Evaluation of Commercial‐scale Approaches for Cryopreservation of White Crappie, Pomoxis annularis, Sperm

Abstract

Crappie, Pomoxis spp., are popular game fish throughout North America and are produced by public and private hatcheries. However, production is limited by a lack of information on tank culture and induced spawning methods. Development of techniques for storage of sperm and in vitro fertilization would increase flexibility in spawning. Therefore, techniques for sperm cryopreservation were examined in white crappie, Pomoxis annularis. Sperm from adult wild white crappie were used to evaluate sperm extender, cryoprotectant agent and concentration, and cooling technique based on post‐thaw sperm motility. Percent egg fertilization was also compared between sperm stored in the two best cryopreservation protocols and two different osmotic activator solutions. Sperm were cryopreserved using treatment combinations of two extenders (350 mOsmol/kg Hanks' balanced salt solution [HBSS] and 350 mOsmol/kg Ca2+free HBSS) and two cryoprotectants (dimethyl sulfoxide [DMSO] and methanol) at concentrations of 5, 10, and 15% that were cooled at four different rates: 5, 10, 20, and 40 C/min. Post‐thaw sperm motility and fertilization rates indicated white crappie sperm can be cryopreserved using either extender, cryoprotectants of either 5% DMSO or 10% methanol, and cooling at 40 C/min. A follow‐up experiment demonstrated sperm in suspensions on ice retained viability after overnight transport.

A generalized approach for sperm cryopreservation in the genus Pomoxis: Sperm cryopreservation and fertilization efficiency of black‐stripe black crappie, Pomoxis nigromaculatus

“…In comparison, the present study found black‐stripe black crappie sperm with 5% DMSO was more effective at fertilizing white crappie eggs (38%) than sperm cryopreserved in 10% methanol (22%). Although not directly comparable due to higher sperm:egg ratios used by Culpepper et al (2018) (3.19 × 10 5 vs. 1.59 × 10 5 in the present study), this study had higher fertilization rates in white crappie eggs than those of Culpepper et al's study. Interestingly, black‐stripe black crappie sperm was more effective at fertilizing white crappie eggs than white crappie sperm.…”

“…Motility was estimated within 10 s of sperm activation using computer-assisted sperm analysis (CASA) (HTM-CEROS, version 14 Build 013, Hamilton Thorne Biosciences, Beverly, MA). System settings for assessing progressive motility were minimum contrast, 60; minimum cell size, 2 μm; number of frames for recording, 100; average-path velocity cut-off, 25 μ/s; straight-line velocity cut-off, 1 μ/s ( Culpepper et al, 2018 ). Three measurements, each from a different field of view, were collected for each sample and the average initial motility calculated.…”

“…For centrarchids, fertilization rates using cryopreserved bluegill sperm ranged from 15 to 50%, depending on cryoprotectant ( Bates, Williams, Lang, & Tiersch, 2005 ). Fertilization rates using cryopreserved white crappie sperm yielded relatively low fertilization (13%) ( Culpepper et al, 2018 ), indicating a need for refinement of fertilization techniques using cryopreserved sperm. Cryopreservation techniques are not commonly applied to produce hybrids in fishes, but have been used successfully ( Gharrett, Smoker, Reisenbichler, & Taylor, 1999 ), sometimes with similar or higher fertilization rates to fresh sperm ( Chao, Chao, Liu, & Liao, 1987 ).…”

“…This would reduce the need for collecting and maintaining difficult-to-locate broodstock and increase fertilization percentages when using cryopreserved sperm. Therefore, the objectives of this study were to investigate whether (a) black-stripe black crappie sperm could be cryopreserved using methods established for white crappie ( Culpepper et al, 2018 ), (b) compare the use of two cryoprotectants (i.e., 5% DMSO and 10% methanol) for black-stripe black crappie sperm, and (c) determine optimal egg volume (i.e., sperm:egg ratios) for fertilization using cryopreserved black-stripe black crappie sperm. These studies also offer insight on the generalization of cryopreservation across species, and practical application to support germplasm repository development.…”

White Crappie ( Pomoxis annularis ) latency period following hormone administration is predictable and independent of diel injection time

“…The development of protocols for these species led to interest in the development of protocols for crappie, although most crappie production has been done extensively in ponds (Morris & Clayton, 2009) with variable techniques and success (Culpepper & Allen, 2016b). However, the development (Al‐ablani & Phelps, 1997; Arslan & Phelps, 2004; Gomelsky et al, 2002) and recent refinement (Cuevas‐Uribe et al, 2009; Culpepper & Allen, 2016b) of techniques for hormone induced spawning, tank holding procedures, out‐of‐season spawning techniques (Culpepper & Allen, 2016a) and sperm cryopreservation (Culpepper et al, 2018; Shirley et al, accepted) have allowed for more intensive crappie spawning techniques to be studied.…”