In practice, two injections of gonadotropin‐releasing hormone (GnRH) vaccine are recommended for pig immunocastration for effective outcomes. The present study aimed to investigate the expressions of cytochrome P450 aromatase (P450arom) and anti‐Müllerian hormone (AMH) in testes, testicular length and testicular histomorphometry of the fattening pigs receiving the first injection of GnRH vaccine 6 weeks earlier than the standard protocol. Based on vaccination protocol, 24 pigs were equally divided into three groups: T1 was vaccinated at 15 and 19 weeks of age, T2 received vaccine at 9 and 19 weeks of age and C remained intact. P450arom and AMH expressions were analysed using immunohistochemistry and Western blot. The results revealed that testicular length was highest in C pigs, but not different between T1 and T2 groups (6.5 ± 0.2 versus 6.9 ± 0.3 cm, p = .538). Histomorphometry demonstrated that the height of spermatogenic epithelia, the diameter of seminiferous tubules and the number of seminiferous tubules between T1 and T2 groups were not different (p > .05). For P450arom, immunohistochemistry revealed that H‐score of C group was significantly higher than that of both T1 and T2 groups. Western blot analysis showed that C group possessed the densest protein band. Moreover, H‐score between T1 and T2 groups was not significantly different. Protein band intensity between both groups was not apparently different. As for AMH, C pigs had significantly lower H‐score than both T1 and T2 pigs. Furthermore, T2 pigs possessed significantly higher H‐score than T1 pigs. Western blot analysis showed that the most intense protein band was found in T2 group. In summary, GnRH vaccine affected testicular development and functions. The first injection could be performed either at 9 or 15 weeks of age since both protocols contributed to comparable results in aspect of testicular length, histomorphometry and expressions of P450arom and AMH.

This study aimed to investigate the immunoexpression of Ki-67 protein, androgen receptor (AR), and estrogen receptor beta (ERβ) in testicular tissues of male pigs immunocastrated using GnRH vaccine (Improvac™, Zoetis Co., Ltd., Thailand) with different times. Totally, 30 male pigs were classified by castration protocol into three groups: T1 (n = 10) consisted of pigs immunocastrated at 14 and 18 weeks of age, T2 (n = 10) included pigs immunocastrated at 9 and 19 weeks of age, and C (n = 10) contained intact pigs. The results revealed that testicular length of pigs in C was longer than that of both T1 (8.1 ± 0.76 vs 6.5 ± 0.5 cm, p < 0.001) and T2 (8.1 ± 0.76 vs 6.9 ± 1.0, p = 0.007). Spearman correlation coefficients showed negative correlation between testicular length and H-score of AR (r = -0.38, p = 0.037), as well as positive correlation between testicular length and Ki-67 index (r = 0.602, p < 0.001). Generally, mean Ki-67 index and mean H-scores of AR and ERβ of pigs in T1 were not different from those in T2 (p > 0.05). However, mean Ki-67 index and mean AR H-scores of T1 and T2 were significantly different from C group (p < 0.05). In summary, the immunocastration significantly affected testicular length, including expressions of Ki-67, AR, and ERβ in pig testes. Moreover, the duration between two shots of GnRH vaccine could be extended from 4 to 10 weeks without difference in Ki-67 protein, AR, and ERβ immunoexpressions.

The purpose of this study was to examine the expression of estrogen receptor alpha (ERα) in the testicular tissues of male fattening pigs injected with the first gonadotropin-releasing hormone (GnRH) vaccine (Improvac™, Zoetis, Thailand) for immunocastration 6 weeks earlier than the standard protocol. All pigs (n=24) were divided into three groups on the criterion of immunocastration protocol: A (n=8) was injected with GnRH vaccine at 15 and 19 weeks old, B (n=8) received GnRH vaccine at 9 and 19 weeks old, and C (n=8) remained untreated. Expression of ERα was investigated using an immunohistochemistry, appraised by an image analysis application (3DHISTECH, Budapest, Hungary), and reported as a H-score. The results revealed that testicular histoarchitecture of the immunocastrated pigs was less developed than that of the intact pigs. ERα was localized both in the seminiferous tubules and interstitial areas of all groups. ERα H-score of the C pigs was lowest (5.49±4.17) among groups and significantly lower than that of both A and B groups (P<0.05). However, the H-score of ERα between A and B groups was not different from each other (21.41±12.61 vs 23.91±11.47, P>0.05). In summary, the first injection of GnRH vaccine either at 9 or 15 weeks of age contributed to a similar result of testicular ERα expression.