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This study explored the migration of follicular fluid (FF)-derived extracellular vesicles (EVs) of the uterine environment to the bloodstream and their interaction with neutrophils in vivo and in vitro. For the in vivo experiment, six Nellore heifers (Bos indicus) received an intrauterine infusion seven days after ovulation with 1X PBS only (sham group; n=1), 1X PBS stained with lipophilic dye PKH26 (control group; n=2), or FF-derived EVs stained with PKH26 (treated group; n=3). Plasma was collected at 0, 10, 30, 60-, 180-, 360-, 720-, and 1440-min post-infusion to obtained EVs for analysis by nano flow cytometry. Labeled EVs were present in the bloodstream at 30- and 60-min post-infusion in the treatment group. Additionally, plasma derived-EVs from all groups were positive for Calcein-AM, Alix, Syntenin, and Calnexin, which confirm the presence of EVs. The second experiment utilized the plasma-derived EVs from the heifers from 30 and 60 min timepoints to evaluate if neutrophils can uptake EVs in vitro. As results, it was possible to observe the presence of labeled EVs in neutrophils treated with plasma derived-EVs from the treatment group. In summary, our results suggest that labeled EVs can migrate from the uterine environment rapidly and interact with circulating immune cells in bovine.

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The aim of this work was to evaluate pregnancy rates (PR) and ovulatory characteristics of Nelore cows receiving PGF2α at the time of AI (artificial insemination) in a progesterone(P4)/estradiol-based timed-AI protocol. We also compared the effects of PGF2α treatment at AI in cows inseminated with conventional or sex-sorted semen, with the absence or expression of estrus. In experiment 1, a total of 701 suckled, multiparous Nelore cows from two commercial beef farms were submitted to the same protocol. All cows received a 12.5 mg (IM) injection of dinoprost tromethamine (Dinoprost; Lutalyse®; PGF treatment) at days 7 and 9 of a timed-AI protocol. Following P4 device removal (day 11; D11), AI was performed 48 h later with conventional or sex-sorted semen from two different sires. At AI, cows received an additional dose of 12.5 mg (IM) of Dinoprost (PGF treatment) or 2.5 mL (IM) of sterile saline (Control). Estrus behavior was determined at D11 by activation of an estrus detection device (Estrotect®). The overall PR was 32.8% (n = 348) at Farm 1 and 42.3% (n = 353) at Farm 2 (P = 0.01). Despite PR differences between farms, the same factors affected PR at Farms 1 and 2. Body condition score (P = 0.02), estrus behavior (P = 0.01), and type of semen (P < 0.001) were factors affecting PR. Conventional semen had a 2.73x greater chance of successful pregnancy than sex-sorted semen. Cows displaying estrus had a 2.5x greater chance of successful pregnancy than cows that did not display estrus. No treatment effect (P = 0.67) was detected in cows receiving conventional or sex-sorted semen. However, there was a tendency (P = 0.08) for an interaction between treatment (PGF or control) and estrus behavior (estrus or no estrus). PGF2α at the time of AI tended to increase PR of cows that did not display estrus (P < 0.10). In experiment 2, 29 suckled, multiparous Nelore cows were compared using B-mode and Doppler ultrasongraphy to assess the ovulatory characteristics of cows receiving the 12.5 mg (IM) injection of Dinoprost (PGF treatment) or saline solution (control) at D11. No significant effects of PGF2α treatment at D11 were observed in follicular characteristics and/or ovulation performance. It was concluded that fertility of sex-sorted semen was lower than conventional semen, regardless of the PGF2α treatment. The 12.5 mg treatment of Dinoprost at AI did not accelerate the occurrence of ovulation; however, it was interesting to note that PGF2α treatment at timed-AI appeared to increase the fertility of cows that did not display estrus, independent of semen type.

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Reproductive failure and pregnancy loss in cattle are some of the largest economic burdens to cattle producers and one of most perplexing factors influencing management decisions. Pregnancy loss may occur at any point during gestation with the largest percentage of loss occurring in the first 30 days and, subsequently, decreasing as the pregnancy progresses. Losses may be attributed to numerous factors, predisposed issues or environmental conditions such as nutritional stressors or disease. From a research perspective, determining the exact causes of pregnancy loss or embryonic mortality in cattle have been difficult, due to limitations of accurately determining early gestation pregnancy status. Until methods that precisely determine embryo success early in gestation are available, our understanding of in vivo pregnancy loss will lack clarity necessary to develop management strategies to decrease such loss. In this review, we will briefly discuss the pivotal periods of pregnancy loss affecting beef and dairy cattle, methods and technologies to determine pregnancy status and embryo viability and potential opportunities to decrease reproductive failure.

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Two experiments were conducted to evaluate if an additional prostaglandin F2α (PG) injection during an estradiol-based estrous synchronization treatment regimen affects pregnancy rates resulting from fixed-time artificial insemination (FTAI) in Nelore cows. In Experiment 1, 1039 cows were administered estradiol benzoate and an intravaginal progesterone releasing device (CIDR) on d -11, an injection of PG on d -4, with CIDR removal and administration of estradiol cypionate and eCG occurring on d -2, and FTAI on d 0. Cows were administered an additional injection of PG (PG2) or saline (PG1) on d -2. Percentage pregnancy per FTAI on d 30 was greater (P = 0.01) in cows of the PG2 than PG1 group (54.5 % and 46.6 %, respectively). In Experiment 2, there was use of the same treatment regimen for estrous synchronization of 934 cows as that for Experiment 1 followed by FTAI, with there being diagnosis of whether cows had or did not have a corpus luteum on d -4. Response to estrous synchronization, largest follicle diameter at FTAI, and pregnancy per FTAI were greater (P ≤ 0.05) in cows of the PG2 than PG1 group with a body condition score (BCS) < 5.0 at FTAI (81.2 % and 72.6 %, 11.9 and 11.2 mm, 55.5 and 45.6 %; respectively). These treatment responses did not differ (P ≥ 0.18) in cows with BCS ≥ 5.0. Collectively, results indicate that treatment with PG2 increased pregnancy per FTAI in B. indicus cows deficient in body energy reserves, by enhancing follicle development and estrous synchronization response.

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Changes in circulating progesterone (P4) and estradiol (E2) during proestrus produce dynamic changes in endometrial function and pituitary release of gonadotropins. Independent and combined effects of P4 and E2 on endometrium and pituitary were evaluated. In a preliminary study, an exogenous hormone model of proestrus was created by removal of corpus luteum and follicles ≥5 mm followed by gradual removal of intravaginal P4 implants during 18 h and treatment with increasing doses of estradiol benzoate during 48 h to mimic proestrus using high E2 (n = 9) or low E2 (n = 9). Decreased P4, increased E2, and increased endometrial area (EA) simulated proestrus in high-E2 cows and this was used subsequently. The main experiment used a 2 × 2 factorial design with: high E2 and low P4 (n = 11); high E2 and high P4 (n = 11); low E2 and high P4 (n = 11); low E2 and low P4 (n = 10). At 48 h, gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) and follicle stimulating hormone (FSH) release was determined. Variables were analyzed using PROCMIXED of Statistical Analysis System. The EA increased dramatically during 48 h only in high-E2 and low-P4 cows. For FSH, high-E2 cows had greater area under the curve (AUC) and FSH peak after GnRH than low E2, with mild negative effects of high P4. For LH, concentration at peak and AUC were 2-fold greater in high E2 compared to low-E2 groups, with low P4 also 2-fold greater than high-P4 groups. Thus, maximal changes in uterus and pituitary during proestrus depend on both low P4 and high E2, but different physiologic responses are regulated differently by E2 and P4. Changes in endometrium depend on low P4 and high E2, whereas GnRH-induced FSH secretion primarily depends on high E2, and GnRH-induced LH secretion is independently increased by high E2 or reduced by high P4.

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We aimed to evaluate the accuracy of Interferon-tau stimulated genes (ISG) abundance in peripheral blood polymorphonuclear cells (PMNs) on D20 after fixed-time artificial insemination (FTAI; D0) as a pregnancy diagnosis method against CL evaluation by Doppler ultrasonography and progesterone (P4) concentrations on D20, as well as Pregnancy Associated Glycoproteins (PAG) concentrations on D25. Additionally, we evaluated the potential of ISG abundance in PMNs as pregnancy loss predictors. Nelore heifers (n = 103) and cows (n = 144) underwent estrous synchronization and were artificially inseminated on D0. Pregnancy was diagnosed by B-mode ultrasonography on D30 and D70, and after the final diagnosis, females were classified in four groups: Pregnant; Non-pregnant; Functional CL on D20 but non-pregnant (CL-NP) and Pregnancy loss between D30 and D70 (PL). After determining cutoff values, the Sensitivity (SE), Specificity (SP), Positive Predictive Value (PPV), Negative Predictive Value (NPV) and Accuracy (ACC) were determined for each method. All methods were classified as significant (P < 0.05) predictors of pregnancy. Both ISG expression and PAG concentrations were greater (P < 0.05) in pregnant females than in non-pregnant and CL-NP females but did not differ (P > 0.05) from the PL group. ISG15 expression was greater (P < 0.05) in heifers than in cows, but this difference was not found in OAS1 expression and PAG concentrations. All the methods evaluated were proven to be adequate predictors of pregnancy, but greater accuracies were obtained through PAG concentrations and Doppler-US, due to the decreased number of false positive and false negative results.

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Reproductive failure and pregnancy loss in cattle are some of the largest economic burdens to cattle producers and one of most perplexing factors influencing management decisions. Pregnancy loss may occur at any point during gestation with the largest percentage of loss occurring in the first 30 days and, subsequently, decreasing as the pregnancy progresses. Losses may be attributed to numerous factors, predisposed issues or environmental conditions such as nutritional stressors or disease. From a research perspective, determining the exact causes of pregnancy loss or embryonic mortality in cattle have been difficult, due to limitations of accurately determining early gestation pregnancy status. Until methods that precisely determine embryo success early in gestation are available, our understanding of in vivo pregnancy loss will lack clarity necessary to develop management strategies to decrease such loss. In this review, we will briefly discuss the pivotal periods of pregnancy loss affecting beef and dairy cattle, methods and technologies to determine pregnancy status and embryo viability and potential opportunities to decrease reproductive failure.(AU)

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The objective of this study was to evaluate a combination of best management practices strategy for steer calves grazing tall fescue pastures with a range of toxicity. The experiment was conducted over 2 grazing seasons (fall 2015 for 91 d and spring 2016 for 84 d). Steers (n = 80 within season, body weight [BW] = 197.0 ± 15.43 kg [fall] and 116.9 ± 4.88 [spring]) were stocked at 2.45 and 4.1 calves/ha in fall and spring, respectively, to 16 pastures with varying levels of toxicity based on interim ergovaline (EV) concentration within season. Pastures were assigned to either mineral (MIN, n = 8) only management (MGMT) or a cumulative MGMT (CM, n = 8). The CM treatment included an implant containing 40-mg trenbolone acetate, 8-mg estradiol, and 29-mg tylosin tartrate (Component TE-G with Tylan, Elanco Animal Health, Greenfield, IN), 150 mg/calf daily monensin (Elanco Animal Health), and 1% BW of a 50:50 corn gluten feed:soybean hull supplement (as-is basis). Data were analyzed within season using pasture as the experimental unit. For fall and spring, the EV concentration was 1,476 ± 883.2 and 1,173 ± 620.6 ppb, respectively, and ranged from 90 to 2,180 ppb. During the fall, forage allowance did not differ (P = 0.76) between CM and MIN. In the spring, however, forage allowance only differed for the month of June (P ≤ 0.05, 2.55 vs. 3.22 ± 0.177 kg DM/kg BW, for MIN and CM, respectively). In the fall, average daily gain (ADG) responded to the simple effects of EV (P = 0.01) and MGMT (P < 0.001), and ADG for MIN steers was explained by ADG = 0.41 - 0.000064 × EV, whereas ADG for CM was explained by ADG = 1.05 - 0.000064 × EV. In the spring, there was an EV × MGMT interaction (P = 0.03) for ADG. For MIN, ADG = 0.80 - 0.000278 × EV, whereas for CM, ADG = 0.94 + 0.000001835 × EV. In spring, the ADG response to CM relative to MIN increased as EV increased. The CM strategy resulted in lower blood urea nitrogen than MIN in fall and spring (P < 0.01), but prolactin and serum Cu were not affected by MGMT in either season. In conclusion, performance was improved within the fescue belt by implementing feeding strategies using implants, ionophores, and supplementation, but a detailed economic analysis is warranted. Further research is needed to evaluate CM programs under varied stocking rates and in combination with dilution of endophyte-infected fescue pastures with nontoxic grasses or legumes.

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This review summarizes strategies to increase overall pregnancy rates to TAI protocols, and potential areas for improvement in reproductive management of Nelore cows. Low BCS at calving and postpartum BCS loss negatively impacted percentage of pregnant females to TAI, with primiparous cows being the most sensitive to the effects of low BCS during the postpartum period. The current reproductive management recommended for replacement heifers (with TAI on day 0) consist of intravaginal progesterone device insertion from day -35 to -23, 0.6 mg i.m. injection of estradiol cypionate on day -23, insertion of another intravaginal progesterone device and 2.0 mg i.m. injection of estradiol benzoate on day -11, 12.5 mg i.m. injection of dinoprost tromethamine on day -4, intravaginal progesterone device removal and 0.6 mg i.m. injection of estradiol cypionate on day -2, and TAI on day 0. Supplementing corn for 41 days after the first TAI of the breeding season increased pregnancy to a second AI in primiparous cows, increased final proportion of pregnancy in primiparous cows, but decreased the final proportion of pregnancy in mature cows. In turn, supplementing melengestrol acetate or Ca salts of PUFA during the expected time of luteolysis was beneficial to pregnancy rates. Vaccinating cows against BoHV-1, BVDV, and Leptospira spp., particularly when both doses were administered before TAI, improved cow reproductive performance. Cow temperament has direct implications not only on reproductive efficiency of B. indicus females, but also on overall production efficiency in cow-calf system based on B. indicus cattle. Lastly, concentration of pregnancy-associated glycoproteins was driven by the ability of pregnancy maintenance and by sire used at TAI, whereas exploring this relationship might be interesting to improve sire fertility regarding late embryonic loss in Nelore females.

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This review summarizes strategies to increase overall pregnancy rates to TAI protocols, and potential areas for improvement in reproductive management of Nelore cows. Low BCS at calving and postpartum BCS loss negatively impacted percentage of pregnant females to TAI, with primiparous cows being the most sensitive to the effects of low BCS during the postpartum period. The current reproductive management recommended for replacement heifers (with TAI on day 0) consist of intravaginal progesterone device insertion from day -35 to -23, 0.6 mg i.m. injection of estradiol cypionate on day -23, insertion of another intravaginal progesterone device and 2.0 mg i.m. injection of estradiol benzoate on day -11, 12.5 mg i.m. injection of dinoprost tromethamine on day -4, intravaginal progesterone device removal and 0.6 mg i.m. injection of estradiol cypionate on day -2, and TAI on day 0. Supplementing corn for 41 days after the first TAI of the breeding season increased pregnancy to a second AI in primiparous cows, increased final proportion of pregnancy in primiparous cows, but decreased the final proportion of pregnancy in mature cows. In turn, supplementing melengestrol acetate or Ca salts of PUFA during the expected time of luteolysis was beneficial to pregnancy rates. Vaccinating cows against BoHV-1, BVDV, and Leptospira spp., particularly when both doses were administered before TAI, improved cow reproductive performance. Cow temperament has direct implications not only on reproductive efficiency of B. indicus females, but also on overall production efficiency in cow-calf system based on B. indicus cattle. Lastly, concentration of pregnancy-associated glycoproteins was driven by the ability of pregnancy maintenance and by sire used at TAI, whereas exploring this relationship might be interesting to improve sire fertility regarding late embryonic loss in Nelore females.(AU)

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