151 EFFECT OF ASCORBIC ACID ON OXIDATIVE STRESS AND ITS THERMOPROTECTANT ROLE ON IN VITRO EMBRYONIC DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) EMBRYOS

The most important factors that lead to stress in farm animals are oxidative and thermal stress, leading to reduced reproductive efficiency. Oxidative stress leads to an increase in proportion of reactive oxygen species, whereas heat stress affects the physiology of animals, which lowers the conception rates of dairy cattle. In vitro culture systems have been enhanced by manipulating media with various supplements such as vitamins, growth factors, and antioxidants that have overcome these problems. Ascorbic acid has been shown to play an antioxidant role in many species such as sheep, goat, and pigs. Keeping this in mind, this study was conducted to investigate the effect of supplementation of in vitro-matured (IVM) and/or in vitro-cultured (IVC) media with ascorbic acid and evaluate its antioxidant role in in vitro development of buffalo embryos. Immature oocytes were collected from visible surface follicles (2 to 8 mm in diameter) in slaughterhouse buffalo ovaries and subjected to IVM, IVF, and IVC in a humidified CO2 incubator at 38.5°C. Ascorbic acid was supplemented to IVM [TCM-199 + 10% featl bovine serum (FBS) + 1 µg mL−1 oestradiol-17β + 5 µg mL−1 pFSH + 0.81 mM sodium pyruvate + 0.68 mM l-glutamine + 50 µg mL−1 gentamicin sulfate] at 50 or 100 µM or IVC (mCR2aa + 0.6% BSA + 10% FBS+ 50 µg mL−1 gentamicin sulfate) at 50 µM or both IVM and IVC media at 50 µM. Oocytes without ascorbic acid were treated as the control group. Cleavage and blastocyst rate was improved when 50 µM (66.67 ± 2.27; 16.67 ± 1.26%) ascorbic acid was supplemented to IVM medium but no significant difference (P < 0.05) was observed at 100 µM (54.04 ± 2.20; 6.16 ± 0.37%) as compared with the control (62.77 ± 2.71; 10.67 ± 0.24%), respectively. When 50 µM ascorbic acid was supplemented in IVM, IVC, or both media, though cleavage rate (66.67 ± 2.27; 69.09 ± 3.22; 66.67 ± 2.23%) was similar in 3 groups, a significant increase was observed in blastocyst rate (16.67 ± 1.26; 20.18 ± 0.86; 28.57 ± 0.37%) when both media were supplemented, respectively. To evaluate the thermoprotectant effect, 4 groups were taken: group 1 without and group 2 with ascorbic acid supplementation, oocytes were given heat treatment at 39.5°C initially for 12 h during IVM; group 3 without and group 4 with ascorbic acid supplementation, oocytes were given heat treatment at 40.5°C initially for 12 h during IVM. No significant difference in developmental rate was observed at elevated temperature of 39.5°C or 40.5°C as compared with the control. Relative mRNA abundance of heat stress-related genes, HSP 70.1 and HSP 70.2, was nonsignificantly higher in oocytes matured at 39.5°C or 40.5°C after supplementation with ascorbic acid as compared to control. Relative mRNA abundance of BAX decreased at 50 µM and increased at 100 µM ascorbic acid compared with control, whereas BID showed similar results between control and treatment. Regarding anti-apoptotic gene expression, significantly higher expression was observed in MCL1 for 50 µM and lower for 100 µM ascorbic acid, and a similar nonsignificant trend was observed for BCL-XL. Developmental genes GDF9 and BMP15 showed a nonsignificant increase in 50 µM, and a nonsignificant decrease in the 100 µM supplemented group as compared with the control. Oxidative stress-related genes SOD and GPX showed a nonsignificant decrease in treated groups as compared to control. From above results, it was concluded that ascorbic acid had an anti-oxidant as well as thermoprotectant role in developmental competence that increased the potential for generation of large domestic animal in vitro embryos for research and applied technologies such as cloning and transgenesis.

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74 Treatment with Melatonin During In Vitro Culture Enhances Porcine Parthenogenetically Activated Embryo Development

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab74 ◽

2018 ◽

Vol 30 (1) ◽

pp. 175

Author(s):

G. A. Kim ◽

J.-X. Jin ◽

S. Lee ◽

A. Taweechaipaisankul ◽

B. C. Lee

Keyword(s):

Oxidative Stress ◽

Embryo Development ◽

Developmental Competence ◽

Free Radical Scavengers ◽

Control Group ◽

Cleavage Rate ◽

Cell Numbers ◽

Significant Difference ◽

Blastocyst Rate

Melatonin and its metabolites are powerful antioxidants and free radical scavengers. Because porcine embryos are vulnerable to oxidative stress in vitro, the addition of various protective chemicals to the culture medium, including melatonin, has been explored. The aim of this study was to investigate the effect of melatonin on in vitro developmental competence of porcine parthenogenetically activated (PA) embryos. Immature cumulus–oocyte complexes (COC) were collected and cultured in medium comprising TCM-199 supplemented with 10 ng mL−1 epidermal growth factor, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μL mL−1 insulin, transferrin selenium solution 100×, 10% porcine follicular fluid, 10 IU mL−1 eCG, and 10 IU mL−1 hCG for 44 h. Then, COC were denuded and PA with electrical stimulation, and PA embryos were cultured in porcine zygote medium 5 (PZM-5) supplemented with melatonin at increased concentrations (10−9, 10−7, 10−5 M) at 39°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 7 days. Subsequent embryo development, including cleavage rate, blastocyst rate, and blastocyst cell numbers, was compared between groups (mean no. of embryos; control, 27.14; 10−9 M, 28.86; 10−7 M, 27.71; 10−5 M, 26.43). The experiments were repeated 7 times for each treatment group. Statistical analyses of all data were performed using one-way ANOVA with Dunn’s multiple comparison test. Results are expressed as the mean ± SEM and all differences were considered significant at P < 0.05. No apparent effect on cleavage rate of melatonin treatment of various concentrations was noted. Blastocyst cell number did not show any significant difference between groups. However, the potential of PA oocytes to develop into blastocysts was significantly higher in the group supplemented with 10−9 M melatonin compared with the control group (35.44 ± 3.84 v. 24.71 ± 1.59) and other melatonin treated groups (10−5 M, 21.35 ± 2.82; 10−7 M, 24.01 ± 2.31; P < 0.05). These indicated that treatment with 10−9 M melatonin in embryo culture might reduce the oxidative stress properly compared with other concentrations, which results in improvement of blastocyst rate formation. In conclusion, treatment with 10−9 M melatonin positively promoted the blastocyst formation rate of porcine PA embryos with no beneficial effects on their blastocyst cell numbers or cleavage rate. This study was supported by the National Research Foundation (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

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A Comparative Study of the Antioxidative Effects of Helichrysum italicum and Helichrysum arenarium Infusions

Antioxidants ◽

10.3390/antiox10030380 ◽

2021 ◽

Vol 10 (3) ◽

pp. 380

Author(s):

Katja Kramberger ◽

Zala Jenko Pražnikar ◽

Alenka Baruca Arbeiter ◽

Ana Petelin ◽

Dunja Bandelj ◽

...

Keyword(s):

Oxidative Stress ◽

High Performance ◽

Radical Scavenging Activity ◽

Radical Scavenging ◽

Morphological Type ◽

Mass Spectrometry Analysis ◽

In Vitro Assays ◽

Spectrometry Analysis ◽

Stress Related Genes

Helichrysum arenarium (L.) Moench (abbrev. as HA) has a long tradition in European ethnomedicine and its inflorescences are approved as a herbal medicinal product. In the Mediterranean part of Europe, Helichrysum italicum (Roth) G. Don (abbrev. as HI) is more common. Since infusions from both plants are traditionally used, we aimed to compare their antioxidative potential using in vitro assays. Two morphologically distinct HI plants, HIa and HIb, were compared to a commercially available HA product. Genetic analysis using microsatellites confirmed a clear differentiation between HI and HA and suggested that HIb was a hybrid resulting from spontaneous hybridization from unknown HI subspecies. High-performance liquid chromatography–mass spectrometry analysis showed the highest amounts of hydroxycinnamic acids and total arzanol derivatives in HIa, whereas HIb was richest in monohydroxybenzoic acids, caffeic acids, and coumarins, and HA contained the highest amounts of flavonoids, especially flavanones. HIa exhibited the highest radical scavenging activity; it was more efficient in protecting different cell lines from induced oxidative stress and in inducing oxidative stress-related genes superoxide dismutase 1, catalase, and glutathione reductase 1. The antioxidative potential of HI was not only dependent on the morphological type of the plant but also on the harvest date, revealing important information for obtaining the best possible product. Considering the superior properties of HI compared to HA, the evaluation of HI as a medicinal plant could be recommended.

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Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽

10.1017/s0967199416000228 ◽

2016 ◽

Vol 24 (6) ◽

pp. 890-899 ◽

Cited By ~ 4

Author(s):

A.L.S. Guimarães ◽

S.A. Pereira ◽

M. N. Diógenes ◽

M.A.N. Dode

Keyword(s):

Ascorbic Acid ◽

Cell Number ◽

Total Cell ◽

Control Group ◽

Bovine Embryo ◽

Total Cell Number ◽

Embryo Production ◽

Embryo Size ◽

Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

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Follicular development, morphological integrity, and oxidative stress in bovine preantral follicles cultured in vitro with ascorbic acid

Zygote ◽

10.1017/s0967199421000903 ◽

2021 ◽

pp. 1-7

Author(s):

Larissa Zamparone Bergamo ◽

Denis Vinicius Bonato ◽

Camila Bizarro-Silva ◽

Francieli Gesleine Capote Bonato ◽

Tamires Korchovei Sanches ◽

...

Keyword(s):

Oxidative Stress ◽

Ascorbic Acid ◽

Culture Medium ◽

Bos Taurus ◽

Follicular Development ◽

Ovarian Follicles ◽

Bos Taurus Indicus ◽

Bonferroni Test ◽

And Oxidative Stress

Summary The aim of this study was to evaluate the follicular development, morphological integrity, and oxidative stress of preantral ovarian follicles from Bos taurus indicus females grown in vitro with ascorbic acid. Ovaries (n = 20) from Bos taurus indicus females were collected, fragmented, and were cultured in vitro for 6 or 12 days in minimum essential medium (MEM), or MEM supplemented with 50 or 100 ng/ml ascorbic acid, with an extracellular matrix of agarose gel, in an incubator at 38.5°C; every 2 days, 100% of the culture medium was replaced. The data were analyzed using the chi-squared test and/or Fisher’s exact test. In the event of a significant effect, the proportions were compared using a 2 × 2 proportion test. The oxidative stress analysis data were submitted to analysis of variance followed by the Bonferroni test. Values were considered significant when P ≤ 0.05. The addition of 100 ng/ml of ascorbic acid to the in vitro culture medium of preantral ovarian follicles from bovine females promoted follicular development, was efficient in maintaining morphological integrity, as well as the stability of reactive oxygen species, after 6 days of in vitro culture.

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Tissue-specific effect of ascorbic acid supplementation on the expression of cytochrome P450 2E1 and oxidative stress in streptozotocin-induced diabetic rats

Toxicology Letters ◽

10.1016/j.toxlet.2006.05.009 ◽

2006 ◽

Vol 166 (1) ◽

pp. 27-36 ◽

Cited By ~ 25

Author(s):

Taeho Ahn ◽

Chul-Ho Yun ◽

Doo-Byoung Oh

Keyword(s):

Oxidative Stress ◽

Ascorbic Acid ◽

Cytochrome P450 ◽

Specific Effect ◽

Diabetic Rats ◽

Cytochrome P450 2E1 ◽

Tissue Specific ◽

Acid Supplementation ◽

And Oxidative Stress ◽

Ascorbic Acid Supplementation

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Protective Effect of Coconut Oil Meal Phenolic Antioxidants against Macromolecular Damage: In Vitro and In Vivo Study

Journal of Chemistry ◽

10.1155/2020/3503165 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

A. N. Karunasiri ◽

C. M. Senanayake ◽

H. Hapugaswatta ◽

N. Jayathilaka ◽

K. N. Seneviratne

Keyword(s):

Oxidative Stress ◽

Protective Effect ◽

Stress Responses ◽

Coconut Oil ◽

Oral Feeding ◽

Phenolic Antioxidants ◽

Significant Difference ◽

Macromolecular Damage

Coconut oil meal, a cheap by-product of coconut oil production, is a rich source of phenolic antioxidants. Many age-related diseases are caused by reactive oxygen species- (ROS-) induced damage to macromolecules such as lipids, proteins, and DNA. In the present study, the protective effect of the phenolic extract of coconut oil meal (CMPE) against macromolecular oxidative damage was evaluated using in vitro and in vivo models. Sunflower oil, bovine serum albumin (BSA), and plasmid DNA were used in the in vitro study, and thiobarbituric acid reactive substances (TBARS), protein carbonyl, and nicked DNA were evaluated as oxidation products. The inhibitory effect of CMPE against H2O2-induced macromolecular damage was evaluated using cultured HEp-2 cells. The results indicate that CMPE inhibits macromolecular damage both in vitro and in vivo. In addition, CMPE regulates redox status of HEp-2 cells under oxidative stress conditions by maintaining higher reduced glutathione levels. There was no significant difference in the expression of glutathione peroxidase in stressed and unstressed cells suggesting that CMPE regulates the cellular oxidative stress responses without affecting the expression of oxidative stress response genes. Oral feeding of Wistar rats with CMPE improves the serum and plasma antioxidant status without causing any toxic effects.

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Metallothionein-I/II Knockout Mice Aggravate Mitochondrial Superoxide Production and Peroxiredoxin 3 Expression in Thyroid after Excessive Iodide Exposure

Oxidative Medicine and Cellular Longevity ◽

10.1155/2015/267027 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 5

Author(s):

Na Zhang ◽

Lingyan Wang ◽

Qi Duan ◽

Laixiang Lin ◽

Mohamed Ahmed ◽

...

Keyword(s):

Oxidative Stress ◽

Urinary Iodine ◽

In Vitro Study ◽

Superoxide Production ◽

Mitochondrial Oxidative Stress ◽

Mitochondrial Superoxide ◽

Iodine Level ◽

Significant Difference

Purpose. We aim to figure out the effect of metallothioneins on iodide excess induced oxidative stress in the thyroid.Methods. Eight-week-old MT-I/II knockout (MT-I/II KO) mice and background-matched wild-type (WT) mice were used. Mitochondrial superoxide production and peroxiredoxin (Prx) 3 expression were measured.Results. In in vitro study, more significant increases in mitochondrial superoxide production and Prx 3 expression were detected in the MT-I/II KO groups. In in vivo study, significantly higher concentrations of urinary iodine level were detected in MT-I/II KO mice in 100 HI group. Compared to the NI group, there was no significant difference existing in serum thyroid hormones level in either groups (P>0.05), while the mitochondrial superoxide production was significantly increased in 100 HI groups with significantly increased LDH activity and decreased relative cell viability. Compared to WT mice, more significant changes were detected in MT-I/II KO mice in 100 HI groups. No significant differences were detected between the NI group and 10 HI group in both the MT-I/II KO and WT mice groups (P>0.05).Conclusions. Iodide excess in a thyroid without MT I/II protection may result in strong mitochondrial oxidative stress, which further leads to the damage of thyrocytes.

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Effects of menopause and tibolone on antioxidants in postmenopausal women

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1258/0004563053857941 ◽

2005 ◽

Vol 42 (3) ◽

pp. 220-223 ◽

Cited By ~ 21

Author(s):

Perin Vural ◽

Cemil Akgül ◽

Mukaddes Canbaz

Keyword(s):

Oxidative Stress ◽

Ascorbic Acid ◽

Postmenopausal Women ◽

Plasma Concentrations ◽

Lipid Peroxide ◽

Lipid Peroxides ◽

Thiol Groups ◽

Plasma Ascorbic Acid ◽

Total Thiol ◽

Significant Difference

Background: Oxidative stress has been implicated in the pathogenesis of ageing and menopause, and can arise through the increased production of lipid peroxides and/or a deficiency of antioxidant defence. Aim: To investigate the effects of the menopause and tibolone treatment (2.5 mg/day for six months) on plasma antioxidants and lipid peroxidation. Methods: Plasma concentrations of ascorbic acid, α-tocopherol, total thiol groups, caeruloplasmin, erythrocyte glutathione (GSH) and malondialdehyde (MDA) were measured in 24 postmenopausal and 24 premenopausal healthy women. Results: Data analysis indicates a significant decrease in plasma ascorbic acid, α-tocopherol, total thiol groups, caeruloplasmin, erythrocyte GSH and a significant increase in lipid peroxides (expressed as MDA concentrations) in postmenopausal women. There was no significant difference between control and study groups in the mean plasma caeruloplasmin concentrations. It was found that there is a significant increase in α-tocopherol and significant decrease in lipid peroxide concentrations in postmenopausal after tibolone treatment. Conclusions: The menopause is associated with an increase in oxidative stress and a decrease of some antioxidants, such as ascorbic acid, α-tocopherol, total thiols and erythrocyte GSH. Tibolone treatment leads to a decrease in concentrations of plasma lipid peroxide, probably by stimulating direct and indirect mechanisms of tocopherol regeneration and increasing plasma concentrations of vitamin E. However, due to the relatively small numbers involved this study can be regarded as a pilot. Further studies performed on a larger scale are necessary to establish the exact mechanisms of tibolone in inhibiting oxidative stress in postmenopausal women.

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