Intracytoplasmic sperm injection in sturgeon species: A promising reproductive technology of selected genitors

Abstract

Sturgeons are the most endangered species group and their wild populations continue to decrease. In this study, we apply intracytoplasmic sperm injection (ICSI), an assisted reproductive technology, for the first time in endangered and critically endangered sturgeons. Using various egg-sperm species combinations we performed different ICSI experiments with immobilized pre- or non-activated spermatozoa, single or many, fresh or cryopreserved. Then we evaluated the fertilization success as well as the paternity of the resultant embryos and larvae. Surprisingly, all experimental groups exhibited embryonic development. Normal-shaped feeding larvae produced in all egg-sperm species-combination groups after ICSI using single fresh-stripped non-activated spermatozoa, in one group after ICSI using single fresh-stripped pre-activated spermatozoa, and in one group after ICSI using multiple fresh-stripped spermatozoa. ICSI with single cryopreserved non-activated spermatozoa produced neurula stage embryos. Molecular analysis showed genome integration of both egg- and sperm-donor species in most of the ICSI transplants. Overall, ICSI technology could be used as an assisted reproduction technique for producing sturgeons to rescue valuable paternal genomes.

Keywords: assisted reproduction; embryo; intracytoplasmic sperm injection; larva; sturgeon.

Figure 1

Electrophoretogram of 395_AB and 153_HH markers in 14 ICSI produced embryos. The marker 395_AB amplifies 395 bp band only when the sample contains Siberian and/or Russian sturgeon nuclear DNA while the marker 153_HH amplifies 153 bp band only when a sample contains nuclear DNA of beluga. Numbers correspond to the embryos and larvae of Table 2. Siberian sturgeon fin (Sib) and beluga fin (Bel) are used as positive controls 100–1500 bp ladder.

Figure 2

Electrophoretogram of 395_AB and 247_AR markers in 10 ICSI produced transplants. The marker 395_AB amplifies 395 bp band only when a sample contains Siberian and/or Russian sturgeon nuclear DNA and the marker 247_AR amplifies 247 bp band only when a sample contains nuclear DNA of sterlet. Numbers correspond to the embryos and larvae of Table 2. Russian sturgeon fin (Rus) and sterlet fin (Sterl) are positive controls 100–1500 bp ladder.

Figure 3

Electrophoretogram of 153_HH and 247_AR markers in 14 produced embryos. The marker 247_AR amplifies 247 bp band only when the sample contains nuclear DNA of sterlet, while the marker 153_HH amplifies the 153 bp band only when the sample contains nuclear DNA of beluga. Sterlet fin (Sterl) and beluga fin (Bel) are positive controls. The numbers 25–33, 35–38 are corresponding to transplants after ICSI of single non-activated cryopreserved beluga spermatozoa into sterlet's eggs. Number 34 is a control embryo produced after in vitro fertilization of cryopreserved beluga spermatozoa with sterlet eggs 100–1500 bp ladder.