Asian Pacific Journal of Reproduction

: 2017  |  Volume : 6  |  Issue : 2  |  Page : 85--88

Effect of using ascorbic acid and cysteamine supplementation on in-vitro development of buffalo embryos

Al-shimaa Al-H H l-Naby1, KarimaGh M Mahmoud2, Gamal AM Sosa1, Mahmoud EA Abouel-Roos1, Youssef F Ahmed2,  
1 Department of Theriogenology, Faculty of Veterinary Medicine, Benha University, Egypt
2 Department of Animal Reproduction and A.I., National Research Center, Dokki, Giza, Egypt

Correspondence Address:
KarimaGh M Mahmoud
Department of Animal Reproduction and A.I., National Research Center, Dokki, Giza, Postal code 12311, Affiliation I.D. 60014618


Objective: To improve in vitro embryo production in buffalo by supplementation of L-ascorbic acid during maturation and development (experiment 1) and combination with another antioxidant as cysteamine (experiment 2). Methods: Two experiments were performed, the first one aimed to evaluate the different concentrations (0, 25, 50, 100 μM) of L-ascorbic acid on embryo developmental rate of buffalo oocytes. The L-ascorbic acid was added to the maturation and culture media. In the second experiment, oocytes were cultured in media with two type of antioxidant (ascorbic acid + cysteamine) or ascorbic acid only. Results: There was a significant increase in cleavage rate at 25, 50 μM than 100 μM and control group. But, the blastocyst rate was higher at 50 μM ascorbic acid than other concentrations (0, 25, 100 μM). Supplementation of ascorbic acid and cysteamine to maturation and cultured media improved embryo development than ascorbic acid alone. Conclusions: Using of 50 μM L-ascorbic acid during in vitro maturation and development improve the developmental competence of buffalo oocytes, this effect was increase with the presence of cysteamine.

How to cite this article:
l-Naby AsA, Mahmoud KM, Sosa GA, Abouel-Roos ME, Ahmed YF. Effect of using ascorbic acid and cysteamine supplementation on in-vitro development of buffalo embryos.Asian Pac J Reprod 2017;6:85-88

How to cite this URL:
l-Naby AsA, Mahmoud KM, Sosa GA, Abouel-Roos ME, Ahmed YF. Effect of using ascorbic acid and cysteamine supplementation on in-vitro development of buffalo embryos. Asian Pac J Reprod [serial online] 2017 [cited 2023 Feb 4 ];6:85-88
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 1. Introduction

Buffalo oocytes and embryos had abundance lipid content[1]. Therefore, they were likely to be more sensitive to oxidative damages lead to low embryo livability and production[2]. In efforts to curtail these damaging effects, the extrinsic protection of embryo mainly involved hypotaurine, taurine and ascorbic acid antioxidants. While embryo intrinsic protection involved enzymes as superoxide dismutase, glutathione peroxidase and gamma glutamyl-cysteine synthetase[3], and other extracellular small molecules scavengers like vitamins A, C, E and also sulphur compounds which including reduced glutathione, hypotaurine, taurine and cysteamine[4].

In the oocyte, a critical intracellular concentration of ascorbic acid would be necessary for normal cytoplasmic maturation[5] and embryo developmental competence[6],[7]. Ascorbic acid deficiency results in infertility in the female which associated with ovarian atrophy, follicular atresia, and the premature resumption of meiosis[8]. Vitamins C (L-ascorbic acid) and E ( α-tocopherol and derivatives) were well-known antioxidants and were recorded to be the primary free radical scavengers in mammalian cell membranes[9]. So, the addition of ascorbic acid to the maturation medium maintained alpha-tocopherol levels during IVM, possibly because of ongoing functionality of vitamin E naturally present in the COC membrane[10]. A complex relationship existed between vitamin E and vitamin C. In vitro, vitamin C regenerated vitamin E molecules by tocopherol from tocopheroxyl radicals[11], that had undergone free radical attack and might also spare vitamin E from oxidation by reacting directly with free radicals[12].

Cysteamine might reduce cysteine to cystine that increased oocyte glutathione synthesis[13]. Cysteamine supplementation was recorded to improve nuclear maturation rates in buffalo[14] by enhancing synthesis of GSH[15] and improve male pronucleus formation[16]. Combination of ascorbic acid with vitamin E was previously reported to improve in vitro culture system, but no studies were reported on combination of ascorbic acid with cysteamine. So, this work aimed to improve in vitro embryo production in buffalo by supplementation of media with ascorbic acid with combination with cysteamine.

 2. Materials and methods

2.1. Experimental designs

Experiment 1: Effect of L-ascorbic acid concentrations on buffalo embryo development. Oocytes were cultured in TCM-199 media with different concentration (0, 25, 50, 100 μM) of ascorbic acid in CO2 incubator at 38 °C during maturation and development.

Experiment 2: Evaluation the combination effect of L-ascorbic acid with cysteamine (double antioxidant substance) on cleavage and blastocyst rate. Group of oocytes were matured and cultured in media supplemented with combination of 50 μM L-ascorbic acid and 50 μM cysteamine. The second group of oocytes were matured, cultured in media with L-ascorbic acid but without cysteamine.

2.2. Chemicals

Tissue culture medium (TCM-199) and fetal calf serum were purchased from Gibico company. Cysteamine (M-6500, Sigma), Ascorbic acid (A-5960, Sigma) and chemicals used in vitro fertilization were from Sigma Company.

2.3.Oocytes recovery and maturation

Ovaries were collected from slaughtered buffalo, then they were aspirated to a syringe of PBS with 0.3% bovine serum albumin and 100 μg/mL streptomycin sulfate and 100 IU/mL penicillin. Oocyte maturation was carried out according to Mahmoud[17]. The oocytes were cultured in maturation medium consisted ofTCM-199, 10% calf serum, L-ascorbic acid, cysteamine and gentamycin (50 μg/mL). Then oocytes were incubated at 38.5 °C, with 5% CO2 in air, and 95% humidity for 24 h.

2.4. In vitro fertilization and culture

Fertilization was performed according to Niwa and Ohgoda[18]. Two straws of frozen semen were thawed in a water bath at 35-37 °C for 1 min. The spermatozoa were washed at 800 g for 10 min in BO medium[19] free from BSA containing10 μg/mL heparin and 2.5 mM caffeine by centrifugation. The pellets were diluted with BO medium with 20 mg/mL bovine serum albumin to reach the concentration of spermatozoa to 12.5 × 106 sperm/mL. Oocytes were washed in BO containing with 10 mg/mL BSA before introducing into sperm suspension. The spermatozoa and oocytes were co-cultured for 4-5 h in 5% CO2, 38.5 °C and 95% humidity. Then, the oocytes were washed several times in TCM-199 to remove attached spermatozoa and incubated in TCM-199 with 10% serum. Cleavage was assessed after 3 d of culture and the morula and blastocyst stages was determined on day 5 and 7, respectively.

2.5. Statistical analysis

Data in the first experiment were statistically analyzed by ANOVA using SPSS for Windows version 16.0. Means were compared by Duncan’s Multiple Range Test. The t-test was performed to compare between means in the second experiment. Differences were considered to be significant at P<0.05.

 3. Results

3.1. Effect of L-ascorbic acid concentrations on buffalo embryo development

The mean proportion of buffalo oocytes matured, cultured in media with different concentration (0, 25, 50, 100 μM) of ascorbic acid was given in [Table 1]. There was a significant (P<0.01) increase in cleavage rate of 25 μM and 50 μM of ascorbic acid than control and 100 μM groups. While the embryo cleavage rate was not significantly differ between 25 μM and 50 μM of ascorbic acid groups. The mean number of cleavage rate was higher (P<0.01) in 50 μM and (P< 0.05) in 25 μM than 100 μM of ascorbic acid supplementation group.{Table 1}

The blastocyst rate after exposure to different concentrations (0, 25, 50, 100 μM) of ascorbic acid was evaluated in [Table 1]. The percentage of blastocyst rate was significantly higher in 25 μM (P<0.01), 50 μM (P<0.001), 100 μM (P<0.05) ascorbic acid groups than control. The highest blastocyst yield was observed at 50 μM ascorbic acid treatment. The blastocyst rate was nearly similar in 25 μM and 100 μM ascorbic acid supplementation.

3.2. The effect of combination between ascorbic acid and cysteamine on developmental competence of buffalo oocytes

Data regarding the combination effect between ascorbic acid (50 μM) and cysteamine (50 μM) on cleavage and blastocyst rate was illustrated as mean±SE. The total number of embryo for ascorbic acid with cysteamine was 80 while for the ascorbic acid without cysteamine was 92. There was significant (P<0.05) increase in embryo cleavage rate of media supplemented with ascorbic acid (52, (65.20%±0.98%)] and cysteamine than media supplemented with ascorbic acid only (53, (56.81%±2.19%)). Also, blastocyst rate was higher significantly (P<0.01) in ascorbic acid [10, (12.31%±0.64%)] and cysteamine combination than in ascorbic acid only [7, (7.20%±0.75%)]. So the combination between two antioxidant substances gave better embryonic developmental rate than single supplementation.

 4. Discussion

In our study, the percentage of embryo cleavage rate significantly increased at 25 μM and 50 μM of L-ascorbic acid than 100 μM and control groups. Moreover, blastocyst rate was highly significant at 50 μM than other concentrations 25 μM, 100 μM and control groups. It was shown that the optimum concentration of vitamin C was 50 μg/mL during IVM of porcine oocytes[20], this concentration not only improved blastocyst rates, total cell numbers but also, reduced apoptotic indices and the high concentration (100 μg/mL) of vitamin C induced high apoptotic index. Miclea[21] and Huang[22] mentioned that the treatment with 50 μg/mL vitamin C resulted in increasing pregnancy rate and enhancing the development somatic cell nuclear transfer embryos in pigs. Moreover, Castillo-Martin[23] mentioned that L-ascorbic acid supplementation in culture and/or vitrification media, enhanced survival rates of porcine blastocyst.

The effect of ascorbic acid and its concentration as a supplement in in-vitro maturation and in vitro culture mediums were recorded previously. The beneficial effect of vitamin C addition might be a scribed to an improved the culture condition and oocytes cytoplasmic maturation by reduction the intra cellular oxidative status for embryo development[7],[20]. Moreover, Eppig[24] and Wang[6] mentioned that the addition of ascorbic acid into the culture media prevented follicular apoptosis in rat and mouse follicles, and also improved the blastocyst production.

It was suggested that vitamin C at high concentration might act as a pro-oxidant that had deleteriously effect on oocyte maturation and cumulus cell viability[25]. In sheep, Natarajan[26] stated that there was a relation between the oxygen levels in culture environment and concentration of vitamin C to achieve optimal embryo development. It was observed that 50 μM L-ascorbic acid in embryo culture medium at 5% oxygen levels and 100 μM of ascorbic acid at 20% oxygen levels were resulted in a significant increasing the morula and blastocysts rates compared to control. While supplementation of embryo culture media with 400 μM, 500 μM L-ascorbic acid resulted in significant retardation for the development of ovine embryos.

In contrast to our results, Cordova[27] stated that the presence of L-ascorbic acid to in-vitro maturation of prepubertal bovine oocytes exerted no effect on maturation rates and significantly reduced the proportion of embryos developing to blastocyst stage. Also, the presence of alpha-tochopherol or ascorbic acid in the maturation medium of bovine oocytes failed to modify nuclear maturation rate and obtained blastocysts[28],[29]. The same results were observed in rat[8] and porcine[5] oocytes. Also, in dromedary she camel, Elsayed[30] stated that there was no significant difference in the maturation rate of in-vitro matured oocytes in medium containing vitamin C and the control group.

To our knowledge, this work was the first investigation to study the positive effect of combination between L-ascorbic acid and cysteamine added to maturation media. Our study was proved that benefits effect of using two antioxidant substances during oocytes maturation and culture. The cleavage rate was significantly (P<0.05) increased by using 50 μM ascorbic acid and 50 μM cysteamine combinations than using ascorbic acid only. Ocampo and Ocampo[31] mentioned that 50 μM cysteamine supported the maturation, fertilization, subsequent embryonic development in vitro.

Some studies reported the synergistic relationship between vitamin C and vitamin E in culture medium. It was expected that the combination of vitamin E and C improved embryonic developmental rate more than vitamin E alone[11],[32]. This was disagreement with Olson and Seidel Jr.[12] who stated that co-supplementation of α-tocopherol or L-ascorbic acid at 100 μM had deleterious effect on bovine culture system. It may be due to the higher concentration used in their work. In contrast, Saikhun[33] stated that culture medium supplemented with either α-tocopherol or L-ascorbic acid at a concentration of 250 μM increased the quality of IVF buffalo derived embryos, blastocyst rate and blastocyst cell number.

In conclusion, 50 μM L-ascorbic acid was the best concentration for in vitro produced buffalo embryos. Higher cleavage and blastocyst rate were achieved by using combination of ascorbic acid and cysteamine.

Conflict of interest statement

The authors declare that they have no conflict of interest.


1Thiyagarajan B, Valivittan K. Ameliorating effect of vitamin E on in vitro development of pre-implantation buffalo embryos. J Assist Reprod Genet 2009; 26: 217-225.
2Mahmoud KGhM, Nawito MF. Influence of serum source on buffalo in vitro embryonic development with reference to heat inactivation. Livestock Production Sci 2005; 96: 285-289.
3Ozturkler Y, Yildiz S, Gungor Ö, Pancarci M, Kacar C, AriUÇ. The effects of L-ergothioneine and L-ascorbic acid on the in vitro maturation and embryonic development of sheep oocytes. Kafkas Univ Vet Fak Derg 2010; 16(5): 757-763.
4Noda Y, Matsumoto H, Umaoka Y, Tatsumi K, Kishi J, Mori T. Involvement of superoxide radicals in the mouse two-cell block. Mol Reprod Dev 1991; 28: 356-360.
5Tatemoto H, Ootaki K, Shigeta K, Muto N. Enhancement of developmental competence after in vitro fertilization of porcine oocytes by treatment with ascorbic acid 2-O-alpha-glucoside during in vitro maturation. Biol Reprod 2001; 65: 1800-1806.
6Wang X, Falcone T, Attaran M, Goldberg JM, Agarwal A, Sharma R. Vitamin C and Vitamin E supplementation reduce oxidative stress–induced embryo toxicity and improve the blastocyst development rate. Fertil Steril 2001; 78: 1272-1277.
7Suzuki C, Yoshioka K, Sakatani M, Takahashi M. Glutamine and hypotaurineimprove intracellular oxidative status and in vitro development of porcine pre-implantation embryos. Zygote 2007; 15: 317-324.
8Takami M, Preston SL, Toyloy VA, Behrman HR. Antioxidants reversibly inhibit the spontaneous resumption of meiosis. Am J Physiol 1999; 276: 684-688.
9Niki E, Yamamoto Y, Takahashi M, Komuro E, Miyama Y. Inhibition of oxidation of bio membranes by tocopherol. Annal New York Acad Sci 1989; 570: 23-31.
10Halliwell B, Gutteridge JMC. The antioxidants of human extracellular fluids. Arch Biochem Biophys 1990; 280: 1-8.
11Machlin LJ, Gabriel E. Interactions of vitamin E with vitamin C, vitamin B12, and zinc. Ann N Y Acad Sci 1980; 355: 98-107.
12Olson SE, Seidel Jr GE. Culture of in vitro-produced bovine embryos with Vitamin Eimprove development in vitro and after transfer to recipients. Biol Reprod 2000; 62: 248-252.
13Issels RD, Nagele A, Eckert KG, Wilmanns W. Promotion of cysteine uptake and its utilization for glutathione biosynthesis induced by cysteamine and N-acetil-cysteine. Biochem Pharmacol 1988; 37: 881-888.
14Singhal S, Prasad S, Singh B, Prasad JK, Gupta HP. Effect of including growth factors and antioxidants inmaturation medium used for in vitro culture of buffalo oocytes recovered in vivo. Anim Reprod Sci 2008; 113(1-4): 44-50.
15Gasparrini B, Sayoud H, Neglia G, Matos DG, Donnay I, Zicarelli L. Glutathione synthesis during in vitro maturation of buffalo (Bubalus bubalis) oocytes: Effects of cysteamine on embryo development. Theriogenol 2003; 60: 943-952.
16Anandi T, Kumar D, Chauhan MS, Manik RS, Palta P. Cysteamine supplementation of in vitro maturation medium, in vitro culture medium or both media promotes invitro development of buffalo (Bubalus bubalis) embryos. Reprod Fertil Dev 2008; 20: 253-257.
17Mahmoud KGhM. Cytogenetic studies on in-vitro fertilization in buffaloes.Ph.D. [Thesis]. Cairo: Cairo University.
18Niwa K, Ohgoda O. Synergistic effect of caffeine and heparin on in-vitro fertilization of cattle oocytes matured in culture. Theriogenol 1988; 30: 733-741.
19Brackett BG, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biolof Reprod 1975; 12: 260-274.
20Kere M, Siriboon C, Lo NW, Nguyen NT, JuJ C. Ascorbic acid improves the developmental competence of porcine oocytes after parthenogenetic activation and somatic cell nuclear transplantation. J Reprod Develop 2013; 59(1): 78-84.
21Miclea I, Zhan M, Rusu A, Ghiuru F, Miclea V. The effect of several ascorbic acid concentrations on swine oocyte maturation and embryo culture. Universitatea de tiinţe Agricoleşi Medicină Veterinară Iaşi Lucrări Ştiinţifice 2008; 52: 35-40.
22Huang Y, Tang X, Xie W, Zhou Y, Li D, Zhou Y, et al. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos. Biochem Biophys Res Commun 2011; 411(2): 397-401.
23Castillo-Martín M, Yeste M, Soler A, Morató R, Bonet S. Addition of L-ascorbic acid to culture and vitrification media of IVF porcine blastocysts improves survival and reduces HSPA1A levels of vitrified embryos. Reprod Fertil Dev 2014; 27(7): 1115-1123.
24Eppig JJ, Hosoe M, O’Brien MJ, Pendola FM, Requena A, Watanabe S. Conditions that affect acquisition of developmental competence by mouse oocytes in vitro: FSH, insulin, glucose and ascorbic acid. Mol Cell Endocrinol 2000; 163: 109-116.
25Nadri B, Zeinoaldini S, Kohram H. Ascorbic acid effects on in vitro maturation of mouse oocyte with or without cumulus cell. Afri J Biotechnol 2009; 8: 5627-5631.
26Natarajan R, Bhawani SM, Munuswamy D. Effect of L-ascorbic acid supplementation at different gaseous environments on in vitro development of preimplantation sheep embryos to the blastocyst stage. Anim Reprod 2010; 7(1): 21-28.
27Cordova B, Morato R, Izquierdo D, Paramio T, Mogas T. Effect of the addition of insulin-transferrin-selenium and/or L-ascorbic acid to the in vitro maturation of prepubertal bovine oocytes on cytoplasmic maturation and embryo development. Theriogenol 2010; 74: 1341-1348.
28Blondin P, Coenen K, Sirard MA. The impact of reactive oxygen species on bovine sperm fertilizing ability and oocyte maturation. J Androl 1997; 18: 454-460.
29Dalvit G, Llanes SP, Descalzo A, Insani M, Beconi M, Cetica P. Effect of alpha-tocopherol and ascorbic acid on bovine oocyte in vitro maturation. Reprod Dom Anim 2005; 40: 93-97.
30Elsayed MA, Taha NA, Hammam AMM, Sawiress FAR. Effect of antioxidant supplementation on in vitro maturation of camelusdromedaries oocytes. Nat Sci 2015; 13(2): 17-21.
31Ocampo LC, Ocampo MB. Improved developmental competence of swamp buffalo oocytes matured in the presence of cysteamine. J Agric Technol 2015; 11(1): 31-40.
32Miclea I, Pacal N, Hettig A, Zhan M, Miclea V. Alpha-tocopherol and ascorbic acid combinations influence the maturation of sheep oocytes. Sci Papers: Anim Sci Biotechnol (Lucrari Stiintifice: Zootehnie si Biotehnologii) 2012; 45: 310-313.
33Saikhun K, Faisaikarm T, Ming Z, Lu KH, Kitiyanant Y. Tocopherol and L-ascorbic acidincrease the in vitro development of IVM/IVF swampbuffalo (Bubalus bubalis) embryos. Animal 2008; 2: 1486-1490.