The cloning between different animals known as interspecific somatic cell nuclear transfer (iSCNT) was carried out for endangered species. The iSCNT has been characterized by a poor success rate due to several factors that influence the formation of the SCNT in various cytoplasms. The cell cycle of the transferred somatic cell, the passage number of the cultured somatic cell, the mitochondria oocytes, and their capabilities are among these factors. This study investigates the role of the passage number of the Arabian Oryx somatic cell culture when transplanted to an enucleated domestic cow oocyte and embryo development in vitro. The fibroblast somatic cell of the Arabian Oryx was cultured for several passage lanes (3–13). The optimal passage cell number was found to be 10–13 Oryx cell lines that progressed to various cell stages up to the blastula stage. There was some variation between the different passage numbers of the oryx cell line. The 3–9 cell line did not show a good developmental stage. These could be attributed to several factors that control the iSCNT as stated by several investigators. More investigation is needed to clarify the role of factors that affect the success rate for the iSCNT.
Enucleated, metaphase II sheep oocytes fused with 8- or 16-cell embryonic blastomeres were employed in the first report of embryonic nuclear transfer in mammals [1,2]. Following that, other attempts to clone cattle and other animals were made. Numerous examples of effective nuclear transfer utilizing donor cells have been published for sheep [3,4,5], cows [6,7], rabbits [8,9], pigs , mice , monkeys , goats [13,14], and camel [15,16]. At the morula and blastocyst stages, donor cells were also revealed to be totipotent and reprogrammable by cytoplasmic factors in cow  and rabbits . American researchers successfully injected the somatic cells of threatened animals (Bos gaurus) into an enucleated oocyte, obtained embryo at blastula stage and transferred to a recipient’s cow . In addition, Saikhun et al.  also succeeded to inject buffalo somatic cells into an enucleated oocyte from a species cow (Bos indicus) . Furthermore, Ikumi et al.  succeeded in transferring a somatic cell from a whale into an in vitro cloned embryo.
The endangered grey wolf was successfully cloned from somatic cells obtained from the dead carcass . In wild goats, somatic cells were transferred into the oocyte of local goats . Although the use of intra- or interspecies somatic cell nuclear transfer (SCNT) has resulted in the generation of cloned offspring in a variety of mammalian species, the efficiency of somatic cell cloning remains often very or extremely low. Therefore, extensive investigations are needed to identify the determinants of SCNT-mediated cloning, which can be responsible for ameliorating the efficiency of this modern-assisted reproductive technology. Among these determinants, the most important role is played by: (1) the source of nuclear donor somatic cells (NDSCs) [23,24,25,26], (2) the abilities of nuclear recipient oocytes to be artificially activated [26,27,28], and (3) molecular interactions between mitochondrial and nuclear DNA molecules in SCNT-derived embryos [29,30,31,32]. It is also worth noting about the remarkable role of: (4) the abilities of donor cell nuclear genes to epigenomically reprogram their transcriptional activities in somatic cell-cloned embryos [33,34,35] and (5) the molecular quality and survival rates of the in vitro cultured NDSCs and SCNT-derived embryos [36,37,38,39]. Taking the above-indicated determinants affecting the efficiency of somatic cell cloning, the research is required to be undertaken to improve the epigenetic reprogrammability (i.e., dedifferentiability) of donor cell nuclei in interspecies SCNT-derived embryos that have been reconstructed with somatic cells stemming from endangered or extinct mammalian species. The technique of transferring somatic cells from threatened wild species of mammals such as the Arabian Oryx is essential in their preservation. Also, the cytoplasm of cow oocytes supports the development of embryos resulting from SCNT of different types of animals . Cow and Arabian Oryx are both members of the family Bovidae. The cloning of Arabian Oryx via the injection of the enucleated oocyte from cow somatic cells is realistic.
The Arabian Oryx is the smallest member of the Oryx genus and is found across the Arabian Peninsula. They vanished from the wild in the early 1970s, but zoos and private custodians preserved them. The Arabian Oryx was categorized as endangered by the International Union for Conservation of Nature in 1986 . In addition, it was the first mammal to be reclassified as vulnerable after being declared extinct in the wild in 2011 . This study aims to produce cloned Arabian Oryx embryos by transferring different passages of fibroblast somatic cells from Arabian Oryx into enucleated cow oocytes.
2 Materials and methods
Both chemicals, media and hormones, were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA), unless otherwise specified.
2.2 Ovaries, Oocyte, and maturation
Ovaries were obtained from a local slaughterhouse in Riyadh and transported in 0.9% Sodium Chloride to the laboratory during 1–2 h. The oocytes were extracted from the ovarian follicles using a 19-gauge needle connected to syringe containing 0.5 mL of tissue culture medium (Caisson Lab. Inc., Smithfield, USA). Cumulus oocyte complexes (n 706) with more than three layers of cumulus cells and homogenous cytoplasm (Figure 1a) were matured in TCM-199 at 39°C and 5% CO2 in atmospheric air at high humidity for 24 h as mentioned in our previous research .
2.3 Zona dissection with micropipettes
Cumulus-enclosed oocytes were placed on a 35 mm Petri plate with hyaluronidase enzyme (300 IU/1 mL) after in vitro maturation period. Cumulus cells were manually removed from oocyte by using a glass Pasteur pipette. Then, for zona dissection, only the oocytes with an extruded first polar body (FPB) would be utilized. The directing of the oocyte was controlled by the cutting pipette. When the polar body is at 12 o’clock, an oocyte position with the holding pipette, a slot or slit in the zona pellucida (ZP) was made with the cutting pipette on the opposite side of oocyte (Figure 1c) .
2.4 Oocyte enucleation
Cutting pipettes were used to softly spin the oocyte to extract a little part of cytoplasm contained in the plasma membrane immediately beneath the FPB of oocytes outside the ZP. Enucleation was ascertained by separating the resultant from the oocyte and cultured primarily in TCM-199. Earle’s salt is mixed with 10% fetal bovine serum and 20 μg/2 mL Hoechst for 30 m (Sigma B2261). The ooplasm was then subjected to UV light to check for the existence of a metaphase II stage .
2.5 Preparation of fibroblast donor somatic cells from Arabian Oryx
Oryx ears were utilized to acquire fibroblast donor cells, which have a normal diploid karyotype, and were used to transduce cell nuclei by lanes 3–13. The fibroblast cells were cultured in Dulbecco’s modified minimal basic 0.5 μg/mL Penicillin streptomycin sigma and 10% fetal bovine serum at 39°C and 5% (CO2) in atmospheric air at high humidity. The cells were grown to 80% confluence and NT cells were separated by 0.25% trypsin-EDTA solution (Gibco 25200) (Figure 1e) .
2.6 Analysis of cell cycle by flow cytometry
Flow cytometry was used to determine the cell cycle of Oryx cells. Cells were extracted by centrifugation, washed once with PBS after being treated with three passages, and incubated for 6–8 h. The cells were then fixed in chilly 70% alcohol for 30 m. After that, the cell suspensions were incubated with RNase for 30 min in the dark. By using a Beckman Coulter flow cytometer, 10,000 events of PI-stained cells were recorded in the FL4 Log channel using a 675 nm band-pass filter (Cytomics FC 500, Beckman Coulter, Fullerton, CA, USA). CXP capture and analysis software was used to compute the cell cycle stages and analyze the data (Figure 2).
2.7 Nuclear transfer, fusion, and activation
One somatic cell from an oryx fibroblast was inserted by polar body dissection pipette into the perivitelline space of every enucleated cows’ oocytes. Immediately following NT at room temperature, the cell fusion was done via a single alternating current pulse of 0.2 kV/cm for one second followed by a single DC pulse of 2.5 kV/cm for 50 s. After fusing, 5 μM ionomycin was employed to activate selected fused SCNT at room temperature for 5 min. Then, they were cultured in SOF medium supplemented with 2 mg/500 μL BSA, 20 μL/2 mL MEM (50×), 20 μL/2 mL MEM (100×), 50 μg/mL Penicillin streptomycin, 5 μg/500 μL Cycloheximide using a 35 mm Petri dish coated by mineral oil that has been tried on embryos, for 6 h at 39°C with 5% CO2. All steps of this protocol follow the method described in a previous study (Figure 1f) .
2.8 In vitro culture of iSCNT embryos
After 6 h of activation, iSCNT embryos were cultured for 42 h in SOF medium supplemented with 2 mg/500 L BSA., 20 μL/2 ml MEM (50×), 20 μL/2 mL MEM (100×) 50, μg/2 mL Penicillin streptomycin using Petri dishes 35 mm coated by mineral oil at 39°C for 6 h with mixture gas 90, 5, 5% of N2, CO2, O2, respectively (Figure 1g–l) [4,43].
2.9 Statistical analysis
All results were recorded and analyzed using the Mine Tab INSTAT program. The Blastocyst and cleavage rates were analyzed by using the Chi-square test.
In this study, the collected oocytes (n 706) injected with different passage lanes 3–13 of donor cells showed different developmental rates of iSCNT Oryx – Cow embryos. From passage 3 up to 9, was no considerable rate of development, which illustrates the collected oocyte numbers, with enucleation rate, fusion rate, embryo cleavage rate, and blastula development rate. The rate of the different passage 10 to 13 was 80, 91.2, 66.3, and 84%, respectively. The fusion rate of the different passage 10 to 13 was 43, 61.4, 71, and 70%, respectively (Table 1).
|Passage number||No. of oocytes||Enucleation rate%||Fusion rate%||Cleavage rate%||Blastocyst rate%|
|Passage 10||190||125 (80)||35 (43)||(8) 14%||(5) 62%*|
|Passage 11||140||114 (91.2)||70 (61.4)||(25) 57%*||(0) 0%|
|Passage 12||271||112 (66.3)||80 (71.4)||(8) 16%||(1) 12%|
|Passage 13||105||142 (84)||100 (70.4)||(17) 48%*||(0) 0%|
*Significant difference at p < 0.01 compared to the others without star at the same type of passage or columns.
In the passage from 10 to 13, the cleavage rate of the iSCNT embryos injection with the different passage was 14, 57, 16, and 48%, respectively. The cleavage rate of the iSCNT in passages 11 and 13 indicated a considerable increase (p < 0.01) than passage lines 10 and 12 (Table 1).
While passage line 10 produced a better blastula rate compared to the other passage lines, it was significantly higher (p < 0.01) than passage lines 11, 12, and 13 (Table 1). In general, the total embryos at the blastocyst stage were five embryos (62%) from passage 10 and one embryo (12%) from passage 12, while no embryos developed up to the blastocyst stage from passages 11 and 13 (Table 1).
The failure of cloning has been related to several variables, one of which is a greater rate of oocyte degeneration. This might be owing to the kind of somatic cell employed since most of the donor cells were from skin or cumulus cells, which are not in the G0 stage, or the electrical current used to fuse the somatic cell with the oocytes, which could damage the ova’s cytoplasm. In addition, due to the long term of the fibroblast cell, the rate of development of the SCNT may be affected by in vitro culture .
This work demonstrated the ability of the cytoplasm of domestic cows to reprogram adult Arabian Oryx fibroblast nuclei. Interspecies somatic cell nuclear transfer embryos have the potential to develop to the blastocyst. The development rate of iSCNT rebuilt Arabian Oryx–Cow embryos can be obtained at a low rate as per Li et al. . SCNT efficiency was substantially lower when embryonic germ (EG) cells with lower passage numbers (3–5) were employed as donor cells than when fetal fibroblast donor cells were used. Higher passage EG cells (numbers 9–12) produced similar blastocyst rates, implying that higher passage EG cells are better suited for SCNT. This agrees with our result regarding the use of passes more than 10. The SCNT development was dramatically reduced when lower passage (numbers 3–5) porcine EG cells were used as donor cells, but higher passage (numbers 9–12) EG cells produced a greater blastula rate. However, these results disagree with Jin et al.  which they got better-cloned embryo development from the first passage cells compared to 4–6 passages.
There are several factors reported about early and late passages [45,46,47], which affect the proliferation of donor somatic cell culture, such as the abnormalities in chromosomes, DNA damage, and DNA methylation. The bulk of iSCNT research has focused on establishing whether an oocyte can reprogram a foreign species’ nucleus and support the initial cleavage divisions. Fewer studies, on the other side, have looked at the variables that cause embryonic failure in less taxonomically various species during the preimplantation stages . Two of the key studied challenges in iSCNT include failure to reprogram the somatic cell and incompatibility between the nucleus and the cytoplasm [49,50]. Incompatibilities between the nucleus and the cytoplasm have been found to cause harmful cellular processes as an outcome of species variation in studies on and nuclear hybrid embryos. Larger species’ divergence lengths, according to some theories, may hinder communication between the nucleus and cytoplasm components such as the mitochondrial genome [51,52].
Many studies have shown the molecular that may cause the failure of the development of the embryos it produces by iSCNT [53,54,55]. In our research, we studied two species known to produce related species (Bovidae species) through interspecies cloning, which indicates some similarities between the two species.
Furthermore, the timing of DNA replication appears to vary among SCNT embryos . In addition, the cytoplasm of donated oocytes for iSCNT is from different species which have different regulations for DNA demethylation of somatic cells [57,58].
The findings of this study revealed information about some of the factors that contribute to the development of Arabian Oryx – Cow iSCNT embryos in certain passages and another passage failure in others. The reduced rate of cleavage and blastocyst formation in iSCNT might be attributed to mitochondrial function, which causes embryo development failure during the early stages of division in embryos [53,59]. The results of our study showed the best cleavage and blastula rate obtained from passages 10 and 12. There may be other reasons that are recommended for more investigation such as gene expression of the Cow–Oryx iSCNT.
Our current investigations have proved the occurrence of intergroup variability in the in vitro developmental outcomes of interspecies (oryx → bovine) cloned embryos generated using somatic cell lines at different passage numbers (between 10th and 13th passage). In contrast, the use of somatic cell lines between passages 3 and 9 has resulted in remarkable alleviations in the in vitro developmental capabilities of oryx → bovine iSCNT-derived embryos to reach the blastocyst stage. The intergroup differences between the extracorporeal developmental competencies of oryx → bovine cloned embryos might be evoked by a variety of factors that affected the iSCNT efficiency. Among them, one of the most important factors seems to be molecular cooperation between nuclear and mitochondrial genomes in interspecies cloned embryos [32,39,60]. The successful developmental rates of such embryos appear to be arising from epigenomic reprogrammability of foreign (xenogeneic) somatic cell nuclear genomes in iSCNT-derived embryos [61,62,63,64,65,66,67]. For those reasons, additional intensive studies are needed to clarify how the high impact of the abovementioned molecular factors is exerted on the ex vivo development of oryx → bovine iSCNT-derived embryos.
The Project for funding this work number (RSP-2021/232) at King Saud University, Riyadh, Saudi Arabia.
Funding information: The authors sincerely acknowledge the Researchers Supporting Project for funding this work number (RSP-2021/232) at King Saud University, Riyadh, Saudi Arabia.
Author contributions: Conceptualization, A.A.A. and M.G.A.; methodology, A.A.A. and R.A.A; validation, A.R.A., A.A.A.; formal analysis, A.A.A.; investigation, A.A.A. and M.G.A.; data curation, A.R.A.; writing – original draft preparation, A.A.A. and A.R.A.; writing – review and editing, A.A.A., A.R.A.; visualization, A.R.A., A.A.A., and M.Q.A.; supervision, A.R.A., and M.Q.A. All authors have read and agreed to the published version of the manuscript.
Conflict of interest: The authors declare that they have no conflict of interest.
Ethical approval: All of the experiments were conducted according to the Guidelines for the Institutional Animal Care and Use Committee of the Zoology Department, College of Science at King Saud University.
Data availability statement: The authors confirm that the data supporting the findings of this study are available within the article.
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