Production of F₁Offspring with Vitrified Sperm from a Live-Bearing Fish, the Green SwordtailXiphophorus hellerii

Abstract

This study reports the first production of offspring with vitrified sperm from a live-bearing fish Xiphophorus hellerii. The overall goal of this study was to develop streamlined protocols for integration into a standardized approach for vitrification of aquatic species germplasm. The objectives were to (1) estimate acute toxicity of cryoprotectants, (2) evaluate vitrification solutions, (3) compare different thawing methods, (4) evaluate membrane integrity of post-thaw sperm vitrified in different cryoprotectants, and (5) evaluate the fertility of vitrified sperm. Nine cryoprotectants and two commercial vitrification additives were tested for acute toxicity and glass forming ability, alone and in combination. Two vitrification solutions, 40% glycerol (Gly) and 20% Gly + 20% ethylene glycol (EG) in 500 mOsmol/kg Hanks' balanced salt solution (HBSS), were selected for vitrification of 10 lL sperm samples using inoculating loops plunged into liquid nitrogen. Samples were thawed at 24°C (one loop in 5 lL of HBSS or three loops in 500 lL of HBSS). Samples thawed in 500 lL were concentrated by centrifugation (1000 g for 5 min at 4°C) into 5 lL for artificial insemination. Offspring were produced from virgin females inseminated with sperm vitrified with 20% Gly + 20% EG and concentrated by centrifugation.

2-Methyl-2,4-pentanediol gas sensor properties of nano-SnO2 flat-type coplanar gas sensing arrays at low detection limit

“…Crispe [10] found that only 82 % of the human monocytes still appeared viable after incubation at 3 × 10 −1 M of MPD. Nacci et al [11] determined that MPD had exceptionally toxic effect to sperm which had also been confirmed by Cuevas-Uribe et al [8]. Procter [12] reported that MPD could depress the central nervous system and had a toxic action on the kidneys as well as the liver.…”

“…Because of those special properties, MPD has become a very important compound. MPD is widely used as a chemical intermediate [2], a solvent for perfume and cosmetics [3,4], an excipient in the formulation [5], an organic additive to promote the phase transition [6], antibacterial and antifungal agent [7], a cryoprotectant [8] and a single most successful agent promoting crystallization of biological macromolecules [1], and so on [9]. MPD is regarded as a high volume chemical (HVC) with production exceeding 0.5 × 10 6 kg annually in the United States [3].…”

Standardized Assessment of Thin‐film Vitrification for Aquatic Species

“…Small fishes include ornamental aquarium species and important biomedical models Medaka ( Oryzias latipes ), Xiphophorus spp., and Zebrafish ( Danio rerio ) (Yang and Tiersch 2009). Small fishes are also disproportionately observed on lists of imperiled species and are in need of germplasm repositories for conservation (Cuevas-Uribe et al 2011a; Cuevas-Uribe et al 2011b). In addition, vitrification does not require specialized equipment and can be done in remote locations such as in the field where endangered fishes occur in the wild.…”

“…First is the lack of devices specifically designed for use with aquatic species, forcing researchers to experiment with devices designed for other uses such as vitrification of human embryos (Kása et al 2016). Other options are to use devices not designed for vitrification, such as microbiology inoculation loops (Cuevas-Uribe et al 2011b; Kuwayama 2007). In addition, there are a large number of technical variables that are not standardized in this research, making it problematic or impossible to directly compare vitrification results among studies.…”

Three-dimensional printing with polylactic acid (PLA) thermoplastic offers new opportunities for cryobiology

“…Such devices are intrinsic to cryobiology research, but pose the inherent problems of dealing with cryogenic temperatures [3]. As such, these devices are often expensive and designed for highly specialized purposes (such as freezing of human embryos [6]) which reduce their utility for adaptation for other applications (such as vitrification of fish sperm [2]). The paucity of existing devices also constrains the starting points for development of new devices, and accordingly cryobiological devices often have to be crafted from simple or readily available materials such as French straws [11].…”