Method for 3D culturing testis cells of bostrychus sinensis to generate sperms and application
A technology of Channa sinensis and testis, applied in 3D culture, biochemical equipment and methods, reproductive cells, etc., can solve problems such as uneven quality of embryos of Channa sinensis, difficult extrusion of semen, and death of precious male parents , to achieve the effect of breaking through the bottleneck of difficult gamete acquisition and breaking through the long maturation cycle
Pending Publication Date: 2020-01-14
SUN YAT SEN UNIV
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Problems solved by technology
However, in the process of artificial reproduction, it was found that the semen of Channa chinensis is difficult to squeeze out, and the testis must be removed by dissecting the male fish, and the semen can be obtained by grinding.
This method has some disadvantages, such as the death of a large number of precious male parents, the difficulty in guaranteeing the quality of sperm, and the uneven quality of the embryos of Channa sinensis
Method used
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Embodiment 1
[0043] 1. Treatment of the testes of Channa sinensis.
[0044] (1) Select a mature male fish of Channa sinensis, dissect out the testis, and place it in sterile PBS.
[0045] (2) Remove blood clots and other impurities on the testis tissue, transfer the testis into new sterile PBS, and wash it twice.
[0046] (3) Transfer the above-mentioned testis into the disinfectant solution, soak and disinfect for 60 seconds.
[0047] (4) Transfer the sterilized testis into sterile PBS, wash it twice for 2 minutes, and remove the residual disinfectant.
[0048] (5) Use ophthalmic scissors to cut the testis into 1mm 3 The size of the testis fragments.
[0049] (6) at 1mm 3 Add 1mL of sterile PBS to testis fragments of different sizes, gently pipette to resuspend, let stand for 2min, wait for the testis tissue to sink to the bottom, and discard the supernatant suspension. Repeat this step 3-5 times until the liquid is clear.
[0050] 2. Preparation of single cell suspension of testis ...
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Abstract
The invention discloses a method for 3D culturing testis cells of bostrychus sinensis to generate sperms. The method for 3D culturing the testis cells of the bostrychus sinensis to generate the spermscomprises the following steps that testes of bostrychus sinensis milters are taken out, cleaned, sterilized and cut into pieces, testis fragments are placed in digestive juice, digestion is carried out on a constant-temperature horizontal shaking table, and a testis cell suspension is prepared; incompletely digestive tissues or large cell clusters are removed through cell mesh screen filtering, filtered cells are subjected to low-speed centrifugation through Percoll gradient, and the testis cells of the bostrychus sinensis after low-speed centrifugal purification are collected; bostrychus sinensis sperm culture mediums and protein additives are added for resuspension, and inoculation is carried out in a 3D cell culture dish for culturing; in the process of 3D culturing, immunofluorescenceis used for detecting germ cells; one half of fresh sperm culture mediums are replaced every 2-5 days, the number of the sperms is counted, and eosin Y dyeing detects the survival rate of the cultured sperms; and the bostrychus sinensis sperms generated by 3D culturing and ripe eggs of the bostrychus sinensis are subjected to in vitro fertilization, and the fertilization rate of an embryo is counted.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method and application for 3D culturing testicular cells of Channa sinensis to produce sperm. Background technique [0002] Germ cells are the only cells in an animal capable of passing on the genetic material it carries to the next generation. Germ cell culture and in vitro induction technology are important scientific propositions in life sciences. In recent years, in mammals, germ cell culture, induction and differentiation models have been successfully established. Through in vitro culture and induction, embryonic stem cells can differentiate into primordial germ cells (PGC). Under certain conditions, PGCs can eventually differentiate into functional haploid gametocytes, namely sperm and eggs. Establishing germ cell in vitro culture induction technology and method is an important way to study germ cell formation, development and differentiation. Moreover, the functional gam...
Claims
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IPC IPC(8): C12N5/076
CPCC12N5/061C12N2500/80C12N2501/115C12N2513/00
Inventor 刘威张洪贾坤同易梅生
Owner SUN YAT SEN UNIV
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