Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin, in vivo (high developmental potential (IV)), in vitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus-conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to in vivo versus in vitro and in vivo versus cloned conceptuses (1153 and 334DEGs respectively) with limited differences between in vitro Vs cloned conceptuses (36 DEGs). The miRNA cargo contained in conceptus-derived EVs was similar between all three groups (426 miRNA in common). Only 8 miRNAs were different between in vivo and cloned conceptuses, while only 6 miRNAs were different between in vivo and in vitro-derived conceptuses. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changed the proteomic content of target cells (96 and 85, respectively) of which mRNAs are altered in the endometrium in vivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy.

Supplementation with long-acting injectable progesterone 3 days after TAI impaired luteal function in buffaloes.

Three experiments were conducted to evaluate the effects of long-acting injectable progesterone (iP4) in buffalo cows. In Experiment 1, ovariectomized buffaloes received 300 mg (iP300) or 600 mg (iP600) of iP4, and serum P4 concentrations were evaluated. In experiment 2, three groups were compared: control or administration of 300 mg of iP4 3 (iP4-D3) or 6 days (iP4-D6) after timed artificial insemination (TAI). On day 16, reproductive tract was recovered for conceptus, endometrium, and corpus luteum (CL) analysis. In experiment 3, pregnancy per AI (P/TAI) and proportion of pregnancy losses were evaluated after administration of 300 mg of iP4 3 (iP4-D3) or 6 days (iP4-D6) after TAI in lactating buffaloes. In experiment 1, serum P4 concentrations remained over 1 ng/mL for ~ 3 days in both groups. The 300 mg dose was used in subsequent experiments. In experiment 2, CL weight and endometrial glands density were decreased, and conceptus length was increased in iP4-D3 compared to control and to iP4-D6 (P < 0.05). Transcript abundance of Prostaglandin F Receptor (FP) and ISG15 in CL and of ISG15 and MX1 in endometrium was greater in iP4-D3 when compared to control and to iP4-D6 (P < 0.05). In experiment 3, there was no difference among experimental groups for P/TAI at D30 and pregnancy losses (P > 0.1); however, iP4-D3 presented a lower P/TAI at day 60 (41.7%) when compared to control (56.8%) and iP4-D6 (57.7%; P = 0.07). In conclusion, administration iP4 at 3 days after TAI affects CL development and consequently decreases final pregnancy outcome in buffaloes.

Effects of Early Weaning on Performance and Carcass Quality of Nellore Young Bulls.

This study compared early weaning (EW; 150 days) with conventional weaning (CW; 240 days) in Nellore young bulls, evaluating performance, carcass characteristics, and meat quality. A total of 74 non-castrated male calves were divided into two weaning strategies: EW (n = 37) and CW (n = 37). During the growth phase, which lasted 454 ± 14 d for EW calves and 359 ± 16 d for CW calves, animals received a protein-energy supplement at a ratio of 5 g per kg of body weight while grazing Brachiaria brizantha cv. Marandu. The animals were managed for an 87d finishing phase in three collective feedlot pens, with a 3-week adaptation protocol, starting with corn silage to a concentrate ratio of 55:45 and reaching a ratio of 30:70 in the final diet. Body weight, average daily gain (ADG), dry matter intake (DMI), feed efficiency (FE), carcass characteristics, and meat quality were evaluated. The EW group was approximately 44 kg lighter than the CW at the time of conventional weaning (p < 0.001). However, this weight difference did not influence ADG, DMI, and FE in the finishing phase. No significant differences were observed in carcass characteristics such as yield percentage, loin area, subcutaneous fat thickness, and meat quality, except for the weight of primal cuts, which was greater in the CW group (p < 0.001). Thus, although calves weaned early are lighter throughout subsequent production phases than those weaned conventionally, performance, efficiency, carcass yield, and meat quality are not affected.

Influence of sub-clinical endometritis on early pregnancy predictors and proinflammatory cytokines in circulating immune cells in dairy cows.

In Experiment 1, PBMC were isolated from cows considered healthy or with SCE (n=6/group) on Days 0 (estrus) and 7 (diestrus) of a synchronized estrous cycle. In Experiment 2, on D21 (D0 was defined as the day of Fixed Timed Artificial Insemination (FTAI), cows were evaluated by ultrasonography to assess luteal blood perfusion and PBMC were isolated. On D32, cows were classified into: healthy pregnant (n=7), pregnant with SCE (n=4), healthy non-pregnant (n=8), and non-pregnant with SCE (n=10). Gene expression of ISGs (ISG15, OAS1, MX1 and IFI6) and proinflammatory cytokines (IL1-ß, TNF-α and IFN-γ) were determined. Expression of ISG15, MX1, IFI6, TNF-α and IFN-γ did not differ between SCE and healthy cows and between Days 0 and 7. Expression of OAS1 and IL1-ß were higher (P=0.02) on Day 7 than Day 0, regardlees of the SCE presence. In Exp.2, ISG15 abundance was 2.5-fold greater (P=0.0008), TNF-α was 2.2-fold greater (P=0.05), and IL1-ß tended (P=0.06) to be 2.4-fold higher in pregnant than non-pregnant cows. Luteal blood perfusion was greater (P=0.01) in pregnant animals. In conclusion, OAS1 and IL1-ß are transcripts upregulated in PBMC at diestrus, regardless of SCE occurrence. Proinflammatory cytokines are not affected by SCE occurrence, but IL1-ß and TNF-α are upregulated in pregnant animals on D21 of pregnancy. ISG15 abundance is a good pregnancy predictor, regardless SCE presence.

Impact of 17ß-estradiol administration at the moment of timed-AI in Nelore cows with small dominant follicle or not showing estrus.

We aimed to evaluate the effects of administering estradiol (E-17ß) at the moment of timed-AI (TAI) on uterine gene expression, estrous expression rate (EER), and pregnancy rate (P/TAI) in Nelore cows with a small dominant follicle (DF) or not showing estrus at TAI. In Experiments 1 and 2 (Exp1, Exp2) cows were submitted to a P4/E-17ß-based protocol (day 0) for synchronization of ovulation. On day 7, devices were removed, cows received 1 mg E-17ß cypionate and 12.5 mg dinoprost. On day 9, cows with DF < 11.5 mm in diameter were split into different groups. In Exp1 (n = 16/group): Control (no treatment), E-2 (2 mg E-17ß) and E-4 (4 mg E-17ß). In Exp2: Control (n = 12); E-2 (n = 14); GnRH (0.1 mg gonadorelin acetate, n = 13); and E-2+GnRH (association of GnRH and E-17ß, n = 13). Between days 9 and 11, endometrial thickness (ET), time of ovulation detection, and EER were recorded. In Exp1, a uterine cytological sample was collected 4 h after treatment to evaluate the transcript expression of receptors for E-17ß (ESR1 and ESR2), oxytocin (OXTR), and P4 (PGR). In Experiment 3 (Exp3), 3829 suckled cows were submitted to a P4/E-17ß-based protocol for TAI. On day 9, devices were removed and cows received 1 mg E-17ß cypionate and 0.4 mg sodium cloprostenol. On day 11, TAI was performed and cows that did not demonstrate estrus received 0.1 mg gonadorelin acetate, and were allocated into two groups: GnRH (n = 368) and E-2+GnRH (2 mg E-17ß; n = 363). In Exp1, plasma E-17ß concentrations increased at 4 h after treatment in a dose-dependent manner but reduced at 12 h. The E-17ß-treated cows had greater transcript abundance for OXTR and lesser for ESR1 and ESR2, and the ET was reduced 12 h after treatment (P < 0.05). No significant difference (P > 0.1) was observed between the E-17ß doses in estrus or ovulation rate. In Exp2, the interval from treatment to ovulation was longer (P < 0.05) in the E-17ß group. GnRH-treated cows showed higher ovulation rates (89 vs. 35 %) compared to cows not treated with GnRH, as E-17ß-treated cows (P < 0.01) had a lower ovulation rate compared to those not receiving E-17ß (44 vs. 78 %). In Exp3, P/TAI was 55 % for cows in estrus. For those not showing estrus, no difference (P > 0.1) in P/TAI was observed between GnRH (34 %) and E-2+GnRH (31 %) groups. Cows with a DF ≥ 11 mm (n = 192) had a greater (P < 0.05) P/TAI (49 %) than those with DF < 11 mm (n = 377; 29 %). In conclusion, E-17ß administration in the moment of TAI modulates the mRNA expression of uterine receptors in cows with a small DF but does not impact the P/TAI compared with GnRH treatment in suckled Nelore not showing estrus previous to TAI.

Accuracy of early pregnancy diagnosis and determining pregnancy loss using different biomarkers and machine learning applications in dairy cattle.

This study aimed to compare the accuracy of IFN-τ stimulated gene abundance (ISGs) in peripheral blood mononuclear cells (PBMCs), CL blood perfusion by Doppler ultrasound (Doppler-US), plasma concentration of P4 on Day 21 and pregnancy-associated glycoproteins (PAGs) test on Day 25 after timed-artificial insemination (TAI) for early pregnancy diagnosis in dairy cows and heifers. Holstein cows (n = 140) and heifers (n = 32) were subjected to a hormonal synchronization protocol and TAI on Day 0. On Day 21 post-TAI, blood samples were collected for PBMC isolation and plasma concentration of P4. The CL blood perfusion was evaluated by Doppler-US. Plasma samples collected on Day 25 were assayed for PAGs. The abundance of ISGs (ISG15 and RSAD2) in PBMCs was determined by RT-qPCR. Pregnancy was confirmed on Days 32 and 60 post-TAI by B-mode ultrasonography. Statistical analyses were performed by ANOVA using the MIXED procedure and GLIMMIX in SAS software. The pregnancy biomarkers were used to categorize the females as having undergone late luteolysis (LL); early embryonic mortality (EEM); late embryonic mortality (LEM); or late pregnancy loss (LPL). The abundance of ISGs, CL blood perfusion by Doppler-US, and concentrations of P4 on Day 21, and PAGs test on Day 25 were significant (P < 0.05) predictors of early pregnancy in dairy cows and heifers. Dairy cows had a greater (P = 0.01) occurrence of LL than heifers, but there was no difference (P > 0.1) for EEM, LEM, and LPL in heifers compared to cows. Cows with postpartum reproductive issues had a greater (P = 0.008) rate of LEM and a lesser (P = 0.01) rate of LPL compared to cows without reproductive issues. In summary, the CL blood perfusion by Doppler-US had the highest accuracy and the least number of false negatives, suggesting it is the best predictor of pregnancy on Day 21 post-TAI. The PAGs test was the most reliable indicator of pregnancy status on Day 25 post-TAI in dairy heifers and cows. The application of machine learning, specifically the MARS algorithm, shows promise in enhancing the accuracy of predicting early pregnancies in cows.

Evolution over the last 40 years of the assisted reproduction technologies in cattle - the Brazilian perspective I - timed artificial insemination.

The reproductive efficiency of the herd is correlated with higher productivity in livestock. Reproduction biotechniques, such as ovulation synchronization protocols, are important to optimize production and accelerate genetic profit in beef and dairy herds. The objective of this review is to describe the evolution over the last 40 years of the artificial insemination (AI) and the timed-AI (TAI) protocols in cattle from a Brazilian perspective. TAI protocols are based on synchronizing emergence of the wave of follicular growth, controlling circulating progesterone (P4) concentrations, stimulating the final growth of the follicle and inducing a synchronized ovulation. Hormonal alternatives that optimize the response at the end of the protocol and strategies to induce final follicle growth and ovulation in categories of females with low expression of estrus are described. Furthermore, the potential positive effect of previous exposure to injectable P4 on fertility of Bos indicus and Bos taurus cows is also discussed.

Efeito de diferentes tempos de equilíbrio na criopreservação de sêmen de garanhões

Objetivou-se com este estudo avaliar a eficiência de diferentes tempos de equilíbrio sobre a viabilidade do sêmen congelado de garanhões. Foram utilizados 15 ejaculados de três garanhões da raça Mangalarga Marchador. O sêmen foi congelado após o resfriamento e permanência a 5C nos seguintes tratamentos: T1 = tempo de equilíbrio de 25 minutos, T2 = tempo de equilíbrio 85 min, e T3 = tempo de equilíbrio com 145 min. A qualidade seminal foi avaliada através da motilidade progressiva, vigor, morfologia espermática, integridade e funcionalidade da membrana plasmática, e teste de termo resistência. A motilidade progressiva e o vigor espermático avaliados pelo teste de termo resistência não diferiram (p > 0,05) entre os tratamentos. A porcentagem de espermatozoides normais através da avaliação da morfologia espermática e a porcentagem de espermatozoides com membrana plasmática íntegra e funcional avaliada respectivamente pelo teste hiposmótico e pelo supravital também não diferiram entre os tratamentos (p > 0,05). Conclui-se que a utilização de tempos de equilíbrio longos (85 ou 145 minutos) previamente ao congelamento do sêmen de equinos não melhora a viabilidade deste, quando comparado ao tempo de equilíbrio de 25 minutos.

Desempenho de novilhas Holandês na produção de embriões F1 Holandês-Zebu, em condições tropicais / Performance of Holstein heifers for F1 Holstein-Zebu embryo production, in tropical conditions

A produção in vivo de embriões bovinos é uma alternativa para a formação e manutenção de rebanhos mestiços F1 destinados à produção de leite. Objetivou-se estudar o potencial de utilização de novilhas da raça Holandês, mantidas em pasto e suplementadas, como doadoras de embrião, nas épocas de verão e inverno. Procederam-se superovulação e coleta de embriões em sete novilhas em cada época. Foram obtidos os dados climáticos, a temperatura retal e a frequência respiratória das doadoras. No verão, observou-se maior índice de temperatura e umidade (ITU) às 6h, 12h e 18h, bem como maior (P0,05) na média de estruturas recuperadas (20,14±17,47 vs. 8,57±9,40), embriões viáveis (14,57±11,91 vs. 7,14±7,24) e congeláveis (13,28±11,39 vs. 6,57±6,24) obtidos no inverno e no verão, respectivamente. Sob as condições estudadas, novilhas da raça Holandês podem ser utilizadas para a produção de embriões F1, tanto no verão, quanto no inverno.

In vivo embryo production (embryo transfer) is an alternative for F1 dairy herds formation and maintenance. The aim of this study was to evaluate the potential of utilization of grazing and supplemented Holstein heifers as embryo donors in summer or winter. Superovulation was performed in seven heifers on each season. Climatic parameters, as well as rectal temperature and respiratory rate of donors were measured. Temperature and humidity index (THI) was higher (p0.05). Holstein heifers can be used as embryo donors in both summer and winter, under the given conditions.

Taxa de concepção de éguas submetidas a duas frequências de palpação retal (24 e 12 horas) e cobertas após a ovulação / Conception rate of mares controlled by two rectal palpation intervals (24 or 12 h)and breeeding after ovulation

Este trabalho foi realizado com o objetivo de estudar a fertilidade de éguas cobertas após a ovulação, segundo um manejo de palpação retal a cada 24 (P1) ou 12 horas (P2), e cobertas por dois garanhões (G1 e G2). O estudo utilizou 84 ciclos estrais de 50 éguas mestiças do tipo militar, distribuídos ao acaso, segundo um esquema fatorial 2 x 2. A taxa de gestação para o primeiro, segundo e terceiro ciclos estrais foram de: 29,41 por cento (5/17) e 50 por cento (4/8) para o grupo P1G1; 25 por cento (5/20), 54,54 por cento (6/11) e 100 por cento (2/2) para o grupo P2G1; 41,66 por cento (5/12) e 50 por cento (2/4) para o grupo P1G2; e de 60 por cento (6/10) para o grupo P2G2, respectivamente. A taxa de gestação total/ciclo, para cada um dos quatro tratamentos, foi de 36 por cento (9/25), 39,40 por cento (13/33), 43,75 por cento (7/16) e 60 por cento (6/10), respectivamente. A taxa de concepção por ciclo estral não diferiu (p>0,05), pelo teste Exato de Fisher. Assim, os dados foram reagrupados levando-se apenas em consideração a freqüência de palpação retal, e obteve-se 39,02 por cento (16/41) e 44,19 por cento (19/43) de taxa de concepção/ciclo e 55,20 por cento (16/29) e 63,30 por cento (19/30) de taxa de concepção total para os intervalos de palpação de 24 e 12 horas, respectivamente.

The objective of the experiment was to study the fertility of mares bred after ovulation, detected by rectal palpation every 24 (P1) or 12 hours (P2), using two stallions (G1 and G2). This study was done with 84 estrus cycles of 50 military crossbred mares, randomly assigned to four treatments. The conception rate for first, second and third estrus cycles were 29.41 percent (5/17) and 50.00 percent (4/8) for group P1G1; 25.00 percent (5/20), 54.54 percent (6/11) and 100.00 percent (2/2) for group P2G1; 41.66 percent (5/12) and 50.00 percent (2/4) for group P1G2; and 60.00 percent (6/10) for group P2G2, respectively. The conception rate for each treatment were 36.00 percent (9/25), 39.40 percent (13/33), 43.75 percent (7/16) and 60.00 percent (6/10). The conception rate by estrus cycle did not differ (p>0.05) by Fisher Test. The data obtained were regrouped considering only the rectal palpation frequency and it was obtained 39.02 (16/41) and 44.19 percent (19/43) for conception rate by estrus cycle and 55.20 percent (16/29) and 63.30 percent (19/30) for total conception rate for 24 and 12 to rectal palpation intervals, respectively.

Taxa de concepção de éguas submetidas a duas frequências de palpação retal (24 e 12 horas) e cobertas após a ovulação / Conception rate of mares controlled by two rectal palpation intervals (24 or 12 h)and breeeding after ovulation

Este trabalho foi realizado com o objetivo de estudar a fertilidade de éguas cobertas após a ovulação, segundo um manejo de palpação retal a cada 24 (P1) ou 12 horas (P2), e cobertas por dois garanhões (G1 e G2). O estudo utilizou 84 ciclos estrais de 50 éguas mestiças do tipo militar, distribuídos ao acaso, segundo um esquema fatorial 2 x 2. A taxa de gestação para o primeiro, segundo e terceiro ciclos estrais foram de: 29,41 por cento (5/17) e 50 por cento (4/8) para o grupo P1G1; 25 por cento (5/20), 54,54 por cento (6/11) e 100 por cento (2/2) para o grupo P2G1; 41,66 por cento (5/12) e 50 por cento (2/4) para o grupo P1G2; e de 60 por cento (6/10) para o grupo P2G2, respectivamente. A taxa de gestação total/ciclo, para cada um dos quatro tratamentos, foi de 36 por cento (9/25), 39,40 por cento (13/33), 43,75 por cento (7/16) e 60 por cento (6/10), respectivamente. A taxa de concepção por ciclo estral não diferiu (p>0,05), pelo teste Exato de Fisher. Assim, os dados foram reagrupados levando-se apenas em consideração a freqüência de palpação retal, e obteve-se 39,02 por cento (16/41) e 44,19 por cento (19/43) de taxa de concepção/ciclo e 55,20 por cento (16/29) e 63,30 por cento (19/30) de taxa de concepção total para os intervalos de palpação de 24 e 12 horas, respectivamente.

The objective of the experiment was to study the fertility of mares bred after ovulation, detected by rectal palpation every 24 (P1) or 12 hours (P2), using two stallions (G1 and G2). This study was done with 84 estrus cycles of 50 military crossbred mares, randomly assigned to four treatments. The conception rate for first, second and third estrus cycles were 29.41 percent (5/17) and 50.00 percent (4/8) for group P1G1; 25.00 percent (5/20), 54.54 percent (6/11) and 100.00 percent (2/2) for group P2G1; 41.66 percent (5/12) and 50.00 percent (2/4) for group P1G2; and 60.00 percent (6/10) for group P2G2, respectively. The conception rate for each treatment were 36.00 percent (9/25), 39.40 percent (13/33), 43.75 percent (7/16) and 60.00 percent (6/10). The conception rate by estrus cycle did not differ (p>0.05) by Fisher Test. The data obtained were regrouped considering only the rectal palpation frequency and it was obtained 39.02 (16/41) and 44.19 percent (19/43) for conception rate by estrus cycle and 55.20 percent (16/29) and 63.30 percent (19/30) for total conception rate for 24 and 12 to rectal palpation intervals, respectively.