In pre-weaned ruminants, the microbiota colonizes rapidly in the rumen after birth and constantly interacts with the host to sustain health and metabolism. The developing microbial community is more malleable, so its manipulation may improve ruminant health and productivity as well as may have long-term effects on ruminants. Hence, understanding the process of rumen microbiota establishment is helpful for nutritional interventions of rumen microbiota in pre-weaned ruminants. This paper reviews the latest advances in the colonization of rumen microbiota while providing insights into the most suitable time for manipulating rumen microbial colonization in early life. In addition, different factors that affect rumen microbiota establishment during the pre-weaned ruminants are discussed in the current manuscript. The purpose of this review is to aid in the development of guidelines for manipulating rumen microbiota to improve animal productivity and health.

Alfalfa silage due to its high protein can lead to easier feeding management, but its high proportion of rumen-degradable protein can reduce rumen nitrogen utilization. Nevertheless, increasing dietary energy can enhance ruminal microbial protein synthesis. Thirty-two Suffolk female sheep were used in this study, with a 2 × 2 factorial arrangement of treatment. The four treatments were a combination of two forage types (alfalfa hay; AH vs. alfalfa silage; AS) and two rumen-degradable starch levels (low RDS; LR vs. high RDS; HR) with a 15 d adaptation and 60 d experimental period. The rumen content and rumen epithelium samples were collected after slaughter. Feeding AS increased the rumen isobutyrate, valerate, ammonia-N (NH₃-N) concentration, urase activity, and papillae height (p < 0.05) and reduced the feed to gain (F:G), rumen bacterial protein (BCP), rumen lactic acid concentration, and papillae width (p < 0.05) of sheep. Increased RDS in the diet improved the daily matter intake, average daily gain, and rumen weight, reduced the F:G, and enhanced the rumen nitrogen capture rate by decreasing total amino acids and the NH₃-N concentration to increase BCP, aquaporins 3 gene, and protein expression. The rumen microbiota also changed as the HR diet reduced the Chao index (p < 0.05). The metabolomics analysis showed that feeding AS upregulated the rumen tryptophan metabolism and steroid hormone biosynthesis, while the purine metabolism, linoleic acid metabolism, and amino acid biosynthesis were downregulated. Furthermore, increased RDS in the diet upregulated rumen lysine degradation and sphingolipid metabolism, while aromatic amino acid biosynthesis was downregulated. Additionally, the correlation analysis results showed that ADG was positively correlated with 5-aminopentanoic acid, and three microorganisms (unclassified_f__Selenomonadaceae, Quinella, Christensenellaceae_R-7_group) were positively correlated with the rumen isobutyrate, valerate, NH₃-N concentration, urase activity, tryptophan metabolism, and steroid hormone biosynthesis and negatively correlated with linoleic acid metabolism and amino acid biosynthesis in sheep. In summary, increased RDS in the diet improved the growth performance and rumen N utilization and reduced bacterial diversity in sheep. The alfalfa silage diet only increased feed efficiency; it did not affect growth performance. Additionally, it decreased rumen nitrogen utilization, linoleic acid, and amino acid biosynthesis. Nevertheless, there were limited interactions between forage and RDS; increased RDS in the AS diet enhanced the nitrogen capture rate of rumen microorganisms for alfalfa silage, with only slight improvements in the purine metabolism, linoleic acid, and amino acid synthesis.

Alfalfa silage has a high proportion of rumen-degradable protein content. Increasing dietary rumen-degradable starch (RDS) can enhance ruminal microbial protein synthesis. This study was conducted to investigate the influence of RDS levels in substrates with alfalfa silage on in vitro rumen fermentation and nitrogen (N) utilization. Rumen fluid was collected and dispensed into anaerobic fermentation bottles, each containing 1 g of substrate and 60 mL of rumen fluid–buffer mixture. The substrate was composed of 40% alfalfa silage and five different RDS levels: 14.85% RDS, 16.40% RDS, 18.67% RDS, 20.21% RDS, and 21.62% RDS. For each RDS level, three replicates were prepared. Each substrate was then incubated at 39 °C for 3, 6, 12, and 24 h. After incubation, the following parameters were measured: gas production, pH, α-amylase activity, ammonia nitrogen (NH₃-N), bacterial protein (BCP), and short-chain fatty acid (SCFA) concentrations were measured. Total gas production increased linearly with increasing RDS levels from 3 to 10 h of incubation (p < 0.01), with no difference observed among five levels after 11 h. At 3 h of incubation, pH decreased linearly with increasing RDS levels (p < 0.05). BCP concentrations and α-amylase activity increased linearly or quadratically with increasing RDS levels (p < 0.01), while the R4 group had the highest concentrations of BCP and the R5 group had the highest activity of α-amylase (p < 0.01). At 6 h of incubation, the NH₃-N concentration decreased linearly or quadratically with increasing RDS levels (p < 0.05), and the α-amylase activity, acetate, propionate, and total SCFA concentrations increased linearly (p < 0.01). The R4 group had the highest activity of α-amylase (p < 0.01), and the R5 group had the highest concentrations of acetate (p < 0.05) and propionate (p < 0.01). At 12 h of incubation, BCP, NH₃-N, and propionate concentrations, as well as α-amylase activity, increased linearly or quadratically with increasing RDS levels (p < 0.05). At 24 h of incubation, the α-amylase activity increased linearly with increasing RDS levels (p < 0.05). The highest multiple-factor associative effects index was observed in the 20.21% RDS substrate, indicating that an RDS level of 20.21% in the alfalfa silage substrate resulted in a desirable rumen N utilization.

The establishment of the rumen microbiota plays an important role in the rumen development. However, little is known about the effects of alfalfa supplementation time on rumen microbiota establishment. Here, a total of 42 Hu lambs, seven-day-old, were chosen for the study. After a week of adjustment, six lambs were sacrificed to establish a baseline. The remaining 36 lambs were randomly split into two groups: one receiving alfalfa hay at 14 days (EAF), the other at 42 days (LAF), both groups received milk replacer and starter pellets. Introducing alfalfa at 14 days of age significantly improved total dry matter intake between 28 and 42 days (p = 0.04) and average daily gain from both 14 to 28 days (p = 0.04) and 28 to 42 days (p < 0.01), but this effect disappears from 56 to 70 days (p > 0.05). At 42 days, the abundances of Naganishia, Ascochyta, and Neosetophoma in the EAF group were significantly higher (p < 0.05) than those in the LAF group (17.8% vs. 3.97, 10.89% vs. 1.77, and 1.27% vs. 0.09%, respectively). At 56 days, the abundances of Ascochyta, Wallemia, and Aspergillus in the EAF group were significantly lower (p < 0.05) than in the LAF group (3.53% vs. 16.40, 8.78% vs. 18.89, and 2.14% vs. 4.69%). At 70 days, Aspergillus abundance in the EAF group was significantly higher (p < 0.05) than in the LAF group (2.69% vs. 0.85%). The LEfSe analysis showed that Methanobrevibacter_smithii was the archaeal biomarker at 14 days in both groups. Methanobrevibacter_sp_AbM4 was enriched at 56 days in the LAF group. Compared to the LAF group, the specific fungal biomarkers in the EAF group included Sporobolomyces and Bullera at 14 days, Naganishia, Didymella, Cleistothelebolus, and Alloleptosphaeria at 42 days, Ascochyta, Neoascochyta, and Alfaria at 70 days. Correlation analysis results showed strong patterns of association both within and between archaea and fungi, which were influenced by alfalfa supplementation time. In summary, alfalfa supplementation at 14 days of age promotes the growth performance of lambs before weaning, and alfalfa supplementation timing significantly affects rumen archaeal and fungal communities and dynamical changes.

Maternal metabolic intensity significantly increases during late gestation and lactation, placing significant stress on cells and tissues. This heightened metabolic demand can lead to inflammatory responses and metabolic disorders, adversely affecting the health of both the mother and her offspring. Diet plays a key role in modulating host health by influencing the gastrointestinal microbiome. This study examined the impact of two roughage sources, corn straw (CS), and alfalfa hay (AH), on ewes and their offspring during late gestation and lactation, with a focus on metabolism, immunity, and the microbiome. Thirty-six multiparous Inner Mongolia cashmere goats, approximately 60 days pregnant, were assigned to CS and AH groups. Samples were collected from the ewes on day 140 of gestation (G140) and day 28 of lactation (L28) for analysis. The results showed that ewes fed AH had reduced body weight loss during lactation (p < 0.05), and increased serum metabolic factors levels (p < 0.05). Additionally, ewes in the AH group exhibited a reduced inflammatory response during both gestation and lactation compared to those in the CS group, as evidenced by a significant decrease in TNF-α and LPS levels and a notable increase in IL-10 (p < 0.05). The rumen microbiomes of ewes in the AH and CS groups exhibited stark differences, with specific microbial markers identified at G140 and L28. Correlation analysis revealed associations between microbiome, volatile fatty acids, cytokines, and metabolic markers. The analysis of the lambs demonstrated that their immune status and microbial composition were significantly influenced by the immune health and microbial community structure of the ewe. Moreover, microbial and immune-related components from the ewes were transmitted to the lambs, further shaping their immune development and rumen microbiota. Overall, different roughage sources during late gestation and lactation had minimal impact on the growth performance of ewes and lambs, given that both diets were iso-nitrogen and iso-energetic. However, ewes fed AH exhibited significant improvements in immune function and overall health for both them and their lambs.

Alfalfa forms and rumen degradable starch (RDS) levels in diets can profoundly affect growth performance and rumen fermentation patterns, this influence may result in variations in rumen microbiota. However, the effects of RDS levels on methanogenic and fungal communities in alfalfa hay (AH) or alfalfa silage (AS) diets, and the interaction between methanogens and fungi with growth performance and rumen fermentation patterns, remain unknown. In this study, a 2 × 2 factorial design resulted in four diets: two alfalfa forms (AH and AS) and two RDS levels (LR: 14.85% DM RDS; and HR: 20.21% DM RDS). We used 32 female Suffolk sheep for the experiment. On day 75 (including a 15-day transition period and a 60-day trial period), rumen content was collected after slaughter to examine the ruminal methanogens and fungi. The AHHR diet reduced the methanogen Chao 1 index compared to the AS diets (P < 0.05), and the Shannon index was lower than in the ASLR diet (P < 0.05). The fungi Chao 1 index was higher in the AH diets than in the ASHR diet (P < 0.05), and the fungi Shannon index was higher in the LR diets than in the HR diets (P < 0.05). The relative abundance of Aspergillus in the AHLR diet was significantly higher than in the AS diets (P < 0.01), and the relative abundance of Occultifur and Meyerozyma were decreased in the AH diets than in the AS diets (P < 0.05). The LEfSe analysis showed that Methanobrevibacter_sp_YE315 and Methanobrevibacter_sp_AbM4 were enriched in the ASLR diet, while Methanobrevibacter_millerae was enriched in the ASHR diet. For the fungal biomarkers, the AHLR diet included Aspergillus, Metschnikowia, and unclassified_f:Stachybotryaceae; the AHHR diet included stachybotrys, Stemphylium, and Cystobasidium; the ASLR diet included unclassified_k:Fungi, Trichothecium, and Psathyrella; and the ASHR diet included Alfaria. The correlation analysis results showed the relative abundance of Methanobrevibacter, Methanoculleus, Penicillium, Cladosporium, and Exophiala and the concentrations of isobutyrate and isovalerate, which may provide deeper insights into the previously observed differences.