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.     

Mdivi-1, mitochondrial fission inhibitor,impairs developmental competence and mitochondrial function of embryos and cells in pigs

Mitochondria are highly dynamic organelles that undergo constant fusion/fission as well as activities orchestrated by large dynamin-related GTPases. These dynamic mitochondrial processes influence mitochondrial morphology, size and function. Therefore, this study was conducted to evaluate the effects of mitochondrial fission inhibitor, mdivi-1, on developmental competence and mitochondrial function of porcine embryos and primary cells. Presumptive porcine embryos were cultured in PZM-3 medium supplemented with mdivi-1 (0, 10 and 50 μM) for 6 days. Porcine fibroblast cells were cultured in growth medium with mdivi-1 (0 and 50 μM) for 2 days. Our results showed that the rate of blastocyst production and cell growth in the mdivi-1 (50 μM) treated group was lower than that of the control group (P < 0.05). Moreover, loss of mitochondrial membrane potential in the mdivi-1 (50 μM) treated group was increased relative to the control group (P < 0.05). Subsequent evaluation revealed that the intracellular levels of reactive oxygen species (ROS) and the apoptotic index were increased by mdivi-1 (50 μM) treatment (P < 0.05). Finally, the expression of mitochondrial fission-related protein (Drp 1) was lower in the embryos and cells in the mdivi-1-treated group than the control group. Taken together, these results indicate that mdivi-1 treatment may inhibit developmental competence and mitochondrial function in porcine embryos and primary cells.     

Evaluation of two commercial PRRSV antibody ELISA kits with samples of known status and singleton reactors

Two commercial PRRSV ELISA kits (IDEXX and Bionote) were evaluated for their sensitivity and specificity using 476 PRRS-positive serum samples collected from 7 animal challenge experiments and 1,000 PRRS-negative sera. Both ELISA kits exhibited 100% sensitivity with sera collected 14 to 42 days post-infection, and the results from the kits were highly correlated (R²=0.9207). The specificity of IDEXX or Bionote kit was 99.9% or 99.7%, respectively. In addition, the Bionote ELISA kit was used to examine 100 sera that were determined to be falsely positive either by IDEXX 2XR or 3XR ELISA, and only 7 of these samples were found to be positive. These results indicate that both ELISA kits exhibited similar levels of sensitivity and specificity and would complement one another for the verification of false-positive samples.     

The effect of fermented apple diet supplementation on the growth performance and meat quality in finishing pigs

The objective of the present study was to investigate the effect of fermented apple diet (FAD) supplementation on the growth performance and meat quality in finishing Berkshires. The FAD was made from dropped apple mixed with rice bran and barley bran. Until 81 ± 1 kg live weight at 133 ± 1 days, the animals were fed a growing diet, after which experimental samples were fixed at 0, 2, 4 and 6% FAD as C, T1, T2 and T3 in the finishing diets. Growth performance, ADG, ADFI and feed efficiency were improved in T1 than other groups. In carcass parameters, carcass weight was higher ( P  < 0.05) in T1 than in other groups. In meat quality, moisture and crude protein contents decreased ( P  < 0.05) by addition of FAD. pH₂₄ and WHC were higher ( P  < 0.05) in T1 than other groups. In sensory evaluation, marbling of fresh meat and tenderness, juiciness, flavor and overall acceptability of cooked meat were improved by the addition of FAD. According to the results of our experiment, FAD can be used for improvement of meat quality parameters.     

MAGNETIC RESONANCE IMAGING OF THE CANINE BRAIN AT 7 T

The purpose of this study was to describe relevant canine brain structures as seen on T2-weighted images following magnetic resonance (MR) imaging at 7 T and to compare the results with imaging at 1.5 T. Imaging was performed on five healthy laboratory beagle dogs using 1.5 and 7 T clinical scanners. At 1.5 T, spin echo images were acquired, while gradient echo images were acquired at 3 T. Image quality and conspicuity of anatomic structures were evaluated qualitatively by direct comparison of the images obtained from the two different magnetic fields. The signal-to-nose ratio (SNR) and contrast-to-noise ratio (CNR) were calculated and compared between 1.5 and 7 T. The T2-weighted images at 7 T provided good spatial and contrast resolution for the identification of clinically relevant brain anatomy; these images provided better delineation and conspicuity of the brain stem and cerebellar structures, which were difficult to unequivocally identify at 1.5 T. However, frontal and parietal lobe and the trigeminal nerve were difficult to identify at 7 T due to susceptibility artifact. The SNR and CNR of the images at 7 T were significantly increased up to 318% and 715% compared with the 1.5 T images. If some disadvantages of 7 T imaging, such as susceptibility artifacts, technical difficulties, and high cost, can be improved, 7 T clinical MR imaging could provide a good experimental and diagnostic tool for the evaluation of canine brain disorders.     

Effect of casein substitution with fishmeal, soybean meal and crustacean meal in the diet of the abalone Haliotis discus hannai Ino

The effect of dietary substitution of casein with fishmeal, soybean meal and crustacean meal on the growth of the abalone Haliotis discus hannai Ino was determined. A 350 g casein per kilogram diet was included into the CS diet. The whole casein was then substituted by: (1) 300 g fishmeal and 200 g soybean meal per kilogram diet (FS), (2) 200 g fishmeal, 200 g soybean meal and 130 g krill meal per kilogram diet (FSK), (3) 200 g fishmeal, 200 g soybean meal and 280 g red crab meal per kilogram diet (FSC) or (4) 200 g fishmeal, 200 g soybean meal and 130 g shrimp head meal per kilogram diet (FSS). In addition, a 50‐g by‐product of green tea per kilogram diet was included in the FS diet to form the FSG diet. Sea tangle (ST)diet was supplied to abalone as a control feed. Weight gain, final shell length and final shell width of abalone fed with the various substitution feeds (FS, FSK, FSC, FSS and FSG) were not different from those obtained with the CS diet. All the formulated feeds, however, produced higher weight gain and final shell width values than the ST diet. The results of this study show that casein can be replaced with a combination of fishmeal, soybean meal, krill meal, crab meal and/or shrimp head meal in the diet without a retardation of growth of abalone.     

Optimal dietary taurine level for growth of juvenile Japanese flounder Paralichthys olivaceus

The requirement for taurine in juvenile Japanese flounder Paralichthys olivaceus was determined by feeding diets containing various levels of taurine and cystine. Test diets supplemented with 0.5, 1.0 and 1.5% of taurine or with 0.5, 1.0 and 1.5% of L -cystine were prepared. The basal diet contained 55% protein from white fish meal. These diets were fed to juvenile Japanese flounder with an initial mean bodyweight of 0.9 g (total length (TL) 48 mm) for 5 weeks. Approximately 1.4% taurine content in the diet was required for optimum growth of juvenile flounder. A positive linear relationship was noted between the content of taurine accumulated in the muscle, liver and brain and the level of taurine in the diet. However, there was no increased taurine content in tissues of fish fed the cystine-supplemented diet. In contrast, the fish fed control and cystine-supplemented diets showed higher contents of cystathionine in the tissues. The concentration of cystathionine in tissues rapidly decreased with an increase of taurine in the diet. It was also observed that for each of the dietary groups, a trace amount of taurine was excreted. These results suggest that the taurine content in the diet affects the sulfur amino acid metabolism of juvenile Japanese flounder, and indicate that juvenile flounder are unable to biosynthesize taurine from cystine.