Abstract
The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.
Highlights
IntroductionFish spermatozoa quality depends on the breeding season, age of the males and manipulation of the gametes in vitro [1,2]
Once the percentage motility of spermatozoa was analyzed in detail, focusing on fast, medium, slow and static spermatozoa (Figure 1B), 81.7% of fast spermatozoa were recorded at 1 h post stripping (HPS)
Significant decreases in the phenotypic characteristics of common carp sperm were detected after aging for 96 h from stripping
Summary
Fish spermatozoa quality depends on the breeding season, age of the males and manipulation of the gametes in vitro [1,2]. Declining spermatozoa quality is directly associated with a decrease in spermatozoa performance resulting in poor fertilizing ability and the quality of the resulting offspring [3,4]. A decline in sperm quality leads to a decrease in energy stores and changes in proteins [5]. Alterations in DNA and membrane integrity, and mitochondrial membrane potential have been noted [2,6,7,8,9]
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