IntroductionReproduction in fish is under the influence of various external factors. The most important factors are photoperiod, water temperature, water quality (dissolved oxygen, pH, hardness, salinity, alkalinity), floods, water currents, tides and cycles of the moon, weather conditions (atmospheric pressure, rainfall), spawning substrates (aquatic plants, pieces of wood, wards), nutrition, diseases, parasites, and the presence of other fish (1). In natural conditions, these fish are mostly female after 2 years of age (2). Seabream (Sparus aurata) has reproductive properties such as protandrous hermaphroditism, asynchronous ovarian development, and multiple daily spawnings (3,4).Newly spawned seabream eggs are approximately 0.9-1 mm in diameter and are transparent. Normally they contain one single oil drop and have a pelagic structure. Since the eggs have a pelagic structure, only the eggs that can float in water are viable and can form embryos (5,6). The egg membrane is transparent and thin, and the micropile hole is about 14 µm (2,7). After the seabream eggs complete their maturation and they are left in water and hydrolyzed, the egg diameter can be up to 1100 µm (8).Since fish spermatozoa are not motile when they leave the testes, they should be activated in species-specific ways (fresh water or sea fish species) (9). For spermatozoa activation, a number of extracellular agents were reported. Flagellar activation is provided by hyperosmotic-osmotic (500-1100 mOsm) medium in sea fish and by hypoosmotic (0-100 mOsm) medium in freshwater fish. In salmonids and sturgeon the most important motility activation factor is potassium (K + ) concentration (10,11). It was stated that in seabream K + and calcium (Ca 2+ ) had no effect on spermatozoa activation and that the optimal osmolarity for this activation was 1100 mOsm/kg (11).Penetration happens quickly after extending because fish spermatozoa are very small in structure; thus, an equilibration step is not required. The toxic effects of cryoprotectants can be minimized by not applying the equilibration stage. In a study conducted on seabream, when dimethyl sulfoxide (DMSO)-added semen was left for equilibration for more than 2 min, fertilization rates were lower (12,13).Fish semen is not appropriate for freezing without extending. The extenders used for freezing sea fish semen are mostly salty (1%-10% concentration) or sugary
