Tomomi Ban-Tokuda

Body fat content and feeding level interact strongly in the short- and medium-term regulation of plasma leptin during underfeeding and re-feeding in adult sheep

Circulating leptin is regulated by food intake in the long, medium and short term; however, little is known about putative remnant effects of these successive regulations at any given time. To clarify this, two experiments were conducted in adult sheep, during which body condition parameters and plasma leptin were measured. During experiment 1, twenty ewes with normal body condition were either well fed (101 % of maintenance energy requirements (MER)) or underfed (41 % MER) for 166 d, then rapidly re-fed (at a mean of 208 % MER) for 3 d. Leptinaemia decreased after 14 d of underfeeding, remained depressed until day 166 and did not increase after 3 d re-feeding, whereas it was increased (+153 %; P < 0.05) by re-feeding the previously well-fed ewes. During experiment 2, twenty-four fat or lean ewes were either well fed (114 % MER) or underfed (52 % MER) for 94 d, and gradually re-fed for 2 d and maintained at a high feeding level (235 % MER) for 9 d. Underfeeding decreased leptinaemia in fat (from 4.19 to 2.63 ng/ml) but not lean ewes, and re-feeding increased leptinaemia after 5 d in lean previously well-fed (+123 %; P < 0.05) but not underfed ewes. In fat ewes, the impact of re-feeding was rapid (+144 %; P < 0.001 at 5 d) in previously well-fed ewes, whereas it was more gradual with a maximum at 11 d (+162 %; P < 0.01) in previously underfed ewes. In conclusion, leptinaemia is modulated by short-term energy intake level in interaction with long-term regulations involving nutritional history and body fatness, suggesting that a biological threshold of adiposity (about 20 %) is necessary to allow short- and medium-term leptin regulation.

Real-Time PCR Assays for Monitoring Anaerobic Fungal Biomass and Population Size in the Rumen

The relationship between copy numbers of internal transcribed spacer 1 (ITS1) and biomass or zoospore count of anaerobic fungi was studied to develop a quantitative real-time PCR-based monitoring method for fungal biomass or population in the rumen. Nine fungal strains were used to determine the relationship between ITS1 copy number and fungal biomass. Rumen fluid from three sheep and a cow were used to determine the relationship between ITS1 copy number and fungal population. ITS1 copy number was determined by real-time PCR with a specific primer set for anaerobic fungi. Freeze-dried fungal cells were weighed for fungal biomass. Zoospore counts were determined by the roll-tube method. A positive correlation was observed between both ITS1 copy number and dry weight and ITS1 copy number and zoospore counts, suggesting that the use of ITS1 copy numbers is effective for estimating fungal biomass and population density. On the basis of ITS1 copy numbers, fluctuations in the fungal population in sheep rumen showed that although the values varied among individual animals, the fungal population tended to decrease after feeding. In the present study, a culture-independent method was established that will provide a powerful tool for understanding the ecology of anaerobic fungi in the rumen.

Detection of Fiber-Digesting Bacteria in the Ceca of Ostrich Using Specific Primer Sets

The purpose of this study was to detect three fibrolytic bacteria, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus, in the cecal digesta of the ostrich (Struthio camelus) by PCR using a species-specific primer set for each 16S ribosomal RNA gene (16S rDNA). Although amplified DNA fragments obtained from each primer set had the expected size, the clone library derived from the amplimer contained non-specific sequences. The F. succinogenes-specific primer set recovered a partial 16S rDNA sequence of an uncultivated Fibrobacter with low similarity (<95%) and distantly related phylogenetic positioning to Fibrobacter sequences deposited in the databases, indicating a novel species of Fibrobacter. The sequence was considered to be identical to a clone detected in our previous experiment. Thus, we confirm that the gastrointestinal tract of the ostrich is one of the habitats of Fibrobacter species. The clone library derived from the R. flavefaciens-specific primer set contained a 16S rDNA sequence with 97% similarity to R. flavefaciens, indicating it could be one of a major fibrolytic bacterium in the ostrich ceca. No R. albus 16S rDNA sequence was found in the clone library of the R. albus-specific primer set.

Ruminal fermentation and microbial ecology of buffaloes and cattle fed the same diet

Although buffaloes and cattle are ruminants, their digestive capabilities and rumen microbial compositions are considered to be different. The purpose of this study was to compare the rumen microbial ecology of crossbred water buffaloes and cattle that were fed the same diet. Cattle exhibited a higher fermentation rate than buffaloes. Methane production and methanogen density were lower in buffaloes. Phylogenetic analysis of Fibrobacter succinogenes-specific 16S ribosomal RNA gene clone library showed that the diversity of groups within a species was significantly different (P < 0.05) between buffalo and cattle and most of the clones were affiliated with group 2 of the species. Population densities of F.succinogenes, Ruminococcus albus and R. flavefaciens were higher until 6 h post-feeding in cattle; however, buffaloes exhibited different traits. The population of anaerobic fungi decreased at 3 h in cattle compared to buffaloes and was similar at 0 h and 6 h. The diversity profiles of bacteria and fungi were similar in the two species. The present study showed that the profiles of the fermentation process, microbial population and diversity were similar in crossbred water buffaloes and crossbred cattle.

Diversity of the formyltetrahydrofolate synthetase (FTHFS) gene in the proximal and mid ostrich colon.

We analysed fragments of the formyltetrahydrofolate synthetase (FTHFS) gene, which encodes a key enzyme in reductive acetogenesis, from the bacterial flora in the proximal (PC) and mid (MC) colon of three ostriches to assess and compare bacterial diversity in this organ. Two clone libraries of FTHFS fragments were constructed from DNA extracted from digesta of the PC and MC, and a total of 46 cloned sequences were analysed from each library. A wide variety of FTHFS sequences were recovered. The coverage of the PC and MC libraries was 90.0% and 83.3%, respectively. Shannon–Wiener index (H’) and Chao1 of the MC library were higher than those of PC library. The sequences from each library were classified into 15 operational taxonomic units (OTUs) and clusters. Only four OTUs in cluster I were distantly related to known acetogens from human feces and rumen, suggesting the presence of the novel acetogens. Phylogenetic analysis suggests that composition of FTHFS sequences differs for the PC and MC.

Diversity and fluctuation in ciliate protozoan population in the rumen of cattle.

The purpose of this study was to investigate the diversity and fluctuation in the ciliate protozoan population in the rumen of cattle. DNA was extracted from the rumen of three ruminally cannulated, crossbred cattle and a polymerase chain reaction (PCR)-derived clone library was constructed, using a specific primer set targeting 18S ribosomal RNA genes of ciliate protozoa. DNA fragments of seven selected clones were validated for standard DNA of the protozoa-specific real-time PCR assay. Furthermore, population fluctuation of ciliate protozoa and methanogens in the cattle rumen was determined by real-time PCR. A total of 60 clones were sequenced, phylogenetically analyzed, and classified into 24 operational taxonomic units (OTUs) based on a 99% similarity criterion. More than 80% sequences were phylogenetically placed in the genus Entodinium. The rest of the sequences were placed in the genus Diploplastron (5%), Dasytricha (8.3%) and Isotricha (3.3%). The results suggest that Entodinium was the dominant group in the rumen of cattle used in this study. The ciliate protozoan population showed no significant change in numbers during the monitoring period and reached a peak at 3 h after feeding. Changes in the protozoa population were lower than those of the methanogens.

Effect of monensin withdrawal on rumen fermentation, methanogenesis and microbial populations in cattle.

This study was designed to obtain information on the residual influence of dietary monensin on ruminant fermentation, methanogenesis and bacterial population. Three ruminally cannulated crossbreed heifers (14 months old, 363 ± 11 kg) were fed Italian ryegrass straw and concentrate supplemented with monensin for 21 days before sampling. Rumen fluid samples were collected for analysis of short chain fatty acid (SCFA) profiles, monensin concentration, methanogens and rumen bacterial density. Post-feeding rumen fluid was also collected to determine in vitro gas production. Monensin was eliminated from the rumen fluid within 3 days. The composition of SCFA varied after elimination of monensin, while total production of SCFA was 1.78 times higher than on the first day. Methane production increased 7 days after monensin administration ceased, whereas hydrogen production decreased. The methanogens and rumen bacterial copy numbers were unaffected by the withdrawal of monensin.

Identification of bovine hibernation-specific protein complex and evidence of its regulation in fasting and aging

Hibernation-specific protein (HP) is a plasma protein that regulates hibernation in chipmunks. The HP complex (HP20c) consists of three homologous proteins, HP20, HP25 and HP27, all produced by liver and belonging to the C1q family. To date, HP20c has not been identified in any mammalian species except chipmunk and ground squirrel hibernators. Here, we report a bovine HP20 gene isolated from liver tissue and aortic endothelial cells. Total homology between bovine and chipmunk variants was 63% at the amino acid level. Gene expression was highest in the liver. Western blot revealed HP20 protein in foetal, newborn, calf and adult serum, with highest concentrations in the adult. Similar proteins were detected in sera of other ruminants but not in humans, bears, mice or rats. Bovine HP20 protein was found mainly in ovaries, stomach, heart, kidneys, lungs, testes and prostate, but not in the skeletal muscle. Native HP20 was purified from bovine adult serum as a complex containing 25 and 27 kDa proteins. Mass spectrometry revealed that these proteins are orthologues of chipmunk HP25 and HP27, respectively. Interestingly, bovine HP20 was highly expressed in cattle serum after fasting. Native bovine HP20c may be a useful tool for investigating HP function.

Effect of supplementation of rice bran and fumarate alone or in combination on in vitro rumen fermentation, methanogenesis and methanogens

This study investigated the effect of fumarate (FUM) and rice bran (RB), alone and together, on in vitro rumen fermentation, methanogenesis and methanogens. In vitro incubation was performed with six media that were either unsupplemented (control) or supplemented with 10% RB, 5 mmol/L FUM, 10% RB + 5 mmol/L FUM, 10 mmol/L FUM, or 10% RB + 10 mmol/L FUM. Methane (CH4 ) production, dry matter digestibility, CH4 per digested dry matter, total short-chain fatty acid (SCFA) production, proportion of SCFA, acetate : proprionate ratio, production of NH3 -N, and population density of rumen microbes were determined. Supplementation with 10% RB + 10 mmol/L FUM yielded a 36% decrease in CH4 production compared to the control. Supplementation of FUM, in the presence or absence of RB, provided increases in total SCFA production and propionate proportion up to 61% and 31%, respectively. Total bacteria, methanogens and protozoa populations were significantly (P < 0.05) decreased with the 10% RB + 10 mmol/L FUM supplementation. The effect of anti-methanogenesis of FUM was enhanced by the addition of RB. Notably, the CH4 production attenuation was achieved by 10% RB + 10 mmol/L FUM without reduction of digestibility or of ruminal fermentation.

Comparative study of plasma leptin concentration between solid ruminal and liquid abomasal feeding in weaned adult sheep.

This experiment was conducted to investigate the difference between ruminal (solid feed, SF) and abomasal (liquid feed, LF) feeding on the plasma leptin concentration in sheep. The experiment consisted of 2 weeks to adapt the animals to SF, 4 weeks of feeding on SF, 2 weeks adaptation to LF, 8 weeks of feeding on LF, 2 weeks of adaptation to SF, and 4 weeks of feeding on SF. The LF directory flowed into the abomasums of sheep by bottle feeding. Plasma leptin concentration before morning feeding was almost constant in the SF periods, whereas it showed between-day variations when measured during the LF periods. Mean plasma leptin levels were higher for LF (7.77 ± 0.76 ng/mL; mean ± SE) than for SF periods (3.95 ± 0.16 ng/mL; mean ± SE). Although plasma leptin concentration did not show any change after feeding in the SF and LF periods, plasma insulin and glucose levels increased within 15 min after liquid abomasal feeding, but not after solid ruminal feeding. The high plasma leptin concentration during the LF periods in weaned sheep could be due to change of digestible energy intake and changes in plasma insulin and glucose levels accompanying the changes in digestive processes and nutrient supply.