Changes in Phenotypes and DNA Methylation of In Vitro Aging Sperm in Common Carp Cyprinus carpio

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.

Bioenergetic Pathways in the Sperm of an Under-Ice Spawning Fish, Burbot (Lota lota): The Role of Mitochondrial Respiration in a Varying Thermal Environment

“…Sperm concentration was evaluated by using a Burker cell hemocytometer (Meopta, Kabelikova 1, Prerov, Czech Republic) and an Olympus BX 50 phase-contrast microscope (200× magnification; Olympus, Shinjuku, Tokyo, Japan) [ 17 ].…”

In vivo and in vitro aging of common carp Cyprinus carpio sperm after multiple hormonal application and stripping of males

“…; Paterna, Spain) at 21°C for different storage times. Following our previous study, the activated spermatozoa were recorded microscopically until the end of their motility (Cheng et al 2021b).…”

“…Using the LIVE/DEAD Sperm Viability Kit (Invitrogen/Thermo Fisher Scienti c Inc.), the ratio of live: dead sperm cells was analyzed by ow cytometry with S3e™ Cell Sorter (Bio-Rad, Hercules, CA, USA). The protocol of ow-cytometer was built and performed based on past research (Cheng et al 2021b). One µL of sperm was added to 2 mL NaCl solution with 0.9% (w/v).…”

“…As a key variable of sperm quality, percentage of sperm motility was recognized as an important variable for successful fertilization (Linhart et al 2000(Linhart et al , 2020bRurangwa et al 2001Rurangwa et al , 2004Gallego et al 2013). It should also be emphasized that fast motile spermatozoa are very important for fertilization (Cheng et al 2021b).…”

“…The sperm motility (60 s post activation) recorded at 48 h after hormonal application was signi cantly better than that 52 and 56 h after hormonal application in pikeperch (Sander lucioperca) (Malinovskyi et 2004). However, both quantity and quality, especially the number of sperm at a higher velocity, are evaluated (Cheng et al 2021b). In our study, sperm concentration was similar at collection at 0.5 or 3 days after hormonal treatment application, but the volume of sperm was much larger when collected at 0.5 days.…”