Reproductive Cycle and Semen Preservation of Yellow Croaker Larimichthys polyactis

Abstract

Studies were performed to find out the reproductive cycle, physico-biochemical properties of semen, mechanism of spermatozoa motility, optimal condition for sperm cold storage and cryopreservation of yellow croaker Larimichthys polyactis. The gonadosomatic index (GSI) of males was lower than that of females. Maximum GSI values for males (5.7%) and females (11.4%) were recorded in May. The breeding season was estimated to be from April to June based on progressive changes in the size of the ovaries and testes, histological examination of the ovaries and testes, oocyte diameter measurements, and GSI calculations. The physical properties of semen in this species were 1.1±0.1 mL/fish in semen volume, 2.5±0.1x10^9 cells/mL in spermatozoa density, 97.8±0.5% in spermatocrit and 2.7±0.3x10^9 cells/fish in total number of spermatozoa. The biochemical properties in seminal plasma of this species were as flows; 148.0±1.0 mmol/L sodium, more than 14.0 mmol/L potassium, 115.5±1.5 mmol/L chloride, 11.7±0.2 mg/dL calcium, 4.5±0.3 mg/dL magnesium, 0.1 mg/dL glucose, 1.0 g/dL total protein, 242.5±0.2 mOsm/kg osmolality and 7.7±0.1 pH. The spermatozoon of yellow croaker consisted of three parts: head without acrosome, mid-piece with three mitochondria and flagellum with '9+2' pattern. The head of spermatozoon in longitudinal section was kidney-shape, and 0.6-0.8 um long and 1.4-1.6 um wide. Optimal spermatozoa velocity (SV), movable spermatozoa ratio (MSR) and duration of spermatozoa motility (DSM) were examined when semen was diluted in artificial seawater (ASW) at a ratio of 1 to 100, with temperature of 10oC and pH 8.0. The spermatozoa of yellow croaker were immotile in distilled water and motile in solution containing different cations and sugars. Maximum SV, MSR and DSM were obtained in each solution containing 0.4 M NaCl, 0.4 M KCl, 0.2 M CaCl2 and 0.2 M MgCl2. The highest SV, MRS and DSM were achieved in each solution containing 0.5 M glucose, sucrose, fructose or mannitol, respectively. This study provides baseline knowledge of the sensitivities of yellow croaker spermatozoa under the effects of pH, temperature, cation and sugar. To find out the optimum cold storage condition of the sperm of yellow croaker, various combinations of factors were tested such as extender (artificial seminal plasma (ASP) or marine fish Ringer's solution (MFRS)), dilution ratios (semen:extender, 1:1, 1:3 or 1:5), temperatures (0, 2 or 4oC), and antibiotics (gentamycin or neomycin with concentrations of 200, 400, 600, 800 or 1000 ppm). The most effective condition for cold storage was ASP extender in dilution ratio of 1 to 3 at 0oC, and the preserved sperm maintained motile for 14 days. The best concentration of antibiotic was 600 ppm of gentamycin or 200 ppm of neomycin, and the preserved sperm retained motile for 26 days. The effects of various extenders and cryoprotectants on MSR, spermatozoa activity index (SAI), SV and DSM of post-thawed spermatozoa were examined. The MSR, SAI, SV and DSM of post-thawed spermatozoa in ASP were higher than those in MFRS and were not significantly different from those of fresh spermatozoa. No significant differences were observed in the motile parameters between fresh sperm and sperm cryopreserved with 10% ethylene glycol (EG). Using the above method, yellow croaker sperm was cryopreserved with extender ASP and 10% EG. As a result, at the spermatozoa/egg ratio of 100,000:1, the fertilization rate and hatching rate of the cryopreserved spermatozoa in liquid nitrogen for 1 week were 45.7±3.2% and 27.2±5.0%, respectively; and those for 1 year were 37.5±4.4% and 27.2±5.0%, respectively. Those results were similar to those of fresh spermatozoa (51.0±3.1% and 36.7±2.2%). However, there was a minor alternation of shape of cyopreserved spermatozoa compared with fresh spermatozoa. Cryopreservation of dead fish sperm showed that it is possible to use for artificial insemination. The male yellow croakers were killed and stored at different temperature regimes of 20oC or 0oC for 6 h. At every two hour interval, sperm from these males were collected by pressing abdomen and used for motility evaluation or cryopreservation. Sperm collected after 6 h from dead male that is stored at 0oC for 6h were tested and the fertilization rate and hatching rate showed 15.0±1.2% and 14.8±1.6%, respectively. This study suggests that germ cells such as spermatozoa of dead fish can be cryopreserved and utilized for restoration of a species.