The aim of this study was to investigate the effect of low-protein diets supplemented with rumen-protected lysine (RPLys) and methionine (RPMet) on growth performance, rumen fermentation, blood biochemical parameters, nitrogen metabolism, and gene expression related to N metabolism in the liver of Holstein bulls. Thirty-six healthy and disease-free Holstein bulls with a similar body weight (BW) (424 ± 15 kg, 13 months old) were selected. According to their BW, they were randomly divided into three groups with 12 bulls in each group in a completely randomized design. The control group (D1) was fed with a high-protein basal diet (CP13%), while bulls in two low-protein groups were supplied a diet with 11% crude protein and RPLys 34 g/d·head + RPMet 2 g/d·head (low protein with low RPAA, T2) or RPLys 55 g/d·head + RPMet 9 g/d·head (low protein with high RPAA, T3). At the end of the experiment, the feces and urine of dairy bulls were collected for three consecutive days. Blood and rumen fluid were collected before morning feeding, and liver samples were collected after slaughtering. The results showed that the average daily gain (ADG) of bulls in the T3 group was higher than those in D1 (p < 0.05). Compared with D1, a significantly higher nitrogen utilization rate (p < 0.05) and serum IGF-1 content (p < 0.05) were observed in both T2 and T3 groups; however, blood urea nitrogen (BUN) content was significantly lower in the T2 and T3 groups (p < 0.05). The content of acetic acid in the rumen of the T3 group was significantly higher than that of the D1 group. No significant differences were observed among the different groups (p > 0.05) in relation to the alpha diversity. Compared with D1, the relative abundance of Christensenellaceae_R-7_group in T3 was higher (p < 0.05), while that of Prevotellaceae _YAB2003_group and Succinivibrio were lower (p < 0.05). Compared with D1 and T2 group, the T3 group showed an expression of messenger ribonucleic acid (mRNA) that is associated with (CPS-1, ASS1, OTC, ARG) and (N-AGS, S6K1, eIF4B, mTORC1) in liver; moreover, the T3 group was significantly enhanced (p < 0.05). Overall, our results indicated that low dietary protein (11%) levels added with RPAA (RPLys 55 g/d +RPMet 9 g/d) can benefit the growth performance of Holstein bulls by reducing nitrogen excretion and enhancing nitrogen efficiency in the liver.

The objective of this study was to assess the effects of gallic acid (GA) on nutrient degradability, gas production, rumen fermentation, and the microbial community and its functions using in vitro fermentation methods. An in vitro experiment was conducted to test GA dose levels (0, 5, 10, 20, and 40 mg/g DM) in the cow’s diet. Based on the results of nutrient degradability, gas production, and rumen fermentation, the control group (0 mg/g DM, CON) and the GA group (10 mg/g DM, GA) were selected for metagenomic analysis to further explore the microbial community and its functions. The degradability of dry matter and crude protein, as well as total gas production, CH₄ production, CH₄/total gas, CO₂ production, and CO₂/total gas, decreased quadratically (p < 0.05) with increasing GA doses, reaching their lowest levels at the 10 mg/g DM dose. Total volatile fatty acid (VFA) (p = 0.004), acetate (p = 0.03), and valerate (p = 0.03) exhibited quadratic decreases, while butyrate (p = 0.0006) showed a quadratic increase with increasing GA doses. The 10 mg/g DM dose group had the lowest levels of total VFA, acetate, and valerate, and the highest butyrate level compared to the other groups. The propionate (p = 0.03) and acetate-to-propionate ratio (p = 0.03) linearly decreased with increasing gallic acid inclusion. At the bacterial species level, GA supplementation significantly affected (p < 0.05) a total of 38 bacterial species. Among these, 29 species, such as Prevotellasp.E15-22, bacteriumP3, and Alistipessp.CAG:435, were less abundant in the GA group, while 9 species, including Aristaeella_lactis and Aristaeella_hokkaidonensis, were significantly more abundant in the GA group. At the archaeal species level, the relative abundances of Methanobrevibacter_thaueri, Methanobrevibacter_boviskoreani, and Methanobrevibactersp.AbM4 were significantly reduced (p < 0.05) by GA supplementation. Amino sugar and nucleotide sugar metabolism, Starch and sucrose metabolism, Glycolysis/Gluconeogenesis, and Pyruvate metabolismwere significantly enriched in the GA group (p < 0.05). Additionally, Alanine, aspartate and glutamate metabolism was also significantly enriched in the GA group (p < 0.05). GA use could potentially be an effective strategy for methane mitigation; however, further research is needed to assess its in vivo effects in dairy cows over a longer period.

Little information is available regarding the effect of supplementing 25-hydroxyvitamin D₃ during the transition period combined with a postpartum oral calcium bolus on Ca homeostasis. The objectives of the current study were to evaluate the effects of 25-hydroxyvitamin D₃ combined with postpartum oral calcium bolus on lactation performance, serum minerals and vitamin D₃ metabolites, blood biochemistry, and antioxidant and immune function in multiparous dairy cows. To evaluate the effects of 25-hydroxyvitamin D₃ combined with oral calcium, 48 multiparous Holstein cows were randomly assigned to one of four treatments: (1) supplementing 240 mg/day vitamin D₃ without a postpartum oral Ca bolus (control), (2) supplementing 240 mg/day vitamin D₃ with an oral Ca bolus containing 90 g of Ca immediately post-calving (Ca + VitD), (3) supplementing 6 g/day 25-hydroxyvitamin D₃ without an oral Ca bolus (25D), and (4) supplementing 6 g/day 25-hydroxyvitamin D₃ with an oral Ca bolus containing 90 g of Ca immediately post-calving (Ca + 25D). Lactation performance during the first 21 days was measured. Blood was collected at the initiation of calving and then 1, 2, 7, 14, and 21 days relative to the calving date. The yield of milk (0.05 < p < 0.10), energy-corrected milk (p < 0.05), 3.5% fat-corrected milk (p < 0.05), and milk protein (p < 0.05) were significantly higher in 25-hydroxyvitamin D₃-treated groups within 3 weeks of lactation than in vitamin D₃-treated cows. The iCa (p < 0.05) and tCa (p < 0.05) were higher in both Ca and 25D + Ca cows than in the control and 25D groups within 48 h. The concentrations of serum tCa (p < 0.05), tP (p < 0.05), and 25-hydroxyvitamin D₃ (p < 0.05) in 25D and 25D + Ca cows were higher than those in control and Ca cows within 21 days postpartum. Feeding 25-hydroxyvitamin D₃ also showed a lower concentration of malondialdehyde (p < 0.05), interleukin 6 (p < 0.05), and tumor necrosis factor-alpha (TNF-α) (p < 0.05), as well as a higher concentration of alkaline phosphatase (p < 0.05), total antioxidant capacity (p < 0.05), and immunoglobulin G (p < 0.05) than vitamin D₃. Supplementing Ca bolus also showed lower concentrations of alanine transaminase (p < 0.05) and TNF-α (p < 0.05). In conclusion, supplementing 25-hydroxyvitamin D₃ during the transition period combined with a postpartum oral calcium bolus improved lactation performance, Ca homeostasis, and antioxidant and immune function of medium-production dairy cows within 21 days postpartum.

Few studies have examined the effects of puerarin (PE) on ruminant parameters and methane production. Therefore, we determined the degradation of PE in the rumen and evaluated the effect of PE on in vitro fermentation, methanogenesis, and microbial community structure. A completely randomized design was used for the in vitro fermentation, and 4 gradient dosages of PE (0 mg/kg, 50 mg/kg, 100 mg/kg, and 150 mg/kg of DM) were applied in this trial. The in vitro fermentation was carried out in three runs at 6 h and 48 h, with four replicates per treatment per time point. Each run included 40 samples: eight treatments × four replicates and eight blank samples. Based on the fermentation results, both the PE treatments and the control group (CON) at 48 h were chosen for further analysis to explore the effects of PE on the bacterial community structure. Meanwhile, we determined the degradation rate and degradation products of PE in vitro ruminal fluid using high-performance liquid chromatography (HPLC). In this trial, PE may be isomerized into daidzin by rumen microorganisms; the in vitro degradation results of PE indicated that 70% of PE was degraded within 6 h, with the degradation rate reaching nearly 85% by 12 h. The concentrations of NH₃-N and microbial crude protein (MCP) significantly increased linearly with the PE doses at 6 h (p = 0.01). The concentrations of MCP (p = 0.02) and propionate (p = 0.04) demonstrated a linear increase with increasing PE doses at 48 h. In contrast to microbial protein (MCP) and propionate, the acetate-to-propionate ratio decreased linearly with increasing PE doses at 48 h (p = 0.05). Additionally, the addition of PE linearly decreased methane production at 48 h (p = 0.01). Meanwhile, the relative abundances of g_UBA1217 (p = 0.03), g_UBA2810 (p = 0.04), and g_Succiniclasticum (p = 0.03) were significantly lower compared with the CON group. The results showed that PE can be degraded by rumen microflora. Furthermore, it can improve rumen fermentation parameters, increase the amount of protein synthesized by rumen microorganisms, and reduce methane production and the acetate-to-propionate ratio. PE could potentially be an effective strategy for methane mitigation; however, further research is needed to assess its in vivo effects in dairy cows over a longer period.

The purpose of this study was to assess how 25-hydroxyvitamin D ₃ affected pre-and post weaning calves’ growth performance, weaning stress, fecal microbial communities and metabolites, and serum metabolites. A total of 30 healthy Holstein heifers were randomly assigned into a control group (6,100 IU VD ₃ / day; n = 15) and 25D group (2,100 IU VD ₃ + 4,000 IU 25(OH)D ₃ /day; n = 15). The findings revealed that 25D considerably improved average daily gain (ADG) and starter intake during the entire experimental period and body weight on d56, 63, while body diagonal length and heart girth on d 63 were greatly increased ( p < 0.05). In contrast to the control group, 25D reduced the levels of IL-1β, IL-6, TNF- α , and D-lactate acid ( p < 0.05), and the higher levels of 25(OH)D ₃ and 1,25(OH) ₂ D ₃ in serum on d 63 ( p < 0.05). LEfSe analysis revealed that on day 56, 25D increased the relative abundance of butyrate producing bacteria (e.g., g_Frisingicoccus and g_Lachnospiraceae_NK4A136 ) and decreased the relative abundance of g_Alistipes associated with pro-inflammation (LDA > 2, p < 0.05). On d 63, 25D further increased the relative abundance of g_Ruminococcus and g_Erysipelotrichaceae_UCG-003 . The metabolomic analysis showed that 25D significantly enriched key pathways in the fecal and serum, including alanine, aspartate and glutamate metabolism, cysteine and methionine metabolism, tryptophan metabolism, valine, arginine and proline metabolism, citrate cycle (TCA cycle), pyruvate metabolism, and fructose and mannose metabolism. The downregulation of fecal amino acid metabolites is consistent with the small intestine’s utilization of dietary amino acids, which may imply that 25D positively influences intestinal absorption and limits fecal amino acid accumulation. Concurrently, the upregulation of serum fumarate, (S)-malate, and citrate promotes energy metabolism. In conclusion, this study demonstrates that 25D improves calf growth and anti-inflammatory responses by modulating hindgut microbial communities, along with their energy and amino acid metabolic pathways. These findings elucidate the microbial and metabolic mechanisms through which 25D enhances calf health and productivity both before and after weaning, and alleviates weaning stress.