Limitações metodológicas para o estudo da nutrição proteica em ruminantes

Abstract

This study consisted of two experiments. The first experiment aimed to evaluate the ruminal degradation and intestinal digestibility of protein from distillers dried grains with solubles (DDGS) coproducts compared with soybean meal (SBM), the primary protein ingredient used in ruminant diets. Four protein sources were evaluated: soybean meal (control feed), corn DDGS (DG1), sorghum DDGS (DG2), and a blended DDGS source derived from corn and sorghum (DG3). Ruminal protein degradation was assessed using the in situ technique, whereas intestinal digestibility was determined using the in vitro three-step procedure. Protein fractions and intestinal digestibility data were analyzed using the PROC MIXED procedure of SAS 9.4. Crude protein (CP; % DM) concentrations were 45.4% for SBM, 35.0% for DG1, 37.8% for DG2, and 33.5% for DG3. Fraction a was lower for DG2 (29.0%) and SBM (16.8%) compared with DG3 and DG1 (40.6% and 42.7%, respectively; P < 0.01), whereas fraction c was greater for DG2 (37.3%; P < 0.01) than for SBM (4.2%), DG1 (17.2%), and DG3 (24.4%). All DDGS sources exhibited lower fraction b values (33.8% for DG2, 35.0% for DG3, and 40.1% for DG1) compared with SBM (79.0%; P < 0.01). The degradation rate of fraction b tended to differ among feed sources (P = 0.07), with the lowest value observed for DG2 (4.8%/h), followed by DG3 (6.1%/h), DG1 (6.5%/h), and SBM (7.8%/h). Rumen undegradable protein (RUP; % of CP) was greater for DG2 (56.8%; P < 0.01) than for SBM (37.4%), DG1 (38.0%), and DG3 (44.1%). However, SBM provided the greatest amount of digestible RUP (35.8% of CP; P < 0.01), followed by DG3 (33.8%), DG1 (32.7%), and DG2 (17.7% of CP). These results demonstrate that evaluating both ruminal degradation characteristics and intestinal digestibility provides a more robust basis for comparing feed ingredients than considering crude protein concentration alone. Such an approach enables a more comprehensive assessment of each ingredient's contribution to metabolizable protein supply and supports more accurate decisions regarding ingredient selection, dietary inclusion rates, and the determination of the true nutritional and economic value of feed ingredients in ruminant diets. The second experiment utilized composite urine samples obtained from three dairy cows to evaluate the effects of dilution level, storage duration, and storage method on the stability of urinary nitrogenous compounds and purine derivatives. Three sample dilution levels were evaluated under three storage conditions. Samples were analyzed for pH, total nitrogen, urea, creatinine, uric acid, and allantoin concentrations at collection (d 0) and after 1, 7, 15, 30, and 60 d of storage. Statistical analyses were conducted using linear mixed-effects models in R (R Core Team) with the lme4, lmerTest, and emmeans packages. Urine pH was significantly affected by storage duration, storage method, and acidification treatment (P < 0.01), with the greatest changes observed under freeze–thaw conditions. Total nitrogen concentration was influenced by storage duration (P < 0.01) but was not affected by sample dilution, with most variation attributed to nitrogen volatilization over time. Urea concentration was primarily affected by storage duration (P < 0.01), remaining relatively stable when samples were stored frozen for up to 30 d. Creatinine concentration was influenced by storage duration, acidification, and storage method (P < 0.02), exhibiting greater stability under refrigeration during short-term storage and greater variability under repeated freeze–thaw cycles. Acidification significantly affected uric acid concentration (P < 0.01), particularly under refrigerated storage, whereas no significant effects were observed under frozen storage conditions. Allantoin concentration was influenced by storage duration, storage method, and acidification treatment (P < 0.01). Overall, the results demonstrate that the stability of nitrogenous compounds and purine derivatives in bovine urine is highly dependent on storage conditions, storage duration, and sample dilution level. These findings highlight the importance of carefully controlling sample handling and storage protocols to ensure the accuracy and reliability of laboratory analyses used to estimate nitrogen utilization and microbial protein synthesis in dairy cows.