Cottonseed meal (CSM) is an important protein feed source for dairy cows. Its inclusion in ruminant diets is limited due to the presence of the highly toxic gossypol though rumen microorganisms are believed to be capable of gossypol degrading and transforming. The objective of the present study was to isolate the gossypol-degrading bacteria from the rumen contents and to assess its potential for gossypol degradation in vitro. A strain named Lactobacillus agilis WWK129 was anaerobically isolated from dairy cows after mixed rumen microorganisms were grown on a substrate with gossypol as the sole carbon source. Furthermore, the strain was applied at 5% inoculum concentration in vitro to continuously ferment CSM at 39 °C for five days, and it presented gossypol degradability as high as 83%. Meanwhile, the CSM contents of crude protein, essential amino acids increased significantly along with the increase of lactic acid yield (p < 0.01). Compared with the original CSM, the fermented CSM contents of neutral detergent fiber and acid detergent fiber was remarkably decreased after the anaerobic fermentation (p < 0.01). In brief, the Lactobacillus strain isolated from the rumen is not only of great importance for gossypol biodegradation of CSM, but it could also be used to further explore the role of rumen microorganisms in gossypol degradation by the ruminants.

Cysteamine (CS) is an essential nutritional regulator that improves the productive performance of animals by regulating somatotropic hormone secretion. To investigate the fattening potential and effects of CS on rumen microbial fermentation, 48 feedlot lambs were randomly assigned to four groups and fed diets supplemented with different CS concentrations (0, 20, 40, and 60 mg/kg BW). An increase in dietary CS concentrations linearly increased the average daily gain (ADG) and dry matter intake (p < 0.05) but decreased the feed-to-gain ratio (p < 0.01). For the serum hormone, increasing the dietary CS concentration linearly decreased somatostatin and leptin concentration (p < 0.01) but linearly increased the concentration of growth hormone and insulin-like growth factor 1 (p < 0.01). Regarding rumen fermentation, ruminal pH, ammonia-N, and butyrate content did not differ among the four treatments, although dietary CS supplementation linearly increased microbial protein and propionate and decreased the amount of acetate (p < 0.05). Furthermore, an increase in dietary CS concentrations quadratically decreased the estimated methane production and methane production per kg ADG (p < 0.05). High-throughput sequencing revealed that increased dietary CS concentrations quadratically increased Prevotella (p < 0.05), and Prevotella and norank_f__norank_o__Clostridia_UCG-014 were positively correlated with growth performance and rumen fermentation in a Spearman correlation analysis (r > 0.55, p < 0.05). Overall, a CS concentration higher than 20 mg/kg BW produced growth-promoting effects by inhibiting somatostatin concentrations and shifting the rumen toward glucogenic propionate fermentation by enriching Prevotella. In addition, Prevotella and norank_f__norank_o__Clostridia_UCG-014 were positively correlated with growth performance in lambs.

Corn silage is an important source of forage, but whether or not bacterial inoculants should be applied is somewhat controversial in ruminant feeding practice. In the present study, chopped whole corn plants treated with a single inoculant of Lactobacillus buchneri (LB), Lactobacillus plantarum (LP), Pediococcus pentosaceus (PP) served as either homofermentation (e.g., lactate only) or heterofermentation (e.g., lactate and acetate) controls and compared with those treated with either a mixture of the lactic acid bacteria (QA: 60% LP, 10%PP, 30% LB) or a mixture of the lactic acid bacteria (QB: 60% LP, 15% PP, 25% LB), to investigate their effects on the fermentation quality, ester-linked phenolic acids, and in vitro digestibility. After 60 day ensiling, the addition of QA exhibited the lowest pH (3.51) with greater lactic acid (LA) production. The ester-linked ferulic acid (FAest) and p-coumaric acid (pCAest) concentrations were significantly decreased during 60 days ensiling. And among all these groups, the LB and QA treated group showed a lower concentration of FAest and pCAest than other groups. After 60 days ensiling, Lactobacillus was the dominant genus in all LAB treated groups. Meanwhile, negative correlations of Bacillus, Bacteroides, Bifidobacterium, Blautia, Prevotella, Ruminococcus, and Roseburia with FAest content after 60 days ensiling occurred in the present study. Komagataeibacter was mainly found in LB and PP addition silages, and presented a significant negative effect with the level of acid detergent fiber (ADF). To explore whether the addition of LABs can improve digestibility of whole corn silage, an in vitro rumen fermentation was conducted using the 60 day ensiled whole corn silages as substrates. The QA addition group exhibited a greater 48 h and 96 h in vitro dry matter and ADF disappearance, greater 48 h gas production and less methane emissions. Even though there were the same neutral NDF levels in corn silages treated with LB and QA after 60 days ensiling, the QA treated silages with lower FAest and pCAest presented higher IVDMD after 96 h and 48 h in vitro fermentation. In brief, the addition of mixed inoculants of 60% LB,10% PP, 30% LB compared with the addition of whichever single HoLAB or HeLAB inoculants, facilitated the release of ester-linked phenolic acids (e.g., ferulic and p-coumaric acids) and remarkably, improved silage quality in terms of sharp pH decline and greater lactate production. Taken together with the improvement in rumen microbial fermentation, the results obtained in the present study provided concrete evidence for the role of mixed LAB application in corn silage preparation for ruminant feeding practices.

Gossypol is a key anti-nutritional factor which limits the feeding application of cottonseed by-products in animal production. A 2 × 4 factorial in vitro experiment was conducted to determine the effect of gossypol addition levels of 0, 0.25, 0.5, and 0.75 mg/g on ruminal fermentation of a high-forage feed (HF, Chinese wildrye hay/corn meal = 3:2) in comparison with a low-forage feed (LF, Chinese wildrye hay/corn meal = 2:3). After 48 h of incubation, in vitro dry matter disappearance was greater in the LF than the HF group, while the cumulative gas production and asymptotic gas production were greater in the HF than the LF group (p < 0.05). Regardless of whatever ration type was incubated, the increasing gossypol addition did not alter in vitro dry matter disappearance. The asymptotic gas production, cumulative gas production, molar percentage of CO₂ and H₂ in fermentation gases, and microbial protein in cultural fluids decreased with the increase in the gossypol addition. Conversely, the gossypol addition increased the molar percentage of CH₄, ammonia N, and total volatile fatty acid production. More than 95% of the gossypol addition disappeared after 48 h of in vitro incubation. Regardless of whatever ration type was incubated, the real-time PCR analysis showed that the gossypol addition decreased the populations of Fibrobactersuccinogenes, Ruminococcus albus, Butyrivibrio fibrisolvens, Prevotella ruminicola, Selenomonas ruminantium, and fungi but increased Ruminococcus flavefaciens, protozoa, and total bacteria in culture fluids in comparison with the control (p < 0.01). Additionally, the tendency of a smaller population was observed for R. albus, B. fibrisolvens, and fungi with greater inclusion of gossypol, but a greater population was observed for F. succinogenes, R. flavefaciens, S. ruminantium, protozoa, and total bacteria. In summary, the present results suggest that rumen microorganisms indeed presented a high ability to degrade gossypol, but there was an obvious detrimental effect of the gossypol addition on rumen fermentation by decreasing microbial activity when the gossypol inclusion exceeded 0.5 mg/g, and such inhibitory effect was more pronounced in the low-forage than the high-forage group.

In plant cell wall, ferulic acid (FA) and p-coumaric acid (pCA) are commonly linked with arabinoxylans and lignin through ester and ether bonds. These linkages were deemed to hinder the access of rumen microbes to cell wall polysaccharides. The attachment of rumen microbes to plant cell wall was believed to have profound effects on the rate and the extent of forage digestion in rumen. The objective of this study was to evaluate the effect of bound phenolic acid content and their composition in corn silages on the nutrient degradability, and the composition of the attached bacteria. Following an in situ rumen degradation method, eight representative corn silages with different FA and pCA contents were placed into nylon bags and incubated in the rumens of three matured lactating Holstein cows for 0, 6, 12, 24, 36, 48, and 72 h, respectively. Corn silage digestibility was assessed by in situ degradation methods. As a result, the effective degradability of dry matter, neutral detergent fibre, and acid detergent fibre were negatively related to the ether-linked FA and pCA, and their ratio in corn silages, suggesting that not only the content and but also the composition of phenolic acids significantly affected the degradation characteristics of corn silages. After 24 h rumen fermentation, Firmicutes, Actinobacteria, and Bacteroidota were observed as the dominant phyla in the bacterial communities attached to the corn silages. After 72 h rumen fermentation, the rumen degradation of ester-linked FA was much greater than that of ester-linked pCA. The correlation analysis noted that Erysipelotrichaceae_UCG-002, Olsenella, Ruminococcus_gauvreauii_group, Acetitomaculum, and Bifidobacterium were negatively related to the initial ether-linked FA content while Prevotella was positively related to the ether-linked FA content and the ratio of pCA to FA. In summary, the present results suggested that the content of ether-linked phenolic acids in plant cell walls exhibited a more profound effect on the pattern of microbial colonization than the fibre content.

Deoxynivalenol (DON) is a type B trichothecene mycotoxin produced by Fusarium fungi. To investigate its ruminal degradability and its effect on rumen fermentation, a 2 × 5 factorial experiment was conducted in vitro with two feed substrates with different forage levels (high forage (HF), forage-to-concentrate = 4:1; low forage (LF), forage-to-concentrate = 1:4) and five DON additions per substrate (0, 5, 10, 15, and 20 mg/kg of dry matter). After 48 h incubation, the DON degradability in the HF group was higher than in the LF group (p < 0.01), and it decreased along with the increase in DON concentrations (p < 0.01), which varied from 57.18% to 29.01% at 48 h. In addition, the gas production rate, total VFA production and microbial crude protein decreased linearly against the increase in DON additions (p < 0.05). Meanwhile, the proportion of CH₄ in the fermentation gas end-products increased linearly, especially in the HF group (p < 0.01). In brief, rumen microorganisms presented 29–57% of the DON degradation ability and were particularly significant under a high-forage substrate. Along with the increasing DON addition, the toxin degradability decreased, showing a dose-dependent response. However, DON inhibited rumen fermentation and increased methane production when it exceeded 5 mg/kg of dry matter.