Culture of Differentiated Adult Rabbit Auricular Chondrocytes

Chondrocytes dedifferentiate to a fibroblast‐like phenotype on plastic surfaces. Dedifferentiation is reversible if these cells are then cultured embedded in gels as alginate, agarose or collagen. Chondrocytes cultured in suspension on a non‐adherent surface are also known to form aggregates of differentiated cells. The knowledge of chondrocyte behavior in culture is relevant for tissue engineering purposes. In this report we describe a simple method to culture differentiated or redifferentiated rabbit auricular chondrocytes on plastic surfaces with a stable phenotype. When chondrocyte aggregates formed in suspension are next seeded on plastic surfaces, most of them attach to the plastic as round or polygonal cells, and this morphological differentiation, confirmed by the presence of type II collagen, is stable for long culture periods. We also report that the addition of aggregates to monolayer cultures of dedifferentiated chondrocytes results in their redifferentiation, as is shown by their morphological changes and the synthesis of type II collagen. Therefore, this simple method can be useful for the study of chondrocyte behavior on plastic surfaces and for redifferentiating previously proliferated chondrocytes in tissue engineering techniques. Furthermore, these results demonstrate that, in addition to culture conditions such as cell isolation method or cell‐density, chondrocyte behavior on plastic depends on the presence or absence of aggregates resulting from the dissociation process.     

Changes of Ca2＋ activated potassium channels and cellular-proliferation in autogenous vein grafts

AIM: To investigate changes of Ca^2＋ activated potassium channels (K_Ca) in autogenous vein grafts. METHODS: The contraction of venous ring was measured by means of perfusion in vitro. The intimal proliferation and proliferation of cultured smooth muscle cells (vascular smooth muscle cells, VSMCs) were observed by the means of computerised image analysis and MTT method, respectively. Furthermore, whole cell mode of patch clamp was used to record K_Ca of VSMCs isolated from autogenous vein grafts. RESULTS: 1 week after transplantation there were no significant differences of contraction and intimal relative thickness between autogenous vein grafts and control. Contraction and intimal relative thickness of autogenous vein graft were significantly increased 2 weeks after transplantation (P＜0.05, n=8 vs control), and they were more enhanced 4 weeks after vein transplantation (P＜0.01, n=8 vs control). TEA (blocker of Ca^2＋ activated potassium channels) increased MTT A₄₉₀ value of VSMCs from femoral vein in a dose-dependent manner (P＜0.05, n=8). K_Ca current density was significantly attenuated in VSMCs from autogenous vein grafts 1-4 weeks after transplantation (P＜0.05, n=5). CONCLUSION: K_Ca was inhibited in autogenous vein graft, which accounted for vasospasm and intimal proliferation.     

Distribution of alpha‐neoendorphin,ACTH (18–39) and beta‐endorphin (1–27) in the alpaca brainstem

Using an immunocytochemical technique, we have studied in the alpaca brainstem the distribution of immunoreactive structures containing prodynorphin (alpha‐neoendorphin)‐ and pro‐opiomelanocortin (adrenocorticotrophin hormone (18–39) (ACTH), beta‐endorphin (1–27))‐derived peptides. No peptidergic‐immunoreactive cell body was observed. Immunoreactive fibres were widely distributed, although in most of the brainstem nuclei the density of the peptidergic fibres was low or very low. In general, the distribution of the immunoreactive fibres containing the peptides studied was very similar. A close anatomical relationship occurred among the fibres containing alpha‐neoendorphin, ACTH or beta‐endorphin (1–27), suggesting a functional interaction among the three peptides in many of the brainstem nuclei. The number of fibres belonging to the prodynorphin system was higher than that of the pro‐opiomelanocortin system. A moderate/low density of immunoreactive fibres was observed in 65.11% (for alpha‐neoendorphin (1–27)), 18.18% (for ACTH) and 13.95% (for beta‐endorphin) of the brainstem nuclei/tracts. In the alpaca brainstem, a high density of immunoreactive fibres was not observed. The neuroanatomical distribution of the immunoreactive fibres suggests that the peptides studied are involved in auditory, motor, gastric, feeding, vigilance, stress, respiratory and cardiovascular mechanisms, taste response, sleep‐waking cycle and the control of pain transmission.     

Effects of quebracho tannin extract on intake,digestibility, rumen fermentation,and methane production in crossbred heifers fed low-quality tropical grass

The aim of this work was to evaluate the effect of quebracho tannins extract (QTE) on feed intake, dry matter (DM) digestibility, and methane (CH₄) emissions in cattle fed low-quality Pennisetum purpureum grass. Five heifers (Bos taurus × Bos indicus) with an average live weight (LW) of 295 ± 19 kg were allotted to five treatments (0, 1, 2, 3, and 4% QTE/kg DM) in a 5 × 5 Latin square design. Intake, digestibility, and total methane emissions (L/day) were recorded for periods of 23 h when cattle were housed in open-circuit respiration chambers. Dry matter intake (DMI), organic matter intake (OMI), dry matter digestibility (DMD), and organic matter digestibility (OMD) were different between treatments with 0 and 4% of QTE/kg DM (P < 0.05). Total volatile fatty acid and the molar proportion of acetate in the rumen was not affected (P < 0.05); however, the molar proportion of propionate increased linearly (P < 0.01) for treatments with 3 and 4% QTE. Total CH₄ production decreased linearly (P < 0.01) as QTE increased in the diet, particularly with 3 and 4% concentration. When expressed as DMI and OMI by CH₄, production (L/kg) was different between treatments with 0 vs 3 and 4% QTE (P < 0.05). It is concluded that the addition of QTE at 2 or 3% of dry matter ration can decrease methane production up to 29 and 41%, respectively, without significantly compromising feed intake and nutrients digestibility.     

<Emphasis Type="SmallCaps">d</Emphasis>-Cloprostenol enhances estrus synchronization in tropical hair sheep

To compare the effects of PGF2α (dinoprost tromethamine) and d-cloprostenol in a two-dose protocol for estrus synchronization in hair sheep during breeding season in Yucatán, México, two experiments were conducted. In experiment 1, 61 cyclic hair sheep were divided into two groups: G1 (control n?=?30), two doses of 50 μg of dinoprost tromethamine IM with 12 days between applications, and G2 (n?=?31), two doses of 50 μg of d-cloprostenol IM at the same time interval. For determination of progesterone levels, 16 ewes from each group were randomly selected. In experiment 2, 70 cyclic hair sheep were assigned at the same treatments (G1 and G2, n?=?35) and 48 h after the second application, the ewes in estrus were detected by two vasectomized rams. Sheep with detected estrus were inseminated, and 45 days after, pregnant animals were identified by ultrasonography. An exact Fisher’s test was performed for the analysis of ewes in estrus (experiments 1 and 2) and number of pregnant ewes (experiment 2); for the comparison of time between end of treatment-estrus presentation, a survival analysis was used. Duration of estrus in hours was analyzed using a generalized mixed model (GLM) ANOVA whereas plasma progesterone concentrations were analyzed by non-linear regression. There were significant differences (P?<?0.05) in the proportion of ewes in estrus upon treatments (G1, 57% vs G2, 87% and G1, 37.1% vs G2, 65.7% in experiments 1 and 2, respectively), and between the end of treatment-onset estrus interval (P?<?0.01), survival curves showed the highest number of sheep in estrus between 40 and 48 h (G1, 43.7?+?8.05 h vs G2, 42.9?+?6.7 h, experiment 1). There were no significant differences (P?>?0.05) in duration of estrus (G1, 42?+?6.1 h, vs G2, 41.1?+?11.2 h, experiment 1) and pregnancy in the ewes that presented estrus, and were inseminated (G1, 38.4% vs 52.1%, experiment 2). With regard to concentrations of progesterone, significant differences (P?<?0.01) were found between treatments, and progesterone levels before the second application of d-cloprostenol were higher. In consideration of the results, it can be concluded that in a two-dose protocol of a luteolytic agent, more ewes presented estrus in response to d-cloprostenol compared to dinoprost tromethamine with similar pregnancy rates.     

Herbage intake of dairy cows in mixed sequential grazing with breeding ewes as followers

This study aimed to evaluate the hypothesis that mixed sequential grazing of dairy cows and breeding ewes is beneficial. During the seasons of spring–summer 2013 and autumn–winter 2013–2014, 12 (spring–summer) and 16 (autumn–winter) Holstein Friesian cows and 24 gestating (spring–summer) and lactating (autumn–winter) Pelibuey ewes grazed on six (spring–summer) and nine (autumn–winter) paddocks of alfalfa and orchard grass mixed pastures. The treatments “single species cow grazing” (CowG) and “mixed sequential grazing with ewes as followers of cows” (MixG) were evaluated, under a completely randomized design with two replicates per paddock. Herbage mass on offer (HO) and residual herbage mass (RH) were estimated by cutting samples. The estimate of herbage intake (HI) of cows was based on the use of internal and external markers; the apparent HI of ewes was calculated as the difference between HO (RH of cows) and RH. Even though HO was higher in CowG, the HI of cows was higher in MixG during spring–summer and similar in both treatments during autumn–winter, implying that in MixG the effects on the cows HI of higher alfalfa proportion and herbage accumulation rate evolving from lower residual herbage mass in the previous cycle counteracted that of a higher HO in CowG. The HI of ewes was sufficient to enable satisfactory performance as breeding ewes. Thus, the benefits of mixed sequential grazing arose from higher herbage accumulation, positive changes in botanical composition, and the achievement of sheep production without negative effects on the herbage intake of cows.     

Evaluation of alternative sanitizers to chlorine disinfection for reducing foodborne pathogens in fresh-cut apple

The risk of undesirable by-products from chlorine disinfection in fresh-cut industries, together with its limited efficacy, has led to a search for alternative agents. The aim of this study was to test several alternative putative antimicrobial substances to reduce Escherichia coli O157:H7, Salmonella spp. and Listeria spp. populations on fresh-cut apple. Carvacrol, vanillin, peroxyacetic acid, hydrogen peroxide, N-acetyl-l-cysteine and Citrox were selected for their results in in vitro assays against E. coli O157:H7 and Listeria spp., to be tested on fresh-cut apple plugs. Apple flesh was inoculated by dipping in a suspension of a mix of the studied pathogens at 10⁶ cfu mL^?1, and then treated with the antimicrobial substances. All treatments were compared to deionized water and a standard sodium hypochlorite treatment (SH, 100 mg L^?1, pH 6.5). Pathogen population on apple plugs was monitored for up to 6 days at 10 °C. Bacterial reductions obtained by peroxyacetic acid (80 and 120 mg L^?1), vanillin (12 g L^?1), hydrogen peroxide (5, 10, 20 mL L^?1) and N-acetyl-l-cysteine (5 and 10 g L^?1) were similar or higher than reduction obtained by SH. In addition, bacterial populations were maintained at low levels throughout storage. No cells of any of the pathogens were detected in the peroxyacetic acid, hydrogen peroxide, Citrox and SH washing solutions after apple treatment. Peroxyacetic acid, hydrogen peroxide and N-acetyl-l-cysteine could be potential disinfectants for the fresh-cut industry as an alternative to chlorine disinfection. However, their effect on sensory quality and effectiveness under commercial processing conditions should be evaluated.