Sperm cryopreservation of a live-bearing fish, Xiphophorus couchianus: male-to-male variation in post-thaw motility and production of F(1) 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 degrees C/min from 5 to -80 degrees C in 250-microL French straws. For artificial insemination, samples were thawed at 40 degrees C for 5 s in a water bath, washed once using fresh HBSS500 by centrifuging at 1000 g for 5 min at 4 degrees C, concentrated into approximately 5 microL, 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.

Fig. 1

The correlation of body length, body weight, testis weight, and body condition of Xiphophorus couchianus males used in this study (N=66). Body condition was calculated as the percentage of body weight (g) to the cube of the body length (cm).

Fig. 2

Sperm motility of individual Xiphophorus couchianus males after thawing (black bars) and before freezing (white bars) in the experiments of 2006 (A) and 2007 (B) along with the gonadosomatic index (percentage of testis weight to the body weight) and body condition factor (percentage of body weight to the cube of the body length) of each male. Males from each year are presented in ranked order (from highest to lowest) of post-thaw motility.

Fig. 3

Genotype (panel A) and phenotype (panels B, C) analyses of parental and interspecies hybrid offspring (8–9 months old) derived from artificial insemination of female X. maculatus with cryopreserved sperm from X. couchianus (Xc). Microsatellite marker Msd037 was used to amplify the genomic DNA isolated from all putative interspecies hybrids with polymerase chain reaction and resulted in a 300-bp amplicon from X. maculatus DNA and a 200-bp amplicon from X. couchianus DNA (Heater et al., 2004). Analysis of each F₁ offspring collected from cryopreserved sperm and controls showed the expected bands at 300 and 200 bp (lanes 7, 8, 9 and 10). The parents exhibited the single band diagnostic of each species (lanes 3, 4, 5 and 6). Blank lanes were the PCR product from a reaction with buffer and primers but without DNA template. Phenotypes of parental and interspecies hybrid identified that the hybrids (pedigree #10916, Panel B) exhibited expression of the Crescent tail pattern (C) originating from X. maculatus (Jp Wild strain) coupled with the distinct vertical barring and more elongated body shape originating from X. couchianus and that the hybrids (pedigree #10929, Panel C) also exhibited expression of the anal red (Ar) and Dot (D) pigment pattern originating from X. maculatus (strain SR) along with the vertical barring pattern and more elongated body shape exhibited by X. couchianus. Analysis of genotype and phenotypes indicate that all of the young collected in this experiment were interspecies hybrid offspring produced from cryopreserved sperm.