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.

Keywords: DNA methylation; common carp; epigenetics; fertilization; fish; milt; sperm aging; sperm quality; sperm storage.

Figure 1

Effect of pooled milt aging (5 males) stored at 1, 24, 48 and 96 h post stripping (HPS) in common carp (mean ± S.D.): (A) percentage of sperm motility evaluated at 15 s of post sperm activation (PSA); (B) percentage of sperm motility from total motility of spermatozoa evaluated at 15 s of PSA: rapid motility (>100 μm/s), medium motility (46 to 100 μm/s), slow motility (10 to 45 μm/s) and static spermatozoa (<10 μm/s); (C) total and motile sperm concentration per mL; (D) curvilinear velocity (VCL) and straight line velocity (VSL) evaluated at 15 s PSA; (E) viability of spermatozoa cells; (F) pH of seminal fluid; (G) osmolality of seminal fluid. Values with different letters within lower- or upper-case letters, are significantly different [p < 0.05, one-way analysis of variance (ANOVA) followed by an LSD test for post hoc multiple comparisons].

Figure 2

Effect of aging milt from five individual males stored at 1, 48 and 96 HPS in common carp (mean ± S.D.): (A) percentage of sperm motility evaluated at 15 s of post sperm activation (PSA); (B) percentage of sperm motility from total motility of spermatozoa evaluated at 15 s of PSA [rapid motility (>100 μm/s), medium motility (46 to 100 μm/s), slow motility (10 to 45 μm/s) and static spermatozoa (<10 μm/s)]; (C) total and motile sperm concentration per ml; (D) curvilinear velocity (VCL), straight line velocity (VSL) evaluated at 15 s of PSA; (E) pH of seminal fluid; (F) osmolality of seminal fluid. Values with different letters within lower or uppercase letters are significantly different (p < 0.05, one-way analysis of variance (ANOVA) followed by an LSD test for post hoc multiple comparisons).

Figure 3

(A) Fertilization (eye stage), (B) hatching and (C) malformation rates (mean ± S.D.) of common carp with a total number of 31,250,000 spermatozoa. Aged milt was stored for 1, 24 and 96 h individually and pooled just prior to fertilization from four males. Controls were fresh milt collected at 24 and 96 h and used as a pool of milt from three males. Eggs were also pooled from four females. Values with the different superscript are significantly different (p < 0.05).

Figure 4

Percent change of differentially methylated regions (DMRs) among the three libraries. The DMRs were calculated by Defiant, and each value is statistically supported by three biological replicates. Asterisks denotes p-value between two groups (** p < 0.01, **** p < 0.0001).