Effects of Novel Oral and Plasmid-Injected KISS1 DNA Vaccines on Immunocastration and in Inhibiting Mutton Odour of Male Goats

Yan-Guo Han, Jia-Yuan Wu, Ri-Su Na, Wei-Jiang Si, Yu-Qin Han, Yan Zeng, Yong-Ju Zhao and Yong-Fu Huang*

College of Animal Science and Technology, Southwest University, Chongqing Key Laboratory of Forage and Herbivore, Chongqing Engineering Research Centre for Herbivores Resource Protection and Utilization, Chongqing, China

ABSTRACT

This study analysed the effect of novel oral and plasmid-injected KISS1 DNA vaccines on immunocastration and in inhibiting mutton odour of male goats. In this study, 20 male goats aged eight weeks were randomly divided into the oral and plasmid-injected KISS1. DNA vaccine groups (groups T1 and T2) and the empty vector groups (groups C1 and C2) for immunisation. Blood samples were collected at 0, 3, 6, 10 and 14 weeks after primary immunisation for the detection of kisspeptin antibodies and testosterone by indirect enzyme-linked immunosorbent assay. Scrotal circumference was detected from week 0 to week 14 after primary immunisation, and the testes were weighed at week 14 after primary immunisation after slaughter. The concentrations of 4-methyloctanoic acid and 4-methylnonanoic acid in waist subcutaneous adipose tissue were detected using gas chromatography. Immunisation of oral and plasmid-injected KISS1 DNA vaccines induced strong antibody response, significantly suppressed testosterone secretion and reduced scrotal circumference and testis weight. It also significantly reduced the concentrations of 4-methyloctanoic acid and 4-methylnonanoic acid in adipose tissue. These findings indicated that immunisation of oral and plasmid-injected KISS1 DNA vaccines can effectively suppress testosterone secretion, testis growth and mutton odour, and the novel oral KISS1 DNA vaccine is more promising in the large-scale preparation of immunocastration vaccines because of simple operations and low cost.

Article Information

Received 12 October 2019

Revised 02 December 2019

Accepted 12 December 2019

Available online 22 April 2020

Authors’ Contribution

Y-GH and Y-FH planned the experiment. Y-GH, J-YW and R-SN executed the experiment and drafted the manuscript. W-JS, Y-QH, YZ and YJ-Z helped in laboratory work, statistical analysis and preparation of manuscript.

Key words

KISS1, DNA vaccine, Steroid hormones, Testis growth, Mutton odour

DOI: https://dx.doi.org/10.17582/journal.pjz/20191012101017

* Corresponding author: H67738337@swu.edu.cn

0030-9923/2020/0004-1603 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

Immunisation against KISS1 DNA vaccines can effectively suppress animal gonad growth and fertility. Kisspeptins, encoded by the KISS1 gene, and their receptor GPR54 play a crucial role on puberty onset and adult fertility by controlling the release of hypothalamic gonadotropin-releasing hormone (GnRH) (Cao et al., 2019; Wassie et al., 2019). KISS1 is a novel target for developing a DNA immunocastration vaccine. Our previous studies showed that immunisation against the plasmid recombinant KISS1 DNA vaccine (pVAX-HBsAg-S-KISS1-54-asd) can inhibit testosterone secretion, testis growth and sexual behaviour in ram lambs (Han et al., 2015, 2018a). To further improve the immunogenicity of the KISS1 DNA vaccine and reduce costs, we developed a novel oral KISS1 DNA vaccine containing tissue plasminogen activator (tPA) signal peptide and two copies of KISS1-54, which is presented by the attenuated Salmonella choleraesuis (C500) (Han et al., 2018b, 2019a, b; Dong et al., 2018). Immunisation of the novel oral KISS1 DNA vaccine can effectively suppress steroid hormone secretion, gonad growth and fertility in mice (Han et al., 2018b). However, the immunocastration effect of the novel oral KISS1 DNA vaccine in large animals remains unclear.

Immunocastration can reduce male animal-associated odours, especially in pigs, goats and sheep. Boar taint is mainly caused by androstenone in the testes and skatole and indole in adipose tissue (Heyrman et al., 2019). Mutton odour in goats or sheep is mainly caused by 4-methyloctanoic acid and 4-methylnonanoic acid in adipose tissue and androstenone in testes (Chen et al., 2012; Gravador et al., 2019). GnRH is a commonly used target for developing immunocastration vaccines. Immunisation against GnRH can effectively reduce boar taint and mutton odour (Heyrman et al., 2019). However, the immunisation effect against the KISS1 DNA vaccine in inhibiting male animal-associated odours remains unclear.

To the best of our knowledge, the novel oral KISS1 DNA vaccine has not been applied to the immunocastration of large animals, and limited information is available regarding the effect of the KISS1 DNA vaccine in inhibiting male animal-associated odours. Therefore, this study detected the effect of novel oral and plasmid-injected KISS1 DNA vaccines on immunocastration and in inhibiting mutton odour of male goats.

Materials and methods

Twenty healthy male Dazu black goats (Ruifeng Modern Agriculture Development Co., Ltd., Chongqing, China) aged eight weeks were used in the study. All animal experiments were approved by the Science and Technology Ethics Committee of Southwest University, China. These ram lambs were randomly divided into four groups, vaccine groups T1 and T2 and control groups C1 and C2 (five lambs each group), on the basis of comparable body weight and scrotal circumference.

Novel oral and plasmid injection types of KISS1 DNA vaccines, C500 (pVAX-tPA-HBsAg-S-2KISS1-54-asd) and pVAX-tPA-HBsAg-S-2KISS1-54-asd, were successfully developed by our laboratory (Han et al. 2018b). Ram lambs in groups T1 and C1 were orally given C500 (pVAX-tPA-HBsAg-S-2KISS1-54-asd) and C500 (pVAX-asd) empty vector bacteria at a dose of 5 × 1011 CFU, respectively. Ram lambs in groups T2 and C2 were intramuscularly injected with 1 mg of pVAX-tPA-HBsAg-S-2KISS1-54-asd and 1 mg of pVAX-asd empty plasmid, respectively. The procedures for oral and plasmid injection types of the KISS1 DNA vaccine were referred to previous studies (Wassie et al., 2019). These ram lambs were boosted twice at an interval of three weeks. Blood samples were collected at weeks 0 (before primary immunisation), 3, 6, 10 and 14 after immunisation by centrifugation at 1157 × g for 10 min.

The titres of specific anti-kisspeptin IgG antibodies were detected by indirect enzyme-linked immunosorbent assay (ELISA) (Han et al., 2015, 2018b). Horseradish peroxidase-labelled rabbit anti-goat IgG secondary antibody (Abbkine, Inc., Redlands, CA, USA) was used in this study. Serum testosterone concentrations were measured by ELISA using commercial kits (Cusabio Biotech, Wuhan, China). The intra- and inter-assay coefficients of variation were less than or equal to 15%, respectively. Scrotal circumference was measured at weeks 0, 3, 6, 10 and 14 after primary immunisation by using a flexible plastic tape in accordance with previous studies (Han et al., 2015). The ram lambs were slaughtered at 14 weeks after the primary immunisation, and their bilateral testes were then weighed.

After slaughter, the ram lambs’ waist subcutaneous adipose tissues were collected and stored at −18 °C until use. To evaluate the mutton odour, the concentrations of 4-methyloctanoic acid and 4-methylnonanoic acid in the subcutaneous adipose tissue were detected by gas chromatography in accordance with a previous study (Chen et al., 2012).

Statistically significant differences (P< 0.05) between groups in terms of anti-kisspeptin antibody, serum testosterone concentrations, scrotal circumference, testis weight and concentrations of 4-methyloctanoic acid and 4-methylnonanoic acid in subcutaneous adipose tissue were analysed with one-way ANOVA using SAS 8.1. The mean values between groups were compared using Duncan’s multiple range test. Data were presented as mean ± standard deviation (SD).

Results

Kisspeptin antibody response was found in vaccine groups (groups T1 and T2) at weeks 3, 6, 10 and 14 after primary immunisation. The anti-kisspeptin antibody titres in groups T1 and T2 were significantly higher than those in groups C1 and C2 at weeks 6, 10 and 14 after primary immunisation, respectively (Fig. 1, P < 0.05); however, no significant difference was observed between groups T1 and T2 (Fig. 1, P > 0.05).

The ram lambs in groups T1 and T2 showed significantly lower serum testosterone concentrations than those in groups C1 and C2 at weeks 6, 10 and 14 after primary immunisation, respectively (Fig. 2, P < 0.05); however, no significant difference was observed between groups T1 and T2 (Fig. 2, P > 0.05).

Scrotal circumference in groups T1 and T2 was significantly lower than that in groups C1 and C2 at weeks 3, 6, 10 and 14 after primary immunisation, respectively (Fig. 3, P < 0.05); however, no significant difference was observed between groups T1 and T2 (Fig. 3, P > 0.05).

The ram lambs in groups T1 and T2 presented significantly lower testis weight than those in groups C1 and C2, respectively (Fig. 4, P < 0.05); however, no significant difference was observed between groups T1 and T2 (Fig. 4, P > 0.05).

The 4-methyloctanoic acid and 4-methylnonanoic acid in the subcutaneous adipose tissue were detected to evaluate the effect of KISS1 gene immunisation on mutton odour. The concentrations of 4-methyloctanoic acid and 4-methylnonanoic acid in groups T1 and T2 were significantly lower than those in groups C1 and C2, respectively (Fig. 5, P < 0.05); however, no significant difference was observed between groups T1 and T2 (Fig. 5, P > 0.05).

Discussion

Immunocastration is an effective measure in suppressing animal reproduction, aggressive behaviour and male-associated odours. Immunisation of the novel oral KISS1 DNA vaccine fused with tPA signal peptide and two copies of KISS1-54 can effectively suppress steroid hormone secretion, gonad growth and fertility in mice (Han et al., 2018b). However, the immunocastration effect of the novel oral KISS1 DNA vaccine in large animals remains unclear. Male animal-associated odours in adipose tissue significantly affect the consumer’s acceptance of the meat (Gravador et al., 2019). Immunocastration against GnRH vaccine can reduce boar taint and mutton odour (Heyrman et al., 2019; Han et al., 2019c). However, the immunisation effect of the KISS1 DNA vaccine in suppressing male animal-associated odours remains unclear. Hence, this study evaluated the effects of the novel oral KISS1 DNA vaccine on immunocastration and in inhibiting mutton odour of male goats.

In this study, immunisation of the novel oral and plasmid-injected KISS1 DNA vaccines effectively suppressed testosterone secretion and testis growth of male goats by inducing the anti-KISS1 antibody. The specific KISS1 antibody response in the oral and plasmid-injected vaccinated groups was found from week 3 to week 14 after primary immunisation and reached a peak from week 6 to week 10. Immunisation of oral and plasmid-injected KISS1 DNA vaccines suppressed testosterone secretion in male goats, which could inhibit testis growth, spermatogenesis and sexual behaviour (Xu et al., 2018). Scrotal circumferences in oral and plasmid-injected vaccinated male goats were lower compared with those of the control groups from week 3 to week 14 after primary immunisation, and testes weight in oral and plasmid-injected vaccinated male goats was also lower compared with control groups, which indicated that immunisation of oral and plasmid-injected KISS1 DNA vaccines suppressed the testis growth of male goats. These results were similar with the immunisation effect of other KISS1 DNA vaccines in suppressing testis growth (Han et al., 2015, 2018a). Our results showed that the novel oral KISS1 DNA vaccine could achieve the same immunocastration effect as the plasmid-injected KISS1 DNA vaccine. The novel oral KISS1 DNA vaccine does not require plasmid extraction and purification, which is easy to operate and saves cost (Han et al., 2019b). Hence, the novel oral KISS1 DNA vaccine is more promising for the large-scale preparation of immunocastration vaccines.

To the best of our knowledge, this study is the first to analyse the immunisation effect of the KISS1 DNA vaccine in suppressing mutton odour. Our results revealed that the immunisation of oral and plasmid-injected KISS1 DNA vaccines effectively reduced the concentrations of 4-methyloctanoic acid and 4-methylnonanoic acid, which are the main compounds responsible for mutton odour in the adipose tissue of male goats (Gravador et al., 2019). Moreover, mutton odour in goats can also be caused by androstenone in the testes (Xu et al., 2018; Heyrman et al., 2019). Our results showed that the testosterone concentrations in oral and plasmid-injected vaccinated male goats were lower compared with those in control groups. These results indicated that immunisation against oral and plasmid-injected KISS1 DNA vaccine could effectively suppress mutton odour of male goats.

Conclusion

Immunisation against oral and plasmid-injected KISS1 DNA vaccine can effectively suppress testosterone secretion, testis growth and mutton odour, and the novel oral KISS1 DNA vaccine is more promising in the large-scale preparation of immunocastration vaccines because of simple operations and low cost. Further studies will focus on the mechanism of the novel oral KISS1 DNA vaccine in immunocastration and suppressing mutton odour.

Acknowledgements

This study was funded by the National Key Research and Development Plan of China (No. 2018YFD0502003), the Chongqing Science and Technology Innovation Special Project (No. cstc2019jscx-gksbX0135, No. cstc2017shms-zdyfX0059) and the Innovation Team Building Program in Chongqing University (No. CXTDG201602004).

Statement of conflict of interest

The authors have declared no conflict of interest.

References

Cao, Y., Li, Z., Jiang, W., Ling, Y. and Kuang, H., 2019. Reprod. Biol. Endocrinol., 17: 65. https://doi.org/10.1186/s12958-019-0511-x

Chen, H., Wang, Y., Jiang, H. and Zhao, G., 2012. Meat. Sci., 92: 715-720.

Dong, F., Luo, X., Yuan, D., Liang, A. and Yang, L., 2018. Pakistan J. Zool., 50: 1875-1883. https://doi.org/10.17582/journal.pjz/2018.50.5.1875.1883

Gravador, R.S., Harrison, S.M., Monahan, F.J., Gkarane, V. and Brunton, N.P., 2019. J. Fd. Sci., 84: 80-85.

Han, Y., Liu, G., Jiang, X., Ijaz, N., Tesema, B. and Xie, G., 2015. Vaccine, 33: 777-782. https://doi.org/10.1016/j.vaccine.2014.12.054

Han, Y.G., Liu, G.Q., Jiang, X.P., Xiang, X.L., Huang, Y.F., Nie, B., Zhao, J.Y., Nabeel, I. and Tesema, B., 2018a. Asian-Australas. J. Anim. Sci., 31: 835-841.

Han, Y., Peng, X., Li, K., Jiang, X., Liu, G., Yang, L., Liang, C., Zhao, Y., Huang, Y., E, G., Zhao, Y., Huang. Y., 2018b. Vaccine, 36: 6631-6639.

Han, Y.G., Peng, X.L., Li, K., Zhao, Y.H.T., Jiang, X.P., E, G.X., Zhao, Y.J., Zhao, Q.T. and Huang, Y.F., 2019a. Pakistan J. Zool., 51: 413-419. https://doi.org/10.17582/journal.pjz/2019.51.2.413.419

Han, Y.G., Ye, J.H., Zhao, Q.T., Huang, Y.J., Li, K., Huang, Y.F. and Xu, Li., 2019b. Pakistan J. Zool., 51: 1711-1719. https://doi.org/10.17582/journal.pjz/2019.51.5.1711.1719

Han, X., Zhou, M., Cao, X., Du, X., Meng, F., Bu, G., Kong, F., Huang, A. and Zeng, X., 2019c. Theriogenology, 131: 32-40.

Heyrman, E., Kowalski, E., Millet, S., Tuyttens, F.A.M., Ampe, B., Buys, N., Wauters, J., Vanhaecke, L. and Aluwé, M., 2019. Res. Vet. Sci., 124: 293-302. https://doi.org/10.1016/j.rvsc.2019.04.010

Wassie, T., Liu, G., Jiang, X., Tesema, B., Han, Y., Zhao, J., Girmay, S. and Ahmad, H.I., 2019. Theriogenology, 125: 193-202. https://doi.org/10.1016/j.theriogenology.2018.10.029

Xu, M., Xu, C., Liu, F., Shen, X., Meng, J., Chen, H., Yang, J., Zhou, P., Gao, R. and Gan, S., 2018. Biol. Reprod., 99: 461-472.