A series of in vitro assays of sperm-egg binding were developed to identify potentially subfertile roosters. Initial assays used either segments of intact hen's egg perivitelline membrane (PVM) placed on a microscope slide or a heat-solubilized extract of PVM (HS-PVM) dried within a flat-bottomed microwell plate, with bound sperm detected with a DNA-specific stain and epifluorescence microscopy. An automated assay was developed using prestained sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate-HS-PVM and enumeration of bound sperm with a fluorometric microwell plate reader. Four populations of chickens differing in fertility were evaluated with the following results: 1) the correlation across lines between in vitro sperm binding and fertility was 0.83 (N = 40; p < 0.0001); 2) correlations with other seminal parameters were low; and 3) the relationship between sperm binding and fertility was not linear, but a threshold plot allowed identification of males with low binding and low fertility. Motile sperm from roosters, turkeys, bulls, humans, mice, rams, and stallions bound in a dose responsive manner. Features of binding were revealed by both scanning and transmission electron microscopy. Use of this assay to cull males whose semen appears normal by traditional modes of analysis but differs in the obligatory trait of sperm-egg binding could be of value to avoid expensive progeny testing.

We report use of an in vitro assay (Barbato et al., 1998: Biol Reprod 58:686–699) to assess binding ability of cauda epididymal mouse sperm to a surrogate zona pellucida and effect of a synthetic peptide (Amann et al., 1999: J Androl 20: 42–46) on fertilization ability in in vitro fertilization (IVF) tests. Sperm from C57Bl/6, CD1, and CF1 mice (4 replicates each) were evaluated for binding ability after exposure to 0 (control) and 80–1280 pM peptide. For control sperm, endogenous binding was C57Bl/6 < CD1 = CF1 (P < 0.05, 1‐way ANOVA). Across all three strains, exposure to > 320 pM peptide increased relative binding of sperm (P < 0.05; 2‐way general linear model; GLM). Strains differed both in basal binding ability and in response to synthetic peptide. To determine if IVF rate increased after exposure of sperm to peptide, ova from B6C3 mice (four replicate pools) were collected after eCG and hCG stimulation. Cumulus–oocyte complexes (COC; 8–15 ova in each of 3–6 drops/treatment) were incubated with hyperactivated C57Bl/6 sperm at ∼1500 sperm per ovum. Data for incubations were corrected for false‐positive classification to yield a better estimate of true cleavage rate, and then related to results observed with a tenfold greater sperm concentration. Relative cleavage rates were 0 peptide (0.48); 420 pM (0.78, P < 0.05); and 840 pM (0.90, P < 0.01; GLM and Tukey tests). IVF rate was increased by exposure of mouse sperm to peptide at concentrations effective in the in vitro assay, and use of peptide allowed use of 1/10 as many sperm. Mol. Reprod. Dev. 57:406–411, 2000. © 2000 Wiley‐Liss, Inc.