Construction Of Oral Kisspeptin Vaccine And It's Efficacy On Reproductive Axis Of Ram Lambs

The castration of male animals raised for meat has been extensively practiced for centuries for fat deposit and the calmer behavior.Usually,it is performed by producers without anesthesia and highly likely painful and nerve-racking events because of testes and scrotal skin innervated by nociceptors.In addition,castration may have temporary injurious effects on the growth,immune system and the condition of the animals.This situation raises the animal welfare concerns to increase the pressure on the animal producers to stop castration without painkiller.Later,scientists started looking for an alternative castration method and developed conjugate of the peptide with a suitable carrier.The gonadotrophic steroid hormone(GnRH)DNA-based vaccines induce strong resistance responses,decreased serum testosterone levels and suppress fertility.The KISS?1 gene product,kisspeptin,stimulates GnRH neuronal signaling via G-protein coupled receptor,GPR-54.Kisspeptin and kisspeptin receptor(GPR-54)have arisen as important upstream regulators of fertility.Kisspeptin is the central regulator of gonadotropins,which stimulate reproductive organ function and the hypothalamic neurons that synthesize GnRH release.The disruption of this signaling pathway causes hypogonadotropic hypogonadism in mammals.As part of this cutting-edge research project,we plan to analyze the efficacy of an oral kisSPeptin recombinant vaccine on the reproductive physiology,behavior of ram lambs and expression level of receptor mRNA.(1)The construction of eukaryotic expression plasmid of C500/pKS-asd.The chemically-synthesized kisspeptin gene was subcloned into pVAXl to construct pVAX1-KISS-1 plasmid,and HBsAg-S gene later subcloned into the PVAX-KISS-1-S-asd plasmid to construct pKS plasmid was stored in the laboratory.Later,the plasmid(pVAX?KISS?1-S-asd was electrically transfected to Salmonella enterica serovar Choleraesuis C500 strain.The positive clones were identified using sequencing and double enzyme digestion,named as C500/pKS-asd.The stain gone through 50 generations in the sterile medium and bacteria genome DNA were extracted.Further,the constancy of plasmid and strain were detected using double enzyme digestion and preserved InvA gene.The strains(C500/?pKS-asd and pVAX-asd)were inoculated with 50?l/Fmg diaminopimelic acid(DAP)into Luria-Bertani(LB)broth and grown at an optical density of 0.5 at 650 nm(OD 650)overnight.Then,the cells were accumulated with the aid of a centrifiuge at 12,000 rpm for 10 min at four degrees Celsius.Prior vaccination to ram lambs,the bacterial number was verified using dilution of the inoculum onto LB agar.Then,the bacterial cells were adjusted spectrophotometrically to 5×1010 CFU/ml dose for both groups.The liquid was eliminated,and the bacterial cells were re-suspended in phosphate-buffered saline(PBS)for vaccinations.(2)Kisspeptin vaccine oral immunization effect on ram castrationTen 56-day old Hu breed ram lambs were randomly grouped into treatment and control groups to receive the experimental recombinant vaccines,C500/pKS-asd or PVAX-asd(aspartate-?semialdehyde dehydrogenase),respectively.The vaccines were orally administered at days 0,and the same treatments were repeated on day 28 and 56 after the primary imnunizatioa At every day-14 interval from primary immunization and end of the research,blood samples were taken and scrotal circumference data recorded for each animal as well as sexual behavior were evaluated.The result showed that the scrotal circumference(19.82 ± 3.18 cm)of the treatment group was significantly lower than control(22.16 ± 4.79 cm)from day 42-98 post-immunization(P<0.05).similarly,testes weight of treatment(91.83 ±1.42 g)and control(118.10±7.29g**,P<0.01)and length of treatment group(6.64±0.11)and control(7.40±0.29*)in the treatment group was lower than control(P<0.05),respectively.The seminiferous trubules of vaccinated animals comprise low densities of spermatogonium,spermatocytes and spermatids compared to the control group.Interestingly,vaccine administration contributed to a sharp decrease(P<0.01)in sexual behavior propensity.These data recommend>that oral immunization of kisspeptin vaccine has the potential for the suppression of gonadal ftunction and sexual behavior of ram lambs.(3)Effects of oral immunization on anti-kisspeptin-antibodies and testosterone serum.Anti?kisspeptin antibody,kisspeptin serum level and testosterone serum levels were measured by indirect ELISA method.Anti-kisspeptin-antibodies were detected in vaccinated group ram lambs on day 42 to the end of the experimental period(day-98)of primary imnunization.Results demonstrated that the concentration of anti-kisspeptin antibodies was higher in the.treated animal compared to control animal(P<0.05,P<0.01).However,sermm testosterone levels were lower in the treatment group than control group(P<0.05).The kisspeptin-54 blood serum concentration in the blood was dramatically reduced in the treatment group compared with the control group(P<0.05).These results revealed that oral kisspeptin immunization could induce antibodies and reduce the testosterone as well as kisspeptin-54 concentration level to achieve immunocastration.(4)Influence of oral kisspeptin immunization on the expression of hormone receptor mRNA in the reproductjion axisTesticular,hypothalamus,pituitary and other tissue samples were taken at day-98.The total RNA was prepared using TRIzol reagent and quantitative real-time polymerase chain reaction(QPCR)was used to analyze the levels of kisspeptin,G protein-coupled receptor-54(GPiR-54)and GnRH mRNA.The expressions of luteinizing hormone receptor(LHR)and follicle stimulating hormone receptor(FSHR)in the pituitary were analyzed.Furthermore,GnRHR mRNA expression in the pituitary was evaluated.Similarly,the luteinizing hormone beta(LH?)mRNA and follicle stimulating hormone beta(FSH?)mRNA,in the testes were analyzed.The levels of testis FSHR and LHR mRNA were highly reduced in the treatment group compared with the control group.However,statistically FSHR was not significant at(P<0.05).In addition,hypothalamic kisspeptin,GPR-54 and GnRH mRNA in the treatment group were notably lower than in control(P<0.01).Moreover,LH-?,FSH-? and GnRHR mRNA expression in the pituitary were significantly reduced in the treatment group(P<0.05,P<0.001).These data suggested that oral kisspeptin immunization can affect the levels of hormone receptors mRNA in the hypothalamus-pituitary-gonadal axis.(5)Effects of oral kisspeptin immunocastration on live weightThe live weight of experimental animals was measured at a day-14 interval jfrom.post-immunization to the end of experimental period.The findings revealed that the:re was no significant difference between group treatment and control.This result showed that oral kisspeptin immunization does not influence the metabolism of ram lambs.The overall results suggested that kisspeptin recombinant oral vaccine regulate and inhibits the reproductive physiology,behavior and expression of hormone receptor mRNA in the ram lambs.