Amino acid supplementation of a simple inorganic salt solution supports efficient in vitro maturation (IVM) of bovine oocytes

Mohammad Bahrami,Margot L Day,Michael B Morris

doi:10.1038/s41598-019-48038-y

Mohammad Bahrami, Margot L Day + Show 1 more

Open Access

https://doi.org/10.1038/s41598-019-48038-y

Copy DOI

Journal: Scientific Reports	Publication Date: Aug 13, 2019
Citations: 17	License type: open-access

Affiliation: University of Sydney

Abstract

Defining oocyte in vitro maturation (IVM) conditions allows for improved reproducibility and efficiency of bovine embryo production. IVM conditions for bovine oocytes have been extensively studied, but beneficial effects of individual supplements remain controversial. This study compared methods of cumulus oocyte complex (COC) isolation, and culture medium requirements, for IVM in order to define optimal conditions. Antral follicles in ovaries were sliced or aspirated to isolate COCs. Brilliant cresyl blue staining of COCs was used to determine the most effective collection technique and the effect of hormones and groups of amino acids in the culture medium was investigated. Our results showed COCs isolated through aspiration had greater meiotic competency to reach MII. Oocyte maturation was achieved with the addition of 1 µg/mL FSH, while estrogen and human chorionic gonadotrophin did not increase the number of MII oocytes. We also provide novel data, that supplementation of a simple inorganic salt solution with L-proline, L-glutamine and essential amino acids in combination, but not individually, resulted in nuclear maturation comparable to TCM199, a more complex medium containing all 20 common amino acids, vitamins, inorganic salts and FBS. Replacement of FBS with BSA in this simplified medium creates a defined medium which provides conditions for IVM that enable reproducible maturation rates.

Highlights

Improving assisted reproductive technologies (ART) is beneficial to cattle production systems[1] and efficient, reliable production of high quality bovine embryos enables genetic improvements in herds to be made, without the need to transport whole animals
Maturation of bovine cumulus oocyte complex (COC) obtained by slicing open follicles on the surface of the ovary or by aspiration of antral follicles was compared to determine the technique that resulted in the higher proportion of MII oocytes
COCs collected by aspiration had higher (51.6 ± 0.9%) nuclear maturation compared to slicing (35.0 ± 3.8%) (n = 3, P < 0.05) (Fig. 1b)

Summary

Introduction

Improving assisted reproductive technologies (ART) is beneficial to cattle production systems[1] and efficient, reliable production of high quality bovine embryos enables genetic improvements in herds to be made, without the need to transport whole animals. Development of IVP bovine embryos from abattoir-acquired ovaries involves the collection of cumulus oocyte complexes (COCs) from antral follicles and subsequent completion of oocyte meiotic maturation in vitro (IVM). A physiological method of selection of the most mature COCs uses brilliant cresyl blue (BCB) to determine glucose-6-phosphate dehydrogenase (G6PDH) activity within the cytoplasm of the oocyte[14]. During their growth phase, oocytes have high G6PDH activity and this enzyme degrades the BCB compound, resulting in a reduction in blue colour[15]. Addition of a large range of FSH concentrations to IVM medium is reported in the literature, from 0.5–20 μg/mL, and these had varying effects on bovine COC maturation. Bovine maturation medium is often supplemented with approximately this concentration of 17β-estradiol[9,13,19] despite reports that addition of 1 μg/mL 17β-estradiol reduces nuclear maturation of bovine oocytes[25] and oocytes of other mammalian species[26]

Objectives

Methods

Results

Conclusion