Tenogenic induction of equine mesenchymal stem cells by means of growth factors and low-level laser technology

Tendons regenerate poorly due to a dense extracellular matrix and low cellularity. Cellular therapies aim to improve tendon repair using mesenchymal stem cells and tenocytes; however, a current limitation is the low proliferative potential of tenocytes in cases of severe trauma. The purpose of this study was to develop a method useful in veterinary medicine to improve the differentiation of Peripheral Blood equine mesenchymal stem cells (PB-MSCs) into tenocytes. PB-MSCs were used to study the effects of the addition of some growth factors (GFs) as TGFβ3 (transforming growth factor), EGF2 (Epidermal growth factor), bFGF2 (Fibroblast growth factor) and IGF-1 (insulin-like growth factor) in presence or without Low Level Laser Technology (LLLT) on the mRNA expression levels of genes important in the tenogenic induction as Early Growth Response Protein-1 (EGR1), Tenascin (TNC) and Decorin (DCN). The singular addition of GFs did not show any influence on the mRNA expression of tenogenic genes whereas the specific combinations that arrested cell proliferation in favour of differentiation were the following: bFGF2 + TGFβ3 and bFGF2 + TGFβ3 + LLLT. Indeed, the supplement of bFGF2 and TGFβ3 significantly upregulated the expression of Early Growth Response Protein-1 and Decorin, while the use of LLLT induced a significant increase of Tenascin C levels. In conclusion, the present study might furnish significant suggestions for developing an efficient approach for tenocyte induction since the external administration of bFGF2 and TGFβ3, along with LLLT, influences the differentiation of PB-MSCs towards the tenogenic fate.     

Use of Non‐invasive Methods for Evaluating the Testicular Biometry in Collared Peccaries (Pecari tajacu Linnaeus, 1758)

The aim of this study was to compare the accuracy of two methods used to estimate testicular volume in the collared peccary. Calliper and ultrasonographic measurements of testicular dimensions (length, width and height) of both testes were taken on five adult collared peccaries. The testicular volume was calculated by Lambert's empiric formula: length (L) × width (W) × height (H) × 0.71, the formula of an ellipsoid L × W × H × 0.52, and Hansen's formula: L × W² × 0.52. The calculated volumes were then compared with the actual ones, which were estimated by water displacement. The mean of true testicular volume was 22.65 ± 1.52 ml. Lambert's formula estimated testicular volume more accurately when ultrasound measurements were taken. However, when the calliper was the methodology used, the results were closest to the true volume, especially when Ellipsoid formula and Hansen's formula were applied, and underestimated the true volumes by 1.53 ± 1.75 ml and 1.53 ± 1.65 ml, respectively. This specific application of technologies in wild animals has the potential to revolutionize the selection process for the collared peccary entering artificial insemination or natural breeding programmes.     

Ultrastructural Characterization of Fresh and Vitrified In Vitro‐ and In Vivo‐Produced Sheep Embryos

The lower results in cryopreservation of in vitro‐produced (IVP) sheep embryos, when compared to the in vivo derived, limits its use. Four groups of blastocyst (BL) were evaluated: fresh IVP (n = 3), fresh in vivo derived (n = 3), warmed IVP cryopreserved in open pulled straws (OPS, n = 3) and warmed in vivo derived cryopreserved in OPS (n = 3). Ultrastructural observation of processed fresh embryos showed a reduced number of microvilli and mitochondria in the IVP ones, as well as a lower number of mature mitochondria, that can be associated with deficient metabolism in IVP embryos, possibly involved in the lower resistance to cryopreservation. Both in vivo‐derived and IVP embryos had a large number of vesicles, with light and dense content. In embryos vitrified by OPS, major changes were observed mainly in IVP embryos with small changes in grade 2 (fair) and high changes in grade 3 (bad) semithin scoring. The main changes associated with cryopreservation included disruption of cellular membranes and poor intracellular preservation, with loss of microvilli and the presence of cellular debris. In conclusion, ultrastructural evaluation of IVP blastocysts cryopreserved in OPS was herein described for the first time, reporting more severe cellular damage in these embryos when compared to those produced in vivo. This is probably associated with a lower cryotolerance that can be related to their lipid content and metabolism.     

Physiological and biochemical changes of females of Piracanjuba,subjected to induced reproduction

The aim of this experiment was to compare the level of fish female stress during induced reproduction with pituitary extract by two different methods, natural and semiextruded. The reproductive efficiency was 62.5% in the seminatural treatment and 100% in the extruded. Obtained egg volume was 5200 ml and 4000 ml, for seminatural and extruded treatments respectively. The mean number of eggs was 46.7 for the seminatural and 52.0 and for the extruded treatment. The percentage of viable eggs was, respectively, 87.2% and 8.17% for the natural treatment and extruded semimethods. Blood samples were collected to quantify cortisol and glucose levels, as well as red cell series and lymphocyte count. Fishes submitted to induction procedures showed elevated cortisol and glucose levels, compared to the control animals. The results for haematocrit, haemoglobin concentration and red blood cell count showed no significant differences among groups. Significant differences found in the number of lymphocytes and monocytes suggest the general adaptation syndrome. Our results suggest the reproductive induction process with extrusion of gametes as a more stressful method than seminatural reproduction process.