To date, no single in vitro assessment can estimate bull fertility. This research was aimed at evaluating the ability of a series of laboratory assessments to assign 50 Holstein Friesian bulls grouped as low (ER-NRR<-1.5), medium (-0.5<ER-NRR<+0.5) and high (ER-NRR≥+1.5) fertility based on estimated relative non-return rates (ER-NRR), to the two categories of low and medium-high fertility. Heterospermic insemination with a Piedmontese reference bull was employed to define an index of competitive binding ability (CBI) to the zona pellucida using fluorochrome-labeled sperm, and a competitive fertility index (CFI) using embryo paternal assignment by single nucleotide polymorphism (SNP). Furthermore, kinetic parameters, membrane integrity and sperm DNA/chromatin integrity (% DFI) were assayed. Low fertility bulls had lesser (P<0.05) values for total motility and membrane integrity, and a greater value of % DFI as compared to medium and high fertility groups. A modest (P<0.001) correlation was reported among ER-NRR and total motility (r=0.30), progressive motility (r=0.26), membrane integrity (r=0.43) and % DFI (r=-0.26). While % DFI alone allowed the identification of 70% lowly fertile bulls, combining membrane integrity, average path velocity and CBI allowed for identification of 78% of the lowly fertile sires. Paternal assignment by SNPs resulted in 96% of successful assignments and could provide an alternative support to microsatellites for in vivo studies based on heterospermic fertilization for estimating fertility.
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