Effects Of Active Immunization Against Gonadotrophin-releasing Hormone On Immune System In Female SD Rats

In recent years,the effects of active immunization against GnRH on animal immune system has attracted more and more attention.However,the relating research data in this field is few.Spleen is crucial immune organ of animals.Is its function affected by active immunization against GnRH?How does it affect?In addition,what is the response of animals immunized with GnRH to the stimulation of foreign antigens?In order to answer the questions above mentioned,In this study,therefore,the GnRH-Tandem-Dimer conjugated to OVA were used as antigen with Specol adjuvant,SD female rats were used as the model to investigate the effects of active immunization against GnRH on immune system and immunological response of GnRH imunized animals to BSA stimulation.The results of this study would enrich the research contents in this field,and provide a more solid theoretical basis for promoting the large-scale application of active immunization against GnRH technology in livestock production practice.（1）36 female SD rats at the age of 4-5 weeks were randomly divided into three groups（n=12）:intact controls（C）with no treatment,bilateral ovariectomy at the age of 7weeks in surgical castrates（SC）and 1 m L emulsified antigen（including 50μg GnRH-TDK）was injected intramuscularly at the age of 9 weeks in immunocastrates（IM）,8 weeks later,the dose and method of the injection were as same as the first immunization.Vaginal smears of female rats in C and IM were observed every day after the first immunization and the booster immunization,respectively.The estrus cycle of female rats were observed for 30 consecutive days.Blood samples from the tail artery were collected in the day of 0wpv（weeks post vaccination）,4 and 8 wpv.All rats were killed in14 wpv.The weight of spleen and ovaries were separated and recorded.The levels of serum E₂ and T were determined by RIA.Serum GnRH-Ab and serum cytokines were detected by ELISA.Total RNA of the spleen was extracted and reversely transcribed into c DNA.The m RNA expression levels of ER-α,GnRH-R and cytokines genes were detected by quantitative real-time PCR.Flow cytometry was used to detect the number of immune-associated cells and T lymphocyte cluster.After active immunization against GnRH,the serum GnRH-Ab were increased,and the GnRH-Ab increased rapidly after the booster immunization,reaching 10.66±0.37ng/m L at the end of the experiment.Compared with C,serum E₂ and T content decreased below the detection threshold in IM.The weight of ovaries in IM decreased to 25.21%compared with C.After the first immunization,compared with C,the proestrus and estrus proportion of estrus in IM were significantly decreased（P<0.01）,and the metestrus and diestrus percentage of estrus in IM were significantly increased（P<0.1%）,and the average days of estrus cycle was extremely prolonged（P<0.01）and the number of estrus cycles significantly decreased（P<0.1%）.The spleen weight and index of IM and SC were significantly lower than that in C（P<0.05）.There was no significant difference in the number of CD3⁺,CD4⁺T,CD8⁺T lymphocytes and NK cells among three groups（P>0.05）.But the number of B lymphocytes decreased significantly in IM and SC（P<0.05）.Compared with C,IM did not affect the serum concentration of IL-1β,IL-2,TNF-α,IFN-γ,IL-4,IL-6 and IL-10.The level of m RNA expression of IL-4 was extremely up-regulated（P<0.05）.However,it did not affect the level of m RNA expression of IL-1β,IL-2,TNF-α,IFN-γ,IL-6 and IL-10（P>0.05）.The levels of IL-1β,TNF-αand IFN-γin serum and the level of m RNA expression of TNF-αin SC were significantly higher than C.The other indexes are similar with IM.（2）36 female SD rats at the age of 4-5 weeks were randomly divided into six groups（n=6）:intact controls（C）,intact controls+BSA（C+BSA）,immunocastrates（IM）,immunocastrates+BSA（IM+BSA）,surgical castrates（SC）and surgical castrates+BSA（SC+BSA）.Bilateral ovariectomy was performed at the age of 7 weeks in the SC and SC+BSA,1 m L emulsified antigen（including 50μg GnRH-TDK）was injected intramuscularly in leg at the 9 weeks of the IM and IM+BSA,8 weeks later,the dose and method of the injection were as same as the first immunization,and no treatment was performed in C.Vaginal smears of female rats in C,C+BSA,IM and IM+BSA were observed every day after the first immunization and the booster immunization,respectively.The estrus cycle of female rats was observed for 30 consecutive days.In 12 wpv,C+BSA,IM+BSA and SC+BSA female rats in leg muscle injection of 1 m L BSA antigen（Specol as adjuvant,including 50μg BSA）.C,IM and SC female rats in the leg muscle injection of 1m L adjuvant.After injection,tail artery blood samples were collected in every 2 weeks.All rats were killed in 20 wpv,the weight of spleen and ovaries were separated and recorded.The serum E₂ and T concentration were detected by the RIA,and the serum anti-BSA antibody concentration was detected by ELISA.Compared with C and C+BSA,the levels of serum E₂ and T in IM and IM+BSA were decreased below the detection threshold.The weight of the ovaries decreased to27.17%and 25.77%compared with C and C+BSA respectively.Compared with C and C+BSA,the proestrus and estrus proportion of female rats in IM and IM+BSA were significantly decreased（P<0.01）,and the metestrus and diestrus percentage of estrus in IM and IM+BSA were significantly increased（P<0.1%）,and the average days of estrus cycle was extremely prolonged（P<0.01）and the number of estrus cycles significantly decreased（P<0.5）.There was no significant difference among six groups of spleen weight and spleen index（P>0.05）.The serum anti-BSA antibody concentration of female rats in 14 wpv,C+BSA,IM+BSA,and SC+BSA had high levels.From 14 wpv to the end of the experiment,the contents of anti-BSA antibodies of the three groups showed no difference（P>0.05）and maintained a high level.In conclusion:Active immunization against GnRH in female rats effectively reduce the serum E₂ and T concentration,stagnate the estrus cycle and inhibit the development of ovaries.Active immunization against GnRH would alter part of parameters relating to immunity（such as the number of B lymphocytes,m RNA expression level of IL-4 in spleen tissue and the content of IL-2 in blood）,and might induce a new Thl/Th2 balance in female rats.The results indicate that active immunization against GnRH would not affect the normal specific immunological response to the stimulation of foreign antigen in female rats.