فهرست مطالب عبد الحسین شاهوردی

In assisted reproductive techniques, the selection of a good quality embryo is an important goal, which requires access to a standard method. Today, there are several methods for evaluating in vitro embryos. The morphological evaluation is based on the cleavage rate, shape and symmetry of the blastomeres in the day 3 and day 5 of development. Also, in the preimplantation genetic diagnosis (PGD) method, embryos are evaluated for genetic diseases, to finally are selected a suitable embryo for transfer to the mother's uterus. Since it is important to achieve a low-risk, and high-precision method for selecting of good quality embryos. Recently, the evaluation of embryonic metabolism using analyzing of the material absorbed and secreted by embryos in culture medium, as a non-invasive assessment, has been proposed, that can predict the embryo growth and development more accurately along with morphological evaluation. Therefore, in this narrative review study, relationship between embryo metabolism and its quality has been discussed as a new approach.

Serotonergic axis has an important role in reproductive process of breeder rooster flocks. The aim of this study was to evaluate semen quality parameters of Ross old broiler breeder roosters, which received serotonin inhibitor (PCPA). In this study, 16 Ross broiler breeder rooster assigned into four groups and received 0, 20, 40 and 80 ppm PAPA during 4 weeks. Then semen samples were collected and frozen. After freezing-thawing process, motion parameters, viability, membrane integrity, morphology, acrosome integrity, mitochondria activity, lipid peroxidation and DNA fragmentation were evaluated. In results, using 20 and 40 ppm PCPA improved total motility, progressive motility, membrane integrity, acrosome integrity, viability and decreased apoptosis rate in frozen-thawed semen samples. Using PCPA did not have any significant effect on morphology, mitochondria activity, lipid peroxidation and DNA fragmentation of semen samples. It seems to using 20 and 40 ppm PCPA could be a suitable method to improve Ross old broiler breeder rooster semen quality parameters after freezing-thawing process.

Sperm associated antigens (SPAGs) play an important role in the incidence of various cancers including breast, lung, liver, and bladder. SPAGs are also important in sperm functions, such as motility. However, it seems that sperm cryopreservation as one of the assisted reproductive techniques (ART), can affect the expression of these genes. This study aimed to evaluate the effect of freezing on the expression of human SPAG 5 and 9 in human spermatozoa.

In the present experimental study, twelve semen samples in terms of normozoospermic parameters were collected from individuals referred to the Royan Institute, and progressive motile sperms were isolated by density gradient centrifugation (DGC). Each sample was divided into two, non-frozen (control) and frozen groups. After rapid freezing and three-day storage in liquid nitrogen, samples were thawed in tap water and incubated for 2 hours of recovery-time in a CO2 incubator. RNA extraction in both groups was performed using TRIzol; and SPAG5 and 9 were evaluated by Real-time PCR technique.

Based on statistical analysis, the expression of SPAG5 decreased significantly in the frozen group compared to the control group (P≤0.05); In contrast, there was no significant difference in the expression of SPAG9 between the control and frozen groups.

Considering the cold shock in the future of cell, a significant reduction in SPAG5 expression in the frozen group may indicate probable influences in the derived fetus.

Nowadays, infertility is one of the most common problems in the world. Statistics show that about 20.2% of Iranian couples are infertile, which is higher than the global average (12-15 percent); and according to the reports, 70% of these disabilities had male factors. New approaches are available to diagnose male infertility problems; one of them is the study of nuclear receptors. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor super family that have been implicated in energy homeostasis, and modulate lipid and glucose metabolism. Although several studies have been confirmed the pivotal roles of these receptors on fatty acid metabolism and female fertility, fewer information exist on PPARs roles in male fertility. Thus, in this review, we at first illustrated the role of PPARs in reproductive tissues. Then, specific studies on the effect of these receptors and their ligands in male reproductive tissues were described. Regarding the crucial role of sperm metabolism and producing sufficient energy for sperm motility in male fertility, the regulation of PPARs expression, particularly the PPARγ isotype on spermatogenesis and sperm movement parameters were investigated, in the following. It seems that the study of these receptors and their role in sperm metabolism, may afford novel research opportunities for the recognition of these receptors and their interactions with other nuclear receptors role in male infertility disorders imputable to metabolic diseases such as obesity and diabetes.

According to the formation and evolution of life along with static magnetic fields,the permanent exposure has given adaptive ability to beings. Therapeutic magnetism is one of the branches of complementary medicine which uses  the low intensity and non-harmful magnetic fields to the body. By studying in infertile couples (20% male factor), the only cause of infertility and in 50% of cases it is considered as an intermediate factor. One of the influential factors in infertility in men is sperm. In the present study, normal specimens in the magnetic field under the intensities of 1,6 and 12 millitesla and at 1,3 and 5 h intervals. Sperm movement rate was evaluated by CASA, as well as sperm viability, by eosin staining of necrosin and morphology by staining Papanicula. The results of this step on normal sperm showed a significant reduction in the sperm movement ,which  that was not affected by the field. Morphological studies also show that sperm motility is not affected by magnetic field.. the survival rate of sperm  was affected by the magnetic field was significantly reduced, and the sperm morphology remained unchanged

Selenium is an essential nutrient for physiological function, including immunity, Reproduction and nervous systems. This element as glutathione peroxidase enzyme cofactor, improve fish growth and propagation index via reducing of free radicals. In the present study, the effect of different levels of organic selenium were evaluated on some antioxidant indices, such as total antioxidant capacity (TAC) and malondialdehyde (MDA) and Reproduction indices including, gonadal development, histology evaluation, fertilization rate and survival of the treated fish larva. Adult goldfish were fed with 0 and 0/5 mg of organic selenium (Selplex) per kg food for 8 weeks, and HCG hormone was injected Intraperitoneally to induce the gonadal development in the treated broodstocks. Results showed that the TAC in blood serum treated with selenium broodstocks was increased and MDA levels were also significantly reduced. Histological studies showed selenium treated testicular tissue broodstocks, the level of mature spermatozoa are significantly higher than the control group and in the ovarian tissue, sexual maturation in the control group and treated group were in stage 2 and 5 respectively. In addition, rates of fertilization (%91/17), deformity (%8/45) and survival (%87) in the Selenium treatment compared to the control group showed significant improvement. Therefore, Significantly higher body weight, antioxidant function, gonadal development in the fish treated with 0.5 mg selenium were shown in compare to the control group, to which no organic selenium was fed.

 One of the major challengs in ovarian tissue transplantation is overcomeing ischemia/ reperfusion injuries. During ischemia–reperfusion processes, oxygen free radicals constitute the most important component that induces damage of the grafted tissues. The aim of this study was to investigate the effect of L-Carnitine (LC) as an antioxidant on heterotopic transplantation of mouse ovarian tissue. In this laboratory experimental study, 5- week old female NMRI mice were divided into four groups: control, transplanted without administration (autograft), sham group (autograft+ saline) and LC group (autograft+ L- carnitine). Left ovarian tissues were transplanted into the Gluteal muscle for 3 weeks. After this time, ovarian tissues from all groups were removed and fixed in formalin for histological studies. Furthermore, rate of Caspase- 3 was assessed by immunohistochemistry test. Lipid peroxidation was assessed by measuring  malondialdehyde (MDA). One-way ANOVA and Tudey test was used to analyze the data using the spss 16 software. Significance was defined as P≤0.0. The study results indicated that total follicular count in transplantedwithout administration and sham groups was significantly lower than the control group (p<0.05), but there was no significant difference between the control and LC groups. In addition, the rate of caspase-3 was decreased in the LC group, but no significant difference existed between all groups (p<0.05). A significant reduction in the concentration of MDA was observed in the LC group than that in the other transplanted groups (p<0.05). In this study, LC could improve the ovarian reserve to some extent, but its effect was not significant.

Spermatogonial stem cells are the undifferentiated cells in the basal membrane of the seminiferous tubules and are able to produce sperm in male testis. Different experimental studies proved the capatility of undifferentiated spermatogonial stem cells to culture under different conditions, after isolation of the testes. In this study mouse neonate testicular cells (5-7 days-old NMRI strain) isolated by digestive enzymes include Collagenase IV, Dispase and DNAse. Then, approximately 106 cells were cultured in both adherent on MEF feeder layer and non-adherent culture system on 1% agarose with condition mediums that contained growth factors. Studies demonstrates that testicular cells cultured in a non-adherent condition (non-adherent colonies) were able to form colonies similar to spermatogonial stem cell colonies in adherent condition (adherent colonies) and clearly expressed germ cell marker. Electron microscope analyses made evident that both types of colonies were similar to localized spermatogonial stem cells on the basement membrane of seminiferous tubules and showed a high nucleus/cytoplasm ratio. Immunocytochemistry assays proved that both of colony expressed germ cell markers β1-Integrin, α6-Integrin and Oct4 positive. Real-time PCR analysis revealed significant differences in the expression of germ cell genes Oct4, Sox-2, CD9 and EPCAM, between these colonies and somatic cells. On the other hand, high expression of Ki67 in these colonies showed that these cells have proliferative characteristics. These results proved that a non-adherent culture system similar to adherent culture could provide a favorable method for in vitro short-term culture of spermatogonial stem-like cell colonies.

The aim of this study was cloning of the beta subunit of human chorionic gonadotropin in an appropriate vector for production of transgenic chicken trrough sperm mediated gene transfer. In this regard, transgenic chicken production tecnology has taken into consideration for having many advantages such as short generation times, the large number of production of offspring and suitable pattern of protein glycosylation. To date, no study has been conducted on the cloning of the beta subunit of human chorionic gonadotropin for rooster sperm. For this purpose, the hormone beta subunit were amplified by a specific primer pairs, and cloned in T vector. The recombinant plasmid was transformed into Competent E. coli cells and colonies that containing recombinant plasmids were selected by colony PCR.The validity of extracted plasmid were analyzed by enzyme digestion and sequencing. The beta chain of T vector was isolated and was cloned again into pcDNA3.1 + expression vector. The results of enzyme analysis and sequencing indicated that recombinant plasmid pCDNA3.1 +/βhCG were cloned with the correct sequence and completely matched up with human chorionic gonadotropin beta subunit gene that can be concluded that it has sutible stracture for sperm mediated gene transfer.

The aim of this study was to investigate the effect of fed fish oil (FO) with or without vitamin E for mothers on the testis cells of male offsprings.
Sixty mature female NMRI mice were divided into different groups: control (CTR; Standard diet(vitamin E 50 mg IU/kg) pre and postnatal period); I) Gavages 0.01 ml/day/mother fish oil (FO)ऴ diet during prenatal period; II) Gavages FOऴ diet during postnatal period; III) consumed VITE(125 mg IU/kg) 2.5 folded greater than standard recommendations(2×)during prenatal period; IV)consumed VITE(2×)diet during pre and postnatal period; V)consumed VITE(2×)diet during postnatal period; VI) Gavages FO咄(2×) diet during prenatal period; VII) Gavage FO咄(2×)diet during postnatal period ;VIII) Gavages FO咄 (2×)diet during pre and postnatal period. After weaning, the testes were collected and histological data were analyzed using SAS software by Duncan test.
testes cells length, width and weight was lower in offspring which their mothers fed FOऴ diet during prenatal, (p The positive effects of supplementation maternal diet by FO with VITE or sole VITE was observed. Thus, antioxidants should be consumed along with omega-3 fatty acids in maternal diet.

Patulin is a toxin from Mycotoxins family that is produced by several species of mold especially Aspergillus, Byssochlamys and Penicillium ecspansom. The presence of Penicillium Ecspansom on apples and its juice causes concern. Patulin has teratogenic on the fetus, genetic mutagenicity and carcinogenicity effects. In this study, we have tried to investigate the effect of this toxin on human sperm and its role as a male factor in men's infertility. The purpose of study Patulin toxin has destructive and harmful effects on human sperm DNA and its excessive amounts in fruit juices. The present study has been done on 40 samples taken from infertile patients referring to Royan Institute. Mobility, pH, morphology, viability and sperm DNA fragmentation were studied before and after the use of Patulin with 5 doses of 0.02, 0.05, 0.16, 0.33 and 0.5 micrograms per milliliter that was diluted with medium HamsF10. The results showed that concentrations of 0.33, 0.5 and 0.05 caused a significant decrease in sperm motility, pH and viability. The concentrations of 0.33 and 0.5 caused a significant damage on sperm DNA but have not affected morphology, the number of positive peroxidase cells and sperm count.

The most important cause of fertilization failure after assisted-reproduction techniques are lack of releasing sperm-associated oocyte activating factors (SAOAFs) and sperm DNA damage. PLCζ is one of the sperm factors that plays important role in oocyte activation. The aim of this study was to compare expression of PLCζ, protamine deficiency (maturity marker of sperm chromatin) and sperm DNA damage between fertile men and oligoasthenoteratozoospermic subfertile men.
In this case-control study, semen samples were collected from 10 subfertile oligoasthenoteratozoospermic and 10 fertile men. Sperm parameters (concentration, motility, morphology), expression of PLCζ, DNA damage, and protamine deficiency were assessed by World Health Organization protocol, flow cytometery, TUNEL and chromomycin A3 staining, respectively.
Percentage of PLCζ- positive sperm, DNA integrity and protamine content significantly was lower in oligoasthenoteratozoospermic men compared to fertile men. In addition, significant correlations were observed between percentage of PLCζ- positive sperm with sperm parameters and percentage of protamine deficiency. A significant correlation was also observed between percentage of DNA fragmentation and protamine deficiency.
In these infertile men, sperm chromatin integrity and percentage of PLCζ-positive sperm decreased extremely. This could be one cause of fertilization failure in these individuals. Therefore, the artificial oocyte activation is recommended for these type of infertile men.

The purpose of this study was to consider the effect of mild oxidative stress period on the frozen-thawed bull semen performance. In this study، 120 minutes was considered for equilibration of sperm before freezing. One µm of Nitric Oxide (NO) at the 0، 45، 90 and 120 cooling period was added to the diluant before cryopreservation. Sperm motion parameters، plasma membrane integrity، acrosome integrity، viability، apoptosis and mitochondria activity were assessed. Induction of oxidative stress with nitric oxide in the T0 and T45 groups led to significant improvement of total (88. 4 ± 2. 8، 84. 7 ± 2. 7) and progressive motility (50. 4 ± 2. 5، 49. 5 ± 2. 5) when compared to other groups. Percentage of plasma membrane integrity and viability were not different in T0 (74. 4 ± 2. 9 and 85. 6 ± 2. 9) and T45 (75. 6 ± 2. 9 and 84. 6 ± 2. 3) groups، where as it was significantly higher when compared with T90 (63. 6 ± 2. 9 and 69. 6 ± 2. 3) and T120 (61. 5 ± 2. 9 and 54 ± 2. 3). Moreover، the highest significant percentage of spermatozoa with active mitochondria was observed in the T0 (82. 5 ± 3. 1) when compared with T45 (65. 7 ± 3. 1)، T90 (42 ± 3. 1) and T120 (43 ± 3. 1). Acrosome integrity and linearity of sperm were not affected by the oxidative stress treatment time. It seems that applying oxidative stress using 1 µM NO would improve post-thawed bull sperm quality at the T0 and T45 time of cooling.

Maturation of oocytes and fertilization in vitro can be considered as one of the most important steps to treat infertility. In this study maturation medium was enriched with Platelet extraction (PL) which has high concentration of growth factors. Meiosis resumption and maturation was monitored after 18 hours of maturation. In this experimental study، oocytes at germinal vesicle stage were obtained from mature NMRI female mice. Maturation medium was αMEM and the control groups had 5-10% FBS and the medium in the experimental groups was enriched by 5، 10% PL and the combination of 5% PL and 5% FBS. Meiosis resumption and maturation were observed after 18 hours. The rate of matured oocytes in the experimental group 5% PL for both COC and DO group was significantly higher than the control groups (P<0. 05). The maturation rate for 5% PL was 61% for the COC group and 72% for the DO group; while this rate for 5% FBS control group was 53% and 50% respectively. PL had a significant effect on meiosis resumption and maturation of oocyte at germinal vesicle stage. Based on these results، PL could be used as a maturation promoting factor.

Fertilization in mammals is dependent on successful connection between the sperm and oocyte plasma membrane. Gametes Binding largely been studied in mice and the crucial role of CD9 a member of tetraspanin family in gametes binding is shown in mice. CD9 found in the inner membrane of the acrosome. And only a small amount of CD9 is expressed on the plasma membrane of mid piece. Inner sperm membrane proteins play an important role in the fertilization process. The aim of this study was to obtain correlation between the percentages of CD9 positive sperm with outcome of intra uterine insemination (IUI).

A total of 120 semen samples from infertile couples referred to Royan Institute who were undergoing IUI treatment were collected. Semen parameters such as motility, morphology, sperm concentration and viscosity were analyzed according to WHO 2010 guidelines. Sperm washed and stained by using anti-CD9 monoclonal antibody. The samples were analyzed by flow cytometry technique.

This study shows that CD9 is detected only in 19% of mature sperm. The result of the ROC area under the curve is equal to 0.446 therefore CD9 is not a good variable predictor for fertility status. (Area=0.446, P=0.482).

There was not a significant difference between semen parameters of Fertile and infertile groups. The results didn’t show correlation between CD9 expression and fertilization in patients who are candidates for IUI.

culture medium can be considered as one of important part in cell culturing and in vitro Folliculogenesis and in recent years lots of research have been focused on improving culture medium. Platelet Layset with high percent of growth factors and micro elements can be effective on oocytes growth and development. This study was done to evaluated whether Follicles and oocytes can survived and growth as mature oocytes in medium which enriched by platelet layset as serum instead of FBS. we used pre-antral follicles with diameter of 100-130µm from prepubertal NMRI mouse and we cultured them for 12 days in different culture medium. We had 4 different culture mediums first and second medium was enriched by 5 and 10% PL as our experimental groups and two groups enriched by 5 and 10% FBS as our control groups. We refreshed our medium every other day and our assessment was done before each refreshments. We evaluated survival rat after one day of culturing and at the end of culturing as well as growth of oocytes in different mediums. oocytes in medium with PL had significant growth and reached to mature size as oocytes in medium with FBS. Oocytes diameter after 12 days reached to 81.66 and 81.35µm in medium with 5 and 10%PL compare to oocytes in 5 and 10% FBS which was 82.69 and 82.19µm respectively p˂0.05. Meanwhile survival rat after one day also was significant and was 93.75% and 97.91% for 5 and 10%PL and 100% and 94.74%for 5 and 10%FBS. platelet layset with high percentage of growth factors and micro elements can be effective on oocytes growth and we can use it as a good replacement for FBS.

One of the challenges facing ovarian transplantation is overcoming the ischemia/ reperfusion injury. When blood supply to the implanted tissue is suboptimal (immediate post implant period)، ultrasound therapy has been shown to increase mass transport. The aim of this study was to investigate the effect of ultrasound on heterotopic transplantation of mouse ovarian tissue.

Adult female NMRI mice were selected and divided into the control، sham and experimental groups. In the experimental group، left ovarian tissue، after auto transplantation into the back muscle، underwent ultrasound exposure with intensity of 0. 3 w/cm2، frequency of 3MHz & pulse mode of 1:4، for five minutes daily. The transplanted ovaries were removed and immediately fixed for histological study. Furthermore، rate of angiogenesis was assessed by immunohistochemistry Cd31 test.

The study results showed that the CD31 angiogenic factor was expressed more in the irradiated animals than in control group animals and ultrasound-therapy resulted in better follicular preservation.

The ultrasound therapy can improve preservation of ovarian follicle. This is probably due to acceleration of angiogenesis and increase in production of growth factors by low intensity pulse ultrasound.

In patients with non-obstructive azoospermia (NOA), vital spermatozoa from the tissue is obtained from testes by enzymatic treatment besides the mechanical treatment.

To increase the sperm recovery success of testicular sperm extraction (TESE), with enzymatic digestion if no sperm is obtained from testis tissue by mechanical method.

Tissue samples were collected from 150 men who presented with clinical and laboratory data indicating NOA by means of TESE and micro dissection TESE methods. Initially, mature spermatozoa were examined for by mechanical extraction technique shredding the biopsy fractions. In cases whom no spermatozoa was observed after maximum 30 min of initial searching under the inverted microscope, the procedure was followed by enzymatic digestion using DNaseI and collagenase type IV. Surgery type, pathology, AZF, karyotype, hormones and testis size were compared in patients.

Of 150 cases with NOA, conventional mincing method extended with enzymatic treatment yielded successful sperm recovery in 13 (about 9%) patients. Comparison of parameters revealed that level of FSH and LH were significantly different (p=0.04 and 0.08 respectively) between two groups that response negative and positive to enzymatic digestion.

The combination of conventional TESE and enzymatic digestion is an effective method to recover spermatozoa. The benefit of the mincing combined with enzyme to sperm retrieval for NOA firstly shorten the mechanical searching time, leading to minimizing further cellular damage as well as exposure to external conditions, and secondly reduce the number of cases with sperm recovery failures. Also, the serum level of FSH and LH are factors that influence the chance of sperm retrieval.

Formaldehyde (H2C = O)، is a member of aldehyde family with a simplest organic molecule، used in various industries. The aim of this experimental study was to investigate the effects of simultaneous formaldehyde and noise exposure on hormonal pituitary-gonadal axis in adult male mice. 46 adult male mice were randomly assigned to one control group and three experimental groups which were exposed to formaldehyde vapor (10 ppm) (F)، noise (100 dB) (N)، and simultaneous formaldehyde with noise (NF)، respectively for 10 days (8 hours a day). At the end of the experiment half of the mice of each group were sacrificed 24 hours after exposure (short-term effects) and the rest were sacrificed 35 days after the end of exposure (long-term effects). Plasma concentrations of testosterone، LH، FSH were measured using the ELISA method. In short-term analysis، the serum testosterone in groups N، F، and NF were 2. 73±0. 24، 2. 58±0. 16، and 2. 03±0. 22، (Ng/ml) respectively which had a significant decrease (p<0. 001) compared to control group (3. 82±0. 35). The LH level in groups F، and NF also showed significant reduction compared to control group (p<0. 05 and p<0. 001). Reduction of serum testosterone and LH level may be potentiated by noise (700-5700 HZ) in the simultaneous exposure to formaldehyde and noise.

In recent years, considerable advances have been made in the field of regenerative medicine. Unlike embryonic stem cells, which pose the problems of ethical concerns and cause severe immunological reactions as well as neoplasma formation after transplantation, umbilical cord blood is a primitive source of mesenchymal stem cells that covers the benefits of both embryonic and adult stem cells. It has been determined that the proliferation capacity of cells is critically linked to the maintenance of the length of telomeres by telomerase activity. Since there is no information accessible regarding the pattern of telomerase activity in UCB-MSCs through several passages, the aim of this study was the evaluation of telomerase activity in UCB-MSCs, as a predisposing factor for cell immortalization. No telomerase activity was detected in UCB-MSCs from several passages applying telomerase rapid amplification protocol (TRAP). Since there is a direct correlation between the activation of telomerase expression and neoplasma formation in adult somatic cells, UCB can be assumed as an excellent source of MSCs for therapeutic application with a high level of safety. According to the histological results, RT-PCR and biochemical assays, MSCs derived from UCB showed high differentiation capacity to bone and cartilage. UCB-MSCs showed very low level of differentiation potential to adipocytes. Our results showed that UCB-MSCs maintain their self-renewal and differentiation potential through several passages. Since a large number of metabolically active cells must be available in cell therapy, high proliferation capacity through several passages is a great advantage for large scale expansion of UCB-MSCs.

The Increase of oxidative stress production a causes after oxygen free radicals one of the factors involved in male infertility. In this study the role of leptin in product reactive oxygen species in seminal plasma in adult rat were evaluated Sixty-five male rats were randomly divided into four groups. The control group receives normal saline and other animal, in experimental groups 2, 3 and 4 were given daily intraperitoneal leptin injections of 5, 10, or 30 mg/kg body weight for 7, 15 and 42 at all. Reactive Oxygen Species Levels and Expression of leptin were measured respectively with DCFH-A and immunohistochemical and sperm parameters were assessed by computer-assisted semen analysis (CASA). Two ways ANOVA with Tukey post hoc test were conducted to compare mean of groups. P-value < 0.05 was considered significant. The results show that sperm count was higher in treated groups than that of control group. These differences were significant (p≥0.05). The interaction between treatment and time showed significant effect on ROS variations.After immune staining leptin were found in the seminiferous, tubules and Leptin receptor were found in the interstitium. the results of this study indicate that leptinadministration by increasing levels of oxygen free radicals, may lead toincrease of sperm count and the number of abnormal sperm in the experimental groups

Cloning is asexual reproduction from of some selected individuals in such a way that the newly produced offspring is like their parent in genetic content. The base of cloning is transferring nucleus (somatic or stem cell) into enucleated oocyte. Considering the purpose, there are two types of cloning: reproductive cloning with purpose of having an offspring that is similar to his parent in genetic content and therapeutic cloning whose main purpose is to useembryo stem cells for curing some diseases especially degenerative ones. This is a multistep process and its steps include: donor cell, the recipient oocyte, nuclear transfer process, embryo culture, and embryo transfer. Cloning can be applied in different areas including genetic engineering, protecting a special species, domestic animal cloning, and therapeutic cells through embryo stem cell nuclear transfer. One of the main purposes of cloning is producing transgenic animals. Transgenesis is the process of transferring the sequence of forign DNA to multicellular organism and making sure that theses sequences will be transferred to the offsprings. Genetic engineering technology has provided a more efficient way for gene transfer in cows, pigs, sheep, goats, chickens, and fish. Transgenic animal reproduction has many different advantages. Some of them include: producing more efficient animals for the purpose of studying about human related diseases, producing animals for the purpose of using medical protein, using their organs for transplantation, producing animals for the purpose of regulating gene expression and the coding sequence. Right applications of gene transfer in farm animals improve the quality and quantity of yield, disease resistance, production of valuable proteins in the milk glands or other organs, genetic modifications of pigs for xenotransplantation and production of new animal models for the time when the rodent cannot be applied for the problem of interest. This paper reviews the recent advances and applications of transgenesis in livestock and their derivative products. We also analyze here some emerging applications of livestock transgenesis in the field of pharmacology, meat and dairy industry, xenotransplantation, and human disease modeling.

There are a growing number of recombinant proteins that have been expressed in milk and one can consider putting any gene of interest under the control of the regulatory elements of a milk protein gene in a dairy farm animal. Nuclear transfer (NT) using donor cells are an efficient means for generation of transgenic animals. In this study in order to produce a transgenic goat with capability of secretion of recombinant human coagulation factor IX (rhcfIX) in the milk, a gene construct (pBC1-hfIX) was made by insertion of (rhcfIX) cDNA after goat beta-casein promoter into the expression cassette. Then, cell lines of goat fetal and adult fibroblasts were transfected by pBC1-hfIX. Fetal fibroblast cells isolated from day 35 to day 40 fetuses. Next, positive clones (the fibroblast cells that contain the gene construct) were selected and confirmed by PCR. The transfected fetal fibroblast cells were allowed to reach confluency and further cultured in 15% FBS in DMEM for an additional 72 h (full confluency) to synchronize G0/G1 cell cycle stage cells. Immature oocytes were obtained from slaughterhouse and matured before enucleation and NT. The reconstructed oocytes electrofused with donor somatic cells, and activated with ionomycin and 6DMAP. Reconstructed embryos were transferred into synchronized recipients. A total of 64 embryos derived from transgenic fetal fibroblast cells were transferred into 12 recipients. Two recipients (16.6%) were confirmed pregnant at Day 42 by ultrasound. Of these, two kids derived from the fetal fibroblast cell line. Presence of the coagulation factor IX gene was confirmed by polymerase chain reaction, sequencing, and fluorescent in situ hybridization analyses. These results demonstrate that goat fetal fibroblasts with human coagulation IX factor gene can direct full-term development following NT. This is the first live born of transgenic farm animal in Iran.

The fate of mouse spermatogonia stem cells’ (SSCs) regulation is controlled by the interplay of signaling networks that either promote self- renewal or induce differentiation. It has been proven that cytokines, growth factors and feeder layers are capable of inducing self- renewal or reducing apoptosis in SSCs, suggesting that the self- renewal signaling pathways and maintenance of cells are promoted by other such triggers as the chemical or physical properties of the Extracellular Matrix (ECM). To investigate the issues, a synthetic polyamide matrix (ultra–web) whose nanofibrillar organization resembles the ECM/basement membrane was employed. Testis cells harvested from the NMRI mouse were cultured on either a nanofiber or a non-nanofiber surface for 10 days. GDNF, bFGF and EGF were taken into account as growth factors. Several tests were conducted on the7th and 10th days past of the culture. Colony assay revealed a higher colony number as well as higher cell number/colony on the nanofiber surface on day 10. Such SSCs markers as? 6, c-kit,? 1, thy-1, OCT-4 and Plzf were also detected using immunoflourecent staining. Reverse Transcription-PCR test revealed the expression of such genes as GFR?-1,Stra8, Vasa,? 6,? 1, thy-1 and OCT-4 for SSCs on the nanofiber and the non-nanofiber surfaces. For functional evaluation, the single cells’ suspension from SSCs colony were microinjected into seminiferous tubules of infertile recipients. Donor germ cells from both groups (nanofiber and non- nanofiber surfaces) were present in the recipient testis. Results indicated that the growth of SSCs on this nanofibrillar surface greatly enhanced proliferation, self- renewal and maintenance in comparison with growth on the non-nanofiber culture surface, despite the presence of growth factors in either of the systems. Thereby it being concluded that a nanofiber surface can provide a suitable microenvironment for SSCs in an in vitro culture system.