| In this paper,Genetic background between Guangxi and Guangdong of Poecilobdella manillensis and effects of blood,compound feed and lysine supplemen on growth,antioxidant indicators,immunity enzyme,activity hirudin and expression of hirudin genes were studied by the methods of feed science,enzymology,molecular biology of poecilobdella manillensis.The experimental results are presented as follows:1.The genetic diveristy of Poecilobdella manillensis of two populations in Yue and Gui revealed by CO? gene and 18 S r DNA Partial sequencesPCR amplification technology were used to detect CO?and partia sequences in18 Sr DNA region of 35 samples,and accomplish the systematical analysis compare of CO?and partial sequences in 18 Sr DNA region.The results showed that 9 variable sites were detected and 9 haplotypes were defined in CO? gene sequences;The haplotype diversity(0.737)and nucleotide diversity(0.00166)of Poecilobdella manillensis in Guangxi were significantly higher than Poecilobdella manillensis in Guangdong(0.638,0.0119).1 variable sites were detected and 3 haplotypes were defined in partia sequences in 18 Sr DNA region;The haplotype diversity(0.154)and nucleotide diversity(0.00014)of Poecilobdella manillensis in Guangxi were higher than Poecilobdella manillensis in Guangdong(0.110,0.00011).The genetic distance among 9 CO? gene haplotypes varied from 0.0015 to 0.0077;and The genetic distance among 3 partia sequences in 18 Sr DNA region gene haplotypes varied from 0.0009 to 0.0018.The proportion of CO I gene predominant haplotype in Guangxi was60%,and The proportion of partia sequences in 18 Sr DNA region gene predominant haplotype in Guangxi was 58.6%.The results suggest that the genetic diversity of Poecilobdella manillensis in Guangxi is higher than Poecilobdella manillensis in Guangdong.2.Effects of compound feed and blood feed on the antioxidant indicators and immunity enzyme of Poecilobdella manillensisActivity hirudin,Antioxidant enzyme(SOD,CAT,MDA)and immunity enzyme(AKP,ACP,LSZ)were determined of young Poecilobdella manillensis.The results show that the activities of hirudin of compound feed group were slightly lower blood feed group and no significant difference(P>0.05).In the intestine,superoxide dismutase(SOD)and catalase(CAT)of compound in feed group were significantly higher than those of in blood feed group(P<0.05),but the content of malonaldehyde(MDA)of compound feed group were slightly lower in blood group.In the crop,activity of superoxide dismutase(SOD)and content of malonaldehyde(MDA)in compound feed group were significantly lower than those of in blood group.In the intestine tissue,the activity of lysozyme(LSZ)of compound feed group was significantly lower blood feed group(P<0.05),while the activities of alkaline phosphatase(AKP)and acid phosphatase(ACP)were significantly higher than blood feed group(P<0.05).In the crop tissue,there was no significant difference between compound feed group and blood feed group in lysozyme(LSZ),alkaline phosphatase(AKP),acid phosphatase(ACP).The activities of lysozyme(LSZ)of compound feed group were slightly lower blood feed group,and the activities of alkaline phosphatase(AKP)and acid phosphatase(ACP)slightly higher blood feed group.The results suggest that there was no effect on the activities of hirudin when fed with compoun feed,the antioxidant capacity increased in the intestinal,and it can reach and exceed the level of blood feeding in AKP,ACP and MDA.3.Screening of Amino acids and determination of hirudin activityThe target amino acids are Lysine,cysteine methionine,taurine,glutamate and histidine.Each experimental diet(The final levels of amino were 1g/kg,2g/kg,3g/kg)was prepared for the experiment of Poecilobdella manillensis.The results showed that The effect of lysine supplementation was the best.The other groups had no or little Feeding after the first eating.In the adding lysine group,the active of hirudin showed a tendency that increased first and then decreased from low concentration group to high concentration group.when the appropriate proportion of added lysine in thecompound feed was 1.4g/kg,The activity of hirudin is the highest(165.6ATU/g),and slightly higher blood feed group.The other add groups was disorder.The results suggest that the feeding condition and hirudin activity were analyzed synt,and lysine was screened as the breeding experiment in the later stage.4.Effects of lysine supplementation on growth and related factors of poecilobdella manillensisEach experimental diet(The final levels of lysine were 0g/kg,0.7g/kg,1.4g/kg,2.1g/kg)was randomly assigned to triplicate in a completely randomized design,The feeding trial lasted for 37 days.Effects of lysine supplement growth,antioxidant indicators,immunity enzyme,activity hirudin and expression of hirudin genes were studied of poecilobdella manillensis.These results showed:(1)Specific growth rate(0.77~0.82)and the weight gain rate(29.76%~32.12%)of compoun feed supplemented lysine group were significantly higher than that of no added Lysine group(P<0.05),and significantly lower blood feed group.The specific growth rate and the weight gain rate were the highest,when the appropriate proportion of added lysine in the compound feed was 1.4g/kg.(2)Activity of SOD no added group were significantly higher than those of in blood feed group(P<0.05)in the intestinal and muscle.with increasing dietary Lysine levels,SOD showed “up-down-up” trend,were significantly higher than blood feed group(P<0.05),and activity of SOD is the highest in the 1.4g/kg.In the intestinal,CAT is the highest in the 1.4g/kg.In the crop and muscle,in the 1.4g/kg,there was no significant difference between added group and blood feed group in the CAT P>0.05).there was no significant difference content of MDA in the difference group(P>0.05).In the crop and muscle,showed “High and low staggered”,The content of MDA is balanced trend of poecilobdella manillensis.The activities of LSZ,AKP and ACP of added group slightly higher blood feed group(P<0.05).In the 1.4g/kg,The activity of the total AKP activity is the highest,the activity of AKP is the highest in the muscle tissue,and significantly higher than the blood control group.(3)Compound in feed group activity of hirudin and relative expressionof hirudin genes were significantly lower than those of in no added group.In the lysine addition experiment,activity of hirudin and relative expressionof hirudingenes showed the same as changing trend,“First increase and then decrease”,When the added concentration is 1.4g/kg,activity of hirudin(285.52±5.68U/g)and relative expressionof hirudin genes(0.76±0.03)were the highest,and there was no significant difference blood feed group(P>0.05).The results suggest that adding lysine in compound feed can effectively improve growth performance,antioxidant ability and immune function,activity of hirudin and relative expressionof hirudin gene was decreased of poecilobdella manillensis.Based on analyses,the lysine leves suitablely were 1.4g/kg in feed of poecilobdella manillensis. |
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