All animal manipulations were conducted in accordance with the guidelines of the Animal Research Committee of Nanjing Agricultural University, China. This study was approved by the Committee of Animal Research Institute, Nanjing Agricultural University, China. Ovaries were collected from prepubertal gilts at a local slaughterhouse, placed in 0.9% physiological saline in a thermos bottle and transported to our laboratory within 2 h. Isolation and selection of cumulus-oocyte complexes (COCs) was as described previously. The medium used for maturation culture was improved TCM-199 supplemented with 75 μg/ml penicillin, 50 μg/ml streptomycin, 0.5 μg/ml FSH, 0.5 μg/ml LH, 10 ng/ml epidermal growth factor (EGF), and 0.57 mM cysteine. Oocytes were cultured in 500 μl of maturation medium covered with a thin layer of mineral oil at 38.5°C for 27 or 44 h in a humidified atmosphere of 5% CO2 in a four-well culture dish (Nunc, Roskilde, Denmark). We used an inverted microscope (200×) to check the polar body, and we rotated the oocytes to ensure proper judgment.
Four well culture dish
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
The four-well culture dish is a laboratory equipment used for in vitro cell culture experiments. It provides a contained environment with four distinct wells, allowing for the simultaneous culturing and observation of multiple cell samples or experimental conditions within a single device.
Automatically generated - may contain errors
Lab products found in correlation
Tcm 199, by Merck Group (1 mentions)
Cysteine, by Merck Group (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Polyvinyl alcohol (pva), by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Antrin r10, by Kyoritsu Seiyaku (1 mentions)
3 protocols using four well culture dish
1
Porcine Oocyte Maturation Protocol
2
Porcine Oocyte In Vitro Maturation
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The experiments were conducted in accordance with the Animal Research institute Committee guidelines of Nanjing Agricultural University, China. This study was approved by the Committee of Animal Research Institute, Nanjing Agricultural University, China. The ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to our laboratory within 2 hours in 0.9% physiological saline and was maintained at 35 °C in a thermos bottle. The ovaries were washed with sterile saline and stored at 37 °C. The cumulus-oocyte complexes (COCs) were aspirated from 2–8 mm antral follicles by using a 10 ml disposable syringe with an 18 G needle. COCs contained in the sediment were washed in the DPBS and the oocytes with an intact and compact cumulus were selected. COCs were washed three times with maturation medium before maturation culture. The medium was an improved TCM-199 (Sigma, St. Louis, MO, USA) supplemented with 75 μg/ml of penicillin, 50 μg /ml of streptomycin, 0.5 μg /ml of FSH, 0.5μg /ml of LH, 10 ng/ml of the epidermal growth factor (EGF) and 0.57 mM cysteine. About 80 oocytes were cultured in 500 μl of maturation medium covered with 200 μl mineral oil for 44 hours at 38.8 °C in a humified 5% CO2 atmosphere in a four well culture dish (Nunc, oskide, Denmark).
3
In Vitro Maturation of Porcine Cumulus-Oocyte Complexes
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In the ovaries, COCs were aspirated from superficial follicles using an 18-guage needle. Next, COCs with multiple layers of unexpanded cumulus cells were justly selected and washed three times in HEPES-buffered Tyrode's medium (TLH) supplemented with 0.05% (w/v) polyvinyl alcohol (PVA; Sigma-Aldrich, St. Louis, MO). Culture of the washed COCs in a four-well culture dish (Nunc, Roskilde, Denmark) containing 500 μL of IVM medium consisting of medium-199 (M-199; Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) porcine follicular fluid, 1 μg/ mL insulin (Sigma-Aldrich), 10 ng/mL epidermal growth factor (EGF, Sigma-Aldrich), 0.6 mM cysteine (Sigma-Aldrich), 0.91 mM pyruvate (Sigma-Aldrich), 75 μg/mL kanamycin (Sigma-Aldrich), 10 IU/mL human chorionic gonadotropin (hCG; Intervet International BV, Boxmeer, Holland) and 80 μg/mL follicle-stimulating hormone (FSH; Antrin R-10, Kyoritsu Seiyaku, Tokyo, Japan) was conducted for 22 h at 39 o C with 5% CO 2 . Subsequently, the in vitro maturated COCs were washed three times with hormone-free IVM medium and additionally cultured in hormone-free IVM medium for 20 h at 39 o C with 5% CO 2 .
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