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Objective: The present study evaluated the efficiency of embryo development and pregnancy of somatic cell nuclear transfer (SCNT) embryos using different source-matured oocytes in Camelus dromedarius.Methods: Camelus dromedarius embryos were produced by SCNT using in vivo- and in vitro- matured oocytes. In vitro embryo developmental capacity of reconstructed embryos was evaluated. To confirm the efficiency of pregnancy and live birth rates, a total of 72 blastocysts using in vitro- matured oocytes transferred into 45 surrogates and 95 blastocysts using in vivo- matured oocytes were transferred into 62 surrogates by transvaginal method.Results: The collected oocytes derived from ovum pick up showed higher maturation potential into metaphase II oocytes than oocytes from the slaughterhouse. The competence of cleavage, and blastocyst were also significantly higher in in vivo- matured oocytes than in vitro- matured oocytes. After embryo transfer, 11 pregnant and 10 live births were confirmed in in vivo- matured oocytes group, and 2 pregnant and 1 live birth were confirmed in in vitro- matured oocytes group. Furthermore, blastocysts produced by in vivo-matured oocytes resulted in significantly higher early pregnancy and live birth rates than in vitromatured oocytes.Conclusion: In this study, SCNT embryos using in vivo- and in vitro-matured camel oocytes were successfully developed, and pregnancy was established in recipient camels. We also confirmed that in vivo-matured oocytes improved the development of embryos and the pregnancy capacity using the blastocyst embryo transfer method.

Several studies have reported methods to estimate the parturition date of dogs using ultrasonographic measurements. However, these prediction models were mainly determined using ultrasonographic measurements of naturally pregnant small- and medium-sized dogs, and no such studies have been performed using dogs carrying cloned fetuses produced via somatic cell nuclear transfer. The present study evaluated the abilities of three reference formulas (Luvoni and Grioni, Milani et al., and Groppetti et al.), all of which were developed using data from naturally occurring pregnancies, to accurately predict the parturition date in surrogates carrying cloned German Shepherd (GS) fetuses. All three formulas were based on the use of inner chorionic cavity diameter (ICC) measurements, obtained via ultrasonography. For evaluation, a total of 54 ICC measurements were collected from 14 pregnant bitches carrying cloned GS fetuses. We found that the clinical accuracy of the breed-specific Groppetti et al. formula was highest among those of the three formulas tested, with 87% and 100% of the estimated parturition dates (calculated based on the ICC measurements) being within 1 and 2 days, respectively, of the actual delivery date. By contrast, the Luvoni and Grioni formula showed relatively low accuracy, and the Milani et al. formula showed higher accuracy than that reported previously for natural pregnancies.

Simple Summary This is the first study to demonstrate the establishment and subsequent analysis of attributes, including the chondrogenic capacity of mesenchymal stem cells (MSCs) from bone marrow (BM) and synovial fluid (SF) from the same donor Camelus dromedarius. MSCs of SF origin were notably more efficient in their chondrogenic capacity and represent a potential source for camel regenerative medicine addressing chondrocyte-related problems. Abstract Mesenchymal stem cells (MSCs) are promising multipotent cells with applications for cartilage tissue regeneration in stem cell-based therapies. In cartilage regeneration, both bone marrow (BM-MSCs) and synovial fluid (SF-MSCs) are valuable sources. However, the cellular characteristics and chondrocyte differentiation potential were not reported in either of the camel stem cells. The in vitro chondrocyte differentiation competence of MSCs, from (BM and SF) sources of the same Camelus dromedaries (camel) donor, was determined. Both MSCs were evaluated on pluripotent markers and proliferation capacity. After passage three, both MSCs showed fibroblast-like morphology. The proliferation capacity was significantly increased in SF-MSCs compared to BM-MSCs. Furthermore, SF-MSCs showed an enhanced expression of transcription factors than BM-MSCs. SF-MSCs exhibited lower differentiation potential toward adipocytes than BM-MSCs. However, the osteoblast differentiation potential was similar in MSCs from both sources. Chondrogenic pellets obtained from SF-MSCs revealed higher levels of chondrocyte-specific markers than those from BM-MSCs. Additionally, glycosaminoglycan (GAG) content was elevated in SF-MSCs related to BM-MSCs. This is, to our knowledge, the first study to establish BM-MSCs and SF-MSCs from the same donor and to demonstrate in vitro differentiation potential into chondrocytes in camels.

Simple Summary Mesenchymal stem cells (MSCs) can be isolated in various types of tissues and exhibit different characteristics. In this study, MSCs were established from skeletal muscle, dermal skin, and adipose tissue from a single Camelus dromedarius donor. We also identified an efficient source for osteoblast differentiation and analyzed biological characteristics. Abstract Mesenchymal stem cells (MSCs) showed in vitro mesoderm-lineage differentiation and self-renewal capacity. However, no comparative study was reported on the biological characteristics of stem cells derived from skeletal muscle (SM-MSCs), dermal skin (DS-MSCs), and adipose tissues (A-MSCs) from a single donor in camels. The present study aimed to evaluate the influence of MSCs source on stem cell characteristics. We evaluated proliferation capacity and mesoderm-lineage differentiation potential from SM-MSCs, DS-MSCs, and A-MSCs. They showed spindle-like morphology after homogenization. The proliferation ability was not significantly difference in any of the groups. Furthermore, the portion of the cell cycle and expression of pluripotent markers (Oct4, Sox2, and Nanog) were similar in all cell lines at passage 3. The differentiation capacity of A-MSCs into adipocytes was significantly higher than that of SM-MSCs and DS-MSCs. However, the osteoblast differentiation capacity of A-MSCs was significantly lower than that of SM-MSCs and DS-MSCs. Additionally, after osteoblast differentiation, the alkaline phosphatase (ALP) activity and calcium content significantly decreased in A-MSCs compared to SM-MSCs and DS-MSCs. To the best of our knowledge, we primarily established MSCs from the single camel and demonstrated their comparative characteristics, including expression of pluripotent factors and proliferation, and in vitro differentiation capacity into adipocytes and osteoblasts.

Simple Summary Somatic cell nuclear transfer (SCNT) is a technique used to reproduce individuals from their somatic cell nucleus, which is commonly known as cloning. In SCNT, viable cell lines are usually established from living animals and preserved for future use. In the present study, tissues were collected and cryopreserved (rather than cells) from a suddenly deceased champion show camel. We established fibroblast cell lines from this decade-old vitrified tissue and used them as nuclear donors. Both the in vitro and in vivo matured oocytes were used to produce cloned embryos. Blastocysts were transferred to synchronized recipients to establish pregnancies. A total of 18 pregnancies (5 from in vitro matured oocytes and 13 from in vivo matured oocytes) were established, and 11 live offspring were born (2 from in vitro matured oocytes and 9 from in vivo matured oocytes). We concluded that fibroblast cell lines could be established from long cryopreserved tissues, and these cells could be used as nucleus donors to clone animals. Abstract Somatic cell nuclear transfer (SCNT) provides a unique opportunity to reproduce animals with superior genetics. Viable cell lines are usually established from tissues collected by biopsy from living animals in the SCNT program. In the present study, tissues were collected and preserved from a suddenly deceased champion camel. We established cell lines from these decade-old tissues and used them as nuclear donors. After 42 h of in vitro maturation, 68.00 ± 2.40% of oocytes reached the metaphase II (M II) stage while 87.31 ± 2.57% in vivo collected oocytes were matured at collection (p < 0.05). We observed a higher blastocyst formation rate when in vivo matured oocytes (43.45 ± 2.07%) were used compared to in vitro matured oocytes (21.52 ± 1.74%). The live birth rate was 6.45% vs. 16.67% for in vitro and in vivo matured oocytes, respectively. Microsatellite analysis of 13 camel loci revealed that all the SCNT-derived offspring were identical to each other and with their somatic cell donor. The present study succeeded in the resurrection of 11 healthy offspring from the decade-old vitrified tissues of a single somatic cell donor individual using both in vitro and in vivo matured oocytes.

To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy-six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P > 0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase-like chromosomes compared to the 4 hr group (P < 0.05). A significantly higher pregnancy rate and a greater number of live born puppies were observed in the 2 hr group (13.0% and 1.1%, respectively) compared to the 4 hr group (0%) (P < 0.05). In total, three surrogate dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family.

The collection of in vivo matured canine oocytes relies on the accurate prediction of ovulation. The present study was designed to develop a protocol for the recovery of in vivo matured canine oocytes based on once daily measurements of serum progesterone (P(4)) concentrations. Blood samples (2 mL) were collected every day at 0900 h, and P(4) concentrations were analyzed using a DSL-3900 ACTIVE Progesterone Coated-Tube Radioimmunoassay Kit. The average number of oocytes at the metaphase II (M II) stage was significantly higher at or after 72 h (6.7 to 7.5) compared to 56 h (1.7) following ovulation. The highest numbers of corpora lutea, and therefore the highest numbers of oocytes, were recovered from bitches with initial ovulatory P(4) concentrations ranging from 6.0 to 8.0 ng/ mL (12.2 and 11.4, respectively) compared to from 4.0 to 4.9 ng/ mL (9.6 and 8.8, respectively; p0.05). The average number of M II oocytes recovered at 84 h from bitches with initial ovulatory P(4) levels of 5.0 to 5.9 ng/mL (7.7) was higher compared to bitches with P(4) levels of 4.0 to 4.9 ng/ mL (3.5) and 6.0 to 8.0 ng/ mL (4.8; p0.05). When oocyte recovery time was adjusted for initial ovulatory P(4) concentration, no significant difference in recovery rates or oocyte quality were observed. In conclusion, once daily measurements of P(4) can be used to predict ovulation in bitches, and oocyte recovery time should be adjusted for initial ovulatory serum P(4) concentrations.

The purpose of the present study on canine somatic cell nuclear transfer (SCNT) was to evaluate the effects of fusion strength, type of activation, culture media and site of transfer on developmental potential of SCNT embryos. We also examined the potential of enucleated bovine oocytes to serve as cytoplast recipients of canine somatic cells. Firstly, we evaluated the morphological characteristics of in vivo-matured canine oocytes collected by retrograde flushing of the oviducts 72 h after ovulation. Secondly, the effectiveness of three electrical strengths (1.8, 2.3 and 3.3 kV/cm), used twice for 20 micros, on fusion of canine cytoplasts with somatic cells were compared. Then, we compared: (1) chemical versus electrical activation (a) after parthenogenetic activation or (b) after reconstruction of canine oocytes with somatic cells; (2) culture of resulting intergeneric (IG) embryos in either (a) mSOF or (b) TCM-199. The exposure time to 6-DMAP was standardized by using bovine oocytes reconstructed with canine somatic cells. Bovine oocytes were used for SCNT after a 22 h in vitro maturation interval. The fusion rate was significantly higher in the 3.3 kV/cm group than in the 1.8 and 2.3 kV/cm treatment groups. After parthenogenesis or SCNT with chemical activation, 3.4 and 5.8%, respectively, of the embryos developed to the morula stage, as compared to none of the embryos produced using electrical activation. Later developmental stages (8-16 cells) were transferred to the uterine horn of eight recipients, but no pregnancy was detected. However, IG cloned embryos (bovine cytoplast/canine somatic cell) were capable of in vitro blastocyst development. In vitro developmental competence of IG cloned embryos was improved after exposure to 6-DMAP for 4 h as compared to 0, 2 or 6h exposure, although the increase was not significantly different among culture media. In summary, for production of canine SCNT embryos, we recommend fusion at 3.3 kV/cm, chemical activation, culture in mSOF medium and transfer of presumptive zygotes to the oviduct of recipient animals. The feasibility of IG production of cloned canine embryos using bovine cytoplasts as recipient of canine somatic cells was demonstrated.

In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2-8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development.

The susceptibility of embryos to reactive oxygen species (ROS) varies in different stages of embryo development. The present study evaluated temporal effects of alpha-tocopherol and L-ascorbic acid on the porcine embryo development, and investigated whether a single or twice supplements of these two antioxidants at a divided concentrations favors the embryo development. In order to determine temporal effects of alpha-tocopherol and/or L-ascorbic acid, 100 microM alpha-tocopherol or 200 microM L-ascorbic acid were supplemented to the North Carolina State University (NCSU)-23 embryo culture media at 0, 48, 96 and 120 h of culture. In another set of experiments, the concentration was divided into two equal halves, i.e., 50 microM alpha-tocopherol and 100 microM L-ascorbic acid, and supplemented twice at 0 and 48, 0 and 96, or 48 and 96 h of culture. Supplementing culture media with 100 microM alpha-tocopherol for the entire culture period of 168 h or starting from the 48 h of culture yielded higher blastocyst percentage compared with the control or starting from the 96 or 120 h of culture. L-Ascorbic acid (200 microM) alone or together with alpha-tocopherol (100 microM) with a single supplement did not affect the frequency of blastocyst formation or number of cells in blastocyst. L-ascorbic acid with a divided supplements yielded higher blastocyst percentage compared with the control. No synergistic effect was observed on embryo development at a single supplement of these antioxidants. Although, at divided supplements higher blastocyst percentage was observed compared with control group, no further beneficial effect was observed compared with alpha-tocopherol or L-ascorbic acid alone. Our results demonstrated that the embryotrophic effects of alpha-tocopherol and/or L-ascorbic acid, in terms of frequency of blastocyst formation and number of cells in blastocyst, depends on the concentration and supplementation timing.

Over the world, canine species, including the gray wolf, have been gradually endangered or extinct. Many efforts have been made to recover and conserve these canids. The aim of this study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes, in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf. In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach for conserving endangered canids.

The present study was conducted to examine the effect of different levels of essential and nonessential amino acid in NCSU-23 medium on the in vitro-produced porcine embryo as it develops from the zygote to the blastocyst stage. Four experiments were performed, each with a completely randomized design involving 5 to 8 replications of treatments. In order to know the effect of nonessential amino acids in NCSU-23 medium, 0, 5, 10 and 20 μl/ml MEM were supplemented there to, (Exp. 1) and the medium was supplemented with same level of essential amino acids (Exp. 2). The combined effect of nonessential (0, 5, 10 and 20 μl/ml MEM) and essential amino acids (0, 5, 10 and 10 μl/ml MEM) in NCSU-23 medium (Exp. 3), first 72 h with non-essential amino acids (at 0, 5, 10 and 20 μl/ml MEM), and last 4 d with essential amino acids with the same level as NEAA (Exp. 4) were examined. The embryo development was monitored and the quality of blastocysts was evaluated by counting the number of total cells and determining the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells. When Eagle's nonessential amino acids (MEM) added to NCSU-23 medium, it significantly increased the likelihood of development to the 2- to 4-cell stage and subsequent blastocyst development. Supplementation of different levels of essential amino acids in the NCSU-23 medium decreased cleavage rate, rate of morula and blastocyst development and the number of ICMs. In the case of the combined effect of essential and nonessential amino acids, better and significant results were found for blastocysts, hatching blastocysts and for ICM numbers which were also dose dependent. With respect to the biphasic effect of nonessential and essential amino acids, nonessential amino acids increased cleavage whereas essential amino acids increased the total cell number. Neither the nonessential nor the essential group of amino acids, on their own, affected blastocyst cell number or the differentiation of cells in the blastocyst. In conclusion, this study determined the role of nonessential and essential amino acids in the culture of the porcine embryo and showed that the embryo requires different levels of amino acids as it develops from the zygote to the blastocyst stage.

Since the only viable cloned offspring born in dogs was a male, the purpose of the present study was to produce female puppies by somatic cell nuclear transfer (SCNT). Adult ear fibroblasts from a 2-month-old female Afghan hound were isolated and used as donor cells. In vivo-matured canine oocytes surgically collected (approximately 72 h after ovulation) from the oviducts of 23 donors were used for SCNT. After removal of the cumulus cells, oocytes were enucleated, microinjected, fused with a donor cell, and activated. A total of 167 reconstructed SCNT embryos were surgically transferred (Day 0) into the oviducts of 12 recipient bitches (average 13.9 embryos/recipient, range 6-22) with spontaneous, synchronous estrous cycles. Three pregnancies were detected by ultrasonography on Day 23, maintained to term, and three healthy female puppies (520, 460, and 520 g), were delivered by Caesarean section on Day 60. These puppies were phenotypically and genotypically identical to the cell donor. In conclusion, we have provided the first demonstration that female dogs can be produced by nuclear transfer of ear fibroblasts into enucleated canine oocytes.

Reactive oxygen species (ROS) generate during electrical activation of oocytes which has detrimental effects on embryo survival when overwhelmed. The present study was designed to investigate the ability of L-ascorbic acid, a novel water soluble antioxidant, to reduce the ROS level in developing embryos and their subsequent effects on embryo development in vitro. The compact cumulus oocyte complexes (COCs) were cultured in tissue culture medium (TCM)-199 supplemented with 10 ng/ml epidermal growth factor, 4 IU/ml pregnant mare serum gonadotropin (PMSG), and human chorionic gonadotropin (hCG) and 10% (v/v) porcine follicular fluid (pFF) for 44 h. After maturation culture, the denuded oocytes were activated with a single DC pulse of 2.0 kV/cm in 0.3 M mannitol solution containing 0.5 mM of HEPES, 0.1 mM of CaCl 2 and 0.1 mM of MgCl 2 for 30 (is using a BTX Electro-cell Manipulator. The activated oocytes were cultured in modified North Carolina State University-23 (mNSCU-23) medium for 168 h. The level of H 2 O 2 in each embryo was measured by the dichlorohydrofluorescein diacetate (DCHFDA) method at 48 h after activation. The blastocyst formation rate was significantly higher when culture medium was supplemented with 50 and 100 μM L-ascorbic acid (31.2 and 38.7%, respectively) compared to non-supplemented (16.1%) group. Accordingly, significantly more cells in blastocyst were found for 50 and 100 μM L-ascorbic acid (50.0 and 56.4, respectively) compared to 0 and 200 μM L-ascorbic acid (36.5 and 39.8, respectively). L-ascorbic acid reduces the H 2 O 2 level in developing embryos in a dose-dependant manner. The H 2 O 2 level (pixels/ embryos) was 191.5, 141.0, 124.0 and 163.3 for 0, 50, 100 and 200 μM L-ascorbic acid, respectively. So, we recommend to supplement 50 or 100 μM L-ascorbic acid in porcine in vitro culture medium.

The objective was to determine optimal concentrations of alpha-tocopherol and l-ascorbic acid for development of porcine embryos derived from in vitro-fertilization (IVF) or somatic cell nuclear transfer (SCNT). The frequency of blastocyst formation in IVF embryos was 17.6, 28.6, 32.4 and 21.4% for control, 50, 100 and 200microM alpha-tocopherol, respectively, whereas in SCNT embryos, the frequency was 12.8, 19.0, 24.8 and 17.7% for corresponding concentrations of alpha-tocopherol. For both IVF and SCNT embryos, there were significantly more cells in blastocysts and the embryos had greater developmental competence when the embryo culture medium was supplemented with 100microM alpha-tocopherol. Although either alpha-tocopherol or l-ascorbic acid reduced the proportion of apoptotic cells in blastocysts, in combination they resulted in rates of apoptosis that were similar to the control group. For IVF embryos, the apoptotic index was 0.09 and 0.11 for alpha-tocopherol or l-ascorbic acid at a concentration of 100microM, respectively. Conversely, when these antioxidants were supplemented together, the apoptotic index increased significantly and was similar to the control group (i.e., 0.17 and 0.21 for combined and control group). For SCNT embryos, the apoptotic index was 0.10 for 100microM for both alpha-tocopherol and l-ascorbic acid, whereas the index was 0.23 and 0.17 for control and combined group. In conclusion, we recommend supplementing porcine embryo culture medium with 100microM alpha-tocopherol or 100microM l-ascorbic (as a second choice).

Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine and/or paracrine growth and/or survival factor for mammalian embryo development. It is known to promote the growth and development of mouse preimplantation embryos. The present study was designed to investigate the effects of IGF-I (50 ng/ml), anti-IGF-I receptor antibody (50 ng/ml) and their combination on porcine preimplantation embryo development. Furthermore, the mechanism underlying the embryotropic effects of IGF-I was evaluated by monitoring the incidence of apoptosis and expression of apoptosis-related genes. In both in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos, culturing with IGF-I increased the rate of blastocyst formation and this embryotrophic effect was neutralized by culturing with IGF-I along with anti-IGF-I receptor (IGF-IR) antibody. Culturing IVF and SCNT embryos with IGF-I significantly increased the number of total cells in blastocysts and decreased the number of apoptotic nuclei. These effects of IGF-I were also neutralized by culturing with IGF-I along with anti-IGF-IR antibody. Expression of the anti-apoptotic Bcl-2 gene was increased, while expression of the pro-apoptotic Bax was decreased in both IVF and SCNT embryos cultured with IGF-I. In both IVF and SCNT embryos, anti-IGF-IR antibody along with IGF-I neutralized the effect of IGF-I on expression of Bcl-2 and Bax genes. In conclusion, the present study demonstrated that IGF-I through its specific receptors improved the developmental competence of IVF and SCNT embryos by decreasing the incidence of apoptosis and regulating apoptosis-related genes in porcine preimplantation embryos.

This study investigated the embryotrophic effects of ethylenediaminetetraacetic acid (EDTA) and hemoglobin (Hb) on porcine preimplantation embryo development. Porcine embryos produced by in vitro maturation/fertilization were cultured for 6 days in modified North Carolina State University-23 medium (mNCSU-23) supplemented with EDTA and/or Hb. In Exp. 1, culturing porcine zygotes with 100 microM EDTA significantly increased cleavage frequencies (85.3%) at 48 h post insemination and the number of inner cell mass (ICM) (9.6+/-5.5) compared to the control (7.0+/-2.8). However, 100 microM EDTA did not improve blastocyst formation compared to 0, 1 or 10 microM EDTA. In Exp. 2, in vitro fertilized oocytes were cultured with 0, 1 or 10 microg/ml Hb. Culturing with Hb did not promote porcine embryo development, but significantly increased the cell numbers of blastocysts in 1 microg/ml Hb compared to 0 or 10 microg/ml Hb. In Exp. 3, culturing embryos with 100 microM EDTA+1 microg/ml Hb significantly improved frequencies of cleavage, blastocyst formation, and total cell numbers in blastocysts compared to the control. Moreover, 100 microM EDTA, 1 microg/ml Hb and their combination reduced reactive oxygen species (ROS) accumulation and decreased the incidence of apoptosis. In conclusion, the present study clearly demonstrated that the combining treatment of EDTA and Hb improved IVF porcine embryo development.

The present study was conducted to examine the comparative efficacy of potassium simplex optimization medium (KSOM) and North Carolina State University (NCSU)-23 medium supplemented with beta-mercaptoethanol (beta-ME) and amino acids (AA) on the developmental competence of porcine in vitro fertilized (IVF) embryos. Four experiments were conducted. KSOM and NCSU-23 medium were used to culture porcine parthenogenetic (Exp. 1) and IVF (Exp. 2) embryos. KSOM and NCSU-23 were equally effective in supporting porcine parthenogenetic and IVF embryo development from the 1-cell stage to blastocysts. The NCSU-23 medium (Exp. 3) and KSOM (Exp. 4) were supplemented with amino acid (AA; 5 microl/ml non-essential amino acids + 10 microl/ml essential amino acids) and/or 10 microM beta-mercaptoethanol (beta-ME). The quality of blastocysts from Exp. 3 and 4 was evaluated by counting the number of total cells and determining the ratio of the inner cell mass (ICM) to trophoectoderm (TE) cells. Supplementing with AA and beta-ME or beta-ME alone in NCSU-23 produced significant (p

Using in vivo-flushed oocytes from a homogenous dog population and subsequent embryo transfer after nuclear transfer, we studied the effects of donor cells collected from 10 different breeds on cloning efficiency and perinatal development of resulted cloned puppies. The breeds were categorized into four groups according to their body weight: small (≤9 kg), medium (>9-20 kg), large (>20-40 kg), and ultra large (>40 kg). A total of 1611 cloned embryos were transferred into 454 surrogate bitches for production of cloned puppies. No statistically significant differences were observed for initial pregnancy rates at Day 30 of embryo transfer for the donor cells originated from different breeds. However, full-term pregnancy rates were 16.5%, 11.0%, 10.0%, and 7.1% for the donor cells originated from ultra-large breed, large, medium, and small breeds, respectively, where pregnancy rate in the ultra-large group was significantly higher compared with the small breeds (P < 0.01). Perinatal mortality until weaning was significantly higher in small breeds (33.3%) compared with medium, large, or ultra-large breeds where no mortality was observed. The mean birth weight of cloned pups significantly increased proportional to breed size. The highest litter size was examined in ultra-large breeds. There was no correlation between the number of embryo transferred and litter size. Taken together, the efficiency of somatic cell cloning and fetal survival after embryo transfer may be affected significantly by selecting the appropriate genotype.

Relaxin, a member of the insulin superfamily, has diverse functions in both reproductive and nonreproductive tissues. The aim of the present study was to evaluate the effects of recombinant relaxin on the in vitro maturation of porcine oocytes and their subsequent embryonic development following in vitro fertilization. Three concentrations of relaxin (1, 10, and 100 ng/ml) were used in the in vitro maturation (IVM) medium [TCM supplemented with 10% (v/v) porcine follicular fluid, 10 ng/ml of epidermal growth factor, 4 IU/ml of pregnant mare serum gonadotropin, and (only for the first 22 hr) 4 IU/ml of human chorionic gonadotropin]. Relaxin was used during the entire IVM period. Nuclear maturation of oocytes was examined under ultraviolet light following staining with bisbenzimide (Hoechst 33342) for 5 min and mounted on a glass slide. The glutathione (GSH) content in oocytes, an important indicator of cytoplasmic maturity, was measured using a micro-glutathione assay. Cryopreserved boar semen was used for in vitro fertilization. Embryos were cultured in modified NCSU-23 medium supplemented with 0.5 mM pyruvate and 5 mM lactate. Although nuclear maturation of oocytes did not vary, the GSH content in oocytes was significantly higher when cultured with 1 ng/ml (7.9 pmol/oocyte) and 10 ng/ml (8.47 pmol/oocyte) compared to a control group. However, no additional beneficial effect was observed when 100 ng/ml of relaxin was added to the IVM medium. A significantly higher rate of blastocyst formation was observed with 10 ng of relaxin (32.4%) compared to the control (14.4%) or 100 ng of relaxin (21.4%). No difference between 1 ng and 10 ng was observed in terms of the blastocyst production rate. The inner cell mass cell numbers in relaxin-treated groups were significantly higher than control, and trophectoderm cell number was the highest in the 10 ng relaxin group. Relaxin (10 ng/ml) can be supplemented in IVM medium to support the maturation of porcine oocytes.

The present study was undertaken to evaluate two activation methods for somatic cell nuclear transfer (SCNT), namely, fusion and simultaneous activation (FSA, fusion medium contains calcium), versus fusion followed by chemical activation (F+CA, fusion medium does not contain calcium), and to evaluate the effects of parity of recipient dogs on the success of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells collected from an 11-year-old female dog named Missy. In the FSA method, oocytes were fused and activated at the same time using two DC pulses of 1.75 kV/cm for 15 microsec. In the F+CA method, oocytes were fused with two DC pulses of 1.75 kV/cm for 15 microsec, and then activated 1 h after fusion by 10 microM calcium ionophore for 4 m and cultured for 4 h in 1.9 mM 6-dimethylaminopurine for postactivation. Activation method had a significant impact on the production efficiency of cloned dogs. There was a significant difference in full-term pregnancy rate and percentage of live puppies between the two methods (6.3% and 38.5% for FSA and F+CA, respectively). In our study, four out of five live offspring produced by F+CA survived versus FSA, which did not result in any surviving puppies. Overall, as few as 14 dogs and 54 reconstructed embryos were needed to produce a cloned puppy. In addition, the parity of recipient bitches had no effect on the success of SCNT in canine species. Both the nullipara and multipara bitches produced live puppies following SCNT-ET.

In an effort to improve the quality ofin vitroproduced porcine embryos, we investigated the effect of brain-derived neurotropic factor (BDNF), a neurotropin family member, onin vitromaturation (IVM) of porcine oocytes. The expression of BDNF and truncated isoforms of its receptor, tyrosine kinase B (TrkB), and p75 common neurotropin receptor was detected in both follicular cells and metaphase-I stage oocytes by RT-PCR. However, mRNA of full-length TrkB was not found in oocytes although it was detected in follicular cells. The expression pattern of BDNF and TrkB was confirmed by immunohistochemistry. Supplementation with BDNF (30 ng/ml) during IVM significantly (P< 0.05) increased the first polar body extrusion and glutathione levels in oocytes, whereas the effect of BDNF on nuclear maturation was diminished when gonadotropin and epidermal growth factor (EGF) were added to the culture media. However, treatment with BDNF (30 ng/ml) along with EGF (10 ng/ml) in the presence of gonadotropin significantly (P< 0.05) increased the developmental competence of oocytes to the blastocyst stage after bothin vitrofertilization (IVF; 29.1% when compared with control, 15.6%) and somatic cell nuclear transfer (SCNT; 13.6% when compared with control, 3%). This appeared to reflect a stimulatory interaction between BDNF and EGF to enhance the cytoplasmic maturation of oocytes to support successful preimplantation development. In conclusion, BDNFenhanced nuclearand cytoplasmic maturation of oocytes by autocrine and/or paracrine signals. Also, when used together with EGF, BDNF increased the developmental potency of embryos after IVF and SCNT, demonstrating an improvedin vitroproduction protocol for porcine oocytes.

Glutathione (GSH) concentrations of oocytes are considered as an important marker of the cytoplasmic maturation. The present study was designed to compare GSH concentrations of in vivo and in vitro matured canine oocytes. In vivo matured oocytes were collected 72 hr after ovulation by flushing fallopian tubes after laparotomy. Ovaries were collected from bitches with different reproductive stages, and collected oocytes were divided into 2 groups according to the size viz.120 microm and120 microm in diameter and cultured for 72 hr in Tissue Culture Medium-199 supplemented with 10% FBS, 2.2 mg/ml sodium bicarbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution in the presence or absence of 50 microM beta-mercaptoethanol. GSH concentrations were determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. GSH concentrations of immature canine oocytes were 2.9 and 3.8, 3.5 and 6.8, and 3.1 and 6.5 pM/oocyte for120 microm and120 microm in diameter oocyte groups at anestrous, follicular and luteal stage, respectively (P0.05). In vivo matured oocytes had significantly higher GSH concentrations compared with in vitro matured oocytes. The GSH content was 19.2 pM/oocyte for in vivo matured oocytes, while 4.1 to 8.1 and 5.7 to 13.2 pM/oocyte for in vitro matured oocytes cultured in the absence or presence of beta-mercaptoethanol, respectively (P0.05). Presence of beta-mercaptoethanol increased GSH synthesis in canine oocytes cultured in vitro, and oocytes collected from follicular and luteal stage was superior to anestrus oocytes.

Various thiol compounds are known to improve cytoplasmic and/or nuclear maturation of oocytes in vitro. The present study examined the effects of two thiol compounds, cysteine (0.1, 0.5, and 1.0 mM) and cysteamine (50, 100, and 200 microM), on cytoplasmic and nuclear maturation of canine oocytes. Oocytes collected from different reproductive stages were cultured in TCM-199 supplemented with 10% fetal bovine serum, 2.2 mg/ml sodium carbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution for 72 h. Data were analyzed by two-way ANOVA after arcscine transformation and protected by Bonferroni post hoc test. The effects of cysteine and cysteamine on canine IVM were varied depending on the reproductive stage of oocyte donor bitches. In the follicular stage, significantly more oocytes reached the metaphase II (M II) stage when cultured with 0.5 or 1.0 mM cysteine (16.7% and 16.9%, respectively) compared to the control (6.2%). In the follicular stage, cysteamine increased oocyte maturation rate upto the M II stage (15.1% to 17.0%) compared to the control (4.4%). Both the 0.5 mM cysteine and 100 microM cysteamine, alone or together, increased the intracellular GSH level of canine oocytes compared to the control. Irrespective of reproductive stage, no further beneficial effects on nuclear or cytoplasmic maturation were observed when 0.5 mM cysteine and 100 microM cysteamine were supplemented together. In conclusion, addition of 0.5 mM cysteine and 100 microM cysteamine to the maturation medium improved IVM of canine oocytes.

This study investigated the effects of culture conditions and somatic cell nuclear transfer (SCNT) protocols on in vitro development of porcine SCNT embryos and on expression patterns of genes involved in stress (heat shock protein 70.2, HSP70.2), trophoblastic function (integrin beta1, ITGB1), metabolism (phosphoglycerate kinase 1, PGK1), apoptosis (BAX), and imprinted gene (insulin-like growth factor 2 receptor, IGF2R). In Experiment 1, supplementing modified North Carolina State University (mNCSU) medium with 10% FBS at Day 4 of culture increased SCNT blastocyst formation (22.9 vs. 10.7%, P0.05), number of inner cell mass cells (13.3+/-4.3 vs. 7.6+/-2.2, P0.05), and total cells (57.9+/-19.5 vs. 36.3+/-8.2, P0.05) in cloned blastocysts. In Experiment 2, using culture medium with 10% FBS, 1.0mM calcium in fusion/activation medium (1.0C), and 7.5mug/mL cytochalasin B treatment (0.1CCB) yielded higher rates (P0.05) of blastocysts (33.6 and 33.3%, respectively) relative to the control (0.1mM calcium fusion medium, 0.1C; 18.3%). Total cell numbers of blastocysts were increased (P0.05) in 1.0C (77.4+/-28.9) compared to the control (58.5+/-22.6). In vitro-derived blastocysts had higher expression levels of BAX and lower levels of HSP70.2, IGF2R compared to their in vivo-derived counterparts. Supplementing culture medium with 10% FBS increased relative abundances of BAX mRNA in SCNT blastocysts relative to in vivo-derived blastocysts. The transcript level of ITGB1 in blastocyst from 0.1CCB was lower than in vivo blastocysts. In conclusion, different culture conditions or SCNT protocols affected in vitro development of SCNT embryos and altered several important genes (BAX, HSP70.2, IGTB1, and IGF2R) compared to conventional in vivo-derived blastocysts.

Insulin-transferrin-selenium (ITS) together has been used in different in vitro maturation system to support in vitro maturation of oocytes. The present study was designed to evaluate the effects of ITS in defined (0.1% PVA) and porcine follicular fluid (10% pFF) supplemented IVM media on the developmental competence of porcine oocytes. Three combinations of ITS, 10 mg/L insulin (Ins), 5.5mg/L transferrin (Tf) and 5 microg/L selenium (Se), 20mg/L Ins, 11 mg/L Tf and 10 microg/L Se, and 30 mg/L Ins, 16.5 mg/L TF and 15 microg/L Se, were used. The data were analyzed by one-way ANOVA and Tukey was used as the post hoc test. Both in the defined and pFF supplemented media, higher concentration of intracellular glutathione was observed in presence of ITS (4.6-4.8, and 6.9-7.1 picomole/oocyte for defined and pFF groups, respectively) compared to the respective control (2.1 and 4.3 picomole/oocyte for defined and pFF group, respectively). ITS decreased polyspermy and increased male pronucleus formation in both the defined and pFF supplemented medium. There was no difference in different treatment groups. The highest frequency of blastocyst formation rate and number of cells in blastocyst following IVF and SCNT was observed in pFF+ITS group (p

Assisted reproductive technology (ART) in dogs largely depends on the in vivo matured oocytes due to lack of a suitable in vitro maturation system. The present study evaluated the technique of flushing fallopian tubes to collect in vivo matured canine oocytes by laparotomy, and determined the effects of seasons, and parity of donor bitches on the success of oocyte retrieval. Oocytes were retrieved from anesthetized bitches by laparotomy. About 7ml of TCM-199 supplemented with HEPES was used to flush each individual fallopian tube. Oocytes were categorized as good, fair, poor, immature or aged based on the nuclear stage, cumulus cell layers, color and homogeneity of ooplasm. Oocytes categorized as being good or fair were considered usable, while poor, aged or immature oocytes were considered unusable for ART. A significantly higher number of oocytes per bitch were retrieved during the spring (11.2) compared to the winter (7.9). The oocyte recovery rates were 89.4, 92.2, 89.7 and 89.3% for spring, summer, autumn, and winter, respectively. The highest percentage of usable oocytes (74.7%) was retrieved during autumn ( P >0.05). The number of oocytes was influenced by the parity of the donor bitch. Significantly more oocytes were collected from the multiparous bitches (10.3) compared to nulliparous bitches (7.7). The percentage of usable oocytes was more in multipara (71.5%) compared to nullipara (64.7%) ( P >0.05). Collection of in vivo produced oocytes by laparotomy represents a potential source of matured oocytes for ART in dogs.