Elsevier

Cryobiology

Volume 63, Issue 1, August 2011, Pages 56-60
Cryobiology

Cryopreservation of sperm from Atlantic halibut (Hippoglossus hippoglossus, L.) for commercial application

https://doi.org/10.1016/j.cryobiol.2011.04.009Get rights and content

Abstract

Development of Atlantic halibut (Hippoglossus hippoglossus) aquaculture will be enhanced with cryopreservation of halibut sperm by ensuring a reliable supply of sperm of desired quality and quantity. To assist in its commercial application, the cryopreservation of large volumes of halibut sperm was investigated. Three cryoprotectants were compared: dimethyl sulfoxide (DMSO), polyethylene glycol (PG) and glycerol (GLY) at two concentrations (10% or 15%). Two salt solutions, Hanks’ balanced salt solution (HBSS) and 0.1 M KHCO3 with 0.125 M sucrose solution (KS) were tested as diluents. Both factors were examined in 1.6 mL volumes. A cryopreservation volume of 4 mL and a low dilution ratio (1:1) were examined separately. Based on motility and fertilization rate, 10% and 15% DMSO diluted with HBSS or KS solution proved to be effective extenders with mean fertilization rates ranging from 52.2 ± 27.2% to 65.8 ± 26.1%; none of which were significantly different from that of the control. Four other extenders, 10% PG or 10% GLY with HBSS or KS, resulted in significantly lower fertilization rates. Use of a 4 mL cryopreservation volume did not exhibit a significant effect on fertilization rate or motility of post-thawed sperm compared to a 1.6 mL volume (P > 0.05); while the use of a dilution ratio of one part sperm with three parts cryopreservation solution (1:3 v/v with sperm concentration of 0.51 ± 0.11 × 1010 cells/ml) had a significantly better preservation effect than using a ratio of 1:1 with sperm concentration of 1.02 ± 0.21 × 1010 cells/ml (P < 0.05). From these results, an optimized protocol for the cryopreservation of Atlantic halibut sperm using a volume as large as 4 mL has been established.

Introduction

Atlantic halibut (Hippoglossus hippoglossus) is among the largest of pleuronectid flatfish and has been identified as an ideal species for farming at higher latitudes because of its high growth rate in the relatively cold northern waters (0–14 °C) [1]. Hatchery production for Atlantic halibut has occurred in Iceland, Norway, Scotland, Canada and more recently Chile [2]. In commercial facilities, Atlantic halibut are spawned by stripping eggs and sperm from individual broodfish. In order to ensure maximum gamete viability, fertilization is generally done within minutes of stripping by mixing the eggs and sperm with sea water. Unfertilized eggs can be held for several days, and sperm for several weeks, with acceptable low level losses in viability provided proper storage techniques are used [3], [4]. These procedures are useful for transportation of gametes but are very limited with respect to holding time relative to cryopreservation.

The Atlantic halibut industry is advancing rapidly with respect to stock development. Wild broodstock are being replaced by F1 and subsequent generations; and the importance of breeding programs for broodstock production is well recognized in order to maintain heterozygosity in domesticated broodstocks [5], [6]. The long-term storage of genetic material afforded by cryopreservation of sperm will greatly facilitate these programs by eliminating temporal and spatial barriers to crosses. It will therefore allow the maximum amount of genetic input to be available for broodstock structuring. Cryopreservation of sperm will also aid with selective breeding for the production of better performing juveniles for grow-out and market. Effective sperm storage permits maximum use of sperm from a limited number of individuals. This could be imperative for the use of sex-reversed sires required for all female production [7], or sires known to be carrying traits that improve production performance [8]. Finally, cryopreservation of sperm will eliminate the problem of asynchronous gamete production that commonly occurs in halibut broodstocks [9], [10], [11].

Early cryopreservation work with Atlantic halibut sperm by Bolla et al. [12] and Billard et al. [13] describes pelleting on dry ice or freezing in straws. These methods were successful but preserved small volumes of sperm (e.g. 20 μL) which would be impractical for large scale insemination at halibut hatcheries. In a spawning season, an excess of 200 L of eggs can be collected in a hatchery. Because of the high natural ejaculate volumes of halibut males, hatcheries typically use a 1:1000 to 1:100 (v/v) ratio of fresh sperm to eggs for fertilization, although a 1:8000 ratio [14] has been proven acceptable in lab scale trials using good quality fresh sperm. Even at the lower sperm/egg ratio stated (1:8000), 1250 pellets (20 μL) would be required in a single season, assuming 200 L of egg production. Similar problems with limited sperm volume have been noted when using classic cryopreservation techniques for other species [15], [16] Procedures for cryopreservation of large volumes of halibut semen are necessary in order to practically apply sperm storage procedures at commercial facilities.

Cryopreservation of large volumes has been successful for several species, such as rainbow trout [17], yellowtail flounder [18], turbot [19], yamú [20] and red sea bream [21]. These techniques used 2 or 5 mL vessels to increase preserved sperm volumes, and proved to be acceptable and profitable in a commercial setting. Alternatively, the volume of preserved sperm can be improved by adopting lower dilution ratios. Although a ratio of sperm to extender of 1:3 has been shown to be optimum for sperm cryopreservation of most species, a 1:1 ratio was also found to be acceptable for the sperm of grey mullet, black porgy, and tilapia [22]. Considering the species-specific optimum parameters for cryopreservation of fish sperm, both vessel volume and optimum dilution ratio should be investigated for the cryopreservation of halibut sperm.

The purpose of this study was to develop commercially applicable cryopreservation techniques for Atlantic halibut sperm either through larger vessel size or lower dilution ratio. The effects of different cryopreservation solutions on the fertilization rate and motility of post-thawed sperm were also assessed.

Section snippets

Fish and sperm sampling

All gametes were obtained from broodstock held at Scotian Halibut Limited, Clarks Harbor, Nova Scotia, Canada. Four male fish were available for sperm collection. In June 2006, sperm was collected from two of these fish; in July 2006, sperm was taken from the other two fish. From August to December when the cryopreserved sperm was tested, fresh sperm was collected from the other available fish to serve as the control. At each collection period, the sperm was gently stripped into 50 mL culture

Results

The sperm density of the four male fish used for cryopreservation were 1.76 ± 0.17 × 1010, 1.77 ± 0.21 × 1010, 1.98 ± 0.19 × 1010, and 2.65 ± 0.30 × 1010 cells/mL (n = 4) with an average density of 2.04 ± 0.42 × 1010. The fertilization rates of post-thawed sperm with eight different solutions (1.6 mL volume and 1:3 dilution ratio, i.e. 0.51 ± 0.11 × 1010 cells/mL) are shown in Table 1. Successful fertilization using thawed sperm was achieved with four of the cryopreservation solutions. The extender, 10% DMSO in KS, gave

Discussion

Three cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (GLY) and polyethylene glycol (PG), were tested for the cryopreservation of halibut sperm in this study. DMSO was the most effective cryoprotectant followed by PG then GLY. These results are not consistent with those obtained by other researchers. Bolla et al. [12] used 12.5% GLY as a cryoprotectant for halibut sperm, and obtained a fertilization rate of 85% with post-thawed sperm. No other cryoprotectants were compared in this

Conclusion

This study has established an effective method for cryopreserving halibut sperm in quantities that are manageable in commercial production. Among the eight extenders tested, 10% and 15% DMSO diluted in HBSS or KS solutions demonstrated high preservation potential for the sperm of Atlantic halibut. Cryopreservation of 4 mL volumes resulted in the same efficiency as the smaller volume of 1.6 mL. The dilution ratio showed a significant effect on the quality of post-thawed sperm with a ratio of 1:3

Acknowledgments

We are grateful to Brian Blanchard for his input in initiating this project and other staff members of Scotian Halibut Limited for their technical assistance in collection of gametes and egg fertilization. Special thanks to Dr. Tony Manning for the review of this manuscript.

References (32)

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Statement of funding: The research was supported from an internal grant from the Institute for Marine Biosciences, National Research Council, Canada.

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