Developmental Ability and Concentration of Hydrogen Peroxide in Bali Cattle Embryos Cultured In Vitro

Bali cattle are native Indonesian cattle widely developed by the farming community in South Sulawesi. One way to continue supporting the productivity of Bali cattle is to utilize ovaries from slaughterhouses by using assisted reproductive technology, namely in vitro embryo production. In general, studies on embryo production have been widely conducted, but those carried out on Bali cattle are still limited in number. In vitro embryo production is still being improved to support the success of embryos that are worthy of transfer (Marsico et al., 2019). The initial development embryo often experiences obstacles and becomes a critical problem for embryo production, resulting in obstacles in the production of embryos in vitro (Medina et al., 2014). Several factors can affect the successful production of embryos in vitro including donors, sperm, heredity and the environment (Ahmed et al., 2015). In addition, different culturalsystems can also affect embryonic development (Abd El-Aziz et al., 2016). In vitro production of Bali cattle embryos resulted in zygote cultures which show varying rates of embryonic cell division indicated by the occurrence of 2-, 4-, 8- and 16-cell embryos on the second day or 48 h after culture (Sonjaya and Hasbi, 2019; Hasbi et al., 2020). The in vitro embryo culture system has not been able to be properly conditioned to resemble in vivo so that it can lead to the increase of reactive oxygen species (Agarwal et al., 2006).

Reactive oxygen species (ROS) are oxygen-derived molecules that act as strong oxidants. ROS produces oxygen free radicals such as hydrogen peroxide (H2O2), hydroxyl radicals (OH) and superoxide anion radicals (O2), and is a by-product of cell metabolism (Takahashi, 2012). Small amounts of ROS are needed in some physiological processes such as maintaining embryonic development (Sies and Jones, 2020), and a small amount of H2O2 can maintain the balance of cell mass (Qian et al., 2016), spermatozoa capacity and acrosome reactions (Du Plessis et al., 2015). An increase in ROS at the intracellular level can cause developmental disorders, apoptosis and embryo death (Giotto et al. 2015; Hu et al., 2015). Oocytes that experience oxidative stress can cause problems during meiosis (Perkins et al., 2016) and increase fragmentation in the embryo (Arias et al., 2012). ROS is also an obstacle to the glycolysis process so the embryo does not optimally utilize glucose as an energy source (Eriani et al., 1994).

The success of in vitro embryo production can be judged by the number of embryos that are able to develop after the fertilization process (Asimaki et al., 2022). The success of embryo culture in vitro is thought to be related to H2O2 concentrations, high H2O2 concentrations can inhibit embryo development. Information regarding the measurement of H2O2 in Bali cattle embryos produced in vitro does not yet exist. This study focuses on measuring the concentration of H2O2 in Bali cattle embryos based on cultures with the same number of cells.

This study used 138 pairs of ovaries with 542 oocytes obtained from slaughterhouse. The cattle ovaries were obtained from slaughterhouse and brought to the laboratory by transport media (0.9% NaCl solution). The oocytes collection was carried out using the slicing method and then were selected using the Olympus SZ51 Japan microscope (only Grade A and B oocytes were used). Grade A has a compact cumulus-oocyte complex (COC) cell surrounded by at least 4-5 layers of cumulus cells and homogeneous cytoplasm. Meanwhile, grade B has a COC surrounded by 2-3 layers of cumulus cells attached to the zona pellucida and homogeneous cytoplasm (Bakri et al., 2016).

The selected oocytes were then washed with phosphate buffered saline (PBS) (Gibco by life technologies, USA) added with 0.2% bovine serum albumin (BSA) (Sigma-Aldrich, USA) matured in a maturation medium consisting of medium 199 (Gibco by life technologies, USA) added with 0.3% BSA, 10 IU/ml Pregnant Mare Serum Gonadotropin (PMSG) (Intergonan, Intervet Deutschland GmbH), 10 IU/ml human chorionic gonadotropin (hCG) (Chorulon, Intervet international BV Boxmeer-Holland), and 50 μg/ml gentamycin (Sigma-Aldrich, USA). Maturation was conducted in the form of drops (80 μl / drop) containing 10-15 per drop and covered with mineral oil (Sigma Chemical Co. St. Louis MO, USA). Maturation was carried out in a 5% CO2 incubator with a temperature of 38.5 oC for 24 h (Hasbi et al., 2017). In this study, oocytes that experienced an expansion of cumulus cells or activation of cumulus cells, were used as an indication of oocyte maturity (Uhde et al., 2018) and continued for fertilization.

The semen used was obtained from the same bull. It was thawed for 20 seconds at 37 oC, was centrifuged for 5 min at 1500 rpm 2 times, split the supernatant and spermatozoa, and then semen was added with fertilization media so that the final concentration of spermatozoa was 1.5×106 cells/ ml (Hall and Glaze, 2014; Hasbi et al., 2020). After that, prepared four drops on a petri dish (80 μL/drop) covered by mineral oil (Sigma Chemical Co. St. Louis MO, USA), equilibrated for 30 min. The matured oocytes were washed 2 times, put into IVF media (Suzuki et al., 2000) and stored in a 38.5oC incubator and a 5% CO2 concentration for 5-6 h.

Oocytes after 5-6 h of fertilization, the oocytes were washed twice using CR1aa culture medium, then transferred in a drop of 80 μl CR1aa culture medium (modification by (Somfai et al., 2010; Sagirkaya et al., 2006) added with 5 mg/ml of BSA, 2.5% FBS and covered by mineral oil (Sigma Chemical Co. St. Louis MO, USA), which then was cultured in an incubator of 5% CO2 at 38.5°C (Meo et al., 2005). The evaluation of embryo division ability is carried out 48 h after culture, then the embryos that develop are re-cultured and as a control, namely embryos that develop are cultured without grouping the number of cells and grouped based on the stages of division, namely embryo 2, 4, 8, and 16-cells. Then the embryo is re-cultured for 48 h and re-evaluated.

Embryos that have been re-cultured for 48 h, namely control and embryos that have been grouped based on the stages of division, then concentration measurements are carried out H2O2 concentration was measured using 2’,7’-dichlorodihydro fluorescein diacetate (DCHFDA) (Sigma) as described by Gustina et al. (2019) with minor modifications. Embryos in each group were incubated for 15 min with a culture medium containing 10 μM of DCHFDA, were washed in a fresh medium before placed on a glass slide and were covered by a cover glass. Fluorescence emission was recorded with a digital camera (Zeiss AxioCam HRc, Germany) which was attached to a fluorescence microscope (Zeiss Axio Imager A2) after excitation at 480 nm and emissions of 510 nm. Fluorescent images were converted into TIFF files using Adobe Photoshop CS3 (Adobe Systems Inc., San Jose, CA), then were analyzed using ImageJ 1.47 software (Sun Microsystems, Inc., California, USA). Fluorescent imagery was measured by counting the number of pixels after color reversal. Fluorescence intensity represents intracellular H2O2 concentration.

The data of embryo developmental ability were analysed descriptively, the H2O2 concentrations were analysed by one-way analysis of variance (ANOVA) and the correlation test was conducted using SPSS 20.00 and significance level of 0.05.

The developmental ability of Bali cattle embryos 48 h after culture varies, namely 2-,4-,8- and 16-cell. Once grouped by the cleavage stages and re-cultured for 48 h, the embryos of Bali cattle that can develop further also vary, as presented in Figure 1. The total number of embryos cultured was 228 embryos consisting of control or no grouping (48 embryos), 2-cells (64 embryos), 4-cells (61 embryos), 8-cells (39 embryos) and 16-cells (16 embryos). The group of embryos cultured of 2-cell was able to develop up to 16-cell by 3.27%. Embryos cultured back from 4-cell 8-cell and 16-cell for 48 h were able to reach the morula. The number of embryos that reached the morula was 15 embryos. Overall, the data of embryo development cultured from a different number of cells for 48 h and continued to be re-cultured for 48 h is presented in Figure 1.

Evaluation of H2O2 concentration was carried out by measuring the intensity of H2O2 concentration in Bali cattle embryos (Fig. 2). The brighter the green color, the higher the concentration of H2O2 in Bali cattle embryos. The results showed that there was no significant influence (P>0.05) between each group of embryonic cells which was re-cultured based on its cell count group and the H2O2 concentration levels in Bali cattle embryos. The concentration of H2O2 in the control group was 168.08±58.77, while the concentration in the 2-cell embryo was 167.88±50.37, 4-cell embryo was 167.20±44.88, 8-cell embryo was 160.68± 45.78 and 16-cell embryo was 150.05±48.75 (Fig. 3).

The testing result on the relationship between the H2O2 concentration and the ability of the embryo to reach the morula stage are determined based on the results of the correlation analysis (Fig. 4). The H2O2 concentration and embryo developmental ability to reach the morula stage were negatively correlated (-0.916) and had a very strong relationship. The H2O2 relationship forms a negative trend, indicating that the lower the concentration of H2O2 in the embryo, the higher the embryo’s ability to reach the morula (Fig. 4).

In the initial development of Bali cattle embryos cultured for 48 h, there were 2-, 4-, 8 and 16-cell cleavage and 42% of which developed from 542 oocytes. The development of Bali cattle embryos cultured in vitro is different from that reported by Martins et al. (2018) who stated that the stage of cattle embryo cell development on the second day only reaches 4-cell division. Marsico et al. (2019) stated that the initial stage of embryonic development is difficult to be carried out because they involve processes that are molecularly dynamically arranged and undergo complex structural changes. The results of grouping after reculturing for 48 h showed that the 2-cell group was unable to develop until morula (Fig. 1A). This indicates that post-fertilization embryo culture is a critical period that determines the rate of division (Mizobe et al., 2017; Medina et al., 2014) and embryonic quality (Milewski et al., 2018). It is further explained by Milewski et al. (2018) that most failures of in vitro embryo development occur between the 2-cell and the blastocyst stage. One of the causes of Bali cattle embryos not being able to develop is the high concentration of H2O2. It is part of ROS that can lead to lipid peroxidation and enzyme inactivation, resulting in cell damage by encouraging the formation of hydroxyl radicals (Sharma et al., 2012). H2O2 contained in Bali cattle embryo cells is the cause of the lack of division in cells. Undeveloped embryo cells occur due to ROS production that exceeds cellular defences resulting in oxidative damage to biomolecular substances such as lipids, proteins, carbohydrates and nucleic acids, consequently inducing structural and functional changes such as lipid hydroperoxide, protein carbonylation and DNA with oxidized bases (7, 8 dihydro-8-oxoguanine) (Torres-Osorio et al., 2019). Mitochondrial DNA (mtDNA) is also known to become more sensitive to oxidative stress (Nissanka and Moraes, 2018). Oxidative damage to mtDNA can cause mutations and alter mitochondrial function and integrity (Han et al., 2013). The results of the study of Tatemoto et al (2000) on pig oocytes cultured without cumulus cells are particularly susceptible to oxidative stress and the incidence of degeneration, the duration of DNA migration, and caspase-3 activity increased significantly in DO (denuned oocyte) exposed to ROS compared to DO cultured without XOD (xanthine oxidase).

The 4-, 8- and 16-cell group showed their ability to reach the morula (Fig. 1B, C, D). Poor embryonic development at the beginning of the culture identifies that the embryo is difficult in reaching the next stage of development. Lee et al. (2012) stated that the speed of early embryo division can be used as an indicator to assess the quality of embryos. It is further believed that cattle embryos undergo genome activation starting at stages 8-16 cell (Krisher, 2004) and 8-cell (Milazzotto et al., 2020), so embryo development up to stage 8-cell depends on the maternal genome carried by oocytes. The process of degeneration in the early development of the embryo is likely due to drastic changes in the synthesis of embryonic proteins, internal signals that turn off maternal control in the process of embryo division and growth control, which may cause the embryo unable to be saved and eventually experience death (Consensus Group, 2020). The development of embryo in vitro 2-cell stage that reaches the blastocyst is influenced by the outside environment and suboptimum cultural conditions to support its development (Xie et al., 2016). Culture media has a great contribution in the early embryonic development, the quality of blastocyst and the number of embryo cells (Medina et al., 2014).

The concentration of H2O2 formed in the embryo depends on high oxygen exposure to the cell. Proper concentration of H2O2 is needed to keep sperm in cattle normally functioning (Rahman et al., 2012). However, high levels of H2O2 can lead to sperm dysfunction (Xiao et al., 2012). Specifically, ROS serves as an intracellular signalling molecule in embryo metabolism (Yang et al., 2018). ROS induces lipid peroxidation in membranes, DNA damage to oocytes and is thought to cause damage to cell division, metabolite transport as well as mitochondrial function (Tamura, 2012).

H2O2 concentrations have a very strong correlation (Fig. 4) with the embryo’s ability to develop to the morula stage. The higher the H2O2 content in Bali cattle embryos, the lesser the embryo’s ability to develop further. Oxidative stress, a cellular condition caused by the accumulation of H2O2, is thought to contribute significantly to the development of damaged embryos (Loren et al., 2017; Tian et al., 2017). Excessive levels of ROS or H2O2 are one of the main determinants of the quality of embryos produced in vivo and in vitro, caused by exposure to high oxygen concentrations, light and increased concentrations of metabolites and substrates (Mata-Campuzano et al., 2012). Oxidative stress caused by the high level of ROS production results in a reduction in ATP production and not only damages the embryo’s development but also triggers apoptotic degeneration (Abd El-Aziz et al., 2016). Excessive oxidative stress or insufficient antioxidant protection in human oocytes and embryos can cause adverse effects on male and female reproduction (Lin and Wang, 2021). Oocytes with H2O2 of more than 90 ng/oocytes undergo apoptosis (Tripati et al., 2009). Additional 30 μM H2O2 to mouse culture medium to induce oxidative stress may reduce mitochondrial membrane potential and decrease mitochondrial activity by up to 40% (Qian et al., 2016). An increase in H2O2 can be detrimental not only to the success rate of inductive assisted reproduction but also result in epigenetic and genetic changes in the embryo, which have an effect on transgeneration (Hardy et al., 2021). Stress tolerance after H2O2 exposure is not mediated by the use of antioxidants such as glutathione (Vandaele et al., 2010) melantonin (Tamura et al., 2012), sericin (Gustina et al., 2019) able to block ROS (Cavallari et al., 2019). The weakness of this study is that the concentration of H2O2 in oocytes after ripening and the concentration of H2O2 in Bali cattle sperm have not been measured. It is suggested that it can be a follow-up research for Bali cattle production in vitro.

The high H2O2 can inhibit the development of Bali cattle embryos that are cultured in vitro.

This research was supported and funded by the Ministry of Research, Technology and Higher Education of Indonesia through PMDSU scholarships (Stipulation Agreement No. 1343/D3/PG/2018) and research grants (Decree No. 57/UN4.22/PT.01.03/2020 with the agreement/contract number 7/AMD/EI/KP.PTNBH/2020).

The authors have declared no conflict of interest.