> research

A radically new approach for active protection of gametes and embryos

A well defined and properly applied high hydrostatic pressure (HHP) stress-followed by a recovery period under optimal culture conditions-may induce a general adaptation syndrome and increases cells' (embryos, gametes, stem cells, other mammalian cells...) survival and developmental competence during the subsequent procedures.

HHP treatment of porcine MII. oocytes resulted in
1) approx. tenfold increase in blastocyst rates                             ->    after activation after vitrification
2) 40-100% increase in blastocyst rates                                       ->    after somatic cell nuclear transfer
3) 100% increase in re-expansion rates                                        ->   after vitrification of cloned blastocysts

HHP treatment of GV stage (porcine) oocytes resulted in
1) 50% increase in blastocyst rates, and
    27% increase in cell numbers of blastocysts                              ->   after in vitro embryo production

HHP treatment of in vitro produced blastocysts (bovine) resulted in
1) 25% increase in re-expansion rates                                          ->   after vitrification

HHP treatment of fresh, extended porcine/bull sperm resulted in
1) 20% increase in motility,
    6% increase in cells with intact morphology , and
    70% increase in litter size (porcine)                                           ->   after cryopreservation
2) 8% increase in litter size (porcine)                                             ->   after insemination

The table below summarizes the results achieved by this technology on different fields:

No.	Biological material treated (Reference)	System, reason	Outcome (treated vs. control)	Reference institute
1.	GV oocyte (porcine) (Ref. M)	To facilitate in vitro system	Blastocyst formation rate: 54 % vs. 36 % Mean cell number of blastocysts: 60 vs. 47	Institute of Genetics and Biotechnology, University of Aarhus , Denmark
2.	MII. Oocyte (porcine) (Ref. A, C, L, N)	To facilitate somatic cell nuclear transfer	Blastocyst rate from reconstructed embryos: 57% vs. 28% ( Yucatan donor cell line) 68% vs. 46% (LW1-2 donor cell line)	Institute of Genetics and Biotechnology, University of Aarhus , Denmark
		To facilitate in vitro system	Survival rate of cryopreserved reconstructed embryos: 62% vs. 30%	Institute of Genetics and Biotechnology, University of Aarhus , Denmark
		To facilitate vitrification	Blastocyst rate from vitrified eggs: ~15% vs. ~ 1%	Institute of Genetics and Biotechnology, University of Aarhus , Denmark
3.	IVF embryo (bovine) (Ref. B, H)	To facilitate vitrification; basic research: gene expression	Survival rate of cryopreserved embryos: 77% vs. 61% embryos hatched	EMBRAPA, Brazil ; UFMG, Brazil ; University of Connecticut , Centre for Regenerative Biology , USA
4.	In vivo produced embryo (mouse) (Ref. I, J, K)	To facilitate cryopreservation; Basic research: gene expression	Survival rate of cryopreserved embryos: 94% vs. 46% embryos upregulation of selected genes after treatment or even after 120 min	St. István University, Faculty of Veterinary Science; Animal Biotechnology Centre , Hungary
5.	Semen (bull, boar) (Ref. D, E, F, G, O)	To facilitate cryopreservation; To improve insemination     basic research: proteomics (2D, mass-spec), protein profile	Ratio of motile cells Bull: 54% vs. 45% Boar: 59% vs. 37% Number of piglets born/sow (pregnancy rate ~ 70% both groups): 9.4-10 vs. 4.4-6.7 Number of piglets born/sow (pregnancy rate ~ 80% both groups): 12.06 vs. 11.09 Ubiquinol-cytochrome c reductase complex core protein 1, perilipin, and carbohydrate-binding protein AWN precursor levels elevated significantly after treatment, and remained elevated after thawing	A.I. Station Klessheim, Austria; A.I. Station Vieselburg, Austria; Bos Genetic, Hungary; Animal Technology Institute, Taiwan
6.	bacteria suspensions ( Lactobacillus plantarum, E.coli)	To facilitate freeze-drying	Living cell number after freeze-drying: E.Coli: 74% vs. 31% L.plantarum: 85% vs. 10%	Corvinus University of Budapest , Faculty of Food Sciences, National Collection Of Agricultural And Industrial Microorganisms (NCAIM)

G:E-Box applies computer controlled high hydrostatic pressure (HHP) in the range of 200-600 bar (200-600 Atm) for a predefined period (usually 30 to 120 min). Oocytes, spermatozoa or embryos are sealed into a container (e.g. 0.25 ml plastic straw) and placed in the chamber that is completely filled with water. After adjustment of parameters the program runs automatically. After the treatment cycle, samples are removed, cultured for a period (one or two hours) in optimal environment, then subjected to the required in vitro treatments.