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.

Asinine milk mitigates stress-mediated immune, cortisol and behavioral responses of piglets to weaning: A study to foster future interventions in humans.

Introduction: The present study assessed whether asinine milk supplementation improved the immune and behavioral responses of piglets during an early life weaning stress event as a model for its future use in humans. Methods: For this, 48 piglets from 4 different litters were used. At 20 days of age, piglets were weighed and allocated with their litter and dam into group pens until 28 days of age. Four piglets from each litter were then randomly assigned to either (1) asinine milk supplementation (n = 16) (2), skimmed cow milk supplementation (n = 16) or (3) no supplementation (n = 16; control group). The supplementations were voluntarily administered for 3 days preweaning and 3 days postweaning using a baby bottle. The effects on the weaning stress response were assessed through salivary cortisol measurements; behavioral tests such as the open field, novel object end elevated plus maze tests; and gene expression of HSD11B1, NR3C1 and IL1B in PBMCs, which was determined by RT-qPCR and normalized to GAPDH and UBB. To test the effect of the supplementations on weight, milk intake, gene expression, and behavior, a randomized block design was used with repeated measurements over time by the PROC MIXED procedure. Results and discussion: The effects on salivary cortisol were determined using the ratio between the morning and afternoon concentrations, considering the time before and after the weaning event. Principal component analysis (PCA) and Fisher's test were performed to evaluate the behavior test data. When comparing salivary cortisol concentrations between the pre- and postweaning periods, there was a difference (p < 0.05) between the supplementation groups in the afternoon period, suggesting that piglets fed asinine milk had lower afternoon cortisol concentrations postweaning than their counterparts. For the behavioral tests, the supplementations had no measurable effects. No difference was between groups pre- and postweaning for the expression of HSD11B2, which codes for an enzyme that breaks down cortisol. However, the expression of NR3C1, which encodes the glucocorticoid receptor, was significantly upregulated in piglets supplemented with cow milk (mean 1.245; p < 0.05). Conclusion: Asinine milk downregulated 1L1B gene expression, which codes for an inflammatory cytokine. In conclusion, these results suggest that supplementation with asinine milk may represent a strategy to diminish the damage associated with an early life event by modulating IL1B expression and reducing salivary cortisol levels in piglets undergoing weaning stress. Further transcriptomic and metabolomic studies may improve our understanding of the molecular pathways that mediate this systemic immune-mediated response.

Housing Conditions and a Challenge with Lipopolysaccharide on the Day of Estrus Can Influence Gene Expression of the Corpus Luteum in Gilts.

The corpus luteum (CL) is a temporary endocrine gland that plays a decisive role in the reproductive physiology of gilts. Recently, it has been suggested that exogenous factors may compromise the normal functioning of the CL. In the present study, we aimed to understand to what extent an acute and systemic challenge with lipopolysaccharide (LPS) on the day of estrus could compromise gene expression of gilts' CLs housed in different welfare conditions. For this, we housed 42 gilts in three different housing systems: crates, indoor group pens, and outdoor housing. Then, we challenged six females from each group with LPS and eight with saline (SAL) on the day of estrus. After slaughtering the gilts on the fifth day after the challenge, ovaries were collected for gene expression analysis by RT-qPCR. Housing system and LPS challenge did not have a significant interaction for any genes evaluated; thus, their effects were studied separately. We identified significant (p < 0.05) downregulation of the angiogenic genes VEGF and FTL1 among LPS-challenged animals. Meanwhile, we also observed upregulation of HSD3B1 gene among LPS-challenged animals. We found that STAR and LHCGR genes were differentially expressed depending on the housing system, which indicates that the environment may affect adaptation capabilities. Our results indicate that an acute health challenge on the estrus day alters CL gene expression; however, the role of the housing system remains uncertain.

Comparison of estradiol benzoate doses for resynchronization of ovulation at 14 days after timed-AI in suckled beef cows.

We aimed to compare the effects of use of 1 or 2 mg estradiol benzoate (EB) associated with an intravaginal progesterone (P4) device for resynchronization of ovulation 14 days after timed-AI (TAI) in suckled beef cows. Nelore cows were submitted to a TAI (D0) and on D14, received an intravaginal P4 device and were randomly assigned to EB-1 group [n = 516] or EB-2 group [n = 510], which that received 1 or 2 mg EB, respectively. Also, cows had the ovaries scanned by ultrasound to detect an active CL on D14. On D22, devices were removed and structural luteolysis was detected by color-Doppler ultrasonography. In cows which underwent luteolysis, the resynchronization protocol was continued and they were submitted to second TAI on D24. Pregnancy diagnosis was performed 30-35 days after first or second TAI. A subgroup [n = 18-19/group] was submitted to daily ovarian ultrasound scanning from D14 to D22. Proportion of cows with an active CL on D14 did not differ (P > 0.1) between EB-1 and EB-2 groups. The proportion of cows with an active CL on D22 and pregnancy per AI (P/AI) at first TAI were greater (P ≤ 0.05) in EB-1 (55% and 51%) than in EB-2 group (48% and 42%). The P/AI at second TAI did not differ (P > 0.1) between EB-1 (47% [106/227]) and EB-2 group (42% [110/259]). Cumulative pregnancy rate was greater in EB-1 (73% [370/508]) than in EB-2 group (64% [322/502]). No difference (P > 0.1) was observed in the proportion of non-pregnant cows with a synchronized follicular wave emergence between EB-1 and EB-2 groups. In conclusion, treatment with either 1 or 2 mg EB associated with an intravaginal P4 device at D14 after TAI are efficient to synchronize a new follicular wave emergence. The decreased P/AI from first TAI observed in the group of cows receiving 2 mg indicates that this dose is not recommended for use in resynchronization programs initiated 14 days after TAI. The use of 1 mg EB associated with a P4 device provides an elevated cumulative pregnancy rate after two TAIs with an interval of 24 days.

Evaluation of ImageJ software in ultrasonic image analysis: Follicular and luteal morphological characteristics of cattle.

This study was conducted to compare the effectiveness of two methodologies in evaluating B- and Doppler-mode ultrasonic images: analysis using ultrasonic software and utilizing a computer with ImageJ software. To determine if ImageJ software utilization is an efficacious alternative to ultrasonic software device- analysis, there were comparisons of values when using the two methods for morphological and vascular characteristics of follicular dynamics and luteal function in 18 crossbred cattle. From day 8 of an ovarian dynamics synchronization treatment regimen period until the time of ovulation (Day 10), B-mode and power-flow ultrasonic cineloops were obtained every 12 h to assess follicular diameter, wall area, and wall blood perfusion area. On Day 14 after ovulation, US cineloops of ovaries were obtained in B mode and power flow to evaluate various morphological and vascular characteristics of the corpus luteum (CL), including luteal diameter, luteal area, and CL blood perfusion area. Cineloops were evaluated and analyzed using ultrasonic software, and in a computer with ImageJ software. To evaluate consistency in results between the two methods, there was evaluation utilizing paired t-test, Pearson correlation coefficient, Bland-Altman plot, and Linear Regression Test procedures to calculate proportion of bias between values for measurements of variables evaluated. Results indicated none of the values for variables before and after ovulation differed (P > 0.05). It, therefore, was concluded that utilization of ImageJ software is an efficacious biomedical technique to analyze ultrasonic images of morphological and vascular characteristics before and after ovulation in cattle.

Early resynchronization of follicular wave emergence among Nelore cattle using injectable and intravaginal progesterone for three timed artificial inseminations.

Follicular dynamics and pregnancy per AI (P/AI) of cattle submitted to resynchronization 13 d after TAI using a progesterone (P4) insert and supplementary injectable progesterone (iP4) were determined. There was synchronization of ovulation timing imposed (Day 0 = expected estrus; Experiment 1). On Day 13, animals were assigned to: control (only P4 insert; 15 cows and 13 heifers) and iP4 (P4 insert+100 mg iP4; 13 cows and 12 heifers) groups, and submitted to daily ovarian ultrasonography and blood collection (Day 13 to Day 22). In Experiment 2, 760 suckled cows and 498 heifers were submitted to a TAI on Day 0 and assigned into control and iP4 groups on Day 13. In animals with luteolysis on Day 22, there was a second TAI on Day 24, and on Day 37 were subjected to the opposite treatments as imposed in first resynchronization procedure. On Day 37, there was pregnancy diagnosis in animals with functional CL. The third TAI was performed on Day 48. Day of follicular wave emergence did not differ between groups and parities. Dominant follicles were larger in cows than heifers, and in animals of the control group on Day 24. Greater P4 concentrations were detected on Day 14 and Day 15 in the iP4-treated animals. Luteolysis occurred earlier in cows than heifers. Overall P/AI percentage as a result of second and third TAIs, regardless of parity, was greater in iP4-treated animals. In conclusion, females treated with a supplementary iP4 had a greater plasma P4 concentration and P/AI, but there was no effect of iP4 treatment on synchrony of timing of follicular wave.

Feasibility and accuracy of using different methods to detect pregnancy by conceptus-stimulated genes in dairy cattle.

Development of new methods for early diagnosis of pregnancy can be important to increase the reproductive efficiency and profitability of dairy herds. The bovine conceptus secretes IFN-τ that stimulates the transcription of several genes in circulating immune cells and extrauterine tissues. The aims of this study were to evaluate the mRNA abundance for pregnancy predictability of a classic gene stimulated by IFN-τ (ISG15) and a novel potential pregnancy marker (LGALS3BP) in peripheral blood mononuclear cells (PBMC), total blood leukocytes (TBL) or milk leukocytes (TML), and cervical cells (CC) on d 20 after timed artificial insemination (TAI) in dairy cattle. Eighteen Holstein females (12 cows and 6 heifers) were submitted to an estrous synchronization protocol for TAI (d 0). On d 20 post-TAI, blood samples were collected from coccygeal vessels for isolation of PBMC and in Tempus Blood RNA tubes (Applied Biosystems) for TBL. Samples of CC were collected using a cytological brush, and the TML were isolated from milk samples collected before routine milking. Pregnancy diagnosis was performed on d 30 post-TAI using transrectal ultrasonography, and females were classified as pregnant (n = 8) or nonpregnant (n = 10). Total RNA was extracted and mRNA abundance of target genes (ISG15 and LGALS3BP) was quantified by reverse-transcription quantitative PCR and normalized in relation to reference genes. Data were analyzed by ANOVA using the MIXED procedure of SAS (SAS Institute Inc.). The mRNA abundance of ISG15 was greater in pregnant than in nonpregnant animals for PBMC, TBL, and CC. No difference was detected for TML based on pregnancy status. For LGALS3BP mRNA abundance, no difference was detected between pregnant and nonpregnant animals for PBMC, TBL, and TML, but a tendency for greater abundance in pregnant animals was observed for CC. The fold change for ISG15 in each pregnant cow related to the mean of nonpregnant animals was 2.73 ± 0.31, 3.40 ± 2.17, 1.64 ± 0.29, and 0.005 ± 0.002 for PBMC, CC, TBL, and TML, respectively. The fold change for LGALS3BP in each pregnant cow related to the mean of nonpregnant animals was 0.97 ± 0.38, 1.77 ± 0.39, 0.20 ± 0.08, and 0.70 ± 0.11 for PBMC, CC, TBL, and TML, respectively. The receiver operating characteristic curve analysis indicated that ISG15 abundance predicted pregnancy in PBMC (area under curve, AUC = 0.92) and CC (AUC = 0.77) but not in TBL (AUC = 0.72) or TML (AUC = 0.52). In conclusion, mRNA abundance for ISG15 in PBMC was the best predictor for pregnancy at d 20 post-TAI, whereas TBL and TML were not good predictors of pregnancy on d 20 post-TAI. The mRNA abundance of LGALS3BP was not associated with pregnancy status in any type of cell evaluated.

Effects of equine chorionic gonadotropin administered via the Baihui acupoint on follicular ovarian dynamics and the luteal function of cattle during an ovulation synchronization treatment regimen for fixed-time artificial insemination.

Objectives were to evaluate effects of a smaller than typically used dose of equine chorionic gonadotropin (eCG) during a fixed-time artificial insemination (FTAI) treatment regimen. Transrectal ultrasonic (US) examinations were conducted on dairy cows on Day 0 (D0) and the treatment regimen was initiated with administrations of an intravaginal progesterone (P4) implant, estradiol benzoate (im), and prostaglandin F2α (PGF2α; im). On D8, the P4 implant was removed and PGF2α and estradiol cypionate were administered to all animals. Subsequently, cows were randomly assigned to three groups and eCG was administered to Groups 1, 2, and 3 in doses of 300 (im); 100 (im); and 100 (Baihui acupoint) IUs, respectively. The B-mode and power-flow US cineloops were performed to assess follicular dynamics and evaluate various morphological and vascular characteristics of the corpus luteum. Blood samples were collected to quantify serum P4 concentrations. There were no differences between the ovulation synchronization treatment regimens for all follicular dynamic variables tested; however, cows in Group 3 differed from Group 2 having a larger follicle diameter (FD) on D10 (P = 0.06) and larger preovulatory FD (P = 0.09), as well as a blood perfusion area of the dominant follicle wall on D8 (P = 0.07). There were no differences in responses to the ovulation synchronization treatment regimens for the luteal variables evaluated subsequent to ovulation. In conclusion, the Baihui acupoint was effective as an alternative route for eCG dose reduction when FTAI treatment regimens were imposed without detrimentally affecting values for reproductive variables.

Contagem de folículos antrais em fêmeas Nelore submetidas a inseminação artificial em tempo fixo / Antral follicle count in Nelore females subjected to fixed-time artificial insemination

Objetivou-se categorizar a contagem de folículos antrais (CFA) em fêmeas Nelore e posteriormente determinar o efeito de alguns parâmetros reprodutivos e do escore de condição corporal (ECC) sobre a CFA, assim como, comparar a taxa de concepção entre fêmeas com distintas categorias de CFA submetidas a um protocolo de inseminação artificial em tempo fixo (IATF). Para tanto, 595 fêmeas Nelore foram submetidas a um protocolo para IATF; no D4 do protocolo, foi determinado o diâmetro ovariano (DOV), a presença de corpo lúteo (CL) e a CFA. As inseminações foram executadas utilizando-se sêmen criopreservado. O diagnóstico de gestação foi realizado por ultrassonografia. As fêmeas foram dividas conforme a categoria animal, dias pós-parto (DPP), DOV, presença de CL e ECC. De acordo com a média da CFA, sugeriu-se uma categorização da CFA, a qual considerou baixa CFA ≤32, intermediária CFA entre 32 e 48 e alta CFA ≥48 folículos/animal. A categoria animal, os DPP e o ECC não afetaram a CFA dos animais. Entretanto, houve diferença entre a média de CFA na classe com maior DOV em comparação com as demais. A presença de CL influenciou na CFA dos animais. Em relação à fertilidade, não foi demonstrado efeito da CFA sobre a taxa de concepção das fêmeas submetidas à IATF. Os resultados encontrados sugerem que a CFA parece ser uma característica intrínseca, mantendo-se constante independente do status fisiológico do animal. Houve uma inter-relação positiva entre o DOV e CFA. A presença de CL influenciou a CFA. No entanto, a CFA não afeta a performance reprodutiva em programas de IATF.

This study aimed to categorize the antral follicle count(AFC) in Nelore females and subsequently determine the effect of some reproductive parameters and body condition score (BCS) on the AFC, as well as compare conception rates among Nelore females with different categories of AFC subjected to a fixed-time artificial insemination (FTAI) protocol. For that, 596 Nelore females were submitted a FTAI protocol, D4 protocol females were determined the ovarian diameter (OVD), the presence of corpus luteum (CL) and the AFC. The inseminations were performed using cryopreserved semen. The pregnancy diagnosis was performed by ultrasonography. The females were divided according to animal category (ACT), the postpartum days(PPD), OVD, presence of CL and BCS. According to this the average of the AFC and the largest population follicular intrinsically presented by the Nelore females, it was suggested that a categorization, which was considered low AFC ≤32, intermediate AFC between 32 and 48and high AFC≥48folliclesanimal-1. The ACT, the PPD and the BCS did not affect AFC of the animals, however, there were differences between the mean AFC in the class with the largest OVD compared the others.The presence of CL affected the AFC animals. Regarding fertility has not been demonstrated effect of AFC on conception rates of females inseminated. The results suggest that the AFC seems to be an intrinsic characteristic remained constant regard less of the physiological status of the animal. There was a positive interrelationship between DOV and the AFC. The presence of CL affected the AFC animals. However the AFC doesn't affect reproductive performance in FTAI programs.