# Effects of ascorbic acid and cysteine supplementation on preimplantation embryo development and oxidative stress-related gene expression in dromedary camels

## TL;DR

This study shows that adding ascorbic acid and cysteine to culture media improves embryo development in dromedary camels by reducing oxidative stress and boosting antioxidant gene activity.

## Contribution

The study introduces the novel use of ascorbic acid and cystein supplementation in dromedary camel embryo culture to enhance developmental outcomes.

## Key findings

- Supplementation increased cleavage and blastocyst formation rates compared to the control group.
- Gene expression of antioxidant and anti-apoptotic genes was upregulated in treated groups.
- Combination of ascorbic acid and cysteine showed beneficial effects on embryonic development.

## Abstract

Oxidative stress (OS) is a harmful threat during early preimplantation that compromises embryonic development. Ascorbic acid and cysteine were found to have significant contributions in various physiological processes, including embryonic development and mitigating various stressors, by acting as antioxidants and regulating gene expression. This study evaluated the mitigating impact of ascorbic acid and cysteine addition on OS during in vitro culture (IVC) and subsequent early embryonic developmental stages of the dromedary camel. The ovaries were sourced from a nearby slaughterhouse; only high-quality oocytes were used for in vitro embryo production (IVP). Produced zygotes were in vitro cultured with ascorbic acid, cysteine, or both under a high oxygen level (20%). This study included four experimental groups: an untreated group without antioxidant i.e., control (T1), the 2nd group supplemented with 150 µg/mL ascorbic acid (T2), the 3rd group supplemented with 100 µM cysteine (T3), and the 4th group (T4) enriched with a combination of both antioxidants (150 µg/mL ascorbic acid and 100 µM cysteine). Embryo development was monitored throughout different preimplantation stages. Real-time PCR was used to assess the relative abundance of various genes, including genes that are related to oxidative stress (catalase (CAT), superoxide dismutase (SOD), and thioredoxin (TXN)), apoptosis related genes (B-cell lymphoma 2 (BCL2), and tumor suppressor protein (p53)), and metabolic related gene (glucose transporter 1 (GLUT-1)).

The results indicated an increased cleavage rate (P < 0.05) in T2 (29.41%), T3 (32.77%), and T4 (27.16%) compared to T1 (14.05%). Moreover, the rate of blastocyst formation was increased (P < 0.05) in T2 (24.79%), T3 (21.43%), and T4 (18.52%), compared to T1 (2.89%). However, the rates of blocked embryos at the morula stage were 8.68%, 4.62%, 10.08%, and 8.23% in T1, T2, T3, and T4, respectively. The expression of genes regulating the antioxidant response (CAT, SOD1, and TXN), anti-apoptosis (BCL2), and metabolic activity (GLUT1) was upregulated in the treated groups.

To conclude, the findings of this study clearly illustrate increased cleavage and blastocyst rates with supplementation of ascorbic acid, cysteine, or a combination of both. Furthermore, gene expression data support the positive effects of antioxidant supplementation to IVC media on enhancing embryonic development by promoting the intracellular defense mechanism and inhibition of apoptosis.