Ming Bo Aquatic Co.

Weichanglu, China

Ming Bo Aquatic Co.

Weichanglu, China

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Tian Y.,Chinese Academy of Fishery Sciences | Tian Y.,Laboratory for Marine Fisheries Science and Food Production Processes | Chen Z.,Chinese Academy of Fishery Sciences | Chen Z.,Laboratory for Marine Fisheries Science and Food Production Processes | And 12 more authors.
Cryobiology | Year: 2017

Fish embryo cryopreservation is highly important for the long-term preservation of genomic and genetic information; however, few successful cases of fish embryo cryopreservation have been reported over the past 60 years. This is the first study to use Epinephelus moara embryos from fertilization with cryopreserved sperm as experimental material. Embryos that developed to the 16–22 somite stage and tail-bud stage were treated with the vitrification solution PMG3T according to a five-step equilibration method and cryopreserved at various temperatures and storage duration. Only 19.9 ± 9.2% of 16–22 somite stage embryos and 1.3 ± 1.1% of tail-bud stage embryos survived when cooled at 4 °C for 60 min. In total, 8.0 ± 3.0% of 16–22 somite stage embryos survived when cooled at −25.7 °C for 30 min, 22.4 ± 4.7% of tail-bud stage embryos survived after 45 min of cooling at −25.7 °C, and none survived after 60 min. Only 2.0 ± 2.7% of embryos survived when cryopreserved at −140 °C for 20 min. However, 9.7% of tail-bud stage embryos survived after cryopreservation in liquid nitrogen (−196 °C) for 2 h. Most surviving embryos developed normally. Embryonic volume decreased and spherical segments appeared when embryos were treated with higher concentrations of vitrification solution. Additionally, the volume recovered gradually after rinsing with sucrose and seawater. This is the first estimate of the survival of E. moara embryos and larvae after cryopreservation. These findings provide a foundation for further explorations of fish embryo cryopreservation techniques. © 2017 Elsevier Inc.


Tian Y.,Chinese Academy of Fishery Sciences | Qi W.,Chinese Academy of Fishery Sciences | Qi W.,Shanghai Ocean University | Jiang J.,Chinese Academy of Fishery Sciences | And 7 more authors.
Animal Reproduction Science | Year: 2013

Seven-band grouper Epinephelus septemfasciatus is a protogynous hermaphrodite. The male individuals' number is more less than the female one. Thus, it is necessary for artificial reproduction and crossbreeding to research the sperm cryopreservation of sex-reversed seven-band grouper. In present study, the spermatozoa of sex-reversed immature fish were frozen using the different cryopreserving solutions for cryopreservation. The several factors that may affect the freezing survival rate of seven-band grouper spermatozoa such as the spermatozoa diluent, the concentration and composition of cryoprotective agent have been studied. The results showed that ES1-3 (60g/L glucose+10g/L NaCl+0.5g/L NaHCO3) was significantly better as a diluent compared with MPRS, TS-2 and other series of diluent ES1. The further experiment revealed that the optimal cryoprotectants were 10% dimethyl sulfoxide (DMSO) or 10% 1,2-propylene glycol (PG) with the post-thaw sperm motility was 76.67±0.00% and 75.00±5.00%, respectively. In addition, salinity of seawater is an important motility stimulator because that the highest motility of 96.00±1.73% was obtained at salinity 30‰. In crossbreeding test with fresh unfertilized eggs of kelp grouper Epinephelus moara, the ratio of fertilization and hatchability had no significant differences between the cryopreserved sperm and fresh sperm of seven-band grouper. It is suggested that the frozen sperm of seven-band grouper could be applied in artificial reproduction and crossbreeding. © 2013 Elsevier B.V.


Tian Y.,Chinese Academy of Fishery Sciences | Jiang J.,Chinese Academy of Fishery Sciences | Jiang J.,Shanghai Ocean University | Song L.,Chinese Academy of Fishery Sciences | And 6 more authors.
Cryobiology | Year: 2015

The effects of cryopreservation and the vitrification solution on the embryo hatchability of the seven-band grouper Epinephelus septemfasciatus were evaluated in this study. Six small molecule cryoprotectants (PG, MeOH, Gly, DMF, DMSO and EG) and four macromolecular cryoprotectants (glucose, fructose, sucrose and trehalose) were used to determine the embryo toxicity levels. Results showed that the embryo survival rate was higher when the PM (24% PG + 16% MeOH):Gly ratios were 3:1 and 4:1. Further experiments showed that the embryo survival rates in PMG3S (35% PMG3 + 5% sucrose) and PMG3T (35% PMG3 + 5% trehalose) were relatively higher, which are 29.24 ± 10.81% and 27.01 ± 3.39%, respectively. When treated with PMG3S and PMG3T by using 5-step method, embryos at somite stage and tail-bud stage shrank in the first 6 min and gradually recovered in volume to the original. This indicated the successful permeation of the vitrification solutions into cells. Then, embryos at the embryoid body formation stage, the somite stage and the tail-bud stage were cryopreserved with PMG3S and PMG3T. In total, 82 floating embryos were obtained, 14 of which developed further, with 8 embryos at the tail-bud stage developing to the heartbeat stage, 4 embryos at the body formation stage development to the somite stage, and 2 embryos at the somite stage hatched to larval fish. © 2015 Elsevier Inc.


Tian Y.S.,Chinese Academy of Fishery Sciences | Jiang J.,Chinese Academy of Fishery Sciences | Jiang J.,Shanghai Ocean University | Wang N.,Chinese Academy of Fishery Sciences | And 8 more authors.
Journal of Reproduction and Development | Year: 2015

In order to develop excellent germplasm resources for giant grouper (Epinephelus lanceolatus), cryopreservation of giant grouper sperm was examined in the present study. Firstly, 13 kinds of sperm dilution (ELS1-3, EM1-2, TS-2, MPRS, ELRS0-6) were prepared with physiological salt, sucrose, glucose and fetal bovine serum. The physiological parameters of ELRS3 (ratio of fast motion, ratio of slow motion, time of fast motion, time of slow motion, lifespan and motility) and ELS3 (sperm ratio of slow motion, time of slow motion and motility) were significantly higher than those of the other dilutions (P < 0.05). Secondly, after adding 15% DMSO and 10% FBS to ELRS3 and ELS3, most physiological parameters of frozen sperm were also significantly higher than the other gradients (P < 0.05), and sperm motility was as high as 63.68 ± 4.16% to74.75 ± 12.71% (fresh sperm motility, 80.70 ± 1.37% to 80.71 ± 1.49%). Mixed with the above dilutions, a final volume of 105 ml semen was cryopreserved. Finally, the sperm of giant grouper cryopreserved with cryoprotectants (ELRS3 + 15% DMSO + 10% FBS) was used for electron-microscopic observation and crossbreeding with red-spotted groupers (Epinephelus akaara). The electron-microscopic observation revealed that part of the frozen-thawed sperm was cryodamaged, e.g., flagellum fracturing and mitochondria falling out, while the ultrastructure of sperm membrane, mitochondria and flagellum remained intact. Also, the fertilization and hatchability rates of giant grouper frozen sperm and red-spotted grouper eggs were as high as 94.56% and 75.56%, respectively. Thus, a technique for cryopreservation of giant grouper sperm was successfully developed and applied to crossbreeding with red-spotted grouper eggs. © 2015 by the Society for Reproduction and Development.


PubMed | Chinese Academy of Fishery Sciences and Ming Bo Aquatic Co.
Type: Journal Article | Journal: Cryobiology | Year: 2015

The effects of cryopreservation and the vitrification solution on the embryo hatchability of the seven-band grouper Epinephelus septemfasciatus were evaluated in this study. Six small molecule cryoprotectants (PG, MeOH, Gly, DMF, DMSO and EG) and four macromolecular cryoprotectants (glucose, fructose, sucrose and trehalose) were used to determine the embryo toxicity levels. Results showed that the embryo survival rate was higher when the PM (24% PG + 16% MeOH):Gly ratios were 3:1 and 4:1. Further experiments showed that the embryo survival rates in PMG3S (35% PMG3 + 5% sucrose) and PMG3T (35% PMG3 + 5% trehalose) were relatively higher, which are 29.24 10.81% and 27.01 3.39%, respectively. When treated with PMG3S and PMG3T by using 5-step method, embryos at somite stage and tail-bud stage shrank in the first 6 min and gradually recovered in volume to the original. This indicated the successful permeation of the vitrification solutions into cells. Then, embryos at the embryoid body formation stage, the somite stage and the tail-bud stage were cryopreserved with PMG3S and PMG3T. In total, 82 floating embryos were obtained, 14 of which developed further, with 8 embryos at the tail-bud stage developing to the heartbeat stage, 4 embryos at the body formation stage development to the somite stage, and 2 embryos at the somite stage hatched to larval fish.

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