The Regulating Effects Of Creatine On Skeletal Muscle Growth And Development Of Broiler Chickens

In broilers,skeletal muscle is one of the main organs of the body,which is composed of multi-nucleated myofibers.The growth of skeletal muscle depends on the increase in the number of myofibers and the volume of existing myofibers.However,there was no change in the number of myofibers after hatching,and the growth of skeletal muscle mainly depend on the increase of myofiber volume at post-hatch.Therefore,it’s vital important to increase the number of myofibers by regulating the proliferative ability during embryonic period for the growth performance of broilers at post-hatch.The high concnetration of creatine(Cr)in skeletal muscle,and skeletal muscle accounting for more than 90%of the total Cr content in the body.As the rapid energy buffer,Cr plays an important role in maintaining muscle energy homeostasis.Meanwhile,as a signal molecule,Cr regulate the mammalian target protein of rapamycin(m TOR)pathway,and promote the process of myogenesis.The guanidinoacetic acid(GAA)is a precursor of Cr synthesized in the body.Creatine monohydrate(CMH)as the monohydrate of Cr and the main additive form of Cr.In this study,the supplemented with GAA in diets were firstly used to determine the effects of Cr precursor on growth performances of broilers.Then,the regulatory mechanism of CMH on skeletal muscle growth and development of broilers during embryonic period were further determined by in ovo injection experiment and in vitro trials with chicken primary myoblasts.Meanwhile,the feeding experiment was conducted to determine the effects of in ovo injection of CMH on the growth performance of broilers at post-hatch.Finally,the regulating effects of CMH on protein metabolism and mitochondrial function of chicken myotubes under different nutritional and physiological conditions cellular models(normal,starved and CORT)were further determined.This research provides a theoretical basis for further exploring the growth potential of broilers and improving the feed efficiency of broilers.The main contents are as follows:1.Effects of dietary supplementation with GAA on growth performances of broilersA total of 360 one-day-old male Arbor Acres broiler chickens were randomly allotted into 3 groups with 6 replicates per group.The experimental treatments as follows:control group,A basal diet without GAA;Low dose GAA(L-GAA),0.3 g/kg and 0.4 g/kg GAA were supplemented at the starter(1-21 d)and later stage(22-42 d)respectively;High dose GAA(H-GAA),0.6 g/kg and 0.8 g/kg GAA were supplemented at the starter and later stage respectively.The experiment lasted for 42 days.The results showed as follows:during 1-21days,dietary treatment had no detectable influences(P>0.05)on average BW,ADG,ADFI and F:G.During 22-42 days,compared with L-GAA and control diets,H-GAA treatment significantly increased the average BW and breast muscle ratio at 42 d(P<0.05),and decreased the F:G and abdominal fat ratio(P<0.05).During 1-42 days,H-GAA treatment significantly increased the average BW(P<0.05)than control and L-GAA groups,and decreased the F:G than control(P<0.05).Taken together,these results indicating that H-GAA treatment(starter:0.6 g/kg;later:0.8 g/kg)improved the production performance and the muscle production of broilers.2.Effects of in ovo injection of CMH on skeletal muscle development at embryonic periodSix AA embryos(broilers)with similar body weight were obtained at E11,E13,E15,E17,E19 and D1(about 2 h after hatching and the feathers dried)respectively.Pectoralis major(PM)and biceps femoris(BF)samples were collected to analysis the m RNA and protein expression of these genes related to myogenesis.The results showed that the m RNA and protein expression of PAX3,PAX7,PCNA and Myf5 were decreased with the increase of embryonic age(P<0.001),indicating that the high capacity of myogenesis at early stage,and the nutritional intervention at this period maybe had the better regulatory effects on myogenesis.The research was further determine the appropriate dosage and injection time.The in ovo injection experiment was conducted at E4,E6,E8,E10 and E12,respectively,and the treatments at each time point as follows:negative control(NC,no injection),positive control(PC,0.9%Na Cl),1 mg CMH,2 mg CMH and 4 mg CMH.The relative weight of breast and thigh muscles were analyzed at D1.The results showed that at E4,the 2 mg CMH treatment significantly increased the weight of breast muscle compared with PC group(P<0.05).At E6,the 1 mg CMH treatment significantly increased the weight of breast and thigh muscles(P<0.05)compared with NC and PC groups,and improved the thigh muscle ratio(P<0.05)than PC group.At E12,the 1 mg and 4 mg CMH groups increased the weight and ratio of breast muscle than NC and PC groups(P<0.05).Therefore,the appropriate injection condition in this experiment was determined the 1 mg CMH at E6.The effects of in ovo injection of CMH on skeletal muscle development was further determined,and the experiment was conducted at E6.The treatments as follows:negative control(NC),positive control(PC)and 1 mg CMH.Each group had 100 embryos,and the breast and thigh muscles were collected and weighted at E11,E13,E15,E17,E19 and D1,respectively.The present results showed that at D1,when compared with NC and PC groups,the CMH treatment significantly increased the weight of breast and thigh muscles(P<0.05),as well as the number of myofibers per fascicle(P<0.05)and the cross section area(CSA)of myofiber(P<0.05)in PM and BF.Meanwhile,the results showed that in BF at E11,when compared with NC and PC treatments,CMH group significantly increased the PCNA~+cells in tissue(P<0.05),and enhanced the myogenesis related protein expression of PAX3,PCNA,Myf5 and Myo D(P<0.05),and improved the p-P70S6K and T-P70S6K expression(P<0.05),indicating that CMH activated the m TOR/P70S6K signal pathway.At E17,compared with NC and PC treatments,CMH significantly increased the protein expression of PCNA in PM and Myo D in BF(P<0.05).The present results indicated that in ovo injection of CMH enhanced the myogenesis at embryonic period,and the effect was more obvious in thigh muscle.3.The regulating of CMH on the proliferation process of chicken primary myoblastsThe primary myoblasts were isolated from the E11 BF of broiler embryos.After 24 h in vitro culture,the experimental treatments as follows:0 m M(control),10 m M and 20 m M CMH.After 24 h of treatment,the results showed that as compared with control,the 10 m M and 20 m M CMH treatments significantly enhanced the MHC expression,increased the number of PCNA~+and Edu~+cells,and improved the cell proliferation ability(P<0.05),as well as the protein expression of p-P70S6K and T-P70S6K(P<0.05).Therefore,the effects of CMH on cell proliferation was further verified by rapamycin(a m TOR inhibitor).The results showed that,in the present of rapamycin,the increased expression levels of p-P70S6K and T-P70S6K by CMH were significantly dismissed(P<0.05),compared with CMH treated cells.Meanwhile,the combined treatment of rapamycin and CMH reduced the number of PCNA~+and Edu~+cells,as well as the protein expression of PAX7,PCNA and Myo D compared with CMH treated cells(P<0.001).In general,CMH enhanced the proliferation process of chicken primary myoblasts by activating the m TOR/P70S6K signaling pathway.4.Effects of in ovo injection of CMH on skeletal muscle growth and development of broilers at post-hatchThe in ovo injection experiment was conducted at E6.The experiment treatments as follows:negative control(NC),positive control(PC)and 1 mg CMH.After hatching,64 male broilers were selected from each treatment and randomly divided into 8 replicates with 8 birds per replicate.The experiment lasted for 42 d.The results showed that during 1-21 days,as compared with NC group,CMH treatment significantly decreased the feed-to-gain ratio(F:G)(P<0.05)and increased the ADG(P=0.078)and average BW(P<0.05)at 21 d.During the22-42 and 1-42 days,the ADG,ADFI and F:G had no obvious differences among these treatments(P>0.05).At 21 d and 42 d,CMH treatment significantly increased the weight and ratio of thigh muscle(P<0.05)than NC group,as well as the average myofiber CSA in BF(P<0.05)as compared with NC and PC groups.The above results showed that,in ovo injection of CMH mainly improved the production performance of broilers for 1-21 days,and enhanced the growth of thigh muscle.5.The regulating of CMH on the protein metabolism and mitochondrial function of chicken myotubesThe primary myoblasts were isolated from the E13 PM of broiler embryos.When myoblasts proliferated and differentiated into myotubes,the regulatory mechanism of CMH on protein metabolism and mitochondrial function were determined under normal(2%serum in medium)or starved(no serum in medium)conditions.The experiment treatments as follows:0 m M(control),10 m M and 20 m M CMH.After 24 h of treatment,the results showed that,under normal condition,20 m M CMH treatment improved the protein content of cells(P<0.05),and protein synthesis rate,as well as the p-P70S6K and T-P70S6K expressions(P<0.01)when compared with control.However,there were no obvious differences in ubiquitin proteasome(UP)pathway and mitochondrial function(P>0.05).Meanwhile,200 nm rapamycin attenuated the positive effects of CMH on the protein synthesis rate,p-P70S6K and T-P70S6K expression(P<0.01).The present result indicated that at normal state,CMH enhanced the protein deposition mainly by activating the m TOR/P70S6K signal pathway.In a starvation state,CMH treatment significantly increased the protein content and myotube diameter(P<0.001).However,the protein synthesis rate was not changed by CMH treatment,as well as the m TOR/P70S6K pathway(P>0.05).In contrast,CMH treatment significantly increased the p-Fo XO1/3a expression(P<0.05)and inhibited Atrogin1 expression(P<0.05).At the same time,CMH treatment decreased ROS level(P<0.05),and enhanced the PGC1αexpression(P<0.05).In order to determine the regulatory mechanism of CMH,glucose(5 and 10 m M Glu)was used to verify the effect of CMH.The results showed that in the presence of glucose,the positive effects of CMH on the protein content,Atrogin1 expression,ROS level and PGC1αexpression were abolished(P<0.05).The results suggested that the regulating effects of CMH on muscular protein metabolism and mitochondrial function depends on the cellular energy state.6.Effects of CMH supplementation on the protein catabolism of chicken myotubes under stress challenged conditionThe present study aimed to investigate the effects of CMH on the protein catabolism of chicken myotubes under corticosterone treatment simulates stress condition.The experiment treatments as follows:control,10 m M CMH,0.2μM CORT and 0.2μM CORT+10 m M CMH.The results showed that,compared to control,CORT treatment significantly decreased the myotube diameter and MHC protein expression(P<0.05),and increased 3M-His content in medium(P<0.01),and up-regulated the m RNA expression levels of Mu RF1(P<0.001)and Atrogin1(P<0.001),and Atrogin1 protein level(P<0.01).However,compared with CORT treated cells,CMH combined with CORT increased the myotube diameter(P<0.001)and MHC protein expression(P<0.05),and decreased 3M-His content in medium(P<0.001),as well as the m RNA expression levels of Mu RF1 and Atrogin1(P<0.05),and Atrogin1 protein expression(P<0.05).Collectively,the result indicated that CMH alleviated myotube atrophy induced by CORT via inhibiting UP pathway in chicken myotubes.In conclusion,dietary supplemented with GAA improved the growth performances and muscle production of broilers.In ovo injection of CMH enhanced embryonic myogenesis,cell proliferation and production performance at 1-21 days of post-hatch.Meanwhile,CMH play important roles in protein metabolism and mitochondrial function under different cell physiological conditions.This research highlight theoretical basis for the application of Cr and its precursor in poultry industry.