CN110305836B - Fertilization liquid for cattle in vitro fertilization and use method thereof - Google Patents

Fertilization liquid for cattle in vitro fertilization and use method thereof Download PDF

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CN110305836B
CN110305836B CN201910551341.0A CN201910551341A CN110305836B CN 110305836 B CN110305836 B CN 110305836B CN 201910551341 A CN201910551341 A CN 201910551341A CN 110305836 B CN110305836 B CN 110305836B
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张涌
张景程
王勇胜
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Abstract

The invention discloses a fertilization liquid for cattle in vitro fertilization and a use method thereof, wherein the fertilization liquid comprises an SOF (soy fluoride) basal culture medium without inositol and also comprises the following additives: l-arginine, L-aspartic acid, L-carnitine, epinephrine, penicillamine, hypotaurine, and heparin. The fertilization liquid can effectively ensure the in-vitro fertilization rate of the bovine embryo and the development quality of the fertilized embryo.

Description

Fertilization liquid for cattle in vitro fertilization and use method thereof
Technical Field
The invention relates to the technical field of livestock embryo engineering, in particular to a fertilization liquid for cattle in vitro fertilization and a using method thereof.
Background
The in vitro culture of livestock embryo is a technology with potential research value and commercial value, and can be widely applied to the aspects of embryo in vitro transplantation, animal gene editing and breeding, excellent livestock variety amplification and the like. However, the quality and quantity of the embryos obtained by in vitro culture are often inferior to the embryos which normally develop in vivo, and in the in vitro fertilization of cattle, semen which is preserved by freezing is often used, so that the quality of the sperms during in vitro fertilization is unstable, the concentration is low, the fertilization rate of the embryos is unstable, few fertilized eggs forming pronucleus are formed, the pronucleus fusion and cleavage are further influenced, the in vitro development of early embryos is influenced, and the wide application of the technology is obviously restricted.
Therefore, how to improve the fertilization semen of cattle in vitro and establish a set of stable using method is a problem which needs to be solved urgently by the technical personnel in the field.
Disclosure of Invention
In view of the above, the invention provides a fertilization fluid for cattle in vitro fertilization and a using method thereof, which promote early embryo in vitro development.
In order to achieve the purpose, the invention adopts the following technical scheme:
the fertilization liquid for the in vitro fertilization of the cattle comprises an SOF basic culture medium without inositol and also comprises the following additives: l-arginine, L-aspartic acid, L-carnitine, epinephrine, penicillamine, hypotaurine, and heparin.
The heparin is a chemical active substance of the spermatids, and the L-arginine, the L-aspartic acid, the epinephrine, the hypotaurine and the L-carnitine can better stimulate the in vitro activity of the spermatids; penicillamine can promote sperm to enter eggs, and hypotaurine and L-carnitine can slow down final substances of a cytotoxic acetaldehyde-peroxidation cascade reaction, so that the harmful effect of ROS is limited; the L-arginine also has the protective effects of improving the cell activity and improving the cell morphology.
The seven additives are added into the sperm receiving liquid, so that the sperm motility of the cattle can be effectively improved, and the prokaryotic formation rate of the fertilized embryo and the development quality of the sperm receiving embryo during the in vitro fertilization of the cattle can be improved.
Furthermore, the concentration of the L-arginine is 10-100mg/L, the concentration of the L-aspartic acid is 1-10mg/L, the concentration of the L-carnitine is 10-100 MuM, the concentration of the epinephrine is 0.2-20 MuM, the concentration of the penicillamine is 2-200 MuM, the concentration of the hypotaurine is 1-100 MuM, and the concentration of the heparin is 5-20 mg/L.
Further, the semen also comprises essential amino acids, non-essential amino acids and fatty acid-free bovine serum albumin.
Furthermore, the volume fraction of the essential amino acid is 0.1-2%, the volume fraction of the non-essential amino acid is 0.1-2%, and the concentration of the fatty acid-free bovine serum albumin is 1-100 mg/mL.
The in vitro development quality of the bovine embryo can be further improved by controlling essential amino acids, non-essential amino acids and fatty acid-free bovine serum albumin.
Further, every 100mL of the SOF basal medium comprises 26.168mg of CaCl2·2H2O、9.999mg C6H5Na3O7·2H2O, 2.923mg glutamine, 37.218mg MgSO4·7H2O, 1.000mg phenol red, 53.378mg KCl, 16.195mg KH2PO44.402mg of sodium pyruvate, 210.025mg of NaHCO3629.399mg NaCl and 0.0757mg sodium lactate.
Further, the semen receiving solution also comprises 12 mu g/mL gentamicin and 12 mu g/mL cefuromycin.
Further, a method for using the fertilization fluid for cattle in vitro fertilization, the mature bovine oocyte is placed in the fertilization fluid of any one of claims 1 to 6 to be co-cultured with sperm.
Further, a method for using the receiving liquid for cattle in vitro fertilization, preheating the fertilization liquid for at least 2h, then placing the cattle sperm and the cattle ovum in the receiving liquid, and adding CO2Combining the culture boxes for 8-10 h; then adding the fertilized ovum of cattle into the embryo culture solution, and adding CO2N2Culturing in an incubator, selecting cracked embryo on day 3 after culturing in embryo culture solution, culturing continuously until day 7-8, and discarding un-cracked embryo.
Further, the L-arginine, L-aspartic acid, L-carnitine, epinephrine, penicillamine, and hypotaurine were added to the SOF basal medium without inositol before preheating, and the receiving solution was not covered with paraffin oil.
The L-arginine, the L-aspartic acid, the L-carnitine, the epinephrine, the penicillamine, the hypotaurine and the heparin are added into the receptor fluid, so that the positive effects of the L-arginine, the L-aspartic acid, the L-carnitine, the epinephrine, the penicillamine, the hypotaurine and the heparin serving as sperm motility and environmental culture action factors can be fully exerted, the bovine in-vitro fertilized eggs are efficiently obtained, the bovine in-vitro fertilized embryo development rate, the blastocyst number and the blastocyst cell number are remarkably improved, and the production cost for preparing the embryos.
Further, the bovine in vitro sperm embryo was cultured at 38.5 ℃ with 5% CO2Culturing under saturated humidity.
According to the technical scheme, compared with the prior art, the fertilization liquid for bovine in vitro fertilization and the use method thereof are disclosed and provided, and the fertilization liquid for bovine in vitro fertilization is added with L-arginine, L-aspartic acid, L-carnitine, epinephrine, penicillamine, hypotaurine and heparin, so that the bovine embryo in vitro fertilization rate and the developmental quality of fertilized embryos can be effectively guaranteed.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Sources and preparation of reagents
Gentamicin, cefamycin, inorganic salt, L-arginine, L-aspartic acid, L-carnitine, epinephrine, penicillamine, hypotaurine, heparin and paraffin oil (M8410) are products of sigma company;
the Hepes-free M199 basic culture solution is a product of Thermo company;
special grade Fetal Bovine Serum (FBS) is a product of Gibco corporation;
essential amino acids are products of Thermo Fisher corporation (cat # 1831512);
non-essential amino acids are products of Thermo Fisher corporation (cat # 1958909);
fatty acid-free bovine serum albumin EFAF-BSA is a product of sigma company (Cat. No. A6003-5G);
the insulin-transferrin-selenium additive ITS-G is a product of Thermo Fisher company (the product number is 1865342, the insulin is 1G/L, the transferrin is 0.55G/L, the selenium is 0.00067G/L);
beta-mercaptoethanol is a product of Thermo Fisher corporation (cat # 1922445);
hypotaurine is a product of sigma corporation (cat number H1384);
sodium 1-oleoyl-sn-glycero-3-phosphate is a product of Enzo Life Science, Inc. (Cat. LP 100-0025);
the salidroside is product of Sigma company (product number SMB00072-1 MG);
the SOF basal medium is self-prepared;
others not identified are sigma company products.
a. Oocyte in vitro maturation culture solution
To Hepes-free M199 basal medium was added 10% (v/v) fetal bovine serum, 1% (v/v) ITS-G, 0.075IU/mL HMG, 1. mu.g/mL 17. beta. -estradiol, 10ng/mL EGF, 12ng/mL bFGF and 50ng/mL CXCL 12.
SOF solution, SOFaa solution, mSOFaa solution
SOF solution: 26.168mg CaCl2·2H2O、9.999mg C6H5Na3O7·2H2O, 2.923mg glutamine, 37.218mg MgSO4·7H2O, 49.904mg inositol, 1.000mg phenol red, 53.378mg KCl, 16.195mg KH2PO44.402mg of sodium pyruvate, 210.025mg of NaHCO3629.399mg NaCl, 0.0757mg sodium lactate and deionized water to 100mL, and adjusting the pH to 7.2-7.4 with 1mM NaOH.
SOFaa solution: essential amino acid with volume fraction of 1%, non-essential amino acid with volume fraction of 1%, 6mg/mL fatty acid-free bovine serum albumin, 12g/mL gentamicin and 12 μ g/mL cefuroxime were added with SOF solution as a basic culture solution.
mSOFaa solution (embryo culture): with the SOFaa solution as a basic culture solution, ITS-G with the volume fraction of 1%, 55 mu M beta-mercaptoethanol, 2.5mM hypotaurine, 1-oleoyl-SN-glycerol-3-sodium phosphate salt and 0.1mM salidroside, hyaluronic acid with the volume fraction of 0.5mg/100mL and L-citrulline with the volume fraction of 0.02mg/mL are added during preheating.
c. Liquid receiver (BO liquid)
The BO solution is a solution of SOFaa without inositol as a basic culture solution, and further comprises 63. mu.g/mL L-arginine, 6. mu.g/mL L-aspartic acid, 50. mu. M L-carnitine, 2mM epinephrine, 20mM penicillamine, 10mM hypotaurine, and 10. mu.g/mL heparin.
Method for preparing cattle in-vitro fertilization embryo
a. Maturation of oocytes
The cow ovary is collected from a three-bridge fixed-point slaughter house (collection time is 2015 for 1 month to 2017 for 12 months) in Xian city of Shaanxi province, the ovary is placed in 20-25 ℃ physiological saline containing 200U/ml penicillin and 0.2mg/ml streptomycin in a vacuum flask, and the cow ovary is transported back to a laboratory within 5 hours. After the ovary is returned, cutting off connective tissues, fat and attached oviduct on the surface of the ovary by using a pair of sterilizing scissors, cleaning the ovary for three times in sterile physiological saline, using a 10mL syringe provided with a 12G needle head to draw out oocytes in follicles of 2-8mm on the surface of the ovary, putting the oocytes into a 6cm glass dish, and collecting cumulus-oocyte complexes (COCs) under a solid microscope; after collection, the cells were washed three times in PBS containing 10% fetal bovine serum. CoCs with morphologically normal oocytes were selected for maturation in vitro (as total oocytes).
Selected COCs were washed twice in oocyte in vitro maturation medium, then transferred to a 3cm dish containing 3mL of oocyte in vitro maturation medium (equilibrated one hour in an incubator at 38.5 ℃ C. in advance), and incubated at 38.5 ℃ C. with 5% CO2Culturing for 20-22h under the condition of saturated humidity. The mature COCs were blown repeatedly with a 1000mL pipette to remove cumulus cells that spread out of the oocytes. Blowing and beating to leave only four to five layers of tightly wrapped cumulus cells around COCs, washing in fertilization liquid (BO liquid) for 2 times, washing the blown free cumulus cells, and transferring the oocytes tightly wrapped with the four to five layers of cumulus cells to fertilization liquid drops (placing in an incubator at 38.5 ℃ in advance for balancing for more than 2 hours)) In (1).
b. Thawing, purifying and fertilizing sperm
Taking out frozen semen (collected in cow center of Xian city in 2015 and 9 months) from a liquid nitrogen tank, thawing in 37 deg.C water bath, and making into Percoll layered liquid in a centrifuge tube. Placing 2mL of 90% Percoll liquid at the bottom of a centrifuge tube, carefully adding 2mL of 45% Percoll liquid on the 90% Percoll liquid, placing the thawed sperms on the 45% Percoll liquid, centrifuging at 1500rpm for 20min, carefully sucking about 100 mu L of sperms at the bottom, adding the sperms into the semen acceptor containing the oocytes, and placing the sperms and the eggs in an incubator to combine the sperms and the eggs for 10 h.
c. Culture of fertilized embryos
Treatment group: the mSOFaa solution was added to a four well plate at 500. mu.L per well, covered with 500. mu.L of paraffin oil on the surface, previously at 38.5 ℃ with 5% CO2And balancing in a saturated humidity incubator for at least 2 hours.
Blowing residual cumulus cells and sperms around the ova by using a mouth suction tube or a gun head after fertilization to obtain cattle in-vitro fertilized embryos which are supposed to be fertilized, placing the cattle in mSOFaa solution of a four-hole plate, wherein each hole contains about 40 cells, the temperature is 38.5 ℃, and the CO content is 5 percent2、7％N2And culturing for 72h in a saturated humidity incubator, picking out the fertilized embryos which are not cracked, and returning the fertilized embryos to the incubator to continue culturing for the eighth day (192 h).
Control 1 was fertilized with SOF solution containing 10ug/ml heparin.
The plate expulsion and pronucleus formation of each group were observed and recorded, and the results are shown in table 1.
TABLE 1
Figure BDA0002105557350000061
The results in table 1 show that in vitro fertilization of cattle using the fertilization fluid of the present invention yielded significantly more embryos in the normal prokaryotic state than universal BO fertilization fluid.
Development of each group was observed and recorded, and the test results are shown in table 2.
TABLE 2
Figure BDA0002105557350000071
a. b, the superscripts are different and represent significance, and the significance is detected by chi-square test; the grade AB blastula is a transplantable blastula, the grade C blastula is a non-recommended transplantation blastula, and the blastula ABC classification is evaluated according to the blastula quality scoring standard of the International embryo transfer Association.
The results in table 2 show that the number of transplantable embryos obtained using the fertilization fluid of the invention for cattle in vitro fertilization is significantly greater than that of the universal BO fertilization fluid.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (5)

1. A fertilization fluid for in vitro fertilization of cattle comprising a SOF basal medium free of inositol, wherein the fertilization fluid further comprises the following additives: l-arginine, L-aspartic acid, L-carnitine, epinephrine, penicillamine, hypotaurine, heparin; the concentration of the L-arginine is 63mg/L, the concentration of the L-aspartic acid is 6mg/L, the concentration of the L-carnitine is 50 mu M, the concentration of the adrenaline is 2 mu M, the concentration of the penicillamine is 20 mu M, the concentration of the sub-taurine is 10 mu M, and the concentration of the heparin is 10 mg/L.
2. The sperm-receiving fluid for bovine in vitro fertilization according to claim 1, wherein the sperm-receiving fluid further comprises essential amino acids, non-essential amino acids, and fatty acid-free bovine serum albumin.
3. The sperm-receiving fluid for bovine in vitro fertilization according to claim 2, wherein the volume fraction of essential amino acids is 0.1-2%, the volume fraction of non-essential amino acids is 0.1-2%, and the concentration of fatty acid-free bovine serum albumin is 1-100 mg/mL.
4. The sperm receiving solution for cattle in vitro fertilization according to claim 3, wherein each 100mL of the SOF basal medium comprises 26.168mg CaCl2•2H2O、9.999mg C6H5Na3O7•2H2O, 2.923mg glutamine, 37.218mg MgSO4•7H2O, 1.000mg phenol red, 53.378mg KCl, 16.195mg KH2PO44.402mg of sodium pyruvate, 210.025mg of NaHCO3629.399mg NaCl and 0.0757mg sodium lactate.
5. The semen receiving fluid for cattle in vitro fertilization according to claim 4, wherein the semen receiving fluid further comprises 12 μ g/mL gentamicin and 12 μ g/mL cefuromycin.
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