The use of COLD‐PCR,DHPLC and GeneScanning for the highly sensitive detection of c‐KIT somatic mutations in canine mast cell tumours

The conventional polymerase chain reaction (PCR)/sequencing methods may be poorly suited for the detection of somatic mutations in canine mast cell tumour (MCT) samples owing to limited sensitivity. This study was aimed at establishing novel and more sensitive methods, assessing their limit of detection and comparing their sensitivity with conventional methods.Two different ‘driver’ somatic mutations of c‐KIT, together with the wild‐type counterparts, were cloned in plasmids to prepare standard samples with known concentrations of mutated alleles in a background of wild‐type alleles; the plasmids standards were assayed using either conventional or novel, highly sensitive technique. Conventional PCR/sequencing showed a sensitivity of 50–20%. Conversely, all the novel methods obtained higher sensitivities allowed reaching as low as 2.5–1.2% of the mutated DNA.The study demonstrates that early conventional methods could likely have underestimated the prevalence of KIT mutations of MCTs, therefore affecting the assessment of their relevance in prognosis and tyrosine kinase inhibitor (TKI) treatment effectiveness.     

Flow‐cytometric detection of phenotypic aberrancies in canine small clear cell lymphoma

Histopathology and immunohistochemistry are mandatory to solve the differential between canine low‐grade lymphoma and reactive hyperplasia. However, clinicians and owners often show reluctance toward these invasive tests. However, molecular biology techniques are still not sensitive and specific enough to be regarded as a reliable tool for final diagnosis. In humans, flow cytometry (FC) allows a definitive diagnosis of T‐cell lymphoma based on high prevalence of antigen aberrancies. We describe here the immunophenotype of 26 cases of suspect canine small‐clear cell lymphoma, determined by multi‐colour FC. All cases showed antigen aberrancies and therefore neoplasia was always confirmed. As a consequence, we argue that the combined use of cytology and FC allows solving the differential diagnosis between small clear cell lymphoma and non‐neoplastic reactive conditions when histopathology is not available. Further studies are needed to establish if any aberrancy can be considered indicative of specific histotypes.     

复合益生菌菌液对奶牛产奶量和肠道菌群的影响

为了研究复合益生菌菌液对奶牛产奶量、乳品质量、牛群健康和肠道菌群的影响,选择20头条件相近的健康奶牛随机分为试验组和对照组,采用菌液与饲料混合的方法饲喂牛群,考察其对奶牛产奶量,肠道菌群乳品质量和牛群健康的影响。结果表明,食用复合益生菌液的每头牛比对照组平均每天可以多产2.9kg牛奶,提高产奶量14.8%,差异极显著(P0.01),经济效益显著;复合益生菌菌液可以促进肠道菌群调整,使有益菌-主要是乳酸菌成为优势菌群,并可以抑制霉菌和降解霉菌毒素,降低牛乳中体细胞数,预防隐性乳腺(房)炎。     

Effect of ovarian hormones on maturation of dendritic cells from peripheral blood monocytes in dogs

Previously, we reported that ovarian hormones affect the immune response against E. coli isolated from the dogs affected with pyometra. In order to investigate mechanisms underlying the immune modulation, we examined the effects of ovarian hormones on the generation of dendritic cells (DCs), the most potent antigen presenting cell. DCs were differentiated from peripheral blood monocytes (PBMOs) using a cytokine cocktail. Both estrogen receptor and progesterone receptors were expressed by the PBMOs and immature DCs. When various ovarian hormones were added to the culture for the DC differentiation, progesterone significantly decreased the expression of DC maturation markers, such as CD1a, CD80 and CD86, on mature DCs. Conversely, the addition of estrogen to the cultures increased the expression of CD86, but not other maturation makers. Furthermore, DCs differentiated in the presence of progesterone did not stimulate allogeneic mononuclear cells in PB. Taken together, these results indicate that progesterone diminishes the maturation of DCs, leading to decreased immune responses against invading pathogens.     

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot^TM reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.     

Effects of supplementation with green tea by‐products on growth performance,meat quality,blood metabolites and immune cell proliferation in goats

Forty‐eight castrated male goats were used to determine the effects of feeding green tea by‐products (GTB) on growth performance, meat quality, blood metabolites and immune cell proliferation. Experimental treatments consisted of basal diets supplemented with four levels of GTB (0%, 0.5%, 1.0% or 2.0%). Four replicate pens were assigned to each treatment with three goats per replicate. Increasing dietary GTB tended to linearly increase the overall average weight gain and feed intake (p = 0.09). Water holding capacity, pH and sensory attributes of meat were not affected by GTB supplementation, while cooking loss was reduced both linearly and quadratically (p < 0.01). The redness (linear; p = 0.02, quadratic; p < 0.01) and yellowness (quadratic; p < 0.01) values of goat meat were improved by GTB supplementation. Increasing dietary GTB quadratically increased protein and decreased crude fat (p < 0.05), while linearly decreased cholesterol (p = 0.03) content of goat meat. The proportions of monounsaturated fatty acid, polyunsaturated fatty acid (PUFA) and n‐6 PUFA increased linearly (p < 0.01) and n‐3 PUFA increased quadratically (p < 0.05) as GTB increased in diets. Increasing dietary GTB linearly increased the PUFA/SFA (saturated fatty acid) and tended to linearly and quadratically increase (p ≤ 0.10) the n‐6/n‐3 ratio. The thiobarbituric acid‐reactive substances values of meat were lower in the 2.0% GTB‐supplemented group in all storage periods (p < 0.05). Dietary GTB linearly decreased plasma glucose and cholesterol (p < 0.01) and quadratically decreased urea nitrogen concentrations (p = 0.001). The growth of spleen cells incubated in concanavalin A and lipopolysaccharides medium increased significantly (p < 0.05) in response to GTB supplementation. Our results suggest that GTB may positively affect the growth performance, meat quality, blood metabolites and immune cell proliferation when supplemented as a feed additive in goat diet.     

Yeast cell wall and live yeast products and their combination in broiler diets formulated with weekly ingredient variations

A 6‐week broiler study was conducted to evaluate whether subjecting the intestinal microflora of broilers to the effect of weekly variations in feed ingredients could be ameliorated by the inclusion of yeast‐derived feed additives: a yeast cell wall extract (YCW), live yeast culture (LY) or their combination (YCW + LY). Recent changes in ingredient prices have motivated producers to formulate diets not necessarily based primarily on corn and soya bean meal. Intestinal microflora in birds can vary significantly based on the ingredient composition of their diet, and the make‐up of the flora can influence overall bird performance. Within the three nutrient phases of this study, birds were fed either a traditional corn–soya ingredient profile or a variable‐ingredient regimen, which had weekly changes in the ingredient composition. There were consistent ameliorative effects of the yeast treatments in both the corn–soya and the variable‐ingredient groups throughout all 6 weeks, with the YCW + LY combination showing a reduced effect when compared to either product fed alone. The effectiveness of YCW and LY on ameliorating the effects of weekly ingredient variations appeared most effective during the starter and grower phases, but was less significant during the sixth week.     

Use of genomic recursions and algorithm for proven and young animals for single‐step genomic BLUP analyses – a simulation study

The purpose of this study was to examine accuracy of genomic selection via single‐step genomic BLUP (ssGBLUP) when the direct inverse of the genomic relationship matrix ( G ) is replaced by an approximation of G ^?1 based on recursions for young genotyped animals conditioned on a subset of proven animals, termed algorithm for proven and young animals (APY). With the efficient implementation, this algorithm has a cubic cost with proven animals and linear with young animals. Ten duplicate data sets mimicking a dairy cattle population were simulated. In a first scenario, genomic information for 20k genotyped bulls, divided in 7k proven and 13k young bulls, was generated for each replicate. In a second scenario, 5k genotyped cows with phenotypes were included in the analysis as young animals. Accuracies (average for the 10 replicates) in regular EBV were 0.72 and 0.34 for proven and young animals, respectively. When genomic information was included, they increased to 0.75 and 0.50. No differences between genomic EBV (GEBV) obtained with the regular G ^?1 and the approximated G ^?1 via the recursive method were observed. In the second scenario, accuracies in GEBV (0.76, 0.51 and 0.59 for proven bulls, young males and young females, respectively) were also higher than those in EBV (0.72, 0.35 and 0.49). Again, no differences between GEBV with regular G ^?1 and with recursions were observed. With the recursive algorithm, the number of iterations to achieve convergence was reduced from 227 to 206 in the first scenario and from 232 to 209 in the second scenario. Cows can be treated as young animals in APY without reducing the accuracy. The proposed algorithm can be implemented to reduce computing costs and to overcome current limitations on the number of genotyped animals in the ssGBLUP method.     

树突状细胞甘露糖受体在旋毛虫感染中的变化研究

应用流式细胞术(FCM)检测旋毛虫感染后小鼠肠系膜淋巴结(MLN)中树突状细胞(DC)上甘露糖受体(MR)的影响。培养小鼠骨髓源树突状细胞(BMDC)并负载旋毛虫排泄/分泌抗原(ES抗原),FCM检测BMDC上MR的变化情况。结果显示,感染第7天MLN中DC表面MR出现下调,但在14 d后出现上调,差异显著(P0.05)。体外实验发现,BMDC负载ES抗原后MR出现下调,直到第48小时出现上调,差异显著(P0.05)。本研究证明旋毛虫感染后可以引起树突状细胞上MR的变化,表明MR可能是ES抗原的识别受体。这为研制旋毛虫树突状细胞疫苗提供了支持,并对寄生虫免疫逃避机制的研究提供了思路。