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

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      Scientific Reports
      Nature Publishing Group UK
      Cell biology, Embryology

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          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.

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          Amino acids and immune function.

          A deficiency of dietary protein or amino acids has long been known to impair immune function and increase the susceptibility of animals and humans to infectious disease. However, only in the past 15 years have the underlying cellular and molecular mechanisms begun to unfold. Protein malnutrition reduces concentrations of most amino acids in plasma. Findings from recent studies indicate an important role for amino acids in immune responses by regulating: (1) the activation of T lymphocytes, B lymphocytes, natural killer cells and macrophages; (2) cellular redox state, gene expression and lymphocyte proliferation; and (3) the production of antibodies, cytokines and other cytotoxic substances. Increasing evidence shows that dietary supplementation of specific amino acids to animals and humans with malnutrition and infectious disease enhances the immune status, thereby reducing morbidity and mortality. Arginine, glutamine and cysteine precursors are the best prototypes. Because of a negative impact of imbalance and antagonism among amino acids on nutrient intake and utilisation, care should be exercised in developing effective strategies of enteral or parenteral provision for maximum health benefits. Such measures should be based on knowledge about the biochemistry and physiology of amino acids, their roles in immune responses, nutritional and pathological states of individuals and expected treatment outcomes. New knowledge about the metabolism of amino acids in leucocytes is critical for the development of effective means to prevent and treat immunodeficient diseases. These nutrients hold great promise in improving health and preventing infectious diseases in animals and humans.
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            The lutropin/choriogonadotropin receptor, a 2002 perspective.

            Reproduction cannot take place without the proper functioning of the lutropin/choriogonadotropin receptor (LHR). When the LHR does not work properly, ovulation does not occur in females and Leydig cells do not develop normally in the male. Also, because the LHR is essential for sustaining the elevated levels of progesterone needed to maintain pregnancy during the first trimester, disruptions in the functions of the LHR during pregnancy have catastrophic consequences. As such, a full understanding of the biology of the LHR is essential to the survival of our species. In this review we summarize our current knowledge of the structure, functions, and regulation of this important receptor.
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              The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development effect of beta-mercaptoethanol, cysteine and cystine.

              Supplementation of IVM medium with cysteamine, beta-mercaptoethanol, cysteine and cystine induced bovine oocyte glutathione (GSH) synthesis, but only the effect of cysteamine on the developmental competence of these oocytes was tested. During IVM of sheep oocytes, cysteamine but not beta-mercaptoethanol increased embryo development. However, it is not known how long the high intracellular oocyte GSH levels obtained after IVM with thiol compounds, can be maintained. Thus, the present study was carried out to evaluate the effects of supplementing maturation medium with 100 microM beta-mercaptoethanol, 0.6 mM cysteine and 0.6 mM cystine on 1) intracellular GSH level after IVM, 2) after IVF, 3) in 6 to 8-cell embryos and 4) on embryo development. In oocytes after IVM and in presumptive zygotes after IVF, intracellular GSH levels were significantly higher in the treated groups (P 0.05). Differences in cleavage rates and the percentage of embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) for treated oocytes than for those matured in the control medium. We conclude from the results that the high intracellular GSH levels after induction of GSH synthesis in bovine IVM by thiol compounds remain during IVF and are still present at the beginning of IVC, improving developmental rates. Moreover, the results indicate that this metabolic pathway is an important component of the cytoplasmic maturation process that affects the subsequent steps of in vitro embryo production.
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                Author and article information

                Contributors
                margot.day@sydney.edu.au
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                13 August 2019
                13 August 2019
                2019
                : 9
                : 11739
                Affiliations
                ISNI 0000 0004 1936 834X, GRID grid.1013.3, Discipline of Physiology and Bosch Institute, School of Medical Sciences, Faculty of Medicine and Health, , University of Sydney, ; New South Wales, Australia
                Article
                48038
                10.1038/s41598-019-48038-y
                6692353
                31409817
                15e2dd49-ed95-4992-9b7b-fa39bcbd7b72
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 2 January 2019
                : 18 July 2019
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                © The Author(s) 2019

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                cell biology,embryology
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                cell biology, embryology

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