Water requirements, fertility, and characterization of liver epigenetic markers during periods of negative energy balance and subsequent compensatory growth in beef cattle

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De Moura, Felipe Henrique

Issue Date

2021

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Dissertation

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body composition , DNA methylation , energy requirements , epigenetics , metabolic water , RNA methylation

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Thirteen crossbred Angus × Hereford bulls [29.6 ± 3.4 mo.; initial body weight (BW) = 715 ± 36.1 kg] were group-housed in a feedlot equipped with an automated water system for 30 d (adaption) + 168 d (trial). Data was collected and analyzed following a pre-post repeated measure design. Three dietary regimes were offered: BW maintenance (Phase 1: prior to BW loss and Phase 3: post BW loss); BW loss (Phase 2); and BW gain (Phase 4: compensatory gain). In order to induce a metabolic stress and a full recovery to initial state, each animal acted as its own control. Animals were fed Beardless wheat (Triticum aestivum) hay. Statistical analyses were performed using SAS 9.4 (SAS Inst., Cary, NC). In the first study, the objective was to evaluate how drastic changes in net energy requirements and body composition of breeding bulls could influence their water intake (WI). Environmental variables, individual WI, and BW were recorded daily. Principal component (PC) analyses were used to account for the relationship between environmental variables, animal requirements, body composition changes, and animal performance on WI. The first two PC presented lower discriminatory power among WI, performance, energy requirements, and environmental variables during phases 1 and 3 of BW maintenance. During phases 2 and 4, the first two PC had a high discriminatory power among WI, animal requirements, body composition, and environmental variables. Lower BW (P = 0.006), metabolic BW (P = 0.004), and net energy for maintenance (P = 0.004) observed in bulls at Phase 2, were followed by a drastic reduction in WI compared to Phase 4. In Phase 4, bulls had a high average daily gain which followed the tendency of lower WI (P = 0.085) relative to what was observed for Phase 2, indicating a change in efficiency of water utilization per unit of BW. Water intake for Phases 2 and 4 was primarily driven by animal requirements and secondarily by environmental factors. For animals undergoing BW loss and compensatory growth, WI is primarily driven by animal requirements and secondarily by environmental variables, particularly in animals undergoing nutritional and metabolic stress. Because of this, caution should be taken to avoid oversimplification of water requirements using the factorial approach. In the second study, the objective was to characterize the effects of nutritional status on epigenetic markers, such as DNA methylation and m6A RNA methylation, of bovine sperm. Higher levels of RNA m6A (P = 0.004) and DNA methylation (P = 0.007) of spermatic cells were observed at Phase 2 compared with Phase 1. In Phase 3, sperm RNA m6A methylation levels continued to be higher (P = 0.004), whereas the DNA of sperm cells was similar (P = 0.426) compared with the Phase 1. Growing bulls had a tendency (P = 0.109) of higher RNA m6A methylation levels than mature bulls. Phase 2 altered scrotal circumference (P < 0.001), sperm volume (P = 0.007), sperm total motility (P = 0.004), sperm progressive motility (P = 0.004), total sperm count (P = 0.049), normal sperm (P < 0.001), abnormal sperm (P < 0.001), primary sperm defects (P = 0.039), and secondary sperm defects (P < 0.001). In Phase 3, bulls had scrotal circumference, sperm volume, sperm motility, sperm progressive motility, total sperm count, normal and abnormal spermatozoa, and primary and secondary spermatozoa defects similar to Phase 1 (P > 0.05). Serum concentrations of insulin-like growth factor-1 (IGF-1) and leptin decreased during Phase 2 (P = 0.010) while no differences (P > 0.05) were detected between Phase 3 and 1; growing bulls tended (P = 0.102) to present higher leptin levels than mature bulls. Specific for mature bulls, DNA methylation was positively correlated with leptin concentration (0.569, P = 0.021). Whereas for young bulls, DNA methylation was positively correlated with abnormal spermatozoa (0.824, P = 0.006), primary spermatozoa defect (0.711, P = 0.032), secondary spermatozoa defect (0.661, P = 0.052), and negatively correlated with normal spermatozoa (−0.824, P = 0.006), total sperm count (−0.702, P = 0.035), and sperm concentration (−0.846, P = 0.004). There was no significant correlation (P > 0.05) between RNA m6A and hormones and semen traits. In conclusion, the nutritional status of breeding bulls alters epigenetic markers, such as DNA methylation and RNA m6A methylation, in sperm, and the impact of change seems to be age-dependent. These markers may serve as biomarkers of sperm quality and fertility of bulls in the future. Detrimental effects on sperm production and seminal quality are observed at periods and places when and where environmental and nutritional limitations are a year-round reality and may carry hidden players that may influence a lifetime of underperformance. In the third study, the objective was to characterize the effects of dietary restriction and subsequent ad libitum feeding on body composition and hepatic gene expression of epigenetic regulators of DNA methylation, RNA m6A methylation, and histone acetylation in beef breeding bulls. Bulls undergoing negative energy balance (NEB) decreased (P < 0.001) of empty body weight [EBW; 23.1% (-139.1 kg)], empty body fat [EBF; 39.8% (-85.4 kg)], and empty body protein [EBP; 14.9% (-13.5 kg)]. A full recovery to initial state of EBW, EBF, and EBP was observed at the end of the ad libitum feeding. Body fat changes accounted for 77.1% of daily changes in body energy status, whereas body protein changes accounted for only 22.9% (P < 0.001). Bulls undergoing NEB tended (P ≤ 0.097) to have increased gene expression of epigenetic regulators of RNA m6A methylation (METTL14, VIRMA, and WTAP), increased (P ≤ 0.050) gene expression of epigenetic regulators of DNA methylation (DNMT3A) and histone-acetylation (SIRT3 and SIRT7). Growing bulls had a tendency (P ≤ 0.072) of higher RNA m6A methylation, VIRMA, and WTAP than mature bulls. Effect of diet × age interaction was not detected (P ≥ 0.137) for METTL14, VIRMA, WTAP, DNMT3A, SIRT3 or SIRT7. Growing bulls tended to have greater RNA m6A methylation levels than mature bulls, indicating that, while contemporaneously fed the same diet during periods of undernourishment followed by compensatory growth, age has an impact on this epigenetic mechanism. In conclusion, metabolic status seems to carry a greater impact on regulating bovine hepatic epigenetic mechanisms that modulate gene transcription, such as DNA methylation and histone acetylation, than on epigenetic mechanisms that regulate gene translation, such as RNA m6A methylation. During periods of undernourishment followed by compensatory growth, body fat appears to have a greater impact on epigenetic markers that modulate hepatic gene transcription.

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