Effect of vitamin A supplementation at different gaseous environments on in vitro development of pre-implantation sheep embryos to the blastocyst stage

Abstract

Vitamin A (all-trans retinol) is an important antioxidant whose role in embryo development in vitro and in vivo is well established. Oxidative stress is a major cause of defective embryo development. This study evaluated the effects of all-trans retinol supplementation to maturation and embryo culture media under different gaseous environments on the development of ovine oocytes and embryos in vitro. The percentages of cleavage, morula and blastocyst, total cell count and comet assay were taken as indicators of developmental competence of embryos. In experiments I and II, all-trans retinol at concentrations of 0, 2, 4, 6, 8 and 10 mM were supplemented to the oocyte maturation medium and cultured in an environment of 5% or 20% O 2 respectively. All-trans retinol supplementation (6 mM) to the maturation medium at 5% O 2 levels significantly increased blastocyst yield and total cell number (P , 0.05). Maturation of oocytes in a 20% O 2 environment bettered cleavage rates in the 6 mM supplemented group compared with the control group (P , 0.05). In experiments III and IV, all-trans retinol, at the aforesaid concentrations was supplemented to embryo culture media under a 5% or 20% O 2 environment, respectively. All-trans retinol supplementation to the embryo culture medium at 5% O 2 levels did not yield any significant result whereas the culture at 20% O 2 levels gave significantly higher blastocyst yield in the 6 mM supplemented group compared with the control group (P , 0.01).Keywords: vitamin A, ovine, in vitro fertilisation, oxidative stress, embryo culture ImplicationsThe supplementation of a suitable quantity of all-trans retinol to oocyte maturation medium or embryo culture medium followed by culture under appropriate gaseous environments protects oocytes and embryos from oxidative damage, thereby greatly enhancing the yields of viable embryos. This could be applied in Animal Biotechnology to increase lamb crops from high-yielding breeds of farm animals, which are seasonal breeders or which have reproduction problems and also to propagate endangered animal species.

Antioxidant and developmental capacity of retinol on thein vitroculture of rabbit embryos

“…The natural metabolites of retinol (RT) such as all- trans retinoic acid (t-RA) and 9- cis RA are collectively known as retinoids and are considered as non-enzymatic antioxidants (Guerin et al , 2001). Recent protocols have used these compounds in the culture medium of embryos taking into account their deep effect upon cellular survival, proliferation, differentiation and embryonic morphogenesis (Noy, 2010; Rajesh et al , 2010; Rhinn & Dollé, 2012; Conceição et al , 2015; Conceição et al , 2016). It was found that the addition of retinoids to the culture medium enhances the subsequent preimplantation embryonic development in bovine embryos (Lima et al , 2004; Ahmed et al , 2016), and in goat embryos (Duque et al , 2002a,b; Lima et al , 2006; Chiamenti et al , 2010, 2012; Conceição et al , 2015).…”

Comet assay on thawed embryos: An optimized technique to evaluate DNA damage in mouse embryos

“…We selected 14 studies using the comet assay on animal embryos matching our criteria, out of 118 studies retrieved. In these studies, fresh embryos were all obtained by natural mating (Fabian et al, 2003;Harrouk et al, 2000;Müller et al, 1996;Takahashi et al, 1999;Tranguch et al, 2003;Webster et al, 2000) or IVF (Hwang et al, 2013;Ju et al, 2010;Kitagawa et al, 2004;Natarajan et al, 2010;Rajesh et al, 2010;Sturmey et al, 2009;Takahashi et al, 2000;Valivittan, 2009a, 2009b). This review of the literature shows that there is no standardization of the protocol: Seven different animal species were used; The ZP was removed in 4 studies; The protocols for the transfer of the embryos in the LMP agarose layer were heterogeneous: a minimum of half a blastocyst and a maximum of 20 cleavage embryos were transferred, and the volume of LMP varied from 4 μL to 50 μL; The lysis and electrophoresis protocols were also heterogeneous.…”

“…All of the published comet assay protocols on mice embryos (Fabian et al, 2003;Harrouk et al, 2000;Hwang et al, 2013;Kitagawa et al, 2004;Müller et al, 1996;Natarajan et al, 2010;Rajesh et al, 2010;Sturmey et al, 2009;Takahashi et al, 1999Takahashi et al, , 2000Valivittan, 2009a, 2009b;Tranguch et al, 2003;Webster et al, 2000) used 2 layers of agarose except one (Ju et al, 2010). A glass microscope slide is initially coated with 1% high or normal-melting point (HMP, NMP) agarose (the first agarose layer), and then, the embryos are mixed with a drop of low-melting point (LMP) agarose and placed on the first layer (the second agarose layer).…”

“…The comet assay is also used by researchers for the assessment of DNA damage in sperm and male germ cells (Baumgartner et al, 2009;Perrin et al, 2007;Preaubert et al, 2016) and in oocytes (Berthelot-Ricou et al, 2013, 2011a, 2011bCourbiere et al, 2013;Einaudi et al, 2014). Studies using the comet assay on animal embryos are scarce and use heterogeneous protocols and species (Blerkom et al, 2001;Fabian et al, 2003;Harrouk et al, 2000;Hwang et al, 2013;Ju et al, 2010;Kitagawa et al, 2004;Müller et al, 1996;Natarajan et al, 2010;Rajesh et al, 2010;Sturmey et al, 2009;Takahashi et al, 1999Takahashi et al, , 2000Valivittan, 2009a, 2009b;Tranguch et al, 2003;Tsuda et al, 1998;Webster et al, 2000). As comet assay is a very sensitive test, the analysis of fresh embryos by comet assay requires the proximity between animal facilities and laboratory.…”

Farklı Boğa Spermalarının Sığır Embriyolarının İn Vitro Gelişim Yetkinliği, Blastosist Kalitesi ve Hücre Sayısı Üzerine Etkileri

“…Moreover this reproductive biotechnology has enabled large amounts embryo production of superior breeds in various domestic animals and the production of large numbers of embryos for scientific research purposes from slaughtered and/ or live animals [2,3] . Production of embryos in vitro also has potential for revolutionizing in cattle breeding and husbandry around the globe.…”