Comparison of two doses of eCG for obtaining embryos in llama

The study was developed at the Pampa del Arco Experimental Center of the National University of San Cristóbal de Huamanga, the objective was to evaluate doses of 800 IU (T1) and doses of 1000 IU (T2) of eCG to obtain embryos. Eight adult female llamas were used, distributed four in each treatment (T1 and T2), with a body condition of 2.5, these were evaluated by ultrasound with a 7.5 MHz rectal linear transducer. Hormonal treatments began when the animals had a follicle> 7 mm. The application of eCG was 72 hours after the induction of ovulation of the dominant follicle with GnRH, on day 7 PGF2α was applied, natural mounting was performed plus GnRH (1ml), on day 15 the collection and evaluation of embryos was performed. The results obtained were the pre-ovarian stimulation follicular diameter of 8.3 mm and 9.1 mm for T1 and T2 respectively, the number of pre-ovulatory follicles was 6.33 and 5.50 for T1 and T2 respectively, the diameter of follicles pre-ovulatory was 11.2mm and 10.6 mm for T1 and T2 respectively, average of embryos recovered per donor was 4.66 ± 0.81 and 3.50 ± 0.54 embryos for T1 and T2 respectively, with a significant difference (p <0.05) and the quality of embryos had 35.7% and 38.1% of embryos of excellent quality, 28.6% and 33.3% of good quality, 10.7% and 9 , 5% of regular quality, 10.7% and 0% of poor quality, for T1 and T2 respectively and 14.3% and 19.0% of non-transferable embryos for T1 and T2 respectively, with no statistical difference (p≥0, 05). It is concluded that the number of embryos recovered with a dose of 800 IU of eCG is higher compared to the dose of 1000 IU of eCG (p <0.05) and that there is no association between the applied dose of eCG and the quality of the llama embryos (p≥0.05)

GABAB receptor antagonism from birth to weaning permanently modifies Kiss1 expression in hypothalamus and gonads in mice

Introduction: The kisspeptin gene Kiss1 is expressed in two hypothalamic areas: anteroventral periventricular nucleus/periventricular nucleus (AVPV/PeN) and arcuate nucleus (ARC), and also in gonads. Evidences suggest that gamma-amino butyric acid B receptors (GABAB) signaling can regulate Kiss1 expression. Here we inhibited GABAB signaling from PND2-PND21 and evaluated the hypothalamic-pituitary-gonadal (HPG) axis. Methods: BALB/c mice were treated on postnatal days 2-21 (PND2-PND21) with CGP55845 (GABAB antagonist), and evaluated in PND21 and adulthood: gene expression (qPCR) in hypothalamus and gonads, hormones by radioimmunoassay, gonad histochemistry (H&E), puberty onset, estrous cycles. Results: At PND21, CGP inhibited Kiss1 and Tac2 and increased Pdyn and Gabbr1 in the ARC of both sexes and decreased Th only in female AVPV/PeN. Serum follicle-stimulating hormone (FSH) and testis weight decreased in CGP-males and puberty onset was delayed. In adults, Kiss1, Tac2, Pdyn, Pgr, Cyp19a1, Gad1 were downregulated, while Gabbr1 was upregulated in the ARC of both sexes. In the AVPV/PeN, Kiss1, Th, Cyp19a1 and Pgr decreased while Gad1 increased in CGP-females, whereas Cyp19a1 increased in CGP-males. Serum FSH increased in CGP-males while prolactin increased in CGP-females. Testosterone and progesterone increased in ovaries from CGP-females, in which Kiss1, Cyp19a1 and Esr1 were downregulated while Hsd3b2 was upregulated, together with increased atretic and decreased ovulatory follicles. Testes from CGP-males showed decreased progesterone, increased Gabbr1, Kiss1, Kiss1r, Esr2 and decreased Cyp19a1 and clear signs of seminiferous tubules atrophy. Conclusion: These results demonstrate that appropriate GABAB signaling during this critical prepubertal period is necessary for the normal development of the HPG axis.

Single-Cell Transcriptomics Analysis of Human Small Antral Follicles

Human ovarian folliculogenesis is a highly regulated and complex process. Characterization of follicular cell signatures during this dynamic process is important to understand follicle fate (to grow, become dominant, or undergo atresia). The transcriptional signature of human oocytes and granulosa cells (GCs) in early-growing and ovulatory follicles have been previously described; however, that of oocytes with surrounding GCs in small antral follicles have not been studied yet. Here, we have generated a unique dataset of single-cell transcriptomics (SmartSeq2) consisting of the oocyte with surrounding GCs from several individual (non-dominant) small antral follicles isolated from adult human ovaries. We have identified two main types of (healthy) follicles, with a distinct oocyte and GC signature. Using the CellphoneDB algorithm, we then investigated the bi-directional ligand–receptor interactions regarding the transforming growth factor-β (TGFβ)/bone morphogenetic protein (BMP), wingless-type (MMTV)-integration site (WNT), NOTCH, and receptor tyrosine kinases (RTK) signaling pathways between oocyte and GCs within each antral follicle type. Our work not only revealed the diversity of small antral follicles, but also contributes to fill the gap in mapping the molecular landscape of human folliculogenesis and oogenesis.

Correlation between Pre-Ovulatory Follicle Diameter and Follicular Fluid Metabolome Profiles in Lactating Beef Cows

Induced ovulation of small pre-ovulatory follicles reduced pregnancy rates, embryo survival, day seven embryo quality, and successful embryo cleavage in beef cows undergoing fixed-time artificial insemination. RNA-sequencing of oocytes and associated cumulus cells collected from pre-ovulatory follicles 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the pre-ovulatory gonadotropin surge suggested reduced capacity for glucose metabolism in cumulus cells of follicles ≤11.7 mm. We hypothesized that the follicular fluid metabolome influences metabolic capacity of the cumulus-oocyte complex and contributes to reduced embryo cleavage and quality grade observed following induced ovulation of small follicles. Therefore, we performed a study to determine the correlation between pre-ovulatory follicle diameter and follicular fluid metabolome profiles in lactating beef cows (Angus, n = 130). We synchronized the development of a pre-ovulatory follicle and collected the follicular contents approximately 20 h after GnRH administration. We then performed ultra-high performance liquid chromatography—high resolution mass spectrometry (UHPLC-HRMS) metabolomic studies on 43 follicular fluid samples and identified 38 metabolites within pre-ovulatory follicles of increasing size. We detected 18 metabolites with a significant, positive correlation to follicle diameter. Individual and pathway enrichment analysis of significantly correlated metabolites suggest that altered glucose and amino acid metabolism likely contribute to reduced developmental competence of oocytes when small pre-ovulatory follicles undergo induced ovulation.

Immunological aspects of follicle ovulation and corpus luteum formation in cattle

Ovulation has been described as an inflammatory event, characterized by an influx of leukocytes into the ovulatory follicle and changes in the expression of immune factors in both the theca and granulosa tissue layers. Since information on this process is limited in cattle, our objective was to elucidate the contribution of the immune system to dominant follicle luteinization, ovulation and corpus luteum formation in cattle. Beef heifers (n=50) were oestrous synchronized, slaughtered and ovarian follicular or luteal tissue collected during a 96h window around ovulation. Follicular fluid cytokine concentration, temporal immune cell infiltration and inflammatory status were determined by Luminex multiplex analysis, immunohistochemistry and quantitative real time PCR-analysis, respectively, in pre- and peri-ovulatory follicular tissues. The concentrations of CXCL10 and VEGF-A were highest in pre-ovulatory follicular fluid samples. The pre and peri -ovulatory follicles play host to a broad repertoire of immune cells, including T-cells, granulocytes and monocytes. Dendritic cells were the most abundant cells in ovulatory follicular and luteal -tissue at all times. The mRNA expression of candidate genes associated with inflammation was highest in pre- and peri-ovulatory tissue, whereas tissue growth and modelling factors were highest in the post-ovulatory follicular and early luteal tissue. In conclusion, ovulation in cattle is characterized by the presence of neutrophils, macrophages and dendritic cells in the ovulatory follicle, reflected in compartmentalized cytokine and growth factor expression. These findings indicate a tightly regulated sterile inflammatory response to the LH surge in the ovulatory follicle which is rapidly resolved during early corpus luteum formation.

P–714 Insulin-like growth factor-I as a mediator of the effect of transdermal testosterone in poor responder patients

Study question Is insulin-like growth factor-I (IGF-I) a mediator of the effect of transdermal testosterone (TT) in poor responder (PR) patients? Summary answer IGF-I might be a mediator of the effect of TT in PR patients who undergo an IVF cycle What is known already Many strategies have been tried to improve the results in PR patients. Androgen supplementation with TT is the only that has significantly increased live birth rate in these patients. The mechanism by which TT might influence on the better results remains unclear but it is likely mediated or facilitated by IGF-I. Testosterone increases the number of primordial follicles, increase IGF-I by threefold and increase IGF-I receptor mRNA by fivehold in primordial follicles in primates. Some studies have suggested that IGF-I could be a parameter that reflects the endocrinological environment of mature follicles, which is correlated with oocyte and embryonic quality Study design, size, duration This prospective cohort study of 93 women PR according Bologna criteria treated with TT and IVF/ICSI was conducted between May 2015 and December 2016 Participants/materials, setting, methods Exogenous andogenization with TT for 5 days prior to ovarian stimulation was carried out. Hormonal parameters were evaluated: basal FSH, LH and Estradiol, AMH, IGF–1 pre and post TT. Ultrasound parameterswere also analysed: antral follicle count (AFC) and number of pre-ovulatory follicles the day of HCGr. We compared these parameters according to the ovarian response: adequate (&gt; 4 oocytes) or insufficient (&lt;3 oocytes), as well as the pregnancy was achieved or not. Main results and the role of chance Baseline characteristics of the patients were: 36.9 years, FSH 11.8, AMH 0.86 and RFA 5.3. In 83% of the patients the oocyte retrieval was carried out, obtaining an average of 3.8 MII oocytes and 2.9 embryos of 2pn with a clinical pregnancy rate per transfer of 33.3%. The FORT Test (AFC/pre-ovulatory follicles x100) was 70%, higher than that observed in other studies with patients with PR without TT (55%). In cases in which an insufficient response was obtained (&lt;3 oocytes) or the cycle was canceled, a higher age and FSH and lower AMH were observed (p &lt; 0.05). There were no differences in the rest of the parameters. Evaluating the hormonal and ultrasound parameters depending on whether or not pregnancy was achieved, a significant increase in IGF1 pre and post-TT was observed in the cases of pregnancy (31.5%) compared to those cases where there was no pregnancy (10.9%) (p = 0’01). There were no differences in the rest of the parameters. A significant correlation was found between AMH, AFC and increase in IGF-I levels (p &lt; 0’05). Limitations, reasons for caution This a prospective cohort study with limited number of patients included. Wider implications of the findings: The significant increase in serum levels of IGF–1 in pregnant patients would indicate the existence of a more favorable clinical setting for the administration of testosterone, probably related to a more favorable ovarian reserve as demonstrated by its correlation with serum levels of AMH and with the AF. Trial registration number Not applicable

P–604 Effectiveness comparison of antral follicular count (AFC), follicular-output-rate (FORT), follicle-to-oocyte-index (FOI), oocyte-sensitivity-index (OSI), and follicular-sensitivity-idex (FSI) for predicting clinical pregnancy rates in IVF

Study question Which is better for predicting clinical pregnancy rate : AFC, FORT, FOI, FSI, or OSI? Summary answer Both AFC and OSI can be used to predict clinical pregnancy better than FORT, FOI or FSI. What is known already AFC, FORT, FOI, OSI, FSI can be used to predict clinical pregnancy, but no study compared which one is better Study design, size, duration Retrospective study using data from medical record (2016–2018) Subjects were patients underwent IVF cycle at Aster Clinic in Hasan Sadikin Hospital Bandung. Subjects divided into 2 groups: clinically pregnant that is visible gestational sac on ultrasound (n = 83) and not pregnant (n = 148). Inclusion criteria : antagonist protocols, &lt;45 years, basal follicle stimulating hormone (FSH) ≤ 12 IU/L, ICSI fertilization method, and fresh transfer cycle. Participants/materials, setting, methods AFC categorized &lt; 5 and ≥ 5 (poseidon) FORT=pre-ovulatory follicles(16–20 mm) x 100 divided by AFC(2–10 mm). FOI=oocytes obtained x 100 divided by AFC. OSI=oocytes obtained x 1000 divided by total FSH dose. FSI=pre-ovulatory follicles x 100,000 divided by (AFC x total FSH dose). FORT and FSI divided using percentil 33 and 67. OSI divided into 3 groups by cut-off 1.697/IU for poor-response and 10.07/IU for hyperresponse. FOI divided into 2 groups, ≤ 50% or &gt; 50% Main results and the role of chance Group of AFC ≥ 5 had a significantly higher clinical pregnancy rate than the AFC &lt; 5 group (39.49% vs. 16.67% ; p = 0.009). High and moderate OSI had higher clinical pregnancy rate than low OSI (66.37% vs. 37.72% vs. 25.45% ; p = 0.038). There is a significant negative correlation between OSI and age (–0.454) or total FSH dose (–0.594). There is a significant positive correlation between OSI and AFC (0.625), the number of follicles at trigger (0.792), and oocytes (0.923). There were no significant differences in clinical pregnancy rates between the FORT, FOI, and FSI groups. Limitations, reasons for caution Limitation Retrospective study using medical record data Ultrasound measurement was done by many reproductive gynecology specialist (not 1 person) --- observer bias. Wider implications of the findings: This study found no association between FORT, FOI, FSI on clinical pregnancy. Why? FORT, FSI, FOI use measurement number of follicles at trigger and antral follicle. Differences among observers in interpreting antral follicles and number of follicles at trigger, or inaccurate measurement. No FORT, FOI, and FSI cut off values from previous study. Trial registration number Not applicable

Differential Transcript Profiles in Cumulus-Oocyte Complexes Originating from Pre-Ovulatory Follicles of Varied Physiological Maturity in Beef Cows

Small dominant follicle diameter at induced ovulation, but not at spontaneous ovulation, decreased pregnancy rate, fertilization rate, and day seven embryo quality in beef cows. We hypothesized that the physiological status of the follicle at GnRH-induced ovulation has a direct effect on the transcriptome of the Cumulus-Oocyte complex, thereby affecting oocyte competence and subsequent embryo development. The objective of this study was to determine if the transcriptome of oocytes and associated cumulus cells (CC) differed among small (≤11.7 mm) and large follicles (≥12.7 mm) exposed to a GnRH-induced gonadotropin surge and follicles (11.7–14.0 mm) exposed to an endogenous gonadotropin surge (spontaneous follicles). RNA sequencing data, from pools of four oocytes or their corresponding CC, revealed 69, 94, and 83 differentially expressed gene transcripts (DEG) among oocyte pools from small versus large, small versus spontaneous, and large versus spontaneous follicle classifications, respectively. An additional 128, 98, and 80 DEG were identified among small versus large, small versus spontaneous, and large versus spontaneous follicle CC pools, respectively. The biological pathway “oxidative phosphorylation” was significantly enriched with DEG from small versus spontaneous follicle oocyte pools (FDR < 0.01); whereas the glycolytic pathway was significantly enriched with DEG from CC pools obtained from large versus small follicles (FDR < 0.01). These findings collectively suggest that altered carbohydrate metabolism within the Cumulus-Oocyte complex likely contributes to the decreased competency of oocytes from small pre-ovulatory follicles exposed to an exogenous GnRH-induced gonadotropin surge.

Cionin, a vertebrate cholecystokinin/gastrin homolog, induces ovulation in the ascidian Ciona intestinalis type A

Cionin is a homolog of vertebrate cholecystokinin/gastrin that has been identified in the ascidian Ciona intestinalis type A. The phylogenetic position of ascidians as the closest living relatives of vertebrates suggests that cionin can provide clues to the evolution of endocrine/neuroendocrine systems throughout chordates. Here, we show the biological role of cionin in the regulation of ovulation. In situ hybridization demonstrated that the mRNA of the cionin receptor, Cior2, was expressed specifically in the inner follicular cells of pre-ovulatory follicles in the Ciona ovary. Cionin was found to significantly stimulate ovulation after 24-h incubation. Transcriptome and subsequent Real-time PCR analyses confirmed that the expression levels of receptor tyrosine kinase (RTK) signaling genes and a matrix metalloproteinase (MMP) gene were significantly elevated in the cionin-treated follicles. Of particular interest is that an RTK inhibitor and MMP inhibitor markedly suppressed the stimulatory effect of cionin on ovulation. Furthermore, inhibition of RTK signaling reduced the MMP gene expression in the cionin-treated follicles. These results provide evidence that cionin induces ovulation by stimulating MMP gene expression via the RTK signaling pathway. This is the first report on the endogenous roles of cionin and the induction of ovulation by cholecystokinin/gastrin family peptides in an organism.

148 Effects of the Immunization Against Gonadotropin-releasing Hormone in Gilts and Boars Under Commercial Conditions

Our objective was to determine the effects of immunocastration on growth performance, carcass characteristics and reproductive tract traits of gilts and boars. A total of 1,584 gilts and boars (PIC 337 x Camborough®, 6.11±0.29 kg, 20.64±0.81 days of age) were blocked by weaning group, and the gilt pens were randomly allotted to 1 of 2 treatments, which consisted of: T1) non-immunocastrated gilts, T2) immunocastrated gilts; whereas the boar pens were alloted to: T3) immunocastrated barrows. There were a total of 12 pens per treatment for T1 and T2, and 24 pens per treatment for T3, with 33 pigs per pen. The immunization against gonadotropin-releasing hormone (administrated with Vivax®, Zoetis, São Paulo, SP, Brazil) was given at 108±1.16 and 143±1.16 days of age (21 days before harvest). Growth performance was assessed from day 143 of age until harvest. Performance data were analyzed using the GLIMMIX procedure of SAS with pen as the experimental unit, and carcass and reproductive tract data were analyzed with pig as the experimental unit. Initial (day 143) and final (day 164) body weights were greater (P &lt; 0.05) for T3 than T1 or T2, with no evidence for differences between T1 and T2 (P &gt; 0.05). There were no evidence for treatment differences (P &gt; 0.05) for average daily gain and feed efficiency. Average daily feed intake was reduced (P &lt; 0.05) for T1 compared to T2 or T3. Hot carcass weight and lean content were greater (P &lt; 0.05) and backfat thickness was thinner (P &lt; 0.05) for T3 compared to T1 or T2, but no evidence for treatment differences (P &gt; 0.05) were observed for loin depth. The ovary weights and the percentage of ovulatory follicles were greater (P &lt; 0.05) for T1 compared to T2. Results of this study show no evidence for differences for immunocastration on growth performance of gilts or barrows. However, immunocastration influenced gilts’ reproductive tract traits.