Artificial Fertilization by Intracytoplasmic Sperm Injection in a Teleost Fish, the Medaka (Oryzias latipes)1

Abstract

Intracytoplasmic sperm injection (ICSI) is a technique that has been successfully used for assisting reproduction in mammals. However, this method is still not reliable in nonmammalian species, including teleosts. We succeeded in producing medaka individuals by ICSI with a rate of 13.4% (28 hatched embryos out of 209 eggs fertilized by ICSI), the best value reported so far in teleosts, including zebrafish and Nile tilapia. Although the technique was based on that developed for mammalian eggs, some critical modifications were made to adjust it to the medaka egg, which has a thick and hard envelope (the chorion) and a single sperm entry site (the micropyle). Medaka ICSI was performed by injecting a demembranated spermatozoon into an egg cytoplasm through the micropyle 10-15 sec after egg activation induced by a piezo-actuated vibration, the site and timing of sperm penetration being consistent with those in normal fertilization in medaka. To increase the efficiency of ICSI in medaka, we found that the fertilization by ICSI should precisely mimic the fertilization by insemination with intact sperm, both spatially and temporally. The success rate of ICSI was highly variable in batches of eggs (ranging from 0% to 56%), suggesting that the conditions of eggs are important factors in stabilizing the production of individuals by ICSI. The success in medaka ICSI provides a basis for future research to understand the basic mechanisms in gamete biology of teleosts as well as for development of new technology that can yield valuable applications in fisheries science.

Assessment of nutritional supplementation in phospholipids on the reproductive performance of zebrafish, Danio rerio (Hamilton, 1822)

“…Moreover, it is important for further studies to check the lipid and fatty acid profile of the zebrafish egg yolk as well as the spermatozoa plasma membrane. The perivitelline space develops after egg activation, and is shown in zebrafish that a large perivitelline space is related to spawning quality, with unhealthy eggs having a narrow perivitelline space (Otani et al, 2009;Joo and Kim, 2013). However, for a better understanding of egg quality, further biochemical analyses should be performed as a complement to the morphological parameters, perivitelline space and egg diameter (Cabrita et al, 2008).…”

Motility, morphology, mitochondria and ATP content of diploid spermatozoa from sex-reversed clonal diploid and neo-tetraploid loach,Misgurnus anguillicaudatus

“…Therefore, for the successful fertilization using immotile diploid spermatozoa of sex-reversed clonal diploid male, different type of biotechnology such as ICSI (Intra Cytoplasmic Sperm Injection) is required. ICSI is considered difficult in teleosts, but successful results have been reported in zebrafish (Poleo et al, 2001), tilapia (Poleo et al, 2005) and medaka (Otani et al, 2009). When ICSI techniques are fully established in fishes, immotile diploid spermatozoa can be utilized as a source of diploid gametes for extension of chromosome manipulation.…”

Visualization of primordial germ cells in the fertilized pelagic eggs of the barfin flounder Verasper moseri

“…The first aim of this study was to develop an efficient microinjection method to enable visualization of PGCs during embryogenesis in the barfin flounder; the pelagic egg of this species has a single membrane-bound yolk mass like that of the medaka (Otani et al, 2009). Secondly, we sought to isolate the barfin flounder nos3 gene with the aim of comparing the 3'UTR sequence with zebrafish.…”