Examination of larval malformations in African catfish Clarias gariepinus following fertilization with cryopreserved sperm

Abstract

In our earlier experiments on the cryopreservation of African catfish ( Clarias gariepinus) sperm a high number of malformed individuals were found among the larvae hatched from eggs fertilized with frozenthawed sperm. In our present study we explored one of the possible reasons of this malformation by examining the ploidy of the larvae. Sperm was frozen in a 6% fructose extender containing 10% methanol and 10% DMSO as cryoprotectant. Sperm was drawn to straw of different volumes (0.25, 0.5 and 1.2 ml). Freezing was carried out in liquid nitrogen vapor. Fifteen grams or thirty grams of eggs were used for fertilization. Chromosome preparations were made of non-feeding larvae. Fifty to one hundred larvae were incubated in 0.05% colchicine for 3 h then in a hypotonic 0.075M KCl solution for 25 min. Larvae were fixed in a 3:1 solution of methanol and acetic acid, then cell suspensions were made in 50% acetic acid. The suspensions were spread on slides and stained in 4% Giemsa for 8 min. Chromosome counting and evaluation was done at 1200× magnification on well spread metaphases. A majority of the hatched larvae were diploid, with 56 chromosomes. Surprisingly some of the malformed larvae hatched from eggs fertilized with cryopreserved sperm were haploids. Haploids occurred only when 0.25 or 0.5 ml straws were used for freezing. One possible explanation of haploidy is that the genome of the fertilizing spermatozoon is damaged during the process of freezing thus it can still move and fertilize the egg but its genome does not take part in the development of the embryo. This hypothesis is supported by the fact that no haploids were found among the malformed larvae of the control group which was fertilized with fresh sperm.