211 REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AS A TOOL TO ELUCIDATE THE BIOLOGICAL ACTIVITY OF COMMERCIAL EQUINE CHORIONIC GONADOTROPIN (eCG)
R. H. Alvarez A , B. E. Almeida B , M. T. C. P. Ribela B , F. L. N. Natal A , A. J. F. Melo A and P. Bartolini BA Agency for Agribusiness Technology of São Paulo, Piracicaba, SP, Brazil;
B Biotechnology Department, IPEN-CNEN, São Paulo, SP, Brazil
Reproduction, Fertility and Development 26(1) 219-219 https://doi.org/10.1071/RDv26n1Ab211
Published: 5 December 2013
Abstract
Superovulation in ruminants can be induced with a single injection of equine chorionic gonadotropin (eCG). However, ovarian response is sometimes lower than expected because of, among other factors, the source of eCG used. This study aimed at establishing the physical-chemical profile of commercial eCG, in order to find differences which can be related to their biological activity. Four different commercial eCG products for veterinary use (A, B, C, D) and one eCG chemical reagent from Sigma (St. Louis, MO, USA), here used as reference preparation, were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C4-Grace Vydac 214 TP 54-column (25 cm × 4.6 mm I.D.), with UV detection at 220 nm. All eCG preparations presented at least three peaks with retention times (tR) of ~27(I), 34(II), and 36(III) minutes, with a peak at tR = 27 min common to A, C, D, and Sigma eCG, whereas preparation B did not present this peak. A bioassay test was carried with all of these preparations. Immature 21- to 25-day-old Wistar female rats received the equivalent to 10 IU of eCG of each one of these preparations. Autopsy was performed 48 h later and ovaries were removed and weighed. The average ovarian weight for preparations A, C, D, and Sigma were ~0.0795 ± 0.0107 g, whereas preparation B was 0.035 ± 0.007 g (P < 0.01). Preparation B was not different from saline (0.034 ± 0.002 g). In order to establish which one of these three peaks presented the highest biological activity, a mass equivalent to 10 IU of eCG from peaks I, II, and III of Sigma and of product A were studied. The average ovarian weight of animals injected with material from peak II and III (~0.0285 ± 0.003 g) were similar to that of the control whereas peak I produced ovarian weights of 0.059 ± 0.007 g and 0.075 ± 0.010 g for Sigma and product A, respectively (P < 0.01). These results suggest that the lack of ovarian response to eCG treatments can be related to differences in the physical-chemical profile of commercial eCG products and that RP-HPLC is a fast and reliable tool for detecting these differences.
Supported by FAPESP (Grant 11/13096-0).
