Clinical anatomy of the meniscus in animal models: pros and cons.

Nowadays, despite the possibility to use in vitro or computer models in research, animal models are still essential. Different animal models are available for meniscal repair investigation. Although a unique perfect model for the structure of the human's knee does not exist, the choice of the proper animal model is crucial for a correct research. The principal animal models in the meniscal repair are sheep, goats, pigs and dogs. Each of these has pros and cons for their utilization. Analysing each pro and con is essential for optimizing the choice of the animal model, which depends on the experimental question, avoiding unnecessary waste of resources and minimizing the animal suffering, according to the Russell and Burch's three "Rs" principles (Reduce, Refine and Recycle). In this concise review, we resume the meniscus anatomical features of the main large animals, to help choose the most suitable animal model for subsequent studies on meniscal repair.

Histological assessment of new bone formation with biomimetic scaffold in the presence of bone loss in trauma surgery.

Open reduction and internal fixation (ORIF) surgery may require the use of bone grafts (usually allogeneic). In the context of traumatology surgeries, the use of autologous grafts is almost never used and allogeneic grafts are not always available. In recent years, bone substitutes have been introduced in clinical practice to overcome these limitations. The purpose of this paper is to report two cases in which the use of a bone substitute was used to overcome the bone loss during surgeries of ORIF. Two patients, one with a tibial plateau fracture (Schatzker 6) and one with a proximal humerus fracture (Neer 4), underwent ORIF surgery. In both cases, due to a loss of bone stock, a synthetic bone substitute (OrthOss®) was used. One year after surgery, the complete osseointegration of the synthetic bone substitute was seen, both radiologically and histologically. This bone substitute may represent a safe and effective alternative to autologous bone grafts, avoiding adverse events related to donor-site morbidity.

Decorin age-related variations in the distribution of pig extracellular matrix meniscus.

Menisci act like shock absorbers and transmit load across the tibiofemoral joint by increasing congruency during movements or body weight load. This leads to decreasing the resultant stress on the articular cartilages. The meniscus has a dense extracellular matrix (ECM) composed of water, different types of collagens, and proteoglycans, such as decorin, aggrecan and biglycan. Decorin (DCN) regulates collagen fibrillogenesis acting on collagen fibrils diameter and fibrils orientation to achieve the proper assembly of its network. This work investigates the spatial disposition of this fundamental protein in pig meniscus' matrix by immunohistochemistry and western blot analysis. DCN shows an increasing trend, moving from neonatal to adult pig menisci. Adult meniscus, in porcine species, is the only one that could be considered fully mature and functional, and, even if an increasing trend is seen, no precise phenotypical switch points are seen in the age stages considered in this study.

Age assessment in puppies: Coming to terms with forensic requests.

Age estimation in growing dogs is crucial not only in clinical practice but increasingly so in forensic practice as well. In the last few years, it has assumed great importance for correctly identifying the age of puppies illegally imported to Italy as well as to other European countries. Puppies are, in fact, transported when they are too young to be moved, which can cause both animal/public health and animal welfare issues. Therefore, the movement of animals within the European Community is governed by strict rules, and veterinarians are often required to evaluate the age of the imported puppies in a forensic scenario as accurately as possible. To date, X-ray evaluation of limb bones ossification centers (OCs) is generally accepted as a valid tool to assess the age of puppies. A wealth of information exists on this topic but it is not always easily available. This work is a historical review of the existing literature and proposes two tables illustrating the timelines of limb OCs appearance and closure, coming to terms with forensic requests to evaluate the age of a puppy. The timelines reported indicate the need to improve methodology to enhance the accuracy and to reduce the error in age estimation.

Comparison between different cell sources and culture strategies for tendon tissue engineering.

The aim of this study was to evaluate the effect of an in vitro mechanical stimulation by the use of a bioreactor on an engineered tendon for 7 and 14 days and to analyze the effect of the use of different cell sources: tenocytes, dermal fibroblasts or Adipose-Derived Stem Cells (ASCs), isolated from pig tissues. Histology showed a re-organization of the neo-tissue derived from the three cell populations along the direction of the stimulus. At T7, cells morphology was preserved while an increased cellular suffering at T14 was observed for all cell populations. Tenocytes exhibited higher survival than other cells. A stable immunopositivity for collagen type 1 or 3 at both time points was also observed. In conclusion, dermal fibroblasts and ASCs represent an interesting alternative and in vitro culture with mechanical stimuli may enhance the maturation of a tendon-like tissue.

Small-sized newborn dogs skeletal development: radiologic, morphometric, and histological findings obtained from spontaneously dead animals.

BACKGROUND: Very little is known about neonatal skeletal development in small-sized purebred dogs. In order to improve this knowledge, 27 spontaneously dead puppies belonging to small-sized breeds were enrolled in this study for radiologic, histological and morphometric investigations. RESULTS: The appearance of the limb secondary ossification centers and the onset of their formation were clearly observed by x rays and confirmed by histological evidences. Radiographic and anatomic measurements of limb bones length and skull length and width were positively correlated with body weight and age of the subjects and the body weight was positively correlated with radius bone mineral density, as demonstrated by dual-energy x-rays absorptiometry. CONCLUSIONS: These data provided original information on the growth of newborn small-sized breed dogs, and suggest that cadavers may be useful to study skeletal development.

Cartilage canals in newborn dogs: histochemical and immunohistochemical findings.

Cartilage canals (CCs) are microscopic structures involved in secondary ossification centers (SOCs) development. The features of CCs were investigated in the humeral and femoral proximal epiphyses of small-sized newborn dogs (from premature to 28 days after birth) with histochemical and immunohistochemical approaches. Masson's Trichrome revealed a ring-shaped area around CCs, which changes in colour from green (immature collagen) to red (mature collagen) as ossification progresses; perichondrium staining always matched the ring colour. Safranin-O was always negative. Immunohistochemical analysis revealed immunopositivity for both collagen type I and V around the CCs; collagen type II was negative. CCs count showed a tendency to be higher in the humerus than in the femur. This work enlightened for the first time changes in composition of CCs surrounding matrix during SOCs development in dogs, paving the way to further investigations.

Large-scale chromatin morpho-functional changes during mammalian oocyte growth and differentiation.

Mammalian oocyte development is characterized by impressive changes in chromatin structure and function within the germinal vesicle (GV). These changes are crucial to confer the oocyte with meiotic and developmental competencies. In cow, oocytes collected from early and middle antral follicles present four patterns of chromatin configuration, from GV0 to GV3, and its progressive condensation has been related to the achievement of developmental potential. During oogenesis, follicular cells are essential for the acquisition of meiotic and developmental competencies and communicate with the oocyte by paracrine and gap junction mediated mechanisms. We recently analyzed the role of gap junction communications (GJC) on chromatin remodeling process during the specific phase of folliculogenesis that coincides with the transcriptional silencing and sequential acquisition of meiotic and developmental capabilities. Our studies demonstrated that GJC between germinal and somatic compartments plays a fundamental role in the regulation of chromatin remodeling and transcription activities during the final oocyte differentiation, throughout cAMP dependent mechanism(s).

Effect of vitrification of feline ovarian cortex on follicular and oocyte quality and competence.

Cryopreservation of ovarian cortex has important implications in the preservation of fertility and biodiversity in animal species. Slow freezing of cat ovarian tissue resulted in the preservation of follicular morphology and in the follicular development after xenografting. Vitrification has been recently applied to ovarian tissues of different species, but no information is available on the effect of this method on feline ovarian cortex. Moreover, meiotic competence of fully grown oocytes isolated from cryopreserved tissue has not been reported. The aim of this study was to evaluate the effect of vitrification of feline ovarian cortex on follicular morphology and oocyte integrity, as well as meiotic competence. A total of 352 fragments (1.5-2 mm(3) ) were obtained from ovarian cortical tissues: 176 were vitrified and 176 were used fresh as control. Histological evaluation of fresh and vitrified fragments showed intact follicles after cryopreservation procedures with no statistically significant destructive effect from primordial to antral follicles. After IVM, oocytes collected from vitrified ovarian fragment showed a higher proportion of gametes arrested at germinal vesicle (GV) stage compared to those isolated from fresh control tissue (33.8% vs 2.9%; p < 0.001). However, oocytes isolated from vitrified tissues were able to resume meiosis, albeit at lower rate than those collected from fresh tissues (39.8% vs 85.9%; p < 0.00001). Vitrification induced changes in the organization of cytoskeletal elements (actin microfilaments and microtubules) of oocytes, but significantly only for actin network (p < 0.001). Finally, chromatin configuration within the GV was not affected by the cryopreservation procedure. Our study demonstrated that vitrification preserves the integrity of ovarian follicles and that oocytes retrieved from cryopreserved tissue maintain the capability of resuming meiosis. To our knowledge, this has not previously been reported in the cat.

Expression of progesterone receptor membrane component-1 in bovine reproductive system during estrous cycle.

Several reports suggest the participation of progesterone receptor membrane component 1 (PGRMC1) in progesterone signaling in the reproductive system. This study aimed at investigating the presence and localization of PGRMC1 in bovine ovary, oviduct and uterus, during the follicular and luteal phases of the estrous cycle. In the ovary, PGRMC1 has been detected in surface germinal epithelium, granulosa cells, theca cells and in the germinal vesicle of the oocytes at all stages of folliculogenesis. In the corpus luteum the expression of PGRMC1 was influenced by the stage of the estrous cycle. In the oviducts and in the uterus horns, PGRMC1 was immunolocalized in the luminal epithelium, in the muscle layer cells and in the endothelial cells. In the uterus, PGRMC1 was intensely localized also in the glandular endometrium. However, in the oviducts and in the uterus horns, the localization of PGRMC1 was independent on the stage of the estrous cycle and on whether evaluating the ipsilateral or the contralateral organ. In conclusion, the present immunohistochemical study showed that PGRMC1 is located in various compartments of the bovine female reproductive organs. With the exception of the corpora lutea, PGRMC1 localization showed similar pattern during different stages of the estrous cycle.

The endothelial nitric oxide synthase/nitric oxide system is involved in the defective quality of bovine oocytes from low mid-antral follicle count ovaries.

In a previous survey concerning cows of reproductive age, we demonstrated that oocytes isolated from ovaries with <10 medium antral follicles of 2 to 6 mm in diameter (low ovaries; Lo) show less developmental competence than oocytes collected from ovaries with >10 medium antral follicles (high ovaries; Hi). The aim of the present study was to evaluate whether a defective endothelial nitric oxide synthase/nitric oxide (eNOS/NO) system and vasculature in healthy medium antral follicles is likely to reduce oocyte competence from Lo ovaries. Thus, experiments were conducted to 1) immunolocalize eNOS protein during folliculogenesis; 2) quantify eNOS protein/vasculature in the follicle wall; and 3) verify if NO donor, S-nitroso acetyl penicillamine (SNAP) administration during in vitro maturation affects developmental competence of oocytes isolated from Lo ovaries. Endothelial nitric oxide synthase protein was detected in granulosa and theca cells, as well as in blood vessels from primordial to antral follicles. Quantitative analysis indicated that in medium antral follicles from Lo ovaries, eNOS protein expression and vasculature were reduced (P < 0.05). The addition of SNAP improved blastocyst and hatching rates of oocytes from Lo ovaries, promoting a percentage similar to oocytes from Hi ovaries, and reduced the percentage of apoptotic nuclei in in vitro-produced blastocysts (P < 0.05). Results from our study suggest that in bovine ovaries with small mid antral follicle number, a defective eNOS/NO system is related to a reduced follicle vasculature and may affect oocyte quality, thus inducing a premature decline of fertility.

Topographic comparative study of paranasal sinuses in adult horses by computed tomography, sinuscopy, and sectional anatomy.

Clinical and radiographic investigations of paranasal sinuses in horses are difficult due to the complex anatomy of these regions, the lack of patognomonic symptoms, and the low sensitivity of conventional diagnostic techniques. The aim of this study was to produce an anatomical atlas to support computed tomography (CT) and sinuscopy of the paranasal sinuses of the adult horse. Transverse, sagittal, and dorsal CT images were acquired, and sinuscopy with both rigid and flexible endoscopes was performed. The heads were frozen and sectioned using a band saw, with the cuts aligned as close as possible with the CT transverse slices. Each CT image was compared with its corresponding anatomical section and sinuscopy image to assist in the accurate identification of specific structures.

Cryopreservation of immature bovine oocytes to reconstruct artificial gametes by germinal vesicle transplantation.

Joining immature gamete cryopreservation and germinal vesicle transplantation (GVT) technique could greatly improve assisted reproductive technologies in animal breeding and human medicine. The present work was aimed to assess the most suitable cryopreservation protocol between slow freezing and vitrification for immature denuded bovine oocytes, able to preserve both nuclear and cytoplasmic competence after thawing. In addition, the outcome of germinal vesicle transfer procedure and gamete reconstruction was tested on the most effective cryopreservation system. Oocytes, isolated from slaughterhouse ovaries, were stored after cumulus cells removal either by slow freezing or by vitrification in open pulled straws. After thawing, oocytes were matured for 24 h in co-culture with an equal number of just isolated intact cumulus enclosed oocytes, and fixed in order to evaluate the stage of meiotic progression and cytoskeleton organization. Our results showed that after warming, vitrified oocytes reached metaphase II (MII) in a percentage significantly higher than oocytes cryopreserved by slow freezing (76.2% and 36.5% respectively, p < 0.05). Moreover, vitrification process preserved the organization of cytoskeleton elements in a higher proportion of oocytes than slow freezing procedure. Therefore vitrification has been identified as the elective method for denuded immature oocytes banking and it has been applied in the second part of the study. Our results showed that 38.3% of oocytes reconstructed from vitrified gametes reached the MII of meiotic division, with efficiency not different from oocytes reconstructed with fresh gametes. We conclude that vitrification represents a suitable method of GV stage denuded oocyte banking since both nuclear and cytoplasmic components derived from cryopreserved immature oocytes can be utilized for GVT.

Localization of DNA methyltransferase-1 during oocyte differentiation, in vitro maturation and early embryonic development in cow.

DNA methyltransferase-1 (Dnmt1) is involved in the maintenance of DNA methylation patterns and is crucial for normal mammalian development. The aim of the present study was to assess the localization of Dnmt1 in cow, during the latest phases of oocyte differentiation and during the early stages of segmentation. Dnmt1 expression and localization were assessed in oocytes according to the chromatin configuration, which in turn provides an important epigenetic mechanism for the control of global gene expression and represents a morphological marker of oocyte differentiation. We found that the initial chromatin condensation was accompanied by a slight increase in the level of global DNA methylation, as assessed by 5-methyl-cytosine immunostaining followed by laser scanning confocal microscopy analysis (LSCM). RT-PCR confirmed the presence of Dnmt1 transcripts throughout this phase of oocyte differentiation. Analogously, Dnmt1 immunodetection and LSCM indicated that the protein was always present and localized in the cytoplasm, regardless the chromatin configuration and the level of global DNA methylation. Moreover, our data indicate that while Dnmt1 is retained in the cytoplasm in metaphase II stage oocytes and zygotes, it enters the nuclei of 8-16 cell stage embryos. As suggested in mouse, the functional meaning of the presence of Dnmt1 in the bovine embryo nuclei could be the maintainement of the methylation pattern of imprinted genes. In conclusion, the present work provides useful elements for the study of Dnmt1 function during the late stage of oocyte differentiation, maturation and early embryonic development in mammals.

Relationship between growth hormone concentrations in bovine oocytes and follicular fluid and oocyte developmental competence.

In the last few years, several works suggest that Growth Hormone (GH) is involved in follicular development and oocyte maturation. These actions may reflect endocrine roles of pituitary GH and also account for local autocrine or paracrine activities of GH produced in reproductive tissue. This study was aimed to verify whether the developmental competence of bovine female gametes might be related to ovarian GH. We evaluated the localisation and distribution of GH in the cumulus oocytes complexes (COCs) and the concentration of GH in the oocytes and in the follicular fluids (FF) from ovaries classified on the basis of the follicles number. Oocytes retrieved from ovaries with more than 10 follicles of 2 to 5 mm in diameter (High ovaries, Hi) show higher rate of maturation and blastocyst formation than those retrieved from ovaries with less than 10 follicles (Low ovaries, Lo). At the same time we measured Estrogen (E2) and Progesterone (P4) concentrations in FF, to relate oocytes quality, GH concentration and follicle health. GH localization in COCs and oocytes was performed by indirect immunofluorescence and its concentration within the ooplasm was evaluated by microspectrophotometer analysis. GH, E2 and P4 concentrations in FF were measured by an Enzyme Linked ImmunoSorbent assay (ELISA). We observed a positive, diffuse signal at cytoplasmic level in most of the cumulus cells, with no differences between COCs collected from Hi and Lo ovaries. On the contrary, GH level was significantly higher in the oocytes collected from Lo ovaries than in those recovered from Hi ovaries. Finally we found that also GH level in the FF was inversely related to the oocytes developmental capability. We suggest that the increase of GH in the oocytes and in the FF derived from Lo ovaries might be interpreted as attempt of the follicular environment to improve ovarian activity and in turn oocytes developmental competence in a autocrine-paracrine manner. Moreover, E2, and P4 levels in FF suggest that, in our model, atresia processes are also involved in oocyte developmental capability and that the highest level of GH may represent a local reaction to these phenomena.

Cytoplasmic changes and developmental competence of bovine oocytes cryopreserved without cumulus cells.

The cryopreservation of female gametes is still an open problem because of their structural sensitivity to the cooling-and-freezing process and to the exposure to cryoprotectants. The present work was aimed to study the effect of vitrification on immature bovine oocytes freed of cumulus cell investment before freezing. To verify the feasibility and efficiency of denuded oocyte (DO) cryopreservation, the cytoplasmic alterations eventually induced either by cell removal or by the vitrification process were analyzed. In particular, the migration of cortical granules and Ca++ localization were studied. In addition, the localization and distribution of microtubules and microfilaments in immature fresh and vitrified DOs were evaluated. Finally, to establish whether the removal of cumulus cells influenced developmental competence, DOs were thawed after vitrification, matured in vitro and fertilized; then presumptive zygotes were cultured to reach the blastocyst stage. The results indicate that mechanical removal of cumulus cells from immature bovine oocytes does not affect their maturation competence but reduces the blastocyst rate when compared with intact cumulus oocyte complexes (COCs). The findings indicate further that the vitrification process induces changes of cytoplasmic components. However, the composition of the manipulation medium used to remove cumulus cells plays a crucial role in reducing the injuries caused by cryopreservation in both cytoplasmic and nuclear compartments. In fact, the presence of serum exerts a sort of protection, significantly improving both oocyte maturation and blastocyst rates. In conclusion, we demonstrate that denuded immature oocytes can be vitrified after cumulus cells removal and successfully develop up, after thawing, to the blastocyst stage, following in vitro maturation and fertilization.

Bovine somatotropin administration to dairy goats in late lactation: effects on mammary gland function, composition and morphology.

We investigated the effects of bovine somatotropin (bST) on mammary gland function and composition in the declining phase of lactation in goats. Sixteen Saanen goats, 180 +/- 11 days in milk (DIM), were divided equally into control and treated groups. The treated group received 120 mg/2 wk of slow-release bST for three cycles. Milk yield, milk composition, milk clotting measures, and plasmin-plasminogen activator activities were recorded weekly. Milk Na and K were determined in individual milk samples collected weekly during the third cycle. Blood samples were collected weekly during the second cycle and the plasma analyzed for nonesterified fatty acids (NEFA), glucose, and urea. At the end of the 6 wk, three goats from each group were slaughtered, and the udders were removed. Mammary gland weight, composition, and total DNA content were determined. The histological effects of bST on mammary tissue were investigated. The analyzed parameters included numbers of alveoli, corpora amylacea, apoptotic cells, and laminin fibronectin distribution and localization. An extensive morphological analysis on the epithelial and stromal components was performed. Milk yield was significantly higher in the treated group, fat content was not affected, but protein and nonprotein nitrogen were lower in treated goats milk. Treatment with bST did not influence milk pH but reduced coagulation time. Plasmin and plasminogen activator activities were not affected. Milk K levels were higher and the Na/K ratio was lower in treated animals. Plasma glucose, NEFA, and urea were unaffected. Mammary gland weight and total DNA were higher in treated than control animals, suggesting that with advancing lactation bST treatment maintains cells. Fat, protein, and collagen content of the mammary tissue did not differ between the groups. Treatment with bST significantly increased the number of lactating alveoli (LA) and significantly reduced the number of regressing alveoli (RA) and corpora amylacea, both within and outside the alveolar lumen. Laminin and fibronectin localization were not affected, and very few apoptotic cells were found in both treated and control samples. Our findings suggest that bST administration to dairy goats in late lactation can modulate mammary gland activity and improve lactation persistency; this is associated with maintained total mammary parenchyma weight and lactating alveoli.

Cellular and molecular mechanisms mediating the effects of polychlorinated biphenyls on oocyte developmental competence in cattle.

Polychlorinated biphenyls (PCBs) can interfere with normal reproductive functions acting as endocrine disruptors. Aroclor-1254 (A-1254), is a pool of more than 60 congeners used for in vitro studies because its composition is representative of PCBs environmental pollution. We previously demonstrated that the exposure of bovine oocytes to A-1254 during in vitro maturation (IVM) was detrimental not only to the maturation process but also induced a significant increase of polyspermy and a reduction of developmental competence. Therefore, we investigated whether A-1254 acts on two processes that occur during IVM and may be related with its negative effects: maternal mRNA polyadenylation and cortical granules (CGs) migration and exocytosis. Bovine cumulus-oocyte complexes (COCs) were exposed to 0.1 microg/ml of A-1254 during IVM, a level of exposure known to affect oocyte maturation, fertilization, and developmental competence. Oocyte exposure to A-1254 altered the poly(A) tail length of 5 out of 10 genes examined. PCBs effect on mRNA polyadenylation was different depending on the gene considered and resulted either in a shorter or in a longer poly(A) tail. At the end of maturation, Aroclor treated oocytes presented clustered CG in a significantly higher percentage than the control group. In addition, CG exocytosis after 8 hr of fertilization occurred at significantly lower extent in zygotes derived from the exposed group compared to control. Our results indicated that the lower developmental competence of oocytes exposed to PCBs during IVM can be related to the interaction of these contaminants with mechanisms regulating maternal mRNA storage in the ooplasm and normal CGs function.

Oocyte developmental competence after in vitro maturation depends on the persistence of cumulus-oocyte comunications which are linked to the intracellular concentration of cAMP.

Oocyte and cumulus cells are interconnected by an extensive network of gap junctions (GJ) formed by connexin 43. In the present study the functional and morphological status of GJ was investigated during in vitro maturation of bovine oocytes using media known to induce different rates of maturation and developmental competence as well as different levels of cumulus expansion and intracellular concentrations of cAMP. GJ functional condition was studied by microinjection of the fluorescent dye Lucyfer Yellow in cumulus-enclosed oocytes and the intracellular distribution of connexin 43 was examined by immunofluorescence immunocytochemistry. Both functional and morphological analysis of GJ between oocytes and cumulus cells indicated that high maturation and development rates are accompanied by the prolonged persistence of permeable communications, which, however, are independent from cumulus expansion. On the contrary, the premature interruption of such communications was linked to low maturation and development. When these results were correlated to the measurements of intracellular levels of cAMP it was observed that cumulus expansion, GJ permeability and good quality maturation require the highest levels of cAMP. Intermediate cAMP levels determine GJ permeability and good quality maturation in the absence of cumulus expansion while below a threshold level of cAMP, oocyte maturation is defective with a premature interruption of communications through GJ and lack of cumulus expansion which lead to poor development.