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Background: Somatic cell nuclear transfer (SCNT) is a prominent technology that can preserve superior genetic traits of animals and expand the population in a short time. Hematological characters and endocrine profiles are important elements that demonstrate the stability of the physiological state of cloned animals. To date, several studies regarding cloned camels with superior genes have been conducted. However, detailed hemato-physiological assessments to prove that cloned camels are physiologically normal are limited. In this study, We evaluated the hemato-physiological characteristics of cloned male and female dromedary camels (Camelus dromedaries). Methods: Therefore, we analyzed variations in hematological characteristics and endocrine profiles between cloned and non-cloned age-matched male and female dromedary camels (Camelus dromedaries). Two groups each of male and female cloned and non-cloned camels were monitored to investigate the differences in hemato-physiological characteristics. Results: All the animals were evaluated by performing complete blood count (CBC), serum chemistry, and endocrinological tests. We found no significant difference between the cloned and non-cloned camels. Furthermore, the blood chemistry and endocrine profiles in male and female camels before maturity were similar. Conclusions: These results suggest that cloned and non-cloned camels have similar hematological characteristics and endocrine parameters.

Monoamniotic twins develop when a blastocyst spontaneously splits its progenitor cells, and each group of progenitor cells independently grows to become an individual. It is the rarest type of twin pregnancy and usually has significant developmental or congenital abnormalities, a higher rate of abortion, perinatal morbidity, and mortality. There is no information regarding monoamniotic twins in livestock species. Here, we reported a spontaneous abortion of monoamniotic twins in a dromedary camel at 278 days of gestation. Gonadorelin acetate (100 μg) was injected intramuscularly to induce ovulation in the recipient. A 7 days-old embryo produced by somatic cell nuclear transfer was transferred transcervically to the recipient. Early pregnancy was confirmed by an elevated level of serum progesterone followed by ultrasonography at 22 and 44 days after embryo transfer. A single sac was observed on 22 days while twins were evident 44 days after embryo transfer. Pregnancy was periodically monitored by the tail-up phenomenon. A ruptured fetal sac was observed on the ground having two fetuses. On autopsy, full-grown fetuses were found. Their bodies were separated. There was no congenital anomaly or any malformation in the fetuses. According to the reported chronology in human twins, we hypothesized that the blastocyst splitted before 13 days as it was monoamniotic and not conjoined. If the embryo splits within 4 to 8 days, it develops two amniotic sacs, and splitting after 13 days develops conjoined fetuses. To the authors’ knowledge, this is the first reported case of monoamniotic twin abortion in dromedary camels. This report will increase awareness among practicing veterinarians and camel breeders about twin abortions.

Propagation of transgenic animals by germline transmission using assisted reproductive technologies such as in vitro fertilization (IVF) is the most efficient way to produce transgenic colonies for biomedical research. The objective of this study was to generate transgenic puppies from a founder dog expressing the mutated human amyloid precursor protein (mhAPP) gene. Experiment I assessed the characteristics of the semen prepared by freshly diluted, swim-up, and Percoll gradient methods using a computer-assisted semen analyzer (CASA). Motile and progressively motile sperm counts were higher in the Percoll gradient samples (p

Objective The present study analyzed the influence of co-transferring embryos with high and low cloning efficiencies produced via somatic cell nuclear transfer (SCNT) on pregnancy outcomes in dogs. Methods Cloned dogs were produced by SCNT using donor cells derived from a Tibetan Mastiff (TM) and Toy Poodle (TP). The in vivo developmental capacity of cloned embryos was evaluated. The pregnancy and parturition rates were determined following single transfer of 284 fused oocytes into 21 surrogates and co-transfer of 47 fused oocytes into four surrogates. Results When cloned embryos produced using a single type of donor cell were transferred into surrogates, the pregnancy and live birth rates were significantly higher following transfer of embryos produced using TP donor cells than following transfer of embryos produced using TM donor cells. Next, pregnancy and live birth rates were compared following single and co-transfer of these cloned embryos. The pregnancy and live birth rates were similar upon co-transfer of embryos and single transfer of embryos produced using TP donor cells but were significantly lower upon single transfer of embryos produced using TM donor cells. Furthermore, the parturition rate for TM dogs and the percentage of these dogs that remained alive until weaning was significantly higher upon co-transfer than upon single transfer of embryos. However, there was no difference between the two embryo transfer methods for TP dogs. The mean birth weight of cloned TM dogs was significantly higher upon single transfer than upon co-transfer of embryos. However, the body weight of TM dogs did not significantly differ between the two embryo transfer methods after day 5. Conclusion For cloned embryos with a lower developmental competence, the parturition rate and percentage of dogs that remain alive until weaning are increased when they are co-transferred with cloned embryos with a greater developmental competence.

Abstract Animal cloning has been popularized for more than two decades, since the birth of Dolly the Sheep 25 years ago in 1996. There has been an apparent waning of interest in cloning, evident by a reduced number of reports. Over 1500 dogs, representing approximately 20% of the American Kennel Club’s recognized breeds, have now been cloned, making the dog (Canis familiaris) one of the most successfully cloned mammals. Dogs have a unique relationship with humans, dating to prehistory, and a high degree of genome homology to humans. A number of phenotypic variations, rarely recorded in natural reproduction have been observed in in these more than 1000 clones. These observations differ between donors and their clones, and between clones from the same donor, indicating a non-genetic effect. These differences cannot be fully explained by current understandings but point to epigenetic and cellular reprograming effects of somatic cell nuclear transfer. Notably, some phenotypic variations have been reversed through further cloning. Here we summarize these observations and elaborate on the cloning procedure.

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 vitro-matured 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.

The umbilical cord acts as the critical lifeline of the developing fetus by providing nutrients and oxygen to it. Umbilical cord abnormalities are considered the leading cause of stillbirth in humans, but information on stillbirths associated with umbilical cord abnormalities is very scant in the clinical practice of animals. Here, we described a case of fetal demise in camels indicated to be caused by fetal death from strangulation by its umbilical cord, which is commonly known as the nuchal cord. A pregnant camel at its 36 weeks of gestation spontaneously aborted a single fetus. The camel was 5 years old and nullipara. A 6-day-old cloned embryo was transferred transcervically to the recipient. Pregnancy was confirmed 50 days after embryo transfer by ultrasonography, and the pregnant camel was maintained under a standard nutritional plan. The neck of the aborted fetus was strangulated tightly by a double loop of the umbilical cord. There was no congenital anomaly or other malformation in the fetus. We concluded that the nuchal cord was tightly coiled around the neck of the fetus and interfered with the blood flow in the fetus by collapsing the umbilical vein and subsequently causing fetal death and abortion. To the authors' knowledge, this is the first reported case of a nuchal cord in camels.

The present study investigated the effect of superstimulation to improve in vitro embryo production in the Gulf area, where the temperature is high. Holstein cows were classified into the control and superstimulation groups. Superstimulation was induced with a single intramuscular injection of pregnant mare serum gonadotropin (PMSG; 2500 IU) on day 14 of the estrus cycle (day 0; estrus). The development of follicles was evaluated by ultrasonography of the ovaries daily. At 40 h after the PMSG injection, oocytes were collected by the ovum pick-up (OPU) technique. OPU was performed at the same stage of the estrus cycle in the control group as in the superstimulation group. The number of follicles with a diameter of more than 6 mm and the number of retrieved cumulus-oocyte complexes were significantly higher in the superstimulation group than in the control group. Furthermore, the maturation rate was higher in the superstimulation group than in the control group. Cloned embryos were produced by somatic cell nuclear transfer using matured oocytes. The cleavage and blastocyst formation rates were significantly higher in the superstimulation group than in the control group. In conclusion, a single injection of PMSG can facilitate the efficient production of cloned cow embryos.

Abstract Background Genetic engineering technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides a powerful tool for developing disease models and determining gene functions. Recent interests in canine cancer models have highlighted the necessity of developing genetic engineering tools for dogs. In this study, we attempted to generate optimized CRISPR/Cas9 system to target canine tumor protein 53 (TP53), one of the most crucial tumor suppressor genes, to establish TP53 knockout canine cells for canine cancer research. Results We constructed CRISPR/Cas9 vectors using each of three TP53 gene-targeting guide RNAs (gRNAs) with minimal off-target potential. After transfection, we obtained several clones of TP53 knockout cells containing “indel” mutations in the targeted locus which had infinite cellular life span, resistance to genotoxicity, and unstable genomic status in contrast to normal cells. Of the established TP53 knockout cells, TP53KO#30 cells targeted by TP53 gRNA #30 showed non-cancerous phenotypes without oncogenic activation both in vitro and in vivo. More importantly, no off-target alteration was detected in TP53KO#30 cells. We also tested the developmental capacity of TP53 knockout cells after application of the somatic cell nuclear transfer technique. Conclusions Our results indicated that TP53 in canine cells was effectively and specifically targeted by our CRISPR/Cas9 system. Thus, we suggest our CRISPR/Cas9-derived canine TP53 knockout cells as a useful platform to reveal novel oncogenic functions and effects of developing anti-cancer therapeutics.

Recent advances in somatic cell nuclear transfer (SCNT) in canines facilitate the production of canine transgenic models. Owing to the importance of stable and strong promoter activity in transgenic animals, we tested human elongation factor 1α (hEF1α) and cytomegalovirus (CMV) promoter sequences in SCNT transgenic dogs. After transfection, transgenic donor fibroblasts with the hEF1α-enhanced green fluorescence protein (EGFP) transgene were successfully isolated using fluorescence-activated cell sorting (FACS). We obtained four puppies, after SCNT, and identified three puppies as being transgenic using PCR analysis. Unexpectedly, EGFP regulated by hEF1α promoter was not observed at the organismal and cellular levels in these transgenic dogs. EGFP expression was rescued by the inhibition of DNA methyltransferases, implying that the hEF1α promoter is silenced by DNA methylation. Next, donor cells with CMV-EGFP transgene were successfully established and SCNT was performed. Three puppies of six born puppies were confirmed to be transgenic. Unlike hEF1α-regulated EGFP, CMV-regulated EGFP was strongly detectable at both the organismal and cellular levels in all transgenic dogs, even after 19 months. In conclusion, our study suggests that the CMV promoter is more suitable, than the hEF1α promoter, for stable transgene expression in SCNT-derived transgenic canine model.

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.

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.

Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly (88.9→114.4). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT.

To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF), and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average) were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes) but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT) process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process, which may eventually lead to problems with embryonic or placental defects.

Granulocyte-colony stimulating factor (G-CSF), a pleiotropic cytokine, belongs to the hematopoietic growth factor family. Recent studies have reported that G-CSF is a predictive biomarker of oocyte and embryo developmental competence in humans. The aim of our study was to determine whether CSF3 and its receptor (CSF3R) were expressed in porcine maternal reproductive tissues (oviduct and uterus), cumulus cells, and embryos and to investigate the effects of human recombinant G-CSF (hrG-CSF) supplementation during in vitro culture (IVC) on the developmental competence of pre-implantation embryos. To do this, we first performed reverse-transcription polymerase chain reaction (RT-PCR). Second, we performed parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT) to evaluate the embryonic developmental potential after hrG-CSF supplementation based on various concentrations (0 ng/mL, 10 ng/mL, 50 ng/mL, and 100 ng/mL) and durations (Un-treated, Days 0-3, Days 4-7, and Days 0-7) of IVC. Finally, we examined transcriptional levels of several marker genes in blastocysts. The results of our study showed that CSF3 transcript was present in all samples we assessed. CSF3-R was also detected, except in cumulus cells and blastocysts from PA. Furthermore, 10 ng/mL and Days 0-7 were the optimal concentration and duration for the viability of in vitro embryonic development, especially for SCNT-derived embryos. The rate of blastocyst formation and the total cell number of blastocysts were significantly enhanced, while the number and index of apoptotic nuclei were significantly decreased in optimal condition groups compared to others. Moreover, the transcriptional levels of anti-apoptotis- (BCL2), proliferation- (PCNA), and pluripotency- (POU5F1) related genes were dramatically upregulated. In conclusion, for the first time, we demonstrated that CSF3 and CSF3R were expressed in porcine reproductive organs, cells, and embryos. Additionally, we determined that hrG-CSF treatment improved porcine embryonic development capacity in vitro.

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.

Somatic cell nuclear transfer (SCNT) is an alternative potential tool for the conservation of endangered. In this study, somatic cells were collected from a purebred 9-month-old male mastiff and an 11-month-old female mastiff. Oocytes that had been matured in vivo were retrieved from outbred dogs by laparoscopy. We used cycling cells as donor cells for SCNT. A total of 289 oocytes were reconstructed with each male or female somatic cell and then fused/activated simultaneously by electrical stimulation. Finally, 224 embryos were transferred to 16 recipients that had been synchronized naturally. The efficiency of delivery of cloned dogs (7.1%) was threefold higher than in previous reports. Moreover, one surrogate delivered four identical cloned female Tibetan Mastiff puppies; another three surrogates each delivered triplets. Microsatellite analysis demonstrated the genotypic identity of the cloned puppies. Thus, our study has demonstrated techniques that improve significantly the overall efficiency of SCNT in the canine species.

To determine whether exogenous amino acids affect gene transcription patterns in parthenogenetic porcine embryos, we investigated the effects of amino acid mixtures in culture medium. Parthenogenetic embryos were cultured in PZM3 medium under four experimental conditions: 1) control (no amino acids except L-glutamine and taurine); 2) nonessential amino acids (NEAA); 3) essential amino acids (EAA); and 4) NEAA and EAA. The rate of development of embryos to the four-cell stage was not affected by treatment. However, fewer (P

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.