Fumio Ebara

Cellular conditions for intramuscular fat deposition in Japanese Black and Holstein steers

The experiment was conducted to study the development of intramuscular fat in Japanese Black (JB) compared to Holstein (HS) steers and to find breed differences for fat depot development and distribution in the carcass under equal feeding conditions. Additional to slaughter samples, biopsy samples of longissimus muscle (LM) and subcutaneous fat, taken at 10, 14, 18, and 22 months of age, were used for histological and molecular investigations. Japanese Black steers stored about 14% more fat in the LM (P = 0.001), resulting in larger marbling flecks (P < 0.001). Muscle fibers and intramuscular adipocytes in both breeds responded to the high energy feeding with significant enlargement, which was faster in JB. Histograms of intramuscular adipocytes size showed a shift toward larger cells during growth, but also the abundance of small, developing adipocytes. This development was accompanied by a correlated up-regulation of adipogenic genes until 22 months of age.

Technical note: Determination of cell-specific gene expression in bovine skeletal muscle tissue using laser microdissection and reverse-transcription quantitative polymerase chain reaction1

Skeletal muscle is a very heterogeneous tissue consisting of diverse cell types with specific transcription profiles. Therefore, the measured mRNA abundance of a certain cell type marker is influenced by the transcriptional activity as well as by the usually unknown number of contributing cells in the sample. In studies on the transcriptional activity of adipogenic genes, as indicators for the development of intramuscular adipocytes, an altered number of adipocytes or respective progenitor cells can mask changes in transcriptional activity. To overcome this problem, we started to use laser microdissection to isolate RNA of adipocytes and muscle fibers separately for downstream analysis. Even muscle fiber types can be collected and analyzed separately. Laser microdissection in combination with biopsy techniques enables gene expression studies of particular cell types during the life cycle of an animal. First experiences using laser microdissection for adipogenic gene expression studies in bovine skeletal muscle are described, and the influence of sample preparation and future challenges are discussed.

Effect of nutrient intake on intramuscular glucose metabolism during the early growth stage in cross-bred steers (Japanese Black male × Holstein female).

The objective was to investigate the impact of nutrient intake during the early growth period on the expression of glucose metabolism-related genes in skeletal muscle of cross-bred cattle. From 1.5 to 5 months of age, group H (n=7) animals were intensively fed a high-protein and low-fat milk replacer [crude protein (CP) 28%; ether extracts (EE) 18%; max: 2.0 kg, 12 l/day], and group R (n=7) animals were fed a restricted amount of normal milk replacer (CP 25%; EE 23%; max 0.5 kg, 4 l/day). From 6 to 10 months of age, group H cattle were fed a high-nutrition total mixed ration mainly prepared from grain feed, and group R cattle were fed only roughage. Blood samples were taken from each animal at three biopsy times (1.5, 5 and 10 months of age), and the blood plasma concentration of glucose and insulin was analysed. In glucose concentration, there were no significant differences; however, the concentrations of insulin were higher in group H than in group R at 5 and 10 months of age. Muscle samples were taken by biopsy from longissimus thoracis muscle (LT) at 1.5, 5 and 10 months of age. We analysed mRNA expression levels using the quantitative real-time polymerase chain reaction (PCR) assay for glucose transporters (GLUT1 and GLUT4), insulin receptor, phosphatidylinositol 3-kinase (PI-3K), protein kinase B (PKB, also known as Akt), hexokinase 1 (HK1) and tumour necrosis factor alpha (TNFα). Although no differences were detected at 1.5 and 5 months of age, at 10 months of age, GLUT1, HK1 and TNFα mRNA expression levels were significantly higher in group H than in group R. These results suggested Glut1 that affects insulin-independently mediated glucose uptake was more responsive to improved nutrition during early growth stage than GLUT4 that insulin-dependently mediated glucose uptake in LT of cattle.

Introduction of foreign gene into the gonads via primordial germ cells originated from germinal crescent of chicken embryos.

Abstract Ebara, F. and Fujihara, N. 2000. Introduction of foreign gene into the gonads via primordial germ cells originated from germinal crescent of chicken embryos. J. Appl. Anim. Res., 18: 33–40. An exogenous DNA (MiwZ, containing lacZ gene) introduced into germinal crescent region (GCR) of the early chicken embryos, from which primordial germ cells (PGCs) originated, was confirmed to be successfully transferred to the gonads of embryos via the PGCs. The DNA was injected into the GCR at stages 3–5 or 9.11. Manipulated eggs were incubated until hatching, and the injected DNA was detected by the methods of X-gal staining and polymerase chain reaction (PCR) analysis. No significant differences (P>0.05) were observed in the level of expression of injected DNA between the two stages of embryonic development. While obvious difference (P<0.05) was found in the expression rates between the right and left gonads, showing higher percentage of DNA expression in the left gonads than that in the right ones, regardless of the embryonic sex. Thus, the exogenous gene introduced into the GCR successfully migrated to the gonads and remained for long time in the gonads after hatching.

In vivo gene transfer into chicken embryos via primordial germ cells using green fluorescent protein hybrid DNA

injected through it into the subgerminal cavity of the blastoderm. Hatchability after injection according to method A ranged from 4·5% to 16·0% of the treated embryos and 9·8% on average. This is similar to the data of other authors who used the same method (Petitte et al., 1990; Thoraval et al., 1994). Higher hatchability was obtained by Naito et al. (1991) and Kino et al. (1997), i.e. 8·6%–40·3% and 25·6%– 40·7%, respectively. However these authors used another method of incubation and transferred 4-day-old embryos to a large recipient surrogate eggshell. In our study the use of method B resulted in markedly better (from 25·0% to 50·0%) hatchability compared with method A. The mean hatchability (41·0%) calculated from six replications was found to be close to the best results reported by Naito et al. (1991) and Kino et al. (1997). It should be pointed out that method B is considerably easier than the transfer of embryos to surrogate eggshells that was used by Naito et al. (1991) and Kino et al. (1997). The influence of the origin of donor BCs on the hatching rate of the chicken chimeras was also investigated. Hatchability was variable and depended on the origin of BC donor embryos. The best result (56·1%) was obtained after injection of cells originating from the Greenleg Partridge breed of chicken. This native Polish breed of layers has been kept under closed selection for 50 generations. It was interesting to note that good hatchability was accompanied by very high percentage of somatic chimeras. Out of 23 hatched chicks, as many as 20, i.e. 86·9%, demonstrated black spots in the plumage, which is a phenotypic marker of chimerism. The experimental findings demonstrate that modification of the injection technique and an adequate choice of recipient and donor of BCs can achieve a substantial improvement in production of somatic chimeras.