Effect of trehalose on sperm cryopreservation

 of Acipenser schrenckii and preliminary study 

on the mechanism of its cryoinjury

Abstract: Cryopreservation is of importance for the genetic improvement of resources, conservation of species diversity, and breeding of improved varieties in aquaculture industry. The techniques of sperm cryopreservation have been established and optimized in numerous kinds of sturgeons; however, the motility, fast movement, and durations of frozenthawed semen were significantly lower than those of fresh semen.  In this study, we added various concentrations of trehalose or sucrose as cryoprotectants in the extender for sperm cryopreservation of Acipenser schrenckii and evaluated the motility, fast movement, and duration of the treated groups and the control group (fresh sperm). The extender diluted with 10% MeOH was mixed with sperm at a ratio of 1∶2 for sperm cryopreservation.The diluted semen was dispensed into 2 mL cryovials, placed into cloth bags, suspended 15 cm above nitrogen vapor for 10 min, then 5 cm above nitrogen vapor for 5 min, and finally stored in liquid nitrogen. After thawing at 37 ℃, the motility, fast movement and durations of frozenthawed semen was observed and recorded under the microscope, compared with fresh semen.  Results showed that ST5 solution (1 mM NaCl, 20 mM Tris, 60 mM trehalose, pH 8.0) worked most effectively during sperm cryopreservation of A. schrenckii. The motility of frozenthawed spermatozoa was(26.67±3.32)%, significantly lower than that of fresh spermatozoa. The fast movement of fresh spermatozoa could last (93.17 ± 4.34) s, while frozenthawed spermatozoa could last (75.63 ± 6.05) s. The duration of fresh spermatozoa ［(246.61 ± 2.29) s］ had no significant difference with frozenthawed spermatozoa ［(239.35 ± 1.49) s］. To explore the mechanisms of cryoinjury in A. schrenckii semen, we fixed the fresh and frozenthawed spermatozoa with 2.5% glutaraldehyde and observed their ultrastructure by using transmission electron microscope (TEM) and scanning electron microscope (SEM). Meanwhile, enzyme activities were compared between fresh and frozenthawed sperm. Results showed that mature spermatozoa were comprised of acrosome, head, midpiece and flagellum. The length of acrosome, head, midpiece was (0.71 ± 0.08) μm, (0.71 ± 0.08) μm, and (0.87 ± 0.31) μm. The width of acrosome, head, midpiece was (0.76 ± 0.06) μm, (0.71 ± 0.08) μm, and (0.73 ± 0.16) μm. It had an obvious acrosome, which was also observed in other sturgeon species. The mitochondria was located in midpiece for the energy supplement during tail movement. Flagellum was composed of central fibers and peripheral fibers, which formed a typical “9+2” structure of axoneme. Cryopreservation caused the disruption of membrane and organelles, including the disruption of plasmalemma, nuclear membrane, cristae of mitochondria and flagellum membrane, acrosome damage, swelling midpiece, cytoplasmic dispersion, and mitochondria and flagellar fracture. The activities of enzymes involved in energy metabolism, containing total adenosine triphosphatase (total ATPase), creatine kinase (CK), succinate dehydrogenase (SDH), and lactate dehydrogenase (LDH), were measured in A. schrenckii sperm before and after cryopreservation. Results showed that the activities of all four enzymes in spermatozoa significantly dropped (P<0.05). The activities of total ATPase, CK, SDH, LDH in spermatozoa in cryoprotectant group decreased from (7.45±1.98) U·mL-1, (4.98 ± 0.97) U·mL-1, (14.50 ± 2.12) U·mL-1, and (10 850.11±184.82) U·L-1 to (2.33±0.20) U·mL-1, (2.13±0.83) U·mL-1, (3.38±1.04) U·mL-1, and (5 588.09±75.35) U·L-1. At the same time, the activities of enzymes in spermatozoa without cryoprotectant dropped to (0.70 ± 0.02) U·mL-1, (2.72 ± 0.39) U·mL-1, (2.83 ± 1.52) U·mL-1, and (4 030.20±1 093.12) U·L-1. The activities of total ATPase and CK in seminal plasma significantly increased(P<0.05), while the activities of SDH and LDH increased but without significant differences. The activities of total ATPase, CK, SDH, LDH in seminal plasma in cryoprotectant group increased from (0.76 ± 0.05) U·mL-1, (0.33 ± 0.14) U·mL-1, (1.56±0.31) U·mL-1, and (5 469.80±131.19) U·L-1 to (3.85 ± 1.62) U·mL-1, (1.56±0.18) U·mL-1, (4.33±2.17) U·mL-1, and (5 618.09±216.88) U·L-1. Meanwhile, the activities of enzymes in seminal plasma without cryoprotectant dropped to (4.60±1.46) U·mL-1, (3.59±0.51) U·mL-1, (29.30±5.88) U·mL-1, and (9 133.11±1 023.54)U·L-1.  In addition, the antioxidant activities containing catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSHPX), and glutathione reductase (GR) were measured in A. schrenckii sperm before and after cryopreservation. Results showed that GR activity in spermatozoa significantly increased while the activities of the other three antioxidants significantly dropped (P<0.05). The activities of total CAT, SOD, GSHPX, GR in spermatozoa in cryoprotectant group changed from (20.94 ± 0.48) U·mL-1, (129.91 ± 2.77) U·mL-1, (52.35 ± 13.03) U·mL-1, and (38.32 ± 17.71) U·L-1 to (4.96 ± 0.72) U·mL-1, (94.59 ± 5.49) U·mL-1, (13.39 ± 7.12) U·mL-1, and (132.44 ± 17.90) U·L-1. At the same time, the activities of enzymes in spermatozoa without cryoprotectant changed to (0.65 ± 0.27) U·mL-1, (78.77 ± 4.90) U·mL-1, (6.14 ± 4.21) U·mL-1, and (166.801 ± 41.39) U·L-1. The activities of CAT and GR in seminal plasma significantly changed (P<0.05), while the activities of GSHPX and SOD increased but without significant differences. The activities of total CAT, SOD, GSHPX, GR in seminal plasma in cryoprotectant group increased from (0.51 ± 0.34) U·mL-1, (94.74 ± 2.37) U·mL-1, (4.22±1.84) U·mL-1, and (161.58 ± 37.57) U·L-1 to (1.84 ± 0.35) U·mL-1, (96.28±2.69) U·mL-1, (10.89±3.41) U·mL-1, and (86.01±7.90) U·L-1. Meanwhile, the activities of enzymes in seminal plasma without cryoprotectant dropped to (17.09 ± 1.70) U·mL-1, (124.10±1.65) U·mL-1, (45.01 ± 7.19) U·mL-1, and (2.01 ± 1.39) U·L-1.  The activities of enzymes including total ATPase, CK, SDH, LDH, CAT, SOD, GSHPX) significantly decreased in frozenthawed spermatozoa (P<0.05), while GR activity significantly increased (P<0.05). These results indicated the damages in psychological functions, intracellular environment, antioxidative system, and energy supplement, which might affect the motility of A. schrenckii spermatozoa.  Compared with the group supplemented with cryoprotectants, the activities of total ATPase, CK, CAT, SOD and GR in the group without cryoprotectants were significantly changed (P<0.05), suggesting the effective protection of trehalose in cryoprotectants on frozenthawed sperm. The observation of spermatic ultrastructure damage and variations of antioxidants and enzymes involved in energy metabolism in frozenthawed spermatozoa could be used to evaluate the quality of fish semen. Furthermore, the results of this study would provide a theoretic basis for the parametric optimization to improve the quality of frozenthawed sperms, and shed some light on the exploration on mechanism of cryoinjury in fish spermatozoa.

Keywords: Acipenser schrenckii; spermatozoa; cryopreservation; trehalose; cyroinjury