Morphometric developmental pattern of bovine handmade cloned concepti in late pregnancy.

Cloning procedures often interfere with conceptus growth and life ex utero, in a set of symptoms known as abnormal offspring syndrome (AOS). The aim of the present study was to compare the developmental pattern of in vivo-derived (IVD), IVF-derived and handmade cloning-derived (NT-HMC) Day 225 bovine concepti using established procedures. Pregnancy diagnosis was performed on Day 30 following blastocyst transfer on Day 7. Conceptus morphometry was assessed by ultrasonography on Day 51, and on Day 225 pregnant cows were killed for morphological examination of concepti. Pregnancy outcome was similar between groups, with greater pregnancy losses in the first trimester (70.6%) and smaller fetuses on Day 51 in the NT-HMC group than in the IVD (14.3%) and IVF (20.0%) groups. However, NT-HMC-derived concepti were twofold larger on Day 225 of gestation than controls. A higher frequency (63.5%) of placentomes larger than the largest in the IVD group was observed in the NT-HMC group, which may be relevant to placental function. Conceptus traits in the IVF group were similar to the IVD controls, with only slight changes in placentome types. Morphological changes in cloned concepti likely affected placental function and metabolism, disrupting the placental constraining mechanism on fetal growth in mid- to late pregnancy.

Immunolocalization of proteins in the spermatogenesis process of canine.

Spermatogenesis is a process in which differentiated cells are produced and the adult stem cell population-known as spermatogonial stem cells (SSCs)-is continuously replenished. However, the molecular mechanisms underlying these processes are not fully understood in the canine species. We addressed this in this study by analysing the expression of specific markers in spermatogonia of seminiferous tubules of canine testes. SSCs at different stages of reproductive development (prepubertal and adult) were examined by immunohistochemistry and flow cytometry. Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRA1), deleted in azoospermia-like (DAZL) and promyelocytic leukaemia zinc finger (PLZF) were expressed in SSCs, while stimulated by retinoic acid gene 8 (STRA8) was detected only in undifferentiated spermatogonia in prepubertal testis and differentiated spermatogonia and spermatocytes in adult canine. Octamer-binding transcription factor 4 (OCT4) showed an expression pattern, and the levels did not differ between the groups examined. However, C-kit expression varied as a function of reproductive developmental stage. Our results demonstrate that these proteins play critical roles in the self-renewal and differentiation of SSCs and can serve as markers to identify canine spermatogonia at specific stages of development.

Transplantation of amniotic membrane-derived multipotent cells ameliorates and delays the progression of chronic kidney disease in cats.

Chronic kidney disease (CKD) is a common clinical condition in domestic cats, characterized by tubulointerstitial, vascular and glomerular inflammation and severe fibrosis. Studies in rodent model of induced CKD have shown a decrease and stabilization of the clinical condition. In this study was evaluated the safety and effect of intrarenal and intravenous infusion of allogeneic mesenchymal stem cells (AMSCs) derived from feline amniotic membrane in cats with naturally occurring CKD. Cat AMSCs were harvested after mechanical and enzymatic digestion of amnion. A healthy cat received intrarenal injection of AMSCs guided by ultrasound in both kidneys (5 × 105  cells/kidney). Nine cats with CDK received repeated intravenous infusions of AMSCs (2 × 106 cells × 2 treatments). The clinical parameters of healthy cat did not change, but sedation and general anaesthesia was required. The number of interventions stressed the animal, and he developed transient haematuria after AMSC injection. Cats with CDK registered a significant improvement of renal function (decrease in serum creatinine and urine protein concentrations and increase in urine specific gravity). The kidney architecture and morphology did not change following the treatment. The feline AMSCs have a renoprotective effect and improve renal function in cats with naturally occurring CKD, stabilizing the clinical condition and disease progression. Thus, intravenous injection of AMSCs may be an important tool to provide welfare in cats with chronic kidney disease.

Characterization of teratogenic potential and gene expression in canine and feline amniotic membrane-derived stem cells.

The biosafety of innovative procedures that utilize stem cells in regenerative medicine has been addressed in several studies. Previous work has showed no tumour formation following the use of feline and human amniotic membrane-derived stem cells (AMSCs). In contrast, tumour formation was observed when canine AMSCs were utilized. These findings suggested that feline and human, but not canine, AMSCs are suitable for cell transplantation trials. This study aimed to further evaluate the feasibility of utilizing canine AMSCs for transplantation purposes as well as for felines. We tested teratoma formation following cell injection into BALB/c nude mice and then assessed expression of haematopoietic, mesenchymal, tumorigenic, pluripotency and cellular regulation markers using flow cytometry and qPCR. The use of canine AMSCs did not result in macroscopic tumour formation as determined 60 days after transplantation. The immunophenotypic characterization by flow cytometry revealed expression of mesenchymal markers (CD73 and CD90) and expression of the pluripotent marker OCT4 and SOX2. Quantitative PCR analysis revealed that there were no differences in the patterns of gene expression (CD34, CD73, OCT4, CD30 and P53) between canine and feline AMSCs, with the exception of the expression of SOX2 and CD90.

Buffalo (Bubalus bubali) Late Embryo and Foetus Development: A Morphological Analysis.

Many researches describe the embryonic developmental features in domestic animals; however, in farm animals, they are scarce. Most farm animal studies are related to assisted reproduction and embryos transfer techniques. But, morphological features and size measure to estimate the age gestation are rarely reported in literature. Thus, in this study, we described the developmental changes in the bubaline (Bubalus bubali) concepts from 21 to 60 days of gestation. Our results revealed that buffalo embryos similar morphological characteristics similar to other mammalian species. Also, similarities between bovine and bubaline persist; except on foetal stages when buffalos have a faster development than bovine. Therefore, buffalo's gestation period exhibits some varieties and accurate embryo age is more difficult. Yet, when we use a combination of the crown-rump, macroscopic analysis and alizarin red, it is possible to describe better the whole embryogenesis stages of the buffalo and which can contribute for future reproduction researches and applications in veterinary practice.

Controversial results of therapy with mesenchymal stem cells in the acute phase of canine distemper disease.

Distemper disease is an infectious disease reported in several species of domestic and wild carnivores. The high mortality rate of animals infected with canine distemper virus (CDV) treated with currently available therapies has driven the study of new efficacious treatments. Mesenchymal stem cell (MSC)-based therapy is a promising therapeutic option for many degenerative, hereditary, and inflammatory diseases. Therefore, the aim of this study was to characterize stem cells derived from the canine fetal olfactory epithelium and to assess the systemic response of animals infected with CDV to symptomatic therapy and treatment with MSCs. Eight domestic mongrel dogs (N = 8) were divided into two groups: support group (SG) (N = 5) and support group + cell therapy (SGCT) (N = 3), which were monitored over 15 days. Blood samples were collected on days 0, 6, 9, 12, and 15 to assess blood count and serum biochemistry (urea, creatinine, alanine transferase, alkaline phosphatase, gamma-glutamyl transferase, total protein, albumin, and globulin), and urine samples were obtained on days 0 and 15 for urinary evaluation (urine I). The results showed a high mortality rate (SG = 4 and SGCT = 2), providing inadequate data on the clinical course of CDV infection. MSC therapy resulted in no significant improvement when administered during the acute phase of canine distemper disease, and a prevalence of animals with high mortality rate was found in both groups due to the severity of symptoms.

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.

Generation of bovine (Bos indicus) and buffalo (Bubalus bubalis) adipose tissue derived stem cells: isolation, characterization, and multipotentiality.

Adult stem cells are known for their plasticity and their potential to differentiate into several different cell types; these characteristics have implications for cell therapy and reproductive biotechnologies. In this study, we report on the isolation and characterization of mesenchymal stem cells (MSC) derived from bovine and buffalo adipose tissue. Cells isolated using enzymatic digestion of bovine and buffalo adipose-tissue biopsy samples were grown in vitro for at least 15 passages, verifying their capacity to proliferate. These cells were also subjected to immunophenotypic characterization for the presence of CD90, CD105, and CD79, and the absence of CD45, CD34, and CD73, which are positive and negative markers of MSC, respectively. To prove their multipotency, the cells were induced to differentiate into three different cell types, chondrocytes, osteoblasts, and adipocytes, which were stained with tissue-specific dyes (Chondrogenic-Alcian Blue, Osteogenic-Alizarin Red, and Adipogenic-Oil-Red O, respectively) to confirm differentiation. Gene expression analysis of pluripotency-related genes was also conducted. Our results suggest that adipose tissue from bovines and buffalos can be used as a source of MSC, making adipose tissue-derived cells an interesting option for cell therapy and regenerative medicine. Additionally, these findings have implications for reproductive biotechnology because the use of MSC as nuclear donors has been linked to an increase in the efficiency of nuclear transfer.

Semen analysis of Golden Retriever healthy dogs and those affected by muscular dystrophy.

This study aimed to characterise canine flow cytometry semen analysis, as well as seminal reactive oxygen species dosage using the Golden Retriever breed as model of study. Moreover, we searched for the influence of muscular dystrophy in Golden Retriever dogs on semen parameters. Thirty-seven semen samples were obtained from healthy Golden Retrievers (n = 15) and from muscular dystrophy affected dogs (n = 22). Sperm-rich fractions were analysed by standardised breeding soundness examination in addition to the assay of fluorescence assisted cell sorting for acrosome integrity, mitochondrial activity and DNA fragmentation. Volume of ejaculate, per cent of motile spermatozoa and vigour were similar between groups; there were no differences in the per cent of minor and major defects. Integrity of acrosomal membrane, mitochondrial potential and sperm DNA fragmentation had no significant differences between groups either. Animals from control group had higher concentration of spontaneous seminal oxidative species in comparison with affected animals. Dogs affected by dystrophy had seminal parameters similar to those observed in healthy dogs except for the lower concentration of oxidative species. Future studies aiming to establish reference values for canine seminal parameters should be considered preferably with distinction of breeds.

Stem cells and regenerative medicine in domestic and companion animals: a multispecies perspective.

Since their original isolation, the majority of the work on embryonic stem cells (ESC) has been carried out in mice. While the mouse is an outstanding model for basic research, it also has considerable limitations for translational work, especially in the area of regenerative medicine. This is due to a combination of factors that include physiological and size differences when compared to humans. In contrast, domestic animal species, such as swine, and companion animal species, such as dogs, provide unique opportunities to develop regenerative medicine protocols that can then be utilized in humans. Unfortunately, at present, the state of knowledge related to, and availability of, ESC from domestic animals vary among species such as pig, horse, dog and cat, and without exception lags significantly behind the mouse and human. It is clear that much still needs to be discovered. The 'stem cell-like' cell lines being reported are still not satisfactorily used in regenerative medicine, due to reasons such as heterogeneity and chromosomal instability. As a result, investigators have searched for alternate source of cells that can be used for regenerative medicine. This approach has uncovered a range of adult stem cells and adult progenitor cells that have utility in both human and veterinary medicine. Here, we review a range of stem cells, from ESC to induced pluripotent stem cells, and discuss their potential application in the field of regenerative medicine.

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.

Characterization of yolk sac proteins of Bos indicus cattle embryos.

The yolk sac is an embryonic membrane that is essential for the embryo's initial survival in many mammals. It also plays an important role in the production of proteins necessary for development. We studied proteins of the yolk sac in bovine embryos at up to 40 days of gestation. We examined the yolk sac of 17 bovine embryos at different gestational periods, measuring α-fetoprotein, α-1-antitrypsin, and transferrin. This experiment was carried out by Western blot technique, associated with electrophoresis on a 6% sodium dodecyl sulfate polyacrylamide gel. Mouse monoclonal antibody anti-human-α-fetoprotein, mouse antibody anti-human-transferrin and rabbit polyclonal anti-human-α-1-antitrypsin were used as primary antibodies, and conjugated peroxidase as a secondary antibody. We detected the three proteins in some of the yolk sac samples; however, the bands in some specimens (samples) were weak, maybe a result of poor antigen-antibody reaction, since the antibodies used in this study were not specific to bovine proteins. The fact that weak bands appeared might be due to a weak cross-reaction.

Canine fibroblasts expressing human transcription factors: what is in the route for the production of canine induced pluripotent stem cells.

The aim of this study was to further clarify the mechanisms involved in inducing pluripotency using canine foetal fibroblast cells. The two pluripotency-related transcription factors, OCT4 and SOX2, coupled to a fluorescent reporter gene were transduced, individually or in combination, using a lentiviral system. Stable transgenic cell lineages were obtained and canine cells showed to be highly responsive to the integration and expression of human SOX2 and OCT4, also depending on the amount of virus used for incubation. Such positive results are essential for the establishment of pluripotency induction through the incorporation of known transcription factors into the genome of somatic cells.

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.

Morpho-physical recording of bovine conceptus (Bos indicus) and placenta from days 20 to 70 of pregnancy.

The study is based on 141 pregnant Bos indicus cows, from days 20 to 70 post-insemination. First, special attention was given to the macroscopically observable phenomena of attachment of the conceptus to the uterus, i.e. the implantation, from about days 20 to 30 post-insemination up to day 70, and placentome development by growth, vascularization and increase in the number of cotyledons opposite to the endometrial caruncles. Secondly, as for the conceptuses, semiquantitative, statistical analyses were performed of the lengths of chorio-allantois, amnion and yolk sac; and the different parts of the centre and two extremes of the yolk sacs were also analysed. Thirdly, the embryos/foetuses corresponding to their membranes were measured by their greatest length and by weight, and described by the appearance of external developmental phenomena during the investigated period like neurulation, somites, branchial arcs, brain vesicles, limb buds, C-form, pigmented eye and facial grooves. In conclusion, all the data collected in this study from days 20 to 70 of bovine pregnancy were compared extensively with corresponding data of the literature. This resulted in an 'embryo/foetal age-scale', which has extended the data in the literature by covering the first 8 to 70 days of pregnancy. This age-scale of early bovine intrauterine development provides model for studies, even when using slaughtered cows without distinct knowledge of insemination or fertilization time, through macroscopic techniques. This distinctly facilitates research into the cow, which is now being widely used as 'an experimental animal' for testing new techniques of reproduction like in vitro fertilization, embryo transfer and cloning.

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

Transplacental transfer of iron in the water buffalo (Bubalus bubalis): uteroferrin and erythrophagocytosis.

The objectives of this investigation were to understand transplacental transport of iron by secreted uteroferrin (UF) and haemophagous areas of water buffalo placenta and clarify the role(s) of blood extravasation at the placental-maternal interface. Placentomes and interplacentomal region of 51 placentae at various stages of gestation were fixed, processed for light and transmission electron microscopy, histochemistry and immunohistochemistry. Haemophagous areas were present in placentomes collected between 4 and 10 months of pregnancy. Perl's reaction for ferric iron was negative in placentomes, but positive in endometrial glands. Positive staining for UF indicated areas in which it was being taken up by phagocytosis and/or fluid phase pinocytosis in areolae of the interplacentomal mesenchyme, with little staining in endometrial stroma. Imunohistochemistry detected UF in trophectoderm of haemophagous regions of placentomes and in other parts of the foetal villous tree, but the strongest immunostaining was in the epithelial cells and lumen of uterine glands. Ultrastructural analyses indicated that erythrophagocytosis was occurring and that erythrocytes were present inside cells of the chorion that also contained endocytic vesicles and caveolae. Results of this study indicate that both the haemophagous areas of placentomes and the areolae at the interface between chorion and endometrial glands are important sites for iron transfer from mother to foetal-placental tissues in buffalo throughout pregnancy.

A critical evaluation of the use of ozone and its derivatives in dentistry.

OBJECTIVE: The therapeutic application of ozone and its derivatives in the dental field has been used for many purposes. However, there has yet to be a consistent evaluation of the outcomes, due to the lack of standardization of the treatment operating procedures. MATERIALS AND METHODS: The keywords "ozone", "ozonated", "ozonation" "ozonized", "ozonization", "dentistry", "periodontology", "oral surgery", "oxygen-ozone therapy" were used to perform a literature review using PubMed, Cochrane, Google Scholar, Zotero databases with the temporal restriction for manuscripts published between 2010 and 2020. Clinical trials and case reports of good, neutral, as well as negative results related to ozone treatment specifications were evaluated. DISCUSSION: A better understanding of the mechanisms of action of this bio-oxidative therapy could open new horizons related to the personalization of treatments and the quality of dental care. The critical condition to achieve these goals is an improved knowledge of the qualitative/quantitative characteristics of ozone and its derivatives.

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.

Identification of three distinguishable phenotypes in golden retriever muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a human disease characterized by progressive and irreversible skeletal muscle degeneration caused by mutations in genes coding for important muscle proteins. Unfortunately, there is no efficient treatment for this disease; it causes progressive loss of motor and muscular ability until death. The canine model (golden retriever muscular dystrophy) is similar to DMD, showing similar clinical signs. Fifteen dogs were followed from birth and closely observed for clinical signs. Dogs had their disease status confirmed by polymerase chain reaction analysis and genotyping. Clinical observations of musculoskeletal, morphological, gastrointestinal, respiratory, cardiovascular, and renal features allowed us to identify three distinguishable phenotypes in dystrophic dogs: mild (grade I), moderate (grade II) and severe (grade III). These three groups showed no difference in dystrophic alterations of muscle morphology and creatine kinase levels. This information will be useful for therapeutic trials, because DMD also shows significant, inter- and intra-familiar clinical variability. Additionally, being aware of phenotypic differences in this animal model is essential for correct interpretation and understanding of results obtained in pre-clinical trials.