Effect Of Feed Restriction On Morphology And Function Of Myofibers And Satellite Cells In Gastrocnemius Of Broiler Chickens And Its Mechanism Involved

The present study comprises three series of experiments designed to describe the effect of feed restriction during different developmental stages on morphology and function of myofibers and satellite cells,and to elucidate the possible underlying mechanisms involved in such effect,using broiler chickens as model.(1) the body and muscle growth retardation and alterations in myofiber hypertrophy and myofiber type transformation induced by early(1～14 d) and late(50～63 d) feed restriction were compared to reveal the role of nutritional deficiency during early posthatch development in programming the morphology and function myofibres in gastrocnemius;(2) the serum levels of hormones,as well as the hepatic and muscle expression of genes involved in GH/IGF-1 and thyroid hormones targeting,protein metabolism and myofiber type transformation,as well as cell apoptosis,were determined to explore the possible mechanism underlying the effect of feed restriction on muscle growth;(3) satellite cells were isolated from muscle of control,restricted, and refed chickens and cultured in vitro to elucidate the effect of in vivo dietary intervention on proliferation and gene expression of satellite cells and their responses to different levels of thyroid hormone in vitro.1 Effect of feed restriction on morphology of gastrocnemius muscle in broiler chickens1-day-old(1 d) broiler chickens were allocated randomly into control(Con), early feed restriction(EFR) and late feed restriction(LFR) groups.Chickens of Con group were fed ad libitum throughout the experiment for 9 weeks,while chickens of EFR and LFR groups were subjected to intermittent feed restriction with feed provided on alternate days for the first and the last 2 weeks of their posthatch life, respectively.Chickens from each group were sacrificed at 14 d and 63 d.Serum thyroid hormone concentration were measured and the lateral gastroenemius muscle were taken for measuring the cross-sectional areas and densities of each type of myofibers using H.E staining,myosin ATPase staining and myofiber typing by relative quantification of mRNA expression for three different types of myosin heavy chain(MyHC):slow myofiber MyHC(SM MyHC),red fast-twitch myofiber MyHC (FRM MyHC) and white fast-twitch myofiber MyHC(FWM MyHC),in order to investigate the influence of feed restriction during different developmental stages on muscle growth.The results showed that 1) Chickens of EFR group exhibited consistently lower body weight throughout the experiment,while chickens of LFR group only showed a transient decrease in body weight on 56 d but caught up with their counterparts in Con group by 63 d.However,the lateral gastrocnemius muscle weights in both EFR and LFR groups were significantly lower compared with those in Con group of the same age.2) The serum T₃,T₄ levels were significandy lower in EFR on 14 d while only T₃ remained low by 63 d.LFR did not affect serum T₃ or T₄ levels.3) LFR group had no effect on the cross-sectional areas of the total myofibers while the cross-sectional areas of fast-twitch myofibres were both decreased in EFR group on both 14 d and 63 d.4) EFR group exhibited significantly higher SM and FRM MyHC mRNA expression but lower FWM MyHC mRNA compared with their control counterparts on 14 d.On 63 d however,a significant down-regulation was observed in EFR group for SM MyHC expression,whereas no significant differences were detected for both FRM and FWM MyHC mRNA expression between EFR and Con groups.5) Histological study by ATPase staining revealed that the percentage of slow myofibers increased on 14 d while decreased on 63 d in EFR group,and that of the fast myofibers increased significantly on 63 d.6) No alteration in percentages of slow and fast fibers was observed for LFR group compared with the control in both measurements.These results indicate that 1) the influences of early and late feed restriction on gastrocnemius muscle growth in broilers are different;2) early feed restriction may delay the conversion from slow-tonic myofiber to fast-twitch myofiber and inhibit myofibers' hypertrophy,resulting in retarded muscle growth that may account for the decreased body weight,demonstrating more profound and lasting effects on broiler growth;3) thyroid hormones might participate in mediating such programming effect.2 Mechanisms involved in the effect of feed restriction on gastrocnemius muscle growth in broiler chickensIn order to explore the mechanisms underlying the effect of feed restriction on gastrocnemius muscle growth,pituitary,liver and the lateral gastrocnemius muscle were taken and abundances of mRNA for genes involved in GH/IGF-1 and thyroid hormones targeting(growth hormone(GH),growth hormone receptor(GHR), insulin-like growth factor-â… (IGF-â… ),type-â… IGF receptor(IGFR-â… ),thyroid hormone receptorα(TRα) and thyroid hormone receptorβ(TRβ)),protein metabolism and myofiber typing(peroxisome proliferator-activated receptor-γcoactivator-1(PGC-1), calcineurin,nuclear factor of activated T cells(NFAT) and calpain3)),cell proliferation or apoptosis(Pax7,B cell lympfoma/leukemia 2(Bcl-2),Bcl-2 associated X protein(Bax)) were determined by RT-PCR with 18S rRNA as an internal-standard. In addition,genomic DNA was isolated from 14 d lateral gastrocnemius and subjected to electrophoresis for the evaluation of apoptosis(DNA Ladder),and fluorescent immunostaining of PCNA were carded out for morphological identification of proliferating satellite cells.The results showed that 1) chickens of EFR group exhibited significantly lower GHR mRNA expression in liver,significantly lower IGF-â… but higher IGFR-â… mRNA expression in gastrocnemius muscle at 14 d,and significantly higher TRαin liver, significantly higher GHR and IGFR-â… mRNA expression in gastrocnemius muscle at 63 d.2) Bax mRNA expression and Bax mRNA/Bcl-2 mRNA were significantly higher in EFR group at 14 d;and 3) the density of PCNA positive cells was significanfly lower in EFR group.4) LFR group did not show significant alterations in mRNA of any genes investigated except higher GHR mRNA expression in liver at 63 d.5) No changes in PGC-1,Calcineurin,NFAT,Calpain3 mRNA expression were detected in group ERF and LRF on either ages.These results indicate that 1) early feed restriction exerts metabolic programming effect involving changes of mRNA expression for the target genes of GH/IGF-1 and thyroid hormone,while late feed restriction has only transient and minor influence. Distinct mechanisms seem to apply for the effects of feed restriction during different periods of life.2) Early feed restriction inhibited proliferation of satellite cells and stimulated apoptosis in gastrocnemius muscle of broiler chickens at 14 d,indicating that satellite cells may be the target for this programming effect of early nutritional deficiency on gastrocnemius muscle growth.3 Effect of early feed restriction and refeeding on satellite cells activity and endocrine-related genes expression on satellite cells in gastrocnemius muscle of Broiler Chickens To elucidate whether the programming effect of early feed restriction on myofiber development is achieved via targeting on satellite cells,1 d broiler chickens were allocated randomly into control(14 days feed ad libitum)(Con),14 days feed restriction(14FR) and 12 days feed restriction folllowed by 2 days refeeding (12FR2R) groups.14FR and 12FR2R groups were subjected to intermittent feed restriction with feed provided on alternate days for the first 14 days and 12 days, respectively.Satellite cells were isolated from the lateral gastrocnemius of each group at 14 d,RNA of isolated satellite cells were extracted and abundances of GHR,IGF-â… , IGFR-â… ,TRα,Bcl-2 and Bax mRNA were determined by real-time quantitative PCR usingβ-actin as an internal-standard.Morphological changes of isolated satellite cells from each group were observed for 3 days in culture and MTT assay was performed to assess the cell viability.T₃ was added to the culture of satellite cells from each group at the level of 12 ng/mL(physiological level),3 ng/mL and 6ng/mL to investigate the changes of satellite cells viability exposed to different concentrations of T₃.The results showed that 1) IGF-â… and GHR mRNA expression were decreased in 14FR group,while IGFR-â… mRNA expression increased.2) The expression of TRαmRNA was significantly increased in 14FR group compared to Con and 12FR2R groups,and there was no significant difference between other two groups.3) Bax mRNA/Bcl-2 mRNA was significantly lower in 12FR2R group than in 14FR group. 4) Satellite cells of Con group exhibited highest viability,while that of 14FR group showed the lowest viability with that of 12FR2R group in between.5) Satellite cells of 14FR group showed no response to any concentrations of T₃,while cells in Con group increased viability when treated with 12 ng/mL T₃ for 48 h but not 72 h. 12FR2R group seems to respond to lower T₃ at 6 ng/mL for 48 h and 72 h.These results indicate that 1) early feed restriction may inhibit the proliferation and stimulate apoptosis of satellite ceils,and refeeding may partly restore it.2) Genes involved in GH/IGF-1 and thyroid hormone targeting may mediate this effect,and 3) early feed restriction may increase the sensitivity or reception of satellite cells to T₃ after refeeding,contributing to compensatory muscle growth.