Recent studies suggest that bone marrow stromal cells are a potential source of osteoblasts and chondrocytes and can be used to regenerate damaged tissues using a tissue-engineering (TE) approach. However, these strategies require the use of an appropriate scaffold architecture that can support the formation de novo of either bone and cartilage tissue, or both, as in the case of osteochondral defects. The later has been attracting a great deal of attention since it is considered a difficult goal to achieve. This work consisted on developing novel hydroxyapatite/chitosan (HA/CS) bilayered scaffold by combining a sintering and a freeze-drying technique, and aims to show the potential of such type of scaffolds for being used in TE of osteochondral defects. The developed HA/CS bilayered scaffolds were characterized by Fourier transform infra-red spectroscopy, X-ray diffraction analysis, micro-computed tomography, and scanning electron microscopy (SEM). Additionally, the mechanical properties of HA/CS bilayered scaffolds were assessed under compression. In vitro tests were also carried out, in order to study the water-uptake and weight loss profile of the HA/CS bilayered scaffolds. This was done by means of soaking the scaffolds into a phosphate buffered saline for 1 up to 30 days. The intrinsic cytotoxicity of the HA scaffolds and HA/CS bilayered scaffolds extract fluids was investigated by carrying out a cellular viability assay (MTS test) using Mouse fibroblastic-like cells. Results have shown that materials do not exert any cytotoxic effect. Complementarily, in vitro (phase I) cell culture studies were carried out to evaluate the capacity of HA and CS layers to separately, support the growth and differentiation of goat marrow stromal cells (GBMCs) into osteoblasts and chondrocytes, respectively. Cell adhesion and morphology were analysed by SEM while the cell viability and proliferation were assessed by MTS test and DNA quantification. The chondrogenic differentiation of GBMCs was evaluated measuring the glucosaminoglycans synthesis. Data showed that GBMCs were able to adhere, proliferate and osteogenic differentiation was evaluated by alkaline phosphatase activity and immunocytochemistry assays after 14 days in osteogenic medium and into chondrocytes after 21 days in culture with chondrogenic medium. The obtained results concerning the physicochemical and biological properties of the developed HA/CS bilayered scaffolds, show that these constructs exhibit great potential for their use in TE strategies leading to the formation of adequate tissue substitutes for the regeneration of osteochondral defects.

Mesenchymal stem cells have a great potential for application in cell based therapies, such as tissue engineering. Adipose derived stem cells have shown the capacity to differentiate into several lineages, and have been isolated in many animal species. Dog is a very relevant animal model to study several human diseases and simultaneously an important subject in veterinary medicine. Thus, in this study we assessed the potential of canine adipose tissue derived stem cells (cASCs) to differentiate into the osteogenic and chondrogenic lineages by performing specific histological stainings, and studied the cell passaging effect on the cASCs stemness and osteogenic potential. We also evaluated the effect of the anatomical origin of the adipose tissue, namely from abdominal subcutaneous layer and from greater omentum. The stemness and osteogenic differentiation was followed by real time RT-PCR analysis of typical markers of mesenchymal stem cells (MSCs) and osteoblasts. The results obtained revealed that cASCs exhibit a progressively decreased expression of the MSCs markers along passages and also a decreased osteogenic differentiation potential. In the author's knowledge, this work presents the first data about the MSCs markers profile and osteogenic potential of cASCs along cellular expansion. Moreover, the obtained data showed that the anatomical origin of the adipose tissue has an evident effect in the differentiation potential of the ASCs. Due to the observed resemblances with the human ASCs, we conclude that canine ASCs can be used as a model cells in tissue engineering research envisioning human applications.

Carcass composition and meat quality traits were evaluated in 55 suckling kids (27 males and 28 females) from Serrana (S), Bravia (B) and Serrana · Bravia (S·B) crossbred genotypes. Kids were slaughtered at 8-11 kg of live weight according to ''Cabrito de Barroso-PGI'' specifications and carcasses' left sides were totally dissected. Dressing percentage (based on ELW) did not vary between genotypes and sexes. Genotype B carcasses have better conformation, expressed in higher compactness index and muscle/bone ratio. Sex had no effect on the composition of dissected carcass but females deposited more internal fat than males. S genotype had significantly less muscle content and higher dissectible fat compared to B and S·B genotypes, suggesting differences in maturity stages. The carcass' bone content (20.4-21.4%) did not differ significantly between genotypes. The longissimus thoracis et lumborum (LTL) and gluteobiceps (GB) muscles were used for meat quality determinations. Genotype had a significant effect on meat traits and fatty acid composition of the analysed muscles: B genotype and LTL muscle showed lower final pH, S·B genotype had darker and more red muscles, GB muscle had a higher shear force value and lower collagen solubility. Few sex effects were observed on meat quality traits as well as on fatty acid composition. Average percentage of desirable fatty acids in kids was superior to 60% with male S genotype displaying a lower value. Genotypes B and B·S, males and GB muscle had more favorable PUFA:SFA ratios.

Ultrasonic measurements were taken on 46 sheep using a real-time ultrasound machine equipped with 2 probes (5 and 7.5 MHz). Measurements of subcutaneous fat thickness (SC) and muscle LM depth (MD) and area (MA) were taken at 2 locations: over the 13th thoracic vertebra (SC13, MD13, and MA13, respectively) and at the interval between the third and fourth lumbar vertebrae (SC34, MD34, and MA34, respectively). Fat thickness was also measured over the third sternebra of the sternum. The relationship between carcass and in vivo ultrasound measurements was high for all the measurements (r(2) between 0.54 and 0.96, P < 0.01). Concerning MD and SC, the 7.5 MHz probe estimates were consistently more precise than the 5-MHz estimates (r(2) increased between 0.09 and 0.13), but the reverse occurred with the MA estimates, although to a lesser extent. Estimates of carcass composition for muscle, subcutaneous fat, intermuscular fat, internal fat, and total fat based on BW explained a large amount of variation in muscle (87%), subcutaneous fat (85%), intermuscular fat (79%), internal fat (74%), and total fat (87%). In most cases (55 of 70) the introduction of one ultrasound measurement in addition to BW in the multiple regression equations further improved the explanation of variation for weight of carcass tissues, internal fat, and total fat. For carcass muscle estimation, the ultrasound measurements of muscle provided an increase of r(2) between 0.05 and 0.10 (P < 0.01). The SC13 and SC34 gave the best improvements in estimating subcutaneous fat, intermuscular fat, internal fat, and total fat (r(2) increased between 0.05 and 0.17; P < 0.01). Prediction of the proportions of the carcass components (internal and total fat from BW) was clearly lower than the prediction of the absolute amounts of these traits. Inclusion of one or more ultrasound measurements in addition to BW increased the predictive ability of the equations. Both probes were useful to estimate carcass muscle depth and area and fat depth, but the 7.5-MHz probe showed a greater ability to estimate depth. For all traits, the stepwise procedure demonstrated that the best fit was obtained with BW and one or more ultrasound measurement with the 7.5-MHz probe.

Twenty-two Serrana goats were studied through two successive estrous cycles in order to characterize their follicular dynamics during the breeding season. The ovaries of the goats were scanned daily by realtime ultrasonography and all follicles ≥3 mm were measured and classified. The data were classified by the number of follicular waves per goat to test the hypothesis that temporal and morphological differences between the last follicular wave of an ovary, irrespective of ovulation, will affect the selection of the next ovulatory wave.The mean interovulatory interval was 20.7 ± 1.0 days (mean ± S.D.). Three to five waves per estrous cycle were observed and 61.3% (19/31) of cycles had four waves. In estrous cycles with four waves, the day of onset of the first, second, third and fourth wave was 1.4 ± 1.0, 6.9 ± 1.4, 11.6 ± 1.8 and 16.8 ± 1.6, respectively. No differences (P > 0.05) were found between the day of onset of the first and second waves for estrous cycles with three, four or five waves. However, the day of onset of the third and fourth waves occurred later when the number of waves per estrous cycle increased (P < 0.001). The duration of the interwave interval (time between the day of onset of two consecutive waves) was longer when the second wave was ovulatory. The length of the growth phase (2.4 ± 0.9 days) and size (5.9 ± 0.7 mm) of the dominant follicle in the second wave were lower (P < 0.01) than for the first wave (3.3 ± 1.2 days and 6.6 ± 0.9 mm, respectively) and the fifth wave (4.1 ± 1.2 days and 7.5 ± 1.0 mm, respectively). Within pairs of ovaries, the onset of the last wave occurred later (P < 0.05) and was less variable in ovulatory ovaries (day 16.8 ± 1.4, n = 20) than in anovulatory ovaries (day 15.1 ± 3.7, n = 20). The length of the growing phase was longer (P < 0.001) in the last waves of ovulatory ovaries (3.1 ± 0.9 days) than in the last waves of anovulatory ovaries (1.7 ± 0.8 days). These results support the hypothesis that the day of onset of the ovulatory wave is related to or, at least, conditioned by the luteolysis and the decrease in plasma progesterone. * Corresponding author. Tel.: +35 1259350417; fax: +35 1259350480. J. Simões et al. / Animal Reproduction Science 95 (2006) 16-26 17 In summary, the estrous cycle of Serrana goats is characterized by sequential follicular wave growth with a great variability in their onset and duration, with the exception of the ovulatory wave. The temporal and morphological differences observed in the last wave of estrous cycle provide strong evidence for the role of progesterone in their regulation.