Detection of oocyte perivitelline membrane-bound sperm: a tool for avian collection management

The success and sustainability of an avian breeding programme depend on managing productive and unproductive pairs. Given that each breeding season can be of immeasurable importance, it is critical to resolve pair fertility issues quickly. Such problems are traditionally diagnosed through behavioural observations, egg lay history and hatch rates, with a decision to re-pair generally taking one or more breeding seasons. In pairs producing incubated eggs that show little or no signs of embryonic development, determining fertility is difficult. Incorporating a technique to assess sperm presence on the oocyte could, in conjunction with behaviour and other data, facilitate a more timely re-pair decision. Detection of perivitelline membrane-bound (PVM-bound) sperm verifies successful copulation, sperm production and sperm functionality. Alternatively, a lack of detectable sperm, at least in freshly laid eggs, suggests no mating, lack of sperm production/function or sperm-oviduct incompatibility. This study demonstrated PVM-bound sperm detection by Hoechst staining in fresh to 24-day-incubated exotic eggs from 39 species representing 13 orders. However, a rapid and significant time-dependent loss of detectable PVM-bound sperm was observed following incubation of chicken eggs. The PCR detection of sperm in seven species, including two bacterially infected eggs, demonstrated that this method was not as reliable as visual detection using Hoechst staining. The absence of amplicons in visually positive PVMs was presumably due to large PVM size and low sperm count, resulting in DNA concentrations too low for standard PCR detection. In summary, this study demonstrated the feasibility and limitations of using PVM-bound sperm detection as a management tool for exotic avian species. We verified that sperm presence or absence on fluorescence microscopy can aid in the differentiation of fertile from infertile eggs to assist breeding managers in making prompt decisions for pair rearrangements. This protocol is currently used to manage several breeding pairs in San Diego Zoo global avian conservation programmes.