Development of an Effective Cryopreservation Protocol for Blue Catfish Oogonia

Abstract

Long‐term storage of oogonia and germ‐line stem cells provides an alternative to the limitations associated with cryopreserving eggs of important fish species. These cell types are less vulnerable to the stresses of freezing. Cryopreservation has enormous potential for aquaculture advancement, but protocols must be developed for each species and cell type since its success hinges on various input factors. Blue Catfish Ictalurus furcatus were selected as the test species in this study because of the need to improve fry production of Blue Catfish ♂ × Channel Catfish I. punctatus ♀ hybrids, which can be facilitated by storing oogonia in gene banks. Our objective was to develop a freezing protocol for oogonia of this species. We tested different permeating and nonpermeating cryoprotectants, concentrations of these agents, and freezing rates. We proved that all three factors influenced postthaw recovery of oogonia. Of the permeating cryoprotectants, 1.0 M dimethyl sulfoxide resulted in the most live cells with the highest viability percentages, and adding 0.2 M lactose with 10% egg yolk further improved the results. There were also specific interactions in which the effects of concentration and freezing rate varied among the cryoprotectant treatments. The most effective freezing rate was −1.0°C/min, and cell viability was reduced at −2.5°C/min and −5.0°C/min. From these results, we propose adding 1.0 M dimethyl sulfoxide with 0.2 M lactose and 10% egg yolk to cryomedia and freezing it at a rate of −1.0°C/min. By developing a cryopreservation protocol for a commonly cultured catfish, this work may guide the development of protocols for other species of interest.

Effect of Seasonality for Optimization of Germ Cell Extraction from Mature Blue Catfish

“…Germ cell isolation and quantification.-The external body of each Blue Catfish was sterilized with 70% ethanol. Fish were then dissected, and germ cells were extracted from testes or ovaries according to methods described by Shang et al (2015), Abualreesh et al (2020Abualreesh et al ( , 2021aAbualreesh et al ( , 2021b, and Hettiarachchi et al (2020). Following extraction, the stem cells were quantified according to standardized protocols (Shang et al 2015;Abualreesh et al 2020Abualreesh et al , 2021aAbualreesh et al , 2021bHettiarachchi et al 2020).…”

“…Spermatogonia A are usually observed as a single cell and rarely make clusters, while spermatogonia B can always be seen in a cluster (Shang et al 2015(Shang et al , 2018Abualreesh et al 2020). Oogonia were distinguished from the other cell types based on size (12-15 μm) and having only one distinct nucleus (~7-8 μm) (Abualreesh et al 2021a).…”

“…Oogonia were distinguished from the other cell types based on size (12–15 μm) and having only one distinct nucleus (~7–8 μm) (Abualreesh et al. 2021a).…”

Advancing aquaculture: Production of xenogenic catfish by transplanting blue catfish (Ictalurus furcatus) and channel catfish (I. punctatus) stem cells into white catfish (Ameiurus catus) triploid fry

“…On a daily basis, four sexually immature channel catfish (mean ± SEM length = 25.3 ± 5.9 cm, weight = 399.5 ± 100.3 g) and four blue catfish (mean ± SEM length = 33.5 ± 5.2 cm, weight = 488.2 ± 84.3 g) were selected (n = 4) and immediately euthanized by blunt force trauma to the center of the head followed by pithing, to avoid any potential effects on cell viability caused by MS-222. After being euthanized and gonad extraction, blue catfish stem cell (BSCs) and channel catfish stem cell (CSCs) isolation were performed according to the standard protocols described by Hettiarachchi et al, Shang et al,and Abualreesh et al [15,[25][26][27][28][29]. In brief, blue and channel catfish gonads were separately placed on a sterile petri dish (100 mm × 15 mm) containing 5 mL of Hanks' Balanced Salt Solution [(HBSS, SH30048.24, GE Healthcare Life Sciences) supplemented with 1.0 μg/mL NaHCO 3 (Church & Dwight Co., NG) and 100 unit/mL penicillin-streptomycin (115140-122, Life Technologies)].…”

Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality Controls for Commercial Applications

“…Between these, the use of DMSO, which is a cryoprotectant that has the ability to penetrate into the cell, gives the best rate on both motility and fertilization rate. Although egg yolk does not have penetrating properties, success has been observed in its use with lactose [108]. DMSO; has been identified as the cryoprotectant substance with the most successful results for cryopreservation of spermatogonial stem cells of Oncorhynchus mykiss, type A spermatogonia of I. furcatus [109], ovary of C. carpio [110], and oogonia [108].…”