Effects Of Injection And Oral GnRH On Artificial Induction Of Production And Changes Of Intestinal Micro Flora In Small Yellow Croaker(Larimichthys Polyactis)

The successful artificial reproduction of Larimichthys polyactis should rely on oxytocin,previous studies shown that LHRH-A2 can effectively induce Larimichthys polyactis spawning,but the effect of oxytocin needs to be improved.The intraperitoneal injection is the general artificial hormone administration method in traditional,which will lead to the excessive stress and death of breeding fish,and resulting in the decrease of breeding population.Moreover,the traditional artificial expend manpower and material resources during the spawning.Therefore,it is urgent to develop new low-cost and effective oxytocin and change the mode of oxytocin.In the first experiment,in order to provide theoretical reference for the preparation of oral oxytocin,three groups of different GnRH concentrations and particle sizes were conducted to study the effects of different indexes on the drug-loading performance of microcapsules.The next experiment is to evaluate the effect of gonadotropin-releasing hormone(GnRH)as an exogenous hormone on artificially induced spawning and the composition of the intestinal flora of small yellow croaker(Larimichthys polyactis).We compared the luteinizing hormone(LHRH-A2,2.4μg/kg)injection group,gonadotropin-releasing hormone(GnRH,75 μg/kg)injection group and gonadotropin-releasing hormone(6 mg/kg)oral group effection of ratification,saline injection group as control.Analyze each group spawning situation,ovarian tissue physiological changes and serum hormone levels,and comprehensively.In addition,the structural changes of intestinal flora were comprehensively analyzed by high-throughput sequencing.The main research results are as follows:1.Adding a certain amount of oxytocin into sodium alginate solution,chitosan and sodium alginate can be combined by ionic crosslinking.Set three groups of different GnRH concentration gradients were respectively at 0.2 mg/mL,0.4 mg/mL and 0.8 mg/mL,Three groups of drug-loaded microspheres with different particle sizes were prepared by the method of cusp granulation,and the particle sizes respectively obtained were 3 mm,4 mm and 4.5 mm.The optimal GnRH concentration and particle size for the encapsulation efficiency and drug loading rate of the prepared microspheres respectively were 0.8 mg/mL and 4.5 mm.The preparation method is simple to operate,and the prepared GnRH-containing microspheres are regular spherical,uniform particle size,high strength and good elasticity.2.Study on the effect of inducing artificial spawning of Larimichthys polyactis by injection and oral GnRH.The results showed that there was no significant difference between GnRH injection group and LHRH-A2 injection group in effect time,spawning duration,fertilization rate and hatching rate,but the total spawning amount and floating egg weight increased significantly(P<0.05);The total number of eggs laid and the weight of floating eggs in GnRH oral group were significantly lower than those in LHRH-A2 injection group,but there was no significant difference in fertilization rate and hatching rate.Further analysis of the spawning process of Larimichthys polyactis through tissue physiology showed that the ovarian development in the control group was mainly in stageⅢ,and LHRH-A2 injection group,GnRH injection group and oral administration group could induce the ovarian development from stage Ⅲ to stage Ⅴ,and promote the oocytes to mature and excrete from stage Ⅲ and Ⅳ.After treatment with oxytocin in each experimental groups,serum luteinizing hormone(LH),follicle stimulating hormone(FSH)and GnRH increased rapidly,and remained at a high level until the Post-spawning,and the hormone levels in all treatment groups showed the same trend.3.16S rRNA high-throughput sequencing was used to compare and analyze the changes of intestinal flora of Larimichthys polyactis during spawning with different kinds of oxytocin.In the spawning process of Larimichthys polyactis,results showed 12528 OTUs and 481 core OTUs;The control group,LHRH-A2 and GnRH treatment groups respectively accounted for 38.75%,26.65%and 36.60%of the total OTUs.In the spawning stage,the species richness and diversity of the control group were higher,while those of the GnRH treatment group were lower.In the Post-spawning stage,the species richness and diversity of GnRH treatment group were higher,while that of A2 treatment group was lower.There was no significant difference in the composition of most intestinal flora samples in each group.At the phylum level,Firmicutes,Proteobacteria and Bacteroides are the dominant flora.In the control group,there was no significant correlation between α-diversity index and hormone content.However,there was a significant correlation in A2 treatment group.In GnRH treatment group,Chao1 index had no significant correlation with spawning and hormone content,but Shannon,Simpson and Observed_species index were significantly correlated with spawning and GnRH content.The common dominant classes in hormone treatment group which are strongly related to egg production and hormone content are Bacteroides.To sum up,injection of GnRH can effectively induce the spawning of Larimichthys polyactis,and the effect of induction is not significantly different from that of injection of LHRH-A2;oral GnRH can also induce spawning of Larimichthys polyactis,but the spawning volume and floating egg weight are significantly lower than those of the hormone injection group;The method and dose of administration need to be further optimized;the study revealed the changes of oxytocin on the composition of the intestinal flora of female Larimichthys polyactis,which provides a reference for further study of the intestinal flora of Sciaenidae and healthy breeding.