| Objective: Animal castration is still a problem which needs to be solved in the livestock production. Because the sequela is left from traditional operation, and the safety of the people and livestock is threatened during the process of operation and so on. The shortcomings from castration may be overcome with the immunocastration. So our current research was designed to study the preparation of immunocastration vaccine and its immunization effect and mechanism. Methods: (1) Synthetic GnRH-I hexamer gene was cloned into pMAL-c4x vector of maltose binding protein fusion expression system, and the recombinant plasmid was transformed into Escherichia coli (E.coli TB1). The over-expression and purification of MBP-GnRH-I6 were inducted with IPTG and polysaccharide resin ( amylose resin) affinity chromatography, respectively. The molecular weight and purity were analyzed by SDS-PAGE and HPLC, respectively; reactogenicity was identified using Western blot. (2) The healthy newborn Duroc×Landrace×Large White boars at the same age were assigned randomly to three groups of the following treatments: MBP-GnRH-I6 immunization, conventional surgical castration within 7 days of birth (negative control group ) and MBP immunization( positive control group). MBP-GnRH-I6 or MBP was emulsified in phosphate buffered saline (PBS) and f Al(OH)3 adjuvant. The first immunization was administered by intramuscular injection of 2mL emulsion (containing 1mg MBP-GnRH-I6). The booster injection was given by the same route and dose. Blood samples were taken via the jugular vein and refrigerated overnight at 4 oC. The samples were then centrifuged at 200g for 20 minutes at 4 oC. Serum was harvested and stored at -80 oC. Scrotal longitudinal length was measured using a caliper. The anti-GnRH antibodies and concentrations of testosterone in serum were determined by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. After the slaughter,the right testicles were used, and epididymides were removed. Testis weight was recorded, and the external size of testis was measured using a caliper. Testis tissue was fixed in formalin and sectioned at 5μm-thick. The slices were stained with hematoxylin and eosin (H & E). Morphological changes in the testes were evaluated under a microscope. Five sections of each pig testis were randomly selected, and the number of germ cells at different developmental stages in seminiferous tubules was counted using a morphologic analysis software. The immunocastration effect of MBP-GnRH-I6 was proved. (3) After the slaughter, hypothalumuses, anterior pituitary glands and testes were removed, frozen immediately in liquid nitrogen, and transported to the laboratory within 1 hour and stored at -80°C. The changes of hypothalamic GnRH-I and GnRH-I receptor mRNA, GnRH-IR, FSHβand LHβmRNA in pituitary and GnRH-IR, FSH receptor, LH receptor, 3βHSD and 17βHSD mRNA in testis were analyzed by using real time quantitative PCR. The ultrastructure of pituitary gonadotropin cells, testicular germ cells, Sertoli cells and Leydig cells were observed by transmission election microscopy. The objective was to investigate the effect of on reproductive hormones and its receptor gene of reproductive axis and the changes of ultrastructure in pituitary and testes with active immunization against MBP-GnRH-I6 in boars. (4) The body weights were measured at 1 d before immunization and slaughter, respectively. Additonally, the concentrations of pGH and IGF-I in serum were determined by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. Potential correlation between testosterone and IGF-I in serum was analyzed with Pearson correlation. Moreover, mechanism underlying the fast growth in immunocastration pigs and positive control was explored. (5) The concentrations of backfat androstenone and 3-methylindole were determined by HPLC. The boar taint of meat in MBP-GnRH-I6 immunized boars was explored. Results: (1) MBP-GnRH-I6 was over-expressed solubly in E.coli TB1, and highly purified MBP-GnRH- I6 fusion protein (purity:97.68% ) was obstained by polysaccharide resin affinity chromatography. The molecular weight was consistent with the theoretical value (50kDa), and Western blot showed that MBP-GnRH-I6 fusion protein was specificly reacted to GnRH-I. (2) Active immunization with MBP-GnRH-I6 increased the serum concentration of anti-GnRH antibodies (P<0.05) and reduced the serum concentration of testosterone (P<0.05) as compared with controls. Scrotal longitudinal length of boars immunized with MBP-GnRH-I6 increased slowly and was shorter than that of MBP immunized control animals (P<0.05). The weight and size of the testes from MBP-GnRH-I6 immunized boars were significantly lower than those from MBP immunized boars (P<0.05). Testes from boars immunized with MBP-GnRH-I6 showed a severe hypoplasia in seminiferous tubules. Stratified epithelium layers of the spermatogonium, primary spermatocytes, and spermatids were depleted and sperm were not observed. Furthermore, the number of germ cells at every developmental stage in seminiferous tubules of MBP-GnRH-I6 immunized boars was significantly less (P<0.01) than that of positive control boars. (3) Hypothalamic GnRH-IR and GnRH-I mRNA expression was not significant between MBP-GnRH-I6 immunized boars, positive control group and negative control animals. But the expression of FSHβmRNA, LHβmRNA in pituitary was obviously lower than the positive and negative control group (P<0.05). The study of ultrastructure showed that active immunization with MBP-GnRH-I6 reduced the number of granules of pituitary gonadotropin cell. Basement membrane of testicular seminiferous tubule was hyperplasia, and the number of mitochondria in primary spermatocytes was significantly decreased, and chromatin was massive in sperm nucleus and distributed around the nuclear membrane, and the number of cytoplasmic organelles in cells decreased. In Sertoli cells, the mitochondrial vacuolation and rupture was observed. In Leydig cell, a small number of mitochondrial vacuolation was also found, the number of lysosomes increased within the cytoplasm. (4) Active immunization against MBP-GnRH-I6 increased the body weight compared to negative control (P<0.05), but did not affect the level of pGH in serum of boars (P>0.05). However, IGF-I concentration of serum in MBP-GnRH-I6 immunized boars was significantly lower than that of the positive control group at 17~21 weeks of age (P<0.05), and markedly higher than that of the negative control animals at 21~25 weeks of age (P<0.05). Serum testosterone was positively correlated with IGF-I. (5) Active immunization against MBP-GnRH-I6 decreased the level of androstenone in backfat of boars, which were similar to the negative control animals that the androstenone content was below the detection limit. But the level of 3-methylindole was not affected in MBP-GnRH-I6 immunized boars (P>0.05). Conclusions: (1) A genetically engineered body GnRH-I6 was successfully constructed, and the MBP-GnRH-I6 was obtained with high purity and good reactogenicity. (2) MBP-GnRH-I6 immunized boars maintained low serum testosterone levels and atrophic testicular, indicating a successful immunocastration. (3) Active immunization against MBP-GnRH-I6 reduced the expression of pituitary FSHβmRNA, LHβmRNA and GnRH-IR mRNA and testicular FSH receptor, LH receptor, 3βHSD and 17βHSD mRNA, and damaged the ultrastructure of the pituitary gonadotropin cell, Sertoli cell, Leydig cell and sperm cell. The results revealed that the active immunization against MBP-GnRH-I6 affect reproductive function by reducing the number of reproductive hormone and its receptor mRNA, as well as destroying the cell ultrastructure in the reproductive axis in boars. (4) Active immunization against MBP-GnRH-I6 increased body weight (P<0.05) and the level of serum IGF-I (P<0.05), and its pathway may control serum IGF-I by serum testosterone in boars. (5) Active immunization against MBP-GnRH-I6 reduced the level of backfat androstenone and eliminated the boar taint. So the meat quality was improved. |
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