Willingness-to-Pay for Genetic Attributes in Aquaculture Industries

The genetic make-up of fish stocks is an important factor in aquaculture production. Choice-based conjoint analysis is used to determine importance of genetic improvements to grow-out producers and an estimated willingness-to-pay for selected attributes. Results from a national survey of aquaculture producers, reveal growth rate as the most important attribute.Resource /Energy Economics and Policy,

Cryopreservation of Sperm of Spotted Seatrout (Cynoscion nebulosus)

Cryopreservation of fish sperm has applications in preserving genetic resources from stocks of endangered fishes, replenishing fisheries, reducing the number of males needed in hatchery situations, and allowing repeated spawning of specific males. As part of a larger study on artificial breeding of sciaenid fishes, we developed procedures for collection, handling, refrigerated storage, and cryopreservation of spotted seatrout sperm. Hanks\u27 balanced salt solution (HBSS) was used as an extender for collection and storage of sperm. Sperm motility in relation to graded concentrations of HBSS was used to determine the osmolality at which sperm were activated. Based on these findings, HBSS was prepared at 201 mOsm/kg as an extender for sperm storage. To determine if ions present in HBSS were involved in sperm activation, separate activating solutions were prepared by the addition of NaCl, CaCl2, KCl, Na2HPO4, or MgSO4 to aliquots of a stock glucose solution (185 mOsm/kg). The chemicals were added at the concentration of each found in 1-x HBSS. Only the glucose solution containing 8 g/l NaCl(424 mOsm/kg) produced activation of sperm. We also evaluated four chemicals as cyroprotectants: methanol, glycerol, dimethyl sulfoxide (DMSO), and n,n-dimethyl acetamide. Two freezing rates were evaluated by placing samples at either of two heights within a nitrogen vapor shipping dewar. The highest post-thaw motilities were in 10% DMSO with an average retention of 60% of initial motility at the lower position in the dewar, and 37% at the upper position. A third freezing rate was produced using a computer-controlled freezer programmed for a rate of -45ºC/min, yielding a retention of initial motility of 31%. Our freezing and transport of cryopreserved sperm in shipping dewars demonstrate the utility of this procedure for field applications

Cryopreservation of Sperm of Spotted Seatrout (Cynoscion nebulosus)

Cryopreservation of fish sperm has applications in preserving genetic resources from stocks of endangered fishes, replenishing fisheries, reducing the number of males needed in hatchery situations, and allowing repeated spawning of specific males. As part of a larger study on artificial breeding of sciaenid fishes, we developed procedures for collection, handling, refrigerated storage, and cryopreservation of spotted seatrout sperm. Hanks\u27 balanced salt solution (HBSS) was used as an extender for collection and storage of sperm. Sperm motility in relation to graded concentrations of HBSS was used to determine the osmolality at which sperm were activated. Based on these findings, HBSS was prepared at 201 mOsm/kg as an extender for sperm storage. To determine if ions present in HBSS were involved in sperm activation, separate activating solutions were prepared by the addition of NaCl, CaCl2, KCl, Na2HPO4, or MgSO4 to aliquots of a stock glucose solution (185 mOsm/kg). The chemicals were added at the concentration of each found in 1-x HBSS. Only the glucose solution containing 8 g/l NaCl(424 mOsm/kg) produced activation of sperm. We also evaluated four chemicals as cyroprotectants: methanol, glycerol, dimethyl sulfoxide (DMSO), and n,n-dimethyl acetamide. Two freezing rates were evaluated by placing samples at either of two heights within a nitrogen vapor shipping dewar. The highest post-thaw motilities were in 10% DMSO with an average retention of 60% of initial motility at the lower position in the dewar, and 37% at the upper position. A third freezing rate was produced using a computer-controlled freezer programmed for a rate of -45ºC/min, yielding a retention of initial motility of 31%. Our freezing and transport of cryopreserved sperm in shipping dewars demonstrate the utility of this procedure for field applications

Design and Cost Analysis of a Self-contained Mobile Laboratory for Commercial-scale Aquatic Species Cryopreservation

© Copyright by the World Aquaculture Society 2018 Although aquatic species cryopreservation protocols have been studied around the world over the past 60 yr., germplasm repository development efforts and commercialization have begun only recently. The goal of this project was to develop a self-contained mobile laboratory for on-site high-throughput cryopreservation of aquatic species. The objectives of this study were to: (1) identify how a mobile laboratory would function in different operational scenarios, (2) customize an enclosed cargo trailer to function as a mobile laboratory, (3) evaluate the laboratory layout and ability of cryopreservation equipment to operate from generator power, and (4) document the investment costs for private and public groups to integrate a mobile laboratory into an existing cryopreservation facility at three levels of automation and estimate the total cost per trip based on hypothetical assumptions for two scenarios (aquaculture production and repository development). There were three operational designs identified for the mobile laboratory: (1) self-contained work inside the unit using generator power, (2) work inside the unit using external facility power, and (3) using the equipment inside of a host facility. The investment costs for a base-level mobile laboratory ranged between US$5670 and US$5787 for private groups and between US$5208 and US$5315 for public groups. With the addition of a range of automated processing equipment, total investment costs ranged from US$13,616 to US$103,529 for private groups and US$12,494 to US$94,891 for public groups. The total cost per trip to cryopreserve sperm of 59 blue catfish, Ictalurus furcatus, males to produce 6300 0.5-mL French straws was estimated to range from US$6089 to US$14,633 for private and between US$5703 and US$16,938 for public groups depending on the level of automation. Total cost per trip to cryopreserve sperm of 500 males of five different species in the genus Xiphophorus to produce 641 0.25-mL French straws was estimated to range from US$6653 to US$7640 for private and US$7582 to US$8088 for public groups depending on level of automation. Overall, a commercial-scale mobile laboratory was developed that can assist current germplasm activities and support future repository and industry development, and the layout information provided can help others to design and build comparable units

Preserving and Using Germplasm and Dissociated Embryonic Cells for Conserving Caribbean and Pacific Coral

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (∼5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had∼50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (−196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems

The role of alkalinization-induced Ca2+ influx in sperm motility activation of a viviparous fish Redtail Splitfin (Xenotoca eiseni)

Mechanisms regulating sperm motility activation are generally known in oviparous fishes, but are poorly understood in viviparous species. The mechanism of osmotic-shock induced signaling for oviparous fishes is not suitable for viviparous fishes which activate sperm motility within an isotonic environment. In addition, the presence of sperm bundles in viviparous fishes further complicates study of sperm activation mechanisms. The goal of this study was to establish methodologies to detect intracellular Ca2+ signals from sperm cells within bundles, and to investigate the signaling mechanism of sperm activation of viviparous fish using Redtail Splitfin (Xenotoca eiseni) as a model. Motility was assessed by classification of bundle dissociation and computer-assisted sperm analysis, and intracellular Ca2+ was assessed using the fluorescent probe Fura-2 AM. Bundle dissociation and sperm motility increased with extracellular Ca2+ and pH levels. Intracellular Ca2+ signals were detected from sperm within bundles, and increased significantly with extracellular Ca2+ and pH levels. Major channel blockers known to inhibit Ca2+ influx (NiCl2, ruthenium red, GdCl3, SKF-96365, nimodipine, verapamil, methoxyverapamil, mibefradil, NNC 55-0396, ω-Conotoxin MVIIC, bepridil, and 2-APB) failed to inhibit Ca2+ influx, except for CdCl2, which partially inhibited the influx. We propose a novel mechanism for motility regulation of fish sperm: an alkaline environment in the female reproductive tract opens Ca2+ channels in the sperm plasma membrane without osmotic shock, and the Ca2+ influx functions as a second messenger to activate motor proteins controlling flagella movement

An impedimetric sensing probe based on printed circuit board technology for monitoring in cryobiology applications

Cryopreservation of living cells is an effective tool for protection, maintenance, and distribution of genetic resources, which involves exposure to cryogenic temperatures and requires precise control over various parameters to avoid potential cell damages. Hundreds of protocols have been reported for cryopreservation of aquatic species, but replicating them is challenging without a reliable monitoring technique during a cryopreservation process. In this work, we aim to use electrical impedance as a monitoring parameter to assist standardization of cryopreservation processes and reporting. Specifically, this paper reports an impedance sensing probe compatible with cryogenic temperatures and conventional containers in cryopreservation of aquatic species based on printed circuit board technology its characterization in cryopreservation conditions including different sperm extenders (buffer) compositions and concentrations, presence of cryoprotectant, and multiple cooling rates. The developed probe based on printed circuit board (PCB) technology shows a capability of measuring conditions during cryopreservation differentiating among samples with different buffer contents and cryoprotectants. The probe also demonstrates the capability to distinguish different cooling regimes and detect phase change phenomena. This PCB-based sensing platform provides quantitative impedance measurement data during the cryopreservation process at sample preparation, cooling, and while frozen. Technology such as this offers opportunities for improving the reproducibility of protocols generated by the aquatic species community and can be made widely available as open hardware

Inducible expression of green fluorescent protein within channel catfish cells by a cecropin gene promoter

The activity of an insect promoter of the cecropin B gene (Cec B) was investigated using green fluorescent protein (gfp) as a reporter in cells of channel catfish (Ictalurus punctatus). The expression vector pQZ-1 containing the Cec B promoter and a modified gfp cDNA sequence was delivered by lipofection to three catfish cell types: fibroblast and leukocyte cell lines, and primary cultures of leukocytes. No resistance genes were included in the vector for selection of GFP-expressing cells. The GFP mRNA was detected in all three cell types with 5 to 10 times higher concentrations observed in leukocytes than in fibroblasts. Expression was enhanced with the addition of irradiated Flavobacterium columnare (7.0 x 106 cells/ml) or Escherichia coli LPS (125 μg/ml). Quantitative RT-PCR showed GFP mRNA reached maximum levels 24 h after bacterial challenge in fibroblast cells, and at 10-12 h after LPS challenge in fibroblasts and leukocytes. The number of fibroblasts expressing GFP increased by 0.8%, and the average of green fluorescence intensity increased by 52.8%, whereas the increase in leukocytes was 0.13% in cell number and 3.4% in fluorescence intensity. These results suggest that the transcription of the Cec B promoter in channel catfish cells exhibited an inducible pattern and could be placed under the control of the immune system (in vivo). The mechanisms for endogenous activation of the Cec B promoter and for production of gfp RNA in unchallenged cells remain to be studied