| As previous researches indicated that immunization with inhibin DNA vaccines neutralize the endogenous inhibin could increase FSH secretions,improve the number of follicles,ovulation and conception rate.Our laboratory developed the attenuated S.choleraesuis-mediated inhibin DNA vaccine C501(pVAX-asd-IS),it has been proved to be a safe tool for increasing ovarian follicular development and improving reproductive traits in mice.Comparing with naked DNA vaccination,the production of the C501(pVAX-asd-IS)is easier and relatively low cost.Moreover,the vaccine was equipped with the asdˉ balanced lethal chromosome–plasmid system and without antibiotic resistance gene is safe for animals and human,depict that this vaccine may have direct application in large animal industry.But in different species,the efficacy of inhibin DNA vaccine was influenced by some factors such as vaccination approaches,the dose and times of immunization.Although the safety that using live bacterial as a DNA vaccine delivery vector has been confirmed in mice,there were few previous studies with regard to the safety using live bacteria as a DNA vaccine delivery vector for the surrounding environment.Therefore,in this study,we used the different dose of intramuscular injection and the different times of nasal immunization of inhibin DNA vaccine C501(pVAX-asd-IS),to evaluate the effect of inhibin DNA vaccine on follicular development,ovulation and fertility of crossbred buffaloes in breeding season.Subsequently,comparison on the safety and efficacy of intramuscular and nasal immunized with C501(pVAX-asd-IS)on ovarian responses and fertility of crossbred buffaloes in non-breeding season,and coupled with synchronization protocols,analyzed the pattern of follicular development and ovulation in crossbred buffaloes during the breeding and non-breeding season,which would provide a foundation for optimizing inhibin gene vaccine and increasing the fertility of crossbred buffalo.The main contents and results of this study are as follows:1.Intramuscular injection with inhibin DNA vaccine for improving follicular development and ovulation in crossbred buffaloesA total of 158 crossbred buffaloes divided into four groups and were intramuscular injected with 1010(T1,n=41),109(T2,n=37),108(T3,n=37)or 0(C,n=43)CFU /ml inhibin vaccine C501(pVAX-asd-IS)in 10 ml PBS on day 0 and 14,respectively.All animals were administered with 1000 IU PMSG on day 28,0.5mg PGF2α on day 30 and 200μg GnRH on day 32.The results showed intramuscular immunized with C501(pVAX-asd-IS)elicited significant immune responses,and group T1 have the highest anti-inhibin IgG antibody titers.The number and diameter of large follicles(≥10mm)as well as ovulatory follicles in group T1 was significantly greater than group C(P<0.05).The growth speed of dominant follicles in group T1 was significantly faster than groups T3 and C(P<0.05),resulting in a greater estrus,ovulation and conception rate in buffaloes with positive antibodies.These results demonstrate that intramuscular immunized with the bacterial delivered inhibin vaccine at high-dose of 1×1010 CFU /ml,could enhance follicular development,improve the proportion of estrus,ovulation and fertility in crossbred buffaloes.2.Nasal immunization with inhibin DNA vaccine for improving follicular development and ovulation in crossbred buffaloesA total of 134 crossbred buffaloes were divided into four groups: Group1(G1,n=34),group 2(G2,n=35)and group 3(G3,n=31)were nasal immunized twice a day with 10 ml of 1×1010 CFU /ml of the C501(pVAX-asd-IS)vaccine for 5,3 and 1 day,respectively.Control group(n=34)was nasal immunized with 10 ml PBS for 5 days.All animals were immunized twice with an interval of 14 days and administered with 200μg of a GnRH analogue on day 28,0.5mg PGF2α on day 35 and 200μg of the same GnRH analogue on day 37.TAI was performed at 18 and 24 h after the second-GnRH treatment.Nasal immunized with C501(pVAX-asd-IS)evidently induced antibodies against inhibin at 14 and 28 d after primary immunization,and the anti-inhibin IgG antibody titers in G1 were significantly higher(P < 0.01)than G3.Following booster immunization,the anti-inhibin antibody titers in G1 gradually increased.After the second-GnRH treatment,the growth speed of the dominant follicles in G1 was significantly faster(P < 0.05)than G3 and control group.The number and diameter of large follicles(≥10mm)as well as ovulatory follicles in G1 was the greatest in all groups,resulting in a greater estrus,ovulation and conception rate in G1.These results demonstrate that nasal immunization with C501(pVAX-asd-IS)for 5 days,coupled with the Ovsynch protocol,could be used as an alternative approach to improve reproductive performance in crossbred buffaloes.3.The efficacy of intramuscular and nasal immunized with inhibn DNA vaccine in improving ovarian responses and fertility of crossbred buffaloes in non-breeding seasonThe experiment was based on(n = 81)multiparous crossbred buffaloes(River ? Swamp)with body condition score ranging(BCS)between 3.0-4.0 were randomly divided into three groups: intramuscular immunization(IM,n=29)were intramuscular immunized with10 ml of 1010 CFU /ml inhibin vaccine C501(pVAX-asd-IS)for 3days;nasal immunization(NI,n=27)were nasal immunized twice a day with the same dose of the inhibin vaccine for 5 days;Group C(n=34)was intramuscular immunized with 10 ml PBS for 5 days.All animals were immunized twice with an interval of 14 days and administered with 1000 IU PMSG on day 28,0.5mg PGF2α on day 30 and 200μg GnRH on day 32.The results showed intramuscular and nasal immunized with the inhibn vaccine could induce effective immune responses in crossbred buffaloes,and the anti-inhibin IgG antibody titers have no significant difference between two immunized groups.But group IM have the greater positive rate of anti-inhibin antibodies than group NI(82.8% vs.74.1%).Compared to the control group,the number and diameter of large follicles(≥10mm)as well as ovulatory follicles in group IM were increased significantly.Intramuscular immunized with C501(pVAX-asd-IS)increased the proportion of estrus,ovulation and conception rate of crossbred buffaloes in non-breeding season.Buffaloes with positive anti-inhibin antibodies showed significantly greater conception rate compared with the buffaloes with negative anti-inhibin antibodies(56.8% vs.27.0%;P< 0.01).These results demonstrate that intramuscular and nasal immunized with the inhibin vaccine induce effective immune responses,but the effects induced by the intramuscular injection were better than the nasal immunization,increased the number and diameter of ovulatory follicular,enhanced estrus and ovulation of crossbred buffaloes and improved conception rate in non-breeding season.4.The safety of intramuscular and nasal immunized with inhibn DNA vaccine in crossbred buffaloes.A total of 81 crossbred buffaloes were divided into three groups: intramuscular immunization(IM,n=29)were intramuscular immunized with10 ml of 1010 CFU /ml inhibin vaccine C501(pVAX-asd-IS)for 3days;nasal immunization(NI,n=27)were nasal immunized twice a day with the same dose of the inhibin vaccine for 5 days;Group C(n=34)was intramuscular immunized with 10 ml PBS for 5 days.All animals were immunized twice with an interval of 14 days.Take the temperature and heart rate of crossbred buffaloes after immunization,and faecal,soil and water samples originating from immunized buffaloes and surrounding environment were collected.The results showed the temperature and heart rate of crossbred buffaloes in each group have no significant difference after immunization.The target gene(invA gene)was not detected by PCR amplification in faecal,soil and water samples,indicating that the surrounding environment was not contaminated by recombinant bacteria,and intramuscular and nasal immunization of C501(pVAX-asd-IS)was safe to crossbred buffalo. |
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