Method for improving the quality of sheep embryos in vitro by using the neuropeptide Y

Abstract

The application discloses an application method of a neuropeptide Y in improving the quality of sheep in-vitro embryos, and the application method comprises the following steps: (1) obtaining ovary of a sheep slaughtered on the same day; (2) respectively aspirating small follicles (1-2 mm) and large follicles (3-5 mm) on the surface of the ovary to culture ovum cells; (3) blowing and stripping the ovum cells to remove cumulus cells after the ovum cells are cultured in-vitro for 24 hours; (4) adding the ovum cells into in-vitro fertilization liquid to continue culturing for 24 hours; (5) counting the cleavage rate of the fertilized ovum, and transferring the fertilized ovum to embryo culture liquid to continue culturing, and counting the blastocyst rate after 144-168 hours. By adding the NPY polypeptide into the in-vitro maturation culture liquid of the sheep small follicle ovum cells, the blastocyst rate of the embryos can be obviously improved.

Description

Technical Field

[0001] This invention belongs to the field of sheep reproductive bioengineering technology, and specifically discloses a method for applying neuropeptide Y (NPY) to improve the quality of sheep in vitro embryos. The main method involves treating sheep oocytes with NPY, thereby demonstrating a new use of NPY in promoting the quality of sheep in vitro embryos. Background Technology

[0002] Neuropeptide Y (NPY) is a C-terminal aminolated neurotransmitter peptide containing 36 amino acids, with a high tyrosine content. NPY is one of the most evolutionarily conserved peptides among species. NPY is generally considered to be the most abundant peptide in the central and peripheral nervous systems. It is known that NPY can regulate immune cell transport, T helper cell differentiation, cytokine secretion, natural killer cell activity, phagocytosis, and reactive oxygen species production. NPY also participates in the regulation of feeding behavior, energy homeostasis, memory retention, and reproductive behavior. NPY is present in the ovary, but it is unclear whether it affects follicle growth and development. However, there are currently no reports of NPY being used in sheep in vitro embryo production. Summary of the Invention

[0003] The purpose of this invention is to improve the quality of in vitro embryos from small sheep follicles by treating them with NPY polypeptide during the in vitro embryo production process, mainly by significantly increasing the blastocyst rate.

[0004] The technical solution for realizing the present invention is as follows: The application method of neuropeptide Y in improving the quality of sheep in vitro embryos includes the following steps: (1) Obtain the ovaries of sheep slaughtered on the same day, place them in physiological saline containing 1000 IU / mL penicillin and 1000 IU / mL streptomycin at a temperature of 30-35℃, bring them back to the laboratory within 3-5 hours, wash them with physiological saline 3-4 times, and set them aside; (2) Aspirate follicular fluid from small and large follicles on the surface of the ovary using a syringe needle pre-absorbed with 2 mL of oocyte aspiration fluid. Under a microscope, collect cumulus-oocyte complexes (COCs) in the follicular fluid. Wash the COCs 3-5 times with in vitro maturation solution and add them to in vitro maturation solution containing NPY. Mature and culture in a CO2 incubator at 5% CO2, 95% air, and 38.6℃ for 22-24 hours. The small follicles are 1-2 mm in size and the large follicles are 3-5 mm in size. (3) Repeatedly blow and aspirate the COCs obtained in step (2) to remove the cumulus cells around the oocytes to form naked eggs. Add 25-30 oocytes to a droplet containing in vitro fertilization fluid for later use. (4) Add the thawed frozen semen to the bottom of a test tube containing in vitro fertilization solution, incubate in a CO2 incubator for 20-25 minutes, collect the upper layer of sperm, centrifuge at 1500 rpm for 4-5 minutes, then discard the supernatant, and use the sperm precipitate at the bottom for density counting, at a ratio of 2-4 × 10⁻⁴. 6 The solution was added to the in vitro fertilization droplets in step (3) at a density of 1 oocyte / mL and incubated with the oocytes. (5) Take out the fertilized eggs 20-22h after fertilization in step (4), wash them 3-4 times in in vitro culture medium (IVC), and culture them in in vitro culture medium for 24-26h. Pick out the oocytes that have not cleaved, count the cleavage rate, and continue to culture for 144-168 hours to count the blastocyst rate.

[0005] Furthermore, the preferred concentration of NPY is 50 nM.

[0006] The NPY polypeptide used is a commercially available synthetic polypeptide protein with the following amino acid sequence: YPSKPDNPGDDAPAEDLARYYSALRHYINLITRQRY.

[0007] Beneficial effects: By adding NPY polypeptide to sheep in vitro maturation culture medium, this invention can significantly improve the blastocyst rate of sheep small follicle in vitro embryos, exploring a new approach for embryo production. Detailed Implementation

[0008] Example 1: Application method of using NPY to improve the quality of sheep in vitro embryos. The amino acid sequence of NPY polypeptide is: YPSKPDNPGDDAPAEDLARYYSALRHYINLITRQRY. The steps of the application method are as follows: (1) Obtain the ovaries of sheep slaughtered on the same day, place them in physiological saline containing 1000 IU / mL penicillin and 1000 IU / mL streptomycin at a temperature of 30-35℃, bring them back to the laboratory within 3-5 hours, and wash them with physiological saline 3-4 times; (2) Use a syringe needle pre-absorbed with 2 mL of oocyte extraction fluid to aspirate follicular fluid from small follicles (1-2 mm) and large follicles (3-5 mm) on the surface of the ovary. Under a microscope, collect the cumulus-oocyte complex (COCs) from the collected follicular fluid. Wash the COCs 3-5 times with in vitro maturation solution and add them to in vitro maturation solution with added NPY. Mature and culture in a CO2 incubator at 5% CO2, 95% air, and 38.6℃ for 22-24 h. (3) The COCs obtained in step (2) are repeatedly blown and sucked to remove the cumulus cells around the oocytes to form naked eggs. 25-30 oocytes are added to a droplet containing in vitro fertilization fluid for later use. (4) Add the thawed frozen semen to the bottom of a test tube containing in vitro fertilization solution, incubate in a CO2 incubator for 20-25 minutes, collect the upper layer of sperm, centrifuge at 1500 rpm for 4-5 minutes, then discard the supernatant, and use the sperm precipitate at the bottom for density counting, at a ratio of 2-4 × 10⁻⁴. 6 The solution was added to the in vitro fertilization droplets in step (3) at a density of 1 oocyte / mL and incubated with the oocytes. (5) Take out the fertilized eggs 20-22h after fertilization in step (4), wash them 3-4 times in in vitro culture medium (IVC), and culture them in in vitro culture medium for 24-26h. Pick out the oocytes that have not cleaved, count the cleavage rate, and continue to culture for 144-168 hours to count the blastocyst rate.

[0009] The solutions involved are as follows: Ovulation retrieval fluid: TCM199-hepes + 1 mg / mL PVA + 0.7 mg / mL heparin sodium; In vitro maturation fluid (IVM): TCM199-HCO3 + 10% FBS + 0.05 IU / mL FSH + 0.05 IU / mL LH + 1 μg / mL estradiol + 24.2 μg / mL sodium pyruvate + 100 IU / mL penicillin + 100 IU / mL streptomycin; In vitro fertilization fluid (IVF): SOF + 20% FBS + 6 IU / mL heparin sodium + 100 IU / mL gentamicin; In vitro culture medium (IVC): SOF + 3 mg / mL BSA; Synthetic fallopian tube fluid (SOF): 6.29 mg / mL NaCl + 0.534 mg / mL KCl + 0.162 mg / mL KH2PO4 + 0.6 μL / mL sodium lactate + 0.089 mg / mL MgSO4 + 2.1 mg / mL NaHCO3 + 0.0357 mg / mL sodium pyruvate + 0.299 mg / mL CaCl2·2H2O.

[0010] Experimental results and data validation: A set of data was used to validate the effectiveness of adding NPY.

[0011]

[0012] Note: Different lowercase letters in the same row indicate significant differences (P < 0.05).

[0013] Conclusion: The results in the table above indicate that adding 50 nM NPY peptide to the in vitro maturation medium of sheep small follicle oocytes can significantly improve the quality of sheep small follicle oocytes, as evidenced by a significant increase in the cleavage rate and blastocyst rate of in vitro embryos.

Claims

1. A method for improving the quality of sheep in vitro embryos using neuropeptide Y, characterized in that: The method includes the following steps: (1) Obtain the ovaries of sheep slaughtered on the same day, place them in physiological saline containing 1000 IU / mL penicillin and 1000 IU / mL streptomycin at a temperature of 30-35℃, bring them back to the laboratory within 3-5 hours, wash them with physiological saline 3-4 times, and set them aside for use. (2) Aspirate follicular fluid from small and large follicles on the surface of the ovary using a syringe needle pre-absorbed with 2 mL of oocyte aspiration fluid. Examine the cumulus-oocyte complex (COCs) in the follicular fluid under a microscope. Wash the COCs 3-5 times with in vitro maturation solution and add them to in vitro maturation solution containing NPY (neuropolypeptide Y). Mature and culture in a CO2 incubator at 5% CO2, 95% air, and 38.6℃ for 22-24 hours. The small follicles are 1-2 mm in size and the large follicles are 3-5 mm in size. (3) Repeatedly blow and aspirate the COCs obtained in step (2) to remove the cumulus cells around the oocytes to form naked eggs. Add 25-30 oocytes to a droplet containing in vitro fertilization fluid for later use. (4) Add the thawed frozen semen to the bottom of a test tube containing in vitro fertilization solution, incubate in a CO2 incubator for 20-25 minutes, collect the upper layer of sperm, centrifuge at 1500 rpm for 4-5 minutes, then discard the supernatant, and use the sperm precipitate at the bottom for density counting, at a ratio of 2-4 × 10⁻⁴. 6 The solution was added to the in vitro fertilization droplets in step (3) at a density of 1 oocyte / mL and incubated with the oocytes. (5) Take out the fertilized eggs 20-22h after fertilization in step (4), wash them 3-4 times in in vitro culture medium (IVC), and culture them in in vitro culture medium for 24-26h. Pick out the oocytes that have not cleaved, count the cleavage rate, and continue to culture for 144-168 hours to count the blastocyst rate.

2. The method for improving the quality of sheep in vitro embryos using neuropeptide Y according to claim 1, characterized in that: The NPY concentration was 50 nM.

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