Sperm Repository for a Breeding Program of the Eastern Oyster Crassostrea virginica: Sample Collection, Processing, Cryopreservation, and Data Management Plan

Abstract

The Eastern oyster Crassostrea virginica (Family Ostreidae) is one of the most important fishery and aquaculture species in the U.S. and is a keystone species for coastal reefs. A breeding program was initiated in 2019 to support the fast-growing aquaculture industry culturing this species in the Gulf of Mexico. Oysters from 17 wild populations in embayment along the U.S. Gulf of Mexico coast from southwest Florida to the Matagorda Bay, Texas were used as broodstock for the program to maximize genetic diversity in the base population. A sperm repository of the broodstock was established to support the breeding project. The goal of this study was to demonstrate the sperm sample collection, processing, cryopreservation, and the data management plan involved in the establishment of a sperm germplasm repository of base populations. The supporting objectives were to: (1) develop a data management plan for the sperm repository; (2) streamline the procedure for sample collection, processing, and cryopreservation; (3) incorporate sperm quality analysis into the procedure, and (4) archive the cryopreserved samples as a repository for future use in the breeding program. This sperm repository included a total of 102 male oysters from the 17 collection sites (six oysters per site). A data management plan was developed with six categories, including sample collection, phenotype, fresh sperm, genotype, cryopreservation, and post-thaw sperm, as guide for data collection. Sperm collection was accomplished by strip spawn, and fresh sperm production, motility, and fertility were recorded for quality analysis. Cryopreserved sperm samples were sorted, labelled, archived, and stored in liquid nitrogen for future use. Post-thaw motility (1-30%) and plasm membrane integrity (15.34-70.36%) were recorded as post-thaw quality parameters. Overall, this study demonstrated a streamlined procedure of oyster sperm collection, processing, and cryopreservation for establishing a sperm repository that can serve as a template for construction of oyster germplasm repositories for breeding programs.

Keywords: Gulf of Mexico; aquaculture; breeding; cryopreservation; eastern oysters Crassostrea virginica; germplasm; sperm repository.

Figure 1

Schematic map showing Eastern oyster Crassostrea virginica 17 collection sites (in order of decreasing longitude) along the Gulf of Mexico). Florida: Corrigan Reef (CR), Lone Cabbage Reef (LR), Seahorse Key (SK), Oyster Bay (OB), Alligator Harbor (AH) and Pensacola Bay (PB). Alabama: Alonzo Landing (AL) and Cedar Point (CP). Mississippi: Pascagoula (PS). Louisiana: Sister Lake (SL) and Lake Calcasieu (LC). Texas: Lake Sabine (LS), West Galveston Bay (WG), East Galveston Bay (EG), West Matagorda Bay (WM), East Matagorda Bay (EM) and San Antonio Bay (SA).

Figure 2

The procedure of sperm collection, processing, cryopreservation, and post-thaw quality analysis. (1) Phenotype measurement (from left to right): lining oysters, height and length standard, width standard, height, length, width, and whole-body weight. (2) Fresh sperm collection: sampling a piece of gonad, observation for sex determination, dissection of testis, filtering sperm suspension, sperm suspension in 50 mL tubes, determination of sperm concentration and motility, sampling of adduct muscle for genotype, fertility test of fresh sperm. (3) Cryopreservation process: mixing with cryomedium, packaging, sealing, arranging on freezing rack, loading into freezer, removing samples to liquid nitrogen after cooling to −80 °C, sorting samples, and storage in dewars. (4) Post-thaw sperm analysis: thawing sample straw, releasing sample into a 1.5 mL tube, post-thaw motility, dilution of post-thaw sample (100×), filter through 20 µm screen, staining with SYBR-14/propidium iodide for membrane integrity analysis, and analysis using flow cytometer.

Figure 3

Schematic procedure for Eastern oyster Crassostrea virginica sperm collection and processing for breeding and cryopreservation. Parameters recorded during the process are reported below each individual step.

Figure 4

Photographs of gonad development condition of male Eastern oysters Crassostrea virginica included in the sperm repository. One male is presented out of the six males from each of the 17 collection sites along the Gulf of Mexico coast. One example of a fully developed gonad condition of an Eastern oyster (collected from Cedar Key in Florida in April 2021) is present here as a comparison.

Figure 5

Fertilization rate (%) observed using fresh sperm of Eastern oysters Crassostrea virginica (n = 6, each male was used to cross two females) from different collection sites in the Gulf of Mexico. Groups labeled with the same letters (a–f) are not statistically different (p > 0.050).

Figure 6

Post-thaw sperm quality analyses in Eastern oysters Crassostrea virginica from 17 collection sites along the Gulf of Mexico. (A). Post-thaw sperm motility (PTSM, %); (B). Post-thaw sperm concentration (PTSC, cells/mL), and (C). Post-thaw sperm membrane integrity (PTSMI, %). Groups labeled with the same letters (a–f) are not statistically different (p > 0.05).