Bovine Immunoglobulin M as a Major Factor Contributing to Serum-Induced Head-to-Head Agglutination in Bovine Spermatozoa1

One ejaculate from each of 5 bulls was used to characterize the factor from heifer serum which causes head-to-head agglutination in bovine spermatozoa. Serum from 10 virgin heifers was pooled, heat-treated and fractionated by Sepharose 6B chromatography. The fractions causing head-tohead agglutination and those which did not cause head-to-head agglutination were identified, pooled and lyophiized. The agglutinating fraction, nonagglutinating fraction or whole serum were added to 30 × 106 spermatozoa. Agglutinating fractions caused higher (P<0.01) head-to-head agglutination than nonagglutinating fractions at all protein concentrations tested and the agglutinating fraction caused higher head-to-head agglutination than whole serum on a mg protein/30 × 106 spermatozoa basis. A second experiment was conducted to purify bovine immunoglobulin M (IgM) and compare the dose response of the purified IgM to those of whole serum, agglutinating fractions, nonagglutinating fraction, bovine IgG1, IgG2 and porcine IgM. Procedures used to isolate bovine IgM resulted in recovery of IgM with a purity of >90%. This protein precipitated with monospecific goat anti-bovine IgM and gave only single precipitin bands with goat anti-bovine serum via agarose immunoelectrophoresis and Ouchterlony double immunodiffusion. Semen from a bull producing semen of average quality from Experiment 1 was used to determine the degree of head-to-head agglutination caused by purified bovine IgM, porcine IgM, whole serum, agglutinating fraction, nonagglutinating fraction, bovine IgG1 and lgG2. Total protein per 30 × 106 sperm required to induce half-maximal head-to-head agglutination was 0.49 mg, 1.14 mg and 2.8 mg for purified bovine IgM, agglutinating fraction and whole serum, respectively. The total IgM per 30 × 106 sperm required to induce half-maximal head-to-head agglutination was 0.45 mg, 0.21 mg and 0.11 mg for purified bovine IgM, agglutinating fraction and whole serum, respectively. Porcine IgM was as effective as bovine IgM in causing head-to-head agglutination, but neither IgG1 nor IgG2 caused head-to-head agglutination. There were no differences in acrosomal maintenance among treatments and levels of protein added. These data suggest that IgM is a major factor in serum responsible for causing head-to-head agglutination in bovine spermatozoa.