Stem cell factor supports migration in canine mesenchymal stem cells

Adult Mesenchymal Stem Cells (MSC) are cells that can be defined as multipotent cells able to differentiate into diverse lineages, under appropriate conditions. These cells have been widely used in regenerative medicine, both in preclinical and clinical settings. Initially discovered in bone marrow, MSC can now be isolated from a wide spectrum of adult and foetal tissues. Studies to evaluate the therapeutic potential of these cells are based on their ability to arrive to damaged tissues. In this paper we have done a comparative study analyzing proliferation, surface markers and OCT4, SOX9, RUNX2, PPARG genes expression in MSC cells from Bone marrow (BMMSC) and Adipose tissue (ASC). We also analyzed the role of Stem Cell Factor (SCF) on MSC proliferation and on ASCs metalloproteinases MMP-2, MMP-9 secretion. Healthy dogs were used as BMMSC donors, and ASC were collected from omentum during elective ovariohysterectomy surgery. Both cell types were cultured in IMDM medium with or without SCF, 10% Dog Serum (DS), and incubated at 38 °C with 5% CO2. Growth of BMMSCs and ASCs was exponential until 25–30 days. Flow citometry of MSCs revealed positive results for CD90 and negative for CD34, CD45 and MCH-II. Genes were evaluated by RT-PCR and metalloproteinases by zymografy. Our findings indicate morphological and immunological similarities as well as expression of genes from both origins on analyzed cells. Furthermore, SCF did not affect proliferation of MSCs, however it up-regulated MMP-2 and MMP-9 secretion in ASCs. These results suggest that metalloproteinases are possibly essential molecules pivoting migration.     

Effects of protein-energetic supplementation frequency on growth performance and nutritional characteristics of grazing beef cattle

Two experiments were conducted to evaluate reduced supplementation frequencies for grazing beef cattle in rainy season. In experiment 1, evaluating the nutritional parameters, four rumen-cannulated Nellore bulls (BW = 410 kg) were used. In experiment 2, evaluating animal performance, 48 Nellore bulls (BW = 358 kg) were used. The treatments were as follows: mineral supplement (MS) alone and MS plus protein-energy supplement provided 3×, 5× and 7×/week. Supplementation frequency did not affect (P > 0.05) intake and digestibility. Average daily gain was greater (P < 0.001) to supplementation compared with MS. The supplementation 5×/week resulted in greater weight gain per hectare (9.24) and higher economic returns during the study period (1.64%) compared to other supplementations. Supplementation 5×/week increased animal performance and positively influenced economic returns.     

Feeding behavior of feedlot lambs fed diets containing levels of cassava wastewater

In this study, we evaluated the effects of including cassava wastewater in the diet on the feeding behavior of feedlot lambs in 35 male uncastrated Santa Inês × Dorper crossbred lambs at an approximate age of 3 months, with an average live weight of 20.0?±?3.4 kg. Diets were formulated with hay of cassava shoots (roughage) and a concentrate based on corn and soybean, with a roughage:concentrate ratio of 50:50, plus inclusion of cassava wastewater at the levels of 0, 12, 24, 36, or 48 g/kg of the total diet. Feeding behavior was evaluated between the 46th and 52nd days of the experiment. Increasing cassava wastewater levels in the diet reduced (P?<?0.05) the intakes (kg/day) of dry matter and neutral detergent fiber as well as the efficiency of rumination (g/cud and g/h) of dry matter and neutral detergent fiber. The other behavioral parameters were not affected by wastewater inclusion in the diet. Therefore, the inclusion of up to 48 g/kg of cassava wastewater on fresh matter of diets is not recommended for feedlot lambs.     

Meta‐analysis of the energy and protein requirements of hair sheep raised in the tropical region of Brazil

The objective of this study was to estimate, through mathematical models, energy and protein requirements for maintenance and gain of hair sheep raised in the tropical region of Brazil. To determine the equation parameters, a meta‐analysis of seven independent experiments of nutrient requirements was performed, comprising a total of 243 experimental units (animals), which were conducted under tropical conditions, using hair sheep in growing and finishing phases and endowed of the following quantitative data for each animal: body weight (BW ), empty body weight (EBW ), average daily gain (ADG ), empty body gain (EBG ), heat production (HP ), metabolizable energy intake (MEI ), retained energy (RE ), metabolizable protein intake (MPI ) and body protein content. The regression equations generated were as follows: for Net Energy for maintenance, (NE _m): ; for Net Energy for gain, (NE _g): ; for Metabolizable Protein for maintenance,(MP _m): MPI _(g/day) = 24.8470 (±7.3646) + 560.28 (±99.6582) × EBG _(kg/day); for Net Protein for gain, (NP _g): . The NE _m requirement was 0.246 MJ EBW^?0.75 day^?1. The metabolizable energy for maintenance requirement was 0.391 MJ EBW^?0.75 day^?1. Considering an ADG of 100 g, the NE _g requirement ranged from 0.496 to 1.701 MJ/day for animals with BW ranging from 10 to 40 kg respectively. The efficiencies of use of the metabolizable energy for maintenance and gain were 0.63 and 0.36 respectively. The MP _m requirement was 3.097 g EBW^?0.75 day^?1. Considering an ADG of 100 g, the NP _g requirement ranged from 12.4 to 10.5 g/day for animals with BW ranging from 10 to 40 kg respectively. The total metabolizable energy and protein requirements were lower than those reported by the NRC and AFRC systems. Thus, our results support the hypothesis that nutrient requirements of hair sheep raised in tropical regions differ from wool sheep raised in temperate regions. Therefore, the use of the equations designed in this study is recommended.     

Effect of resistant starch on the intestinal health of old dogs: fermentation products and histological features of the intestinal mucosa

The effects of resistant starch (RS) intake on nutrient digestibility, microbial fermentation products, faecal IgA, faecal pH, and histological features of the intestinal mucosa of old dogs were evaluated. The same formulation was extruded in two different conditions: one to obtain elevated starch cooking degree with low RS content (0.21%) and the other lower starch cooking with high RS content (1.46%). Eight geriatric Beagles (11.5 ± 0.38 years old) were fed each diet for 61 days in a crossover design. Food intake, nutrient digestibility, fermentation products, faecal pH, and faecal IgA were examined via variance analysis. Histological results of intestinal biopsies were assessed via Wilcoxon test for paired data. The morphometric characteristics of large intestine crypts were evaluated via paired t tests (p < .05). Protein, fat, and energy digestibilities were higher for the low‐RS diet (p < .05). Dogs receiving the high‐RS diet had lower faecal pH and higher values for propionate, butyrate, total volatile fatty acids, and lactate (p < .05). No differences between diets were found in the histological parameters of the gut mucosa, and only a tendency for deeper crypts in the descending colon was observed for dogs fed the high‐RS diet (p = .083). The intake of a corn‐based kibble diet manufactured with coarse ground raw material and low starch gelatinization to obtain 1.4% of RS affected microbial fermentation products and faecal pH and tended to increase crypt depth in the descending colon of old dogs.     

A fast,low‐cost and efficient method for the diagnosis of sperm DNA fragmentation in several species

Sperm DNA fragmentation is a condition that interferes directly in the reproductive efficiency. Currently, there are several methods for assessing the sperm DNA integrity, such as Alkaline Comet, TUNEL and Sperm Chromatin Structure Assay. However, many of these techniques are laborious and require high‐precision equipment. Thus, the development of new techniques can optimize the evaluation of sperm DNA damage. Therefore, the aim of this study was to standardize the toluidine blue (TB) stain technique for the analysis of DNA fragmentation of dog, cat, bull, stallion and ram spermatozoa. For this purpose, we used six animals of each specie (n = 30), in reproductive age. Sperm was collected by different methods according to the particularities of each species, and such samples were divided into two aliquots: a sperm sample was kept at 5°C (considered as intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation by exposure to ultraviolet light for 4 hr. Samples were then mixed with the intact sample to obtain known and progressive proportions of sperm with fragmented DNA (0%, 25%, 50%, 75% and 100%). Semen smears were performed and subjected to staining with TB. Blue‐stained spermatozoa were considered to have DNA fragmentation. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for dog, cat, ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA in several species.     

Melatonin reduces apoptotic cells,SOD2 and HSPB1 and improves the in vitro production and quality of bovine blastocysts

Effects of adding different concentrations of melatonin (10^?7, 10^?9 and 10^?11 M) to maturation (Experiment 1; Control, IVM  + 10^?7, IVM  + 10^?9, IVM  + 10^?11) and culture media (Experiment 2; Control, IVC  + 10^?7, IVC  + 10^?9, IVC  + 10^?11) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10^?9 M) from Experiments 1–2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM  + 10^?9, IVC  + 10^?9, IVM /IVC  + 10^?9). In Experiment 1, maturated oocytes from Control and IVM  + 10^?9 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC  + 10^?7 (43.5%; 56.7%) and IVC  + 10^?9 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC  + 10^?9 group compared to Control (3.8 ± 0.6; 3.6% versus 6.1 ± 0.6; 5.3%). In Experiment 3, the IVC  + 10^?9 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM /IVC  + 10^?9 group (52.6%; 3.0 ± 0.5 versus 46.0%; 5.4 ± 1.0). The IVC  + 10^?9 treatment also had a higher mRNA expression of antioxidant gene (SOD 2) compared to the Control, as well as the heat shock protein (HSPB 1) compared to the IVM  + 10^?9. Reactive oxygen species production was greater in the IVM /IVC  + 10^?9 treatment group. In conclusion, the 10^?9 M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality.     

Pedotransfer functions to assess adsorbed phosphate using iron oxide content and magnetic susceptibility in an Oxisol

Adsorbed phosphate in soils can be chemically extracted; however, this process is both time‐consuming and not cost‐effective if large numbers of samples have to be analysed. Indirect assessment of adsorbed phosphate by pedotransfer functions (PTFs) can help optimize fertilizer strategies. This study aimed to evaluate the spatial variability of adsorbed phosphate (P_ads), iron oxides and magnetic susceptibility (MS) in oxisols and to calibrate PTFs to predict P_ads. A total of 308 soil samples were collected from Hapludox and Eutrudox soils formed from sandstone in Brazil. The contents of clay (196–607 g/kg), iron oxides (40–165 g/kg), MS (1.2–29 × 10^?6 m³/kg) and P_ads (327–842 mg/kg) were in the range of typical values for these highly weathered soils. This study showed that the attributes studied were spatially dependent. Geomorphic surfaces enabled understanding of spatial variability and helped to develop a more efficient sampling scheme to calibrate PTFs. Moreover, the adsorbed phosphate in these oxisols could be predicted by a PTF using iron oxides and MS as predictors. The MS attribute enabled the most accurate prediction (concordance coefficient = 0.95, root‐mean‐square error = 46 mg/kg and relative improvement in root‐mean‐square error = ?4.12) of spatial variability through PTF compared to other predictors.     

Effect of Caffeine Added to the Activating Solution on Sperm Motility of Fresh and Thawed Semen of Pacu,Piaractus mesopotamicus,and Curimba,Prochilodus lineatus

The aim was to evaluate the effect of different concentrations of caffeine added in activating solution over sperm motility in fresh and thawed semen of pacu, Piaractus mesopotamicus, and curimba, Prochilodus lineatus. The activating solutions were prepared with sodium bicarbonate solution of 0.76% (NaHCO₃) and caffeine was added at concentrations of 2.5, 5.0, 10.0, and 20.0 mM. As control, a solution of NaHCO₃ 0.76 without caffeine was used. Eight males of pacu and 20 males of curimba were used. Aliquots of 200 μL of semen were diluted in 800 μL extender solution (DMSO 10% and BTS 5%), placed in 0.5 mL straws and cryopreserved for 7 d in a liquid nitrogen tank. There was a linear increase in sperm motility for fresh semen of pacu, and for curimba fresh and thawed semen (P < 0.05), due to the increase in the concentration of caffeine. There was a quadratic response for duration of motility for thawed semen of pacu and for fresh semen of curimba (P < 0.05), respectively. These results indicate that addition of caffeine in the activator solution can improve sperm motility parameters, however, is dependent on the species and concentration used.