Age-specific variations in hematological and biochemical parameters in middle- and large-sized of dogs

Aging triggers cellular and molecular alterations, including genomic instability and organ dysfunction, which increases the risk of disease in mammals. Recently, due to the markedly growing number of aging dogs in the world, as much as 49% in total number of pet dogs, it is necessary to improve and maintain their quality of life by understanding of the biological effects of aging. Therefore, the aim of this study was to determine specific biomarkers in aging dogs as a means of defining a set of hematological/biochemical biomarkers that influence the aging process. Blood samples were collected from younger (1–3 years) and older (7–10 years) dogs of middle/large size. The hematological/biochemistry analysis was performed to evaluate parameters significantly associated with age. Enzyme-linked immunosorbent assay was used to target growth hormone (GH)/insulin growth factor-1 (IGF-1), one of the main regulators of the aging process. Declining levels of total protein and increased levels of glucose in young dogs was observed regardless of their body size. Notably, a significantly high concentration of GH and IGF-1 in the younger dogs compared to the older dogs was found in middle/large-sized dogs. GH and IGF-1 were also found at significantly high levels in large-sized dogs compared to middle-sized dogs, suggesting a similar trend to that of elderly humans. Consequently, glucose, total protein, GH, and IGF-1 were identified as potential biomarkers for regulating the aging process in large/middle-sized dogs. These findings provide an invaluable insight into the mechanism of aging for the field of aging research.

Age-specific variations in hematological and biochemical parameters in middle- and large-sized of dogs

Aging triggers cellular and molecular alterations, including genomic instability and organ dysfunction, which increases the risk of disease in mammals. Recently, due to the markedly growing number of aging dogs in the world, as much as 49% in total number of pet dogs, it is necessary to improve and maintain their quality of life by understanding of the biological effects of aging. Therefore, the aim of this study was to determine specific biomarkers in aging dogs as a means of defining a set of hematological/biochemical biomarkers that influence the aging process. Blood samples were collected from younger (1–3 years) and older (7–10 years) dogs of middle/large size. The hematological/biochemistry analysis was performed to evaluate parameters significantly associated with age. Enzyme-linked immunosorbent assay was used to target growth hormone (GH)/insulin growth factor-1 (IGF-1), one of the main regulators of the aging process. Declining levels of total protein and increased levels of glucose in young dogs was observed regardless of their body size. Notably, a significantly high concentration of GH and IGF-1 in the younger dogs compared to the older dogs was found in middle/large-sized dogs. GH and IGF-1 were also found at significantly high levels in large-sized dogs compared to middle-sized dogs, suggesting a similar trend to that of elderly humans. Consequently, glucose, total protein, GH, and IGF-1 were identified as potential biomarkers for regulating the aging process in large/middle-sized dogs. These findings provide an invaluable insight into the mechanism of aging for the field of aging research.

Age-specific variations in hematological and biochemical parameters in middle- and large-sized of dogs

Iodixanol supplementation during sperm cryopreservation improves protamine level and reduces reactive oxygen species of canine sperm

The objective of this study was to analyze the protective effects of iodixanol on dog spermatozoa during cryopreservation. The optimal concentration of iodixanol, 1.5%, was determined using fresh spermatozoa and was applied in the following experiments. The 1.5% iodixanol group showed significantly increased sperm motility from that in the control (p < 0.05). Lower mitochondrial reactive oxygen species (ROS) modulator (ROMO1) and pro-apoptotic gene (BAX) expressions, together with higher expressions of protamine-2 (PRM2), protamine-3 (PRM3), anti-apoptotic B-cell lymphoma-2 (BCL2), and sperm acrosome associated-3 (SPACA3) genes were detected in the iodixanol-treated group. In addition, decreased protamine deficiency and cryocapacitation were observed in the treatment group. Our results show that supplementation with 1.5% iodixanol is ideal for reducing production of ROS and preventing detrimental effects during the canine sperm cryopreservation process, effects manifested as increased motility and reduced cryocapacitation in frozen-thawed spermatozoa.

Health and temperaments of cloned working dogs

Dogs serve human society in various ways by working at tasks that are based on their superior olfactory sensitivity. However, it has been reported that only about half of all trained dogs may qualify as working dogs through conventional breeding management because proper temperament and health are needed in addition to their innate scent detection ability. To overcome this low efficiency of breeding qualified working dogs, and to reduce the enormous costs of maintaining unqualified dogs, somatic cell nuclear transfer has been applied in the propagation of working dogs. Herein, we review the history of cloning working dogs and evaluate the health development, temperaments, and behavioral similarities among the cloned dogs. We also discuss concerns about dog cloning including those related to birth defects, lifespan, and cloning efficiency.

Establishment and identification of cell lines from type O blood Korean native pigs and their efficiency in supporting embryonic development via somatic cell nuclear transfer

Due to their similarities with humans in anatomy, physiology, and genetics miniature pigs are becoming an attractive model for biomedical research. We aim to establish and evaluate blood type O cells derived from Korean native pig (KNP), a typical miniature pig breed in Korea. Ten cell lines derived from 8 KNP piglets and one adult female KNP (kidney and ear tissues) were established. To confirm the presence of blood type O, genomic DNA, fucosyltransferase (FUT) expression, and immunofluorescence staining were examined. Additionally, fluorescence-activated cell sorting and somatic cell nuclear transfer were performed to investigate the normality of the cell lines and to evaluate their effectiveness in embryo development. We found no significant bands corresponding to specific blood group A, and no increase in FUT expression in cell lines derived from piglets No. 1, No. 4, No. 5, No. 8, and the adult female KNP; moreover, they showed normal levels of expression of α 1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase. There was no significant difference in embryo development between skin and kidney fibroblasts derived from the blood type O KNPs. In conclusion, we successfully established blood type O KNP cell lines, which may serve as a useful model in xenotransplantation research.

Clinical assessment after human adipose stem cell transplantation into dogs

Adipose tissue-derived stem cell (ASCs) are an attractive source of stem cells with therapeutic applicability in various fields for regenerating damaged tissues because of their stemness characteristics. However, little has reported on evaluating adverse responses caused by human ASC therapy. Therefore, in the present study, a clinical assessment after human ASC transplantation into dogs was undertaken. A total of 12 healthy male dogs were selected and divided into four groups: saline infusion, saline bolus, ASC infusion, and ASC bolus groups. Physical assessment and blood analysis were performed following ASC transplantation, and the concentrations of angiogenic factors, and pro- and anti-inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). There were no adverse vital sign responses among the dogs. Blood analyses revealed no remarkable complete blood count or serum chemistry results. ELISA results for angiogenic and anti-inflammatory factors including matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were significantly higher in the two ASCs groups than in the controls. In conclusion, this study demonstrated that transplantation of human ASCs produced no adverse effects and could be used safely in dogs. In addition, human ASCs could be involved in modulating secretions of angiogenic factors including MMP9, VEGF, bFGF, and HGF and anti-inflammatory factor IL-10.

Mineralized deposits in the uterus of a pig without pregnancy loss

Herein, we describe a case of uterine calcification in the uterus of a pig without pregnancy loss. The recipient underwent cloned embryo transfer and Cesarean section for safe delivery of cloned piglets. During the Cesarean section, 4 white, star-like, (2 × 2 × 2) cm, calcified structures were found within the endometrial cavity. Despite dystrophic calcification around the placenta, healthy cloned piglets were produced successfully. To our knowledge, this is the first reported case of dystrophic calcification occurring within the uterus in a pregnant pig.

Isolation and molecular identification of Vibrio harveyi from the look down fish (Selene vomer)

Gradual mortality of look down fish (Selene vomer) was observed in a private aquarium in Seoul, showing abnormal swimming behavior and lethargy. A bacterial pathogen from kidney was cultured, identified, and confirmed as Vibrio harveyi using Vitek System 2 and 16S rRNA gene sequencing. A predominant bacterial strain, SNUVh-LW2 was proved to be most closely related to isolates from China by phylogenetic analysis with minimum evolution method. Also, tetracycline was considered as the most sensitive antibiotic agent via antibiotic usceptibility test. The group of fish was treated according to the diagnostic result and no more mortality was observed.

Transgenesis for pig models

Animal models, particularly pigs, have come to play an important role in translational biomedical research. There have been many pig models with genetically modifications via somatic cell nuclear transfer (SCNT). However, because most transgenic pigs have been produced by random integration to date, the necessity for more exact gene-mutated models using recombinase based conditional gene expression like mice has been raised. Currently, advanced genome-editing technologies enable us to generate specific gene-deleted and -inserted pig models. In the future, the development of pig models with gene editing technologies could be a valuable resource for biomedical research.

Reproductive ability of a cloned male detector dog and behavioral traits of its offspring

In 2007, seven detector dogs were produced by somatic cell nuclear transfer using one nuclear donor dog, then trained and certified as excellent detector dogs, similar to their donor. In 2011, we crossed a cloned male and normal female by natural breeding and produced ten offspring. In this study, we investigated the puppies' temperaments, which we later compared with those of the cloned parent male. The results show that the cloned male had normal reproductive abilities and produced healthy offspring. All puppies completed narcotic detector dog training with a success rate for selection of 60%. Although the litter of cloned males was small in this study, a cloned male dog bred by natural mating produced puppies that later successfully completed the training course for drug detection. In conclusion, cloning an elite dog with superior genetic factors and breeding of the cloned dog was found to be a useful method to efficiently procure detector dogs.

Learning, memory and exploratory similarities in genetically identical cloned dogs

Somatic cell nuclear transfer allows generation of genetically identical animals using donor cells derived from animals with particular traits. To date, few studies have investigated whether or not these cloned dogs will show identical behavior patterns. To address this question, learning, memory and exploratory patterns were examined using six cloned dogs with identical nuclear genomes. The variance of total incorrect choice number in the Y-maze test among cloned dogs was significantly lower than that of the control dogs. There was also a significant decrease in variance in the level of exploratory activity in the open fields test compared to age-matched control dogs. These results indicate that cloned dogs show similar cognitive and exploratory patterns, suggesting that these behavioral phenotypes are related to the genotypes of the individuals.

Cloned foal derived from in vivo matured horse oocytes aspirated by the short disposable needle system

Transvaginal ultrasound-guided follicle aspiration is one method of obtaining recipient oocytes for equine somatic cell nuclear transfer (SCNT). This study was conducted: (1) to evaluate the possibility of oocyte aspiration from pre-ovulatory follicles using a short disposable needle system (14-G) by comparing the oocyte recovery rate with that of a long double lumen needle (12-G); (2) to investigate the developmental competence of recovered oocytes after SCNT and embryo transfer. The recovery rates with the short disposable needle vs. the long needle were not significantly different (47.5% and 35.0%, respectively). Twenty-six SCNT embryos were transferred to 13 mares, and one mare delivered a live offspring at Day 342. There was a perfect identity match between the cloned foal and the cell donor after analysis of microsatellite DNA, and the mitochondrial DNA of the cloned foal was identical with that of the oocyte donor. These results demonstrated that the short disposable needle system can be used to recover oocytes to use as cytoplasts for SCNT, in the production of cloned foals and for other applications in equine embryology

Intrapancreatic ectopic splenic tissue found in a cloned miniature pig

Somatic cell nuclear transfer (SCNT) is a cost-effective technique for producing transgenic pigs. However, abnormalities in the cloned pigs might prevent use these animals for clinical applications or disease modeling. In the present study, we generated several cloned pigs. One of the pigs was found to have intrapancreatic ectopic splenic tissue during histopathology analysis although this animal was grossly normal and genetically identical to the other cloned pigs. Ectopic splenic tissue in the pancreas is very rare, especially in animals. To the best of our knowledge, this is the first such report for cloned pigs.

Identification of abnormal gene expression in bovine transgenic somatic cell nuclear transfer embryos

This study was conducted to investigate the expression of three genes related to early embryonic development in bovine transgenic cloned embryos. To accomplish this, development of bovine transgenic somatic cell nuclear transfer (SCNT) embryos was compared with non-transgenic embryos. Next, mRNA transcription of three specific genes (DNMT1, Hsp 70.1, and Mash2) related to early embryo development in transgenic SCNT embryos was compared between transgenic and non-transgenic SCNTs, parthenogenetic embryos, and in vitro fertilization (IVF) embryos. Transgenic SCNT embryos showed significantly lower rates of development to the blastocyst stage than non-transgenic ones. To investigate normal gene expression, RNA was extracted from ten blastocysts derived from parthenogenesis, IVF, non-transgenic, and transgenic SCNT embryos and reverse-transcribed to synthesize cDNA. The cDNA was then subjected to PCR amplification and semi-quantified. More DNMT1 mRNA was detected in the transgenic SCNT group than the other three groups. Hsp 70.1 mRNA was detected in the IVF embryos, while lower levels were found in SCNT and parthenogenetic embryos. Mash2 mRNA was present at the highest levels in transgenic SCNT embryos. In conclusion, the higher levels of methylation and lower protein synthesis after heat shock in the transgenic SCNT embryos expected based on our results may cause lower embryonic development.

Relationship between pregnancy rate and serum progesterone concentration in cases of porcine embryo transfer

The level of P4 at the time of embryo transfer (ET) is important. P4 concentrations and numbers of corpora lutea for 126 recipients were evaluated. Nuclear transfer embryos were transferred into 126 surrogates. 11 maintained their pregnancy until full-term delivery, 17 miscarried, and implantation failed in 98 animals. P4 levels in the full-term group were significantly different from those of the pigs that aborted or in which implantation failed (p < 0.05). However, the numbers of corpora lutea were not significantly different. These findings indicate that the concentration of progesterone can be an important factor for successful ET in pigs.

Proposed Motor Scoring System in a Porcine Model of Parkinson's Disease induced by Chronic Subcutaneous Injection of MPTP

Destruction of dopaminergic neurons in the substantia nigra pars compacta (SNpc) is a common pathophysiology of Parkinson's disease (PD). Characteristics of PD patients include bradykinesia, muscle rigidity, tremor at rest and disturbances in balance. For about four decades, PD animal models have been produced by toxin-induced or gene-modified techniques. However, in mice, none of the gene-modified models showed all 4 major criteria of PD. Moreover, distinguishing between PD model pigs and normal pigs has not been well established. Therefore, we planned to produce a pig model for PD by chronic subcutaneous administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), neurotoxin. Changes in behavioral patterns of pigs were thoroughly evaluated and a new motor scoring system was established for this porcine model that was based on the Unified Parkinson's Disease Rating Scale (UPDRS) in human PD patients. In summary, this motor scoring system could be helpful to analyze the porcine PD model and to confirm the pathology prior to further examinations, such as positron emission tomography-computed tomography (PET-CT), which is expensive, and invasive immunohistochemistry (IHC) of the brain.

Quercetin improves the in vitro development of porcine oocytes by decreasing reactive oxygen species levels

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 microg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 microg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 microg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 microg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.

Quercetin improves the in vitro development of porcine oocytes by decreasing reactive oxygen species levels

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 microg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 microg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 microg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 microg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.

Functional improvement of porcine neonatal pancreatic cell clusters via conformal encapsulation using an air-driven encapsulator

Transplantation of islet cells into diabetic patients is a promising therapy, provided that the islet cells are able to evade host immune rejection. With improved islet viability, this strategy may effectively reverse diabetes. We applied 2% calcium alginate to generate small and large capsules to encapsulate porcine neonatal pancreatic cell clusters (NPCCs) using an air-driven encapsulator. After encapsulation, the viability was assessed at 1, 4, 7, 14 and 28 days and secretion of functional insulin in response to glucose stimulation were tested at days 14 and 28. Selective permeability of the small alginate capsules was confirmed using various sizes of isothiocyanate-labeled dextran (FITC-dextran). Encapsulation of NPCCs was performed without islet protrusion in the small and large capsules. The viability of NPCCs in all experimental groups was greater than 90% at day 1 and then gradually decreased after day 7. The NPCCs encapsulated in large capsules showed significantly lower viability (79.50 +/- 2.88%) than that of naive NPCCs and NPCCs in small capsule (86.83 +/- 2.32%, 87.67 +/- 2.07%, respectively) at day 7. The viability of naive NPCCs decreased rapidly at day 14 (75.67 +/- 1.75%), whereas the NPCCs encapsulated in small capsules maintained (82.0 +/- 2.19%). After 14 and 28 days NPCCs' function in small capsules (2.67 +/- 0.09 and 2.13 +/- 0.09) was conserved better compared to that of naive NPCCs (2.04 +/- 0.25 and 1.53 +/- 0.32, respectively) and NPCCs in large capsules (2.04 +/- 0.34 and 1.13 +/- 0.10, respectively), as assessed by a stimulation index. The small capsules also demonstrated selective permeability. With this encapsulation technique, small capsules improved the viability and insulin secretion of NPCCs without islet protrusion.