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Investigations pertaining to spermatogonial stem cells (SSCs) have led to the use of these cells in a variety of fields including infertility treatments, production of transgenic animals, and genome editing. The aim of the present study was to investigate the plausibility of regenerating spermatogenesis in infertile roosters by transplanting transfected SSCs into testes. Spermatogonial stem cells were isolated and cultured for seven days. Afterward, pDB2, a plasmid vector carrying a reporter gene, GFP, was transfected into the SSCs. Transfected SSCs were transplanted into the left testis of infertile roosters. Tissue samples from the recipients' testes were obtained six weeks after the transplantation and transplanted SSCs were observed in the basement membrane. After eight weeks, GFP-positive spermatozoa were observed in collected semen from the recipient roosters and GFP gene in spermatozoa was confirmed using PCR. The recipient roosters were mated with hens. Hatchlings were visually checked and their tissue samples were tested by PCR to identify transgenesis but both of them were negative. Overall, it seems that regeneration of spermatogenesis in roosters via transfected SSCs is possible but more studies are need to produce recombinant proteins by this way.

Maintaining of female fertility is strictly dependent on proper hormonal regulation. Granulosa cells (GCs) are components of ovarian follicles, and they are important in paracrine regulation of the ovary. Preovulatory follicle GCs are responsible for production of estrogens to the ovary microenvironment and lead to the LH surge. Proper functioning of GCs is necessary to ensure appropriate conditions for oocyte development, maturation, ovulation and its release to the oviduct. Long-term in vitro culture of GCs show significant stem-like characteristics. Understanding the molecular processes underlying GCs differentiation towards different cell lineages may reveal other possible stem cell markers. A transcriptomic analysis of short-term primary in vitro cultured GCs, which were isolated from porcine preovulatory follicles was the major focus of the study. The ontological groups herby considered are associated with endodermal and epithelial tissues. Results were and compare to freshly isolated GC cells. 6 the most reduced expression: HSD17B1, DAPL1, NEBL, MAL2, DAB1, ITM2A were chosen for analysis. These genes have been response for processes associated with GCs development and differentiation towards endodermal and epithelial tissues, which make them important for further consideration.

Telomerase activity is highly correlated to the proliferation capacity and immortality of cells. To evaluate the possibility of continuous culture, myoblasts were isolated from the Pectoralis thoracicus muscle of newborn turkeys and maintained in 2D (adherence based) and suspension cultures. Furthermore, adherent myoblasts were differentiated into myotubes. Telomerase activity was evaluated in all types of obtained cultures. The expression of telomerase related genes, including TERT1, TERT2, dyskerin, as well as myogenesis related genes, including myogenin, MyoD, MRF1 and MRF5 were measured. Telomerase bands were detected in both adherent and suspended cells, but they were not detected in samples from rat muscle. Myotube differentiation caused a significant reduction in the expression of TERT1, TERT2 and Dyskerin, while MyoD, Myogenin and MRF4 were upregulated in myotubes vs. myoblasts. Long-term culture of suspended myoblasts caused a significant increase in TERT1 levels, with no significant change in expression of myogenesis related genes. Overall, the results show that myoblasts are able to grow in suspension without losing their myogenic properties. Furthermore, upregulation of TERT1 indicates continued proliferation of myoblasts and generation of enough daughter cells necessary for in vitro meat production. Running title: Telomerase activity and myogenic properties of cultured Turkey satellite cells

ADSCs are readily accessible and widely available. Isolated through a minimally invasive procedure from adipose depots, they can be found at diverse body location, where they served various functions, including energy homeostasis. They can be obtained upon surgeries from otherwise waste tissues, like after excision of fat tissue or liposuction. In addition, due to the possibility to isolate many ADSCs, in vitro proliferation can be performed in a short time period, resulting in cells showing more predictable results[1]. For this study ADSCs were obtained from waste material following routing sterilization procedures of dogs. This study aimed to analyse the expression of MSC specific markers before and after in vitro differentiation of ASCs. Three positive and three negative markers were analysed, CD105, CD73, CD90, CD34, CD14 and CD45. There were significant differences detected in the expression of all of the genes, with most of them exhibiting notable downregulation. The only exception, CD14 showed major upregulation after the process of differentiation. These changes confirm the success of differentiation, as well as suggest that this process significantly lowers the stem-like ability of ASCs. This knowledge should serve as a reference for further molecular and clinical studies, possibly aiding the understanding of the internal mechanisms governing the differentiation and stemness of ASCs, to enable their widespread and safe application in regenerative medicine. Running title: Mesenchymal markers during ASC osteogenic differentiation

Modern science is becoming increasingly committed to environmentally friendly solutions, mitigating the impact of the developing human civilisation on the environment. One of the leading fields aimed at sustainable agriculture is in vitro meat production. Cellular agriculture aims to provide a source of animal-free meat products, which would decrease worldwide nutritional dependency on animal husbandry, thereby reducing the significant impact of this industry on Earth’s climate. However, while some studies successfully produced lab-based meat on a small scale, scalability of this approach requires significant optimisation of the methodology in order to ensure its viability on an industrial scale. One of the methodological promises of in vitro meat production is the application of cell suspension-based bioreactors. Hence, this study focused on a complex transcriptomic comparison of adherent undifferentiated, differentiated and suspension-cultured myosatellite cells, aiming to determine the effects of different culture methods on their transcriptome. Modern next-generation sequencing (RNAseq) was used to determine the levels of transcripts in the cultures’ cell samples. Then, differential expression and pathway analyses were performed using bionformatical methods. The significantly regulated pathways included: EIF2, mTOR, GP6, integrin and HIFα signalling. Differential regulation of gene expression, as well as significant enrichment and modulation of pathway activity, suggest that suspension culture potentially promotes the ex vivo-associated loss of physiological characteristics and gain of plasticity. Therefore, it seems that suspension cultures, often considered the desired method for in vitro meat production, require further investigation to fully elucidate their effect on myosatellite cells and, therefore, possibly enable their easier scalability to ensure suitability for industrial application.

One of the major reasons for implantation failure and spontaneous abortion is a high incidence of preimplantation chromosomal aneuploidy. Lapatinib simultaneously inhibits EGFR and HER2, leading to apoptosis. We hypothesized a higher sensitivity for aneuploid cells in preimplantation embryos to lapatinib based on reports of aneuploid cell lines being sensitive to some anticancer drugs. Late 2-cell mouse embryos were treated with lapatinib after determining a nontoxic dose. Morphologies were recorded 24, 48, and 60 hours later. The effect of lapatinib on the aneuploidy rate was evaluated by studying blastocyst cells using FISH. Although the rate of development to 8-cell and morula stage was higher in the control group (p < 0.05), there was no difference in development to the blastocyst stage at the same studied intervals between lapatinib-treated and control groups (p = 0.924). The mean number of cells in morula and blastocyst stages were not different between the groups (p = 0.331 and p = 0.175, respectively). The frequency of aneuploid cells and diploid embryos was, respectively, significantly lower and higher in lapatinib-treated embryos, (p < 0.001). Since lapatinib treatment reduced the aneuploidy rate without impact on the development of mouse preimplantation embryos to the blastocyst stage and number of total cells, lapatinib seems useful for prevention of preimplantation aneuploidy in in vitro fertilization.

Objective: Retrieval of high quality follicles and oocytes from transplanted ovaries is essential for higher fertility preservation efficiency. The effect of vascular endothelial growth factor (VEGF) was evaluated on the survival rate of preantral follicles following ovarian transplantation. Material and Methods: Prepubertal female mice were divided to 6 groups including: control (C), transplanted with no VEGF treatment (T) and transplanted with different dosages of VEGF [0.5 µg/mL (TV1), 1 µg/mL (TV2), 2 µg/mL (TV3), and 4 µg/mL (TV4)]. Twenty-one days later, the left ovaries were removed and transplanted on gluteal muscle. Each dose was injected directly into transplanted ovary. Twenty-one days after transplantation, the ovaries were taken, and follicles and cumulus-oocyte-complexes (COCs) were released using 26-gauge needles with a stereo microscope. The number of healthy COCs, matured oocytes, and in vitro developed embryos after fertilization in vitro were evaluated to determine the best dose of VEGF. Follicle number and follicular growth was evaluated relative to the dose of VEGF provided. Transplantation and VEGF treatment with the best dose was performed as mentioned above and in vitro follicle growth in transplanted ovaries was compared with opposite ovaries (OPP). Results: COC retrieval was significantly lower in the transplanted groups compared with the control group (p

Avian transgenesis has been considered as a useful tool for many purposes including improvement of the poultry industry, production of recombinant proteins and developmental biology. Due to the importance of the poultry industry in human nutrition, many efforts have been made for the improvement of the poultry industry in the aspect of egg and meat production for higher quantity or quality. After the development of transgenic technology the idea of transgenic poultry was raised for the application of this technology in the poultry industry. Due to the important role of recombinant proteins and humanized antibodies for medical and diagnostic purposes, mass production of these biological macromolecules is very important in human health. Development of transgenic technology in poultry science possibly helps to improve the efficiency of poultry industry productions and it may cause the development of different branches of avian industry with novel productions.

One of the most important factors affecting survival rate of ovarian follicles during transplantation period is proper vascular development. The objective of the study was to evaluate the effect of vascular endothelial growth factor (VEGF) on auto-transplanted ovarian tissue. Twenty-one-day-old female mice (n = 30) transplanted as control group and 21-day-old female mice (n = 40) were divided into 4 groups that were treated with 0.5, 1, 2 and 4 µg/mL of VEGF directly injected to auto-transplanted ovarian tissue. Twenty-one days after transplantation, mice were treated with 7.5 IU pregnant mare serum gonadotropin and human chorionic gonadotropin. Transplanted ovaries were removed and sections were prepared from transplanted tissues for staining. The most effective dosage of VEGF on transplanted tissue was determined over H&E (hematoxylin and eosin) staining results. Slides were compared using TUNEL staining and CD31 assay for the most effective dosage. The percentages of preantral and antral follic...

Selenium binds some enzymes such as glutathione peroxidase and thioredoxin reductase, which may be activated in biological infections and oxidative stress. Chemical and physical methods for synthesizing nanoparticles, apart from being expensive, have their own particular risks. However, nanoparticle synthesis through green chemistry is a safe procedure that different biological sources such as bacteria, fungi, yeasts, algae and plants can be the catalyst bed for processing. Synthesis of selenium nanoparticles (SeNPs) by macro/microorganisms causes variation in morphology and shape of the particles is due to diversity of reduction enzymes in organisms. Reducing enzymes of microorganisms by changing the status of redox convert metal ions (Se2-) to SeNPs without charge (Se0). Biological activity of SeNPs includes their protective role against DNA oxidation. Because of the biological and industrial properties, SeNPs have wide applications in the fields of medicine, microelectronic, agriculture and animal husbandry. SeNPs can show strong antimicrobial effects on the growth and proliferation of microorganisms in a dose-dependent manner. The objective of this review is to consider SeNPs applications to various organisms.

The protective effect of amifostine against cyclophosphamide (CP) was evaluated on mouse oocytes.Female mice were divided into four groups as follows: group1: cyclophosphamide (CP) (75 mg/kg, i.p) injection, group2: amifostine (250 mg/kg, i.p) injection, group3: amifostine (250 mg/kg, i.p) administered prior to CP (75 mg/kg, i.p) injection, Control group with injection of saline. About 21 days after injection, in vitro maturation (IVM) of oocytes was recorded. Furthermore the percentage of aneuploid oocytes was determined.In the CP group IVM rate was significantly decreased and aneploidy rate was significantly increased when compared to other groups (p 0.05). With the administration of Amifostine prior to CP injection IVM rate was increased and aneploidy rate was decreased.Depletion in IVM rate with administration of CP is due its adverse effects on oocyte quality. Amifostine administration prior to CP injection appears to modulate deleterious effects of CP on oocytes.

This research studies the effect of water-alcohol Papaver rhoeas L. (P. rhoeas) extract on mouse ovaries and in vitro development (IVD) of oocytes. Different dosages of P. rhoeas extract (25, 50, 100, 200 mg/kg body weight) were injected intraperitoneally (i.p.) during a period of 12 days. Following superovulation, the numbers of ovulated oocytes, the rates of in vitro fertilization, IVD and the cellularity of blastocysts were recorded. Additionally, effect of the best dosage on ovarian follicle population and the ability of immature oocytes to mature in vitro were evaluated. Administration of 200 mg/kg significantly increased the percentage of 4–8 cells, morula and blastocyst embryos compared to the control group (p

This study investigated the cytoprotective effects of amifostine against the adverse effects of cyclophosphamide relying on ovarian cell death markers and the fertilization rate of the surviving follicles as a late outcome of the study. Combined pretreatment of amifostine with cyclophosphamide enabled partial recovery of antiapoptotic messenger RNA (mRNA) expression levels while a decrease in expression of BAX and Casp3 were identified. The pretreatment of amifostine to cyclophosphamide significantly reduced the proportion of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive primary and preantral follicles (P < .001 and P < .05, respectively). These findings were comparable with the results obtained from protein expression of cleaved caspase 3. The fertilization rate showed a significant capability of amifostine to improve fertilization potency of oocytes exposed to cyclophosphamide (P < .01). In conclusion, administration of amifostine prior to cyclophosphamide might serve as a promising protocol to improve female fertility.

To study assisted reproductive technology (ART) protocols including superovulation, in vitro fertilization (IVF) and in vitro development (IVD) for BALB/cJ mice in comparison with a common ART protocol for NMRI mice.Adult NMRI and BALB/cJ mice were superovulated using a 48 h G-interval. In order to find a more suitable G-interval for the BALB/cJ strain, G-intervals including 44, 46 and 50 h were also examined. Superovulation rates were recorded in all groups. IVF rate of BALB/c oocytes in T6 and mHTF media were compared. IVD rates of BALB/cJ zygotes in mHTF, T6 and G1VIn BALB/cJ mice the highest superovulation rates were observed with 44-46 h G-intervals. However, with a 48 h G-interval, superovulation rates were significantly lower in BALB/cJ compared to NMRI mice (Superovulation with 48 h G-interval and using T6 during all in vitro procedures produces embryos more efficiently for NMRI mice than for BALB/cJ mice. For BALB/cJ mice, a protocol including superovulation with a 44-46 h G-interval, using mHTF during IVF and G1V

The aim of this study was to investigate the survival and development of premature follicles and oocytes from a vitrified-transplanted ovary in a murine experimental model.The 14-day-old mice were unilaterally ovariectomized and the separated ovaries were vitrified by cryotop. After 2 weeks the ovaries were warmed and autotransplanted into the gluteus superfiscialis muscle. After 3 weeks, these ovaries (vit-trans), the ovaries from the opposite side (OPP), and 7-week fresh mouse ovaries as sham and control group (7 week-fresh), were recovered and examined histologically and by TUNEL test.All 4 vitrified-autotransplanted ovaries had developing follicles. Primordial, primary, preantral and antral follicles were found in all three groups (7 week-fresh, OPP and vit-trans). The rate of apoptosis by TUNEL test was similar in all groups and no significant difference was found between vitrified-transplanted ovarian tissue and controls.These data demonstrate successful autotransplantation of vitrified whole mouse ovaries, manifested by the presence of all stages of folliculogenesis. According to the results of this experiment, heterotopic autotransplantation of whole cryopreserved ovary provides the opportunity for follicle development at all stages. However, further experiments are required to improve the efficiency of autotransplantation of cryopreserved ovaries to obtain better results.

This experiment examined the effect of Papaver rhoeas L. extract on in vitro maturation, in vitro fertilization (IVF) and subsequent developmental competence of mouse oocytes. Cumulus oocyte complexes (COCs) at germinal vesicle stage were collected from female Naval Medical Research Institute (NMRI) mouse ovaries. The COCs were transferred to maturation medium supplemented with different concentrations of P. rhoeas extract. Two trials were carried out to examine the effect of low concentrations (0, 10, 15, 20, 25 μg/ml) and high concentrations (0, 50, 100, 200 μg/ml) of the extract. The maturation rate was recorded. After IVF, embryos were cultured and their developmental process was monitored for 96 h. Maturation rate and blastocyst formation improved by using low concentrations of the extract; however, no significant increase was observed when compared to the control group. In addition no significant differences were observed in the fertilization rates of oocytes treated with both low and high concentrations compared to the control group. However, among higher concentrations, 100 μg/ml, P. rhoeas extract significantly increased both the in vitro maturation rate and in vitro developmental (IVD) competence when compared to the control group (P