In this video protocol, we demonstrate in detail, a culture system for growing bovine oocytes. The system supports the oocytes viability in culture for up to five days and allows for their differentiation. Using this culture system, oocytes collected from small antral follicles, which are normally not used in standard protocols for in vitro in the production, can be grown to increase the availability of fertilizable gametes.
This exploitation of the wider reserve, can provide new options for the genetic salvage of threatened species, of the bovine family, or of local breeds at risk of genetic erosion. To begin, prepare 15 milliliters of basic culture medium and supplement it according to manuscript directions. Next, prepare three milliliters of holding medium by adding five micromolar cilostamide to the basic culture medium and pour it in a 35 millimeter Petri dish.
Prepare long IVCO medium by supplementing basic culture medium according to manuscript directions. Add 200 microliters of long IVCO medium to each well of a 96-well coated plate. Then fill the four edges with sterile water to compensate for evaporation and to maintain appropriate humidity during culture.
Incubate the 96-well plate and the holding medium at 38.5 degrees celsius and 5%carbon dioxide with maximum humidity. Wash the ovaries four times in sterile saline, maintained at 26 degrees celsius. Then aspirate all follicles that are more than two millimeter in diameter, with an 18 gauge needle connected to an aspiration pump.
Lace the aspirated ovaries in a beaker with sterile saline maintained at 26 degrees celsius. Place one ovary at a time on a sterile PTFE cutting board, and use a surgical number 22 blade to cut slices of ovarian cortex that are 1.5 to two millimeters thick, and parallel to the major axis of the organ. Lace the slices of ovarian cortex in a sterile glass Petri dish, with dissecting medium on a warm plate, at 38.5 degrees celsius.
Lace one ovarian cortex slice in a 60 millimeter glass Petri dish, with two to three milliliters of M199D under a dissection microscope, select the follicles that are between 5 and two millimeters. Identify the healthy non-atretic follicles under the stereo microscope, by observing morphological parameters, such as a uniformly bright translucent appearance, extensive vascularization, and a regular granulosa layer. While atretic follicles have a gray opaque appearance, and are poorly vascularized, with a dark COC inside.
Discard the atretic follicles and process all the others. Use the surgical blade to remove the ovarian tissue surrounding the follicle on one side, until it is exposed. Then use a 26 gauge needle to carefully make a slit in the exposed follicle wall, which will release the follicular content comprising the COC, follicular fluid, and clumps of cells.
Identify the COC and examine it for cumulus integrity, zona pellucida integrity and homogeneity of the cytoplasm. If these criteria are fulfilled, aspirate the COC with a P20 pipette. Lace the isolated COC in M199D cilostamide, and continue the isolation procedure for 30 minutes.
After selecting COCs, as previously described, prepare 16 drops with 20 microliters of M199D cilostamide in a 60 millimeter Petri dish, and place one healthy COC into each drop. Use an inverted microscope attached to a camera to measure the oocyte diameter, excluding the zona pellucida. With a clear visualization of the oocyte, make two perpendicular measurements, assure that the mean of the two measurements is within a range of 100 to 110 micrometers.
It can be difficult to precisely measure the oocytes diameter, due to the companion cumulus cells. COCs that have oocytes with smaller or larger diameter, or that don't have a rounded oocyte, or those with oocytes that are not measurable, should be discarded. Transfer the selected COCs into a 35 millimeter dish containing M199H medium, and keep them in the incubator at 38.5 degrees celsius and 5%carbon dioxide with maximum humidity, make sure that the overall working time does not exceed two hours.
Once the selection and collection procedures are completed, transfer one COC into the center of each well of the previously prepared 96-well plate. Incubate the plate for five days at 38.5 degrees celsius and 5%carbon dioxide with maximum humidity, refresh the medium every other day. To renew the medium, replace 100 microliters of old medium with 100 microliters of fresh long IVCO medium, do this under the stereo microscope to avoid moving the COCs.
At the end of the long IVCO, analyze the COCs morphology. If they have a compact cumulus cell investment with no sign of cumulus expansion, or cell degeneration, classify them as class one. If the COCs have a compact cumulus cell investment with no sign of cumulus expansion or cell degeneration, and with one or more antrum-like formations, classify them as class two.
Class three COCs, show several layers of cumulus cells with no sign of cumulus expansion, some disaggregated cells in the outer layer of the cumulus cells and no antrum-like formation. Classify the COCs as class four if they show abundant loss of cumulus cells extending for more than 50%of the oocyte surface, as well as signs of cell degeneration and cell debris. At the end of long IVCO the gross morphology of the COC is changed.
And four classes were identified based on the appearance of the cumulus cells. Overall, 74 oocytes in five biological replicates were analyzed, of which, 9.45%were discarded from further evaluation. Classes one, two, and three were judged healthy, while class four showed clear signs of degeneration, such as the absence of complete layers of cumulus cells surrounding the oocytes.
These COCs were considered unsuitable to undergo downstream procedures in a prospective IVP setting. Assessment of the meiotic stage at the end of the long IVCO showed that a significantly higher percentage of the oocytes remained arrested at the immature stage, with the chromatin still enclosed within the GV.When chromatin condensation within the GV was investigated as a marker of gain of competence, the transition of the chromatin configuration to more condensed stages, namely GV two and GV three, was observed in 59%of the oocytes. A small percentage resumed meiosis reaching metaphase one stage, or degeneration.
These results demonstrate that L-IVCO culture supports the oocyte viability, while preventing meiotic resumption for five days. At the end of the long IVCO, the COCs can be used in downstream procedures of in vitro embryo production, namely, in vitro maturation, fertilization, and embryo culture. These steps are necessary to provide proof of concept that the oocyte have acquired a higher developmental competence.
Besides holding the potential of increasing the amount of fertilizable oocytes, the long IVCO culture system is a tool for scientists who are interested in dissecting the cellular and molecular processes that regulate the formation of a competent gamete.
