Core microbiota for nutrient digestion remained and ammonia utilization increased after continuous batch culture of rumen microbiota in vitro.

Introduction: This study aimed to investigate the digestive function, urea utilization ability, and bacterial composition changes in rumen microbiota under high urea (5% urea in diet) over 23 days of continuous batch culture in vitro. Methods: The gas production, dry matter digestibility, and bacterial counts were determined for the continuously batch-cultured rumen fluid (CRF). The changes in fermentation parameters, NH3-N utilization efficiency, and microbial taxa were analyzed in CRF and were compared with that of fresh rumen fluid (RF), frozen rumen fluid (FRF, frozen rumen fluid at -80°C for 1 month), and the mixed rumen fluid (MRF, 3/4 RF mixed with 1/4 CRF) with in vitro rumen fermentation. Results: The results showed that the dry matter digestibility remained stable while both the microbial counts and diversity significantly decreased over the 23 days of continuous batch culture. However, the NH3-N utilization efficiency of the CRF group was significantly higher than that of RF, FRF, and MRF groups (p < 0.05), while five core genera including Succinivibrio, Prevotella, Streptococcus, F082, and Megasphaera were retained after 23 days of continuous batch culture. The NH3-N utilization efficiency was effectively improved after continuous batch culture in vitro, and Streptococcus, Succinivibrio, Clostridium_sensu_stricto_1, p.251.o5, Oxalobacter, Bacteroidales_UCG.001, and p.1088.a5_gut_group were identified to explain 75.72% of the variation in NH3-N utilization efficiency with the RandomForest model. Conclusion: Thus, core bacterial composition and function retained under high urea (5% urea in diet) over 23 days of continuous batch culture in vitro, and bacterial biomarkers for ammonia utilization were illustrated in this study. These findings might provide potential applications in improving the efficiency and safety of non-protein nitrogen utilization in ruminants.

The effect of early colonized gut microbiota on the growth performance of suckling lambs.

The early colonized gut microbiota during the newborn period has been reported to play important roles in the health and immunity of animals; however, whether they can affect the growth performance of suckling lambs is still unclear. In this study, a total of 84 newborn lambs were assigned into LF-1 (top 15%), LF-2 (medium 70%), and LF-3 (bottom 15%) groups according to their average body weight gain at 30 days of age. Fecal samples of lambs (LF) as well as feces (MF), vagina (VAG), colostrum (COL), teat skin (TEAT) samples of ewes, and the air sediment (AIR) in the delivery room were collected 72 h after birth, and then the 16S rRNA gene was sequenced on the Illumina MiSeq platform. The results showed that the early colonized gut microbiota had a significant effect on the growth performance of suckling lambs with alpha and beta diversity (p < 0.05), and we observed that the contribution of early colonized bacteria on the growth performance of lambs increased with age (from BW30 at 25.35% to BW45 at 31.10%; from ADG30 at 33.02% to ADG45 at 39.79% by measuring the relative effects of factors that influence growth performance). The early colonized gut microbiota of suckling lambs with high growth performance was similar to that in VAG, MF, and AIR (p < 0.05). With the RandomForest machine learning algorithm, we detected 11, 11, 6, and 4 bacterial taxa at the genus level that were associated with BW30, BW45, ADG30, and ADG45 of suckling lambs, respectively, and the correlation analysis showed that Butyricicoccus, Ruminococcus_gnavus_group, Ruminococcaceae_Other, and Fusobacterium could significantly affect the growth performance (BW30, BW45, ADG30, and ADG45) of suckling lambs (p < 0.05). In conclusion, the early colonized gut microbiota could significantly affect the growth performance of suckling lambs, and targeting the early colonized gut microbiota might be an alternative strategy to improve the growth performance of suckling lambs.

Rumen-Protected Lysine and Methionine Supplementation Reduced Protein Requirement of Holstein Bulls by Altering Nitrogen Metabolism in Liver.

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.

Average Daily Gain in Lambs Weaned at 60 Days of Age Is Correlated with Rumen and Rectum Microbiota.

Colonization of gastrointestinal microbiota in mammals during early life is vital to host health. The objective of this study was to investigate whether lambs with high and low ADG have a different rumen and rectum microbial community. Thus, we investigated potential relationships between rumen and rectum microbiota and average daily gain (ADG) in weaned lambs. Sixteen lambs with similar body weights (7.63 ± 1.18 kg) were selected at 30 days of age. At 60 days of age, lambs were weaned, and ADG was calculated from 60 to 90 days. Then, two groups were generated: higher ADG (HG, 134.17 ± 13.48 g/day) and lower ADG (LG, 47.50 ± 19.51 g/day). Microbiota was evaluated at 30, 60, and 90 days of age. The final live weight and ADG at 90 days of age was higher (p < 0.05) in the HG group compared to the LG group. The maturity of bacterial and fungal communities was increased (p < 0.05) in the HG group for the 30 days vs. 90 days comparison and 60 days vs. 90 days comparison. Linear discriminant analysis effect size (LEfSe) analysis revealed a total of 18 bacterial biomarkers that are ADG-specific in the rumen and 35 bacterial biomarkers in the rectum. Meanwhile, 15 fungal biomarkers were found in the rumen and 8 biomarkers were found in the rectum. Our findings indicated that ADG is related to the rumen and rectum microbiota in lambs.

LncGSAR Controls Ovarian Granulosa Cell Steroidogenesis via Sponging MiR-125b to Activate SCAP/SREBP Pathway.

Long non-coding RNAs (lncRNAs) have been shown to play important roles in livestock fecundity, and many lncRNAs that affect follicular development and reproductive diseases have been identified in the ovary. However, only a few of them have been functionally annotated and mechanistically validated. In this study, we identified a new lncRNA (lncGSAR) and investigated its effects on the proliferation and steroidogenesis of ovine granulosa cells (GCs). High concentrations of glucose (add 33.6 mM glucose) caused high expression of lncGSAR in GCs by regulating its stability, and lncGSAR overexpression promoted GCs proliferation, estrogen secretion, and inhibited progesterone secretion, whereas interference with lncGASR had the opposite effect. Next, we found that the RNA molecules of lncGSAR act on MiR-125b as competitive endogenous RNA (ceRNA), and SREBP-cleavage-activating protein (SCAP) was verified as a target of MiR-125b. LncGASR overexpression increased the expression of SCAP, SREBP, and steroid hormone-related proteins, which can be attenuated by MiR-125b. Our results demonstrated that lncGSAR can act as a ceRNA to activate SCAP/SREBP signaling by sponging MiR-125b to regulate steroid hormone secretion in GCs. These findings provide new insights into the mechanisms of nutrient-regulated follicle development in ewes.

Age-Related Changes in the Ruminal Microbiota and Their Relationship With Rumen Fermentation in Lambs.

The rumen microbiota is vital for the health and growth performance of the host animal, mainly due to its role in the fermentation of ingested feed within the rumen. Attaining a better understanding of the development of the bacterial community and fermentation in the rumen can provide the theoretical basis for regulating feed utilization. This study analyzed the development of rumen bacteria in lambs from birth to 4 months of age using 16S-rRNA amplicon sequencing data and studied its relationship with ruminal fermentation. Serum levels of metabolites were monitored at 30, 60, 90, and 120 days of age, and the RandomForest approach was used to determine age-related changes in rumen bacteria. Levels of blood metabolites, ruminal fermentation, the rumen bacterial community and its functions were all affected by the age of the lambs (P < 0.05). Based on the Bray-Curtis distance within the age groups of the rumen microbiota, the similarity increased sharply after the lambs were weaned at 60 days of age (P < 0.05). The similarity between the samples collected from birth to 90 days of age and those collected at 120 days of age, increased after 20 days of age, reaching a maximum at 90 days vs. 120 days (P < 0.05). Some age-associated changes in the microbial genera were correlated with changes in the concentrations of volatile fatty acids and the levels of microbial crude protein in the rumen, including positive correlations between main volatile fatty acids and the genera of Prevotella 1, Lachnospiraceae NK3A20 group, Ruminococcus gauvreauii group, Ruminococcaceae UCG-014, and Ruminococcus 2 (P < 0.05). These results indicated that the microbial community and the function of rumen was not well-established before 20 days of age, so there is a degree of plasticity in the rumen bacterial community during the first 20 days of post-natal development in lambs, and this might provide an opportunity for interventions to improve rumen fermentation and, thus, increase their growth performance.

Analysis of Nutrient Composition, Rumen Degradation Characteristics, and Feeding Value of Chinese Rye Grass, Barley Grass, and Naked Oat Straw.

The current study was designed to investigate the chemical composition, rumen degradation characteristics, and feeding value of three roughages commonly used in Asia as ruminant feed, including Chinese rye grass (CRG), barley grass (BG), and naked oat straw (NO). Four Holstein Friesian cows equipped with permanent rumen fistulas were chosen for experimental trials in the current study. The nylon bag method was carried out to measure the crude protein (CP), acid detergent fiber (ADF), ruminal degradability of dry matter (DM), and neutral detergent fiber (NDF). Our analysis revealed that the contents of CP in the CRG (9.0%) and BG (8.9%) were higher than in the NO (5.94%). The contents of NDF in the CRG (64.97%) and NO (63.83%) were lower than in the BG (67.33%), and the content of ADF in the CRG (37.03%) was lower than in the BG (37.93%) and NO (38.28%). The ED values of DM in the NO and CRG were significantly higher (p < 0.001) than in the BG. The effective degradability (ED) values of NDF were the highest in the CRG and lowest in the NO (p < 0.001). In addition, the ED values of ADF were the highest in the CRG and lowest in the BG (p < 0.001). The ED value of CP in the CRG was significantly higher than that in the BG and NO (p < 0.001). The estimated total digestible nutrients (TDN) (54.56%) and DM degradation rate (DDM) (60.06%) of the CRG were higher than those of BG and NO. In addition, the expected DM intake (DMI), estimated relative feed value (RFV), and estimated relative feed quality (RFQ) of the BG were lower than those of the CRG and NO. Altogether, based on our findings, we concluded that the nutritional quality, feeding value and effective rumen degradation rate of CRG were better than of BG and NO.

Adsorbents Reduce Aflatoxin M1 Residue in Milk of Healthy Dairy Cow Exposed to Moderate Level Aflatoxin B1 in Diet and Its Exposure Risk for Humans.

This study investigated the effect of moderate risk level (8 µg/kg) AFB1 in diet supplemented with or without adsorbents on lactation performance, serum parameters, milk AFM1 content of healthy lactating cows and the AFM1 residue exposure risk in different human age groups. Forty late healthy lactating Holstein cows (270 ± 22 d in milk; daily milk yield 21 ± 3.1 kg/d) were randomly assigned to four treatments: control diet without AFB1 and adsorbents (CON), CON with 8 µg/kg AFB1 (dry matter basis, AF), AF + 15 g/d adsorbent 1 (AD1), AF + 15 g/d adsorbent 2 (AD2). The experiment lasted for 19 days, including an AFB1-challenge phase (day 1 to 14) and an AFB1-withdraw phase (day 15 to 19). Results showed that both AFB1 and adsorbents treatments had no significant effects on the DMI, milk yield, 3.5% FCM yield, milk components and serum parameters. Compared with the AF, AD1 and AD2 had significantly lower milk AFM1 concentrations (93 ng/L vs. 46 ng/L vs. 51 ng/L) and transfer rates of dietary AFB1 into milk AFM1 (1.16% vs. 0.57% vs. 0.63%) (p < 0.05). Children aged 2-4 years old had the highest exposure risk to AFM1 in milk in AF, with an EDI of 1.02 ng/kg bw/day and a HI of 5.11 (HI > 1 indicates a potential risk for liver cancer). Both AD1 and AD2 had obviously reductions in EDI and HI for all population groups, whereas, the EDI (≥0.25 ng/kg bw/day) and HI (≥1.23) of children aged 2-11 years old were still higher than the suggested tolerable daily intake (TDI) of 0.20 ng/kg bw/day and 1.00 (HI). In conclusion, moderate risk level AFB1 in the diet of healthy lactating cows could cause a public health hazard and adding adsorbents in the dairy diet is an effective measure to remit AFM1 residue in milk and its exposure risk for humans.

Effects of Age, Diet CP, NDF, EE, and Starch on the Rumen Bacteria Community and Function in Dairy Cattle.

To understand the effects of diet and age on the rumen bacterial community and function, forty-eight dairy cattle at 1.5 (M1.5), 6 (M6), 9 (M9), 18 (M18), 23 (M23), and 27 (M27) months old were selected. Rumen fermentation profile, enzyme activity, and bacteria community in rumen fluid were measured. The acetate to propionate ratio (A/P) at M9, M18, and M23 was higher than other ages, and M6 was the lowest (p < 0.05). The total volatile fatty acid (TVFA) at M23 and M27 was higher than at other ages (p < 0.05). The urease at M18 was lower than at M1.5, M6, and M9, and the xylanase at M18 was higher than at M1.5, M23, and M27 (p < 0.05). Thirty-three bacteria were identified as biomarkers of the different groups based on the linear discriminant analysis (LDA) when the LDA score >4. The variation partitioning approach analysis showed that the age and diet had a 7.98 and 32.49% contribution to the rumen bacteria community variation, respectively. The richness of Succinivibrionaceae_UCG-002 and Fibrobacter were positive correlated with age (r > 0.60, p < 0.01) and positively correlated with TVFA and acetate (r > 0.50, p < 0.01). The Lachnospiraceae_AC2044_group, Pseudobutyrivibrio, and Saccharofermentans has a positive correlation (r > 0.80, p < 0.05) with diet fiber and a negative correlation (r < -0.80, p < 0.05) with diet protein and starch, which were also positively correlated with the acetate and A/P (r > 0.50, p < 0.01). The genera of Lachnospiraceae_AC2044_group, Pseudobutyrivibrio, and Saccharofermentans could be worked as the target bacteria to modulate the rumen fermentation by diet; meanwhile, the high age correlated bacteria such as Succinivibrionaceae_UCG-002 and Fibrobacter also should be considered when shaping the rumen function.

The Rumen Bacterial Community in Dairy Cows Is Correlated to Production Traits During Freshening Period.

The rumen microbiome plays a vital role in providing nutrition to the host animal, thereby influencing ruminant production. Despite its importance, it is not fully understood how variation in the ruminal bacteria community composition influences dry matter intake (DMI), milk yield and ruminal fermentative parameters in dairy cows, especially during freshening period. Here, we hypothesized that during early lactation, high DMI cows having a different ruminal microbiota than low DMI cows, and that this difference persists over time. To test this, we enrolled 65 fresh and determinzed their DMI using an auto-feed intake recording system. Fourteen days after calving, the 10 animals with the lowest (LFI) and the 10 animals with the highest (HFI)-average DMI were selected for further analysis. Rumen fluid was collected from these two cohorts at 1 (Fresh1d) and 14 days (Fresh14d) after calving and their ruminal microbiota were assessed using 16S rRNA sequencing. Volatile fatty acid (VFA) concentrations were also quantified. Comparison of the ruminal microbiotas between Fresh1d and Fresh14d showed that Fresh14d cows had a significantly higher relative abundance of VFA-producing microbes (P < 0.05), such as Prevotella_7 and Succinivibrionaceae_UCG-001. This was commensurate with the concentrations of acetate, propionate, butyrate, valerate and total VFAs, were also significantly (P < 0.05) increased in Fresh14d cows. We also found that the differences in the ruminal microbiota between LFI and HFI cows was limited, but DMI significantly altered (P < 0.05) the relative proportion of bacteria in the families Coriobacteriaceae, and Succinivibrionaceae. Furthermore, specific operational taxonomic units belonging to the Anaeroplasma was significantly (P < 0.05) correlated with DMI and milk yield. Taking together, our findings provide a framework for future studies of freshening period cow that seek to better understand the role of the ruminal microbiota during this critical period in the lactation cycle.

Dynamic changes of the fecal bacterial community in dairy cows during early lactation.

The dynamics of the community structure and composition of the dairy cow fecal bacterial communities during early lactation is unclear, therefore this study was conducted to characterize the fecal bacterial communities in dairy cows during early lactation using 16S rRNA gene sequencing. Feces were sampled from 20 healthy fresh Holstein dairy cows on day 1 (Fresh1d group) and day 14 (Fresh14d group) after calving. After calving, cows were fed the same fresh diet. The dominant phyla Firmicutes and Proteobacteria were decreased (P ≤ 0.01) with lactating progress and phyla Bacteroidetes were increased (P = 0.008) with lactating progress and dietary transition. At family level, the predominant families were Ruminococcaceae (35.23%), Lachnospiraceae (11.46%), Rikenellaceae (10.44%) and Prevotellaceae (6.89%). A total of 14 genera were different between fecal samples from Fresh1d and Fresh14d, included the predominant genera, such as Ruminococcaceae_UCG-005 (P = 0.008), Rikenellaceae_RC9_gut_group (P = 0.043) and Christensenellaceae_R-7_group (P = 0.008). All fecal bacterial communities shared members of the genera Ruminococcaceae_UCG-005, Bacteroides and Rikenellaceae_RC9_gut_group. These findings help to improve our understanding of the composition and structure of the fecal microbial community in fresh cows and may provide insight into bacterial adaptation time and dietary in lactating cows.

The day-to-day stability of the ruminal and fecal microbiota in lactating dairy cows.

In this study, we examined differences between the microbiota of the ruminal fluid (DR) and feces (DF) from five lactating dairy cows over three consecutive days using 16S rRNA gene sequence-based analysis. Results showed significant differences between the microbial communities of the DR and DF. In particular, the relative abundance of the phyla Firmicutes and Actinobacteria was significantly lower (q < 0.001) in DR compared with DF, while the relative abundance of Bacteroidetes was significantly higher in DF than that of DR (q < 0.001). A significantly higher relative abundance of the genera Bifidobacterium, 5-7N15, Clostridium, Epulopiscium, SMB53, Turicibacter, Dorea, Roseburia, and Akkermansia was observed in the DF, while a higher relative abundance of the genera Prevotella, Butyrivibrio, CF231, RFN20, and Succiniclasticum was observed in the DR. A further analysis using the functional prediction program PICRUSt showed that sequences belonging to the 5-7N15, Akkermansia, Bifidobacterium, Clostridium, Dorea, Epulopiscium, Roseburia, and Turicibacter were significantly and positively correlated with glycan biosynthesis and metabolism, while CF231, Prevotella, RFN20, and Succiniclasticum were significantly and positively correlated with amino acid, lipid, carbohydrate, other amino acid, cofactors, and vitamins metabolism. No significant differences were observed across the three consecutive days in either the DR or DF ecosystems, with no significant differences in the diversity or abundance at the phylum and genus levels suggested that there is a limited day-to-day variability in the gut microbiota.

Effects of Saccharomyces Cerevisiae Fermentation Products on the Microbial Community throughout the Gastrointestinal Tract of Calves.

The effect of Saccharomyces cerevisiae fermentation products (SCFP) on improving growth and health of calves could be attributed to the ability of SCFP to modulate the microbiota in the gastrointestinal tract (GIT). However, the changes in microbial community along the gut in calves supplemented with SCFP have not been investigated extensively. The aims of this study were to investigate the effect of SCFP on microbial communities in each sites of GIT using high-throughput sequencing technique. Fifteen Holstein male calves were used and randomly assigned to 1 of the 3 treatments including a calf starter containing 0 (Control, CON), 0.5 (SCFP1) or 1% SCFP (SCFP2, Original XPC, Diamond V, Cedar Rapids, IA, USA) of dry matter from day 4 to 56. The supplemented calves were fed with an additional 1 g/d SCFP (SmartCare, Diamond V, Cedar Rapids, IA, USA) in milk from day 2 to 30. Rumen fluid was sampled at day 28 of age via esophageal tube. All calves were slaughtered and gastrointestinal samples collected on day 56. Inclusion of SCFP increased the microbial species richness in the large intestine. The SCFP also affected the bacterial community at an early age in the rumen and later in rectum microbiota. Supplementation of SCFP stimulated colonization by fibrolytic bacteria (Lachnospiraceae and Ruminococcaceae) in rumen and large intestine, respectively. No differences were found between SCFP1 and SCFP2. This is the first study to analyze the effect of SCFP on bacterial community of the GIT microbiota in calves. The results provide the basic bacterial community information, which helps us understand the mechanism of action of SCFP for improving the health and performance of pre-weaning calf.

Low Concentration of Antibiotics Modulates Gut Microbiota at Different Levels in Pre-Weaning Dairy Calves.

The aim of this study was to investigate the effect of feeding milk replacer (MR) with two different antibiotics treatments on the gut microbiota of pre-weaning calves. Twelve (12) Holstein male calves at 1-day-old were randomly assigned to: milk replacer without antibiotics (CON), milk replacer plus low cocktail of antibiotics (LCA) concentration (penicillin 0.024 mg/L, streptomycin 0.025 mg/L, tetracycline 0.1 mg/L, ceftiofur 0.33 mg/L), and milk replacer plus a low concentration of single antibiotic (LSA; ceftiofur 0.33 mg/L). All the calves were harvested at 35-day-old, and the digesta from the ileum and colon was collected in addition to fecal samples. Samples were analyzed by 16S rRNA gene using Illumina MiSeq platform. Results showed that there were significant differences among treatments in the ileum, where LCA significantly reduced the relative abundance of Enterobacteriaceae (P = 0.02) especially Escherichia-coli (P = 0.02), while LSA significantly reduced the relative abundance of Comamonas (P = 0.02). In the colon and rectum, LSA treatment was significantly enriched with the class Bacilli, whereas the control group was significantly enriched with Alloprevotlla (P = 0.03). However, at the family level in the rectum LCA and LSA significantly reduced the relative abundance of Acidaminococcaceae (P = 0.01). Moreover, at the genera level in the colon, LSA significantly increased Prevotellaceae_Ga6A1_ group (P = 0.02), whereas in the rectum both of treatments reduced the relative abundance of Phascolarctobacterium (P = 0.01). In conclusion, the overall low cocktail of antibiotics concentration induced changes at different taxonomic levels; specifically the decrease in Escherichia-coli which might subsequently reduce the incidences of diarrhea in calves.

Ecological Restoration of Antibiotic-Disturbed Gastrointestinal Microbiota in Foregut and Hindgut of Cows.

Antibiotically disturbed gastrointestinal microbiota needs a long period time to be restored to normal, which may cause a series of problems to the host. The understanding of restoration of the biased microbiota by antibiotics remains largely unknown. Here, we investigated the microbiota shift in foregut (rumen) and hindgut (rectum) of lactating cows after antibiotics exposure as well as after antibiotics withdrawal with (Microbiota transplantation, MT group) or without (Control, CON group) microbiota transplantation. We were able to demonstrate that microbiota in both foregut and hindgut significantly changed after 3 or 14 days of antibiotics exposure, and the changes persisted over long period of time (>18 days) after withdrawing the antibiotics. We further observed a faster restoration of microbiota in both foregut and hindgut of MT group than CON group, microbiota in foregut was mainly benefited from microbiota transplantation by restoring the alpha-diversity as well as within-group similarity, while microbiota in hindgut was primarily benefited from microbiota transplantation by reestablishing the co-occurrence network (nodes number, edges number, density, modularity as well as closeness centrality). These results together expanded our understanding of restoration of the biased microbiota by antibiotics, and may also be instructive to deal with the delayed microbiota restoration at least in cows.

Effects of a wide range of dietary forage-to-concentrate ratios on nutrient utilization and hepatic transcriptional profiles in limit-fed Holstein heifers.

BACKGROUND: Improving the efficiency of animal production is a relentless pursuit of ruminant producers. Energy utilization and partition can be affected by dietary composition and nutrient availability. Furthermore, the liver is the central metabolic intersection in cattle. However, the specific metabolic changes in the liver under conditions of limit-feeding remain unclear and require further study. The present study aimed to elucidate the effects of a wide range of dietary forage:concentrate ratios (F:C) on energy utilization, and identify potential changes in molecular metabolism by analyzing hepatic transcriptional profiles. Twenty-four half-sib Holstein heifers were fed four F:C diets (20:80, 40:60, 60:40, and 80:20 on a dry matter basis), with similar intake levels of metabolizable energy (ME) and crude protein. Liver biopsy samples were obtained and RNA sequencing was conducted to identify the hepatic transcriptomic changes. Moreover, the ruminal fermentation profiles, growth characteristics, and levels of metabolites in the liver and plasma of the heifers were monitored. RESULTS: The proportion of acetate showed a linear increase (P < 0.01) with increasing dietary forage levels, whereas the proportion of propionate showed a linear decline (P ≤ 0.01). Lower levels of average daily gain and feed efficiency (P < 0.01) were observed in heifers fed high levels of forage, with a significant linear response. Using the Short Time-series Expression Miner software package, the expression trends of significant differentially expressed genes (DEGs) were generally divided into 20 clusters, according to their dynamic expression patterns. Functional classification analysis showed that lipid metabolism (particularly cholesterol and steroid metabolism which were in line with the cholesterol content in the liver and plasma) was significantly increased with increasing dietary forage levels and slightly reduced by the 80% forage diet. Nine DEGs were enriched in the related pathways, namely HMGCS1, HMGCR, MSMO1, MVK, MVD, IDI1, FDPS, LSS, and DHCR7. CONCLUSIONS: The ruminal fermentation and feed efficiency results suggest that different mechanisms of energy utilization might occur in heifers fed different F:C diets with similar levels of ME intake. Increased cholesterol synthesis from acetate might be responsible for the reduced efficiency of energy utilization in heifers fed high-forage diets.

Effect of Dietary Forage to Concentrate Ratios on Dynamic Profile Changes and Interactions of Ruminal Microbiota and Metabolites in Holstein Heifers.

A better understanding of global ruminal microbiota and metabolites under extensive feeding conditions is a prerequisite for optimizing rumen function and improving ruminant feed efficiency. Furthermore, the gap between the information on the ruminal microbiota and metabolites needs to be bridged. The aim of this study was to investigate the effects of a wide range of forage to concentrate ratios (F:C) on changes and interactions of ruminal microbiota and metabolites. Four diets with different F:C (80:20, 60:40, 40:60, and 20:80) were limit-fed to 24 Holstein heifers, and Illumina MiSeq sequencing and gas chromatography time-of-flight/mass spectrometry were used to investigate the profile changes of the ruminal microbes and metabolites, and the interaction between them. The predominant bacterial phyla in the rumen were Bacteroidetes (57.2 ± 2.6%) and Firmicutes (26.8 ± 1.6%), and the predominant anaerobic fungi were Neocallimastigomycota (64.3 ± 3.8%) and Ascomycota (22.6 ± 2.4%). In total, 44, 9, 25, and 2 genera, respectively, were identified as the core rumen bacteria, ciliate protozoa, anaerobic fungi, and archaea communities across all samples. An increased concentrate level linearly decreased the relative abundance of cellulolytic bacteria and ciliates, namely Fibrobacter, Succinimonas, Polyplastron, and Ostracodinium (q < 0.05), and linearly increased the relative abundance of Entodinium (q = 0.04), which is a non-fibrous carbohydrate degrader. Dietary F:C had no effect on the communities of anaerobic fungi and archaea. Rumen metabolomics analysis revealed that ruminal amino acids, lipids, organic acids, and carbohydrates were altered significantly by altering the dietary F:C. With increasing dietary concentrate levels, the proportions of propionate and butyrate linearly increased in the rumen (P ≤ 0.01). Correlation analysis revealed that there was some utilization relationship or productive association between candidate metabolites and affected microbe groups. This study provides a better understanding of ruminal microbiota and metabolites under a wide range of dietary F:C, which could further reveal integrative information of rumen function and lead to an improvement in ruminant production.

Comparison of rumen bacteria distribution in original rumen digesta, rumen liquid and solid fractions in lactating Holstein cows.

BACKGROUND: Original rumen digesta, rumen liquid and solid fractions have been frequently used to assess the rumen bacterial community. However, bacterial profiles in rumen original digesta, liquid and solid fractions vary from each other and need to be better established. METHODS: To compare bacterial profiles in each fraction, samples of rumen digesta from six cows fed either a high fiber diet (HFD) or a high energy diet (HED) were collected via rumen fistulas. Rumen digesta was then squeezed through four layers of cheesecloth to separate liquid and solid fractions. The bacterial profiles of rumen original digesta, liquid and solid fractions were analyzed with High-throughput sequencing technique. RESULTS: Rumen bacterial diversity was mainly affected by diet and individual cow (P > 0.05) rather than rumen fraction. Bias distributed bacteria were observed in solid and liquid fractions of rumen content using Venn diagram and LEfSe analysis. Fifteen out of 16 detected biomarkers (using LEfSe analysis) were found in liquid fraction, and these 15 biomarkers contributed the most to the bacterial differences among rumen content fractions. CONCLUSIONS: Similar results were found when using samples of original rumen digesta, rumen liquid or solid fractions to assess diversity of rumen bacteria; however, more attention should be draw onto bias distributed bacteria in different ruminal fractions, especially when liquid fraction has been used as a representative sample for rumen bacterial study.

Net Phosphorus Requirements of Dorper×Thin-tailed Han Crossbred Ram Lambs.

A comparative slaughter trial was conducted to estimate the phosphorus (P) requirement for maintenance and growth of crossbred lambs of Dorper with a Chinese indigenous sheep breed, thin-tailed Han sheep. Thirty-five Dorper×thin-tailed Han crossbred, noncastrated ram lambs (20.3±0.22 kg of shrunk body weight (SBW)) were used. Seven lambs were randomly chosen and slaughtered at 20 kg SBW as the baseline group for measuring initial body composition. Another seven lambs were also randomly chosen and offered a pelleted mixed diet for ad libitum intake and slaughtered at 28 kg SBW. The remaining 21 sheep were randomly divided into 3 groups with 7 sheep each and subject to the same diet of either 70 or 40% of ad libitum intake. The 3 groups were slaughtered when the sheep fed ad libitum reached 35 kg of SBW. Body P contents were determined after slaughter. The results showed that the net P requirement for maintenance was 30.0 mg/kg of empty body weight (EBW) or 23.4 mg/kg body weight (BW), and the P requirement for growth decreased from 5.3 to 5.0 g/kg of EBW gain as the lamb grew from 20 to 35 kg. The net P requirement for growth of Dorper×thin-tailed Han crossbred ram lambs was lower than that of sheep adopted by the American nutritional system.