknown. Therefore, to determine the effects of BMP4 on proliferation and steroidogenesis of GC and its interaction with gremlin or CTGF, experiments were conducted using bovine GC cultures. In vitro, BMP4 (30 ng/mL) inhibited (P < 0.05) follicle-stimulating hormone (FSH) plus insulin-like growth factor 1 (IGF1)-induced progesterone and estradiol production by large- and small-follicle GC but the inhibitory effect of BMP4 on estradiol production was much more pronounced in large-follicle GC. In small-follicle GC, BMP4 had no effect (P > 0.10) on IGF1-induced proliferation, but gremlin inhibited (P < 0.05) cell proliferation and estradiol and progesterone production in IGF1 plus FSH-treated GC. In large-follicle GC, BMP4 (10-30 ng/mL) increased (P < 0.05) GC numbers and gremlin (100 ng/mL) blocked this effect. In large-follicle GC, CTGF inhibited (P < 0.05) FSH plus IGF1-induced progesterone and estradiol production, and CTGF blocked the stimulatory effect of BMP4 on GC proliferation. These results indicate that BMP4, gremlin, and CTGF inhibit GC aromatase activity and progesterone production. Also, the stimulatory effect of BMP4 on GC proliferation and the inhibitory effects of BMP4 on GC steroidogenesis are more pronounced in large versus small follicles.

237 Incidence and Implications of Disparate Uterine and Ovarian Development Observed Among Heifers Evaluated During the Peripubertal Period

Rapid development in the reproductive tract occurs in the peripubertal period, with variable rates of development of reproductive tissues potentially resulting in perceived disparities in ovarian and uterine development at pre-breeding examination. Data collected from 22,173 heifers evaluated over five consecutive breeding seasons (2014–2018) were analyzed retrospectively to assess relationships between observed disparities in uterine and ovarian development and the timing of conception within the breeding season. Pre-breeding evaluations were conducted 35–45 days prior to breeding. Weight, hip height, and pelvic area were recorded. A single veterinarian conducted all reproductive evaluations via rectal palpation, and heifers were assigned individual scores for ovarian (2=infantile, 3=no significant structures, 4=large follicle and/or corpus luteum) and uterine (2=infantile, 3=mid-sized, distended tract, 4=well-vascularized, distended or coiled tract) development respectively. Heifers received artificial insemination (AI) based on detected estrus following the 14-day MGA-PG protocol. Pregnancy diagnosis was performed via transrectal ultrasonography. Pregnancy rates to AI (P/AI) were greater for scores 4-4 (n = 14,567) in comparison with 3-3 (n=3,941; P < 0.0001) or 2-2 (n = 44; P < 0.0001) and likewise were greater (P < 0.0001) for scores 3-3 in comparison with 2-2. Heifers with disparate scores of 2–3 (n = 38) did not differ (P = 0.53) from those with disparate scores of 3-2 (n = 53) with respect to P/AI, but either disparity was associated with greater (P < 0.01) P/AI relative to scores of 2-2 and reduced P/AI relative to scores of 3-3 (P < 0.01). Scores of 4-3 (1,698) and 3-3 did not differ (P >0.44) in P/AI, but P/AI tended (P = 0.06) to be greater among scores 3–4 (1,832) in comparison with 3-3. Similar (P > 0.33) P/AI was observed among heifers scored 4-4, 4-3, or 3–4. In summary, disparate ovarian and uterine development impacted timing of conception within the breeding season among heifers with lower tract scores. Such disparities were observed among 0.4% of heifers evaluated.

Variables Affecting Veterinary Students’ Ability to Accurately Interpret Ovulation in Live Mare Palpation

In a veterinary medicine curriculum, students’ hands-on practice is essential but is still considered one of the major deficiencies in veterinary schools in Europe. After theoretical and basic practical training, students, under the control of experienced veterinarians (supervisors), monitored the reproductive cycle of embryo recipients by transrectal palpation and ultrasound. To evaluate the skills of students, the question “Has she ovulated?” was posed when a dominant follicle ≥ 35 mm was recorded in the previous day’s examination and a score of 1 or 0 was assigned in the case of a correct or incorrect answer (test palpation), respectively. Study 1 involved the retrospective evaluation of 3,509 test palpation records of 43 students (31 females, 12 males) and showed a statistically significant positive correlation between the number of test palpations performed and the proportion of correct answers. There was a statistically significant effect of the number of test palpations performed by each student, their gender, and the season on the correct answers. When performing > 50 test palpations, a statistical difference between gender was observed ( p < .05). Study 2 involved the prospective evaluation of 687 records on 52 standardbred or thoroughbred recipient mares collected from nine right-handed female students. The different mares, breed, occurrence of ovulation on the left or right ovary, and the presence of one or more large follicle(s) per ovary had no effect on the correct answers ( p > .05). Individual students’ performances were statistically different ( p < .05), ranging from 60% to 92%.

Steroidal Regulation of Oviductal microRNAs Is Associated with microRNA-Processing in Beef Cows

Information on molecular mechanisms through which sex-steroids regulate oviductal function to support early embryo development is lacking. Here, we hypothesized that the periovulatory endocrine milieu affects the miRNA processing machinery and miRNA expression in bovine oviductal tissues. Growth of the preovulatory follicle was controlled to obtain cows that ovulated a small follicle (SF) and subsequently bore a small corpus luteum (CL; SF-SCL) or a large follicle (LF) and large CL (LF-LCL). These groups differed in the periovulatory plasmatic sex-steroid’s concentrations. Ampulla and isthmus samples were collected on day four of the estrous cycle. Abundance of DROSHA, DICER1, and AGO4 transcripts was greater in the ampulla than the isthmus. In the ampulla, transcription of these genes was greater for the SF-SCL group, while the opposite was observed in the isthmus. The expression of the 88 most abundant miRNAs and 14 miRNAs in the ampulla and 34 miRNAs in isthmus were differentially expressed between LF-LCL and SF-SCL groups. Integration of transcriptomic and miRNA data and molecular pathways enrichment showed that important pathways were inhibited in the SF-SCL group due to miRNA control. In conclusion, the endocrine milieu affects the miRNA expression in the bovine oviduct in a region-specific manner.

PSXII-35 The Periovulatory Endocrine Milieu Affects the Composition of the Oviductal Fluid in Beef Cows

Our main objective was to compare the composition of the oviductal fluid (OFL) between cows of high and low receptivity to the embryo. A model for receptivity based on the manipulation of the size of the preovulatory follicle (POF) was used to compare the composition of the OFL. Using this model, it has been proved that the oviduct of high receptivity cows has differences in morphology, gene expression, and extracellular matrix remodeling when compared to low receptivity cows. Cycling, non-lactating, multiparous Nelore cows (n = 14) were presynchronized before receiving cloprostenol (large follicle [LF] group) or not (small follicle [SF] group), along with a progesterone (P4) device on Day (D) ─10. Devices were withdrawn, and cloprostenol administered 42–60 h (LF) or 30–36 h (SF) before GnRH agonist treatment (D0). As a result, higher estrogen concentrations, corpora lutea, and progesterone concentrations were also observed in the LF group in comparison to the SF group. Four days after GnRH-induced ovulation, OFL was collected. Quantitative mass spectrometry was used to determine the concentration of 21 amino acids, 21 biogenic amines, 40 acylcarnitines, 76 phosphatidylcholines, 14 lysophosphatidylcholines, 15 sphingomyelins, 29 hexoses, and 17 prostaglandins and related compounds in the OF. MetaboAnalyst 3.0 was used to identify which metabolites better explained the separation of experimental groups and which could potentially be used as markers of receptivity. After multivariate and PLS analysis, samples of the LF and SF were divided clearly into two non-overlapping clusters. The most influential variables to separate the two groups included: Glutamate, Leucine, four phosphatidylcholines, three lysophosphatidylcholines, and arachidonic acid. Univariate analyses further confirmed these results. There were statistical differences in the concentration of 31 metabolites (P ≤ 0.05) between groups. We concluded that the composition of the OFL is different between cows with contrasting receptivity and fertility status.

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."

Does a Large (>24 mm) Follicle Yield a Competent Oocyte/Embryo?

<b><i>Aim:</i></b> To evaluate the effect of large follicular size (≥24 mm) at day of oocyte retrieval on oocyte/embryo quality. <b><i>Patients and Methods:</i></b> A cohort study was conducted in a single tertiary medical center between July 2018 and May 2019. Before ultrasound-guided follicular aspiration, follicles were measured and divided into 2 groups according to their maximal dimensional size: large: ≥24 mm and normal: &#x3c;24 mm. Microscopic examination of the follicular aspirates was performed by an embryologist. Each follicle aspirated was evaluated for oocyte maturation, oocyte fertilization, and embryo quality. <b><i>Results:</i></b> 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 (<i>p</i> = 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%]; <i>p</i> = 0.062). <b><i>Conclusion:</i></b> While a nonsignificant decrease in oocyte recovery rate was found in follicles ≥24 mm, the zygote and TQE per follicle were comparable.

Effect of Sweet Potato Vine on the Onset of Puberty and Follicle Development in Chinese Meishan Gilts

This study was conducted to evaluate the effects of sweet potato vine on the onset of puberty and the follicular development in the ovaries of Chinese Meishan gilts. A total of 20 Meishan gilts (initial body weight at 30 ± 0.18 kg) were randomly fed a control (CON) or sweet potato vine (SPV) supplemented diet until 19 days following the third estrous. Sweet potato vine was instead of part of basal diet with the same amount of energy and protein in the sweet potato vine group. The results indicate that gilts fed with sweet potato vine reached puberty 9.4 days later. The development of ovaries was enhanced by sweet potato vine supplementation, characterized by an increase (p < 0.05) in the relative weight of the ovaries and the number of large follicles (>5 mm). Sweet potato vine supplementation increased (p < 0.05) the total superoxide dismutase (T-SOD) and reduced (p < 0.05) the concentration of malondialdehyde (MDA) in the serum of the gilts. Also, the expression of superoxide dismutase-1 (SOD1) and luteinizing hormone receptor (LHR) mRNA in the granulosa cells from the large follicle (>5 mm) of gilts in the SPV group were increased (p < 0.05) as compared with the CON group. These results indicate that gilts fed with sweet potato vine exhibited delayed puberty as well as improved follicular development, which may contribute to the reproductive performance of Chinese Meishan gilts.

Effect of large follicle puncture on IVF-ET outcome in patients with unsynchronized follicle maturationcan

Objective This retrospective study was conducted to explore causes of unsynchronized follicular maturation (UFM) and analyze the effects of large follicle puncture on embryo quality and pregnancy outcome. Methods Clinical features and controlled ovulation hyperstimulation (COH) were compared between the puncture group (n = 48) and the control group (n = 2545). We analyzed the COH process with in vitro fertilization during fresh cycle embryo transfer with different clinical pregnancy outcomes. We compared clinical characteristics and COH process of patients in the clinical pregnancy (n = 774) and non-clinical pregnancy (n = 527) groups. Finally, factors related to pregnancy outcomes were analyzed using multivariate logistic regression analysis. Results Age, level of estradiol on down-regulation day, and initial gonadotropin dose were significantly higher in the puncture group than in the control group. We detected significant differences in age, infertility, and body mass index (BMI) between the clinical and non-clinical pregnancy groups. Age, BMI, and endometrial thickness on the day of human chorionic gonadotropin administration were the independent factors influencing pregnancy outcome. Conclusions Patient’s age and level of anti-Müllerian hormone were the main factors causing UFM in patients undergoing COH. Large follicle puncture had no significant effect on pregnancy outcome.

Influence of follicle size on bovine oocyte lipid composition, follicular metabolic and stress markers, embryo development and blastocyst lipid content

This study assessed the lipid composition of oocytes from different follicle sizes and compared the expression of lipid-related genes and follicular fluid (FF) molecules between groups. We also investigated the functional consequences of differences on embryo development and blastocyst lipid deposits. Oocytes and FF were recovered from different follicle sizes. Oocytes from small (≤5mm) and large (≥6mm) bovine follicles were used to produce Day 7 expanded blastocysts (Day7Ex) and blastocysts that only became expanded at Day 8 (Day8Ex) after insemination. Oocytes from &gt;8mm follicles had the highest lipid content. Few oocyte phospholipid variations were identified between groups. Very long chain fatty acid elongase 6 (ELOVL6) mRNA abundance was reduced in larger follicle-derived oocytes compared with the ≤2mm group. Increased levels of glucose, reactive oxygen species, glutathione and superoxide dismutase activity were also identified in FF from larger follicles. Large follicle-derived embryo development and lipid content of Day7Ex were greater than those derived from small follicles. Day8Ex had greater lipid deposition than Day7Ex. Oocytes and blastocysts exhibited follicle size-specific lipids. Large-follicle oocytes had increased lipid content and became Day7Ex with greater lipid deposition whereas delayed blastocoel expansion associated with a prolonged period of culture determined the lipid accumulation of Day8Ex. The FF microenvironment of large follicles seems to favour embryo development.