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Abstract
The present study is an endeavor for profound exploration of the influence of supplementing the vitrification medium with sugars ( sucrose or trehalose ) and / or macromolecules; including polyvinylpyrilidone ” PVP ” and polyethylene glycol ” PEG ”, upon bovine oocytes ( either immature or mature ) and embryonic stages ( morulae and blastocyts ). Attention was also directed to determine the most suitable stage to be vitrified in terms of its ability to undergo post - thawing development as well as the optimal treatment including sugar and polymer to improve post - thawing biological activities.
For this purpose, a total number of 5760 oocytes were recovered, using the slicing procedure, from 790 slaughterhouse ovaries. The recovered COCs were classified according to their morphological criteria into 3 grades; I, II and III. The good quality recovered oocytes ( grades I and II ) were selected to undergo in vitro maturation. The maturation medium was supplemented with gentamycin sulfate, FCS in addition to hormonal combination of FSH, hCG and E2. Following maturation, excellent and good mature bovine oocytes were used for fertilization then the zygotes were placed in culture medium for further development to morulae and blastocysts. In the respect, IVM, IVF and IVC were done in CO2 incubator adjusted at 39°C, 5 % CO2 and 95 % relative humidity.
During the aforementioned steps, oocytes ( immature or mature ) and embryonic stages ( morulae and blastocyts ) were subjected to vitrification by equilibration of cells in vitrification solution ( VS ) containing 10 % DMSO + 10 % EG ( as permeating cryoprotectants ) in TCM-199 as base medium ” BM ” for 2 min. at 22 ° C followed by their transfer into final VS containing 20 % DMSO + 20 % EG in the same BM for 30 seconds then cells were loaded in 0.25 ml straws that were sealed before their plunging into liquid nitrogen.
Oocytes and embryos were divided into 7 groups whereas the final vitrification solution was supplemented with no sugars and macromolecules ( control group ), sucrose, trehalose, PVP + sucrose, PVP + trehalose, PEG + sucrose and PEG + trehalose groups. The rate of post-thawing morphologically normal cells, live oocytes % as well as in vitro developmental rates were recorded in order to compare between the different treatments and record the best one that can be used for vitrification in relation to the developmental stage ( immature, mature oocytes as well as embryos ).
Analysis of data reveals that :
As compared with the control group, addition of sugars ( either sucrose or trehalose ) improves the post - thawing studied parameters. In addition, sucrose is better to be added to the VS of oocytes while trehalose is for embryos.
The best stages to be vitrified, in the presence of sugars alone, are mature oocytes with sucrose and compact morulae with trehalose.
As sugars are investigated to be indispensable for vitrification of oocytes and embryos, the vitrification solution, containing sugars, was supplied with different combinations of non permeating macromolecules ( PEG and PVP ). In this respect, combination of sugars and polymers markedly enhanced post - thawing viability compared with the control group. Furthermore, PVP and sucrose are ideal additives for vitrification of oocytes ( either immature or mature ) while PEG with trehalose followed by PVP with trehalose are convenient for embryos ( morulae and blastocysts ).
Among oocytes, mature ones are the ideal cells to undergo vitrification, if the VS is supplied with PVP and sucrose, as they yield the highest post - thawing cleavage rate as well as morula and blastocyst development. On the other side, among embryonic stages, compact morulae ( 7 days embryo ) are highly recommended to be vitrified in the presence of PEG with trehalose followed by PVP with trehalose as the post - thawing developmental rate is greater than that of blastocysts.