Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility

The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresi s (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a bre eding score for each stallion. Each score was calculated by dividing the nu mber of conceptions by the number of breedings for each stallion for four s uccessive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n=6) were collected with a Missouri-style artificial vagina. Imm ediately after collection, the semen sample was filtered and the gel fracti on removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pellete d sperm cells. Two-dimensional PAGE of the seminal plasma was performed und er denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P<0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P<0.05, r(2) = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were fo und to be negatively correlated (P<0.05, r(2) = 0.762, 0.730, 0.775 respect ively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasm a protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion se minal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to be tter insight into the nature of subfertility or infertility in the horse, a s well as to indicate better cryopreservation strategies. (C) 1999 by Elsev ier Science Inc.