Evaluation of embryo-foetal biometry and its correlation with parturition date in Toy Poodle bitches.

Estimating the parturition date in dogs is challenging due to their reproductive peculiarities that. Ultrasonographic examination serves as a tool for studying embryo/foetal biometry and estimating the time of parturition by measuring foetal and extra-foetal structures. However, due to reproductive differences among various dog breeds, such estimates may have a non-significant pattern, representing inaccuracies in the estimated date of birth. This study aimed to monitor pregnant Toy Poodle bitches and establish relationships between ultrasonographically measured foetal and extra-foetal dimensions and the remaining time until parturition. Eighteen pregnant Toy Poodle bitches were subjected to weekly ultrasonographic evaluations and measurements of the inner chorionic cavity diameter, craniocaudal length (CCL), biparietal diameter (BPD), diameter of the deep portion of diencephalo-telencephalic vesicle (DPTV), abdominal diameter, thorax diameter (TXD), placental thickness and the renal diameter (REND). These parameters were retrospectively correlated with the date of parturition and linear regressions were established between gestational measurements and days before parturition (DBP). All analyses were conducted using the Statistical Package for Social Sciences (IBM® SPSS®) program at a 5% significance level. The foetal measurements that showed a high correlation (r) and reliability (R2) with DBP were BPD [(DBP = [15.538 × BPD] - 39.756), r = .97 and R2 = .93], TXD [(DBP = [8.933 × TXD] - 32.487), r = .94 and R2 = .89], DPTV [(DBP = [34.580 × DPTV] - 39.403), r = .93 and R2 = .86] and REND [(DBP = [13.735 × REND] - 28.937), r = .91 and R2 = .82]. This statistically validates the application of these specific formulas to estimate the parturition date in Toy Poodle bitches.

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.

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.

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.

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.

Effect of epigenetic modification with trichostatin A and S-adenosylhomocysteine on developmental competence and POU5F1-EGFP expression of interspecies cloned embryos in dog.

Adult canine fibroblasts stably transfected with either cytomegalovirus (CMV) or POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) were used to investigate if pre-treatment of these donor cells with two epigenetic drugs [trichostatin A (TSA), or S-adenosylhomocysteine (SAH)] can improve the efficiency of interspecies somatic cell nuclear transfer (iSCNT). Fluorescence-activated cell sorting (FACS), analyses revealed that TSA, but not SAH, treatment of both transgenic and non-transgenic fibroblasts significantly increased acetylation levels compared with untreated relatives. The expression levels of Bcl2 and P53 were significantly affected in TSA-treated cells compared with untreated cells, whereas SAH treatment had no significant effect on cell apoptosis. Irrespective of epigenetic modification, dog/bovine iSCNT embryos had overall similar rates of cleavage and development to 8-16-cell and morula stages in non-transgenic groups. For transgenic reconstructed embryos, however, TSA and SAH could significantly improve development to 8-16-cell and morula stages compared with control. Even though, irrespective of cell transgenesis and epigenetic modification, none of the iSCNT embryos developed to the blastocyst stage. The iSCNT embryos carrying CMV-EGFP expressed EGFP at all developmental stages (2-cell, 4-cell, 8-16-cell, and morula) without mosaicism, while no POU5F1-EGFP signal was observed in any stage of developing iSCNT embryos irrespective of TSA/SAH epigenetic modifications. These results indicated that bovine oocytes partially remodel canine fibroblasts and that TSA and SAH have marginal beneficial effects on this process.

Comparative Development of Embryonic Age by Organogenesis in Domestic Dogs and Cats.

The precise determination of the embryonic chronology is very important in reproductive biotechnologies, especially in estimating embryonic age. Thus, there is a need for greater knowledge and standardization for determining the chronology of embryonic development and functional morphology. We describe aspects of embryonic development in two domestic carnivores to add knowledge about organ peculiarities and for application in veterinary practice, in prenatal development and in the biotechnology fields. We found that the development of differential characteristics of embryonic organs occurs in the first trimester of pregnancy for both species. Thus, using the combination of the crown-rump length, macroscopic analysis and optical microscopy, it is possible to predict gestational age more precisely in animals that lack a defined breed and establish an embryonic pattern.

Cats and dogs: two neglected species in this era of embryo production in vitro?

During the last decades, in vitro fertilization (IVF) has become a routine technique in most domestic animals. However, in the dog the technique has lagged behind, with to date not a single pup born after IVF. In cats, healthy kittens have been born, but in fewer numbers than in cattle and horses. In pet animals, research in reproduction has mainly been focused on contraception, although recently, the introduction of new drugs especially marketed for cats and dogs will probably expand fertility research in carnivores towards the previously neglected area of assisted reproduction. In particular, the dog remains a real challenge for the reproductive biologist, due to the low meiotic capacity of canine follicular oocytes. In cats, oocyte maturation is less of a problem and embryo production rates comparable to those of cattle can be achieved. The domestic cat is a valuable model for endangered felids and it can even be used as a recipient for wild felid embryos. In this short review, we list some of the problems associated with the implementation of IVF in dogs and cats in relation to their reproductive characteristics, and we discuss the state-of-the-art of IVF in several other domestic species such as cattle, horses and pigs.

Domestic carnivore's development: detection of Oct-4, a pluripotency marker, in pharyngeal arches.

Very few carnivore's embryology is reported mainly restricted to old literature without new technique analyses. Also, their development focuses on pharyngeal arches and stem cell sources and the high capacity for differentiation from those cells to generate embryonic tissue. We aimed to use immunohistochemistry to prove the potentiality of these stem cell niches. The results were to highlight the timetable for the development of dogs and cats, the proper formation of pharyngeal arches and the description of these cells on first and second arches since 17-25 days of pregnancy. After that, the differentiation process is reduced.

Inherited liver shunts in dogs elucidate pathways regulating embryonic development and clinical disorders of the portal vein.

Congenital disorders of the hepatic portal vasculature are rare in man but occur frequently in certain dog breeds. In dogs, there are two main subtypes: intrahepatic portosystemic shunts, which are considered to stem from defective closure of the embryonic ductus venosus, and extrahepatic shunts, which connect the splanchnic vascular system with the vena cava or vena azygos. Both subtypes result in nearly complete bypass of the liver by the portal blood flow. In both subtypes the development of the smaller branches of the portal vein tree in the liver is impaired and terminal branches delivering portal blood to the liver lobules are often lacking. The clinical signs are due to poor liver growth, development, and function. Patency of the ductus venosus seems to be a digenic trait in Irish wolfhounds, whereas Cairn terriers with extrahepatic portosystemic shunts display a more complex inheritance. The genes involved in these disorders cannot be identified with the sporadic human cases, but in dogs, the genome-wide study of the extrahepatic form is at an advanced stage. The canine disease may lead to the identification of novel genes and pathways cooperating in growth and development of the hepatic portal vein tree. The same pathways likely regulate the development of the vascular system of regenerating livers during liver diseases such as hepatitis and cirrhosis. Therefore, the identification of these molecular pathways may provide a basis for future proregenerative intervention.

Folliculogenesis, ovulation and endocrine control of oocytes and embryos in the dog.

Reproductive physiology in dogs is quite unusual compared with that in other mammalian species. The peculiarities include the presence of numerous polyoocyte follicles, the ovulation of an immature oocyte (GV stage, non-fertilizable) and a peri-ovulatory period during which concentrations of circulating progesterone are particularly high. The aim of this review is to examine the unusual aspects of the reproductive physiology of dogs and how this relates to the clinical biology of this species.

Cryopreservation of canine embryos and resulting pregnancies.

The assisted reproductive techniques used in dogs have strictly limited utility when compared with other mammals. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen has been only readily available method for improved breeding for companion and working dogs. Recently, successful cryopreservation of embryos and subsequent embryo transfer with a non-surgical technique in dog was reported. Application of embryo cryopreservation and transfer technology could contribute to breeding management in companion dogs, working dogs including guide dogs and drug-detecting dogs and quarantine dogs. Such technology would also facilitate the transportation and storage of genetic materials and aid in the elimination of vertically transmitting diseases in the dog.

Ex vivo recovery of preimplantatory embryos in bitches.

The present study was conducted to investigate the timing of preimplantatory development in the dog and to evaluate the efficiency of flushing oviducts and uterine horns to collect embryos. Among the embryonic structures collected between day 8 and day 12 after ovulation, 43 % were at the 1-16 cells stage, 23% were at the morula stage and 34% at the blastocyst stage. Our collection method yielded to a recovery rate of 61.3 %, and 7.1 ± 0.7 embryos were harvested per bitch. In addition, the ovulation rate reached 11.6 ± 0.8 per bitch. The first morulae were observed from day 9 post-ovulation, while the first blastocyst appeared from day 10. Two-thirds of the collected morulae-blastocysts were obtained between the 11th and the 12th day after ovulation. To the moment, we suggest this is the best period to harvest canine embryo for cryopreservation.

Early canine pregnancy--a battle for successful growth and angiogenesis.

Currently, no early pregnancy marker has been identified in the bitch. However, significantly decreased concentrations of heat-shock protein (HSP) 70 and increased activity of matrix metalloproteinases (MMP) 2,9 were detected in serum from bitches during the pre-implantation period between days 5 and 13 after mating, that is, 2-3 days after ovulation as determined by the measurement of progesterone and vaginal cytology. Especially during the implantation period and thereafter (days 15-55), high serum concentrations of antibodies against desmin are present, which is believed to indicate or regulate decidualization. Pre-implantation embryos express mRNA for enzymes and cytokines, known to promote and regulate trophoblast growth, and some intrauterine changings like the increased activity of MMP 2,9 in maternal endometrium are dependant on the presence of embryos. Some mechanisms that protect canine embryos from attack by the maternal immune system can also be identified. The embryos express CD4, a receptor known to interact with immune cells. They, furthermore, do not express MHC I and II, which might prevent them from being recognized as foreign antigen. Pre-implantation embryos express FasL, which probably renders them able to destroy Fas-bearing cytotoxic T cells. Furthermore, the uterus during pre-implantation and implantation expresses cytokines that modulate the intrauterine milieu towards a predominance of Th2 cells. During pre-implantation and implantation, an increased uterine expression of platelet activating factor (PAF) and PAFR, vascular endothelial growth factor (VEGF) and EGFR2 as well as epithelial growth factor (EGF) is characteristic. Towards placentation, the upregulation of leukaemia inhibiting factor (LIF) and at placentation the expression of insulin-like growth factor(IGF)2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) are striking. Progesterone receptor (PR) appears to be downregulated inside the uterus except at placentation sites, presumably where it is essential for maintenance of pregnancy. In addition, receptor-bound P4 regulates the activity of MMP 2,9. Apoptosis seems to be a further regulatory mechanism. Expression of Fas and FasL mRNA in uterine tissue is maximum until implantation, both factors then decreased significantly. These changings might indicate increased endometrial apoptosis and defence against maternal cytotoxic T cells, probably promoting trophoblast invasion. In human decidual stromal cells, GnRH is involved in the regulation of apoptosis, which is proposed to be similar in pregnant bitches, as GnRH-R is expressed at canine implantation sites. Our work investigating immunological changes in pregnant bitches has elucidated aspects of the complex physiology of implantation but raises important questions about the mechanisms involved.

Cryopreservation of canine embryos.

The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer, and cryopreservation of gametes have contributed considerably to the development of biomedical sciences in addition to improving infertility treatments in humans as well as the breeding of domestic animals. However, ARTs used in canine species have strictly limited utility when compared with other mammalian species, including humans. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen to date is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind. We describe here the successful cryopreservation of embryos and subsequent embryo transfer in dogs. Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. When the 4-cell to morula stage of cryopreserved embryos were nonsurgically transferred into the uteri of nine recipient bitches using a cystoscope, five recipients became pregnant and four of them delivered a total of seven pups. The cryopreservation of embryos in canine species will facilitate the transportation and storage of genetic materials and will aid in the elimination of vertically transmitted diseases in dogs. In addition, this technique will contribute to the improved breeding of companion and working dogs such as guide dogs, drug-detecting dogs, and quarantine dogs.

Dogs cloned from adult somatic cells.

Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.

Immunohistochemical profile of odontogenic epithelium in developing dog teeth (Canis familiaris).

Tumors of the jaw bones and oral soft tissue are relatively common lesions in dogs. The aim of this study was to find cell markers to differentiate odontogenic epithelium from nonodontogenic epithelium for future research on the pathogenesis and pathology of odontogenic neoplasms in dogs. Keratin 14 and 19 staining was observed in odontogenic and nonodontogenic epithelium, whereas amelogenin and p75 neurotrophin receptor immunoreactivity was observed in certain odontogenic epithelial cells at various stages of development but not in other epithelial cells. Calretinin staining was observed in the alveolar epithelial cells directly overlying the developing tooth germ in 28 of 39 sections (71.8%), as well as the dental laminae in 30 of 35 sections (85.7%) and Serres rests in 24 of 28 sections (85.7%). Focal positivity was detected in the respiratory mucosa, some hair follicles, and fusion epithelium of the palate, but no calretinin staining was observed in other oral epithelial cells; therefore, calretinin has potential to be utilized as a marker to differentiate odontogenic form nonodontogenic epithelium.

High embryonic recovery rates with in vivo and ex vivo techniques in the bitch.

The embryonic collection techniques in dogs present a vast methodological variation and low recovery rates. The objectives were to compare and describe two techniques as to the recovery of canine embryos, on the 12th day after the first mating or artificial insemination. Embryos were recovered through uterine horn flushing in vivo, before performing the ovariohysterectomy (OHE) (Group 1; n = 9) or ex vivo, immediately after the OHE (Group 2; n = 9). In total, 43 and 47 embryonic structures were recovered in Groups 1 and 2, respectively. There was no significant difference (p > 0.05) between groups on recovery rates (72.8% and 81.0%, respectively). We inferred that both in vivo and ex vivo techniques allow a high rate of embryonic recovery; in the collection technique prior to the OHE, it is essential to carefully handle the reproductive system during the trans-surgical period and that the 12th day (D12) after the first mating/artificial insemination is an efficient option for the high recovery rate of morulae and blastocysts.

Early development and putative primordial germ cells characterization in dogs.

Previously, three distinct populations of putative primordial germ cells (PGCs), namely gonocytes, intermediate cells and pre-spermatogonia, have been described in the human foetal testis. According to our knowledge, these PGCs have not been studied in any other species. The aim of our study was to identify similar PGC populations in canine embryos. First, we develop a protocol for canine embryo isolation. Following our protocol, 15 canine embryos at 21-25 days of pregnancy were isolated by ovaryhysterectomy surgery. Our data indicate that dramatic changes occur in canine embryo development and PGCs specification between 21 to 25 days of gestation. At that moment, only two PGC populations with distinct morphology can be identified by histological analyses. Cell population 1 presented round nuclei with prominent nucleolus and a high nuclear to cytoplasm ratio, showing gonocyte morphology. Cell population 2 was often localized at the periphery of the testicular cords and presented typical features of PGC. Both germ cell populations were positively immunostained with anti-human OCT-4 antibody. However, at day 25, all cells of population 1 reacted positively with OCT-4, whereas in population 2, fewer cells were positive for this marker. These two PGCs populations present morphological features similar to gonocytes and intermediate cells from human foetal testis. It is expected that a population of pre-spermatogonia would be observed at later stages of canine foetus development. We also showed that anti-human OCT-4 antibody can be useful to identify canine PGC in vivo.

Canine embryo-derived stem cells and models for human diseases.

Embryonic stem (ES) cells are pluripotent and permanent cell lines which can differentiate into cell types of all the three germ layers. These features imply multiple opportunities for clinical applications in tissue engineering and regenerative medicine. Most of our knowledge on the biology and technology of ES cells is derived from studies with mouse ES cells. While appropriate for proof-of-principle studies, the mouse model has limitations in its application in translational, pre-clinical studies. This is particularly true for studies evaluating the safety and efficacy of stem cell therapies. For this purpose, large animal models more closely mimicking important aspects of human anatomy, physiology and pathology than mouse models are urgently needed. In this context, the dog is an excellent candidate: the plethora of different dog breeds offer a large phenotypic and genetic variability, which can be exploited increasingly well due to the advanced status of the dog genome project and the rapidly growing box of genomic tools. Recently, the first pluripotent canine embryo-derived stem cells have been described, further increasing the potential of the dog as a model system for regenerative medicine. Although these cells express alkaline phosphatase, NANOG and OCT4, and can be differentiated in vitro towards endoderm-, mesoderm- and ectoderm-lineages (typical features of human and mouse ES cells), their in vivo differentiation capability, i.e. formation of teratomas in immunodeficient mice or contribution to chimeric animals, remains to be demonstrated. Here, we discuss the features of reported canine embryo-derived cells and their potential applications in basic and translational biomedical research.