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    • chemical m Thirteen multiparous German Holstein cows in thei

    2021-11-24

    Thirteen multiparous German Holstein cows, in their second to fourth lactation, were selected from a previous study (Schäff et al., 2012) based on their individual peak in plasma BHB concentrations in wk 2 or 3 postpartum: high BHB (H-BHB; 1.05 to 2.57 mmol/L, mean 1.59 mmol/L; n = 8) and low BHB (L-BHB; 0.43 to 0.76 mmol/L, mean 0.65 mmol/L; n = 5) concentrations. The cows had a milk yield of more than 10,000 kg/305 d in a previous lactation, and were selected according to their DGAT1 genotype (K232A) to exclude differences in fat metabolism based on this trait. Cows were housed in tiestalls and were fed twice daily (0630 and 1530 h) 1 of 3 different TMR (Supplemental Table S1 for further information on the diet; https://doi.org/10.3168/jds.2016-11021) corresponding to their physiological state; that is, far-off dry period (wk 7 to 4 before expected calving; 5.9 MJ of NEL/kg of DM; CP 126 g/kg of DM), close-up dry period (wk 3 until calving; 6.5 MJ of NEL/kg of DM; CP 137 g/kg of DM), and lactation (7 MJ of NEL/kg of DM; CP 163 g/kg of DM) according to the recommendations of German Society of Nutritional Physiology (GfE, 2001). All procedures were conducted in agreement with the recommendations for the use of animals as experimental subjects of the State Government in Mecklenburg-West Pomerania (Registration No. LALLF M-V/TSD/7221.3-2.1-021/09). Blood samples were taken weekly, starting at d −34 relative to parturition until slaughter at d 40, from the jugular vein into EDTA-containing tubes (no. 4550036; Greiner Bio-One, Frickenhausen, Germany) and centrifuged at 1,565 × g for 20 min at 4°C. The obtained plasma was stored at −80°C until analyzed. Liver tissue samples were taken by biopsy at d −34, −17, 3, 18, and 30 relative to parturition and by sampling at slaughter (d 40). Tissue obtained was immediately frozen and stored at −80°C for further analysis. Further details on animal management and samples collection, including data on BHB concentrations and chemical m balance, were published by Schäff et al. (2012, 2013). Data on protein extraction, SDS-PAGE, Western blotting, and immunohistochemistry are provided as supplemental data (https://doi.org/10.3168/jds.2016-11021). All data were analyzed using SAS software (version 9.3, SAS Institute Inc., Cary, NC). Because of very low FFAR1 protein detection (at the detection limit or no signal) in 3 H-BHB cows, quantification was not possible within a linear range and therefore these animals were omitted from the statistical analysis (H-BHB: n = 5; L-BHB n = 5). The MIXED procedure of SAS for repeated measures was used, considering group, time, and their interactions as fixed effects. As covariance structures the spatial power or ante-dependence were used. The Tukey-Kramer test was applied for multiple comparisons of means. In addition, Spearman correlation statistics with Fisher's z transformations for the calculation of the 95% confidence limits were calculated for FFAR1 and FFAR2 versus blood metabolites and the liver fat content (LFC) without grouping by BHB. Statistical significance was accepted at P < 0.05 and trends toward significances were considered at P < 0.10. The analysis of FFAR1 and FFAR2 in liver revealed specific protein bands of ∼31 and 50 kDa, respectively. The use of antigen-specific blocking peptides confirmed the specificity of both antibodies (Supplemental Figure S1; https://doi.org/10.3168/jds.2016-11021). As shown in Figure 1A, H-BHB animals had lower hepatic FFAR1 abundance (P = 0.011) than L-BHB cows over the entire peripartal period. Changes over time were observed as a trend (P = 0.096). In contrast, abundance of FFAR2 protein tended to be higher in H-BHB cows than in L-BHB cows (Figure 1B; P = 0.055). In addition, the abundance of FFAR2 increased over time (Figure 1B; P = 0.025). No interactions between group and time were found for either protein. The calculation of the correlations revealed a positive correlation of FFAR1 with plasma glucose concentrations antepartum (r = 0.68, P = 0.004) as well as postpartum (r = 0.49, P = 0.009) but negative correlations with BHB postpartum (r = −0.46, P = 0.016) and LFC (r = −0.40, P = 0.040). In contrast, FFAR2 was correlated negatively with glucose postpartum (r = −3.5, P = 0.041) but positively correlated with BHB (r = 0.52, P = 0.001; Supplemental Table S2; https://doi.org/10.3168/jds.2016-11021). For further information on blood metabolites, insulin, LFC as well as energy balance, please check Supplemental Table S3 (https://doi.org/10.3168/jds.2016-11021).