Animals
The research was performed at the Research Institute of Fish Culture and Hydrobiology of the Faculty of Fisheries and Protection of Waters, University of South Bohemia, Czech Republic. Handling of common carp broodstock was performed following earlier (Linhart et al. 2015). Before application of hormonal treatment and gamete collection, three males (2 to 3 years old, 1.5, 1.6 and 1.7 kg bw) and one female (8.3 kg bw) in optimal physical condition were anaesthetized in a solution of 2-phenoxyethanol (1:1,000 v/v). To avoid injury or trauma of the fish, sperm collection was performed as gently as possible throughout the whole experiment.
Experimental design
The whole experiment was based on the use of 3 males from a recirculation aquaculture system (RAS). The fish had very good sperm production and motility parameters and were in a healthy condition as observed from previous experiments. Males were treated 3 times with carp pituitary (CP) dissolved in 0.9% (w/v) NaCl solution. CP of 1.5 mg/kg body weight was injected similar males total of 3 times with 3 days apart of experiment. Sperm was stored in vivo in the male body cavity (21 oC) for 0.5 days (fresh sperm) and 3 days (old sperm) days after CP application and then collected. Everything was simply synchronized so that after the 1st collection of old sperm (i.e., after 3 days of the 1st storage of sperm in vivo at body cavity of fish) we applied the 2nd and in 3 days later the 3rd dose of CP, so the whole experiment with CP application, sperm storage in vivo and sperm collection lasted 9 days. All is explained in detail in Schema 1. Sperm was immediately diluted with a carp extender (Cejko et al. 2018) 1:1 after collection and stored in vitro for 0, 3 and 6 days sperm storage (DSS) at 0 to 2°C. Diluted sperm (6 mL each from three individual males) was stored in 50 mL containers in an aerobic environment.
The variables: percentage of sperm motility, number of motile spermatozoa (× 109 mL− 1), percentage of fast motile spermatozoa, number of fast motile spermatozoa (× 109 mL− 1), curvilinear velocity (VCL, µm/s), straight-linear velocity (VSL, µm/s), percentage of live spermatozoa, number of live spermatozoa (× 109 mL− 1), sperm concentration (× 109 mL− 1 ), volume of sperm (mL), number of total spermatozoa (× 109) and number of total motile spermatozoa (× 109), were recorded and calculated in this study. The fertilization and hatching level with embryo malformation rates were performed in the case of three males with fresh sperm 1,2 and 3 and old sperm 1 and 2 stored in vivo followed with in vitro 0 to 6 DSS of storage sperm (see Schema 1).
Examining phenotypic characteristics of spermatozoa
Sperm motility and velocity
With the addition of 0.25% Pluronic F-127 (catalog number P2443, Sigma-Aldrich; used to prevent sperm from sticking to the slide), distilled water was used as an activating medium (pH 7.0 to 7.5). All sperm was kept on ice during the whole experiment. The diluted sperm sample was mixed into 10 µL of the activation medium using needles, and spermatozoa were activated on a chamber SpermTrack-10® (Proiser R + D, S.L.; 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). The sperm variables including the percentage of motile sperm, VCL, VSL and spermatozoa rate with rapid motility (> 100 µm/s) were analyzed by computer-assisted sperm analysis (CASA). Based on the microscope and adaptor set and digital video camera operated at 25 fps, scales were calibrated in Integrated Semen Analysis System (ISAS) at 15 s post-activation. Escala X and Escala Y were both set up to 1.4 µm when using 10 × lens on a negative phase-contrast condenser microscope. Quantitative analyses of all samples were conducted in triplicate.
Concentration of spermatozoa
Combined with the sperm number, the concentration of sperm from each male was evaluated at 0, 3 and 6 DSS. Twelve squares in a Bürker cell hemocytometer (Marienfeld, Germany) were counted for each sample. Under an Olympus microscope BX 41 (4009), the sperm concentration, which was expressed as × 109 spermatozoa per mL, was determined by using an optical phase-contrast condenser and an ISAS digital camera (PROISER, Spain). In order to count spermatozoa numbers precisely and clearly, the Bürker cell hemocytometer was covered after adding 10 µL of diluted samples (990 µL of 0.9% NaCl + 10 µL of sperm) with two repetitions. The duration of sample placement in a Bürker cell hemocytometer was about 3 min to sediment the spermatozoa.
Sperm viability analysis
Using the LIVE/DEAD Sperm Viability Kit (Invitrogen/Thermo Fisher Scientific Inc.), the ratio of live: dead sperm cells was analyzed by flow cytometry with S3e™ Cell Sorter (Bio-Rad, Hercules, CA, USA). The protocol of flow-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). The sperm solution was then supplemented with 5 µL of propidium iodide (PI; Sigma-Aldrich, St Louis, MO, USA). It was then vortexed for a few seconds and incubated on ice in the dark for at least 15 min. More than 10,000 spermatozoa were separated and analyzed by the S3e Cell Sorter. In this system, the membrane of the damaged cells was fluorescent. Therefore, high PI fluorescent signals were considered as dead cells, and low signals as live cells. The data were processed using ProSortTM software. Based on the ratio of low: high PI fluorescent cells, the percentage of live and dead sperm cells was calculated (Horokhovatskyi et al. 2018).
Fertilization, hatching and larval malformation rates
The sperm samples stored in vivo and in vitro, included fresh 1 (6 DSS), fresh 2 (3 DSS) and fresh 3 (0 DSS), and old 1 (3.5 DSS) and old 2 (0.5 DSS) (Schema 1) from three individual males (together making 15 samples) were used for fertilization. Prior to fertilization, the concentration of spermatozoa (already diluted with carp extender, 1:1) from all of the 15 samples (three males) was measured (see Table 1).
Ten females in good physical condition were selected and treated with CP. After a 3 days period of acclimation in the hatchery at 20°C, ovulation of the females was induced using CP dissolved in 0.9% (w/v) NaCl solution; 0.5 mg/kg bw was treated as a priming dose and a second dose of 2.7 mg/kg bw was administered 12 h later. Eggs were collected into plastic bowls 12 h after the second hormonal treatment. The eggs, considered to be of greatest quality from one individual female, were visually selected and used for experiments. The criteria for selection of eggs were those with a darker coloration (i.e., the darker the more desirable), without a lot of ovarian fluid and without any eggs undergoing decomposition. The eggs were stored in an incubator and covered by parafilm under aerobic conditions at 19°C within 30 min prior to fertilization.
Five g of c. 4000 eggs of the greatest quality female were fertilized with the appropriate volume of sperm, which was pipetted to the bottom of a beaker (25 mL), placed on a shaking device at 21 oC. To achieve good fertilization, sufficient spermatozoa were used for performing the experiment with a ratio of eggs: spermatozoa = 1: 500,000. Appropriate volumes of sperm from three males used for fertilization were shown in Table 1. Then 10 mL of activation solution (5 mM KCl, 45 mM NaCl and 30 mM Tris, pH 8.0, 160 mOsm/kg) (Perchec et al. 1996) were pipetted into the beaker and kept on a shaking device with a speed of 250 rpm/min for 1 min. Subsequently, 400 fertilized eggs were gently distributed into four Petri dishes (9 cm in diameter and 1.5 cm in depth), for the current fertilization assay (the other fertilized eggs were used for another study).
Table 1
The concentration and volume of spermatozoa prior to fertilization (Concentration of spermatozoa denoted as sperm diluted by carp extender (1:1).
Males Sample | Male 1 | Male 2 | Male 3 |
Concentration (×109/mL) | Volume (mL) | Concentration (×109/mL) | Volume (mL) | Concentration (×109/mL) | Volume (mL) |
Fresh sperm 1 | 12.10 | 0.17 | 11.60 | 0.17 | 11.10 | 0.18 |
Fresh sperm 2 | 10.60 | 0.19 | 10.50 | 0.19 | 10.0 | 0.20 |
Fresh sperm 3 | 10.30 | 0.19 | 9.20 | 0.22 | 9.50 | 0.21 |
Old sperm 1 | 10.40 | 0.19 | 12.80 | 0.16 | 12.00 | 0.17 |
Old sperm 2 | 12.10 | 0.17 | 11.70 | 0.17 | 12.30 | 0.16 |
Each group of four Petri dishes was placed in four plastic boxes (13.5 cm × 10 cm × 6.5 cm) filled with dechlorinated water (300 mL) (Cheng et al. 2021b). The room temperature was controlled at 21.5°C by air-conditioning. After fertilization and distribution of eggs into the Petri dishes, if more than three eggs adhered together, the cluster was removed using a needle and plastic pipette to avoid future embryonic death accompanied by the development of fungi. Then the total remaining number of eggs was counted and considered as the original total number of eggs. At 48 h post-fertilization, non-developing embryos were removed, and eye-stage embryos (fertilized ones) were counted during the water exchange. After the eye-stage, dechlorinated water was changed completely and daily up to hatching. Until hatching, the normal and malformed larvae (abnormal body size and shape, Zi et al. 2018) were manually counted directly over the next 4 days of incubation at 23.5°C. Fertilization and total hatching rates were calculated as the ratio of all remaining eye-stage embryos at 48 h post-fertilization and hatched larvae at 4 days post-fertilization from the initial number of eggs per Petri dish. All larvae with unusual body proportions, and irregular body axis and head, were considered as malformed larvae. The malformation rate was calculated from total hatched larvae in each petri dish.
Data evaluation and statistical analysis
Using the Levene’s test, the data distribution homogeneity of dispersion was assessed. A Shapiro-Wilk normality test was used to evaluate whether a parametric or non-parametric test was more appropriate in analyses. In experiments with more than two independent experimental groups, the differences among the variables were analyzed by one- and two-way ANOVA with LSD and Tukey’s HSD tests or Kruskal-Wallis with Dunn’s test for multiple comparisons.
Principal component analysis (PCA) was used to identify new variables and/or principal components. A set of values of linearly uncorrelated variables, called PCs or Dims, was extracted from a set of possibly correlated variables using orthogonal transformation. Usually, the new variable with the largest possible variance was regarded as the first PC. After extraction of the first PC, the other succeeding component with the highest variance was obtained. Thus, the variance and information content of each PC diminished consecutively. All the results are presented as mean ± S.D. in bar graphs. All analyses were performed at a significance level of P < 0.05 in R software 3.3.2 (Core R Team, 2019).