Supplementation of bovine embryo culture medium with L‐arginine improves embryo quality via nitric oxide production

Abstract

Nitric oxide (NO) is a cell-signaling molecule that regulates a variety of molecular pathways. We investigated the role of NO during preimplantation embryonic development by blocking its production with an inhibitor or supplementing in vitro bovine embryo cultures with its natural precursor, L-arginine, over different periods. Endpoints evaluated included blastocyst rates, development kinetics, and embryo quality. Supplementation with the NO synthase inhibitor N-Nitro-L-arginine-methyl ester (L-NAME) from Days 1 to 8 of culture decreased blastocyst (P < 0.05) and hatching (P < 0.05) rates. When added from Days 1 to 8, 50 mM L-arginine decreased blastocyst rates (P < 0.001); in contrast, when added from Days 5 to 8, 1 mM L-arginine improved embryo hatching rates (P < 0.05) and quality (P < 0.05) as well as increased POU5F1 gene expression (P < 0.05) as compared to the untreated control. Moreover, NO levels in the medium during this culture period positively correlated with the increased embryo hatching rates and quality (P < 0.05). These data suggest exerts its positive effects during the transition from morula to blastocyst stage, and that supplementing the embryo culture medium with L-arginine favors preimplantation development of bovine embryos.

Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells

“…Many groups worldwide have achieved blastocyst rates similar to the ones reported here (∼40 %), indicating that co-culture with stem cells was as efficient as current IVEP protocols in bovines, most of them using supplemented IVC medium [10,[50][51][52][53][54]. Therefore, further identification of molecules produced by stem cells for embryo development could help to improve IVEP not only in bovine but also in other species such as humans.…”

“…Embryo production was undertaken as described by Santana et al [10]. Briefly, cumulus-oocyte complexes (COCs) were recovered by follicular aspiration from abattoir ovaries.…”

“…Briefly, cumulus-oocyte complexes (COCs) were recovered by follicular aspiration from abattoir ovaries. Only compact COCs with good cytoplasm were selected for in vitro maturation for 24 h in tissue culture medium (TCM) 199 (Gibco BRL, USA) supplemented with 2.2 g/L sodium bicarbonate, 11 μg/mL pyruvate, 50 μg/mL gentamicin, 10 % FBS, 0.5 μg/mL follicle-stimulating hormone (FolltropinBioniche Animal Health, Canada), and 5.0 μg/mL luteinizing hormone (Lutropin-Bioniche Animal Health, Canada) as described by [10].…”

“…Spermatozoa were diluted to a final concentration of 2 × 10 6 cells/mL in 80-μL IVF droplets of Tyrode's albumin-lactate-pyruvate (TALP) medium [10]. Between 15 and 20 expanded COCs were placed into sperm-containing droplets for 24 h. Following IVF, groups of 20 presumptive zygotes were directly transferred to 100-μL drops of synthetic oviductal fluid (SOF) medium supplemented with 5 % FBS, 50 μg/mL gentamicin, and 6 mg/ mL bovine serum albumin (BSA) for in vitro culture in an incubator during 7 days without any medium replacement.…”

“…Despite great advances in the design of defined and semidefined embryo culture media [4][5][6], researchers still seek new media additives and somatic cell-based co-culture systems in an attempt to obtain higher yields of better quality embryos [7][8][9][10]. Presumably, somatic cells in a co-culture system may remove toxic components and reactive oxygen species, as well as Capsule Co-culture of in vitro produced bovine embryos with adult adipose tissue-derived bovine stem cells improved blastocyst development and quality.…”