Adding Essential Amino Acids at a Low Concentration Improves the Development of In Vitro Fertilized Porcine Embryos

Abstract

Abstract. The aims of this study were to investigate improvements to the pig preimplantation embryo culture system using in vitro produced embryos. For experiment 1, the optimum time to change the medium from NCSU23 containing 0.6 mM glucose, 0.2 mM pyruvate, 5.7 mM lactate and nonessential amino acids to NCSU23 containing 5.6 mM glucose and both essential and nonessential amino acids was examined. There were no statistically significant differences in blastocyst rates or cell number when the medium was changed at 48, 72 or 96 h, although there was a consistent trend for the 96 h treatment to produce fewer blastocysts with fewer cells. For experiment 2, the addition of essential amino acids at either a 1:50 or a 1:100 dilution of the purchased stock solution for day 1 to 6 or for days 3 to 6 only was investigated. Adding essential amino acids at a 1:50 dilution for day 3 to 6 significantly reduced the blastocyst rate and adding them at a 1:50 dilution from day 1 to 6 significantly reduced both the blastocyst rate and blastocyst cell number compared to when it was added at a 1:100 dilution. Embryos were produced by IVF, cultured for 6 days and good quality blastocysts were transferred into 6 synchronized pseudopregnant recipients (24 to 35 blastocysts per recipient) resulting in 4 pregnancies and 21 live birth piglets. These results show that adding essential amino acids at a 1:100 dilution provided the best culture conditions and the blastocysts produced were able to attain full term development after transfer. Key words: Blastocysts, Essential amino acids, In vitro culture, Piglets, Porcine (J. Reprod. Dev. 55: [373][374][375][376][377] 2009) he pig is becoming increasingly important as a biomedical research tool [1] and shows great promise as a model for embryonic stem cell technologies. The capacity to reliably generate embryonic stem cell lines is essential to using the pig as such a model. While in vivo produced embryos are ideal for producing cell lines, collecting them is expensive and time consuming. An alternative is to use embryos produced entirely in vitro. Obviously, a critical component in generating in vitro embryos is the culture medium in which they are grown.Several reports have investigated improving two commonly used porcine embryo culture media, North Carolina State University 23 and 37 (NCSU23 and NCSU37;[2]) [3][4][5]. As originally formulated, these media contain only glucose as the primary energy substrate. These reports found that providing pyruvate and lactate for the early preimplantation period (approximately 48 to 73 h post insemination) and only glucose for the remainder results in improved development and embryonic quality.Amino acids have been grouped by Eagle [6] into essential (EAA) and non-essential (NEAA), and are normally added to culture media as prepared concentrated stock solutions at a dilution of 1:50 for EAA and 1:100 for NEAA. Early cleavage development was found to be stimulated by the addition of NEAA to the culture medium but inhibited by EAA in both the mouse [...

Development of Culture Conditions for the Isolation of Pluripotent Porcine Embryonal Outgrowths from In Vitro Produced and In Vivo Derived Embryos

“…Previous attempts to isolate ES cells from in vitro produced pig embryos have been unsuccessful [11,32] possibly because these contained too few ES cell progenitor cells as a consequence of the culture system used. However recent improvements in the porcine preimplantation culture conditions used by our laboratory have resulted in increases in cell number which have resulted in acceptable pregnancy rates and litter sizes following their transfer to recipient animals [33].…”

“…Previous attempts to isolate ES cells from in vitro produced pig embryos have been unsuccessful [11,32] possibly because these contained too few ES cell progenitor cells as a consequence of the culture system used. However recent improvements in the porcine preimplantation culture conditions used by our laboratory have resulted in increases in cell number which have resulted in acceptable pregnancy rates and litter sizes following their transfer to recipient animals [33].In conclusion, we have developed a new method for the isolation of porcine embryonic stem cells. This method can be used for in vitro and in vivo derived embryos and differs from the methods developed previously for pigs in that it results in the establishment of homogeneous outgrowths consisting of pluripotent progenitor cells from which putative ESC lines can be established following vitrification.…”

Sonic hedgehog supplementation of oocyte and embryo culture media enhances development of IVF porcine embryos

“…The porcine animal model has been broadly applied in many aspects of biochemical research (Prather et al 2003), and porcine embryos hold great promise for embryonic stem cell technologies (Beebe et al 2009). Despite efforts that have been made to optimize the in vitro production (IVP) system of embryos, the yield and the quality of IVP embryos are still low when compared with their in vivo-produced counterparts (Abeydeera et al 1998, Ock et al 2007.…”

Progesterone improves porcine in vitro fertilisation system

“…Several reports have investigated improving two commonly used porcine embryo culture media, NCSU-23 and 37 (Raychoudhury and Suárez, 1991;Kikuchi et al, 2002;Karja et al, 2004;Beebe et al, 2009). Very few experiments have studied the use of hormones in the maturation of porcine oocytes (Eroglu, 1993;Bing et al, 2001;Dode and Graves, 2002).…”