Identification of hepatic progenitor cells in the canine fetal liver.

The liver plays essential roles in human and animal organisms, such as the storage, release, metabolism, and elimination of various endogenous or exogenous substances. Although its vital importance, few treatments are yet available when a hepatic failure occurs, and hence, the use of stem cells has arisen as a possible solution for both human and veterinary medicines. Previous studies have shown the existence of hepatic progenitor cells in human fetuses that were positive for EpCAM and NCAM. There is limited evidence, however, further identification and characterization of these cells in other species. Considering the similarity between dogs and humans regarding physiology, and also the increasing importance of developing new treatments for both veterinary and translational medicine, this study attempted to identify hepatic progenitor cells in canine fetal liver. For that, livers from canine fetuses were collected, cells were isolated by enzymatic digestion and cultured. Cells were characterized regarding morphology and expression of EpCAM, NCAM, Nestin, and Thy-1/CD90 markers. Our results suggest that it is possible to identify hepatic progenitor cells in the canine fetal liver; however, for therapeutic use, further techniques for cellular isolation and culture are necessary to obtain enriched populations of hepatic progenitors from the canine fetal liver.

Foetal development of endocrine organs in dog.

The canine adenohypophysis starts to be identifiable from 25 day of pregnancy. ACTH-immunoreactive cells migrate until day 38 after which the number of ACTH-producing cells increases but their distribution does not change. The STH- and LH-producing cells first appear on day 38 of pregnancy. The primordium of the adrenal glands appears as a slender structure on day 27 and forms the definitive cortical structure on day 35. The histological pattern of the foetal adrenal cortex differs from the post-natal structure in so far as the three cortical zones (definitive zone, transitional zone and foetal zone) extend from the outside towards the inside of gland. The mass of foetal and neonatal adrenals is more than 10 times larger than the adult adrenals relative to body weight. The cortisol concentration in the amnion is slightly lower than in the allantois but the foetal serum cortisol concentration is significantly higher than in the maternal and foetal fluid compartments. The thyroxine concentrations in the allantois and amnion fluids exceed the foetal serum concentrations except in the ninth week of pregnancy, but thyroxine levels in foetal fluids and serum are below the physiological levels of adult animals. The exocrine and endocrine functions of the pancreas develop and act in parallel. Pancreatic cells are first detected at 30 days when the branched structure is clearly detectable immunohistochemically, and at that time, insulin-positive ß-cells and α-cells are visible as well. The foetal serum glucose concentration exceeds the healthy adult range, but the glucose concentration in the allantois and amnion fluid remains below the physiological blood glucose concentration of mature dogs. The insulin concentration in the allantois fluid greatly exceeds the foetal serum and amnion insulin concentrations.

Uterine and umbilical resistance index and fetal heart rate in pregnant bitches of different body weight.

The aim of this study was to compare uterine and umbilical artery blood flow and fetal heart rate (FHR) in small, medium and large body weight (BW) dogs during the second half of pregnancy. Purebred pregnant bitches were assigned to one of the following groups according to their BW: small (S; ≤10 kg), medium (M; 11-25 kg) and large (L; >25-45 kg). Uterine and umbilical Doppler and M-mode ultrasonography was conducted every 10 days from Day 30-60 (Day 0 = first day of gestation). From Day 40, uterine and umbilical artery resistance index (RI) progressively and differentially decreased in the three groups (P < 0.01) being less in L than S bitches (P < 0.01). Litter size but not maternal BW (P > 0.1) affected uterine RI on Days 40 (r = 0.39; P < 0.01) and 50 (r = 0.41; P < 0.01). Conversely, on Day 60, maternal BW (r = 0.61; P < 0.01) had an effect on uterine RI while litter size did not (P > 0.1). Fetal heart rate increased from Day 30-50 and decreased to the time of parturition (P < 0.01) without differences among groups at any time point (P > 0.1). Uterine and umbilical blood flow differentially increased throughout mid- and late-pregnancy in breeds with large and small BW. These differences were affected by litter size on Days 40 and 50, and by maternal BW on Day 60. Conversely, during this same period, FHR did not vary among BW groups. Physiological variations should be considered when gestational ultrasonic examination is interpreted in different BW bitches.

Molecular characterization of nephron progenitors and their early epithelial derivative structures in the nephrogenic zone of the canine fetal kidney.

Nephron progenitors (NPs) and nephrogenesis have been extensively studied in mice and humans and have provided insights into the mechanisms of renal development, disease and possibility of NP-based therapies. However, molecular features of NPs and their derivatives in the canine fetal kidney (CFK) remain unknown. This study was focused to characterize the expression of potential markers of canine NPs and their derivatives by immuno-fluorescence and western blot analysis. Transcription factors (TFs) SIX1 and SIX2, well-characterized human NP markers, were expressed in NPs surrounding the ureteric bud in the CFK. Canine NPs also expressed ITGA8 and NCAM1, surface markers previously used to isolate NPs from the mouse and human fetal kidneys. TF, PAX2 was detected in the ureteric bud, NPs and their derivative structures such as renal vesicle and S-shaped body. This study highlights the similarities in dog, mouse and human renal development and characterizes markers to identify canine NPs and their derivatives. These results will facilitate the isolation of canine NPs and their functional characterization to develop NP-based therapies for canine renal diseases.

In vitro production of canine blastocysts.

Though blastocyst production in vitro has been successful in several animal species, a culture system to produce viable and normal canine blastocysts in vitro remains to be established. In this study, we report the development of an in vitro culture system for canine preimplantation embryos produced via parthenogenetic activation (PA) and somatic cell nucleus transfer (SCNT). Our results show that the medium developed by us, named "Qingdao Agricultural University's (QAU)-4 medium", successfully breaks the developmental arrest observed at the eight-cell stage in canine embryos grown in other culture systems. The blastocysts produced in QAU-4 displayed normal blastocyst structures, including a clear inner cell mass and blastocyst cavity. We also found that blastocyst formation in PA embryos cultured in QAU-4 medium was quite high, though this was not so in the case of SCNT embryos. However, supplementation of QAU-4 medium with 100 nM of scriptaid caused a sharp increase in blastocyst formation in SCNT embryos. After culture, hatched blastocysts were also observed to successfully adhere to collagen-coated dishes, where further growth and differentiation occurred. To our knowledge, this is the first in vitro canine preimplantation embryo culture system that can successfully produce canine blastocysts.

The timeline development of female canine germ cells.

During the sex differentiation, the primordial germ cells (PGCs) pass through a differentiation, becoming spermatogonial cells in males and oocytes in females. In this phase, there is difference in gene expression and differentiation potency between males and females. Specific cell markers have been essential in the PGC meiosis beginning and become oocyte cells. However, there are few studies about germline in domestic animals. The domestic dog (Canis lupus familiaris) is an interesting animal model to be used in the investigation about the mammal development because it has several biochemical and physiological similarities to humans. In addition, some additional investigations about dogs may contribute to a better understanding of the biology and genetic components, improving clinical veterinary and zoological sciences. Here, we elucidated by immunofluorescence and quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), the dynamics of the expression of pluripotent (POU5F1 and NANOG) and germline (DDX4, DAZL and DPPA3) markers that are very important in the development of female canine germ cells during 35-50 days post-fertilization (dpf). The female canine germ cells were positive for pluripotent markers during middle developmental period. The number of DDX4, DAZL and DPPA3 cells increased along the germ cell maturation from 45 to 50 dpf. We provided an expression analysis of the pluripotent and germline markers in paraffin sections using the middle and later periods in female canine germ cells. The results can contribute the understanding about the timeline of each marker along the maturation of female canine germ cells. These results have a great significance to demonstrate the germ cell profile changes because it may allow the development of protocols about in vitro germ cell derivation.

Is the biparietal diameter of fetuses in late gestation too variable to predict readiness for cesarean section in dogs?

Correct assessment of readiness for cesarean section is essential for timing elective cesarean section during late pregnancy in the bitch. In humans, biparietal diameter is sufficiently precise and accurate and used in a clinical setting daily. The objectives of this study were to determine whether fetal biparietal diameter in late gestation in the dog could be used to predict readiness for cesarean section by having reached a minimum cut-off value and to correlate the biparietal diameter to birth weight. The biparietal diameter of 208 puppies in 34 litters from 31 English bulldog bitches and 660 puppies in 78 litters from 70 Boerboel bitches were measured immediately after delivery by cesarean section, performed at full term, using digital calipers. At the same time the birth weight of the same 208 English bulldog puppies and 494 of the same Boerboel puppies in 59 litters from 54 bitches was measured by means of an electronic scale. With a cesarean section, all the puppies in a litter are delivered simultaneously and readiness for cesarean section must be determined for a litter. The minimum, median and maximum biparietal diameter varied from 21.1 to 47.8, 32.9 to 50.0 and 34.2-58.2 mm, respectively, among English bulldog litters and from 18.4 to 48.7, 35.5 to 49.7 and 39.8-54.3 mm among Boerboel litters. This large variation suggests that biparietal diameter is too variable within and among litters to be useful as a means of determining readiness for cesarean section.

Early results on canine fetal kidney development: Ultrasonographic evaluation and value in prediction of delivery time.

To date there have been no studies that describe the ultrasonographic evaluation of kidney development in canine fetuses. The aim of this prospective and longitudinal study was to monitor fetal kidney development with ultrasound and use fetal kidney measurements as a complementary biometric index for estimation of gestational age. Ultrasonographic examinations were performed on 15 clinically healthy pregnant bitches every four days from 30th day of pregnancy, until visualization of the fetal renal pelvis was no longer possible. Four distinct periods of ultrasonographic canine fetal kidney development were defined. Kidney length and renal pelvis distention were measured on longitudinal plane images. The fetal kidney ranged from 0.40 cm to 2.30 cm in length, and diameter of the pelvis ranged from 0.06 cm to 0.17 cm, however by the end of gestation the renal pelvis was no longer dilated and so its diameter could not be measured. Statistical analysis confirmed a relationship between gestational age and fetal kidney growth. Ultrasonographic evaluation of fetal kidney development is simple to perform. There is a strong correlation between gestational age and kidney length, which allowed generation of an equation to estimate delivery date with high sensitivity between 48 and 52 days of pregnancy. Fetal organ development can be considered complete when the renal pelvis is no longer dilated, this finding can assist the ultrasonographer in staging the gestation by prompting examination for fetal intestinal motility which begins at the same gestational age. Measurement of fetal kidney length can be used in conjunction with other methods to estimate gestational age and predict delivery time.

Use of a GnRH synthetic analog (buserelin) for estrous induction in female dogs / Utilização de análogo sintético de GnRH (buserelina) na indução do estro em cadelas

The aim of this study was to evaluate the effectiveness of a GnRH synthetic analog, as an estrous inducer in female dogs when administered during the anestrous phase, and to evaluate the pregnancy rate achieved through natural copulation. For this purpose, ten female dogs of different breeds were used. The subjects received buserelin by intramuscular injections at a dose of 2,1mcg when female dogs weighed up to 10kg (Group 1) and of 4,2mcg when the dogs weighed above 10kg (Group 2). Of the ten subjects, only three presented estrus after a single injection of buserelin: two dogs from Group 1 and one dog from Group 2 on average 7±1.29 days. The remaining seven dogs were given a second dose of buserelin, equal to the first administration. Of these, three belonged to Group 1 and four to Group 2. Four of these dogs exhibited clinical signs of estrus within, on average 9±7.3 days from the second injection. The seven female dogs that did enter estrus were fertilized successfully through natural copulation. The administration of buserelin was effective in inducing estrus in female dogs during the anestrous phase, with a maximum of two administrations.(AU)

Objetivou-se avaliar a eficácia de um análogo sintético ao GnRH como indutor de estro em cadelas em anestro e a taxa de prenhez por meio de cópula natural. Para isso, foram utilizadas 10 fêmeas caninas de diferentes raças. Cadelas de até 10kg de peso (grupo 1) foram submetidas à administração de buserelina por via intramuscular, na dose de 2,1mcg, e cadelas acima de 10kg (grupo 2) foram submetidas à mesma medicação, porém na dose de 4,2mcg. Das fêmeas em anestro, apenas três apresentaram estro com apenas uma aplicação, sendo duas do grupo 1 e uma do grupo 2, em 7±1,29 dias, em média. Em sete cadelas foi administrada mais uma dose de buserelina; destas, quatro eram pertencentes ao grupo 2 e três eram do grupo 1. Os sinais de estro ocorreram, em média, após 9±2,73 dias da segunda aplicação. As sete cadelas que manifestaram estro foram fertilizadas por meio de cópula natural. A administração de buserelina é eficiente para a indução de estro, em cadelas em anestro, em, no máximo, duas aplicações.(AU)

Development and in vivo validation of tissue-engineered, small-diameter vascular grafts from decellularized aortae of fetal pigs and canine vascular endothelial cells.

BACKGROUND: Tissue engineering has emerged as a promising alternative for small-diameter vascular grafts. The aim of this study was to determine the feasibility of using decellularized aortae of fetal pigs (DAFPs) to construct tissue-engineered, small-diameter vascular grafts and to test the performance and application of DAFPs as vascular tissue-engineered scaffolds in the canine arterial system. METHODS: DAFPs were prepared by continuous enzymatic digestion. Canine vascular endothelial cells (ECs) were seeded onto DAFPs in vitro and then the vascular grafts were cultured in a custom-designed vascular bioreactor system for 7 days of dynamic culture following 3 days of static culture. The grafts were then transplanted into the common carotid artery of the same seven dogs from which ECs had been derived (two grafts were prepared for each dog with one as a backup; therefore, a total of 14 tissue-engineered blood vessels were prepared). At 1, 3, and 6 months post-transplantation, ultrasonography and contrast-enhanced computed tomography (CT) were used to check the patency of the grafts. Additionally, vascular grafts were sampled for histological and electron microscopic examination. RESULTS: Tissue-engineered, small-diameter vascular grafts can be successfully constructed using DAFPs and canine vascular ECs. Ultrasonographic and CT test results confirmed that implanted vascular grafts displayed good patency with no obvious thrombi. Six months after implantation, the grafts had been remodeled and exhibited a similar structure to normal arteries. Immunohistochemical staining showed that cells had evenly infiltrated the tunica media and were identified as muscular fibroblasts. Scanning electron microscopy showed that the graft possessed a complete cell layer, and the internal cells of the graft were confirmed to be ECs by transmission electron microscopy. CONCLUSIONS: Tissue-engineered, small-diameter vascular grafts constructed using DAFPs and canine vascular ECs can be successfully transplanted to replace the canine common carotid artery. This investigation potentially paves the way for solving a problem of considerable clinical need, i.e., the requirement for small-diameter vascular grafts.

Clinical use of fetal measurements to determine the whelping day in German shepherd breed bitches.

The aim of this work was to use a linear regression model previously developed in a pilot study to calculate days before parturition (DbP) using inner chorionic cavity (ICC), biparietal diameter (BPD), crown-rump length (CRL), body diameter (BD) and deep portion of telencephalic vesicle (DPTV) in German shepherd dogs (GSD) with known ovulation day and then to test that model in bitches with unknown ovulation day. In our current study, a model for GSD bitches published in a previous report, proved satisfactory for ICC [DbP=44.76-(4.34×ICC)] and BPD [DbP=38.65-(12.86×BPD)]. We therefore used their model, but developed a new one for CRL, BD and DPTV. For ICC and BPD, we tested accuracy for more than 35days before parturition (ICC) and more than 15days before parturition (BPD). Measurements were taken on at least two fetuses during each ultrasound recording (US) of 22 GSD bitches with known (n=16) and unknown (n=6) ovulation days. The accuracy of the above model was 77-100% for ICC and 83-96% for BPD with a precision of ±1 and ±2days, respectively. Accuracy increased significantly when US was performed more than 35days before parturition for ICC and more than 15days before parturition for BPD. BD and CRL were the most accurate parameters (R2=0.95 and 0.85). In bitches with unknown ovulation day, BD accuracy was 71.4-100% with a precision of ±1day and ±2days, respectively. CRL and DPTV were less accurate (±1day, 60%; ±2days, 80% accuracy).

Estimation of fetal lung development using quantitative analysis of ultrasonographic images in normal canine pregnancy.

We investigated the quantitative analysis of sonographic images to predict fetal lung maturity of the canine foetus in normal pregnancy. Twelve bitches were recruited in the present study. Serial ultrasonographic exams were performed at three pre-determined time periods corresponding to the pseudoglandular (40-48 days of pregnancy), canalicular (49-56 days of pregnancy) and saccular phase (57-63 days of pregnancy) of lung development. Mean grey level (MGL) and the standard deviation of the histogram (SDH) of fetal lung and liver sonographic images were measured with dedicated software. The lung-to-liver ratio (LLR) for both parameters was also calculated. Measurements were taken on the two caudal-most foetuses and then averaged. SDH did not show any statistically significant difference between the three time periods in the lungs or in the liver. MGL measured in the lungs significantly increased in the first period and reached a plateau during the last two periods. Liver echogenicity was constant during the first two periods and significantly increased during the last week of gestation. The LLR of MGL significantly decreased during the last week of pregnancy. The LLR was a very good test to detect fetal lung maturity (area under the receiver operator curve (AUROC) = 0.875); using a cut-off value of LLR < 1.541, sensitivity was 83.33% and specificity was 83.33%, positive likelihood ratio = 5. LLR of MGL is an accurate test to estimate lung development in normal canine pregnancies.

The bald and the beautiful: hairlessness in domestic dog breeds.

An extraordinary amount of genomic variation is contained within the chromosomes of domestic dogs, manifesting as dramatic differences in morphology, behaviour and disease susceptibility. Morphology, in particular, has been a topic of enormous interest as biologists struggle to understand the small window of dog domestication from wolves, and the division of dogs into pure breeding, closed populations termed breeds. Many traits related to morphology, including body size, leg length and skull shape, have been under selection as part of the standard descriptions for the nearly 400 breeds recognized worldwide. Just as important, however, are the minor traits that have undergone selection by fanciers and breeders to define dogs of a particular appearance, such as tail length, ear position, back arch and variation in fur (pelage) growth patterns. In this paper, we both review and present new data for traits associated with pelage including fur length, curl, growth, shedding and even the presence or absence of fur. Finally, we report the discovery of a new gene associated with the absence of coat in the American Hairless Terrier breed.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'.

Development of the independent function of fetal thyroid glands in the dog in connection with iodothyronine concentrations in pregnant bitches, fetal fluids, and fetal serum.

Thyroxine (T4) and triiodothyronine (T3) concentrations in pregnant and nonpregnant bitches were measured. The allantoic and amniotic fluid samples were collected separately in the third week of pregnancy, and fetal blood samples were collected in the fourth week of pregnancy. There was no difference between T4 results in the pregnant and nonpregnant animals, but the measured serum concentrations exceeded the healthy range for normal adults. Serum T4 concentrations were lower in the fetus than those in adults (P < 0.01). Fetal T4 concentrations continuously increased and reached 13.38 ± 6.19 nmol/L before birth. The fetal serum T4 concentrations were lower than the T4 concentrations in allantoic and amniotic fluid until the seventh week, and the fetal serum T3 concentrations were lower than those in fetal fluids throughout the pregnancy (P < 0.01). Maximum T3 concentrations in allantoic and amniotic fluid exceeded the concentrations in the fetal and maternal serum. It is conceivable that the considerable differences between maternal and fetal serum T4 concentrations in healthy animals are explained by the T4 impermeability of the placenta. Extremely high maternal T4 (193.5 nmol/L) in 1 bitch was associated with T4 concentrations under the detection limit in the fetal fluids and serum suggesting an inhibitory effect. The T4 concentrations in all the fetal fluids and serum were under the detectable concentration that can be defined by 3.0 nmol/L in that bitch. We have demonstrated that fetal thyroid glands start functioning independently at the same time as thyroid cell formation in the dog, but the overproduction of maternal T4 may have a suppressive effect on fetal iodothyronine production.

The promise of dog cloning.

Dog cloning as a concept is no longer infeasible. Starting with Snuppy, the first cloned dog in the world, somatic cell nuclear transfer (SCNT) has been continuously developed and used for diverse purposes. In this article we summarise the current method for SCNT, the normality of cloned dogs and the application of dog cloning not only for personal reasons, but also for public purposes.

Preimplantation development of cloned canine embryos recovered by hysterectomy or surgical uterine flushing and subsequent pregnancy outcomes.

Dog cloning offers a substantial potential because of the advancements in assisted reproductive technology and development of the human disease model in line with the transgenic technique. However, little is known about the development of the canine cloned embryo during the preimplantation period. The aim of this study was to investigate the most efficient method and time for collecting cloned canine preimplantation embryos and to ascertain the developmental timeline of cloned canine embryos. Two hundred cloned embryos were created and transferred into 11 surrogates. The preimplantation stage cloned embryos were then collected on Days 7, 8, and 9 using an ovariohysterectomy or the Foley balloon catheter method. The recovery rate of reconstructed embryos was 63.6% and 60.6% for the ovariohysterectomy and Foley balloon catheter methods, respectively. Although significant differences were observed in the early developmental stages (one-cell and 16-cell stages), no significant difference was observed in the blastocyst stage. Significantly higher blastocyst rate was observed when the embryos were collected on Day 8 (11.4%) than on Day 7 (0.0%; P < 0.05). At the proximal uterine horn on Day 7, no embryos at any stage were found, whereas on Days 8 and 9, blastocysts were found. We have observed a 63% initial pregnancy rate at 25 to 30 days after embryo transfer and a 50% full-term pregnancy rate, whereas 6.3% of the puppies were born, and 5.5% were born live among the total transferred embryos. Our results suggest that cloned embryos can develop to blastocysts by Day 8, and full-term pregnancy can be achieved after embryo transfer in canine.

Development of the cardiorespiratory system in dogs from days 16 to 46 of pregnancy.

Dogs have been studied for several reasons, such as the genetic improvement, their use as experimental models, in zoonotic research, cell therapy and as a model for human diseases. However, many features relating to the embryonic development of dogs remain unknown because of the absence of embryological studies. Considering the importance of the cardiorespiratory system in the development of embryos, the aim of this study was to investigate the development of the main cardiorespiratory organs of dog embryos and foetuses with estimated gestational ages from 16 to 46 days using macro- and microscopic descriptions. On day 16 of development, the neural tube and crest were formed, the anterior and posterior neuropore closure had begun and the somites had developed. Between days 22 and 27 of gestation, the lung buds and the initial formation of the primary bronchi and heart chambers were observed. The heart chambers exhibited the endo-, myo- and epicardial layers but did not have obvious differences in thickness among each other. Between days 41 and 46 of gestation, the nasal conchae and septa and trachea were formed, which exhibited characteristic epithelia. The lung formation and lobation were complete. The heart and major vessels exhibited mature histological architecture when their anatomical development was complete. The results of this study contribute to a more accurate definition of the embryonic and foetal developmental stages in dogs.

Can maternal-fetal hemodynamics influence prenatal development in dogs?

The goals of this study were to report embryonic and fetal ultrasound changes and compare blood flow of uteroplacental and umbilical arteries of normal and abnormal conceptus. Accordingly, from the day of mating or artificial insemination, all fetuses in 60 pregnancies were evaluated weekly. According to the ultrasound findings, the gestational age was determined and the conceptuses were divided into normal or abnormal (embryonic and fetal abnormalities). The two-dimensional ultrasound assessment consists of measuring and evaluating the echogenicity of conceptus and extra-fetal structures. Doppler velocimetry measured the resistivity index (RI) and pulsatility index (PI) of uteroplacental and umbilical arteries. Two-dimensional and Doppler measurements were expressed as mean and standard deviation. Differences between normal and abnormal groups were subject to Mann-Whitney test (P<0.05). Of 264 fetuses, 15.90% showed embryonic abnormalities (resorption) and 5.68% presented fetal abnormalities (congenital abnormalities, fetal underdevelopment and fetal death). We observed a reduced diameter and abnormalities in the contour of gestational vesicle, lack of viability, increased placental thickness, increased fluid echogenicity and increases in RI and PI of uteroplacental arteries of conceptuses with embryonic resorption between the 2nd and 4th weeks. Fetuses with abnormalities showed changes in the flow of uteroplacental and umbilical arteries prior to visualization of two-dimensional alterations and different vascular behavior according to the classification of the change. Results show that ultrasound is efficient for the detection of embryonic and fetal abnormalities. When combined with Doppler ultrasound, it allows early detection of gestational changes, as well as hemodynamic changes, in conceptuses with abnormalities, which may influence their development.

Endothelial differentiation of canine yolk sac cells transduced with VEGF.

Yolk sac (YS) is the site of blood-cell production where primitive erythroid cells originate and complete their maturation. YS is a source of precursor cells, however its differentiation potential and suitability for cell therapies are not well described. YS can be a cell source when neovascularization is required. This study characterized YS canine cells, transduced with VEGF, to analyze then using Immunocytochemistry, flow cytometry and real time PCR. Immunocytochemistry: positive expression for CD105, PCNA, VEGF and vWF, flow cytometry for CD105, VEGF, PCNA, OCT-4 and RT-qPCR for VEGF, CD31, CD105, PCNA and FLT - 1, indicating that these cells have characteristics of endothelial progenitor and pluripotency. After transduction, the YS cells changed their morphology and showed endothelial-like cells. We suggest, because of their cell surface phenotype as well as their capacity to differentiate into endothelial-like cells, that canine YS represents a source of cells for neovascularization therapies.

Altering histone acetylation status in donor cells with suberoylanilide hydroxamic acid does not affect dog cloning efficiency.

Although dog cloning technology has been applied to conservation of endangered canids, propagation of elite dogs, and production of transgenic dogs, the efficiency of cloning is still very low. To help overcome this problem, we evaluated the effect of treating donor cells with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on dog cloning efficiency. Relative messenger RNA expressions of the bax1/bcl2 ratio and Dnmt1 in fibroblasts treated with different concentrations (0, 1, 10, 50 µM) of SAHA and durations (0, 20, 44 hours) were compared. Treatment with 1 µM for 20 hours showed significantly lower bax1/bcl2 and Dnmt1 transcript abundance. Acetylation of H3K9 was significantly increased after SAHA treatment, but H4K5, H4K8 and H4K16 were not changed. After SCNT using control or donor cells treated with SAHA, a total of 76 and 64 cloned embryos were transferred to seven and five recipients, respectively. Three fetuses were diagnosed in both control and SAHA-treated groups by ultrasonography 29 days after the embryo transfer, but there was no significant difference in the pregnancy rate (4.2% vs. 4.3%). In conclusion, although SAHA treatment as used in this study significantly decreased bax1/bcl2 and Dnmt1 transcripts of donor nuclei, as well as increased H3 acetylation, it was not enough to increase in vivo developmental competence of cloned dog embryos.