Krzysztof Goryczko

Androgenesis in rainbow trout using cryopreserved spermatozoa: the effect of processing and biological factors.

In addition to producing homozygous lines for biomedical and genomic research and monosex stocks for commercial purposes, androgenesis is the biotechnology considered most promising and reliable for recovering complete nuclear genome information from cryopreserved fish cells. That is because procedures of cryopreserving spermatozoa, contrary to procedures for oocytes or entire eggs, are being well developed. Application of androgenesis in genome banking programs addresses the needs of both the aquaculture industry (safeguard for valuable strains and lines) and natural resource conservation (in vitro protection of endangered species or populations). The present study was focused on successful production of an androgenetic rainbow trout stock using cryopreserved spermatozoa. Our report constitutes the first time a full factorial analysis of processing and biological factors affecting androgenesis efficacy has been presented. Also for the first time, the survival of androgenetic individuals during 2 years of life was recorded. Remarkably high survival rates were observed in one of the two experiments-up to 42.5 +/- 2.8% of hatched larvae, 22.5 +/- 0.1% of swim-up larvae and 10.5% of androgenetic alevins 0.4 g. Mortality rates in androgenetic groups were high especially during the first 6 months. In all, 114 androgenetic individuals (0.9%) survived 2 years. Cryopreservation of spermatozoa generally did not affect androgenesis efficiency significantly, however, this effect was significantly dependent on the method of ploidization shock and on the duration of treatment. Significant interactions were revealed between the irradiation dose and the magnitude of pressure applied, and between the treatment of sperm and duration of pressure shock. Individual variability of spermatozoa donors significantly affected androgenesis efficiency regardless of their genetic (outbred or inbred) origin. Genetic source of the oocytes, contrary to spermatozoa, proved to be an important factor. Following findings of other researchers that androgenesis using cryopreserved spermatozoa is possible, we demonstrated that viable stock could be successfully established from cryopreserved nuclear genome information. Complex statistical analysis of previously developed procedures resulted in information-rich data regarding factorial interactions helpful for developing protocols in genome-restoration programs.

Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved spermatozoa

The effects of extender composition and equilibration time on fertilizing ability of cryopreserved spermatozoa from rainbow trout, Oncorhynchus mykiss, were investigated. In addition, enzyme activity in supernatants from thawed sperm was assessed. The use of the two extenders: Erdahl & Grahams + 10% DMA (dimethyl acetamide) + 10% egg yolk and 0.3 M glucose + 10% DMA yielded the highest post-thaw fertilization rates. We observed interactions between extender constituents and the equilibration of diluted semen. This indicates a multifactorial effect of the extender constituents on spermatozoal resistance against injuries. The 10-min equilibration of spermatozoa in extender before freezing generally lowered the fertilization ability of spermatozoa, except for DMA-based extenders. The addition of egg yolk to the extender was generally beneficial, especially in DMA- and DMSO-based extenders. The use of low-density lipoprotein fraction showed no advantage to full-yolk or free-of-yolk extenders. Aspartate aminotransferase and lactate dehydrogenase leakage from damaged spermatozoa correlated negatively with the ability of cryopreserved spermatozoa to fertilize eggs. Each factor tested, when analyzed separately, did not give general information about its effect on the fertilization ability of cryopreserved sperm. The multifactorial analysis of the important factors in cryopreservation of trout spermatozoa showed their cumulative effect. This is the most likely reason for divergent information reported elsewhere on the effect of various factors in the cryopreservation of rainbow trout spermatozoa.

Formation of chromosome aberrations in androgenetic rainbow trout Oncorhynchus mykiss

Residues of maternal nuclear DNA in the form of chromosome fragments were observed in the healthy and morphologically normal androgenetic rainbow trout Oncorhynchus mykiss. A hypothetical model for formation of chromosome re-arrangements caused by the incomplete maternal nuclear DNA inactivation in the androgenetic rainbow trout was proposed in the present paper.