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A practical in vitro sperm-egg binding assay that detects subfertile males
G F Barbato1, P G Cramer, R H Hammerstedt
1Department of Poultry Science, Pennsylvania State University, University Park 16802, USA.
Abstract:
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.
Insights
Developing in vitro sperm-egg binding assays can identify subfertile roosters. This method accurately predicts fertility, aiding in early detection and culling of males with poor reproductive potential.
Area of Science:
- Animal Science
- Reproductive Biology
- Biotechnology
Background:
- Subfertility in roosters significantly impacts poultry production efficiency.
- Traditional semen analysis methods may not detect all causes of reduced fertility.
- Sperm-egg binding is a critical step in natural fertilization.
Purpose of the Study:
- To develop and validate in vitro assays for assessing rooster sperm-egg binding.
- To correlate in vitro sperm binding efficiency with in vivo fertility in chickens.
- To establish a tool for identifying potentially subfertile males early.
Main Methods:
- Development of in vitro assays using intact perivitelline membrane (PVM) or heat-solubilized PVM (HS-PVM).
- Detection of bound sperm using DNA-specific stains and epifluorescence microscopy.
- Automation of the assay using fluorescently labeled HS-PVM and a fluorometric plate reader.
Main Results:
- A high correlation (0.83) was observed between in vitro sperm binding and fertility across different chicken lines.
- Correlations between sperm binding and other seminal parameters were low.
- A threshold analysis identified males with low sperm binding and low fertility, even with normal semen appearance.
Conclusions:
- In vitro sperm-egg binding assays are effective predictors of rooster fertility.
- These assays can identify subfertile males missed by traditional analyses.
- The assay offers a valuable tool for selective breeding and improving flock fertility.