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Basic Culture Kit for Porcine Embryos in vitro

Background

This is a basic culture kit for porcine embryos in vitro containing porcine oocyte/embryo collection medium (POE-CM), basic medium for porcine oocyte maturation (POM), porcine fertilization medium (PFM), defined medium for porcine embryos (PZM-5), dbcAMP concentrated solution-100X (dbcAMP-100X) and Reproplate. This kit can efficiently produce transferable embryos (blastocysts) under a low oxygen condition (5%O2/5%CO2/90%N2, 39℃) without the coculture of cumulus/granulose cells.
This kit can support to increase the blastomere numbers of blastocysts and the formation of hatched blastocysts.

Kit component

  1. Porcine oocyte/embryo collection medium (POE-CM), Catalog No.:CK020, 100 ml x 3
  2. Basic medium for porcine oocyte maturation (POM), Catalog No.:CK021, 25 ml x 2
  3. Porcine fertilization medium (PFM), Catalog No.:CK023, 100 ml
  4. Defined medium for porcine embryos (PZM-5), Catalog No.:CK024, 25 ml
  5. dbcAMP concentrated solution-100X (dbcAMP-100X), Catalog No.:CK027, 0.3 ml
  6. Reproplate, Catalog No.:CK028, 10 plates x 3

In vitro production of porcine embryos

Oocyte collection and oocyte maturation culture

  1. Wash porcine ovaries se veral times with PBS (-) or saline at room temperature (about 25℃) and collect cumulus-oocyte complexes (COCs) by aspirating from ovarian follicles (diameter=3-6 mm).
  2. Transfer the follicular fluid containing COCs to centrifuge tube and stand it for 20 min.
  3. Remove the supernatant and dilute the precipitation with POE-CM medium at room temperature. Dispense the precipitate to Petri dish. Harvest and select the oocytes that the cumulus cells adhere to more than two or three layers (cumulus-oocyte complexes;COCs) under the stereomicroscope (Fig.1).
  4. Wash the selected COCs twice with 1st POM medium prepared in Reproplate. Put 15-20 COCs in one drop of 1st POM medium and then, incubate for 20-22 hours.
  5. Wash the COCs three times with 2 nd POM medium prepared in Reproplate after 20-22 hours of the maturation culture. Transfer the COCs into the drop of 2nd POM medium and incubate for additional 20-24 hours.

Sperm preparation and in vitro fertilization

  1. Keep warm PFM medium (about 20 ml) and 80% and 50% isotonic Percoll solution in a thermostat bath at 38℃.
  2. Thaw frozen sperm*1 in the straw with warm water at 38℃. Suspend them into 50% Percoll solution.
  3. Overlay the sperm suspension gently on the top of 80% Percoll solution. Then, centrifuge at 700 x g for 20 min (35℃ or room temperature).
  4. Aspirate the supernatant and add about 7 ml PFM medium to it and gently mix. Centrifuge it at 500 x g for 5 min.
  5. Repeat 4.
  6. Aspirate the supernatant and add 0.5-1 ml PFM medium to it and mix. Count the numbers of sperm with a hemacytometer.
  7. Dilute sperm suspension with PFM medium at the concentrations of 2-20×106/ml*2.
  8. Prepare the drops of gas-phase equili brated PFM medium in the Reproplate and a dd 50 ul of sper suspension (final sperm concentrations: 1-10×106/ml*2) in each drop.
  9. Wash the COCs twice with PFM me dium in the Reproplate after the in vitro maturation. And then, transfer 15-20 COCs into the drop containing sperm suspension and incubate for 10~20 hours*2.

*1 Please contact us if you would like to use fresh spermatozoa.
*2 The optimal sperm concentration and the incubation time of insemination are variable for different sperm preparation. Please make sure to determine these appropriate conditions before the experiments.


Embryo culture in vitro

  1. Wash fertilized oocytes once with POE-CM medium at 38℃ after in vitro fertilization. Transfer them into 1ml POE-CM medium of 15 ml centrifuge tube and remove cumulus cells of them by mixing with vortex mixer for 4 min.
  2. Collect the denuded embryos (Fig.2) from centrifuge tube. Remove the fertilized oocytes with strongly attached cumulus cells and/or with degenerated cytoplasm.
  3. Wash the selected fertilized oocytes twice with POE-CM medium. W ash them twice with PZM-5 medium in the Reproplate.
  4. Transfer 20-30 embryos into each drop of PZM-5 medium in the Reproplate and incubate them. They develop to morula and blastocyst after 4-5 days after in vitro fertilization (Fig.3).
  5. Wash morula and blastocyst twice with PBM medium in the Reproplate. Transfer 10-20 of those into each drop of PBM medium and incubate them. They develop to expanded blastocysts and hatched blastocysts after an additional 1-2 days of culture.

Fig.1 Cumulus-oocyte complexes
just after the collection
Fig.2 Denuded embryos Fig.3 Blastocysts

Product List

Product Name Cat# Quantity Price

Complete culture kit for porcine embryos in vitro

CSR-CK030 1KIT

¥ 48,000
$ 640
€ 480

Porcine oocyte/embryo collection medium

CSR-CK020 5*100ML

¥ 10,000
$ 134
€ 100

Basic medium for porcine oocyte maturation

CSR-CK021 3*25ML

¥ 16,000
$ 214
€ 160

Porcine fertilization medium

CSR-CK023 2*100ML

¥ 20,000
$ 267
€ 200

Defined Medium for Porcine Embryos(PZM-5)

CSR-CK024 3*25ML

¥ 16,000
$ 214
€ 160

dbcAMP concentrated solution-100X

CSR-CK027 0.5ML

¥ 8,000
$ 107
€ 80

Reproplate

CSR-CK028 10*10PLATE

¥ 47,000
$ 627
€ 470

High performance basic medium for porcine oocyte maturation

CSR-CK022 3*25ML

¥ 20,000
$ 267
€ 200

Defined medium for late stage porcine embryos

CSR-CK025 2*25ML

¥ 14,000
$ 187
€ 140

Sperm diluent for in vitro fertilization

CSR-CK026 25ML

¥ 7,000
$ 94
€ 70

Basal culture kit for porcine embryos in vitro

CSR-CK029 1KIT

¥ 43,000
$ 574
€ 430

References
  • Mito T, Yoshioka K, Nagano M, Suzuki C, Yamashita S, Hoshi H. Transforming growth factor-??in a defined medium during in vitro maturation of porcine oocytes improves their developmental competence and intracellular ultrastructure. Theriogenology 2009; 72: 841-850.
  • Suzuki C, Yoshioka K. Effects of amino acid supplements and replacement of polyvinyl alcohol with bovine serum albumin in porcine zygote medium. Reprod Fertil Dev 2006; 18: 789-795.
  • Suzuki C, Yoshioka K, Sakatani M, Takahashi M. Glutamine and hypotaurine improves intracellular oxidative status and in vitro development of porcine preimplantation embryos. Zygote 2007; 15: 317-324.
  • Yoshioka K, Suzuki C, Tanaka A, Anas IMK, Iwamura S. Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod 2002; 60: 112-119.
  • Yoshioka K, Suzuki C, Itoh S, Kikuchi K, Iwamura S, Rodriguez-Martinez H. Production of piglets derived from in vitro-produced blastocysts fertilized and cultured in chemically defined media: effects of theophylline, adenosine, and cysteine during in vitro fertilization. Biol Reprod 2003; 69: 2092-2099.
  • Yoshioka K, Suzuki C, Onishi A. Defined system for in vitro production of porcine embryos using asingle basic medium. J Reprod Dev 2008; 54: 208-213.
  • Mito T, Yoshioka K, Noguchi M, Yamashita S, Hoshi H. Recombinant human follicle-stimulating horm ones and transforming growth factor-alpha enhance in vitro maturation of por cine oocytes. Mol Repro d Dev 2013;80: 549-560.
  • Mito T, Yoshioka K, Yamashita S, Suzuki C, N oguchi M, Hoshi H. Glucose and glycine synergistically enhance the in vitro development of porcine blastocysts in a chemically defined medium. Reprod Fertil Dev 2012; 24: 443-450.

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.