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United States Department of Agriculture

Agricultural Research Service

Research Project: Development of New Technologies and Methods to Enhance the Utilization and Long-Term Storage of Poultry, Swine and Fish Gametes and Embryos

Location: Animal Biosciences and Biotechnology Laboratory

Title: The inner perivitelline layer sperm-hole assay: use of filter paper rings for the isolation of the perivitelline layer overlying the germinal disc and new observations on its morphology

Authors
item Bakst, Murray
item Eastridge, Janet
item Malecki, Irek -

Submitted to: Journal of Applied Poultry Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 1, 2013
Publication Date: January 8, 2014
Citation: Bakst, M.R., Eastridge, J.S., Malecki, I.A. 2014. The inner perivitelline layer sperm-hole assay: use of filter paper rings for the isolation of the perivitelline layer overlying the germinal disc and new observations on its morphology. Journal of Applied Poultry Research. 23:121-128.

Interpretive Summary: The inner perivitelline layer (IPL) sperm-hole assay provides an objective assessment of the number of sperm penetrating the hen’s ovum at the germinal disc (GD). That number will provide insight into numerous parameters concerning fertility including, but not limited to, the probability of the ovum being fertilized (true fertility), the duration of fertility, an assessment of mating activity, the impact of inseminating in vitro stored semen, and the influence of spiking a breeder flock. In this paper, we discuss the use of a filter paper ring (filter-ring) to isolate and process the IPL overlying the germinal disc (PL-disc) in five species of domestic birds. The morphology of the PL-disc is also described to assist those using the sperm-hole assay during the sperm hole counting. The filter-ring approach was successfully used with chicken, turkey, duck, goose, and Japanese quail eggs; the PL-disc remained intact and stretched across the filter-ring and sperm-holes, when present, were clearly discernible. Morphologically, the PL-disc appeared to be a silhouette of the GD including the germinal vesicle observed in follicular oocytes. The fibers forming the IPL had a honey-comb like arrangement that in faintly stained slides could be mistaken for actual sperm-holes. Given its diversity of applications and relative simplicity using the filter-ring, it is recommended that the sperm-hole assay be part of a quality control program at the hatchery. Already used in the scientific community, this filter-ring method has the additional advantage that the blastoderm may be collected before the staining process.

Technical Abstract: Sperm must penetrate the hen’s ovum (egg yolk) for fertilization. The inner perivitelline layer (IPL) is a fibrous protein mesh surrounding the ovum that the sperm attach to and then digest a hole through at the onset of the fertilization process. This process of sperm attachment and digestion of the IPL is done in a restricted region on the ovum’s surface called the germinal disc. Scientists have discovered that by isolating the IPL from laid eggs and counting the sperm holes, they are able to determine the probability of the ovum being fertilized (true fertility), the duration of fertility, provides an assessment of mating activity, the impact of inseminating in vitro stored semen, and the influence of spiking (adding new males) within a breeder flock. While important information to the commercial poultry producer, the main draw-back of using this procedure is that it takes too much time to prepare the IPL prior to examination. In this study, we describe a technique to isolate the IPL that is simple, fast, and applicable to several different domestic and non-domestic bird species. In addition we provide detailed information on the gross appearance and microscopic structure and of the IPL. This information will be very useful to commercial poultry companies as a quality control measure and to anyone doing fertility research with birds.

Last Modified: 10/25/2014
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