C. G. Paniagua-Chavez | Geology Reports

USGS2006

Assessment of gamete quality for the eastern oyster (Crassostrea virginica) by use of fluorescent dyes

Evaluation of sperm motility is the single most widely used parameter to determine semen quality in mammals and aquatic species. While a good indicator for fresh sperm viability, post-thaw motility is not always effective at predicting fertilizing ability. Techniques using fluorescent dyes can assess functionality of mammalian sperm, but have not been widely applied in aquatic organisms. The eastern oyster Crassostrea virginica is an important mollusk in the United States, and cryopreservation protocols have been developed to preserve sperm and larvae to assist research and hatchery production. In this study, protocols were developed to assess sperm cell membrane integrity and mitochondrial function by flow cytometry and to assess viability of eggs by fluorescence microscopy. The fluorescent dyes SYBR 14 and propidium iodide (PI) (to assess membrane integrity) and rhodamine 123 (R123) (to assess mitochondrial membrane potential) were used to evaluate the quality of thawed oyster sperm previously cryopreserved with different cryoprotectant and thawing treatments. Membrane integrity results were correlated with motility of thawed sperm and mitochondrial membrane potential with fertilizing ability. Fluorescein diacetate (FDA) was used to assess cytotoxicity of cryoprotectant solutions and post-thaw damage to oyster eggs. The results indicated that membrane integrity (P = 0.004) and thawing treatments (P = 0.04), and mitochondrial membrane potential (P = 0.0015) were correlated with motility. Fertilizing ability was correlated with cryoprotectant treatments (P = 0.0258) and with mitochondrial membrane potential (P = 0.001). The dye FDA was useful in indicating structural integrity of fresh and thawed eggs. Exposure of eggs, without freezing, to dimethyl sulfoxide yielded higher percentages of stained eggs and fertilization rate than did exposure to propylene glycol (P = 0.002). Thawed eggs were not stained with FDA (<1%) and larvae were not produced.

Cryobiology↗

USGS2005

Cryopreservation of sperm of red abalone (Haliotis rufescens)

Abalone culture, a developing industry in Baja California, Mexico, would benefit from genetic improvement and controlled breeding. The use of cryopreserved sperm would allow germplasm availability, and this study was designed to develop sperm cryopreservation protocols for red abalone Haliotis rufescens . The acute toxic effects of the cryoprotectants dimethyl sulfoxide (DMSO), propylene glycol (PG), and glycerol (GLY) were assessed after suspending sperm in different concentrations, whereby cryoprotectant treatments of 10% DMSO and 10% GLY equilibrated for 10 min yielded the highest range of motile sperm in preliminary freezing trials and were used for cryopreservation studies. To determine effective cooling rates, three freezing chambers were tested. Replicate samples of sperm from 4 males were placed in 0.5-mL French straws and frozen using a commercial freezing chamber (CFC) used for bull sperm, a programmable rate chamber (PRC), and a manually controlled styrofoam chamber (MCC). For the CFC, the cooling rate was 16°C/min, from 4°C to −140°C. For the PRC and MCC, it was 1°C/min, from −20°C to −30°C. The samples were held at −30°C for 5 min before being plunged into liquid nitrogen (−196°C) for storage, and each sample was thawed in a water bath at 45°C for 8 s. The quality of thawed sperm was determined by estimating percent motility, evaluating membrane integrity using a dual-staining technique and flow cytometry, and estimating fertilization rate. Statistical analyses were performed using 2-way ANOVA where chamber and treatment were the independent variables. Sperm quality parameters were independent. For motilities, a significant interaction was noted between the cryoprotective treatment and the chamber type, whereby motilities for DMSO and GLY were higher ( P = 0.0055) using MCC. Membrane integrities were significantly lower after using the PRC than the CFC or the MCC ( P = 0.0167). The highest post-thaw motility (48 ± 7%) was found using sperm suspended in 10% glycerol and frozen in the MCC. The highest percent of intact membranes (56 ± 11%) was for sperm suspended in 10% glycerol and frozen in the CFC. The highest fertilization rate (29 ± 10%) was with samples frozen with 10% glycerol in the CFC. The use of cryopreserved sperm from red abalone provides an alternative breeding option for culture and the protocols delineated are the first developed for this species.

Baja California↗