Bovine sperm treatment method and bovine intracytoplasmic sperm injection method

A treatment method and sperm technology, applied in the field of single sperm oocytoplasmic microinjection, can solve the problems of destroying the formation of the spindle body, difficulty in injection, and damage to the cytoskeleton

Pending Publication Date: 2021-09-10
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing sperm treatment methods mainly focus on removing the sperm acrosome and sperm plasma membrane, but the sperm tail is long, making injection difficult, causing cytoskeleton damage and disrupting spindle formation
Later, some researchers injected only the sperm head to reduce the difficulty of micromanipulation, but many researchers questioned this, suggesting that the absence of centrosomes in sperm head embryos is similar to that of parthenogenetic embryos, which may lead to unknown growth and development defects

Method used

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  • Bovine sperm treatment method and bovine intracytoplasmic sperm injection method
  • Bovine sperm treatment method and bovine intracytoplasmic sperm injection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The processing method of the bovine sperm in the present embodiment, comprises the steps:

[0025] 1) Prepare standard 10mmol / L NaOH solution and 10mmol / L hydrochloric acid solution;

[0026] 2) Take one tube of frozen bovine spermatozoa, which contains about 100 μL bovine sperm stock solution, put it in warm water at 38°C, and thaw it for 15-20 seconds;

[0027] 3) Release the thawed thin tube frozen semen into a 1.5mL PE tube containing 1mL PBS at 37°C for washing, and centrifuge for 5min after washing at 1500rpm;

[0028] 4) Remove the supernatant, add 1 mL of new PBS solution, and repeat step 3);

[0029] 5) Remove the supernatant, add 100 μL of PBS solution and mix evenly to obtain bovine sperm treatment solution;

[0030] 6) Take 10 μL bovine sperm treatment solution and 100 μL NaOH solution, mix evenly, and incubate at room temperature for 1 hour;

[0031] 7) After incubation, add hydrochloric acid solution to adjust the pH value of the mixture to 7.2.

[003...

Embodiment 2

[0034] The processing method of the bovine sperm in the present embodiment, comprises the steps:

[0035] 1) Prepare standard 9mmol / L NaOH solution and 9mmol / L hydrochloric acid solution;

[0036] 2) Take a tube of frozen bovine semen (about 100 μL bovine sperm stock solution), put it in warm water at 38°C, and thaw it for 15-20 seconds;

[0037] 3) Release the thawed thin tube frozen semen into a 1.5mL PE tube containing 1mL PBS at 37°C for washing, and centrifuge for 5min after washing at 1500rpm;

[0038] 4) Remove the supernatant, add 1 mL of new PBS solution, and repeat step 3);

[0039] 5) Remove the supernatant, add 100 μL of PBS solution and mix evenly to obtain bovine sperm treatment solution;

[0040] 6) Take 10 μL bovine sperm treatment solution and 100 μL NaOH solution, mix evenly, and incubate at room temperature for 1 hour;

[0041] 7) After incubation, add hydrochloric acid solution to adjust the pH value of the mixture to 7.2.

Embodiment 3

[0043] The processing method of the bovine sperm in the present embodiment, comprises the steps:

[0044] 1) prepare standard 11mmol / L NaOH solution and 11mmol / L hydrochloric acid solution;

[0045] 2) Take one tube of frozen beef thin tube semen, put it in warm water at 38°C, and thaw it for 15-20 seconds;

[0046] 3) Release the thawed thin tube frozen semen into a 1.5mL PE tube containing 1mL PBS at 37°C for washing, and centrifuge for 5min after washing at 1500rpm;

[0047] 4) Remove the supernatant, add 1 mL of new PBS solution, and repeat step 3);

[0048] 5) Remove the supernatant, add 100 μL of PBS solution and mix evenly to obtain bovine sperm treatment solution;

[0049] 6) Take 10 μL bovine sperm treatment solution and 100 μL NaOH solution, mix evenly, and incubate at room temperature for 1 hour;

[0050] 7) After incubation, add hydrochloric acid solution to adjust the pH value of the mixture to 7.2.

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Abstract

The invention relates to a bovine sperm treatment method and a bovine intracytoplasmic sperm injection method, and belongs to the technical field of intracytoplasmic sperm injection. According to the bovine sperm treatment method, firstly, the sperm is treated by using a weakly alkaline solution, and therefore, the tail part of the sperm is shortened by 1 / 3-1 / 2; and then, the sperm of which the tail part is shortened by 1 / 3-1 / 2 is selected and is injected into the oocytes. After the bovine sperm treatment method disclosed by the invention is adopted for carrying out the treatment, only the tail part of the sperm is shortened by 1 / 3-1 / 2, the sperm still retains the centrosome, the integrity of the sperm is structurally retained, the method is more similar to the change of sperm capacitation in a natural fertilization process, the embryonic development potential is improved, and the development rate of microinjection embryos is improved. In addition, the tail part of the sperm treated by the bovine sperm treatment method is shortened by 1 / 3-1 / 2, and shortening of the tail part can lower the difficulty of microinjection, improve the operation speed of injection and improve the efficiency of intracytoplasmic sperm injection.

Description

technical field [0001] The invention relates to a method for processing bovine sperm and a bovine single sperm ovum cytoplasmic microinjection method, belonging to the technical field of single sperm oocytoplasmic microinjection. Background technique [0002] Micro-fertilization was developed in the late 1980s and has become a relatively cutting-edge artificial assisted reproductive technology. It bypasses the physiological process of normal fertilization and directly injects a single processed sperm into the egg with the help of a micro-manipulation instrument. In the cytoplasm of the mother cell (MII), the sperm and egg are combined and the fertilization process is completed. As an important technology of embryo engineering, microfertilization is used for the utilization of excellent male animal sperm, factory production of improved embryos, sperm-mediated breeding of transgenic organisms, research on fertilization mechanism, protection of wild animals and endangered anima...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076C12N15/89
CPCC12N5/061C12N15/89
Inventor 朱肖亭滑留帅施巧婷金磊张子敬赵彩艳吕世杰王二耀辛晓玲冯亚杰
Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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