Evaluation of cryopreservation methods for in vitro produced bovine embryos

English: The objective of this study was to evaluate four cryopreservation techniques for in vitro produced bovine embryos, and to select the best method for practical application. The cryopreservation methods investigated were three vitrification methods and a slow freezing method. This study was done at the ARC-Animal Improvernent Institute in conjunction with the University of the Orange Free State (Department of Animal Science). Embryos were obtained by the IVM, IVF and IVC of bovine follicular oocytes. A total of 136 early blastocysts, blastocysts and expanded blastocysts were randomly assigned to four different treatment groups. In the conventional slow freezing method, the IVP bovine embryos were first held in ViGro™Holdingplus medium before being transferred to 1.5M ViGro™EG Freezeplus medium (TMT 4). In this technique, the IVP embryos were loaded into 0.25ml straws. The straws containing the embryos were immediately placed into a programmable freezer (CL-863 cryo-chamber) at -6°C. Straws were seeded after a 5 minutes equilibration period. Embryos were initially cooled from -6 "C to -30°C at a rate of 0.3 °C/min. Thereafter, from -30°C to -33°C the rate was changed to 0.1 °C/min. After the target temperature was reached, straws were immediately transferred to liquid nitrogen. Vitrification of IVP bovine embryos was performed according to the following procedures: Embryos were initially placed in 10% EO in ViGro™Holdingplus medium for 5 minutes (Equilibration I), thereafter in 40% EO + 0.3M trehalose in ViOro ™Holdingplus medium for 5 minutes (Equilibration 11), both at room temperature. Embryos were then transferred to vitrification solutions, containing 40% EO (TMT 1); 40% EG + 0.3M trehalose (TMT 2); 40% EG + 0.3M trehalose + 20% PVP (TMT 3) in ViGro TM Holdingplus. Embryos were then loaded into 0.25ml straws, and plunged directly into liquid nitrogen (LN2). The straws were vertically stored in liquid nitrogen (- 196°C) until thawing and evaluation took pl
Afrikaans: Die doel van die studie was om vier kriopreserverings tegnieke vir in vitro geproduseerde beesembrio's te evalueer en die die beste metode te selekteer vir praktiese toepassing. Die kriopreserveringsmetodes ondersoek, het bestaan drie vitrifikasiernetodes en 'n stadige stapsgewyse metode. Die studie IS uitgevoer by die LNR Diereverbeteringsinstituut in samewerking met die Universiteit van die Oranje Vrystaat (Departement Veekunde). Embrios is verkry deur IVM, IVB en IVK van follikulêre beesoosiete. 'n Totaal van 136 vroeë blastosiste, blastosiste en laat blastosiste was ewekansig toegedeel tot die vier behandelingsgroepe. In die konvensionele stapsgewyse bevriesingsmetode is die embrios in 'n Vigro TM houmedium geplaas en daarna in 1.5 M Vigro TM EG freezeplus medium oorgeplaas - behandeling 4 (TMT4). Met die tegniek is die embrios in 0.25 ml strooitjies gelaai en direk in 'n programmeerbare bevriesingsapparaat (CL 863 eryoehamber) by 'n temparatuur van -6°C. geplaas Na 'n ekwilibrasieperiode van 5 minute is die strooitjies geinduseer vir vinnige ysvorming. Embrios is inisieel afgekoel teen' n tempo van 0.3 "Czminuut tot 'n temparatuur van -30°C, daarna van -30 °C tot en met -33°C teen 'n tempo van 0.1 "Czminuut. Nadat die teikentemperatuur bereik is, is die strooitjies direk in vloeibare stikstof gedompel. Strooitjies is vertikaal gestoor in vloeibare stikstof (-196 QC) tot en met ontdooiing en evaluasie. Vitrifasie van embrios is gedoen volgens die volgende prosedures. Embrios is aanvanklik geplaas in 'n 10% EG in Vigro TM houmedium vir 'n periode van 5 minute (ekwilibrasie J), daarna in 40% EG + 0.3 M trehalose in Vigro TM houmedium vir 5 minute (ekwilibrasie II). Beide stappe is by kamertemperatuur uitgevoer. Embrios is daarna oorgeplaas na vitrifikasie oplossings bevattende 40% EG (TMTI); 40% EG + 0.3 TM M trehalose (TMT2); 40% EG + 0.3 M trehalose + 20% pyp (TMT3) in ViGro Houmedium. Embrios is gelaai in 0.25 strooitjies en direk in vloeibare stikst
Professional Development Project (PDP)
University of the Free State, Department of Animal Science