Study On The Changes Of Serum Free Amino Acids And The Related Metabolic Mechanisms In Laying Pigeons During One Egg-laying Cycle

Due to the broodiness of pigeons,there will be a certain interval period for egg-laying,so the efficiency of the egg-laying rate is very low,which restricts the production of the laying pigeon industry.Previous studies reported the egg-laying cycle(ELC)of pigeons can be shortened by adding amino acids(AA)in the feed.However,both at home and abroad,research on laying pigeons is insufficient,and the data of the AA requirements of laying pigeons are scarce.Therefore,in the present study,we aim to investigate the dynamic changes of the serum free amino acids of pigeons within an ELC and the underlying mechanisms by analyzing the serum biochemistry,serum hormone and related gene expression in different tissues,to improve the pigeon egg-laying rate and provide basic data for other related studies.The study mainly consists of 3 parts.1.Dynamic changes of serum free amino acids in laying pigeons during an ELCThis experiment adopts the"2 x 7 factors"of two-factor factorial design,with age(6months and 12 months)and egg production interval(7 time points during one ELC)as factors.Thirty-five 6-months-old(6-mo)laying pigeons(American Silver King)were randomly selected and divided into 7 groups according to the 7 time points during one ELC,as follows:D1,the day as the first-egg laying;D2,the day after the first-egg laying day;D3,the day as the second-egg laying(SEL);D4,one day after the SEL;D7,four days after the SEL;D10,seven days after the SEL;D13,ten days after the SEL.Blood samples were collected from a wing vein in the afternoon(17:00-18:00)for AA analysis.Meanwhile,thirty-five 12-month-old(12-mo)laying pigeons were also randomly selected and used to collect blood samples to compare the variations of serum AA levels between the 6-mo and12-mo laying pigeons during one ELC.The results showed that serum AA of two ages laying pigeons showed a similar variation pattern during one ELC.A point in time within an ELC has a significant effect on most AA.In both two ages,the values of Lys,Met,Leu,Phe,Tyr,Ser and Ala peaked at the D2,decreased at the D3 and maintained the low level until the D13.A parental age effect was observed in the contents of Lys(P<0.05),Met(P<0.001),Ile(P<0.05),Leu(P<0.001),Val(P<0.01),Phe(P<0.001),Glu(P<0.001)and Ala(P<0.001),but not in the contents of total amino acids(TAA).There was a significant interaction of time point age for the contents of Leu,Tyr,Asp and Glu.These results showed that serum AA in laying pigeons increased significantly before laying,and then decreased quickly after laying.2.The relationship between feed intake and serum metabolites with amino acid changes of laying pigeons during an ELCIn order to analyze the mechanism of the increase of serum AA in laying pigeons,feed intake in one ELC of laying pigeons was recorded.The results showed that there was no significant change in feed intake in one ELC of laying pigeons,suggesting that the increase of AA not as a result of the increasing feed intake,but due to changs in vivo.Therefore,another thirty-five 6-mo laying pigeons(American Silver King)were randomly selected and divided into 7 groups according to the 7 time points,as Experiment 1 described.Immediately following the 12-h feed deprivation,all birds were sacrificed to collect samples and then analyzed.The results showed that the serum TAA peaked at D2,began to decline at D3,and gradually recovered to peak at D13,similar to the change pattern of the non-fasted pigeons.Serum contents of Lys,Cys,Ile,Val,Phe,Asp,Glu and Ala remained high from D2 to D3,then decreased and finally increased to a higher level at D13.Meanwhile,serum TG reached the highest value at D13 and decreased to the lowest value at D4(P<0.05).Serum Glu and Ca levels remained high at D1 and D13,but decreased to a lower level at D4(P<0.05).Serum PRL level gradually increased from D1-D7,and D7reached the peak value(P<0.05)and then decreased gradually to the lowest level at D13(P<0.05).Serum E2 level reached its maximum at D13 and was significantly higher than at other time points in one ELC(P<0.01).At D7,the contents of TP and ALB in liver of pigeons reached the highest value(P<0.05),and GLB content reached the maximum at D2(P<0.05).TC,LDL and TG showed an upward trend from D2 to D13,and all reached the maximum value at D13(P<0.05).After analyzing the correlation between these biochemical indexes with AA,it was found that TG and Ca were positively correlated with most AA.These results indicate that there are many physiological changes in laying pigeons during one ELC,which may be closely related to the changes of serum AA.3.The relationship between amino acid transport and synthesis genes with amino acid changes of laying pigeons during an ELCIn order to further investigate the mechanism of changes of serum AA,the tissue morphology of the liver,ovary,and oviduct of those pigeons was observed and m RNA expressions of amino acids transporters,amino acids synthase-related genes and hormone receptor-related genes in these three tissues were analyzed.The results showed that:(1)In addition,different degrees of lipid droplets appeared in the liver from D7 to D13.The lumen diameter,fold width and fold length of the oviduct magnum at D1,D2 and D13 were significantly higher than other time points.(2)The relative m RNA expression of amino acids transporters:SNAT2 was upregulated in oviduct at D2(P<0.05).The relative m RNA expression of LAT4 was significantly increased in both liver and ovary at D1.The expression of B~0AT1 in liver and ovary was reached the highest value at D13(P<0.01)or D3(P<0.01).EAAT3 was detected in the liver(P<0.01)and ovary(P<0.05)with a higher level at D2.In the ovary,the relative m RNA expression of CAT1(P<0.05)and y~+LAT2(P<0.01)significantly increased at D3.(3)Amino acids synthase:In liver,the relative m RNA expression of GDH1 reached the maximum at D13(P<0.05).In ovary,ASL levels remained high leve at D10 and D13(P<0.05).The expression of PDH and SHMT2 were up-regulated in D3(P<0.05).The expression of GDH1 at D4 was significantly higher than other time points(P<0.05).In oviduct,PDH and SHMT2 reached maximum values at D2(P<0.05).(4)Related hormone receptor genes:In liver,the relative expression of FSHR reached the highest level at D13(P<0.01),and the relative m RNA expression of ER(P<0.05)and GHR(P<0.01)reached the highest values in D3 or D2.In ovary,both PRLR and ER showed the highest relative expression at D2(P<0.01).In oviduct,the expression levels of both PRLR and ER were the highest at D1,and the lowest at D4(P<0.01).The expression of FSHR was up-regulated at D2,then decreased to a lower level,and then increased to a higher level at D10(P<0.01).The results of correlation analysis showed that some genes had different correlations with the changes of AA in these three tissues.These results suggest that the large amount of AA during egg production process is partly from serum transport by amino acids transporters and partly from de novo synthesis in vivo,which may be regulated by hormones.To sum up,during the laying process,laying pigeons need a large of AAs,some of which are transported from blood,and some of which are dependent on synthesis in vivo.This process is regulated by various factors,such as hormones,etc.,and adding specific AA into the feed of laying pigeons may improve their laying efficiency.