The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.
The maintaining of oocyte potential viability after longterm storage represent a tool of great opportunity, as it would improve domestic animal breeding by genetic selection programs, contribute to preserve biodiversity through wildlife species conservation, and increase the research in biotechnology. Juvenile oocyte vitrification was a lot shorter in the generation interval in breeding programs. Vitrifaction by ultrarapid cooling and warming is considered a standard approach in cattle oocyte preservation.
However, in sheep this procedure is still considered relatively new and a standard method is lacking. In this video, we describe a protocol for the vitrification of sheep oocyte, collected from both juvenile and adult donors, and in vitro matured to prior to cryopreservation. The protocol include all the procedures from in vitro maturation, vitrification, warming, and post-warming culture.
After recovering the Cumulus oocytes complex, where matured in vitro for 24 hours in static a condition that include for juvenile oocyte, the supplementation with hundred micromolar of trehalose in maturation medium. Following in vitro maturation, the oocytes were denuded of Cumulus cells mechanically by gently pipetting and examined under stereo microscope, with 60x magnification. The selection of the oocytes to submit at the vitrification procedures represent the first important step to warranty the successful application of this procedure.
Only those with the uniform cytoplasm, with homogeneous distribution of lipid droplets, integrity of the all Emma wide and continuous pivotal in space, compact zona pellucida the outer diameter of about 19 micrometers and showing the extrusion of the first polar body, and thus at the M2, stage must be selected. Vitrification was performed using the minimum essential volume method, with a Cryotops device. After selection, a group of five juvenile oocytes were conditioned at 38.5 Celsius degrees in base medium for two minutes.
The oocytes were then dehydrated by three minutes exposure to a calibration solution. The loading of the oocyte in the vitrification device used is an important and critical step. Cryotop uses a polypropylene strip attached to a holder.
In this method, the oocytes in the vitrification solution, less than 0 point milliliter are rapidly loaded with a glass capillary on top of the film strip. Then the solution must be removed, leaving behind a thin layer enough to cover the cells to be cryopreserved. Before being loaded in Cryotop device and directly plunged into liquid nitrogen within 30 seconds.
For warm interior biological temperature, the content of each vitrification device was transferred from liquid nitrogen into 200 microliter drops of decreasing trehalose solution. Oocytes were transferred set was, dragging the oocytes with a thin, glass pipette, to promote the removal of intracelular cryoprotectants. Finally, the oocytes were pre-breeded in a base medium in incubator in 5%carbon dioxide.
Immediately after warming, and for each time point of post-warming culture, oocytes were morphologically evaluated using an inverted microscope with 100x magnification. The cryo tolerance of oocytes from juvenile donors is lower compared to altered ones with lower membrane integrity after warming. The use of trehalose in maturation medium induced in juvenile oocytes higher membrane integrity, increasing the survival rates after vitrification.
However cleavage, fertilization, and developmental rates of juvenile oocytes were not increased by trehalose supplementation. The use of media with calcium concentration equal to 2.2 milligrams deciliter for the vitrification of juvenile oocytes, should higher fertilization rates compared to oocytes vitrified with calcium concentration, but no differences were found enabling production. By comparing post-warming culture on different durations, we showed that after four hours of culture oocyte collected from older twos are able to recover the energetic bonds and microtubular setup, and to restore the developmental competence with higher cleavage and blastocytes waste.
Mitochondrial activity was higher in vitrified warm juvenile oocytes after four hours of post-warming culture, compared to other time points. Mitochondrial distribution parting also changed during six hours of post warming culture. Moreover, reactive oxygen species intracellular levels were significantly lower in juvenile oocytes at two hours of post-warming culture compared to 0, 4, and 6 hours.
However, and in contrast with what found in other oocytes, the rates of spontaneous parthenogentic activation increased during post-warming culture in juvenile oocytes. One of the main advantages of the proposed method is it includes all the steps from oocyte collection to in-vitro maturation, vitrification, and warming. Moreover it includes a post-warming culture period to allow oocyte recovery from the damages incurred during the vitrification procedure.
Choosing the optimum time for fertilization is challenging, and it may impact on the outcome of the vitrification program. It should also be considered that, unlike slow freezing, vitrification is a manual technique, and is thus operator dependent. For this reason, a major challenge is the need for skilled technicians and researchers who take into consideration the operator effect in the evaluation of the outcome of the vitrification program.
Further studies from our lab will allow in attempting to standardize oocyte vitrification procedure and selection, and in better tailoring media composition to the needs of the juvenile oocyte. A disregard about the use of culture and character and antioxidant we offer promising opportunities.
