Effects Of Active Immunization Against Three Different GnRH-like Peptide Constructs On Reproductive Performance In Male Rats

Surgical castration of animals is performed to prevent occurrence of boar taint in pork, aggressive and sexual behavior and uawanted pregnancies in fattening of bulls and heifers. In consequence, the castrated males were easier to handle. Therefore, traditional surgical castration generally results in permanent sterilization. Other disadvantages of surgical castration are the associated trauma, production setback and deaths. These disadvantages make it evident that effective and practical alternatives to surgical castration would be readily adopted in a number of species.Using active immunization against GnRH as a promising alternative to surgical castration, which avoids disadvantages of surgical castration. In the past years, some kinds of GnRH antigens against native GnRH-self peptide were designed and increase immunogenicity, such as the forms of GnRH with D-Lys replacing Gly at position 6 allows G6K-GnRH-tandem(TK) and G6K-GnRH-tandem-dimer (TDK) to conjugate to carrier protein improve their immunogenicity.Therefore, The present study was aimed to comparatively investigate the effects of acitive immunization against three different GnRH-peptide antigens on reproductive traits in Sprague-Dawlry (SD) male rats..Sixty male Sprague-Dawlry(SD) rats (BW 150-210 g) at the age of 5-6 weeks, were randomly allocated five groups (n=12):intact controls (no treatment), surgically castrated or vaccinated against 100 ug TDK, TK-OVA conjugate (TK-OVA) or TDK-OVA conjugate (TDK-OVA) in Specol adjuvant at 7 weeks of age (given booster injection 8 weeks later). All rats were decapitated at 12 weeks of post vaccination (wpv), to collect blood samples (at 0 wpv, every 2 weeks thereafter, and the day of decapitation)for antibody titter and hormone assays. At decapitation, testes and pituitary tissues(recorded the weights of both testes and pituitary and teats volume) were collected to detect the mRNA expressions of reproduction-related genes with real-time fluorescence quantitative PCR, and testes were excised for morphological observation. Results indicated that active immunization against three different GnRH-like peptide antigens all triggered a good antibody response, During the experimental period, serum anti-GnRH antibody titers in TK-OVA goup were comparable to that in TDK-OVA group, but both were significantly higher than that in TDK group(P<0.05). Testes in TK-OVA and TDK-OVA groups were reduce to 30% and 50% of both the average weight and volume of intact controls at decapitation, respectively (p<0.05), and testis histologic evaluation also displayed severe damage of leydig-cell interstitial tissue and absence of spermatids. As expected, the average weight and volume testes in TDK group shown a tendency to be decrased, though the values did not reach a significant level(1.50g Vs 1.63g) (p>0.05), and testis morphology also indicated a reduced number of the spermatogenic germs. Compared to intact males, the serum T, LH and FSH concentrations in TK-OVA and TDK-OVAgroups were significantly decreased 2 week after the primary vaccination, and maintained low until the end of the experiment (p<0.05), the serum hormones in TK-OVA group (LH: 0.60pg/ml, FSH:0.81pg/ml, T:0.20ng/ml) lower than that in TDK-OVA group (LH:1.18pg/ml,FSH:1.35pg/ml,T:0.37ng/ml) at decapitation. In contrast, serum T, LH and FSH concentrations in TDK group were decrased initially after the booster vaccination, and remained significantly thereafter until the end of the experiment (p<0.05), But, during the whole experimental period serum T, LH and FSH concentrations in TDK group (6.75pg/ml,6.86pg/ml,1.24ng/ml, respectively) were significantly high than those of the TK-OVA and TDK-OVA groups. By quantitative PCR analysis, after active immunization against three different GnRH-like peptide antigens,the mRNA expression levels of GnRH receptor, LH-β，FSH-β in the pituitary as well as LH receptor and FSH receptor in the testes shown a significantly down-regulated as compared to control males{all mRNA expression of reproduction-related gens were set act as 1) (p<0.05). In parallel with the changes of serum hormone levels, these tested gene expression in TDK group (0.27,0.37,0.24,0.47,0.62, respectively) were significantly higher than those in the TK-OVA(0.12,12,0.13,0.12,0.21, respectively) and TDK-OVA(0.12,0.15,0.15, 0.18,0.23, respectively) groups. In surgical castrates, the serum LH and FSH levels, and mRNA expression of reproduction-related genes in pituitary and testes were significantly increased after castration (p<0.05). Taken together, these results suggested that:1) active immunization against the three different immunocastrative GnRH-peptide immunogens all could triggered good antibody responses, reduce serum reproductive hormone levels and down-regulate reproductive-related gene expressions, thus suppressing reproductive functions of the male SD rats; 2) D-Lys6-GnRH-tandem-dimer its self, not conjugated to carrier protein, could be considered as alternative immuncastrative antigen.