Genetic variation in resistance of turkeys to experimental infection with Newcastle disease virus.

Seven hundred eighty male and female turkeys representing four genetic lines were challenged in four experiments with the Texas GB strain of Newcastle disease virus (NDV). The lines of turkeys included two randombred control lines (RBC1 and RBC2), a subline (E) of RBC1 selected for increased egg production, and a subline (F) of RBC2 selected for increased 16-week body weight. Mortality in turkeys of subline F (32.5%) was significantly higher than that in turkeys of line RBC2 (15.8%), subline E (17.5%), and line RBC1 (18.4%). At the end of each experiment, surviving birds were tested for antibody to NDV using the enzyme-linked immunosorbent assay (ELISA) and hemagglutination-inhibition (HI) test. Turkeys of subline E and line RBC1 had significantly lower ELISA antibody titers than those of subline F and line RBC2. Subline F had the highest HI antibody titers, followed in decreasing order by lines RBC2 and RBC1 and subline E. No apparent correlation was found between antibody response and mortality after NDV challenge.     

成纤维细胞生长因子21及其受体1、2在小鼠毛囊形成期的定位和表达

本试验旨在通过研究成纤维细胞生长因子21(fibroblast growth factor 21,FGF21)及其受体FGFR1、FGFR2在胚胎小鼠毛囊形成阶段中的定位与表达,从而探究其在小鼠毛囊形成中的作用。试验采用免疫组织化学、实时荧光定量PCR及Western blotting方法研究FGF21、FGFR1、FGFR2蛋白和mRNA在胚胎期13～18d(E13～E18)小鼠皮肤中的表达。结果显示:(1)FGF21蛋白在E13主要表达于表层细胞、基底层细胞及结缔组织中,E14表达于表层细胞和基板,E15在毛芽中有微量表达,E16、E17表达于毛钉中,E18主要表达于未成熟毛囊细胞;FGFR1和FGFR2蛋白在E13～E18于表层细胞、基底层、基板、毛芽、毛钉、未成熟毛囊和结缔组织中均有表达。(2)实时荧光定量PCR及Western blotting结果显示:FGF21mRNA和蛋白在E14相对表达量最高;FGFR1mRNA及蛋白在E16～E17相对表达量较高;FGFR2mRNA及蛋白的相对表达量在E13至E18均呈上升趋势,在E18相对表达量最高。结果提示:在小鼠胚胎期毛囊形成和发育过程中,FGF21可能在诱导毛囊启动过程中发挥一定的作用;FGFR1可能促进毛钉形成;FGFR2可能在毛囊形成和成熟中发挥重要作用。     

The distribution and immunolocalization of fibroblast growth factors (FGFs) in the rat oviduct during early pregnancy and the post-partum period

The mammalian oviduct provides a favourable environment for several reproductive processes, including ovum transport, sperm capacitation, fertilization and pre-implantation embryonic development. This environment is regulated by cyclic ovarian steroids, that is oestrogen, and growth factors. Fibroblast growth factors (FGFs) regulate the differentiation and growth of various cell types in the female genital tract. This study aimed to determine the localization of FGF1, FGF2, FGF receptor 1 (FGFR1) and 2 (FGFR2) in the rat oviduct, by immunohistochemistry, on day 5 of pregnancy and post-partum days 1, 3 and 5, and to demonstrate the possible functions of these proteins during early pregnancy and the post-partum period. On all examination days, cytoplasmic and nuclear FGF1 immunoreactivity was detected in the epithelium lining the infundibulum, ampulla and isthmus of the oviduct. Immunoreactivity was much stronger in the basal bodies of the cilia on the epithelium lining the infundibulum and ampulla. FGF1 immunoreactivity was also detected in stromal cells, myocytes and endothelial cells. Cytoplasmic FGF2 immunoreactivity was observed in the tunica muscularis, vascular myocytes and endothelial cells. While strong cytoplasmic FGF2 immunoreactions were observed in the stromal cells of the lamina propria, the luminal epithelium, some stromal cells and smooth muscle cells displayed a rather weak FGFR1 and FGFR2 immunoreactivity. Immunoreaction intensity did not differ between the periods examined. This study shows that FGF1, FGF2, FGFR1 and FGFR2 are produced by rat oviduct cells during pregnancy and the post-partum period, and reproductive physiology is regulated not only by hormonal mechanisms, but also by growth factors.     

Messenger ribonucleic acid expression of the MyoD gene family in muscle tissue at slaughter in relation to selection for porcine growth rate

Livestock meat production capacity is related to muscle fiber numbers and growth. Muscle fibers develop during early embryonic development from proliferating and differentiating myoblasts. Post-natal muscle growth requires satellite cell proliferation and differentiation. Myoblast and satellite cell proliferation and differentiation is regulated by the genes of the MyoD gene family (myogenin, myf-5, myf-6, and MyoD1). Our aim was to study the mRNA expression of these genes in postnatal muscle tissue in relation to porcine selection for growth rate or leanness. Five boars from a line selected for fast growth (F-line) and five boars from a line selected against backfat thickness (L-line) were slaughtered, and biopsies were taken from 12 muscles. Between-line effects, within-line effects in relation to the performance of the pigs, and muscle-specific effects were studied. Comparing the F-line with the L-line revealed significantly greater myogenin, myf-5, and MyoD1 mRNA expression in some muscles of the F-line. The expression of myf-6 showed a tendency for the opposite effect in some muscles. Muscles were ordered by their muscle-specific growth rate (b-value). Within-line evaluation of the data revealed a systematic muscle effect for the myf-6 expression level in the F-line because higher b-values correlated with increased myf-6 expression level. Backfat thickness was negatively related to myogenin expression in the F-line. A relationship was found between myogenin:MyoD1 mRNA expression ratio and meat color/muscle fiber type composition in the L-line. Furthermore, the myogenin:MyoD1 ratio was greater in muscles from F-line boars than in muscles from L-line boars, which relates to the difference between the lines in muscle fiber type. We conclude that the mRNA levels of the MyoD genes in porcine muscle tissue at slaughter showed different relationships to selection for growth rate when evaluated between selection lines and within selection lines.     

In vitro model of equine muscle regeneration

Equine satellite cells are responsible for muscle healing and regeneration in the mature horse. We describe the in vitro cell culture conditions required for clonal populations of equine satellite cells to undergo both proliferation and differentiation. Our hypothesis is that these in vitro conditions model regeneration of muscle and can be used to evaluate potential therapeutics. In this study, 2 areas of satellite cell response were tested: proliferation of clones induced by growth factors, and fusion induced by culture conditions. Equine satellite cell clones showed differences in their response to growth factors as well as accumulation of cellular protein concentrations. Equine satellite cells proliferate in response to both human and bovine FGF. IGF-1, a powerful mitogen of other satellite cell culture systems, was not as effective for inducing equine satellite cell proliferation. Protein concentrations were also measured in satellite cell cultures. Clones differed in cellular protein produced depending on growth conditions. Conditions inducing differentiation into myotubes was also determined for a 96 well assay and can be used to study the final stage of functioning muscle production. This in vitro model is the first step in identifying potential therapeutics to speed wound healing and promote muscle regeneration in horses.     

Genetic variation in resistance of turkeys to experimental challenge with Pasteurella multocida.

Six hundred fifty-five male and female turkeys representing four genetic lines were challenged in 10 experiments over a 3-year period with a field isolate of Pasteurella multocida. Poults were challenged at 45 days of age with 1 ml of an inoculum containing 1.2 x 10(7) bacteria per ml. The lines of turkeys included two randombred control lines (RBC1 and RBC2), a subline (E) of RBC1 selected for increased egg production, and a subline (F) of RBC2 selected for increased 16-week body weight. The number of days from exposure to severe clinical signs or death for Line F (5.8 days) differed significantly from that of Line E (8.2 days), Line RBC1 (8.0 days), and Line RBC2 (8.2 days). There were no significant differences due to sex of poult for number of days from exposure to severe clinical signs or death. Overall mortality observed was 51.2%. Mortality was highest for Line F (72.1%) and differed significantly from that of the other lines. Mortality among male poults did not differ significantly from mortality among female poults.     

Expression of fibroblast growth factor 1 (FGF1) and FGF7 in mature follicles during the periovulatory period after GnRH in the cow

The aim of this study was to evaluate the expression pattern of mRNA for fibroblast growth factor 1 (FGF1), FGF7, and their receptor variants (FGFR2IIIb) in time-defined follicle classes before LH surge, between LH surge and ovulation, and in the early corpus luteum (CL) in the cow. The ovaries were collected by transvaginal ovariectomy (n=5 cows/group), and the follicles (n=5, one follicle/cow) were classified into the following groups: before GnRH administration (before LH surge); 3-5 h after GnRH (during LH surge); 10 h after GnRH; 20 h after GnRH; 25 h after GnRH (periovulation), and early CL (Days 2-3). The mRNA expression was analyzed by quantitative real-time PCR (RotorGene 3000). The mRNA expression of FGF1 showed no significant differences in the follicle groups examined, but increased significantly at the early CL phase. A transient increase in FGF7 mRNA expression was observed 3-5 h after GnRH and again in the early CL phase. In contrast, the expression of FGFR2IIIb was constant throughout the period from the final growth of the follicle to early CL formation. The results of this study suggest that FGF1 and FGF7 may be involved differently in the process of follicle maturation and CL formation, which is strongly dependent on angiogenesis.     

Genetic variation in response of turkeys to experimental infection with Bordetella avium

One hundred ninety-six male and female turkeys representing two genetic lines were experimentally infected with Bordetella avium. The lines of turkeys included a randombred control line (RBC2) and a subline (F) of RBC2 selected for increased 16-wk body weight. No difference was found between lines RBC2 and F in the number of days to onset of clinical signs, and no mortality due to B. avium infection was observed in either line. Interestingly, however, a significant depression (12%) occurred in body weight of F line poults infected with B. avium, but no significant depression occurred in body weight of RBC2 poults.     

Extracellular matrix proteoglycan decorin-mediated myogenic satellite cell responsiveness to transforming growth factor-beta1 during cell proliferation and differentiation Decorin and transforming growth factor-beta1 in satellite cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation. Decorin, a small proteoglycan in the extracellular matrix, binds to TGF-beta1 and modulates the activity of TGF-beta1 during muscle cell growth and development. However, its interaction with TGF-beta1 and involvement in myogenesis is not well characterized. In the present study, chicken myogenic satellite cells, myogenic precursors for muscle growth and repair, were isolated from the pectoralis major muscle and used to investigate the biological function of TGF-beta1 and decorin during myogenesis. The over-expression of decorin in satellite cells significantly increased cell proliferation, compared to the control cells. Consistent with this result, reducing decorin expression decreased cell proliferation, which suggests a decorin-mediated mechanism is involved in the regulation of myogenic satellite cell proliferation. Satellite cells over-expressing decorin were less sensitive to TGF-beta1 during proliferation, which indicates that decorin may sequester TGF-beta1 leading to increased proliferation. During satellite cell differentiation, the over-expression of decorin induced differentiation by increasing the muscle specific creatine kinase concentration. However, the addition of TGF-beta1 diminished decorin-mediated cell responsiveness to TGF-beta1 during differentiation. Taken together, these results suggest that decorin induces myogenic satellite cell proliferation and differentiation by regulating cellular responsiveness to TGF-beta1. An alternative TGF-beta1-independent pathway may be involved in the regulation of satellite cells by decorin.     

过表达MyBPC1对牛骨骼肌卫星细胞增殖分化的影响

旨在探究肌球蛋白结合蛋白C1（myosin binding protein C1,MyBPC1）对牛骨骼肌卫星细胞增殖与成肌分化的影响,为进一步研究MyBPC1在细胞分化和肌肉发育过程中的调控作用提供依据。本研究利用西门塔尔胎牛原代牛骨骼肌卫星细胞体外诱导成肌分化模型模拟牛骨骼肌的生长发育过程。采用qRT-PCR和Western blot检测MyBPC1的细胞时序表达谱。试验分为两组。在RNA水平每组4个重复,每个重复20 μL;在蛋白水平每组3个重复,每个重复15 μg。采用qRT-PCR和Western blot检测牛骨骼肌卫星细胞转染MyBPC1的过表达效果,并进一步检测细胞增殖期标志因子Pax7、Ki67以及细胞分化期标志因子MyHC、MyOG的表达变化情况,观察牛骨骼肌卫星细胞肌管形成状态。结果,MyBPC1在牛骨骼肌卫星细胞分化前后表达水平存在极显著差异,牛骨骼肌卫星细胞诱导分化后MyBPC1的mRNA和蛋白表达量均极显著高于增殖期（P<0.01）。过表达MyBPC1后,细胞分化形成的肌管数量明显多于对照组,增殖标志因子Pax7的mRNA水平和蛋白表达水平无显著差异,分化标志因子MyHC的mRNA水平和蛋白表达水平极显著高于对照组（P<0.01）。过表达MyBPC1可以促进牛骨骼肌卫星细胞体外成肌分化,为进一步开展MyBPC1对牛骨骼肌卫星细胞的调控机制奠定基础。     

Basic FGF promotes proliferation of ovarian granulosa cells in the laying chickens via FGFR1 and PKC pathway

The proliferating effect of basic fibroblast growth factor (bFGF) on granulosa cells from ovarian pre-hierarchical follicles was evaluated in the laying chickens. Expression of bFGF receptor 1 (FGFR1) from small yellow follicles was determined by immunohistochemistry and RT-PCR. The FGFR1 protein and mRNA were intensively expressed in the granulosa layer. After 8- to 24-h treatment with bFGF (0.1-100 ng/ml), the proliferation of cultured granulosa cells was remarkably enhanced in a dose- and time-dependent manner. The FGFR1 antagonist SU5402 inhibited bFGF-induced cell proliferation. This stimulating effect was further confirmed by 5-bromo-2-deoxyuridine incorporation and terminal transferase dUTP nick end-labelling assay. Immunocytochemistry of protein kinases A (PKA) and C (PKC) showed that the pro-proliferation action of bFGF predominantly activated PKC expression. Meanwhile, the bFGF-induced cell proliferation was significantly promoted by PKC activator PMA and inhibited by PKC inhibitor H(7) (p < 0.05). In addition, the bFGF-elicited cell proliferation was accompanied with increased mRNA expression of the cell cycle-regulating genes including cyclins D1 and E1, cyclin-dependent kinases 2 and 6. In conclusion, bFGF promoted the proliferation of ovarian granulosa cells through binding with FGFR1 and involving PKC pathway in the pre-hierarchical follicles of the laying chickens.     

Effect of insulin and linoleic acid on satellite cell differentiation

Differentiation of rat skeletal muscle satellite cells was studied in vitro. Linoleic acid and insulin, two unrelated compounds that reportedly stimulate differentiation of other types of myogenic cells, were used to examine the regulation of differentiation in satellite cell cultures. As in cultures of chick embryo muscle cells, linoleic acid stimulated fusion but only at low serum concentrations or in defined medium without fibroblast growth factor (FGF). The effects of insulin on differentiation were quite variable, however; at very low cell densities no stimulatory effect was observed. In intermediate and, to a lesser extent, high density satellite cell cultures, the addition of insulin at concentrations between .01 and 1.0 microM stimulated satellite cell fusion. Whenever increases in fusion were observed, however, a parallel increase in cell number was also found. A closer examination of the relationship between differentiation and the presence or absence of mitogenic agents in the medium suggested that a mitogenic signal and the resultant proliferation of cells prevented differentiation. Subsequent experiments indicated that fusion could be induced by lower serum concentration or by removal of FGF, as long as linoleic acid was present in the medium. Therefore, proliferation and differentiation appear to be antagonistic processes in cultured satellite cells. If the rate of proliferation is depressed, either by mitogen removal or by increasing cell density, differentiation is favored. Differentiation can, therefore, be regulated and applied to in vitro studies of satellite cell activity.     

Circulating IGF-I in plasma of growing male and female turkeys of medium and heavy weight lines

Plasma concentrations of insulin-like growth factor-I (IGF-I) were determined in male and female turkeys from a medium weight (RBC2) and a related heavy weight line (F) from 1 to 28 wks of age. At hatch, the concentrations of IGF-I were relatively low and not different between lines or sexes. During the neonatal period (1 to 7 wks), the concentrations of IGF-I increased and were higher in the faster growing F line and in males. During the juvenile period (8 to 15 wks) the concentrations of IGF-I were higher in males but not different between lines. During the preadolescent period (16 to 21 wks), the concentrations of IGF-I were higher in males but was not different between lines in males while the females of the RBC2 line had higher concentrations than females of the F line. During the adolescent period (22 to 28 wks) the concentrations of IGF-I were higher in males but was not different between lines in males while the females of the RBC2 line had higher concentrations than females of the F line. A phenotypic correlation (+.25) between plasma IGF-I and growth rate was present after statistical absorption of model effects during the neonatal period but not at the later ages. We conclude that IGF-I concentration was positively correlated with growth rate during the neonatal period, but that this relationship changed during the preadolescent and adolescent periods so that IGF-I concentrations were not related to growth rate in males but were negatively related to growth rate in females.     

干扰MSTN对牛骨骼肌卫星细胞增殖分化的影响

为研究肌肉生长抑制素（myostatin,MSTN）对牛骨骼肌卫星细胞增殖与成肌分化的影响,本试验以牛骨骼肌卫星细胞体外诱导成肌分化模型为对象,以前期设计合成3个干扰RNA（si-MSTN-1、si-MSTN-2、si-MSTN-3）并对其进行干扰效果筛选为基础,将干扰效果极显著的si-MSTN-2（si-MSTN）转染牛骨骼肌卫星细胞,通过EdU染色法检测干扰MSTN对牛骨骼肌卫星细胞增殖的影响;进一步对干扰MSTN的牛骨骼肌卫星细胞进行体外成肌诱导分化,通过肌管形成状态和分化标志因子综合分析干扰MSTN对牛骨骼肌卫星细胞分化的影响：首先通过显微镜观察牛骨骼肌卫星细胞分化时期的肌管形成状态,然后利用实时荧光定量PCR和Western blotting技术检测牛骨骼肌卫星细胞分化标志因子MyoG和MyHC在mRNA和蛋白水平的表达情况。结果显示,干扰MSTN后,牛骨骼肌卫星细胞中EdU阳性细胞率极显著增加（P < 0.01）,说明下调MSTN表达极显著促进了牛骨骼肌卫星细胞的增殖;牛骨骼肌卫星细胞诱导分化后形成的肌管数量和直径均呈现增大趋势,牛骨骼肌卫星细胞成肌分化标志因子MyHC在mRNA和蛋白水平的表达均极显著高于对照组（P < 0.01）,说明下调MSTN表达能够促进牛骨骼肌卫星细胞的成肌分化过程。本研究结果表明,干扰MSTN可以显著促进牛骨骼肌卫星细胞的增殖及成肌分化过程。本试验结果为进一步开展MSTN对牛骨骼肌卫星细胞成肌分化的调控机制研究提供了参考。     

Meat Science and Muscle Biology Symposium: extracellular matrix regulation of skeletal muscle formation

Skeletal muscle development and growth is a complex process that involves the interaction of muscle cells with their extracellular environment. Because muscle development involves the interaction of the cell surface and extracellular matrix molecules, research focus has been placed on the proteoglycans. Proteoglycans are macromolecules containing a central core protein with attached carbohydrates, called glycosaminoglycans, that are located at both the cell surface and the extracellular matrix. Research focus has been placed on understanding the mechanisms of the membrane-associated heparan sulfate proteoglycans, syndecan-4 and glypican-1, which are both capable of regulating cellular responsiveness to fibroblast growth factor 2 (FGF2). Fibroblast growth factor 2 is a potent stimulator of muscle cell proliferation and a strong inhibitor of differentiation. Studies on syndecan-4 and glypican-1 show that these proteoglycans differentially regulate muscle cell proliferation, differentiation, and cellular responsiveness to FGF2 with syndecan-4 predominantly modulating muscle cell proliferation and glypican-1 modulating differentiation. Site-directed mutagenesis approaches were used to define the effect of the syndecan-4 and glypican-1 covalently attached side chains on their activity. In general, a functional association was found between the glycosaminoglycan and N-glycosylated chains attached to the central core proteins of syndecan-4 and glypican-1 affecting their regulation of muscle cell proliferation, differentiation, and FGF2 responsiveness. Current research efforts are directed at identifying the cellular signaling pathways modulated by syndecan-4 and glypican-1.     

Changes in the Expression Patterns of the Genes Involved in the Segregation and Function of Inner Cell Mass and Trophectoderm Lineages During Porcine Preimplantation Development

In mouse embryos, segregation of the inner cell mass (ICM) and trophectoderm (TE) lineages is regulated by genes, such as OCT-4, CDX2 and TEAD4. However, the molecular mechanisms that regulate the segregation of the ICM and TE lineages in porcine embryos remain unknown. To obtain insights regarding the segregation of the ICM and TE lineages in porcine embryos, we examined the mRNA expression patterns of candidate genes, OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc, in blastocyst and elongated stage embryos. In blastocyst embryos, the expression levels of OCT-4, FGF4 and FGFR1-IIIc were significantly higher in the ICM than in the TE, while the CDX2, TEAD4 and GATA3 levels did not differ between the ICM and TE. The expression ratio of CDX2 to OCT-4 (CDX2/OCT-4) also did not differ between the ICM and TE at the blastocyst stage. In elongated embryos, OCT-4, NANOG, FGF4 and FGFR1-IIIc were abundantly expressed in the embryo disc (ED; ICM lineage), but their expression levels were very low in the TE. In contrast, the CDX2, TEAD4 and GATA3 levels were significantly higher in the TE than in the ED. In addition, the CDX2/OCT-4 ratio was markedly higher in the TE than in the ED. We demonstrated that differences in the expression levels of OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc genes between ICM and TE lineages cells become more clear during development from porcine blastocyst to elongated embryos, which indicates the possibility that in porcine embryos, functions of ICM and TE lineage cells depend on these gene expressions proceed as transition from blastocyst to elongated stage.     

Basic principles of muscle development and growth in meat-producing mammals as affected by the insulin-like growth factor (IGF) system

This presentation aims to describe how the basic events in prenatal muscle development and postnatal muscle growth are controlled by the insulin-like growth factor system (IGF). The prenatal events (myogenesis) cover the rate of proliferation, the rate and extent of fusion, and the differentiation of three myoblast populations, giving rise to primary fibers, secondary fibers, and a satellite cell population, respectively. The number of muscle fibers, a key determinant of the postnatal growth rate, is fixed late in gestation. The postnatal events contributing to myofiber hypertrophy comprise satellite cell proliferation and differentiation, and protein turnover. Muscle cell cultures produce IGFs and IGF binding proteins (IGFBPs) in various degrees depending on the origin (species, muscle type) and state of development of these cells, suggesting an autocrine/paracrine mode of action of IGF-related factors. In vivo studies and results based on cell lines or primary cell cultures show that IGF-I and IGF-II stimulate both proliferation and differentiation of myoblasts and satellite cells in a time and concentration-dependent way, via interaction with type I IGF receptors. However, IGF binding proteins (IGFBP) may either inhibit or potentiate the stimulating effects of IGFs on proliferation or differentiation. During postnatal growth in vivo or in fully differentiated muscle cells in culture, IGF-I stimulates the rate of protein synthesis and inhibits the rate of protein degradation, thereby enhancing myofiber hypertrophy. The possible roles and actions of the IGF system in regulating and determining muscle growth as affected by developmental stage and age, muscle type, feeding levels, treatment with growth hormone and selection for growth performance are discussed.