In the Danio species, interspecific hybridization has been conducted in several combinations. Among them, only the hybrid between a zebrafish (D. rerio) female and a spotted danio (D. nigrofasciatus) male was reported to be fertile. However, beyond these investigations, by means of reproductive biology, gametes of the hybrid have also not been investigated genetically. For this study, we induced a hybrid of the D. rerio female and D. nigrofasciatus male in order to study its developmental capacity, reproductive performance and gametic characteristics. Its hybrid nature was genetically verified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the rhodopsin gene. Almost all the hybrids (36/37) were males, and only one was female. Developing oocytes were observed in the hybrid female, but ovulated eggs have not been obtained thus far. Microscopic observation revealed various head sizes of sperm in the hybrid males. Flow cytometry showed that the hybrid males generated aneuploid sperm with various ploidy levels up to diploidy. In backcrosses between D. rerio females and hybrid males, fertilization rates were significantly lower than the control D. rerio, and most resultant progeny with abnormal appearance exhibited various kinds of aneuploidies ranging from haploidy to triploidy, but only one viable progeny, which survived more than four months, was triploid. This suggested the contribution of fertile diploid sperm of the hybrid male to successful fertilization and development.
Androgenesis is useful for induction of doubled haploids from male genetic resources and contributes to the restoration of individuals from cryopreserved sperm. Here, we determined the suitable conditions for egg in vitro preservation and the suitable dose of UV irradiation for genetic inactivation of the egg nucleus, and established an improved procedure for induction of androgenetic-doubled haploids in zebrafish. The suitable solution for egg preservation was evaluated by the fertilization rate using different types of solutions or conditions. Hank's solution with 0.5% bovine serum albumin (pH8.0) was suitable for the preservation of zebrafish eggs. In addition, we discovered an improvement of fertilization rates by temporal preservation of ovulated eggs in the suitable solution. UV irradiation of eggs at 50-75 mJ/cm induced haploid embryos. Microsatellite genotyping using eight loci revealed the paternity and homozygosity of the putative androgenetic doubled haploids. The yield rate of androgenetic doubled haploids, which were induced by UV irradiation and heat shock, ranged from 0.4% to 10.7%.
Artificial fertilization of cultured fish is essential for seed production using breeding techniques. However, in tuna species, the success rate of artificial fertilization is tremendously low. In this study, it was reported that the adequate procedure for ovulated egg collection and storage for artificial fertilization in kawakawa Euthynnus affinis. The collection of ovulated eggs was attempted using new techniques that disrupt only spawning activity without discontinuing ovulation. The available time to use ovulated eggs was also examined by assessing the optimal preservation process and temperature. As a result, artificial fertilization was effectively executed by assessing spawning time and thoroughly extracting ovulated eggs immediately after ovulation, with a success rate of 70% and an ovulation rate of 51.7%. Ovulated eggs could be stored with small quantities of ovarian fluid to sustain fertility. However, fertility was better preserved with Hanks’ solution. Ovulated eggs with high productivity were achieved 3 h after egg extraction when maintained in Hanks’ solution at 20 °C, leading to a supply of one-cell stage embryo for microinjection treatment constantly by continuously executing artificial fertilization. This systematic procedure permitted selective breeding by 1:1 mating between top-quality parental fish and applying several developmental engineering techniques to kawakawa breeding.
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