iTRAQ-Based proteomic dataset for bovine pre-ovulatory plasma and follicular fluid containing high and low Estradiol.

This is isobaric tags for a relative and absolute quantification (iTRAQ)-Based Proteomic Data on bovine plasma (PL) and follicular fluid (FF) containing high and low pre-ovulatory circulating concentration of estradiol (E2). The PL and FF were collected from nine beef cows that were identified to initiate a new follicular wave on day -4 during synchronization. Follicular dynamics and ovulatory response were monitored using transrectal ultrasonography. Blood samples were collected at slaughter and FF was aspirated from dominant follicles (DF; >10 mm). Estradiol concentrations in PL and FF were measured by radioimmunoassays. Plasma and FF were labeled as containing high E2 (PL HE2 and FF HE2) or low E2 (PL LE2 and FF LE2). Abundant proteins (albumin, IgG, IgA, and alpha-1-antitrypsin) were depleted from the four PL and FF samples. Peptides were labeled with iTRAQ reagents and analyzed using 2-dimentional liquid chromatography ESI-based mass spectrometry. Proteins were identified and quantified using SEQUESTTM search engine embedded in Proteome Discoverer. The proteins matched with at least one unique peptide at minimum 95% confidence were considered positive identifications. Protein expression levels were determined by assigned fold change of >2.0 or <0.5 between any pair from the four sample types. The paired comparisons made were PL HE2 and PL LE2, FF HE2 and FF LE2, PL HE2 and FF HE2, and PL LE2 and FF LE2. Protein Analysis Through Evolutionary Relationships (PANTHER) and Database for Annotation, Visualization and Integrated Discovery (DAVID) were used to classify protein functions. This dataset includes the overview of workflow for identification and quantification of proteins and details on 231 proteins identified which includes 103 up- and down-regulate proteins. This dataset can be useful for further probing of the identified regulated proteins to better understand folliculogenesis and ovulation, particularly in bovine. This dataset is related to the article 'iTRAQ-Based Proteomic Analysis of Bovine Pre-ovulatory Plasma and Follicular Fluid' by P. A. Afedi, E. L. Larimore, R. A. Cushman, D. Raynie, G. A. Perry. Domestic Animal Endocrinology. https://doi.org/10.1016/j.domaniend.2021.106606.

iTRAQ-based proteomic analysis of bovine pre-ovulatory plasma and follicular fluid.

Bovine follicular fluid (FF) creates a unique microenvironment in follicles necessary for follicle growth, oocyte maturation, and estradiol (E2) production. The objective of this study was to analyze changes in proteins in FF and plasma (PL) from animals with high E2 (HE2) or low E2 (LE2) during the preovulatory period. Beef cows were synchronized, and follicular dynamics and ovulatory response were monitored using transrectal ultrasonography. Nine cows were selected and slaughtered, blood samples were collected at slaughter and FF was aspirated from dominant follicles (DF; >10 mm). Abundant proteins (albumin, IgG, IgA, and alpha-1-antitrypsin) were depleted from both PL and FF. Peptides were labeled with iTRAQ reagents and quantified using 2-dimentional liquid chromatography ESI-based mass spectrometry. Estradiol was associated with protein changes in PL and FF. Protein expression changes between FF HE2 and FF LE2 were greater than between PL HE2 and PL LE2. There were 15 up-regulated proteins and 10 down-regulated proteins in FF HE2 compared to FF LE2, and 7 proteins up-regulated and 9 proteins down-regulated in PL HE2 compared to PL LE2. Several of the differentially expressed proteins function in follicle development and were mainly categorized under cellular process and metabolic process. Pathway analysis identified the up- and down-regulated proteins were predominantly associated with the complement and coagulation cascades. The data demonstrate E2 regulates a wide range of reproductive associated proteins in bovine PL and FF and can provide the basis for further investigation of specific processes involved in such regulation.

Influence of post-insemination nutrition on embryonic development in beef heifers.

Previous studies have demonstrated that a decrease in nutrition immediately following AI reduces pregnancy success in beef heifers. The objective of this experiment was to determine if nutrient restriction following AI impacted early embryonic development among non-super ovulated heifers. Beef heifers in eight replications (Rep; Rep 1; n = 14, Rep 2; n = 15, Rep 3; n = 15, Rep 4; n = 14, Rep 5; n = 15, Rep 6; n = 15, Rep 7; n = 25, Rep 8; n = 25) across two locations (UMN, SDSU) were developed in a dry-lot and fed 125% NRC requirements from weaning to timed-AI (d 0). Heifers were timed-AI to a single sire in all replications. Immediately following AI, heifers were assigned, based on age, weight, and estrous response to one of two post-AI nutritional treatments. Half the heifers in each replication continued on the pre-insemination diet, serving as the control treatment (CON) and the remaining heifers were restricted to a sub-maintenance diet (RES). At UMN, heifers in the RES treatment were fed the same diet, but intake was limited to 80% NEm, while at SDSU, DMI remained the same, but diet composition was altered with the addition of straw to reduce NEm to 50% of requirements. On d 6, single embryos were collected nonsurgically and recovered embryos (CON; n = 46, RES; n = 42) were evaluated to determine quality (grade 1-9) and stage (1-4). Embryos were then stained and evaluated to determine the number of dead cells and total blastomeres. In Reps 1 through 6, concentrations of IGF-1 were assessed on d 0 and 6 and progesterone concentrations on d 4 and 6. Data were analyzed using the Mixed procedures of SAS. There were no treatment by Rep or treatment by location interactions for any embryo parameter evaluated, thus all data were pooled. Embryo stage and quality were improved (P < 0.01) in the CON (4.4 ± 0.16, 2.2 ± 0.19, respectively) compared to RES treatment (3.7 ± 0.16, 2.9 ± 0.19, respectively). Embryos in the CON treatment had greater total blastomeres (66.9 ± 5.05; P < 0.01) and tended to have a greater percentage of live cells (P < 0.10; 80.9 ± 4.19%) compared to RES (47.9 ± 5.41; 69.7 ± 4.39%, respectively). Progesterone and IGF-1 concentrations did not differ between treatments. In summary, nutrient restriction for 6 days immediately following AI resulted in poorer quality embryos that were delayed in stage of development, suggesting that immediate changes in nutritional status after insemination can alter early embryonic development.

Using estrus detection patches to optimally time insemination improved pregnancy risk in suckled beef cows enrolled in a fixed-time artificial insemination program.

A multilocation study examined pregnancy risk (PR) after delaying AI in suckled beef cows from 60 to 75 h when estrus had not been detected by 60 h in response to a 7-d CO-Synch + progesterone insert (CIDR) timed AI (TAI) program (d -7: CIDR insert concurrent with an injection of GnRH; d 0: PGF injection and removal of CIDR insert; and GnRH injection at TAI [60 or 75 h after CIDR removal]). A total of 1,611 suckled beef cows at 15 locations in 9 states (CO, IL, KS, MN, MS, MT, ND, SD, and VA) were enrolled. Before applying the fixed-time AI program, BCS was assessed, and blood samples were collected. Estrus was defined to have occurred when an estrus detection patch was >50% colored (activated). Pregnancy was determined 35 d after AI via transrectal ultrasound. Cows ( = 746) detected in estrus by 60 h (46.3%) after CIDR removal were inseminated and treated with GnRH at AI (Control). Remaining nonestrous cows were allocated within location to 3 treatments on the basis of parity and days postpartum: 1) GnRH injection and AI at 60 h (early-early = EE; = 292), 2) GnRH injection at 60 h and AI at 75 h (early-delayed = ED; = 282), or 3) GnRH injection and AI at 75 h (delayed-delayed = DD; = 291). Control cows had a greater ( < 0.01) PR (64.2%) than other treatments (EE = 41.7%, ED = 52.8%, DD = 50.0%). Use of estrus detection patches to delay AI in cows not in estrus by 60 h after CIDR insert removal (ED and DD treatments) increased ( < 0.05) PR to TAI when compared with cows in the EE treatment. More ( < 0.001) cows that showed estrus by 60 h conceived to AI at 60 h than those not showing estrus (64.2% vs. 48.1%). Approximately half (49.2%) of the cows not in estrus by 60 h had activated patches by 75 h, resulting in a greater ( < 0.05) PR than their nonestrous herd mates in the EE (46.1% vs. 34.5%), ED (64.2% vs. 39.2%), and DD (64.8% vs. 31.5%) treatments, respectively. Overall, cows showing estrus by 75 h (72.7%) had greater ( < 0.001) PR to AI (61.3% vs. 37.9%) than cows not showing estrus. Use of estrus detection patches to allow for a delayed AI in cows not in estrus by 60 h after removal of the CIDR insert improved PR to TAI by optimizing the timing of the AI in those cows.

Changes in ovarian function associated with circulating concentrations of estradiol before a GnRH-induced ovulation in beef cows.

These studies were conducted to evaluate causes for differences in circulating concentrations of estradiol before a GnRH-induced ovulation. Beef cows were synchronized by an injection of GnRH on day -7 and an injection of prostaglandin F2α (PGF2α) on day 0. In experiment 1, blood samples were collected every 3 h from PGF2α on day 0 to hour 33 after PGF2α and at slaughter (hour 36 to 42; n = 10). Cows were assigned to treatment group based on circulating concentrations of estradiol (E2): HighE2 vs LowE2. At slaughter, follicular fluid (FF) and granulosa cells were collected from the dominant follicle. In experiment 2, blood samples (n = 30) were collected every 8 h from PGF2α until the dominant follicle was aspirated via ultrasound-guided follicular aspiration to collect FF and granulosa cells (hour 38 to 46). In experiment 1, HighE2 had increased abundance of 3ß-hydroxysteroid dehydrogenase, cytochrome P450 aromatase, and LHR (P ≤ 0.02), and greater concentrations of estradiol and androstenedione (P ≤ 0.02) in the FF. In experiment 2, HighE2 had increased abundance of CYP11A1, 3ß-hydroxysteroid dehydrogenase, cytochrome P450 aromatase, and LHR (P ≤ 0.03) vs either LowE2 or GnRHLowE2. There was a tendency (P = 0.07) for LH pulse frequency to be increased in both the GnRHLowE2 and HighE2 compared with LowE2. HighE2 cows experienced increas in circulating concentrations of estradiol compared with LowE2. In conclusion, animals with greater concentrations of circulating estradiol before fixed-time AI experienced an upregulation of the steroidogenic pathway during the preovulatory period.

Postweaning nutritional programming of ovarian development in beef heifers.

Peripubertal caloric restriction increases primordial follicle numbers at breeding, which may improve reproductive potential. Our hypothesis was that feed restriction was changing primordial follicle number through stimulation of follicle formation via leptin, roundabout axon guidance receptor, homolog 4 (), or or through inhibition of follicle activation via anti-Müllerian hormone (). Heifers ( = 30) were fed a ration consisting of 30% alfalfa hay, 69.8% corn silage, and 0.2% salt as DM. Heifers received the control diet for 42 d before an initial 6 heifers were ovariectomized at 8 mo of age. The remaining 24 heifers were divided into 2 treatment groups. Controls were offered 97.9 g DM/kg BW over the entire feeding period. Stair-step heifers received 67.4 g DM/kg BW for 84 d. Following the 84-d restriction, heifers were stepped up to receive 118.9 g DM/kg BW over a 15-d period and were held at this feeding level 68 d. At the end of the feed restriction (11 mo of age), ovaries were collected from 6 heifers per treatment, and at the end of the refeeding period (13 mo of age), ovaries were collected from 6 heifers per treatment. Plasma leptin concentrations were greater in control heifers than in stair-step heifers at 11 mo of age ( < 0.0001). In histological sections, stair-step heifers had more primordial follicles ( = 0.03) than control heifers at 13 mo of age. There was no difference in secondary or antral follicle numbers between dietary treatment groups or ages. Relative abundance of mRNA in ovarian cortex of control heifers was greater at 13 mo than at 11 mo or before feed restriction (8 mo; = 0.01). Relative abundance of mRNA in stair-step heifers at 13 mo was greater than before feed restriction ( = 0.02) and at 11 mo did not differ from 8 or 13 mo ( = 0.70). Relative abundance of mRNA in the ovarian cortex followed a similar pattern, being greater in stair-step heifers at 11 mo compared with control heifers ( = 0.001). At 13 mo, mRNA did not differ between treatments ( = 0.30). Abundance of mRNA in the ovarian cortex did not change due to dietary treatment or age ( > 0.10). In conclusion, developing heifers on a stair-step compensatory growth scheme resulted in larger ovarian reserve before the onset of breeding, which may have beneficial effects on increasing reproductive lifespan.

Influence of estrus at fixed-time artificial insemination on early embryonic development in beef cattle.

It has been repeatedly demonstrated that estrous expression before fixed-time AI (TAI) results in increased pregnancy success. Therefore, the objective of this experiment was to determine if preblastocyst embryonic developmental characteristics differed from heifers that did or did not exhibit estrus before TAI. Beef heifers (n = 113) were synchronized using the 5-d CO-Synch + controlled internal drug release device with TAI on d 0. Before TAI, estrous expression was assessed twice daily. On d 6, single embryos were collected and visually evaluated to determine quality (International Embryo Transfer Society standards; 1-4, in which 1 = excellent/good and 4 = degenerate) and stage (1-9, in which 1 = unfertilized and 9 = expanded hatched blastocyst). Embryos were stained and evaluated to determine number of dead blastomeres, number of total blastomeres, and number of accessory sperm. Estrous expression before TAI did not affect the percent of embryos recovered (P = 0.59), number of dead cells (P = 0.99), or number of total cells (P = 0.25). However, heifers that exhibited estrus had increased mean (P = 0.03) and median accessory sperm numbers and (P = 0.01) percent live cells when compared with nonestrus heifers. Heifers that exhibited estrus also produced embryos that had a more advanced stage (P = 0.03) and improved quality (P = 0.04) when compared with those heifers not exhibiting estrus. When all heifers were evaluated, there was no correlation between circulating concentration of estradiol at TAI and embryo quality or embryo stage. There was a significant correlation between accessory sperm numbers and embryo quality (P = 0.01) and embryo stage (P < 0.01), such that as accessory sperm numbers increased, embryo quality and stage increased. In conclusion, exhibiting estrus before TAI resulted in improved embryo quality and advanced embryo stage on d 6 and increased the number of accessory sperm associated with the embryo.

Relationship of follicle size and concentrations of estradiol among cows exhibiting or not exhibiting estrus during a fixed-time AI protocol.

Cows exhibiting estrus near fixed-time artificial insemination (AI) had greater pregnancy success than cows not showing estrus. The objective of this study was to determine the relationship between follicle size and peak estradiol concentration between cows that did or did not exhibit estrus during a fixed-time AI protocol. Ovulation was synchronized in beef cows by applying the CO-Synch protocol [GnRH (100 µg) on day-9, prostaglandin F2α (PGF2α; 25 mg) on day-2, and a second injection of GnRH 48 h after PGF2α (day 0)] to both suckled (experiment 1) and nonsuckled (experiment 2) cows. Follicle size (day 0) and ovulation (day 2) was determined by ultrasonography. Blood samples were collected every 3 or 4 h beginning at the time of PGF2α injection (0 h). Estrus was detected by visual observation with the aid of estrus-detection patches, and cows that ovulated were classified as exhibited estrus (n = 46) or did not exhibit estrus (n = 63). In both suckled and nonsuckled cows, there was a positive relationship between all cows (P < 0.05) and among those that exhibited estrus (P < 0.05) between follicle size and peak estradiol concentration, but no linear relationship (P > 0.50) between follicle size and peak estradiol concentration was observed among cows not exhibiting estrus. Cows that exhibited estrus had greater (P < 0.01) peak estradiol concentrations than cows that did not exhibit estrus. Suckled cows exhibiting standing estrus had greater (P < 0.001) preovulatory concentrations of estradiol beginning 6 h (replicate 1) or 4 h (replicate 2) after the injection of PGF2α on day-2 compared with cows not exhibiting standing estrus. Nonsuckled cows exhibiting standing estrus had greater (P < 0.001) preovulatory concentrations of estradiol beginning at the injection of PGF2α on day-2 compared with cows not exhibiting standing estrus. Furthermore, cows that exhibited estrus had an increased (P < 0.01) rate in the rise in concentrations of estradiol following the PGF2α to peak estradiol than cows not exhibiting estrus. In summary, follicle diameter had a positive relationship with peak concentrations of estradiol, but only among cows that exhibited standing estrus, and estradiol increased earlier in cows that exhibited estrus compared with cows that did not.