INTERVENTIONSix dietary crude protein-to-starch metabolizable energy ratios (DPSRs) during pre-weaning (d 4–60) and post-weaning (d 61–180) periods
COMPARISONGroups with different CP levels (20% vs 22% pre-weaning; 18% vs 20% post-weaning) and starch levels (35%, 30%, 25% pre-weaning; 30%, 27%, 24% post-weaning)
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This study investigated how different ratios of crude protein to starch metabolizable energy (DPSRs) in calf starter diets affect growth performance and gastrointestinal microbiota in Holstein bull calves from pre-weaning through post-weaning. While no significant differences in overall growth were observed, heart girth gain and blood urea nitrogen were significantly affected by the protein-starch interaction, and distinct shifts in rumen and cecum microbial communities were detected across different DPSRs. The findings suggest that optimizing the protein-to-starch energy ratio at different developmental stages may benefit calf performance and gut microbiota development.
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**Background:** Protein is essential for calf growth, but the optimal protein content in starter diets remains controversial. Starch is the primary energy source for calves. While the relationship between dietary protein and energy on calf growth is documented, limited information exists on how rumen and cecum microbiomes are affected by different diet structures during pre-weaning and post-weaning periods. This study hypothesized that appropriate crude protein-to-starch metabolizable energy ratios (DPSRs) could improve growth performance and gastrointestinal microbiota.
**Methods:** Forty-eight pre-weaning Holstein bull calves (mean birth weight 39.75 ± 1.58 kg) were assigned to six groups (n=8 each) with different DPSRs from d 4 to d 60 (pre-weaning): A20-35 (20% CP, 35% starch), B20-30 (20% CP, 30% starch), C20-25 (20% CP, 25% starch), D22-35 (22% CP, 35% starch), E22-30 (22% CP, 30% starch), F22-25 (22% CP, 25% starch). From d 61 to d 180 (post-weaning), diets changed to: A18-30 (18% CP, 30% starch), B18-27 (18% CP, 27% starch), C18-24 (18% CP, 24% starch), D20-30 (20% CP, 30% starch), E20-27 (20% CP, 27% starch), F20-24 (20% CP, 24% starch). Calves were housed individually until d 60, then moved to group pens. Body weight, withers height, body length, heart girth, and cannon bone circumference were measured on d 4, 60, and 180. Blood was sampled on d 60 and 180 for TP, ALB, and BUN analysis. Twelve calves were euthanized at d 60 and another 12 at d 180 for rumen fluid and cecum digesta collection. The V3–V4 regions of 16S rRNA genes were sequenced using Illumina MiSeq. Data were analyzed using linear mixed-effects models and one-way ANOVA with Duncan's multiple range test.
**Key Results:** No significant differences were observed in overall growth performance (p > 0.05). For lower protein treatments (20% pre-weaning, 18% post-weaning), group C20-25 showed higher body weight on d 60 (88.89 ± 2.83 kg) and ADG from d 4–60 (0.88 ± 0.05 kg/d), while group B18-27 had the highest ADG from d 61–180 (1.32 ± 0.06 kg/d). For higher protein treatments (22% pre-weaning, 20% post-weaning), group D22-35 had higher d 60 body weight (86.37 ± 2.89 kg) and ADG (0.83 ± 0.05 kg/d) before weaning, while group E20-27 had higher d 180 body weight (229.96 ± 7.06 kg) and ADG (1.24 ± 0.04 kg/d) after weaning. A significant protein × starch interaction was found for heart girth gain on d 180 (p = 0.002), with group B18-27 significantly higher than A18-30 and C18-24 (p < 0.05). Cannon bone circumference gain was significantly affected independently by protein (p = 0.02) and starch (p = 0.01) on d 180. ALB levels were significantly affected by protein and starch independently on d 60 (p < 0.05). Group C20-25 and C18-24 had significantly higher ALB than A20-35 on d 60 and A18-30 on d 180, respectively (p < 0.05). BUN was significantly affected by starch and protein × starch on d 60 (p < 0.05). At the phylum level, Firmicutes dominated the rumen pre-weaning, while Bacteroidetes dominated post-weaning. In the cecum, Firmicutes was consistently more abundant than Bacteroidetes. At the genus level, Prevotella 7 was most abundant in the rumen pre-weaning (>35% average relative abundance), while Ruminococcaceae UCG-005 was highest in the cecum (18–38% average relative abundance). Rumen microbial diversity increased from pre-weaning to post-weaning, while cecal diversity decreased. No significant differences in alpha diversity indices were found. LEfSe analysis identified several biomarkers, including Lactobacillaceae, Streptococcaceae, and Enterobacteriaceae in group A20-35 rumen pre-weaning. Metabolic pathway analysis revealed that in lower CP groups, pathways of aromatic compound degradation, amine and polyamine degradation, and amino acid degradation were common in rumen and cecum microbiomes.
**Clinical Implications:** This study demonstrates that while overall growth performance was not significantly different across DPSRs, specific body measurements (heart girth, cannon bone circumference) and blood indices (ALB, BUN) were affected by the protein-starch interaction. The findings suggest that a lower CP (20%) with lower starch (25%; 218.48 g CP/Mcal starch ME) or higher CP (22%) with higher starch (35%; 170.54 g CP/Mcal starch ME) before weaning, and lower CP (18%) with medium starch (27%; 185.05 g CP/Mcal starch ME) or higher CP (20%) with medium starch (27%; 205.05 g CP/Mcal starch ME) after weaning may provide greater benefits for calf performance. The distinct shifts in rumen and cecum microbial communities in response to different DPSRs highlight the importance of optimizing dietary protein-to-energy ratios at different developmental stages to support gastrointestinal microbiota development and calf health.