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CN-121975725-A - Method for improving IVF embryo transfer development capacity of frozen cattle

CN121975725ACN 121975725 ACN121975725 ACN 121975725ACN-121975725-A

Abstract

The invention provides a method for improving the transfer development capability of frozen bovine IVF embryos, which is characterized in that in the processes of freezing, thawing, late embryo culture and the like of the bovine IVF embryos, high-concentration sodium pyruvate, insulin, TLR2 and Nur77 agonists (Cytosporone B) are singly or jointly added, so that the survival rate and the development capability of the frozen embryos, the pregnancy rate after transfer and the like are further improved. Experiments show that the embryo survival rate and the hatching rate of the combined addition of sodium pyruvate, insulin, TLR2 and Nur77 agonist (Cytosporone B) are obviously higher than those of a double-substance addition group, a single-substance addition group and a control group. The invention lays a foundation for the production of in vitro fertilized embryos and related scientific research in the future, provides an effective, safe and feasible strategy for improving the production efficiency and the embryo quality of in vitro embryos, and has great popularization value.

Inventors

  • ZHAO XUEMING
  • WAN PENGCHENG

Assignees

  • 中国农业科学院北京畜牧兽医研究所

Dates

Publication Date
20260505
Application Date
20251217

Claims (8)

  1. 1. A method for improving the IVF embryo transfer development capability of frozen cattle is characterized in that sodium pyruvate, insulin, TLR2 and Nur77 agonist (Cytosporone B) are added into a front embryo culture solution, a rear embryo culture solution, an embryo pretreatment solution, a vitrification freezing solution and a thawing solution in the production of the in vitro embryo of the cattle, so that the survival rate and the transfer development capability of the IVF embryo of the frozen cattle and the pregnancy rate after transfer are improved; Wherein the insulin is bovine-derived insulin and the TLR2 is bovine-derived Toll-like receptor family member TLR2.
  2. 2. The method of claim 1, wherein the step of determining the position of the substrate comprises, The formulation of the pre-embryo culture solution comprises MCR solution, BSA, essential amino acid (50×) +10 μl/mL and non-essential amino acid (100×) +0.15 mg/mL; The formulation of the later embryo culture solution is that the earlier embryo culture solution is added with 10% FBS; the formulation of the MCR liquid is ：44 μg/mL C 3 H 3 NaNO 3 + 0.33 mg/mL CaCl 2 ·2H 2 O + 0.23 mg/mL KCl + 0.1619 mg/mL KH 2 PO 4 + 0.1626 mg/mL MgCl 2 ·6H 2 O + 6.55 mg/mL NaCl + 2.2 mg/mL NaHCO 3 +0.55 mg/mL calcium galactoate+2 mu L/mL phenol red+0.27 mg/mL glucose; Fertilized eggs from bovine oocytes were fertilized in vitro for 16-18h, first cultured for 48h in a pre-embryo culture broth supplemented with 2-6mM sodium pyruvate, 5-15. Mu.g/mL insulin, 100-400. Mu.g/mL TLR2 and 5-20. Mu.M Nur77 agonist (Cytosporone B), then transferred to a post-embryo culture broth supplemented with 2-6mM sodium pyruvate, 5-15. Mu.g/mL insulin, 100-400. Mu.g/mL TLR2 and 5-20. Mu.M Nur77 agonist (Cytosporone B) for 5d, half-changing every 48, h until 7 th d after fertilization, and then subjected to vitrification freezing and thawing.
  3. 3. The method of claim 2, wherein performing the vitrification freezing process comprises first placing the embryo into an embryo pretreatment liquid with 2-6mM sodium pyruvate, 5-15 μg/mL insulin, 100-400 μg/mL TLR2, and 5-20 μM Nur77 agonist (Cytosporone B) for 30 s, then transferring the embryo into EDFSF liquid for 25 s, and finally sucking the embryo into an OPS tube and then into liquid nitrogen; Wherein, the formula of the embryo pretreatment liquid is 10% (v/v) EG and 10% (v/v) DMSO; The vitrification frozen solution is EDFSF liquid, the formula of the vitrification frozen solution is FSF solution containing 20% (v/v) EG and 20% (v/v) DMSO, and the FSF solution is DPBS solution containing 300 g/L Ficoll, 0.5M sucrose and 20% (v/v) FBS.
  4. 4. A method according to claim 3, wherein the thawing solution consists of thawing solution I, thawing solution II and thawing solution III, 0.5M sucrose solution, 0.25M sucrose solution and 0.125M sucrose solution, respectively, with the addition of 2-6mM sodium pyruvate, 5-15 μg/mL insulin, 100-400 μg/mL TLR2 and 5-20 μΜ Nur77 agonist (Cytosporone B); The thawing method comprises taking OPS tube out of liquid nitrogen, blowing embryo into thawing solution I, incubating for 5 min, transferring to thawing solution II, incubating for 5 min, transferring to thawing solution III, incubating for 5 min, and culturing embryo in late embryo culture solution.
  5. 5. The method of claim 1, wherein the bovine embryo is cultured in vitro at 38.5 ℃ with 5% co 2 .
  6. 6. The method according to any one of claims 1 to 5, wherein sodium pyruvate is added to the pre-embryo culture solution, the post-embryo culture solution, the embryo pretreatment solution, the vitrification frozen solution and the thawing solution at a concentration of 2, 4, 6 mM, and/or, Insulin is added to the pre-embryo culture solution, the post-embryo culture solution, the embryo pretreatment solution, the vitrification freezing solution and the thawing solution at the concentration of 5, 10 and 15 mug/mL, and/or, TLR2 is added into the pre-embryo culture solution, the post-embryo culture solution, the embryo pretreatment solution, the vitrification freezing solution and the thawing solution at the concentration of 100, 200 and 400 mug/mL, and/or, Nur77 agonist (Cytosporone B) was added to the pre-embryo culture solution, the post-embryo culture solution, the embryo pretreatment solution, the vitrification frozen solution and the thawing solution at 5, 10 and 20. Mu.M.
  7. 7. The method of claim 6, wherein sodium pyruvate, insulin, TLR2, and Nur77 agonist (Cytosporone B) are added to the pre-embryo culture broth, the post-embryo culture broth, the embryo pretreatment broth, the vitrification frozen solution, and the thawing solution at concentrations of 4 mM, 10 μg/mL, 200 μg/mL, and 10 μΜ, respectively.
  8. 8. Use of a combination of sodium pyruvate, insulin, TLR2 and Nur77 agonist (Cytosporone B) in the production of bovine in vitro embryos; the application aims at improving the survival rate and the transplanting development capacity of frozen bovine IVF embryos and the pregnancy rate after transplanting.

Description

Method for improving IVF embryo transfer development capacity of frozen cattle Technical Field The invention relates to the technical field of animal embryo engineering, in particular to a method for improving IVF embryo transfer development capability of frozen cattle. Background The in vitro embryo production technology is an important tool for improving the genetic value and reproductive capacity of cattle, and is also an indispensable component of somatic cell cloning, transgenic and other technologies. Niu Tiwai embryo cryopreservation is an important part of the in vitro embryo production process, is a key step for widely breeding and preserving valuable and high-quality animals, is an important means for protecting endangered species or varieties, and is one of strategic ways for preserving and improving the genetic potential of varieties. The current annual proportion of freeze-thaw bovine embryo transfer in other countries is about 60%, which indicates that embryo freezing technology is essential in bovine embryo transfer. And cryopreservation allows embryos to be stored for a long period of time, successfully undergoing commercial exchanges worldwide. In vitro culture (IVP), embryo cryopreservation and embryo transfer have become routine procedures in animal husbandry today. Although in vitro embryo freezing has been successfully applied to in vitro production, pregnancy rates after implantation have been consistently lower than in vivo embryos (Hayashi E, et al 2022). The apoptosis level of the embryo rises linearly after freezing compared with fresh embryos, and freezing triggers the apoptosis pathway of embryo death such that gene expression of apoptosis factors such as Bcl-2 and Bax is changed due to cryopreservation (Vining LM, et al 2021). Vitrification affects mitochondrial function and increases ROS levels, resulting in subsequent loss of embryo developmental capacity (Kandil OM, et al, 2021). Mitochondrial metabolism is closely related to the production and elimination of Reactive Oxygen Species (ROS), which are naturally produced as byproducts by mitochondria during the electron transfer chain of ATP synthesis. However, the endogenous antioxidant system itself is not sufficient to counteract the oxidative imbalance that occurs during in vitro culture and cryopreservation of bovine embryos. These differences lead to sustained elevated ROS levels and mitochondrial dysfunction in IVP-derived bovine embryos (Schreiber Met al, 2025), which are known to promote apoptosis and endoplasmic reticulum stress, thereby negatively affecting embryo quality. Disclosure of Invention The invention aims to provide a method for improving the IVF embryo transfer development capability of frozen cattle. In order to achieve the object of the invention, in a first aspect, the invention provides a method for improving the IVF embryo transfer development capability of frozen cattle, in the production of the in vitro embryo of cattle, sodium pyruvate, insulin, TLR2 and Nur77 agonist (Cytosporone B) are added into a forward embryo culture solution, a later embryo culture solution, an embryo pretreatment solution, a vitrification freezing solution and a thawing solution, so that the survival rate and the transfer development capability of the IVF embryo of the frozen cattle and the pregnancy rate after transfer are improved. Wherein the insulin is bovine-derived insulin and the TLR2 is bovine-derived Toll-like receptor family member TLR2. Bovine insulin (Bos taurus Insulin) is numbered 280829 in NCBI. Bovine TLR2 (Bos taurus Toll-like receptor 2) was numbered 281534 in NCBI and murine Cytosporone B (Nur 77 agonist) was numbered 15370 in NCBI. Further, the formulation of the pre-embryo culture solution is MCR solution +6mg/mL BSA (bovine serum albumin) +20mu.L/mL essential amino acid (50×) +10mu.L/mL non-essential amino acid (100×) +0.15mg/mL glutamine; The formulation of the later embryo culture solution is that the earlier embryo culture solution is added with 10% FBS; the formulation of the MCR liquid is ：44 μg/mL C3H3NaNO3+ 0.33 mg/mL CaCl2·2H2O + 0.23 mg/mL KCl + 0.1619 mg/mL KH2PO4+ 0.1626 mg/mL MgCl2·6H2O + 6.55 mg/mL NaCl + 2.2 mg/mL NaHCO3+0.55 mg/mL calcium galactoate+2 mu L/mL phenol red+0.27 mg/mL glucose; Fertilized eggs from bovine oocytes were fertilized in vitro for 16-18h, first cultured for 48h in a pre-embryo culture broth supplemented with 2-6mM sodium pyruvate, 5-15. Mu.g/mL insulin, 100-400. Mu.g/mL TLR2 and 5-20. Mu.M Nur77 agonist (Cytosporone B), then transferred to a post-embryo culture broth supplemented with 2-6mM sodium pyruvate, 5-15. Mu.g/mL insulin, 100-400. Mu.g/mL TLR2 and 5-20. Mu.M Nur77 agonist (Cytosporone B) for 5d, half-changing every 48, h until 7 th d after fertilization, and then subjected to vitrification freezing and thawing. Further, the vitrification freezing treatment comprises placing embryo into embryo pretreatment solution containing 2-6mM sodium pyruvate, 5-15 μg/mL