Production of F1 Interspecies Hybrid Offspring with Cryopreserved Sperm from a Live-Bearing Fish, the Swordtail Xiphophorus helleri1

Abstract

Despite study of sperm cryopreservation in more than 200 fish species, production of broods from cryopreserved sperm in live-bearing fish has not been demonstrated. This has not been due to a lack of effort, but instead is a result of the unique morphology, biology, and biochemistry of reproduction in viviparous fishes. For example, sperm of Xiphophorus helleri have a cylindrical nucleus, can swim for days after being activated, have glycolytic capabilities, and can reside in the female reproduction tract for months before fertilization. These traits are not found in fishes with external fertilization. The long-standing research use of the genus Xiphophorus has led to development of over 60 pedigreed lines among the 26 species maintained around the world. These species and lines serve as contemporary models in medical research, although they must be maintained as live populations. Previous attempts at establishing sperm cryopreservation protocols for Xiphophorus have not produced live young. To address this we have been studying the parameters surrounding cryobiology of Xiphophorus sperm and applying this information to an improved understanding of internal fertilization and reproduction. Here we report the first successful fertilization and offspring production by cryopreserved sperm in any live-bearing fish. This claim is supported by our use of artificial insemination between two species that yield distinct hybrid offspring to verify paternity via cryopreserved sperm. We provide a practical approach for preservation of valuable genetic resources from live-bearing fish species, a group that is rapidly being lost due to destruction of native habitats.

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…”

“…The artificial insemination procedure used in this study was based on previously published protocols (see details in Yang et al 15 and Dong et al 26 ). In brief, females were anesthetized in 0.01% MS-222 (w/v), and transferred to a Petri dish with the abdomen facing up.…”

“…Cryoprotectant solutions were prepared in HBSS500 at double strength of the final concentrations, followed by mixing with sperm suspension at a ratio of 1:1 for toxicity estimation. Motility was estimated immediately (within 10 s) and at 5,10,15,20,25,30, and 60 min.…”

“…Studies on artificial insemination date back to 1914 when hybrids were produced from X. hellerii and X. maculatus 74 and since then several artificial insemination protocols have been published using fresh sperm [75][76][77] and cryopreserved sperm. 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 ).…”

“…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).…”