Evaluation of the chemiluminescent enzyme immunoassay system for the measurement of testosterone in the serum and whole blood of stallions

Abstract

Testosterone (T) concentration is a useful indicator of reproductive function in male animals. However, T concentration is not usually measured in veterinary clinics, partly due to the unavailability of reliable and rapid assays for animal samples. In this study, a rapid chemiluminescent enzyme immunoassay system (CLEIA system) that was developed for the measurement of T concentration in humans use was validated for stallion blood samples. First, serum T concentrations were measured using the CLEIA system and compared with those measured by a fluoroimmunoassay that has been validated for use in stallions. The serum T concentrations measured by the two methods were highly correlated (r = 0.9865, n = 56). Second, to validate the use of whole blood as assay samples, T concentrations in whole blood and in the serum were measured by the CLEIA system. T concentrations in both samples were highly correlated (r = 0.9665, n = 64). Finally, to evaluate the practical value of the CLEIA system in clinical settings, T concentrations were measured in three stallions with reproductive abnormalities after the administration of human chorionic gonadotropin (hCG). Two stallions with small or absent testes in the scrotum showed an increase in T production in response to hCG administration and one stallion with seminoma did not. In conclusion, the CLEIA system was found to be a rapid and reliable tool for measuring T concentrations in stallions and may improve reproductive management in clinical settings and in breeding studs.

Keywords: Chemiluminescent enzyme immunoassay; Cryptorchidism; Reproductive abnormalities; Stallions; Testosterone.

Fig. 1.

Correlation of serum testosterone (T) concentrations measured by the chemiluminescent enzyme immunoassay (CLEIA) system and by fluoroimmunoassay (FIA). Stallions 1 to 4 were administered 10,000 IU of human chorionic gonadotrophin (hCG) intravenously, and T concentrations in blood samples (n = 56) were measured by the CLEIA system and FIA. T concentrations measured by the two methods were strongly correlated (r = 0.9865).

Fig. 2.

Comparison of testosterone (T) concentrations measured by the chemiluminescent enzyme immunoassay (CLEIA) system and by fluoroimmunoassay (FIA). Four stallions (1 to 4) that presented no clinical signs were administered 10,000 IU of human chorionic gonadotrophin (hCG). T concentrations in serum samples were measured by the CLEIA system and by FIA. In the figures, T concentrations are shown as averages ± standard error of the mean. The left vertical axis shows hourly data and the right vertical axis shows daily data. “*” indicates that the mean of T concentrations is significantly higher than that before administration.

Fig. 3.

Correlation of testosterone (T) concentrations in whole blood and serum as measured by the chemiluminescent enzyme immunoassay (CLEIA) system. Stallions 5 to 8 were administered (i. m.) T enanthate (100 or 250 mg) for one to four days (daily). Blood samples (n = 64) were collected before and after T enanthate administration for 5 to 8 consecutive days. See Table 2 for details of the T enanthate treatment protocol in each stallion.

Fig. 4.

Concentrations of testosterone (T) in whole blood after T enanthate administration. Stallions 5 to 8 were administered T enanthate as shown in Table 2. Arrows indicate administration of T enanthate.

Fig. 5.

Changes in serum testosterone (T) concentrations (ng/ml) after human chorionic gonadotropin (hCG) administration in stallions with reproductive abnormalities. Stallion 9 (5,000 IU) and stallions 10 and 11 (10,000 IU) were administered hCG to determine T responses to hCG administration. See Table 3 for clinical findings of each animal. Stallion 11 was administered hCG twice with a two-month interval.