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Kvalita spermatu ovlivňuje oplození u sladkovodních ryb
CHENG, Yu
Short-term storage of sperm is a widely used technique during artificial fertilization in hatchery practice due to its convenient, inexpensive and practical value. Chapter 2, three egg incubation techniques were compared to supply one simple and efficiency method to incubate fertilized sterlet eggs. The percentage of cleavage, neurulation and hatching rates of embryos and larvae were examined to compare the effects of incubation systems consisting of placing Petri dishes 1) in the hatchery tank, 2) on a table in small incubation box in an air-conditioned room or 3) in a large thermostatic incubator box. No significant differences were found among these egg incubation systems. The experience gained allowed us to practice this simple method of egg incubation in Petri dishes on other fish species such as carp or zebrafish. Chapter 3, attention was paid to processes of egg activation and fertilization, which are highly important issues in fish reproductive biology. In the sterlet, multiple treatment groups with different gradient of spermatozoa number and volume of activation solution were used to fertilize eggs and further determine the minimum number of spermatozoa with optimal volume of activation water. The artificial fertilization experiments revealed that a total of 50,000 spermatozoa in 8 ml or 32 ml of activation water was sufficient for successful fertilization. It was obtained that a very low concentration of 100-1,300 spermatozoa per ml in activation water can achieve efficient fertilization with good quality sperm. Chapter 4, sperm managements including prevent sperm spontaneous movement, artificial seminal plasma composition, use of natural seminal plasma from good sperm were explored. The fertilization optimizations with different methods, spermatozoa: egg ratio and minimum sperm concentration were also examined in zebrafish. Results indicated that extender E400 was efficient during zebrafish sperm storage; a test tube with a well-defined amount of 6,000,000 zebrafish spermatozoa in E400 extender per 100 eggs and 100 ?L of activation solution has proven to be more successful than using a Petri dish. Sperm of European catfish can be collected and stored in immobilization solution for as long as 1 week for successful fertilization. Sterlet sperm with bad quality can be revitalized by incubation with seminal plasma from good sperm. The results help to establishing a sperm in vitro storage method and fertilization procedures in freshwater fishes. Chapter 5, activation solution is one of the key factors providing sperm and egg performance during fertilization. Different activation solutions were employed to test the practical application efficiency in a hatchery jar with sperm that was aged but diluted in artificial seminal plasma. The large-scale artificial fertilization trials indicated that fertilization and hatching rate were high (78-87%) after one week of sperm storage in a carp artificial seminal plasma in activation solutions as hatchery water and Perchec solution. Results offered the practical basic for the further application of aging sperm in a hatchery. Chapter 6, in this study, aged sperm in common carp was used to evaluate the sperm motility and kinematics, sperm viability, osmolality and pH of seminal plasma sperm short-term in vitro storage. DNA methylation analysis based on the whole-genome bisulfite sequencing was also performed to reveal the molecular changes of aging sperm. The results showed that aging spermatozoa in common carp has a considerable negative impact on their performance and as well as artificial fertilization success. the level of methylation at the CpG sites increased significantly with 24 hours post stripping spermatozoa compared to the fresh group, but then reduced significantly at 96 hours post stripping. The results contribute to investigate the sperm phenotype and functional changes in common carp and providing clues for understanding the molecular changes during sperm storage
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