Masaaki Wakita

Molecular characterization of chicken growth hormone secretagogue receptor gene

Synthetic growth hormone secretagogues stimulate growth hormone secretion by binding to a specific receptor, growth hormone secretagogue receptor (GHS-R). In this study, we investigated the cDNA and the genomic structure of chicken GHS-R. Chicken GHS-R gene is composed of two exons separated by an intron. Two GHS-R mRNA species, cGHS-R1a and cGHS-R1a-variant (cGHS-R1aV) are generated by alternative splicing of a primary transcript. cGHS-R1a protein is predicted to have seven transmembrane domains by a high degree of amino acid sequence identity with mammalian and teleost homologs. cGHS-R1aV lacks the transmembrane-6 domain due to a 48 bp deletion. RT-PCR analysis showed widespread tissue distributions of cGHS-R1a and cGHS-R1aV mRNAs with much higher amounts of cGHS-R1a in all the tissues.

Changes in plasma thyroid hormones, luteinizing hormone (LH), estradiol, progesterone and corticosterone of laying hens during a forced molt

1. Circulating concentrations of iodothyronines, luteinizing hormone(LH), estradiol(E2), progesterone and corticosterone were measured in hens before, during, and after a forced molt induced by fasting. 2. Corticosterone increased at the onset of molt, peaked at the maximal molt and returned to pre- and post-molt levels. LH, E2 and progesterone declined during the molt, and the decline was coincident with the cessation of egg production. 3. Thyroxine(T4), triiodothyronine (T3) and reverse triiodothyronine(rT3) increased during the molt. The increases of T4 and T3 were not abolished even if the forced molt was conducted in mild weather.

Thyroid hormones are involved in insulin-like growth factor-I (IGF-I) production by stimulating hepatic growth hormone receptor (GHR) gene expression in the chicken.

Effect of thyroid status on IGF-I production in growing chickens was studied. Serum concentrations of GH were not affected by propylthiouracil (PTU) or thyroxine (T4) treatments, whereas serum IGF-I levels were significantly decreased in PTU-treated chickens. The lowered serum IGF-I levels in the PTU-treated group were completely restored to the control levels by T4 injections. In the liver, the messenger RNA (mRNA) expressions both for GH receptor (GHR) and IGF-I were significantly repressed by PTU treatment, and were restored again by T4 replacement. In addition, the results of analysis on radiolabelled GH binding to the liver membrane were consistent with the levels of hepatic GHR mRNA expression. Serum concentrations of IGF-I were positively correlated with hepatic IGF-I mRNA and GHR mRNA expressions. The correlation coefficient between serum T3 levels and hepatic IGF-I mRNA expressions was also significant. These results indicate that thyroid hormones regulate IGF-I production in the chicken by affecting hepatic GHR expression.

Digestion properties of ammoniated rice straw in the rumen and lower tract of sheep

The effect of ammoniation of rice straw (2 g NH3 per 100 g dry matter (DM)) on digestibilities and fermentation in the rumen and lower tract of sheep was investigated. Four sheep fitted with cannulae in the rumen and the duodenum were used in a 4 × 4 Latin square experiment. Diets were formulated to contain 80% rice straw and 20% supplement. These were an ammoniated rice straw diet (80% ammoniated rice straw +20% formula feed), a urea-supplemented diet (80% untreated rice straw +18.9% formula feed +1.1% urea), a soya-bean meal diet (80% untreated rice straw +11.1% formula feed +8.9% soya-bean meal) and a control diet (80% untreated rice straw +20% formula feed). There was a significant increase (P < 0.05) in apparent rumen organic matter (OM) digestibility in sheep on the ammoniated rice straw diet. Apparent total tract DM and OM digestibilities were increased (P < 0.05) in ammoniated rice straw-fed animals. The digestibility was 11% greater than that of the control. Apparent ruminal neutral detergent fibre (NDF) digestibilities were enhanced (P < 0.05) in animals on the ammoniated rice straw diet and the diet supplemented with soyabean meal. However, digestibility of the ammoniated rice straw diet was 13% greater than that of the diet supplemented with soya-bean meal. Total tract NDF digestibility in the ammoniated diet was 17% greater than that of the control. At all sampling periods, the total volatile fatty acid (VFA) was highest and pH lowest in animals on the ammoniated diet. Ruminal n-butyrate proportion was increased (P < 0.05) in sheep fed the ammoniated diet at 0 h. Total VFA and acetate proportion in rectal samples of animals fed the ammoniated rice straw diet were significantly lower (P < 0.05). However, n-butyrate and n-valerate proportions were increased (P < 0.05). Gram-negative cocci and gram-negative rods appeared to be the predominant bacteria in sheep on rice straw diets. There was a significantly higher (P < 0.05) rectal total bacterial count in sheep on the ammoniated diet. Ruminal cellulolytic bacterial count tended to increase in animals fed the ammoniated diet. Neither bacterial flow at the duodenum nor nitrogen retention differed significantly among treatments.

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.

Effects of the ionophore salinomycin on nitrogen and long-chain fatty acid profiles of digesta in the rumen and the duodenum of sheep

Modes of salinomycin action on digestion with special regard to nitrogen and long-chain fatty acid utilization were investigated in sheep equipped with ruminal and duodenal cannulae. Sheep were fed hay (300 g day−1 as fed basis) and concentrate (500 g day−1 as fed basis) with or without 32 ppm salinomycin. Digesta flow was monitored with a dual marker administration method. Dietary nitrogen flow at the duodenum was higher in salinomycin-fed sheep than in control sheep (8.0 g day−1 vs. 6.2 g day−1, P < 0.05) when the estimation was based on purine bases as a bacterial marker. Slight depression in bacterial growth with salinomycin feeding was observed, even though it was not significant. Sheep on a diet with salinomycin tended to retain more nitrogen, although this was non-significant. Duodenal digesta in sheep fed salinomycin was richer in unsaturated fatty acids, in particular oleic acid, than those in control sheep (23.6% vs. 9.1%, P < 0.01). Ruminal digesta and rumen bacterial samples showed the same trend with duodenal digesta. These results suggest that salinomycin would inhibit ruminal biohydrogenation of unsaturated fatty acids.

Variations in serum levels of insulin-like growth factor-1, growth hormone and thyroid hormones during lactation in dairy cows

1. Serum levels of insulin-like growth factor-1 (IGF-1) in dairy cows declined after parturition, remained low during the period of early lactation with peak milk production and rose gradually until the end of lactation thereafter. 2. Growth hormone (GH) levels in sera of cows changed in parallel with milk yields. 3. Serum thyroxine and triiodothyronine levels during lactation remained fairly constant.

Effect of hypophysectomy on insulin-like growth factor (IGF)-1 binding activity of serum in chickens

1. Hypophysectomy elevated IGF-1 binding activity of serum concurrently with decreases in circulating concentrations of IGF-1 and growth hormone (GH). 2. When sera pooled from each experimental group were chromatographed on a Sephadex G-200 column, IGF-1 binding activity was mainly found in 150 and 30 kilodalton (kDa) proteins. 3. Most of the serum IGF-1 were distributed among 150 kDa protein fractions. 4. Hypophysectomy elevated IGF-1 binding activity of these proteins, especially that of 30 kDa protein. 5. The binding of [125I]IGF-1 to the 30 kDa protein was inhibited by unlabelled IGF-1 but not by insulin, and saturated with a small amount of the ligand compared to the 150 kDa protein. 6. The IGF-1 binding activity of 30 kDa protein was dependent on GH status of chickens.

Physicochemical properties of a reserve polysaccharide from sheep rumen ciliates genus entodinium

Abstract 1. 1. The reserve polysaccharide of sheep rumen ciliates genus Entodinium was extracted with dimethyl sulfoxide and purified as oval granules. It amounted to 7.6% of the dry weight of the protozoa and contained trace amounts of nitrogen and phosphorus. 2. 2. This polysaccharide was stained reddish-purple by iodine and contained only glucose residues. The unit chains, of average chain length 22–25, consisted of α-1,4-linked glucose residues and were jointed by α-1,6-linkages, similar to amylopectin. 3. 3. The infrared spectrum of the protozoal polysaccharide was identical with those of potato amylopectin and shellfish glycogen. The iodized compounds of these polysaccharides had different absorption peaks. The solution of the protozoal polysaccharide showed its characteristic intrinsic viscosity.

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.