Evaluation Of The Efficacy Of Immunocastration Vaccines Composed Of GnRH Peptides

The development and function of boar testis is regulated by Gonadotropin releasing hormone（GnRH）,secreted by hypothalamus,the combination of GnRH and specific receptor in pituitary gland causing the synthesis,secretion and release of luteinizing hormone（LH）and follicle stimulating hormone（FSH）,LH and FSH play a role in testis,regulating testosterone and androsteone secretion.Testosterone is one of the substances that lead to mutton of pork,which is produced by leydig cells under the control of LH,and tansport through the blood circulation to the whole body tissue and accumulates.Another substance that causes mutton is fecal odor.Therefore,by regulating the secretion of LH and FSH can play a role in regulating the content of testosterone and fecal odor in the body.Therefore,combined with the predecessors’experience,we prepared four kinds of immunocastration vaccines,which were composed with GnRH monomer,GnRH dimerⅠ,GnRH dimerⅡand GnRH parallel dimer castration peptide as antigen.After the quality examination of the vaccine,studied the immunocastration effcrt against male SD rats and the male pig’s,which showed certain trend of immunocastration effect,and the indirect Elisa method for detecting GnRH antibody was established,the main research contents were as follows:1.Preparation of GnRH synthetic peptide castration vaccine.The castration peptides of GnRH monomer,GnRH dimerⅠ,GnRH dimerⅡand GnRH parallel dimer were used as antigen,and the Montanide^TM ISA 61VG was prepared as oil adjuvant.The vaccine is tested,the drip experiment is not disperse,the microscopic examination shows that the emulsification effect is good;sterile test shows that the liquid of the sulfur-ethanol acid salt culture medium and the glucose peptone medium is still yellowish-colored,clarified and transparent,and there is no contaminated bacterium.The vaccines prepared can be used for subsequent experimental studies.2.Establishment of Indirect ELISA assay for serum GnRH antibody in rats.Using GnRH peptide as coated antigen in ELISA,the goat anti-rat horseradish peroxidase was used as the second antibody,the best dilution multiple of antibody and second antibody were screened by checkerboard,the standard curve was drawn,and the established method was tested by specificity and repeatability test.The results showed that the best concentration of GnRH synthetic peptide antigen was 1.28μg/m L,and the optimal dilution of serum was 1:51,200,the optimum dilution of the second antibody was 4000,the positive serum antibody concentration was linearly correlated with OD value,and the equation was R~2=0.9976;the variation coefficient of the ELISA board was from 0.78%to 3.16%,and the variation coefficient between the plates was from 3.24%to 7.79%,which showed that the method was good in linearity and repeatability,can be used for the detection of serum sample next.3.Effect of immunological castration of GnRH synthetic peptide vaccine in rats.54 Male SD Rats with 9-week-old were randomly divided into 9 groups,1 were blank control group,2～8 was vaccine immune groups:GnRH monomer single immunization Group,GnRH monomer two times immune group,GnRH dimerⅠsingle immune group,GnRH dimerⅠtwo times immunization group,GnRH dimerⅡin single immune,GnRH dimerⅡin two times immunization group,GnRH tandem dimer single immunization group,GnRH tandem dimer two immunization group,the first immunizaiton is at 9 week old,the boster immunizaiton was at 11 weeks old,the blood was collected every two weeks before and after immunization,and the isolated serum was preserved at-20℃.Indirect Elisa was used to detect serum GnRH antibody titer,the testosterone levels were detected by radioimmunoassay(¹²⁵I),the weight changes in rats were measured,testicular weight was observed,and the changes of tissue structure were studied by the testicular tissue biopsy and H.E.staining.The results showed that compared with the blank control group,the titer of GnRH antibody in the sera of the vaccine-immune group was significantly higher（P<0.01）,and the titer of antibody in two times immunization was high than that of single immunization.Until 43 weeks after immunization,the titer of serum GnRH antibody in GnRH dimerⅠvaccine group was still more than 2.5.The concentration of serum testosterone in the immune group was significantly decreased（P<0.01）,two times of GnRH dimer and GnRH monomer were 100%effective,and the onset time of GnRH two was 2 weeks earlier.This effect was keep at least 30 weeks.The weight of testis in the immune group was significantly lower than that in the blank control group（P<0.01）.There was no significant difference in body weight between the control group and the immune group.At the same time,the results showed that the testis structure was intact in the control group,and the seminiferous cells in seminiferous tubules gradually developed into the lumen and differentiated markedly.The testis of the immune group was obviously atrophic and the diameter of seminiferous tubules was smaller.The results showed that GnRH synthetic peptide vaccine could produce immune castration,GnRH dimerⅠwas more effective,and immunocasttration effect can lasted more than 25 weeks,be selected as immunocastration vaccine in sequent study.4.Effect of immunocastration of GnRH synthetic peptide vaccine in boar.40 Boars of 10weeks age,healthy,weight closly,randomly divided into 4 groups,respectively:blank control group,surgical castration group,Improvac immunization Group（10,14 weeks old two times immunization）,GnRH dimerⅠvaccine immunization Group（10,12 weeks old two times immunization）,before immunized and every two weeks after immunization blood sampling was collected,separates the serum to detect the change of testosterone concentration in serum.The results showed that the levels of serum testosterone in the castrated group were reduced to the serum testosterone levels in the surgical castration group at the 6th week,among them,the Improvac immunization group lasted for 6 weeks,then recovered,and the level of GnRH dimerⅠvaccine immunization group’s serum testosterone remained at the surgical castration level until at least to 14 weeks,which showed that the duration was better.