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Embryonic mortality in cattle is high, reaching 10-40 % in vivo and 60-70 % in vitro. Death of embryos involves reduced expression of genes related to embryonic viability, inhibition of DNA repair and increased DNA damage. In follicular granulosa cells, FGF18 from the theca layer increases apoptosis and DNA damage, so we hypothesized that FGF18 may also affect the oocyte and contribute to early embryonic death. The aims of this study were to identify the effects of FGF18 on cumulus expansion, oocyte maturation and embryo development from cleavage to blastocyst stage using a conventional bovine in vitro embryo production system using ovaries of abattoir origin. Addition of FGF18 during in-vitro maturation did not affect FSH-induced cumulus expansion or rates of nuclear maturation. When FGF18 was present in the culture system, rates of cleavage were not affected however, blastocyst and expanded blastocyst development was substantially inhibited (P < 0.05), indicating a delay of blastulation. The number of phosphorylated histone H2AFX foci per nucleus, a marker of DNA damage, was higher in cleavage-stage embryos cultured with FGF18 than in those from control group (P < 0.05). Furthermore, FGF18 decreased accumulation of PTGS2 and IFNT2 mRNA in blastocysts. In conclusion, these novel findings suggest that FGF18 plays a role in the regulation of embryonic death during the early stages of development by impairing DNA double-strand break repair and expression of genes associated with embryo viability and maternal recognition of pregnancy during the progression from oocyte to expanded blastocysts., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In cattle, 17β-estradiol (E2) is essential for triggering luteolysis via the synthesis of prostaglandin F2α (PGF2α). We aimed to evaluate the effects of E2-treatment on day 15 of the estrous cycle on the transcript abundance of genes involved in the PGF2α synthetic cascade. Nelore heifers (N = 50) were subjected to a hormonal protocol for the synchronization of ovulation. Between days 14 and 23 after estrus, the area (cm 2 ) and blood perfusion (%) of the corpus luteum (CL) and progesterone (P4) plasma concentrations were evaluated daily. On day 15, the heifers were assigned to the Control (2 mL of pure sesame oil, N = 21) or Estradiol group (1 mg of E2 diluted in 2 mL of sesame oil, N = 23). After the treatments at 0 h, uterine biopsies were collected at times 1.5 h (C1.5h, N = 8 and E1.5h, N = 10) or 3 h (C3h, N = 8 and E3h, N = 11); and blood samples were obtained from 0, 3, 4, 6 and 7 h for the measurement of 13,14-dihydro-15-keto-PGF2α (PGFM) concentrations by ELISA. Transcript abundance was determined by RT-qPCR and protein abundance of ESRβ and OXTR was determined by Western Blotting. The Estradiol group showed greater (P < 0.05) concentrations of PGFM at 6 and 7 h compared to the Control group. A progressive decrease in plasma P4 concentrations characterized a hastened functional luteolysis, followed by structural luteolysis in the Estradiol group (P < 0.05). Among the treatment groups, no significant difference was detected for the abundance of PRKCα, PRKCβ, AKR1B1, PTGS2 and ESRα transcripts (P > 0.05). Estradiol treatment decreased the abundance of PLA2G4A, AKR1C4, and ESRβ both 1.5h and 3h after treatment (P < 0.05). The relative expression of PGR and OXTR was greater in E3h compared to the C3h (P > 0.05). Protein abundance did not differ between treatment groups at either experimental times (P > 0.05). Overall, E2 promoted an increase in PGFM concentrations and the hastening of functional and structural luteolysis in Nelore heifers through the upregulation of PGR and OXTR, demonstrating for the first time that the expression of these receptors within 3 h after E2 stimulus was associated with triggering luteolysis in cattle., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The expression of interferon (IFN) stimulated genes (ISGs) in lymphocytes has been used for pregnancy diagnosis in cattle. However, among-cow variability has yielded sub-optimal predictive accuracy. We hypothesized that the expression of ISGs (ISG15, OAS1, RSAD2, CLEC3B, and AKR1B1) in early pregnancy varies according to the proportion of Bos indicus (B. indicus) genetics on females. Multiparous cows were classified in three genetic groups, High Angus (HA; n = 45 [0-33% Brahman influence]), Angus-Brahman (AB; n = 30 [34-67%]), and High Brahman (HB; n = 19 [68-100%]) and submitted to a Select-Synch + CIDR protocol. Cows that displayed estrus (n = 94) were artificially inseminated (Day0; D0). On D19, blood samples were collected to obtain peripheral blood mononuclear cells (PBMC) and measure progesterone (P4) concentrations. On D30, pregnancy diagnosis was performed. The expression of RSAD2 in PBMC of pregnant cows was positively related to the proportion of B. indicus genetics of the groups, but not the expression of ISG15 and OAS1. In pregnant cows, the proportion of B. indicus genetics was negatively associated to circulating levels of P4 concentrations. The P4 concentrations were related positively with RSAD2 expression. ROC curve results determined that for cattle with B. indicus genetics lower than 67%, the CLEC3B and AKR1B1 combination was the most accurate option to predict the outcome of pregnancy. In cows with more than 68% of B. indicus genetics, RSAD2 provided the best accuracy. In conclusion, there is a relationship between the proportion of B. indicus genetics and the ISGs gene expression in PBMC during pregnancy., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Early embryonic mortality caused by maternal-fetal recognition failure in the three weeks after fertilization represents a major cause of reproductive inefficiency in the cattle industry. Modifying the amounts and ratios of prostaglandin (PG) F 2α and PGE 2 can benefit the establishment of pregnancy in cattle. Adding conjugated linoleic acid (CLA) to endometrial and fetal cells culture affects PG synthesis, but its effect on bovine trophoblast cells (CT-1) is unknown. The aim of this study was to determine the effects of CLA (a mixture of cis- and trans-9, 11- and -10,12-octadecadienoic acids) on PGE 2 and PGF 2α synthesis and the expression of transcripts involved with maternal-fetal recognition of bovine trophectoderm. Cultures of CT-1 were exposed to CLA for 24, 48 and 72 h. Transcript abundance was determined by qRT-PCR and hormone profiles were quantified by ELISA. The PGE 2 and PGF 2α concentrations were reduced in the culture medium of CLA-exposed CT-1 compared to that of unexposed cells. Furthermore, CLA supplementation increased the PGE 2 :PGF 2α ratio in CT-1 and had a quadratic effect (P < 0.05) on the relative expression of MMP9, PTGES2, and PTGER4. The relative expression levels of PTGER4 were reduced (P < 0.05) in CT-1 cultured with 100 μM CLA than in the unsupplemented and 10 μM-CLA groups. Treatment of CT-1 with CLA decreased PGE 2 and PGF 2α synthesis but a biphasic effect of CLA was observed on the PGE 2 :PGF 2α ratio and relative abundance of transcripts with 10 μM CLA providing maximal improvements in each endpoint. Our data suggest that CLA may influence eicosanoid metabolic process and extracellular matrix remodeling., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The objective of this study was to compare the efficiency of estradiol benzoate (EB) and 17β-estradiol (E2) associated with progesterone (P4) in a fixed-time artificial insemination (FTAI) protocol. We hypothesized that E2+P4 induces an earlier emergence of a new follicular wave (NFW), improving pre-ovulatory follicle diameter and pregnancy rates to FTAI (P/FTAI). In Exp.1, on Day 0 (D0), all Bos indicus cows (n = 12/group) received an intravaginal P4 device and a dose of PGF2α analogue. On D0, females were randomly assigned to receive EB or E2+P4. On D8.5, P4 intravaginal devices were removed and a dose of PGF2α and EB were administered in all females followed by fixed-timed AI on D10. Between D0 and D10, the dominant follicular growth was determined by ovary ultrasonography exams. On D8.5 and D10 the percentage of color power-Doppler signals in the dominant follicular wall was evaluated. In Exp. 2, 467 females (2-year-old nulliparous [n = 76], primiparous [n = 92] and pluriparous [n = 299]) were subjected to the similar FTAI and assigned to be treated with EB (n = 243) or E2+P4 (n = 224). Pregnancy diagnosis was performed 30 days after FTAI by ultrasonography. The day to emergence of NFW was similar between treatments (EB: 3.7 ± 0.37 vs. E2+P4: 3.3 ± 0.3, P = 0.76). Females treated with E2+P4 presented greater (P = 0.06) follicular growth between the emergence and D9 (1.18 ± 0.07) than those treated with EB (0.97 ± 0.08). There was also a positive effect (P < 0.05) of E2+P4 on diameter of the dominant follicle on D9 (13.0 ± 0.6 vs. 10.9 ± 0.55) and blood perfusion of the follicle wall on D8.5 (49 vs. 40%). There was a treatment by parity category interaction effect on P/FTAI (P < 0.05). Treatment with E2+P4 was advantageous to P/FTAI of primiparous cows (E2+P4: 58% and EB: 30%). However, for nulliparous and pluriparous cows, P/FTAI was similar between treatments (∼50%). In conclusion, in a E2/P4-based protocol for FTAI, E2+P4 is as efficient as EB in inducing new follicular emergence within a similar day range, but it results similar or greater P/FTAI., (Published by Elsevier Inc.)

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., (Copyright © 2022. Published by Elsevier Inc.)

The effects of 17β-estradiol (E2) or estradiol benzoate (EB) on PGF2α release were studied in bred-non-pregnant and pregnant Nelore beef heifers. The day of timed artificial insemination (TAI) was designated day 0 (D0), and a single treatment was given on D14. All heifers also received an intravaginal P4 device on D14, and were randomly assigned to three groups: Control (C, P4 device only, n = 12); E2 (1 mg E2 + 9 mg P4, n = 10); or EB (1 mg, n = 10). Blood samples were collected hourly for 8 hours after treatment (Hours 0-8) to measure plasma concentrations (pg/mL) of a PGF2α metabolite (PGFM). The P4 device was removed on D22 and pregnancy was diagnosed on D28. Pregnancy rate was not different among groups (C, n = 7/12; E2, n = 5/10; EB, n = 5/10). More (P < 0.05) heifers had a CV-identified prominent PGFM pulse (peak of > 100 pg/mL) in E2 group (6/10) than in EB (1/10) and C (0/12) groups. Hourly concentration of PGFM for Hours 0 to 8 showed significant effects of group and hour and an interaction of group by hour but did not show an interaction of group or hour with pregnancy status. In preliminary post-hoc analyses, PGFM concentrations during Hours 0 to 8 and pulse characteristics were analyzed within each pregnancy status. For the non-pregnant heifers, a group-by-hour interaction was detected tentatively indicating an increase (P < 0.005) in PGFM concentrations in E2 group from Hours 4 to 6 and in EB group at Hours 5 and 6. Maximum PGFM concentration during Hours 0 to 8 did not differ (P > 0.1) between E2 (124 ± 23) and EB (110 ± 30) groups, but was greater (P < 0.05) in each group than in C (32 ± 3). Furthermore, PGFM concentrations of pulses at the peak, amplitude, and area under pulse curve (pg/mL/h) were greater (P < 0.05) in E2 group than in C group whereas the EB group did not differ (P > 0.1) from the other groups. For pregnant heifers, no effects of group, hour, or their interaction were detected in PGFM concentrations during the hourly sessions, except that maximum PGFM concentration was greater (P < 0.05) in E2 than in EB and C groups. In addition, the number of prominent pulses was greater in E2 group than in Control or EB groups. In conclusion, PGFM increased earlier and in greater concentration combined for bred-non-pregnant and pregnant heifers treated 14 days after TAI with 1 mg E2 plus 9 mg P4 than with 1 mg EB. Tentatively, a positive effect for each of E2 and EB on PGFM concentrations was attenuated in pregnant heifers., (Copyright © 2021. Published by Elsevier Inc.)

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We aimed to evaluate the treatment with estradiol benzoate (EB) or 17β-estradiol (E2) associated with progesterone (P4) for resynchronization of ovulation 14 days after timed artificial insemination (TAI). In Experiment 1 (Exp. 1), Nelore heifers were submitted to TAI (D0). On D14, the animals received an intravaginal P4 device and were randomly assigned to one of three groups: control (no treatment; n = 17); EB (1  mg EB; n = 17); and E2+P4 (1 mg E2 + 9 mg P4; n = 18). Ultrasonography evaluations were performed daily from D14 to D22 to map follicular and luteal dynamics. On D22, the P4 devices were removed and non-pregnant (NP) animals were determined using corpus luteum blood flow Doppler ultrasonography. In Exp. 2, 1295 beef heifers were resynchronized and randomly allocated to the same experimental groups as described in Exp. 1. On D22, the largest follicle (LF) was measured in NP and a second TAI was performed on D24. In a subset of heifers (n = 337), an estrus detection patch was used between D22 and D24 to monitor estrus expression and the LF was measured at D24. Confirmatory diagnosis of pregnancy was performed between D37-67 and D43-67 after first and second TAI, respectively. In Exp 1, the proportion of heifers with a synchronized follicular wave emergence (from 3 to 5 days after treatment) was greater (P < 0.05) in the EB group (93.8%) than in the control (62.5%) and E2+P4 (64.7%) groups. Structural luteolysis occurred earlier (P < 0.05) in the EB and E2+P4 groups than in the controls. The pregnancy rate after first TAI did not differ (P > 0.1) among the groups at D22 and at confirmatory diagnosis in both experiments. In Exp 2, the potential pregnancy loss between D22 and D37-67 was similar (P > 0.1) in the control (19% [36/185]), EB (15% [28/182]) and E2+P4 (15% [28/184]) groups. The LF diameter (mm) on D22 was greater (P < 0.05) in the control group (11.9 ± 0.1) than in EB (11.3 ± 0.1) and E2+P4 (11.5 ± 0.1). No difference (P > 0.1) was observed in the proportion of heifers detected in estrus, but LF growth rate (mm/day) between D22 and D24 was greater (P < 0.05) in EB group (0.9 ± 0.08) than in control (0.6 ± 0.07) and E2+P4 (0.7 ± 0.09) groups. The pregnancy rate for the second TAI was greater (P < 0.05) in the EB group (47% [94/200]) than in the control (37% [76/203]), but did not differ (P > 0.1) from the E2+P4 group (43% [93/214]). In conclusion, the treatment with 1 mg EB or 1 mg E2 + 9 mg P4 at 14 days post-TAI anticipates luteolysis in NP heifers but does not compromise pregnancy. The EB treatment induces a new synchronized follicle wave emergence and increases the pregnancy rate of resynchronized NP heifers., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Semen biotechniques may impair sperm quality due to excessive production of reactive oxygen species (ROS). Additionally, products of the oxidative reaction, especially involving lipids (e.g., malondialdehyde - MDA), may be even more harmful to sperm. Carnosine, previously reported to be present in seminal plasma of several species, may be a key factor on sperm tolerance to biotechniques by counterattacking the deleterious influence of MDA. Therefore, the aim of this study was to measure the levels of carnosine present in equine seminal plasma and relate these findings with sperm function and oxidative status during cooling and cryopreservation. Thus, semen samples were collected from 40 stallions in duplicate (N = 80) and then submitted to cooling and cryopreservation. Samples were then allocated into groups of high and low tolerance to refrigeration and cryopreservation (bad cooler and good cooler/bad freezer and good freezer, respectively), and in groups of different concentrations of carnosine (High, Medium-high, Medium-low and Low carnosine). Samples were evaluated for sperm kinetics patterns, function of sperm structures and oxidative status. In good cooler samples, it was observed higher concentrations of carnosine (Good cooler: 224.98 ± 19.16 ng/mL; Bad cooler: 159.72 ± 15.99 ng/mL; p = 0.0056), ROS production (Good cooler: 26.40 ± 18.33%; Bad cooler: 18.33 ± 1.84%; p = 0.001) and lipid peroxidation rates (Good cooler: 193.23 ± 18.22 ng/mL; Bad cooler: 131.92 ± 12.25; p = 0.0064). Groups of samples with higher carnosine concentrations had lower levels of malondialdehyde (High: 79.33 ± 6.72 ng/mL; Medium-high: 140.45 ± 11.70 ng/mL; Medium-low: 202.57 ± 16.30 ng/mL and Low: 231.02 ± 32.35 ng/mL; p < 0.05), demonstrating that carnosine was effective in removing lipid peroxidation products. Due to the removal of seminal plasma during the cryopreservation process, no differences occurred in carnosine levels between bad and good freezer groups. In this context, this study provides relevant data for future therapies using carnosine during cryopreservation, aiming to replace the levels lost due to the necessary removal of seminal plasma., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Two experiments were performed to evaluate the reproductive performance of zebu beef cows treated with different doses of eCG at the end of a progesterone (P4)/estrogen based protocol for timed artificial insemination (TAI). In Experiment 1, suckling Bos indicus Nelore cows (n = 261) received, on day 0, a progesterone (P4) intravaginal device (PD) and an injection of 1 mg estradiol benzoate (EB). On day 8, the PD was removed, 500 μg of cloprostenol was injected, and cows were assigned to one of the following groups: Control (no treatment), 300 (300 IU of eCG), 600 (600 IU of eCG), and 900 (900 IU of eCG). On day 9, all cows received 1 mg EB and TAI performed 54-56 h after cloprostenol injection. A pregnancy diagnosis was done by ultrasound scanning 40 days after TAI, and the number of fetuses and calves was recorded at pregnancy diagnosis and at birth. More cows treated with eCG displayed estrus within 48 h after removal of the PD (42.3% vs. 11.6%, P < 0.01), and ovulated more than one follicle (42%, 58/138 vs. 1.8%, 1/54; P < 0.01). This effect on ovulation rate was dose dependent (P < 0.05). The pregnancy rate was affected only by cow parity (primiparous, 25.3% vs. multiparous, 48.9%; P < 0.01). Twin pregnancy was higher (P < 0.01) in cows treated with eCG (42%, 58/138) than controls (0%, 0/54). However, few cows (33.3%) were able to keep both fetuses intact until birth. For evaluation of ovarian characteristics by B-mode and Doppler ultrasonography, 43 Nelore cows were submitted In Experiment 2 to the same four groups described in Experiment 1. Although no difference (P > 0.1) was observed for size and blood perfusion in the pre-ovulatory follicles, corpus luteum was larger and with greater blood perfusion (P < 0.05) in eCG-treated cows. In conclusion, eCG increased the number of double/multiple ovulations in a dose-dependent manner, induced larger and more vascularized corpora lutea, but did not affect the fertility of cyclic or anestrous cows. Although eCG results in twin pregnancies, most of cows underwet embryo/fetus loss and birth a single calf., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs) were determined in water samples collected in two streams and a lake located at Niteroi City, Rio de Janeiro State, Brazil between October 2008 and September 2009. Samples were extracted using liquid-liquid extraction and analyzed using high performance liquid chromatography with fluorescence detection. The limits of quantification were sufficiently low to accomplish PAH determination below the maximum concentration levels established by the Brazilian (50 ng/L) and USEPA legislations, with recoveries larger than 81.6%. Phenanthrene, fluoranthene, pyrene and benz[a]anthracene predominated among PAHs. Total concentrations of PAHs were well correlated with rainfall indicating a possible role of runoff to local pollution of water by PAHs and showed a seasonal variation in wet and dry seasons. Our results highlight the contribution of the widespread streams located around Guanabara Bay to the PAH burden found in its waters., (Copyright © 2012 Elsevier Ltd. All rights reserved.)