(231) Spermatic RXFP2 expression levels and seminal INSL3 concentrations among Japanese Black beef bull ejaculates with different levels of sperm morphological normality

Abstract Authors: Noritoshi Kawate¹, H. D. A. Wimalarathne¹, Kenta Arashi¹, Fumiyuki Iwaki², Mitsuhiro Sakase², Duritahara³, Hiroshi Harayama³

1. Graduate School of Veterinary Science, Osaka Metropolitan University, Osaka, Japan

2. Hokubu Agricultural Technology Institute, Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries, Hyogo, Japan

3. Graduate School of Agricultural Science, Kobe University, Hyogo, Japan

Abstract Text:

Japanese Black (JB) cattle are the most common beef breed in Japan and are renowned for their exceptional meat quality. Some candidate JB sires produce aberrant semen, including morphologically abnormal spermatozoa. Recently, various new semen biomarkers for bulls’ infertility or subfertility have been reported. Insulin-like peptide 3 (INSL3) is secreted from bovine testicular Leydig cells and its receptor (RXFP2) is expressed on the spermatozoa. The objective of this study was to examine the relationship between the RXFP2 expression levels on spermatozoa and INSL3 concentrations in the seminal plasma of fresh semen from beef bulls with different levels of sperm morphological normality. Ejaculates (n = 44) were collected from 21 yearling JB beef bulls and categorized into three groups based on the levels of sperm morphological normality: High (normal morphology ≥ 80%; n = 23), Mid (< 80% & ≥ 65%; n = 10) and Low (< 65%; n = 11). Immunofluorescence with anti-RXFP2 rabbit polyclonal antibody (primary antibody) and Alexa Fluor 488-labeled anti-rabbit IgG goat polyclonal antibody (secondary antibody) was used to determine the localization and expression levels of RXFP2 in spermatozoa. INSL3 and testosterone concentrations in seminal plasma were measured by a time-resolved fluorescence immunoassay and an enzyme immunoassay, respectively. Sperm RXFP2 was detected in the principal and equatorial segments of the acrosomal region, postacrosomal region, and neck in all groups. The levels of RXFP2 in the acrosomal principal segment, postacrosomal region, and neck in the Low group were significantly lower than those in the High and Mid groups, and those in the equatorial segment tended to be lower than those in the High group. The total level of RXFP2 in the Low group was also significantly reduced compared with that in the other two groups. Seminal plasma INSL3 concentrations were significantly higher in the Low group than in the other two groups, whereas testosterone levels did not differ significantly between the groups. In conclusion, the lower expression level of RXFP2 in the sperm head and neck in abnormal bovine semen with a higher percentage of malformation suggests possible links between increased sperm deformity and INSL3 receptor reduction. Higher seminal INSL3 concentrations in abnormal semen are probably related to fewer INSL3 receptors in spermatozoa. Seminal INSL3 concentrations negatively correlated with sperm morphological normality, whereas testosterone levels did not show such a correlation, highlighting the potential value of seminal INSL3 as a biomarker for assessing bull fertility.