Stable long-term germline transmission of GFP transgenic rat via PiggyBac transposon mediated gene transfer.

Transgene silencing provides a significant challenge in animal model production via gene engineering using viral vectors or transposons. Selecting an appropriate strategy, contingent upon the species is crucial to circumvent transgene silencing, necessitating long-term observation of in vivo gene expression. This study employed the PiggyBac transposon to create a GFP rat model to address transgene silencing in rats. Surprisingly, transgene silencing occurred while using the CAG promoter, contrary to conventional understanding, whereas the Ef1α promoter prevented silencing. GFP expression remained stable through over five generations, confirming efficacy of the Ef1α promoter for long-term protein expression in rats. Additionally, GFP expression was consistently maintained at the cellular level in various cellular sources produced from the GFP rats, thereby validating the in vitro GFP expression of GFP rats. Whole-genome sequencing identified a stable integration site in Akap1 between exons 1 and 2, mitigating sequence-independent mechanism-mediated transgene silencing. This study established an efficient method for producing transgenic rat models using PiggyBac transposon. Our GFP rats represent the first model to exhibit prolonged expression of foreign genes over five generations, with implications for future research in gene-engineered rat models.

Investigation of a multicomponent mycotoxin detoxifying agent for aflatoxin B1 and ochratoxin A-induced blood profile in broiler chickens.

Background and Aim: Mycotoxins such as aflatoxin B1 and ochratoxin A (OTA) are secondary metabolites in molds that grow in raw materials or commercial feed. This interaction has a synergistic effect on mortality, body weight, feed intake, embryo abnormalities, egg production, and lymphoid organ atrophy. This study was conducted to determine the effect of a mycotoxin detoxifier on the blood profile of broilers that were given feed contaminated with mycotoxin, such as the number of heterophils, lymphocytes, monocytes, mean corpuscular hemoglobin (MCH), and MCH concentration (MCHC). Materials and Methods: A total of 20 day-old chicks (DOC) of Cobb broilers were given four treatments with five replicates. The number of chickens used in this research was determined using statistical calculations, and the data obtained was homogeneous so that the population was represented. Treatments included negative control with basal feed (C-), positive control with mycotoxins contamination (C+), treatment 1: Mycotoxins contamination and mycotoxin detoxification 1.1 g/kg (T1), and treatment 2: Mycotoxins contamination and mycotoxin detoxification 1.6 g/kg (T2). Mycotoxin contamination comprised 0.1 mg/kg aflatoxin B1 and 0.1 mg/kg OTA. The treatment period for chickens was 28 days, from 8 to 35 days. A battery cage was used in this study. Chickens were kept in a closed, ventilated room and the room temperature (27°C) was monitored during the treatment period. Results: Based on the results of statistical data processing, a significant difference (p < 0.05) was observed between chickens fed mycotoxin-contaminated feed (C+) and chickens not fed mycotoxin-contaminated feed (C-) and chickens given 1.6 g/kg mycotoxin detoxification (T2). Mycotoxin detoxification at a dose of 1.6 g/kg had a significant (p < 0.05) effect on the heterophil, lymphocyte, and heterophil lymphocyte ratio, leukocyte, erythrocyte, and hemoglobin levels of the blood broiler in this experiment. On other parameters such as monocytes, MCH, and MCHC, treatment 2 at dose 1.6 g/kg was the best treatment, although there was no significant effect with C- and T1. Conclusion: The administration of mycotoxin detoxifiers at a dose of 1.6 g/kg increased the number of heterophils and the ratio of heterophil lymphocytes, leukocytes, erythrocytes, and hemoglobin in broilers fed mycotoxin-contaminated feed.

Outlook on genome editing application to cattle.

In livestock industry, there is growing interest in methods to increase the production efficiency of livestock to address food shortages, given the increasing global population. With the advancements in gene engineering technology, it is a valuable tool and has been intensively utilized in research specifically focused on human disease. In historically, this technology has been used with livestock to create human disease models or to produce recombinant proteins from their byproducts. However, in recent years, utilizing gene editing technology, cattle with identified genes related to productivity can be edited, thereby enhancing productivity in response to climate change or specific disease instead of producing recombinant proteins. Furthermore, with the advancement in the efficiency of gene editing, it has become possible to edit multiple genes simultaneously. This cattle breed improvement has been achieved by discovering the genes through the comprehensive analysis of the entire genome of cattle. The cattle industry has been able to address gene bottlenecks that were previously impossible through conventional breeding systems. This review concludes that gene editing is necessary to expand the cattle industry, improving productivity in the future. Additionally, the enhancement of cattle through gene editing is expected to contribute to addressing environmental challenges associated with the cattle industry. Further research and development in gene editing, coupled with genomic analysis technologies, will significantly contribute to solving issues that conventional breeding systems have not been able to address.

Investigation of the General Molecular Mechanisms of Gallic Acid via Analyses of Its Transcriptome Profile.

Gallic acid (GA), a phenolic compound naturally found in many plants, exhibits potential preventive and therapeutic roles. However, the underlying molecular mechanisms of its diverse biological activities remain unclear. Here, we investigated possible mechanisms of GA function through a transcriptome-based analysis using LINCS L1000, a publicly available data resource. We compared the changes in the gene expression profiles induced by GA with those induced by FDA-approved drugs in three cancer cell lines (A549, PC3, and MCF7). The top 10 drugs exhibiting high similarity with GA in their expression patterns were identified by calculating the connectivity score in the three cell lines. We specified the known target proteins of these drugs, which could be potential targets of GA, and identified 19 potential targets. Next, we retrieved evidence in the literature that GA likely binds directly to DNA polymerase ß and ribonucleoside-diphosphate reductase. Although our results align with previous studies suggesting a direct and/or indirect connection between GA and the target proteins, further experimental investigations are required to fully understand the exact molecular mechanisms of GA. Our study provides insights into the therapeutic mechanisms of GA, introducing a new approach to characterizing therapeutic natural compounds using transcriptome-based analyses.

Stretchable zein-coated alginate fiber for aligning muscle cells to artificially produce cultivated meat.

Numerous studies have explored the cultivation of muscle cells using non-animal materials for cultivated meat production. Achieving muscle cell proliferation and alignment using 3D scaffolds made from plant-based materials remains challenging. This study introduces a technique to culture and align muscle cells using only plant-based materials, avoiding toxic chemical modifications. Zein-alginate fibers (ZA fibers) were fabricated by coating zein protein onto alginate fibers (A fibers). Zein's excellent cell compatibility and biodegradability enable high cell adhesion and proliferation rates, and the good ductility of the ZA fibers enable a high strain rate (>75%). We demonstrate mature and aligned myotube formation in ZA fibers, providing a simple way to align muscle cells using plant-based materials. Additionally, cultivated meat was constructed by assembling muscle, fat, and vessel fibers. This method holds promise for the future mass production of cultivated meat.

Current status and future of gene engineering in livestock.

The application of gene engineering in livestock is necessary for various reasons, such as increasing productivity and producing disease resistance and biomedicine models. Overall, gene engineering provides benefits to the agricultural and research aspects, and humans. In particular, productivity can be increased by producing livestock with enhanced growth and improved feed conversion efficiency. In addition, the application of the disease resistance models prevents the spread of infectious diseases, which reduces the need for treatment, such as the use of antibiotics; consequently, it promotes the overall health of the herd and reduces unexpected economic losses. The application of biomedicine could be a valuable tool for understanding specific livestock diseases and improving human welfare through the development and testing of new vaccines, research on human physiology, such as human metabolism or immune response, and research and development of xenotransplantation models. Gene engineering technology has been evolving, from random, time-consuming, and laborious methods to specific, time-saving, convenient, and stable methods. This paper reviews the overall trend of genetic engineering technologies development and their application for efficient production of genetically engineered livestock, and provides examples of technologies approved by the United States (US) Food and Drug Administration (FDA) for application in humans. [BMB Reports 2024; 57(1): 50-59].

Taste receptor type 1 member 3 regulates Western diet-induced male infertility.

Western diet (WD), characterized by a high intake of fats and sugary drinks, is a risk factor for male reproductive impairment. However, the molecular mechanisms underlying this remain unclear. Taste receptor type 1 member 3 (TAS1R3), activated by ligands of WD, is highly expressed in extra-oral tissues, particularly in the testes. Here, we investigated to determine the effects of WD intake on male reproduction and whether TAS1R3 mediates WD-induced impairment in male reproduction. Male C57BL/6 J wild-type (WT) and Tas1r3 knockout (KO) mice were fed either a normal diet and plain water (ND) or a 60 % high-fat-diet and 30 % (w/v) sucrose water (WD) for 18 weeks (n = 7-9/group). Long-term WD consumption significantly impaired sperm count, motility and testicular morphology in WT mice with marked Tas1r3 overexpression, whereas Tas1r3 KO mice were protected from WD-induced reproductive impairment. Testicular transcriptome analysis revealed downregulated AMP-activated protein kinase (AMPK) signaling and significantly elevated AMPK-targeted nuclear receptor 4A1 (Nr4a1) expression in WD-fed Tas1r3 KO mice. In vitro studies further validated that Tas1r3 knockdown in Leydig cells prevented the suppression of Nr4a1 and downstream steroidogenic genes (Star, Cyp11a1, Cyp17a1, and Hsd3b1) caused by high glucose, fructose, and palmitic acid levels, and maintained the levels of testosterone. Additionally, we analyzed the public human dataset to assess the clinical implications of our findings and confirmed a significant association between TAS1R3 and male-infertility-related diseases. Our findings suggest that TAS1R3 regulates WD-induced male reproductive impairment via the AMPK/NR4A1 signaling and can be a novel therapeutic target for male infertility.

Nutritional evaluation of new alternative types of dog foods including raw and cooked homemade-style diets.

BACKGROUND: New alternative types of pet foods such as raw and cooked homemade-style diets containing human food ingredients have been introduced due to a trend of pet humanization and diversification of consumer needs. OBJECTIVES: To evaluate nutritional adequacy of new alternative types of dog foods containing human food ingredients as maintenance diets for dogs. METHODS: Eleven homemade-style foods for adult dogs were purchased from online channel in Korea and analyzed to evaluate nutritional adequacy for adult dogs. Nutrients analyzed included crude protein, amino acids, crude fat, fatty acids, and minerals. RESULTS: Crude protein and amino acids in all products satisfied Association of American Feed Control Officials (AAFCO) requirements. Crude fat in one of 11 products did not meet AAFCO requirements. The most deficient minerals were selenium (10 of 11, 90.9%), copper (five of 11, 45.5%), zinc (five of 11, 45.5%), potassium (three of 11, 27.3%), calcium (three of 11, 27.3%), iron (two of 11, 18.2%), and magnesium (one of 11, 9.1%). Six products were not in the range of the recommended Ca:P ratio in AAFCO dog food maintenance nutrient profiles. CONCLUSIONS: This study performed nutritional evaluation of raw and cooked homemade-style foods as maintenance diets for adult dogs. Some nutritional inadequacies were observed including some minerals, Ca:P ratio, and omega-6:omega-3 fatty acid ratio, although three products (26.2%) satisfied the AAFCO standard except selenium. Overall, the data suggest a need for accurate nutritional adequacy statement for consumers based on proper methods to validate the formula.

Generation of double knockout cattle via CRISPR-Cas9 ribonucleoprotein (RNP) electroporation.

BACKGROUND: Genome editing has been considered as powerful tool in agricultural fields. However, genome editing progress in cattle has not been fast as in other mammal species, for some disadvantages including long gestational periods, single pregnancy, and high raising cost. Furthermore, technically demanding methods such as microinjection and somatic cell nuclear transfer (SCNT) are needed for gene editing in cattle. In this point of view, electroporation in embryos has been risen as an alternative. RESULTS: First, editing efficiency of our electroporation methods were tested for embryos. Presence of mutation on embryo was confirmed by T7E1 assay. With first combination, mutation rates for MSTN and PRNP were 57.6% ± 13.7% and 54.6% ± 13.5%, respectively. In case of MSTN/BLG, mutation rates were 83.9% ± 23.6% for MSTN, 84.5% ± 18.0% for BLG. Afterwards, the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing. Thirteen recipients were transferred for MSTN/PRNP, 4 calves were delivered, and one calf underwent an induction for double KO. Ten surrogates were given double-KO embryos for MSTN/BLG, and four of the six calves that were born had mutations in both genes. CONCLUSIONS: These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied. Finally, MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.

Identification AAVS1-like locus from the porcine genome and site-specific integration of recombinase-mediated cassette exchange using CRISPR/Cas9.

Gene integration at site-specific loci is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor for human and mouse studies. In this study, we found an AAVS1-like sequence (pAAVS1) in the porcine genome using the Genome Browser and designed TALEN and CRISPR/Cas9 to target the pAAVS1. The efficiency of CRISPR/Cas9 in porcine cells was superior to that of TALEN. We added a loxP-lox2272 sequences to the pAAVS1 targeting donor vector containing GFP for further exchange of various transgenes via recombinase-mediated cassette exchange (RMCE). The donor vector and CRISPR/Cas9 components were transfected into porcine fibroblasts. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in was confirmed by PCR. To induce RMCE, another donor vector containing the loxP-lox2272 and inducible Cre recombinase was cloned. The Cre-donor vector was transfected into the pAAVS1 targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR. In conclusion, gene targeting at the pAAVS1 and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.

Correction: Altered Cell Cycle Gene Expression and Apoptosis in Post-Implantation Dog Parthenotes.

[This corrects the article DOI: 10.1371/journal.pone.0041256.].

Triple-conjugated photo-/temperature-/pH-sensitive chitosan with an intelligent response for bioengineering applications.

Hybrid-crosslinked systems, which can be formed using heat and visible light, are significant for improving the stability of hydrogels under physiological conditions. However, several challenges for their practical application remain, such as shrinking under culture medium conditions or the neutral pH in the small intestine. Therefore, a multi-sensitive hydrogel with response to external conditions has been designed and prepared, which could be employed as a biopolymer ink formulation for three-dimensional printing in bioengineering applications. When exposed to body temperature and visible light, the N-succinyl hydroxybutyl methacrylated chitosan (NS-HBC-MA) undergoes a sol-gel phase transition. The NS-HBC-MA hydrogel exhibits pH-responsive swelling, effectively preventing shrinkage at a neutral pH. Furthermore, NS-HBC-MA hydrogel demonstrates excellent biocompatibility and biodegradability. This study demonstrates that the NS-HBC-MA hydrogel has significant potential for various applications, including wound healing, delivery systems, and tissue engineering.

Supplement of secreted recombinant low molecular weight human fibroblast growth factor 2 in culture media enhances in vitro bovine maturation.

With the annual increase in in vitro bovine embryo production, understanding oocyte maturation is becoming more important. Previous studies have shown that oocyte maturation can be improved by adding bovine additives to in vitro maturation media. Among the additives, human fibroblast growth factor 2 (hFGF2) is well known for its positive influence on the growth rate and quality of cells and oocytes. However, the effect of LMW-hFGF2, one of the isoforms of hFGF2, on bovine in vitro maturation has not yet been identified. Therefore, the goal of this study was to elucidate the effect of LMW-hFGF2 on bovine oocyte maturation. Vectors expressing LMW-hFGF2 were cloned and transfected into cells. Afterward, secretion of LMW-hFGF2 from cells was confirmed, and used to assess the effect LMW-hFGF2 on cells and bovine oocytes. LMW-hFGF2 improved bovine oocyte maturation and embryo developmental competence. Laboratories can use LMW-hFGF2 in bovine oocyte culture media to improve in vitro embryo production success rates.

Germline transmission of MSTN knockout cattle via CRISPR-Cas9.

Although the production of several founder animals (F0) for gene editing in livestock has been reported in cattle, very few studies have assessed germline transmission to the next generation due to the long sexual maturation and gestation periods. The present study aimed to assess the germline transmission of MSTN mutations (-12bps deletion) in MSTN mutant F0 male and female cattle. For this purpose, oocytes and semen were collected after the sexual maturation of MSTN cattle, and embryos produced by in vitro fertilization were analyzed. In addition, the embryos were subjected to additional gene (PRNP) editing using electroporation. Embryos produced by in vitro fertilization with MSTN male and female cattle were transferred to a surrogate, and one calf was successfully born. MSTN heterozygous mutation was shown by sequencing of the F1 calf, which had no health issues. As a further experiment, using electroporation, additional gene-edited embryos fertilized with the MSTN male sperm showed a high mutation rate of PRNP (86.2 ± 3.4%). These data demonstrate that the cattle produced through gene editing matured without health issues and had transmitted MSTN mutation from the germ cells. Also, additional mutation of embryos fertilized with the MSTN male sperm could enable further mutagenesis using electroporation.

Application of transposon systems in the transgenesis of bovine somatic and germ cells.

BACKGROUND: Several DNA transposons including PiggyBac (PB), Sleeping Beauty (SB), and Tol2 have been applied as effective means for of transgenesis in many species. Cattle are not typically experimental animals, and relatively little verification has been presented on this species. Thus, the goal here was to determine the applicability of three transposon systems in somatic and embryo cells in cattle, while also investigating which of the three systems is appropriate for each cell type. Green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, the GFP-positive cells or blastocysts were observed through fluorescence, while the transfection efficiency was calculated by FACS. RESULTS: In bovine somatic cells, the PB (63.97 ± 11.56) showed the highest efficiency of the three systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Conversely, Tol2 (75.00%) and SB (70.00%) presented a higher tendency in the embryonic cells compared to PB (42.86%). CONCLUSIONS: These results demonstrate that these three transposon systems can be used in bovine somatic cells and embryos as gene engineering experimental methods. Moreover, they demonstrate which type of transposon system to apply depending on the cell type.

Differences in hormone levels around parturition in Hanwoo cattle (Bos taurus coreanae) following artificial insemination and embryo transfer.

BACKGROUND: With unique genetic traits, Hanwoo cattle (Bos taurus coreanae) are well-adapted to the Korean environment. However, their perinatal mortality rate is 2%-3%, which imposes an economic burden. OBJECTIVE: Due to insufficient data on hormonal changes around parturition, the timing of parturition is often predicted subjectively; few studies have examined hormones in Hanwoo cattle. We measured the changes in various hormones around parturition, to seek an objective predictor of parturition time. METHODS: In 14 female Hanwoo cattle, we measured progesterone, prolactin and cortisol concentrations daily in jugular vein blood samples, beginning 6 days before parturition until 7 days after parturition. Conception was induced in five animals using artificial insemination. Nine animals received embryo transfer. RESULTS: During parturition, the concentration of progesterone decreased significantly in the embryo transfer group (n = 9) and in the total population (n = 14); it did not change significantly in the artificial insemination group (n = 5). Prolactin concentration increased on the day of parturition but did not differ significantly among the groups. Cortisol remained constant throughout the study course. CONCLUSION: We concluded that parturition time can be predicted in Hanwoo cattle using progesterone concentration. This knowledge can reduce perinatal mortality, which would help to improve farm income and animal welfare.

Production of MSTN-mutated cattle without exogenous gene integration using CRISPR-Cas9.

Many genome-edited animals have been produced using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology to edit specific genes. However, there are few guidelines for the application of this technique to cattle. The goal of this study was to produce trait-improved cattle using the genome-editing technology CRISPR-Cas9. Myostatin (MSTN) was selected as a target locus, and synthetic mRNA of sgRNA and Cas9 were microinjected into fertilized bovine embryos in vitro. As a result, 17 healthy calves were born, and three of them showed MSTN mutation rates of 10.5%, 45.4%, and 99.9%, respectively. Importantly, the offspring with the 99.9% MSTN mutation rate had a biallelic mutation (-12 bps) and a double-muscling phenotype. In conclusion, we demonstrate that the genome-editing technology CRISPR-Cas9 can produce genetically modified calves with improved traits.

Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts.

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

Mitofusin-2 modulates the epithelial to mesenchymal transition in thyroid cancer progression.

Here, we investigated the potential roles of Mitofusin-2 (MFN2) in thyroid cancer progression. MFN2 regulates mitochondrial fusion/division in cells and plays an important role in various aspects of cell metabolism. MFN2 might involve in cell cycle regulation, apoptosis, and differentiation, and it might play a role as a tumor suppressor in carcinogenesis. We evaluated the prognostic impacts of MFN2 expression in thyroid cancer by analyzing TCGA data. In vitro and in vivo, MFN2 was knocked out using CRISPR/Cas9 or siRNA, and MFN2 was stably overexpressed in two thyroid cancer cell lines (Cal62 and HTH83). TCGA analysis revealed that MFN2 expression was lower in thyroid cancer than in normal tissues and significantly associated with a degree of differentiation, RAS mutations, and less lymph node metastasis. MFN2 expression was significantly correlated with cell adhesion molecules and epithelial to mesenchymal transition (EMT) in a gene-set enrichment assay. MFN2 knock-out (KO) in Cal62 and HTH83 cells using CRISPR/Cas9 or siRNA significantly promoted cell migration and invasion in vitro. The same trends were observed in MFN2 KO mouse embryonic fibroblasts (MEFs) compared to those in the controls (MFN2 WT MEFs). Conversely, MFN2 overexpression in cancer cell lines greatly inhibited cell migration and invasion. However, there was no difference in colony formation and proliferation in Cal62 and HTH83 cells after modulating MFN2, although there were significant differences between MFN KO and WT MEFs. EMT-associated protein expression was induced after MFN2 KO in both cancer cell lines. The mechanistic results suggest that MFN2 might modulate EMT through inducing the AKT signaling pathway. EMT-associated changes in protein expression were also confirmed by modulating MFN2 in xenograft tumors. Thus, MFN2 acts as a tumor suppressor in thyroid cancer progression and metastasis by modulating EMT.

Development of in-vitro maturation protocol for rat oocytes; under simple culture vs co-culture with cumulus cell monolayer and its developmental potential via Parthenogenetic/artificial activation.

BACKGROUND: Murine is the most abundantly used as laboratory animal models. There has been a tremendous amount of research including; their evolution, growth, physiology, disease modeling as well as genomic mapping. Rats and mice are the most widely used among them. Although both rats and mice fall under the same category still both are different a lot too. As regarding in vitro maturation and development mouse studies are well established as compared to rats which still lies in the early phase of development. So, we tried to figure out rat oocytes in vitro maturation and their developmental potential by performing 3 experiments i.e. superovulation, in vitro Maturation as simple culture (COC's only), and COC's & cumulus cells co-culture, which later further developed using parthenogenetic activation after IVM. Female Sprague Dawley rat 3-4 week used for these studies, we hyper-stimulated their ovaries using PMSG and hCG 150 IU/kg each. After that, we collected ovaries via dissection and retrieved oocytes. We matured them in TCM 199 supplemented with FSH, Estrogen, EGF, and Pyruvate. After maturation, we activated them using two types of activators i.e. Ethanol 7%, Ionomycin. After that, we saw and compared their developmental potential in vitro. RESULTS: Oocytes matured in COC's and Cumulus cell monolayer co-culture (59% ± 4*) showed significantly more even growth and extrusion of the first polar body as compared to the COC's only culture (53.8 ± 7%*). While oocytes activated using Ionomycin showed more promising development until 8 cells/blastocyst level compared to ethanol 7%. CONCLUSION: we concluded that COC's and cumulus monolayer co-culture is better than COC's only culture. Cumulus monolayer provides extra aid in the absorption of nutrients and supplements thus providing a better environment for oocytes growth. Also, we concluded that matured oocytes showed more developmental capacity after activation via ionomycin compared to ethanol.