Genomic imputation and evaluation using high-density Holstein genotypes.

Genomic evaluations for 161,341 Holsteins were computed by using 311,725 of 777,962 markers on the Illumina BovineHD Genotyping BeadChip (HD). Initial edits with 1,741 HD genotypes from 5 breeds revealed that 636,967 markers were usable but that half were redundant. Holstein genotypes were from 1,510 animals with HD markers, 82,358 animals with 45,187 (50K) markers, 1,797 animals with 8,031 (8K) markers, 20,177 animals with 6,836 (6K) markers, 52,270 animals with 2,683 (3K) markers, and 3,229 nongenotyped dams (0K) with >90% of haplotypes imputable because they had 4 or more genotyped progeny. The Holstein HD genotypes were from 1,142 US, Canadian, British, and Italian sires, 196 other sires, 138 cows in a US Department of Agriculture research herd (Beltsville, MD), and 34 other females. Percentages of correctly imputed genotypes were tested by applying the programs findhap and FImpute to a simulated chromosome for an earlier population that had only 1,112 animals with HD genotypes and none with 8K genotypes. For each chip, 1% of the genotypes were missing and 0.02% were incorrect initially. After imputation of missing markers with findhap, percentages of genotypes correct were 99.9% from HD, 99.0% from 50K, 94.6% from 6K, 90.5% from 3K, and 93.5% from 0K. With FImpute, 99.96% were correct from HD, 99.3% from 50K, 94.7% from 6K, 91.1% from 3K, and 95.1% from 0K genotypes. Accuracy for the 3K and 6K genotypes further improved by approximately 2 percentage points if imputed first to 50K and then to HD instead of imputing all genotypes directly to HD. Evaluations were tested by using imputed actual genotypes and August 2008 phenotypes to predict deregressed evaluations of US bulls proven after August 2008. For 28 traits tested, the estimated genomic reliability averaged 61.1% when using 311,725 markers vs. 60.7% when using 45,187 markers vs. 29.6% from the traditional parent average. Squared correlations with future data were slightly greater for 16 traits and slightly less for 12 with HD than with 50K evaluations. The observed 0.4 percentage point average increase in reliability was less favorable than the 0.9 expected from simulation but was similar to actual gains from other HD studies. The largest HD and 50K marker effects were often located at very similar positions. The single-breed evaluation tested here and previous single-breed or multibreed evaluations have not produced large gains. Increasing the number of HD genotypes used for imputation above 1,074 did not improve the reliability of Holstein genomic evaluations.

Stimulation of bull sperm hyaluronidase by polycations.

The activity of bull sperm hyaluronidase (hyaluronate 3-glycanohydrolase, EC 3.2.1.36) is increased by the inclusion of polycations in the assay mixture. At pH 3.8, bovine serum albumin and histone give the greatest stimulation, while protamine sulfate, spermine, spermidine and hyamine 2389 stimulate to a lesser extent. Enzyme activity increases with serum albumin concentration to a nearly constant, high level at serum albumin concentrations greater than 1 mg/ml. Other stimulatory compounds show a similar concentration dependence except that inhibition of enzyme activity occurs at high concentrations of stimulator. The degree of stimulation depends on the pH, sample concentration and substrate concentration. Enzyme preparations with a low protein content give the greatest stimulation, while preparations with a high protein content show little stimulation. The concentration of serum albumin required for maximum stimulation increases with increased hyaluronic acid concentration. The results suggest that the stimulation of sperm hyaluronidase is nonspecific and results from an interaction of the polycation with hyaluronic acid. Since protein in the enzyme preparation substitutes for exogenous stimulator to a varying degree, serum albumin should be included in the assay mixture for sperm and testicular hyaluronidase to assure measurement of maximum enzyme activity.

Isolation of the outer acrosomal membrane from bull sperm.

A procedure is described for subcellular fractionation of bull sperm which allows the isolation of outer acrosomal membrane without the use of detergent. After washing to remove seminal plasma contaminants, the acrosomal membrane is removed by homogenization and separated on a two-step sucrose gradient. The isolated membranes have been characterized by light and electron microscopy and enzyme analysis. While the acrosomal enzymes hyaluronidase and acrosin are bound to the isolated membranes, they represent only a small percentage of the total activity and therefore do not provide reliable marker enzymes for this fraction. Subcellular fractionation of sperm also yields information on the solubility of acrosomal enzymes. Two types of acrosomal enzymes have been identified on the basis of their distribution in gradient fractions. Both alpha-fucosidase and beta-N-acetyl glucosaminidase are concentrated in the soluble fraction of the gradient. In contrast, over 70% of the acrosin and hyaluronidase activity remains associated with the sperm pellet. These differences in solubility of these enzymes may reflect differences in their function in fertilization.