Sperm cryopreservation of a live-bearing fish, Xiphophorus couchianus: Male-to-male variation in post-thaw motility and production of F1 hybrid offspring

Abstract

Fishes of the genus Xiphophorus are well-studied biomedical research models, and some species, such as X. couchianus, are currently listed as endangered in the wild. Sperm cryopreservation in these live-bearing fishes has begun recently. Thus far, live young have been produced with cryopreserved sperm only in one species (Xiphophorus helleri). In this study, the goal was to develop a practical protocol for sperm cryopreservation of Xiphophorus couchianus, and to produce live young with cryopreserved sperm. Sperm were collected by crushing of testis in Hanks’ balanced salt solution at an osmolality of 500 mOsmol/kg (HBSS500), and were cryopreserved with 14% glycerol (v/v) as cryoprotectant at a cooling rate of 20 °C/min from 5 to −80 °C in 250-μL French straws. For artificial insemination, samples were thawed at 40 °C for 5 s in a water bath, washed once using fresh HBSS500 by centrifuging at 1000 g for 5 min at 4 °C, concentrated into ~5 μL, and injected into virgin females of Xiphophorus maculatus. The inseminated females were monitored for 90 days for subsequent discharge of live young. Results from 2006 and 2007 showed considerable male-to-male variation in post-thaw motility (from 1 to 70%). Offspring were produced by cryopreserved sperm in two tanks (of three) at 36 and 66 days after insemination in 2007. Paternity was confirmed via phenotypes (body color) and genotypes (microsatellite genetic marker) of the hybrid offspring. Overall, a practical protocol for sperm cryopreservation and artificial insemination is provided to preserve X. couchianus, which is an important biomedical research model, and also currently listed as an endangered species in the International Union for Conservation of Nature (IUCN) red list.

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

“…Of the 27 species of Xiphophorus, conventional cryopreservation of germplasm yielding offspring has been reported in only 3 species (X. hellerii, X. couchianus, and X. maculatus). [15][16][17] Cryopreservation and artificial insemination efforts in Xiphophorus are faced with four major challenges: (1) limited volume of sperm samples ( < 10 lL); (2) a low percentage (20%-30%) of females…”

“…15,26 Artificial insemination involves the injection of sperm (2-5 lL) into the female reproductive tract and pregnancy monitoring for as long as 90 days because females can store sperm and delay fertilization. 25,78 Success rates for artificial insemination using fresh sperm are usually 20%-30%, 16 although one study reported 50% success by insemination of at least 40 sperm bundles (spermatozeugmata) into the female (a single male can provide as many as 3000 bundles per stripping 79 ). 80 Previously reported success rates for artificial insemination using cryopreserved sperm ranged from 10% to 20%, 15,16 although one study reported as high as 40% fertilization.…”

“…16 Conventional cryopreservation has been applied for the protection of genetic resources in fish and shellfish, but is not always practical for use in remote locations or in developing countries (often the places where preservation is most necessary). Vitrification is considered to provide an alternative to standard cryopreservation and has been used successfully for cryopreservation of spermatozoa, embryos, oocytes, stem cells, and organs from several mammalian species.…”

“…The testes (13.8 -10.3 mg, n = 101) were removed and separated from the surrounding lipid tissues while viewing with a dissection microscope (10 · magnification) and transferred to 1.5-mL centrifuge tubes for weighing. Sperm were released by crushing of the testis in Hanks' balanced salt solution at an osmolality of 500 mOsmol/kg (HBSS500), 16 and diluted to a final concentration of 1 · 10 8 cells/mL unless otherwise stated. Sperm concentration was estimated by use of a hemacytometer (Hausser Scientific), and osmolality was measured with a vapor pressure osmometer (Model 5520; Wescor Inc.).…”

“…12 Despite these limitations, live young have been produced from cryopreserved sperm in X. hellerii, 15 X. couchianus, 16 X. maculatus, 17 and X. variatus (Yang et al, unpublished data) since the first research on sperm cryopreservation of Xiphophorus fishes in 2004. 13 Vitrification is a form of cryopreservation that utilizes rapid cooling rates ( > 1000°C/min compared with < 40°C/min for conventional cryopreservation) and high concentrations of cryoprotectants (40%-60% compared with 5%-15% for conventional cryopreservation) to form glass (noncrystalline ice).…”