The Rumex vesicarius seeds water extract effect in the in vitro fertilization and embryo development of mice as mammalian modelABSTRACT
During IVF and embryo development in vitro there are a lot of free radical oxygen reactive spices (ROS), the lower the successful viable embryo rate. Rumex vesicarius plant is used as food and acts as a biochemical marker and as a scavenger of the free radical (ROS). The aim of this research was to study the role of Rumex vesicarius water seeds extract (RVWSE) on ova (IVF) and embryo development of mice. Water seed extracts of RV were made at a concentration of 10 mg/ml. These extracts were intended to be added to the fertilization medium of mice oocytes. This study showed that the RVWSE treated ova had showed significantly (P<0.05) higher rate of the in vitro fertilization and early cleavage development rate (2-4 cell) stage compared to control. Also, the rate of developed embryo from the cell stage to morula stage of the (RVWSE) treated group were higher (75.2%) compared to the control (62.65%.). Regarding the blastula stage, the control group showed significantly (P<0.05) higher developmental rate (26.35%) compared to the treated group (12.8%). The (10 mg/ml) dosage of supplementation of (RVWSE) in this research were significantly higher on the in vitro fertilization and early cleavage rate but not the blastula stage in mice.
Keywords:
Rumex vesicarius; seed extract; mice; IVF
RESUMO
Durante IVF e desenvolvimento do embrião in vitro, há uma grande quantidade de radicais livres e especiarias reativas ao oxigênio (ROS), o que diminui a taxa de sucesso de embriões viáveis. A planta Rumex vesicarius é usada como alimento e atua como um marcador bioquímico e como eliminador de radicais livres (ROS). O objetivo desta pesquisa é estudar o papel do extrato aquoso de sementes de Rumex vesicarius (RVWSE) no desenvolvimento de óvulos (IVF) e embriões de camundongos. Esses extratos foram destinados a serem adicionados ao meio de fertilização de oócitos de camundongos. Esse estudo mostrou que os óvulos tratados com RVWSE apresentaram uma taxa significativamente maior (P<0,05) de fertilização in vitro e taxa de desenvolvimento de clivagem precoce (2-4 células) em comparação com o controle. Além disso, a taxa de embriões desenvolvidos desde o estágio de duas células até o estágio de mórula do grupo tratado com RVWSE foi maior (75,2%) em comparação com o controle (62,65%). Mas em relação ao estágio de blástula, o grupo de controle mostrou uma taxa de desenvolvimento significativamente (P<0,05) maior (26,35%) em comparação com o grupo tratado (12,8%). A dosagem (10 mg/ml) da suplementação de (RVWSE) nesta pesquisa foi significativamente maior na fertilização in vitro e na taxa de clivagem precoce, mas não no estágio de blástula em camundongos.
Palavras-chave:
Rumex vesicarius; extrato de semente; camundongos; IVF
INTRODUCTION
Rumex vesicarius L (RV) is a medicinal annual plant many uses. Rumex vesicarius L (RV) plant used as food and as a folk medicinal plant in several Asian and Middle eastern countries, Rumex vesicarius (RV) is an edible wild annual plant that is high in minerals, protein, and ascorbic acid (Al-Rumaih et al., 2002). The RV plant has been shown to have anti-liver damage, anticancer, antibacterial, and antioxidant effects, (AlFawaz, 2006). Al Aboody (2015) conducted a study on the analysis of antibacterial and antioxidant in vitro activities of chemical content of Rumex vesicarius L. A study by Hariprasad, and Ramakrishnan (2012), was conducted to evaluate the HPTLC fingerprint sequences components of Ethyl-acetate (Etoh) and aqueous Rumex vesicarius extract. The results showed the Etoh-extracts had nine peaks in 5μl concentration and ten peaks in 10μl concentrations while the aqueous extract showed only 2 peaks in both the concentrations. Also another study by Hariprasad, and Ramakrishnan (2011) were conducted on Rumex vesicarius to assess in-vitro antioxidant activity and perform high performance thin layer chromatography fingerprint analysis of the most active fraction of Rumex vesicarius. Acetone, ethyl acetate, n-butanol, and methanol extracts of R. vesicarius were evaluated for the free radical scavenging activity by studying the inhibition of the level of lipid peroxidation induced by Fe (++)/ascorbate, DNA sugar damage, scavenging of hydrogen peroxide, diphenyl phosphine DPPH radical scavenging activity, total phenolic content, total flavonoids content and total proanthocyanidin. The Rumex vesicarius acts as a biochemical marker and is medically important for the pharmaceutical industry Hariprasad, and Ramakrishnan (2011 ,2012). Younes (2021) carried out an in-vitro study of antioxidant and antiradical potential of semi-purified fractions and biosynthesized silver nanoparticles of Rumex vesicarius. The Rumex vesicarius has antibacterial and antioxidant property effects. Abou Elfotoh et al., 2013 and also Alotibi et al. (2020) conducted a study to evaluate of the antifungal activity of Rumex vesicarius L. and Ziziphus spina-christi L, water extracts and assessment of the morphological changes induced to certain myco-phytopathogens. In addition, Quradha, et al. (2019) studied the chemical composition of essential oil and methanol extract from Rumex nervosus as an antioxidant activity, anticancer, and as antimicrobial activity in vitro. Also, Khan et al. (2014) studied the in vitro antioxidant activities of Rumex vesicarius.
The in vitro fertilization (IVF) widely used as a part of the assisted reproductive technique (ART) for studying the association of gamete cells (sperm/egg) outside of the body, also includes important processes, namely the basic cellular and molecular processes that assist in the fertilization of mammals. But there is an effect on the result of fertilization, which is oxidative stress, (which is a type of reactive oxygen species ROS) that the antioxidants can cause cell damage especially in the in vitro embryo cell culture. Oxidative stress has a detrimental effect on cells, and especially embryonic cells. Free radicals (types of ROS) may be generated during the IVF process, either from external or internal sources from the cell itself, which leads to the occurrence of oxidative stress in the cells and works to weaken the fertilization process, embryo culture, and embryo cleavage cell development rates. The internal sources include gametes and embryo metabolism and cell respiration, while the external sources could be from culture media that have high sources of H2O2, Fe2 and Cu2. Several research have examined some plant extracts in the ART such as Maleki et al. (2014) studied the saffron aqueous extract and its active ingredient, crocin on the in vitro maturation, in vitro fertilization, and in vitro culture of mouse oocytes. Eriani et al., 2017, used the extract of Pandanus conoideus red fruit in the fertilization and development of mice embryo in vitro. The extract of red fruit (ERF/Pandanus conoideus Lam.) contains beta-carotene and alpha-tocopherol, i.e., antioxidant compounds group which can stop the formation of free radicals during in vitro fertilization. Farooq et al. (2020) investigating the antiangiogenic potential of Rumex vesicarius, as an anticancer in cell lines and in the zebrafish embryos’ developmental toxicity.
Gonzales-Daga (2020) and his co-worker studied the effect of the aqueous extract of "Mashua" tropaeolum tuberosum "on sperm quality and its implication in preimplantation embryonic development, as pre-clinical test. Our previous studies indicated the safe use of the Rumex vesicarius treatment in the intact liver, kidney and fertility of mice (Alhimaidi et al., 2020, 2021). Kambhar, in 2014 wrote a review article on the Rumex vesicarius L.(Polygonaceae) its characteristic and uses.
The aim of this study is to indicate the effect of Rumex vesicarius seed water extract (RVSWE) on the in the IVF and embryo cleavage development of mice as a model for mammals.
MATERIAL AND METHODS
All animal procedures and treatments were conducted according to ethics committee guidelines at the Zoology Dept. College of Science, at King Saud University, Riyadh Saudi Arabia.
Preparation of the Rumex Vesicarius seeds water extract (RVSWE): The R. vesicarius (RV) plant collected from different parts around the northeast of Riyadh city area during Feb and April 2020. The collected plant was kept in a dried room at 25-28°C until it dried for 2 weeks. Then, the seeds were separated and ground to a 200g powder. The ground seeds (100g) added to 1 L of distilled water in the separation tube for 48 h with repeated stirring, and the solution was separated via a steam filter. To calculate the concentration of water seed extract of R. vesicarius, a 50 mL beaker was weighed and then filled with 5mL of water seed extract RV filtered fluid and then evaporated in an incubator (60°C for 5 h, the remaining seed water extract in the beaker was weighed and ×100) (Radyy 2017). Afterward, to reach the (10mg/ml) desired concentration of water seed extracts RV, to be add to the fertilization medium of oocytes (Alhimaidi et al.2020, 2021).
Oocytes Collection: Yang female mice (6-8 weeks old), were injected with ovulation hormone (5 international units IU) of equine chorionic gonadotropin (eCG) between 2:00 and 4:00 pm. On the third day (48 h. post eCG first injection), the same female was injected with human chorionic gonadotropin (hCG) 5 IU between 2:00 and 4:00 pm via IP injection. The ovulation was expected to occur around 15-17 hours after the hCG injection, then oocytes would then be collected from the female oviduct ampoule. The next day between 8:00 and 9:00 a.m., mice were anesthetized and dissected, and the oviducts removed, then the ovum ampoule was dissected and removed and placed in the human tubular fluid (HTF). They were covered with mineral oil in the collection petri dish. Directly after ampullae collection, and oocytes were extracted from the ampulla under the oil using the needle of insulin syringe to open the ampulla to extract the oocytes with their cumulus oophorous cell (COCS) into the HTF medium. The dish was then incubated for 15 minutes in an incubator (37 ° C, 5% CO2 in air). (Takeo and Nakagata, 2018).
Spermatozoa collection: The Spermatozoa were collected from male mice (4 to 6 months old), the male was anesthetized and dissected to extract the epididymis which was transferred to a sperm collection dish that contained 200 μl per drop of HTF coated with mineral paraffin oil. An incision was made in each tail of the cauda epididymis using small spring scissors and then the sperm clot was released and collected from the incision in HTF droplets by gently pressing the surface of the caudate epididymis with a dissection needle. Sperm collection dishes were prepared before starting the experiment (before sperm collection) and the dish was placed in an incubator at 37 ° C and 5% CO2 in the air, for 60 minutes prior to insemination. (Takeo and Nakagata, 2018).
in vitro fertilization (IVF): To make the in vitro fertilization (IVF) the fertilization medium was prepared, and equilibration made in the fertilization dish and placed in an incubator (37°C, 5% CO2 in air) for at least 1 hour before transferring the collected oocytes (COCS) into it. To do the insemination a sufficient amount of sperm cell concentration of one million sperm / ml, were add to the collected oocytes COCS drop at the petri dish, the fertilization dish was be placed in an incubator (37°C, 5% CO2 in air) for 6 hours (Takeo and Nakagata, 2018).
Embryo culture: The embryo culture media Potassium optimization medium KOMS dish was be prepared at least 30 minutes before transferring the fertilized oocytes, they were placed in a 5%CO2, incubator for equilibration. After fertilization, the oocytes transferred into 35 mm petri dish containing hyaluronidase enzyme covered with mineral paraffin oil to remove cumulus cells mechanically using glass pasture pipette. The denuded oocytes wewre then transferred to the culture medium, washed twice, and any parthenogenetic oocytes (those with only one pronucleus) were identified and removed, and the fertilized oocytes were be cultured until the blastocyst stage, in a 5% CO2% incubator at 37°C. (Takeo and Nakagata, 2018).
The total of each females’ oocytes and embryo developmental stages were recorded.
The one-way analysis of variance (ANOVA) was used for the means of the collected ova, fertilized and the different developed embryo cleavage stages by using SAS version 1.9 (Raleigh, NC, USA), and were applied for all statistical analysis. All data are expressed as mean standard error of the mean. A probability of p < 0.05 was considered to be statistically significant.
RESULTS
The total collected ova from all female mice were 392 ova, the total of developed ova (1-cell to blastula 34) while forty-five ova were degenerated or not developed oocytes. For control group 200 ova were used, and 192 ova used for the group treated with 10mg/ml Rumex vesicarius seeds water extract (RVSWE). The rate of all fertilized ova and developed embryos for control group were 176/200 (89.0%) with a mean of (25 + 4.01) ova, which were close to the total ova and developed embryos rate (347/ 392= 88%). In addition, the treated group showed about the same rate of fertilized and developed embryos 169/192 (88.21%) with a mean of (24 +3.58) ova, compared to total and control group. The rate of one cell stage of control group 37/178 (21.89%) were not significantly higher compared to the rate of the fertilized treated ova (16.29%) Table 1. The rate of developed embryo from two cell stage to morula stage of the treated (AESRV) ova 115/132 (87.12%) developed embryos, which is significantly higher (P0<05) compared to the control developed embryos 110/149 (73.83%). Regarding the early cleavage rate, the higher developmental rate in the treated group shown in two cell stage (48.48%) while the lowest stage was seen in 8-16 cell stage (2.27%) which is higher compared to the control group (42.5%) and (0.5%) respectively. Regarding the blastula stage the control group showed significantly (P<0.05) higher developmental rate (21.91%) compared to the treated group (10.06%). The degenerating ova of control were (22 oocytes/200 = 11.0%) with a mean of 2.75, and the treated were close to control with (23 oocytes /192= (11.99 %) with a mean of 2.88. Table 2. Figure (1.a,b,c,d) illustrate the photographs of different in vitro developmental stages of mice embryos.
The total and percentage of in vitro fertilization and different embryo development cleavage stages of treated mice oocytes with 10mg/mL of aqua extract of the seed of Rumex vesicarius (AESRV)
The in vitro fertilization IVF and cleavage stages of mice embryos treated with aqua extract of the seed of Rumex vesicarius (AESRV): a.) The in vitro fertilization, b.) 2 cell stage, c.) 4,8-16 cell and morula stages, and d.) blastula stage.
DISCUSSION
The aim and the result of this study was designed to illustrate the role of Aqua extract of the Seed of Rumex vesicarius (AESRV) in in vitro fertilization and embryo development. It showed that the (AESRV) enhanced the ova in vitro fertilized and especially in the early cleavage embryo development, which agrees that the reproductive technology has been explored widely is in vitro embryo production (IVEP). Also, with the development of biotechnology, there is an important role in many fields such as the assisted reproduction technology ART, the agriculture, farming, human health, medicine, and fishery as stated by Erlani et al., 2017. In addition, Gordon (1994) stated the advantages of biotechnology in farming is increasing farming product including such as artificial insemination, embryo transfer, cryopreservation, in vitro fertilization (IVF), spermatozoa sexing, embryo cloning, genetic engineering, in veterinary area and ART in human. The result of our study agrees with Al-Dujaily and Al-Saadi (2009) who studied the effect of Glycyrrhiza glabra water extract (GgE) by using 10% GgE on IVF of mice, their result showed a significant (P<0 .05) increase in the fertilization rate of mice oocytes (53.89%) compared to IVF medium alone in group (36.82%). The addition of 10% GgE to the IVF medium, the number of 2-cell and 4-cell embryonic stages of mice was significantly (P<0.05) higher than that of control mice embryos cultured with IVF medium alone (60% and 60% Vs. 51% and 54%, respectively). In addition, the result of this research agrees with Eriani et al. (2017) study, that mice ova obtained from females that were treated with an extract of red fruit (ERF/Pandanus conoideus Lam. where in vitro fertilization and the embryos grow to morula and blastocyst stage, there was no significant influence on embryo development between control, and treated 0.1mL ERF 0.05mL ERF. This could be due to the lower concentration of the treated groups. But seen from the percentage, ERF dosage 0.1mL was the highest that reached morula and blastocyst phase. Also, Isobe et al. (2012) reported that alpha-tocopherol as an antioxidant has the potential to enhance embryo cell proliferation which led to an increase of the percentage of blastocyst. In this study the embryo was able to cleavage to morula and blastocyst stage. Our results disagree Gonzales-Daga et al. (2020) study where it showed that treatment with Mashua water extract produces a significant reduction in the number of embryos in the morula stage and a non-significant increase in embryos in the indeterminate stage, as well as a reduction in the quality and capacity of the embryos to develop.
CONCLUSION
The use of Rumex Vesicarius seeds water extract (RVSWE) enhances the ova in vitro fertilized and early cleavage rate, but not the final blastula stage. Further studies of different concentrations with the role of their gene expiration are recommended.
ACKNOWLEDGEMENT
The authors sincerely acknowledge the Researcher Support Project (RSP-2024/R232) for funding this work at King Saud University, Riyadh, Saudi Arabia.
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Publication Dates
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Publication in this collection
21 Feb 2025 -
Date of issue
Mar-Apr 2025
History
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Received
12 May 2024 -
Accepted
11 Sept 2024
