Research Article

Effect of Cysteine Supplementation in Maturation Medium on Bovine Embryos Development

Chalothon Amporn1, Somchit Guntaprom1*, Sarawut Duongmawong1, Wilasinee Srisanyong1, Sirikanda Thanasuwan1, Phalita Koonnadilokpot1, Dechawut Bunyaluk2, Juggrid Jugsumrit3, Jakrit Yaeram4, Chompunut Lumsangkul5

1Department of Veterinary Technology, Kalasin University, Kalasin 46000, Thailand; 2Department of Science and Mathematics, Kalasin University, Kalasin 46000, Thailand; 3Surin provincial livestock office, Surin 32000, Thailand; 4Rajamangala University of Technology Isan, Nakhon Ratchasima Campus, Nakhon Ratchasima 30000, Thailand; 5Department of Animal and Aquatic Sciences, Chiangmai University, Chiangmai 50200, Thailand.

Received | August 16, 2022; Accepted | September 19, 2022; Published | February 02, 2023

*Correspondence | Somchit Guntaprom, Department of Veterinary Technology, Kalasin University, Kalasin 46000, Thailand; Email: [email protected]

Citation | Amporn C, Guntaprom S, Duongmawong S, Srisanyong W, Thanasuwan S, Koonnadilokpot P, Bunyaluk D, Jugsumrit J, Yaeram J, Lumsangkul C (2023). Effect of cysteine supplementation in maturation mediumon bovine embryos development. Adv. Anim. Vet. Sci. 11(2):350-354.

DOI | https://dx.doi.org/10.17582/journal.aavs/2023/11.2.350.354

ISSN (Online) | 2307-8316

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Over the past 10 years, the blastocyst from in vitro embryo production (IVEP) are used for the genetic improvement of herds (Stroebech et al., 2015) and also used to generate a nuclear transfer of embryonic stem cells (NT-ESCs) for stem-cell research (Wolf et al., 2017). Moreover, this method, based on the production of safe and healthy food, conservation genetics in endangered species (Folch et al., 2009; Lopez et al., 2020), and an animal model for studying human disease (Sui et al., 2018). However, the efficiency of IVEP obtained through IVM (in vitro maturation), IVF (in vitro fertilization) or IVC (in vitro culture) remains limited (Luciano et al., 2018). IVEP has developed over two decades. There are many factors of procedure success. One of the problem for embryonic development is oxidative stress (OS) since the quality of the oocytes may decrease as a consequence as meiotic arrest is induced in the oocyte (Khazaei and Aghaz, 2017). The IVEP conditions usually increase reactive oxygen species (ROS). ROS production is increased during IVM because oxygen is employed by the oocytes for energy production via the process of mitochondrial oxidative phosphorylation. OS results from an imbalance between ROS and antioxidants. Thus, the enzymatic antioxidants comprise catalase (CAT) and superoxide dismutase stress (SOD) while the non-enzymatic antioxidants comprising glutathione (GSH), cysteamine, vitamin C, and vitamin E have been used to supplement the culture medium to reduce the intracellular level of ROS (Caamano et al., 1998; He et al., 2017). GSH functions protection against oxidative damage, raising embryo development competence (Sandal et al., 2018). Cysteine and cysteamine are precursors of GSH. There are many reports indicating that when antioxidants are introduced to the in vitro maturation of bovine oocyte to supplement the culture media, this caused the high intracellular GSH level and decreased ROS production, raising embryo quality and development (Lee and Fukui, 1996; Hosseini et al., 2009; Hansen and Harris, 2015). Moreover, it was demonstrated that addition of cysteine to IVM medium, increased the maturation rate of bovine oocytes (Rahim et al., 2011). Nabenishi et al. (2012) reported that the supplementation of cysteine to IVM mediumcaused increases of oocytes GSH content and inhibiting the production of oocyte ROS. Therefore, cysteine can be used as a tools to enhance the efficiency of bovine embryo in vitro system. This research sought to examine how the development of IVP of a bovine embryo is influenced by cysteine.

MATERIALS AND METHODS

Oocyte retrieval

The ovaries required for the study were gathered at the abattoir within 3 hours of the animal slaughter and placed in a solution of 0.9% NaCl at a temperature of 37°C for transportation to the laboratory. The recovery of bovine oocytes was achieved by slicing open the ovaries in Dulbecco’s Phosphate Buffered Saline (DPBS) mixed with 2% fetal bovine serum (FBS), Pen/Strep solution (All supplied by Thermo Fisher Scientific Inc., Grand Island, USA), with streptomycin (100 μg/ml) and penicillin (100 U/ml).

In vitro maturation (IVM)

The IVM medium comprised TCM 199 medium and Earle’s salts, L-glutamine, and NaHCO3 (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) with the addition of 10% (v/v) FBS and Pen/Strep solution (Thermo Fisher Scientific Inc., Grand Island, USA), streptomycin (100 μg/ml), penicillin (100 U/ml), 2 IU/ml of hCG (Chorulon, Intervet, Netherlands), 25 μg/ml of FSH (Folltropin® - V, Bioniche Animal Health (A/Asia) Pty. Ltd., Australia), 0.2 mM sodium pyruvate, and 1 μg/ml 17-β-Estradiol (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Cumulus-oocyte complexes (COCs) were cultured in the medium for IVM and the designated levels of cysteine (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) were 0 (Control), 100 µM and 200 μM. COCs which had at least three cumulus cell layers were then rinsed thrice in IVM medium, cultured in 100 μl drops (12-15 COCs/drop) under mineral oil (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for 22 h under a humidified atmosphere of 5% carbon dioxide at a temperature of 38.5°C.

In vitro fertilization (IVF)

Sperm preparation for IVF, frozen semen was thawed and prepared for sperm capacitation. 100 ul of semen added into 1 ml sperm-TALP medium which comprised 100 mM NaCl, 25 mM NaHCO3, 3.1 mM KCl, 0.29 mM NaH2PO4, 21.60 mM Lactic Acid, 10 mM Hepes, 1 ul/ml Phenol red, 0.40 mM MgCl2.6H2O, 2.1 mM CaCl2.2H2O, 6 mg/ml BSA (All supplied by Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), 0.25 mM sodium pyruvate. The motile sperm were selected using the swim-up technique. The sperms were incubated with sperm-TALP medium in a 1.5 ml Eppendorf tube for 1 h at 38.5 °C in 5% CO2 for swim-up. Then the top 0.8 ml from each tube, pooled sperm inside a 15 ml plastic centrifuge tube prior to centrifugation for 5 min at 1,500 rpm twice. The final sperm pellet concentration in fertilization microdroplets was 2 X 106 sperm/ml. Once the oocytes had been cultured for 22 h in IVM media, 12-15 expanded bovine cumulus-oocyte complexes were washed three times then transfer into a fertilization drop (500 ul) of IVF-TALP containing 114 mM NaCl, 3.2 mM KCl, 25 mM NaHCO3, 0.34 mM NaH2PO4, 10 mM Lactic Acid, 1 ul/ml Phenol red, 2 mM CaCl2.2H2O, 0.5 mM MgCl2.6H2O, 6 mg EFAF BSA (All supplied by Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), and 0.25 mM sodium pyruvate, 3 IU/ml Heparin (Leo Pharmaceutical Products, Ballerup, Denmark), 100 U/ml penicillin, 100 μg/ml streptomycin. Incubation of the sperm and oocytes was subsequently conducted for a period of 18-20 h at a temperature of 38.5°C under an atmosphere comprising 5% carbon dioxide.

In vitro culture (IVC)

After 18-20 h of incubation, fertilized oocytes were removed cumulus cells by repeated pipetting of oocytes. Then, washed 3 times and cultured in KSOM medium containing 2.5 mM KCl, 95.0 mM NaCl, 0.35 mM KH2PO4, 0.20 mM MgSO47H2O, 0.20 mM glucose, 10.00 mM Lactic Acid, 0.20 mM sodium pyruvate, 25.0 mM NaHCO3, 1.71 mM CaCl22H20, 1.0 mM L-glutamine, 0.01 mM EDTA, 1.0 mg/ml BSA, 100 μg/ml streptomycin, 100 U/ml penicillin, 5 ul/ml MEM non-essential solution 100x, 10 ul/ml BME essential solution 50x, and 10% FBS (All supplied by Sigma Aldrich, Merck KGaA, Darmstadt, Germany). Embryos were placed in 50 ul drop (5 embryos/drop) in a Petri dish and covered with mineral oil and cultured in an incubator under humid conditions in a 5% carbon dioxide atmosphere at a temperature of 38.5°C. In addition, the embryos underwent culturing and the medium was replaced at each 48 h until 8-cells then cultured in KSOM medium containing 1.5 mM glucose. At 7-9 days after culturing, the blastocysts rate was evaluated.

Statistical analysis

Evaluation of the effects of cysteine in maturation medium was carried out using Duncan’s New Multiple Range Test (DMRT), and analysis of variance (ANOVA) was performed using SAS software. The statistical significance level of 0.05 was employed for all analyses.

RESULTS AND DISCUSSION

The examination of different levels of cysteine showed that percentage of matured oocytes, fertilized oocytes, 8-16 cells developments, blastocyst in 100 µM cysteine treatment were higher than control group. Between treatments, percentage of matured oocytes, fertilized oocytes, 8-16 cells developments, blastocyst in 200 µM cysteine treatment were lower than 100 µM cysteine treatment at a significance level of 0.05, but difference between the 200 µM cysteine treatment and the control group was not found to be significant (Table 1). Figure 1 presents the first polar body, the zygote with two polar bodies, 8-cells, 16-cells, early blastocyst, and blastocyst observed after IVC.

IVEP of bovine embryos has become a technology which has been used for genomic selection and systems biology (Stroebech et al., 2015), but this technology is still being improved. IVM of oocytes is the first step that develop to the blastocyst stage. According to the reports, multiple factors contribute to the maturation of oocytes such as media compositions; growth factors, estradiol, gonadotropin, and antioxidants (Vahedi et al., 2009; Balasubramanian and Rho, 2007). The major factor affecting IVM maybe OS. OS arises whenever an imbalance between oxidants and antioxidants. Under normal conditions, ROS is kept at a normal balance by a defense system consisting of enzymatic

 

Table 1: Maturation of bovine oocytes and embryonic development following various dosages of cysteine supplementation during in vitro maturation

Group	Number of oocytes examined	Matured oocytes (% mean ± S.E)	Fertilized oocytes (% mean ± S.E)	8-16 cells (% mean ± S.E)	Blastocyst (% mean ± S.E)
Control	100	48 (48.00±0.31)b	18 (37.50±1.92)b	9 (18.75±2.81)b	5 (10.42±1.00)b
100 (uM)	100	73 (73.00±3.33)a	40 (54.79±1.10)a	22 (30.14±2.78)a	13 (17.81±0.69)a
200 (uM)	100	52 (52.00±1.00)b	20 (38.46±0.75)b	10 (19.23±0.38)b	5 (9.62±1.68)b

 

a, b the values bearing different superscript with in a column differed significantly (p<0.05).

 

and non-enzymatic antioxidants. But the in vitro system may lack defenses (Combelles, 2009). Therefore, there are many reports that the addition of antioxidants in the medium can lead to the improved development of bovine embryos when produced in vitro. In the present study, the effect of cysteine on bovine IVP. The results of the maturation rate of oocytes cultured with 100 µMand 200 µM cysteine were 73% and 52%, respectively. These results are similar to a previous study that culture with 100 µMand 500 µM cysteine. The matured oocytes were 42.22% and 53.33%, respectively (Rahim et al., 2011). Lee and Fukui (1996) demonstrated that glutamine, glycine, and alanine enhanced bovine embryonic development and blastocyst cell number. Moreover, in our study, the addition of 100 µM cysteine to the culture medium resulted in significant improvements to the rate of maturation as well as the development of the embryo and the blastocyst cell numbers, at a statistical significance level of 0.05. As shown in Table 1, fertilization rates were observed in 54.79±1.10% by 72 h after IVF cells, 8–16 cells were checked in 30.14±2.78% by 96 h after IVF, and 17.81±0.69% developed to blastocyst stage after 216 h of culture. But the addition of 200 µM cysteine to the culture medium did not improve fertilization rate and embryonic development. According to many reports and the results in this study showed that amino acids may play an important role with increased GSH synthesis then reduce the intracellular level of ROS. It may affect oocyte cytoplasmic maturation and embryonic development (Gordan, 2003; Gasparrini et al., 2006). However, the mechanism of amino acids affecting embryonic development requires more study to gain a better understanding.

CONCLUSIONS AND RECOMMENDATIONS

Our results showed that where the IVM media is augmented by the introduction of cysteine, this could result in a percentage increase in matured oocytes, fertilized oocytes, 8–16 cell development and blastocyst stage. These results support the evidence that ROS production is increased during in vitro mammalian embryo production. Moreover, the addition of 100 µM of cysteine supplementation in IVM media was shown to deliver efficiency improvements in the in vitro production of bovine embryos.

ACKNOWLEDGEMENTS

The authors wish to thank Kalasin University, whose provision of the facilities used in this research allowed the study to be completed successfully.

NOVELTY STATEMENT

Our work demonstrated that when antioxidants are introduced to the in vitro maturation of the bovine oocytes to supplement the culture medium, this improved embryonic development. Thus, cysteine has great potential for use in prevention of oxidative stress during in vitro culture.

AUTHOR’S CONTRIBUTION

All the authors contributed to the manuscript. CA, SG, JJ, SD and JY designed research methodology, data collection, statistical analysis, manuscript writing. WS, ST and PK, DB and CL all approved the final document.

Ethical consideration

Approval for the techniques employed during the experiments in this study was granted by the Committee on the Ethics of Animal Experiments of Kalasin University (Approval Number: KSU-AE-005/2022).

Conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Balasubramanian S, Rho GJ (2007). Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos. Anim. Reprod. Sci., 98(3): 282-292. https://doi.org/10.1016/j.anireprosci.2006.03.011

Caamano JN, Ryoo ZY, Youngs CR (1998). Promotion of development of bovine embryos produced in vitro by addition of cysteine and beta-mercaptoethanol to a chemically defined culture system. J. Dairy Sci. 81: 369-374. https://doi.org/10.3168/jds.S0022-0302(98)75586-9

Combelles C (2009). Could oxidative stress influence the in-vitro maturation of oocytes? Reprod. Biomed. Online, 18(6): 864-880. https://doi.org/10.1016/S1472-6483(10)60038-7

Folch J, Cocero MJ, Chesne P, Alabart, JL, Dominguez V, Cognie Y, Roche A, Fernandez-Arias A, Marti JI, Sanchez P, Echegoyen E, Beckers JF, Sanchez Bonastre A, Vignon X (2009). First birth of an animal from an extinct subspecies (Capra pyrenaica pyrenaica) by cloning. Theriogenology, 71: 1026-1034. https://doi.org/10.1016/j.theriogenology.2008.11.005

Gasparrini B, Boccia L, Marchandise J, Di Palo R, George F (2006). Enrichment of in vitro maturation medi µM for buffalo (Bubalus bubalis) oocytes with thiol compounds: Effects of cysteine on glutathione synthesis and embryo development. Theriogenology, 65(2): 275-287. https://doi.org/10.1016/j.theriogenology.2005.05.036

Gordon I (2003). Laboratory production of cattle embryos: Maturing the oocyte, 2th edition. CABI publishing, 44 Brattle Street, Cambridge, MA 02138, USA. https://doi.org/10.1079/9780851996660.0112

Hansen JM, Harris C (2015). Glutathione during embryonic development. Biochim. Biophys. Acta, https://doi.org/10.1016/j.bbagen.2014.12.001

He L, He T, Farrar S, Ji L, Liu T, Ma X (2017). Antioxidants maintain cellular redox homeostasis by elimination of reaction oxygen species. Cell Physiol. Biochem., 44: 532-533. https://doi.org/10.1159/000485089

Hosseini SM, Forouzanfar M, Hajian M, Asgari V, Abedi P, Hosseini L, Ostadhosseini S, Moulavi F, Safahani Langrroodi M, Sadeghi H, Bahramian H, Eghbalsaied Sh, Nasr-Esfahani MH (2009). Antioxidant supplementation of culture mediumduring embryo development and/or after vitrification-warming; which is the most important? J. Assist. Reprod. Genet., 26: 355-364. https://doi.org/10.1007/s10815-009-9317-7

Khazaei M and Aghaz F (2017). Reactive oxygen species generation and use of antioxidants during in vitro maturation of oocytes. Int. J. Fertil. Steril., 11(2): 63-70.

Lee ES, Fukui Y (1996). Synergistic effect of alanine and glycine on bovine embryos cultured in a chemically defined medium and amino acid uptake by in vitro-produced bovine morulae and blastocysts. Biol. Reprod., 55: 1383-1389. https://doi.org/10.1095/biolreprod55.6.1383

Lopes JS, Alcazar-Trivino E, Soriano-Ubeda C, Hamdi M, Canovas S, Rizos D, Coy P (2020). Reproductive outcomes and endocrine profile in artificially inseminated versus embryo transferred cows. Animals, 10: 1359. https://doi.org/10.3390/ani10081359

Luciano AM, Franciosi F, Barros RG, Dieci C, and Lodde V (2018). The variable success of in vitro maturation: Can we do better? Anim. Reprod., 15: 727-736. https://doi.org/10.21451/1984-3143-AR2018-0021

Nabenishi H, Ohta H, Nishimoto T, Morita T, Ashizawa K and Tsuzuki Y (2012). The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress. Zygote, 20(3): 249-259. https://doi.org/10.1017/S0967199411000220

Rahim B, Jalal S. and Yosef N (2011). Effect of cysteine supplementation on in vitro maturation of bovine oocyte. Afr. J. Biotechnol., 10(70): 15830-15833. https://doi.org/10.5897/AJB11.1686

Sandal AI, Ozdas OB and Baran A (2018). In vitro maturation of bovine oocytes: Beneficial effects of cysteamine. J. Dairy Vet. Anim. Res., 7(2): 64-65. https://doi.org/10.15406/jdvar.2018.07.00191

Stroebech L, Mazzoni G, Pedersen HS, Freude KK, Kadarmideen HN, Callesen H and Hyttel P (2015). In vitro production of bovine embryos: Revisiting oocyte development and application of systems biology. Anim. Reprod., 12(3): 465-472.

Sui L, Danzl N, Campbell SR, Viola R, Williams D, Xing Y, Wang Y, Phillips N, Poffenberger G, Johannesson B, Oberholzer J, Powers AC, Leibel RL, Chen X, Sykes M and Egli D (2018). β-cell replacement in mice using human type diabetes nuclear transfer embryonic stem cells. Diabetes, 67: 26–35. https://doi.org/10.2337/db17-0120

Vahedi V, Zeinoaldini S, Kohram H, Farahavar A (2009). Retinoic acid effects on nuclear maturation of bovine oocytes in vitro. Afr. J. Biotechnol., 8(16): 3974-3978.

Wolf DP, Morey R, Kang E, Ma H, Hayama T, Laurent LC, Mitalipov S (2017). Concise review: Embryonic stem cells derived by somatic cell nuclear transfer: A house in the race? Stem Cells, 35: 26-34. https://doi.org/10.1002/stem.2496