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 < 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.

A rapid and robust method for the cryopreservation of human granulosa cells

Human primary granulosa cells (GCs) derived from women undergoing oocyte retrieval can be cultured and used as a cellular model for the study of human ovarian function. In vitro, they change rapidly, initially resembling cells of the preovulatory follicle and then cells of the corpus luteum. They are derived from individual patients, whose different medical history, lifestyle and age lead to heterogeneity. Thus, cells can rarely be ideally matched for cellular experiments or, if available, only in small quantities. We reasoned that cryopreservation of human GCs may be helpful to improve this situation. Previous studies indicated the feasibility of such an approach, but low survival of human GCs was reported, and effects on human GC functionality were only partially evaluated. We tested a slow freezing protocol (employing FCS and DMSO) for human GCs upon isolation from follicular fluid. We compared cryopreserved and subsequently thawed cells with fresh, non-cryopreserved cells from the same patients. About 80% of human GCs survived freezing/thawing. No differences were found in cell morphology, survival rate in culture, or transcript levels of mitochondrial (COX4, OPA1, TOMM20), steroidogenic (CYP11A1, CYP19A1) or cell–cell contact genes (GJA1) between the two groups in cells cultured for 1–5 days. A proteomic analysis revealed no statistically significant change in the abundance of a total of 5962 proteins. The two groups produced comparable basal levels of progesterone and responded similarly to hCG with elevation of progesterone. Taken together, our results show this to be a rapid and readily available method for the cryopreservation of human GCs. We anticipate that it will allow future large-scale experiments and may thereby improve cellular studies with human ovarian cells.

Pregnancy rate in ewes injected with zinc oxide during an estrus synchronization protocol

Objective: To measure the effect of zin injection, during an estrus synchronization protocol, on pregnancy rate in sheep from the “Valle de Mexicali”.Design/methodology/approach: The experimental units were 157 ewes, which wereallocated in five farms (UP): UP1 (n=19), UP2 (n=27) UP3 (n=20) UP4 (n=71) and UP5(n=21). In each farm, the ewes were randomly assigned to one of three treatments:control, z-100 and z-200. The ewes from control groups were subcutaneously injectedwith 4 mL of olive oil as placebo. The ewes from groups z-100 and z-200 weresubcutaneously injected with 100 and 200 mg of zinc oxide. The response variableswere the preovulatory diameter of the largest follicle and pregnancy rate.Results: The differences between experimental groups on diameter of the largestpreovulatory follicle and pregnancy rates were not significant different (p>0.05). Limitations of the study/implications: The ewe’s reproductive response to zincinjection might be affected by the animal mineral status, it is recommended to carry onsupplementation with base on mineral blood concentrations.Conclusion: The subcutaneous injection with 100 or 200 mg of zinc oxide did not affectthe size of the largest preovulatory follicle and pregnancy rate in ewes.

The relationship between digital ultrasonographic image echotexture parameters of the uterus and preovulatory follicle development and ovulation in purebred Arabian mares

Objective assessment of ultrasonographic images is only possible using computer-assisted echotexture analysis. This study investigated the relationship between cyclic digital echotexture analysis of ultrasonographic uterine images, and preovulatory follicle development and ovulation in 48 cyclic purebred Arabian mares during the breeding season. When the mares’ ovarian follicle diameters exceeded 30 mm, ultrasonographic examinations were performed and ultrasonographic images recorded with uterine endometrial edema degrees following follicle development until ovulation day. Echotexture measurements, including mean grayness value (MGV), heterogeneity (HET) and contrast (CON) parameters, were performed on the days when ovarian follicle diameters reached 30 mm (Group 1, n = 12), 35 mm (Group 2, n = 12), 40 mm (Group 3, n = 12) and 45 mm (Group 4, n = 12) in images recorded the day before ovulation and on the day of ovulation. MGV was the lowest in Group 2 and the difference from the other groups was statistically significant (P<0.001). CON was significantly higher in Group 2 than Groups 3 and 4 (P<0.01). However, there was no significant difference compared to Group 1 (P>0.05). Variations in HOM (P<0.05), contrast (P<0.05) and MGV (P<0.01) were statistically significant on the day before ovulation and ovulation day. Thus, uterine ultrasonographic echotexture parameters are associated with preovulatory follicle development in mares, which suggests that these parameters can indicate the day of ovulation in purebred Arabian mares.

Vascular Perfusion of the Preovulatory Follicle and its Relationship in Pregnancy Establishment during Natural and Induced Estrus in Buffaloes

Background: In buffaloes, little attention has been devoted on the haemodynamics of preovulatory follicle (POF) and serum estradiol profiles and their relationship with conception rate following AI at natural and induced estrus. Methods: Sixty healthy pleuriparous graded Murrah buffaloes were selected and divided into two groups, viz., Group I and II. Group I buffaloes were artificially inseminated twice at an interval of 24 hours during natural estrus. Estrus induction was done using PIVD and PGF2α in group II buffaloes and AI was performed 48 h and 72 h after the withdrawal of PIVD. The blood flow of the preovulatory follicle (POF) and early pregnancy were assessed in both the groups by Doppler ultrasonography.Result: On statistical analysis, the mean (± SE) of spectral indices revealed there was an increased blood flow to the POF during induced estrus (Group II) when compared to natural estrus (Group I). The conception rate observed was 66.67 and 58.33 per cent in group I and II, respectively. It is concluded that increased vascular perfusion to POF resulted in almost similar conception rates in induced and natural estrus following AI in buffaloes.

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.

Prostaglandin F2? as ovulatory stimulus in dairy cows and buffaloes raised in the Amazon biome

This study evaluated the effect of a prostaglandin F2? (PGF) analogue as an ovulatory stimulus in dairy cows and buffaloes raised in the Amazon biome. To this end, three experiments were performed in the state of Rondônia, located in the Amazon biome. In Experiment 1, 22 lactating dairy buffaloes received 2 mg of intramuscular (I.M.) estradiol benzoate (EB) on day 0 and an intravaginal progesterone-releasing device (CIDR) from day 0 to day 9 of the protocol. On days 8 and 9, all cows were given 500 ?g of I.M. d-cloprostenol (PGF analogue). On day 10, buffaloes were divided into two groups to receive 500 ?g of PGF (PGF group, n = 8) or no treatment (CTL group, n = 14), respectively. In Experiment 2, 16 lactating crossbred dairy cows (Holstein x Gir) received 2 mg of EB on day 0 and a CIDR insert from day 0 to day 8. On days 7 and 8, all cows were given 500 ?g of d-cloprostenol. On day 9, cows were divided into two groups to receive 500 ?g of d-cloprostenol (PGF group, n = 8) or no treatment (CTL group, n = 8), respectively. In Experiment 3, 16 lactating crossbred dairy cows (Holstein x Gir) were handled and treated similarly as in Experiment 2, although cows did not receive d-cloprostenol on day 8. Single-point outcome variables were analyzed using one-way analysis of variance (ANOVA), while proportions with dichotomous outcomes were analyzed with the chi-square test. In Experiment 1, there was no difference (P = 0.30) in the ovulation rate between groups, and, on average, 68% of the buffaloes ovulated. Moreover, the treatment did not affect the interval to ovulation (P = 0.61) nor the diameter of the preovulatory follicle (P = 0.47). As for Experiment 2, only one cow, from the PG group, did not ovulate. There were no differences between the CTL and PG groups (P = 0.69) in the moment of ovulation, which occurred in average 82 h after CIDR removal. Finally, in Experiment 3, cows treated with PGF ovulated earlier than those in the CTL group (62.5 ± 5.8 and 94.5 ± 13.5 h, respectively; P = 0.05). Collectively, these results suggest that PGF hastens ovulation in lactating dairy cows, whereas no effect was observed in dairy buffaloes.