Metabolism and some characteristics of lactobacilli isolated from the rumen of young calves

Fourteen strains of lactobacilli isolated from the rumen of young calves were studied to determine their biochemical characteristics, growth parameters, metabolism on lactose and sensitivity to 28 antimicrobial agents. Thirteen homofermentative strains belonged to Lactobacillus acidophilus and one heterofermentative strain resembled Lact. fermentum. The relevance of rumen lactobacilli to the nutrition of calves is discussed.     

THE NORMAL FLORA OF THE BOVINE RUMEN. IV. QUALITATIVE STUDIES OF LACTOBACILLI FROM COWS AND CALVES

SUMMARY: Two hundred and four samples of rumen contents were taken from eight fistulated cows receiving various diets, and seventy-one samples from twelve calves. From dilution counts of these samples, 91 isolates of Gram-positive catalase-negative rods were obtained, and by means of physiological and serological tests, 78 (95%) out of 82 lactobacilli were characterized as follows: Lactobacillus fermenti (33 strains), L. acidophilus (21), L. casei (17) and L. plantarum (7). Indications were obtained of progressive establishment of lactobacilli in the rumen of the growing calf, L. fermenti predominating. In the cow, L. acidophilus was generally associated with hay plus concentrates diets and L. casei with silage, whereas with grass small numbers of several species of lactobacilli were found.     

The adherence of lactic acid bacteria to the columnar epithelial cells of pigs and calves

The adhesion of various lactobacilli and streptococci to columnar epithelial cells of pigs and calves were studied, by in vitro methods. The porcine strains isolated most frequently were Lactobacillus delbrueckii, Lact. acidophilus and Lact. fermentum. Thirteen of the 22 lactobacilli were adhesive. All the streptococci isolated belonged to Lancefield's D-group; none of them adhered to pig epithelial cells. The adhesive strains (9 of 22) of calves were identified as Lact. fermentum. Adherence was variable even between strains of the same species. Isolates from plant material, cultured milk and cheese did not adhere to the columnar epithelial cells in vitro. The adhesive porcine strains tolerated low pH and bile acids, which is important for their survival under conditions in the stomach and intestine.     

The Bacterial Flora of the Rumen of Healthy and Bloating Calves

Rumen samples from five healthy milk-fed calves and one healthy calf given a milk-replacer diet containing yeast protein had pH values of 6.5–8.0, total counts of ca. 10¹⁰/ml and viable counts of 10⁸-10¹⁰/ml. The facultatively and strictly anaerobic, and obligately aerobic, components of the floras were enumerated and identified. Similar tests performed on samples from two calves bloating on the yeast diet had pH values of 4.8 and 5.3, and although total and viable counts were of the same order as those from healthy calves, obligately aerobic bacteria were not detected and counts of lactobacilli were elevated. Infusion of the rumen of a calf with yeast diet caused a reduction in pH from 7.3–5.1 and an increase of at least 100-fold in the numbers of organisms capable of forming copious gas from yeast diet. Results suggest that milk or milk-replacer diet normally does not enter the rumen to any extent but when it does, due perhaps to poor palatability, bloat may ensue.     

Multiresistant strains ofEscherichia coli isolated from the rumen of young calves

Five multiresistant strains ofEscherichia coli were isolated from the rumen fluid of young calves. Resistance to tetracycline and ampicillin was found to be associated with the transfer of a 6.4 kbp plasmid present in two investigated strains.     

Anaerobic Cocci from the Bovine Alimentary Tract, the Amino Acids of their Cell Wall Peptidoglycans and those of various Species of Anaerobic Streptococcus

Physiological characteristics of 45 strains of anaerobic cocci isolated from the rumen, duodenum, jejunum, ileum, caecum and faeces of pre-ruminant and ruminating calves and cows were examined. Among these strains were unusually anaerobic strains of Streptococcus bovis , Gram negative cocci resembling Megasphaera elsdenii but not fermenting lactic acid and strains resembling Coprococcus catus. The amino acid composition of the cell wall peptidoglycans of these and strains of anaerobic streptococci was determined.     

Characterization of the rumen microbiota of pre-ruminant calves using metagenomic tools

The temporal sequence of microbial establishment in the rumen of the neonatal ruminant has important ecological and pathophysiological implications. In this study, we characterized the rumen microbiota of pre-ruminant calves fed milk replacer using two approaches, pyrosequencing of hypervariable V3-V5 regions of the 16S rRNA gene and whole-genome shotgun approach. Fifteen bacterial phyla were identified in the microbiota of pre-ruminant calves. Bacteroidetes was the predominant phylum in the rumen microbiota of 42-day-old calves, representing 74.8% of the 16S sequences, followed by Firmicutes (12.0%), Proteobacteria (10.4%), Verrucomicrobia (1.2%) and Synergistetes (1.1%). However, the phylum-level composition of 14-day-old calves was distinctly different. A total of 170 bacterial genera were identified while the core microbiome of pre-ruminant calves included 45 genera. Rumen development seemingly had a significant impact on microbial diversity. The dazzling functional diversity of the rumen microbiota was reflected by identification of 8298 Pfam and 3670 COG protein families. The rumen microbiota of pre-ruminant calves displayed a considerable compositional heterogeneity during early development. This is evidenced by a profound difference in rumen microbial composition between the two age groups. However, all functional classes between the two age groups had a remarkably similar assignment, suggesting that rumen microbial communities of pre-ruminant calves maintained a stable function and metabolic potentials while their phylogenetic composition fluctuated greatly. The presence of all major types of rumen microorganisms suggests that the rumen of pre-ruminant calves may not be rudimentary. Our results provide insight into rumen microbiota dynamics and will facilitate efforts in formulating optimal early-weaning strategies.     

Effects of isovalerate supplements on morphology and functional gene expression of rumen mucosa in pre- and post-weaning dairy calves

Isovalerate supplements could stimulate rumen development by improving morphology and function of rumen mucosa, and then promote the growth of calves. This study was done to evaluate the effects of isovalerate supplements on morphology and functional gene expression of rumen mucosa in dairy calves. In total, 48 Chinese Holstein male calves with 15 days of age and 45.1±0.36 kg of BW were randomly assigned to four groups. The treatments were: control, low-isovalerate, moderate-isovalerate and high-isovalerate with 0, 3, 6 and 9 g isovalerate per calf per day, respectively. Supplementary isovalerate was hand-mixed into milk in pre-weaning calves and into concentrate portion in post-weaning calves. The study consisted of a 15-day-adaptation period and a 60-day-sampling period. Calves were weaned at 60 days of age. Three calves were slaughtered from each of the four treatments at 30, 60 and 90 days of age. The weight of body and stomach were measured, samples of ruminal tissues and blood were analyzed. Total stomach weight, total stomach to BW ratio, rumen wall and keratinized layer thickness, serum growth hormone and IGF-1 for both pre- and post-weaning calves increased linearly with increasing isovalerate supplements. Rumen to total stomach weight ratio, the length and width of rumen papillae, and serum β-hydroxybutyrate increased linearly for post-weaning calves. However, abomasum weight to total stomach weight ratio decreased linearly for both pre- and post-weaning calves. The relative messenger RNA expression for growth hormone receptor, IGF-1 receptor and 3-hydroxy-3-methylglutaryl-CoA synthase 1 in rumen mucosa increased linearly for post-weaning calves. Our results suggested that isovalerate supplements promoted rumen development in a dose-dependent manner. The optimum dose was 6.0 g isovalerate per calf per day.     

Calcium propionate supplementation improves development of rumen epithelium in calves via stimulating G protein-coupled receptors

In the present study, calcium propionate (CaP) was used as feed additive in the diet of calves to investigate their effects on rumen fermentation and the development of rumen epithelium in calves. To elucidate the mechanism in which CaP improves development of calf rumen epithelium via stimulating the messenger RNA (mRNA) expression of G protein-coupled receptors, a total of 54 male Jersey calves (age=7±1 days, BW=23.1±1.2 kg) were randomly divided into three treatment groups: control without CaP supplementation (Con), 5% CaP supplementation (5% CaP) and 10% CaP supplementation (10% CaP). The experiment lasted 160 days and was divided into three feeding stages: Stage 1 (days 0 to 30), Stage 2 (days 31 to 90) and Stage 3 (days 91 to 160). Calcium propionate supplementation percentages were calculated on a dry matter basis. In total, six calves from each group were randomly selected and slaughtered on days 30, 90 and 160 at the conclusion of each experimental feeding stage. Rumen fermentation was improved with increasing concentration of CaP supplementation in calves through the first 30 days (Stage 1). No effects of CaP supplementation were observed on rumen fermentation in calves during Stage 2 (days 31 to 90). Supplementation with 5% CaP increased propionate concentration, but not acetate and butyrate in calves during Stage 3 (days 91 to 160). The rumen papillae length of calves in the 5% CaP supplementation group was greater than that of Con groups in calves after 160 days feeding. The mRNA expression of G protein-coupled receptor 41 (GPR41) and GPR43 supplemented with 5% CaP were greater than the control group and 10% CaP group in feeding 160 days calves. 5% CaP supplementation increased the mRNA expression of cyclin D1, whereas did not increase the mRNA expression of cyclin-dependent kinase 4 compared with the control group in feeding 160-day calves. These results indicate that propionate may act as a signaling molecule to improve rumen epithelium development through stimulating mRNA expression of GPR41 and GPR43.     

Characteristics of isolates of Lactobacillus fermentum from the rumen of sheep

A detailed characterization of four representative Lactobacillus isolates from the rumen was undertaken after tests with API CH50 kits had indicated that they differed from currently recognized species. These isolates are shown to be related to Lactobacillus fermentum , although they differ from the type and reference strains of this species in sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns and electrophoretic mobility of LDH. It seemed that certain heterofermentative lactobacilli could give negative results for some fermentation tests (particularly fructose and lactose) in API kits, whilst showing good growth on the same substrates in conventional tests.     

Comparison of the Phospholipid Composition of Bifidobacterium and Lactobacillus Strains

Phospholipid composition of 10 Bifidobacterium strains of human intestinal origin and of 9 Lactobacillus strains was determined by quantitative two-dimensional thin-layer chromatography. Phospholipids of three Bifidobacterium strains from honey bees and of two strains from bovine rumen liquor were qualitatively investigated. Diphosphatidylglycerol and phosphatidylglycerol were present in strains of both genera. All Bifidobacterium strains contained as specific phospholipids a new polyglycerolphospholipid, compound 15, and its lyso derivatives, earlier detected in B. bifidum var. pennsylvanicus. Also, lyso compounds of diphosphatidylglycerol and alanyl phosphatidylglycerol were only present in this genus in variable amounts. Lysyl phosphatidylglycerol was the only ninhydrin-positive phospholipid in seven Lactobacillus strains. In L. delbrückii and L. helveticus it was absent and partially replaced by an unidentified ninhydrin-negative phospholipid. The differences in phospholipid composition between bifidobacteria and lactobacilli may be another argument to differentiate these two genera.     

Survival of bifidobacteria administered to calves

Twenty-five bifidobacteria were isolated from feces of calves. Isolates were identified, and their functional properties and antimicrobial activity were determined. From 10 strains with suitable properties rifampicin-resistant mutants (RRBs) were prepared and mixture of RRBs was administered to 2-d-old calves. These strains were identified by sequencing as Bifidobacterium animalis ssp. animalis (6 strains), B. thermophilum (2 strains), B. choerinum (1 strain) and B. longum ssp. suis (1 strain). The control group was without probiotic treatment. Survival ability of administered bifidobacteria was monitored in fecal samples by cultivation on modified TPY agar supplemented with mupirocin, acetic acid, and rifampicin. Administered bifidobacteria survived in gastrointestinal tract of calves for at least 60 d. Other bacteria were also determined after cultivation using fluorescence in situ hybridization (FISH). Bifidobacteria and lactobacilli dominated in fecal microflora. Significantly lower amounts of E. coli and higher amounts of bifidobacteria and total anaerobes were found in the treated group relative to the control group.     

Staphylococci associated with the rumen of young and wild ruminants

Staphylococcus warneri, Staph, xylosus, Staph. saprophyticus, Staph. epidermidis, Staph. sciuri subsp. lentus, Staph. sciuri subsp. sciuri and Staph. cohnii subsp. urealyticum were the most frequently occurring staphylococci in the rumen content and wall of young and wild ruminants. Staphylococcus warneri formed a high percentage mainly among 2–9-week-old ruminants. Staphylococcus hominis was found only in mouflons. Staphylococcus gallinarum was detected only in calves. Staphylococcus aureus was the predominant representative of coagulase-positive staphylococci in the rumen of wild ruminants. Most of the strains examined could not be identified as known species.     

Effects of isobutyrate supplementation in pre- and post-weaned dairy calves diet on growth performance,rumen development,blood metabolites and hormone secretion

Isobutyrate supplements could improve rumen development by increasing ruminal fermentation products, especially butyrate, and then promote the growth performance of calves. The objective of this study was to evaluate the effects of isobutyrate supplementation on growth performance, rumen development, blood metabolites and hormone secretion in pre- and post-weaned dairy calves. In total, 56 Chinese Holstein male calves with 30 days of age and 72.9±1.43 kg of BW, blocked by days of age and BW, were assigned to four groups in a randomized block design. The treatments were as follows: control, low-isobutyrate, moderate-isobutyrate and high-isobutyrate with 0, 0.03, 0.06 and 0.09 g isobutyrate/kg BW per calf per day, respectively. Supplemental isobutyrate was hand-mixed into milk of pre-weaned calves and the concentrate portion of post-weaned calves. The study consisted of 10 days of an adaptation period and a 50-day sampling period. Calves were weaned at 60 days of age. Seven calves were chosen from each treatment at random and slaughtered at 45 and 90 days of age. BW, dry matter (DM) intake and stomach weight were measured, samples of ruminal tissues and blood were determined. For pre- and post-weaned calves, DM intake and average daily gain increased linearly (P<0.05), but feed conversion ratio decreased linearly (P<0.05) with increasing isobutyrate supplementation. Total stomach weight and the ratio of rumen weight to total stomach weight tended to increase (P=0.073) for pre-weaned calves and increased linearly (P=0.021) for post-weaned calves, whereas the ratio of abomasum weight to total stomach weight was not affected for pre-weaned calves and decreased linearly (P<0.05) for post-weaned calves with increasing isobutyrate supplementation. Both length and width of rumen papillae tended to increase linearly for pre-weaned calves, but increased linearly (P<0.05) for post-weaned calves with increasing isobutyrate supplementation. The relative expression of messenger RNA for growth hormone (GH) receptor and 3-hydroxy-3-methylglutaryl-CoA synthase 1 in rumen mucosa increased linearly (P<0.05) for pre- and post-weaned calves with increasing isobutyrate supplementation. Blood concentrations of glucose, acetoacetate, β-hydroxybutyrate, GH and IGF-1 increased linearly (P<0.05) for pre- and post-weaned calves, whereas blood concentration of insulin decreased linearly with increasing isobutyrate supplementation. The present results indicated that isobutyrate promoted growth of calves by improving rumen development and its ketogenesis in a dose-dependent manner.     

Effects of protozoa on Methane production in rumen and hindgut of calves around time of weaning

Effects of the presence or absence of ciliate protozoa on methanogenesis in the rumen and hindgut were investigated in young calves during a 7-week period. Ten Holstein calves, aged 7 days, were divided in two groups (n = 5) and fed an increasing amount of a commercial milk replacer and small amounts of a calves starter. One group was inoculated with ciliate fauna on two occasions, week 5 and 6, while the second remained ciliate-free. The absence of protozoa in the rumen decreased rumen empty weight ( ? 23%, P < 0.01), and rumen pool size of N ( ? 36%, P < 0.01) and crude fat ( ? 37%, P < 0.05). Rumen bacteria of non-faunated calves contained a higher proportion of total amino acid-N per 16 g N ( + 3%, P < 0.01) and D-alanine-N per 16 g N ( + 13%, P < 0.05) compared to faunated calves. Further results contain a reference for a higher bacterial mass in the ciliate-free rumen with an increased number of bacteria adherent to rumen mucosa. The CH₄ production in the rumen increased exponentially with the increase in protozoa population size (R² = 0.68). In presence of 46 · 10⁴ protozoa per ml rumen fluid, the in vitro CH₄ production of rumen fluid per mol total VFA was about 34% higher in faunated than in non-faunated calves (P < 0.001). Hydrogen (2H) recovery of rumen fermentation was positively correlated (R² = 0.55) to the CH₄ production rate. Methanogens were attached on rumen mucosa. Methanogenesis, induced by rumen mucosa attached bacteria, was stimulated by ruminal protozoa. In the absence of protozoa in the rumen, the acetate - propionate ratio and butyrate proportion of VFA were reduced. In vivo in the absence of protozoa not only the whole animal CH₄ production ( ? 30%, P < 0.05) but also the digestibility of carbohydrates ( ? 4%, P < 0.05) was reduced. Thereby no difference was observed in the intake of ME per kg DM between the groups. In conclusion, the methanogenesis in the rumen, but not in hindgut, is associated with the development of the ruminal protozoa population. The level of methanogenesis (mol/mol VFA) in the hindgut amounts to 20% of the ruminal methanogenesis.     

Distribution of bifidobacteria in the gastrointestinal tract of calves

Development of gastrointestinal microflora of calves with special reference to bifidobacteria was investigated; fecal bacteria were enumerated in calves aged 3 days to 7 weeks. Bacteria were detected by using selective media, bifidobacteria using modified TPY agar with an addition of mupirocin and acetic acid and by fluorescence in situ hybridization (FISH). Bifidobacteria were dominant group of fecal flora of calves after 7 d of life, constituting 10 % of total bacterial counts. The highest bacterial concentrations were observed in rumen, cecum, and colon, the lowest in abomasum and duodenum. Bifidobacteria and lactobacilli exhibited the highest survival ability during stomach passage and dominated in all parts of the digestive tract. Bifidobacteria counts determined by FISH were significantly higher than those provided by cultivation. Modified TPY agar was highly selective and suitable for bifidobacteria isolation but FISH was shown to be a more precise method for their enumeration. Our results show that gastrointestinal microflora of calves in the milk-feeding period is similar to breast-fed infants with respect to the occurrence of bifidobacteria as a dominant bacterial group. The use of Bifidobacterium strains offers a promising way for providing beneficial effectors for calves in the milk-feeding period.     

Establishment of a spinated type of Diplodinium rangiferi by transfaunation of the rumen ciliates of Japanese sika deer (Cervus nippon centralis) to the rumen of two Japanese shorthorn calves (Bos taurus taurus)

One liter of rumen fluid containing 4.7 x 10(4) ciliates/ml, representing four genera including nine species of ciliates from a Japanese sika deer was inoculated into two unfaunated Japanese shorthorn calves. Two weeks after inoculation, all species originally present in the inoculum were subsequently detected in the rumen fluid of one or both calves. Ciliate densities ranged from 10(5)-10(6) cells/ml over the remainder of the 33-wk experiment. The inoculum contained Diplodinium rangiferi. which lacks caudal appendages, as is characteristic for the species. However, three weeks later, the rumen fluid of both calves contained D. rangiferi, which possesses caudal appendages varying from a single spine to multiple spines with a complicated furcate appearance. The caudal spines of D. rangiferi did not disappear during the experiment, even when the diet of the calves was switched to the ration of sika deer from which the inoculum was obtained.     

Rumen and post-rumen fermentation of spent tea leaf protein and other protein sources studied by the nylon bag method

Spent tea leaf (STL) and seven other protein feeds were subjected to microbial fermentation in nylon bags in the rumen of two fistulated buffalo calves and subsequent acid-pepsin digestion in vitro.The STL showed the lowest protein disappearance rate and rumen solubility at 8 h of fermentation. Polyphenols present in STL appear to offer protection to the protein and reduce rumen fermentation. An over-protection could be responsible for the low solubility of the protein in acid—pepsin solution.The high by-pass ability of STL protein appears to make it a suitable feed for growing calves and milking cows.     

Expression of MCT1, MCT2 and MCT4 in the rumen, small intestine and liver of reindeer (Rangifer tarandus tarandus L.)

The expression of monocarboxylate transporters MCT1, MCT2 and MCT4 in the rumen, small intestine and liver was examined in free-ranging and captive reindeer. In addition, expression of chaperone protein CD147, which is needed for the activity of MCT1 and MCT4, was studied in the rumen of suckling calves. Immunoblotting of cell membrane proteins showed the expression of MCT1 and MCT4, but not that of MCT2 in the rumen of reindeer. In free-ranging reindeer the amount of MCT1 was higher than in the captive ones (P<0.01). Developing rumen of suckling calves expressed MCT1 and MCT4 and positive correlation was found between MCT1 and CD147. Both MCT1 and CD147 correlated also with age in calves less than 10 days. In the small intestine all the isoforms studied were expressed, but the amounts were lower than in the rumen (P<0.05). In the liver MCT1 and MCT2 were found while MCT4 was nearly undetectable. The expression of MCT isoforms in the rumen and small intestine reflects the site of absorption and concentrations of short chain fatty acids (SCFA). In the liver the expression of high affinity transporters, MCT1 and MCT2, is in accordance with almost complete uptake of propionate from portal blood.     

Indigestion in Young Calves. II. The Influence Of Ground Barley,Coarse and Fine Hay

Four experiments comprising 86 calves have been carried out in order to examine the influence of ground barley, coarse and fine hay, when raising young calves on a high lactose milk replacer causing diarrhoea. The frequency of diarrhoea decreased when feeding barley (P < 0.01) or fine hay (P < 0.001), but increased when feeding coarse hay (P<0.01). Barley increased (P < 0.01) live weight gain and carcass weight, reduced (P < 0.001) rumen pH, favoured gram-positive cocci and rods in the rumen, caused hyperkeratosis and gave increased (P < 0.01) empty reticulo-rumen weights, the latter being supposed to be enhanced by hyperkeratosis. The calves ate more (P < 0.001) fine than coarse hay, fine hay being superior (P < 0.01) in promoting growth of the rumen. All hay maintained a predominantly gram-negative rumen flora, but somewhat different from that on the milk replacer, and counteracted (P < 0.01) low pH in the rumen and apparently also hyperkeratosis due to barley. The results with barley indicated no relationship between diarrhoea and hyperkeratosis.