The Effect of A PRID®Delta-Based Estrus Synchronization Protocol On Pregnancy Rates In Cows With No Estrus Symptoms

The present study aimed to determine the effects of progesterone-based gonadotropin-releasing hormone (GnRH), prostaglandin F2α (PGF2α), and equine chorionic gonadotropin (eCG) injections on progesterone profiles and pregnancy rates in cows with no estrus symptoms within 60 days after parturition. A total of 80 cows were included in the study. All animals had the progesterone-releasing device PRID®Delta placed intravaginally for nine days with an injection of GnRH. On the eighth day, PGF2α was injected, and PRID®Delta was removed from the vagina on day nine. Artificial insemination was carried out 60 hours after PRID®Delta removal. In half of the animals (n = 40), 600 IU of eCG was injected when PRID®Delta was removed on the ninth day before artificial insemination 60 hours later. Blood samples were taken from the tail vein on days 0 and 8 to determine progesterone levels. The pregnancy rate in the group that received eCG was 37.5%, while it was 27.5% in those that did not (P = 0.4). While the dominant follicle diameter was 15.5 mm in cows injected with eCG during timed artificial insemination, the follicle diameter was 12.4 mm in cows with no eCG injection (P <0.001). There were no differences in serum progesterone values in blood samples taken until the time of artificial insemination. However, progesterone values in the blood taken during artificial insemination were 0.94 ng/ml in the eCG- group and 0.72 ng/ml in the eCG+ group (P <0.05). As a result, it was determined that eCG injections, in addition to progesterone-based GnRH and PGF2α applications, increased the pregnancy rates in cows without symptoms of estrus. The dominant follicle diameter was larger in cows treated with eCG during artificial insemination; however, follicle size did not increase the pregnancy rate.

Effect of Follicle Diameter and Culture Medium on The in Vitro Maturation of Sheep Oocytes Using Culture Media Supplemented With Different Concentrations of Sucrose in Local Sheep (Ovis Aries)

The current study was conducted in the postgraduate laboratory of the college of Agriculture/University of Al-Muthanna/Department of Animal Production, which aimed to find out the effect of follicle diameter and the culture medium on the percentage of in vitro maturation of sheep oocytes (Ovis Aries). The experiment included in vitro maturation of sheep oocytes aspirated from large, medium and small follicles in different culture media (named A, B and C). In vitro maturation of oocytes was carried out using three culture media that differed only in the concentration of sucrose, which were 0. M, 0.25 M and 0.5 M for A, B and C culture media respectively. The results of the current study showed significant superiority (P ≤ 0.05) of culture medium C than the two media A and B in the percentages of in vitro maturation, which were to 38.0 ± 1.71 %, 27.37±1.47 % and 21.902±0.76 % for C, A and B respectively. The results also indicated significant effect (P ≤ 0.05) of follicle diameter in the percentages of in vitro maturation of sheep oocytes which were 38.0±1.71 %, 29.57±2.06 % and 18.5± 0.27 % for large, medium and small follicles respectively. It could be conclusion from the present study that the follicle diameter and culture medium had a significant effect on the percentage of in vitro maturation of sheep oocytes.

250 Effect of Pre- and Post-ai Nutrition on Ovarian Dynamics, Steroidogenesis, and Expression of Estrus in Beef Heifers

The objective of this study was to evaluate nutrition before and after artificial insemination (AI) on follicular dynamics, expression of estrus, and steroidogenesis. Seventy-nine beef heifers were randomly assigned to one of two dietary treatments (High;155% and Low;86% of maintenance energy) 30d prior to AI. Estrus was synchronized (PG 6-d CIDR protocol) and heifers received AI (d0) 8 to 12h following onset of estrus. On d0, heifers were randomly reassigned diets generating four Pre-x-Post-AI nutritional treatments; High-High (HH, n = 20), High-Low (HL, n = 20), Low-High (LH, n = 19), and Low-Low (LL, n = 20). Heifers remained on new diet treatments until embryo collection (flush; d7-8). Blood samples were collected daily from d-3 to d0, and on d1, 3, 5, 7 and 8 for plasma concentrations of estradiol (E2) and progesterone (P4). Dominant follicle diameter was evaluated on d-3 and d0, and CL diameter was recorded at flush. Statistical analyses were completed in SAS using the MIXED (body weight, E2, P4), GLIMMIX (expression of estrus), and GLM (ovarian parameters, interval to estrus) procedures. There were Pre-AI (P &lt; 0.0001) and Post-AI by time (P &lt; 0.0001) interactions on body weight; heifers in high and low treatments gained and lost weight, respectively, during both periods. Estradiol concentrations increased (P &lt; 0.0001) from d-3 (2.18±0.15pg/mL) to d0 (6.05±0.04pg/mL). Pre-AI diet increased the proportion of heifers in estrus (80±6.3% vs. 59±7.9%; P = 0.05), and dominant follicle size (11.7±1.42mm vs. 10.68±1.33mm; P = 0.0016) in High vs. Low, respectively. Additionally, High Pre-AI heifers had greater P4 after AI compared to Low Pre-AI heifers (4.85±0.37ng/mL vs. 3.53±0.38ng/mL; P = 0.015). Post-AI treatment did not influence concentrations of P4 (P = 0.88). There was no effect of Pre, Post or Pre-x-Post-AI treatment on initial follicle size, follicle growth rate, E2 concentrations, interval to estrus, or CL size at flush (P &gt; 0.10). In conclusion, nutrient restriction before AI negatively impacted ovarian function, steroidogenesis, and expression of estru.

PSXII-20 Ovarian dynamics and fertility in beef cows administered a 5-day or 6-day CO-Synch protocol for timed artificial insemination

The 5-day CO-Synch is an extensively used TAI protocol; however, it requires multiple administrations of prostaglandin F2α (PGF). The objective of the present study was to evaluate the effect of increasing progesterone (P4) device insertion from 5 to 6 days during a TAI protocol on ovarian dynamics and fertility in beef cows. Suckled beef cows (n = 867) at six locations were randomly assigned to either a 5-day or 6-day CO-Synch. Cows received an intravaginal P4 device (CIDR, Zoetis) and 100 µg of gonadorelin acetate (GnRH, Parnell) on d-9 (6-day) or d-8 (5-day). On d-3 CIDR’s were removed, an estrus detection patch applied (Estrotect, Rockway Inc.) and either one (6-day) or two (5-day) doses of PGF (500 µg of cloprostenol sodium, Estroplan, Parnell) was administered. On d0 (72 h after CIDR removal) all cows received 100 µg of GnRH, estrus expression was evaluated, and AI was performed. Pregnancy was determined by ultrasonography 34 days after TAI. Ovarian dynamics were evaluated in a subset of animals (n = 20) between d-3 and d7 to determine preovulatory follicle size, time of ovulation and corpus luteum (CL) size. Data were analyzed using generalized linear mixed models (SAS 9.4). Cows in the 6-day group tended (P = 0.09) to have a larger maximum follicle diameter (16.2±0.4mm) than cows in the 5-day group (15.0±0.6mm). In addition, CL volume on d7 was greater (P = 0.01) in the 6-day (4302±495mm3) than 5-day (2406±455mm3) group. There were, however, no differences (P = 0.15) in the interval between CIDR removal and ovulation. Cows assigned to the 6-day group had greater (P &lt; 0.05) estrus expression [56.1% (215/385) vs 41.5% (161/388)] and pregnancy rate [54.6% (236/438) vs 47.4% (207/435)] than cows in the 5-day group. In conclusion, extension of the period of P4 device insertion in a prolonged proestrus protocol increases preovulatory follicle size, estrous expression, and fertility to TAI.

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.

O-081 High variability of molecular isoforms of AMH in follicular fluid and granulosa cells from human small antral follicles

Study question Is the composition of AMH isoforms different in follicular fluids (FF) and granulosa cells (GCs) from human small antral follicles? Summary answer There is a high viability of AMH isoforms in FFs and GCs. Even between same size follicles from the same women, the isoform composition differs. What is known already Anti Müllerian Hormone (AMH) is a member of the TGF-β superfamily produced by follicular granulosa cells (GCs) in women from late gestation to the end of reproductive live. AMH is suggested to inhibit aromatase (i.e. CYP19) expression and thereby decreasing the conversion of androgens to oestrogens in humans, especially in small antral follicles before dominance is achieved and thereby act as a gatekeeper of ovarian steroidogenesis. However, the exact function and processing of AMH in human follicles is still not clarified. Study design, size, duration This retrospective study measured AMH isoforms in human FF and GCs from small antral follicles using ELISA, Western blot, and immunofluorescence analysis. A total of 41 female adolescents and women aged 15 to 38 years (mean age: 29.7 years), who underwent ovarian tissue cryopreservation (OTC) at the University Hospital of Copenhagen, Rigshospitalet were included between year 2006 and 2020 included. Participants/materials, setting, methods Donated human ovarian medulla tissue were FFs and GCs were obtained in connection with OTC. The following isoforms were evaluated in FFs using ELISA analysis: full-length AMH precursor (proAMH), cleaved associated AMH (AMHN,C), N-terminal pro-region (AMHN), and active C-terminal (AMHC) AMH. Antibodies specific for the N-terminal and the C-terminal AMH were used in both Western blot and immunofluorescence analysis of FFs and GCs. Main results and the role of chance A negative correlation between follicle diameter and the mentioned AMH forms were detected. Moreover, Western blot analysis detected various AMH forms in both FFs and GCs, which did not match the above-mentioned consensus forms suggesting an unknown proteolytic processing of AMH. The presence of these new molecular weight isoforms of AMH differs between individual follicles of identical size from the same woman. Limitations, reasons for caution The study group is limited and the significance of the variable AMH isoforms compositions between follicles cannot not be clarified from this data. Wider implications of the findings Collectively, these data suggest that intrafollicular processing of AMH is complex and variable, and thus, it may be difficult to develop an antibody based AMH assay that detect all AMH isoforms. Furthermore, the variability between follicles suggests that designing a proper standard will be difficult. Trial registration number not applicable

Presynch-Heatsynch: A New Approach to Improve Ovarian and Fertility Responses in Water Buffalo (Bubalus bubalis)

Background: Standard estrus synchronization regimens resulted in variable outcomes in buffaloes. The present study evaluated ovarian and fertility responses following presynchronization in Heatsynch protocol administered in postpartum buffaloes. Methods: In group-I (Presynch-Heatsynch group, n = 30), PGF2α was administered on days -14 and -2. Then, GnRH analogue, PGF2α (Cloprostenol) and Estradiol benzoate were administered on day 0, 7 and 8, respectively followed by fixed-time artificial insemination (FTAI) 48 hours later. In group-II (Heatsynch group, n = 30), rest protocol was same, except that first two PGF2α injections were not administered. Result: The progesterone concentrations differed (P less than 0.01) between the two groups on days -2, 0 and 7. Post-treatment, progesterone profiles were also higher in pregnant compared to non-pregnant buffaloes in both the groups except on day 10. CL diameter differed (P less than 0.01) between groups on days -2, 0 and 7. It was larger in pregnant than non-pregnant buffaloes on day 7 in both the groups. Dominant follicle diameter remained larger on days -2, 0 and 8 in group-I than -II. Buffaloes getting pregnant had a larger (P less than 0.01) dominant follicle size on the day of FTAI in group-I than of group-II. Ovulatory response of 93.33 and 90.00% was observed in group-I and II. The conception rate was higher (66.66 vs. 40.00%; P less than 0.05) in group-I than Group-II. Presynchronization improved reproductive efficiency in Heatsynch treatment and may aid for better fertility in buffaloes.

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.

The New Detection Method of Ovarian Follicle Development Using Digitized Wide Area Measurement

METHODS: The research method used in this research was experimental laboratory with pre-and posttest only control group design. RESULTS: The result shows that the extradiol level which has range of 26.30–31.03 from 28 experimental animals measured, this showed more measurement diameter which has not had measurement addition compare with the wide percentage of measurement. The result shows strong correlation between digitalized measured wide follicles to the changing of estradiol level with value of 0.453. The result of comparation between estradiol level and measured diameter shows weak correlation. This shows that manual measurement of follicle diameter still weak to the changing of estradiol level. CONCLUSION: There is strong correlation between measured wide area follicle used ImageJ applications to the changing of estradiol level compare to the measurement of follicle diameter.

THE FOLLICLEGENESIS IN THE THYROID GLAND IN THE POSTNATAL PERIOD OF ONTOGENESIS UNDER PRENATAL ANTIGENIC INFLUENCE

Antigenic influence at critical terms of ontogenesis can cause significant changes in the child's immune system. It is known that the entry of antigens into the fetus causes premature release of T-lymphocytes from the thymus and their migration to various organs. The paper was aimed at the study of the folliclegenesis of the thyroid gland in postnatal period at norm and after prenatal influence of staphylococcal toxoid. In the experimental research as a material were thyroid glands of Wistar rats aged 1 to 60 days of postnatal development (162 animals), about 6 animals in each group. Three animals groups were studied on 1, 3, 7, 11, 14, 21, 30, 45, 60 days after bith. I gr. - intact animals (norm); ІІ gr. - control, animals which were injected intrauterine 0.9% NaCl solution; III - experimental animals injected with staphylococcal toxoid liquid purified adsorbed (10-14 units of binding in 1 ml, diluted 10 portions) by operation intrauterinely on the 18th day of dated pregnancy. Histological sections 3-5 μm thick were stained by hematoxylin and eosin, histochemicaly by alcian blue and azan staining. Immunohistochemical study was performed according to the protocol recommended for a particular antibody of the manufacturer. Used ki-67 (Ki-67), TTF-1 (8G7G3/1), Fox-1 (A-12) monoclonal antibodies by Santa Cruz Biotechnology, Inc. A set of morphometric studies was performed by microscope Carl Zeiss Primo Star equipped with the Axiocam digital microphoto attachment with using program complex Zeiss Zen 2011. The results were considered reliable at p≤0,05. For processing of statistical material was used the standard software package Microsoft Office Excel and Statistica 10.0.The results were obtained about morphogenesis of rat’s thyroid after intrauterine antigenic action of staphylococcal toxoid. Morphofunctional homeostasis and stromal-parenchymal proportional relationship to thyroid gland closely associated with the activity of immune cells, including special role of lymphocytes, macrophages, and mast cells. Prenatal influence of staphylococcal toxoid led to the formation of a more pronounced structure of the parenchyma and stroma, but they showed signs of functional immaturity after birth. During the sucking period, the simultaneous presence of intra-, extrafollicular, septal and intramural types of folliculogesis is determined, which is a local reaction to systemic antigenic irritation with activation of compensatory-adaptive reactive folliclegenesis. The revealed changes in the process of folliclegenesis, accompanied by venous plethora, the formation of intraorgan diffuse lymphoid tissue and nodules, desquamation of the follicular epithelium, redistribution of the follicle diameter is a reaction to the systemic antigenic effect on the body during the critical period of prenatal development and normalizes by 45 days.Keywords: morphogenesis, thyroid gland, antigen, staphylococcal toxoid, experiment.