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Global warming has a significant impact on the dairy farming industry, as heat stress causes reproductive endocrine imbalances and leads to substantial economic losses, particularly in tropical–subtropical regions. The Holstein breed, which is widely used for dairy production, is highly susceptible to heat stress, resulting in a dramatic reduction in milk production during hot seasons. However, previous studies have shown that cells of cows produced from reconstructed embryos containing cytoplasm (o) from Taiwan yellow cattle (Y) have improved thermotolerance despite their nuclei (n) being derived from heat-sensitive Holstein cattle (H). Using spindle transfer (ST) technology, we successfully produced ST-Yo-Hn cattle and proved that the thermotolerance of their ear fibroblasts is similar to that of Y and significantly better than that of H (p < 0.05). Despite these findings, the genes and molecules responsible for the different sensitivities of cells derived from ST-Yo-Hn and H cattle have not been extensively investigated. In the present study, ear fibroblasts from ST-Yo-Hn and H cattle were isolated, and differentially expressed protein and gene profiles were compared with or without heat stress (hs) (42 °C for 12 h). The results revealed that the relative protein expression levels of pro-apoptotic factors, including Caspase-3, -8, and -9, in the ear fibroblasts from the ST-Yo-Hn-hs group were significantly lower (p < 0.05) than those from the H-hs group. Conversely, the relative expression levels of anti-apoptotic factors, including GNA14 protein and the CRELD2 and PRKCQ genes, were significantly higher (p < 0.05) in the ear fibroblasts from the ST-Yo-Hn-hs group compared to those from the H-hs group. Analysis of oxidative phosphorylation-related factors revealed that the relative expression levels of the GPX1 gene and Complex-I, Complex-IV, CAT, and PGLS proteins were significantly higher (p < 0.05) in the ear fibroblasts from the ST-Yo-Hn-hs group compared to those from the H-hs group. Taken together, these findings suggest that ear fibroblasts from ST-Yo-Hn cattle have superior thermotolerance compared to those from H cattle due to their lower expression of pro-apoptotic factors and higher expression of oxidative phosphorylation and antioxidant factors. Moreover, this improved thermotolerance is attributed, at least partially, to the cytoplasm derived from more heat-tolerant Y cattle. Hence, using ST technology to produce more heat-tolerant H cattle containing Y cytoplasm could be a feasible approach to alleviate the negative impacts of heat stress on dairy cattle in tropical–subtropical regions.

This study aimed to investigate the effects of triiodothyronine (T3)- or dopamine (Dp)-supplemented diets on oxygen consumption by Na+, K+-ATPase activity in broiler chicks. Five groups, each with twenty-four 6-day-old chicks, randomly received one of the five dietary treatments: (1) Basal diet (commercial broiler rations with 23.0% crude protein and 3,133 kcal metabolizable energy/kg) or CON, (2) basal diet plus 0.7 μmol Dp/kg diet or Dp0.7, (3) basal diet plus 2.4 μmol Dp/kg diet or Dp2.4, (4) basal diet plus 1.9 μmol T3/kg diet or T1.9, and (5) basal diet plus 3.8 μmol T3/kg diet or T3.8 from 6 to 14 days of age. There were four replicates per treatment and 120 birds in total. At 14 days of age, three chicks from each replicate of each treatment were pooled into a flock and fed commercial broiler diets until 7 weeks of age. Compared to CON group, birds fed with T3-supplemented diets had lower thyroid, abdominal fat pad, gizzard and pancreas weight, and heavier heart weight adjusted for fasted body weight. Chicks with T1.9 had lower ileal densities at 14 day old compared with those in Dp groups or CON. Chicks with T3.8 exhibited greater duodenal and jejunal O2 consumptions as well as ouabain-sensitive O2 consumptions of jejunum and small intestine (duodenum, jejunum, and ileum) by 46.5%, 58.3%, 40.6%, and 26.4% increases, than those in CON. Partial correlation analysis revealed that the weight and length of the small intestine were negatively correlated with body weight gain. Oxygen consumption in the various small intestinal segments was negatively correlated with their respective densities (mg/mm2). In conclusion, a greater oxygen requirement for maintaining ouabain-sensitive respiration (Na+-K+-ATPase) in the intestine limits energy availability to support gastrointestinal tract growth and, thereby, may result in lower body weight gain.

Abstract Spinal muscular atrophy (SMA) is a single gene disorder affecting motor function in uterus. Amniotic fluid is an alternative source of stem cell to ameliorate SMA. Therefore, this study aims to examine the therapeutic potential of Human amniotic fluid stem cell (hAFSC) for SMA. Our SMA model mice were generated by deletion of exon 7 of Smn gene and knock-in of human SMN2. A total of 16 SMA model mice were injected with 1 × 105 hAFSC in uterus, and the other 16 mice served as the negative control. Motor function was analyzed by three behavioral tests. Engraftment of hAFSC in organs were assessed by flow cytometry and RNA scope. Frequency of myocytes, neurons and innervated receptors were estimated by staining. With hAFSC transplantation, 15 fetuses survived (93.75% survival) and showed better performance in all motor function tests. Higher engraftment frequency were observed in muscle and liver. Besides, the muscle with hAFSC transplantation expressed much laminin α and PAX-7. Significantly higher frequency of myocytes, neurons and innervated receptors were observed. In our study, hAFSC engrafted on neuromuscular organs and improved cellular and behavioral outcomes of SMA model mice. This fetal therapy could preserve the time window and treat in the uterus.

Fallopian tube is essential to fertilization and embryonic development. Extracellular vesicles (EVs) from Fallopian tube containing biological regulatory factors, such as lipids, proteins and microRNAs (miRNAs) serve as the key role. At present, studies on oocytes from porcine oviduct and components from EVs remain limited. We aim to explore the effect of EVs secreted by porcine fallopian tube stem cells (PFTSCs) on oocyte. When the fifth-generation PFTSCs reached 80–90% of confluency, the pig in vitro maturation medium was utilized, and the conditioned medium collected for oocyte incubations. To realize the functions of EVs, several proteins were used to determine whether extracted EVs were cell-free. Field emission scanning electron microscope and nanoparticle tracking analyzer were used to observe the morphology. By next generation sequencing, 267 miRNAs were identified, and those with higher expression were selected to analyze the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment maps. The selected miR-152-3p, miR-148a-3p, miR-320a-3p, let-7f-5p, and miR-22-3p, were predicted to target Cepb1 gene affecting MAPK pathway. Of the five miRNAs, miR-320a-3p showed significant difference in maturation rate in vitro maturation. The blastocyst rate of pig embryos was also significantly enhanced by adding 50 nM miR-320a-3p. In vitro culture with miR-320a-3p, the blastocyst rate was significantly higher, but the cleavage rate and cell numbers were not. The CM of PFTSCs effectively improves porcine oocyte development. The miRNAs in EVs are sequenced and identified. miR-320a-3p not only helps the maturation, but also increases the blastocyst rates.

Objective: Bovine mastitis results in economic loss due to decrease in milk production. Antibiotic ointments are commonly used for treating. However, residue and anti-microbial resistance warranted attention progressively. Fortunately, stem cell anti-inflammatory properties and paracrine expression of cytokines accelerates wound healing and suppresses inflammatory reactions in mastitis. The objective of this study is to use the conditioned-Dulbecco's pluripotent stem cells (DPBS) from amniotic membrane stem cells (AMSCs) in treating bovine mastitis. Materials and methods: The cows with mastitis were divided into two groups. In antibiotic control group, the cows were given tetraneomycin ointment. In conditioned-DPBS of AMSCs treatment group, amniotic membrane was collected for AMSCs after delivery. With expression of surface antigen and potential of tri-linage differentiation, AMSCs were injected into mammary glands. Then, milk was sampled every three days to monitor the effect of both treatments. The quality of milk was measured with pH, titratable acidity, free calcium ions and somatic cell count. Results: Our results demonstrated the Bovine AMSCs expressed CD44, low levels of CD4 and no CD105. Bovine AMSCs demonstrated the differentiation capability in the tri-cell lineages. Mastitis treatment with conditioned-DPBS from AMSCs (experimental group) and conventional antibiotics (control group) showed insignificant difference in pH value and titratable acidity. The level of ionic calcium concentration in the conditioned-DPBS group decreased from 3rd day to 12th day, while the level in the antibiotic group decreased from 0 day to 12th day. The somatic cell number was similar in both groups, which meet the standard of Taiwan milk collection. Conclusion: In conclusion, conditioned-DPBS from bovine AMSCs has the therapeutic potential to treat bovine mastitis and may replace antibiotics therapy in the future.

Glucocorticoid (GC)-induced bone loss is the most prevalent form of secondary osteoporosis. Previous studies demonstrated that long-term incubation of dexamethasone (DEX) induced oxidative stress and mitochondrial dysfunctions, consequently leading to apoptosis of differentiated osteoblasts. This DEX-induced cell death might be the main causes of bone loss. We previously described that DEX induced biphasic mitochondrial alternations. As GC affects mitochondrial physiology through several different possible routes, the short-term and long-term effects of GC treatment on mitochondria in the osteoblast have not been carefully characterized. Here, we examined the expression levels of genes that are associated with mitochondrial functions at several different time points after incubation with DEX. Mitochondrial biogenesis-mediated genes nuclear respiratory factor 1 (Nrf1) and Nrf2 were upregulated after 4-h incubation, and then declined after 24-h incubation, suggesting that mitochondrial biogenesis were transiently upregulated by DEX. In contrast, mitochondrial fusion gene optic atrophy 1 (Opa1) and mitofusin 2 (Mfn2) started to be elevated as the biogenesis started to decrease. Finally, the mitochondrial fission increased and apoptosis becomes prominent. Agree with the mitochondrial biphasic alterations hypothesis, the results suggested an early increase of mitochondrial activities and biogenesis upon DEX stimulation to the osteoblasts. The oxidative phosphorylation and inducible nitric oxide synthase levels increased results in oxidative stress accumulation, leading to mitochondrial fusion, and subsequently fission and triggering the apoptosis. Our results indicated that the primary effects of GC on mitochondria are promoting their functions and biogenesis. Mitochondrial breakdown and the activation of the apoptotic pathways appeared to be the secondary effect after long-term treatment.

Glucocorticoid-induced bone loss is the most common form of secondary osteoporosis. This toxic effect has not been efficiently managed, possibly due to the incomplete understanding of the extraordinarily diverse cellular responses induced by glucocorticoid treatment. Previous literatures revealed that high dose of exogenous glucocorticoid triggers apoptosis in osteocytes and osteoblasts. This cell death is associated with glucocorticoid-induced oxidative stress. In this study, we aimed to investigate the mechanisms of glucocorticoid-induced apoptosis in osteoblasts and examine the responses of osteoclasts to the synthetic glucocorticoid, dexamethasone. We demonstrated the biphasic effects of exogenous glucocorticoid on osteoblastic mitochondrial functions and elevated intracellular oxidative stress in a dose- and time-dependent manner. On comparison, similar treatment did not induce mitochondrial dysfunctions and oxidative stress in osteoclasts. The production of reactive oxygen/nitrogen species was decreased in osteoclasts. The differences are not due to varying efficiency of cellular antioxidant system. The opposite effects on nitrogen oxide synthase might provide an explanation, as the expression levels of nos2 gene are suppressed in the osteoclast but elevated in the osteoblast. We further revealed that glucocorticoids have a substantial impact on the osteoblastic mitochondria. Basal respiration rate and ATP production were increased upon 24 h incubation of glucocorticoids. The increase in proton leak and nonmitochondrial respiration suggests a potential source of glucocorticoid-induced oxidative stress. Long-term incubation of glucocorticoids accumulates these detrimental changes and results in cytochrome C release and mitochondrial breakdown, consequently leading to apoptosis in osteoblasts. The mitochondrial alterations might be other sources of glucocorticoid-induced oxidative stress in osteoblasts.

Objective: Type 1 diabetes is an autoimmune disease that destroys islet cells and results in insufficient insulin secretion by pancreatic β-cells. Islet transplantation from donors is an approach used for treating patients with diabetes; however, this therapy is difficult to implement because of the lack of donors. Nevertheless, several stem cells have the potential to differentiate from islet-like cells and enable insulin secretion for treating diabetes in animal models. For example, placenta is considered a waste material and can be harvested noninvasively during delivery without ethical or moral concerns. To date, the differentiation of islet-like cells from cow-derived placental stem cells (CPSCs) has yet to be demonstrated. Materials and methods: The investigation of potential differentiation of islet-like cells from CPSCs was conducted by supplementation with nicotinamide, exendin-4, glucose, and poly-d-lysine and was detected through reverse transcription polymerase chain reaction, dithizone staining, and immunocytochemical methods. Results: Our results indicated that CPSCs are established and express mesenchymal stem cell surface antigen markers, such as CD73, CD166, β-integrin, and Oct-4, but not hematopoietic stem cell surface antigen markers, such as CD45. After induction, the CPSCs successfully differentiated into islet-like cells. The CPSC-derived islet-like cells expressed islet cell development-related genes, such as insulin, glucagon, pax-4, Nkx6.1, pax-6, and Fox. Moreover, CPSC-derived islet-like cells can be stained with zinc ions, which are widely distributed in the islet cells and enable insulin secretion. Conclusion: Altogether, islet-like cells have the potential to be differentiated from CPSCs without gene manipulation, and can be used in diabetic animal models in the future for preclinical and drug testing trial investigations.

The purpose of this study was to investigate the effects of forskolin on body weight, glucose metabolism and fat cell diameter in high-fat diet-induced obese mice. Four-week-old male mice (C57BL/6) were randomly assigned to 1 of 3 treatment groups: a high-fat diet plus 5% dimethyl sulfoxide (vehicle), high-fat diet plus 2 mg/kg of forskolin (dissolved in 5% dimethyl sulfoxide) and high-fat diet plus 4 mg/kg of forskolin (dissolved in 5% dimethyl sulfoxide). Forskolin or dimethyl sulfoxide was administered intraperitoneally every two days. The results indicated that no significant difference was observed in the body weight, feed intake and serum lipid parameters among groups at 20 weeks of age. The blood glucose levels were significantly reduced in the groups treated with 2 mg/kg of forskolin before glucose tolerance test. Forskolin administration linearly decreased blood glucose levels of high-fat diet-fed mice at 90 min and total area under curve (AUC) after insulin tolerance test. The subcutaneous adipocyte diameter was significantly reduced in the groups treated with 2 mg/kg of forskolin. Forskolin administration linearly reduced the gonadal adipocyte diameter of high-fat diet-fed mice. Forskolin significantly reduced the differentiation of murine mesenchymal stem cells into adipocytes and this was accompanied by a decrease in intracellular triglyceride content and an increase in glycerol concentration in the culture medium. The subcutaneous adipocyte diameter, gonadal adipocyte diameter and total AUC of insulin tolerance test were moderately negatively correlated with the concentration of forskolin in the high-fat diet-induced obese model. These results demonstrate that forskolin can regulate glucose metabolism and reduce fat cell diameter of high-fat diet-fed mice and inhibit the adipocyte differentiation of murine mesenchymal stem cells.

Amniotic fluid-derived progenitor cells (AFPCs) are oligopotent and shed from the fetus into the amniotic fluid. It was reported that AFPCs express stem cell-like markers and are capable of differentiating into specific cell type in in vitro experiments. However, no study has fully investigated the potentiality and destiny of these cells in in vivo experiments. Methods: Ds-red transgenic mice (on Day 13.5 of pregnancy) were transplanted in utero with enhanced green fluorescent protein-labeled mouse AFPC (EGFP-mAFPCs). After birth, baby mice were euthanized at 3-week intervals beginning 3 weeks postnatally, and the specimens were examined by polymerase chain reaction, histology, and flow cytometry. Results: Our results demonstrate the transplantability of mAFPCs into all three germ layers and the potential of mAFPCs in the study of progenitor cell homing, differentiation, and function. Engraftment of EGFP-mAFPCs was detected in the intestine, kidney, muscle, skin, bladder, heart, stomach, etc., at 3 weeks after delivery. Conclusion: This model using EGFP-mAFPCs injected in utero may provide an ideal method for determining the fate of transplanted cells in recipients and these findings may justify a clinical trial of in utero transplantation during gestation for patients who have inherited genetic disorders.

Objective: Liver fibrosis results from the wound healing response to chronic liver damage. Advanced liver fibrosis results in cirrhosis and liver failure, and liver transplantation is often the only option for effective therapy; however, the shortage of available donor livers limits this treatment. Thus, new therapies for advanced liver fibrosis are essential. Materials and methods: Amniotic fluid contains an abundance of stem cells, which are derived from all three germ layers of the developing fetus. These cells do not induce teratomas in vivo and do not pose any ethical concerns. To generate liver fibrosis models, male ICR mice were treated with CCl4 via oral gavage for 4 weeks, and the serum levels of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and albumin were higher than in the control group following chemical induction. To assess the potential of amniotic-fluid-derived stem cells (mAFSCs) to ameliorate liver fibrosis in vivo, mAFSCs were isolated from amniotic fluid of 13.5-day-old transgenic mice, which globally express the fluorescent protein, enhanced green fluorescent protein (EGFP), for tracing purposes (EGFP-mAFSCs). Single cells were injected via the mesentery (1 × 106 cells/mouse) of transplanted mice with liver fibrosis. Results: Four weeks after EGFP-mAFSC transplantation, the serum glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and albumin levels of recipient mice in the EGFP-mAFSC-injected group were significantly decreased when compared with mice in the saline-injected group. Additionally, fibrotic tissues were evaluated using Masson's trichrome staining 4 weeks after cell transplantation. Shrinkage of the fibrotic area was observed in the EGFP-mAFSC-injected group. The tissue-repair effects were also confirmed by hydroxyproline content analysis. Conclusion: The possible repair mechanism from our data revealed that EGFP-mAFSCs may fuse with the recipient liver cells. Overall, EGFP-mAFSCs can ameliorate liver fibrosis in mice, thus providing insight into the future development of regenerative medicine.

Acute myocardial infarction (MI) is a fatal event that causes a large number of deaths worldwide. MI results in pathological remodeling and decreased cardiac function, which could lead to heart failure and fatal arrhythmia. Cell therapy is a potential strategy to repair the damage through enhanced angiogenesis or by modulation of the inflammatory process via paracrine signaling. Amniotic fluid-derived progenitor cells (AFPCs) have been reported to differentiate into several lineages and can be used without ethical concerns or risk of teratoma formation. Since pigs are anatomically, physiologically, and genetically similar to humans, and pregnant pigs can be an abundant source of AFPCs, we used porcine AFPCs (pAFPCs) as our target cells. Intramyocardial injection of AFPCs has been shown to cure MI in animal models. However, intramuscular transplantation of cells has not been extensively investigated. In this study, we investigated the therapeutic potential of intramuscular injection of pAFPCs on acute MI. MI mice were divided into 1) PBS control, 2) medium cell dose (1 × 10 6 cells per leg; cell-M), and 3) high cell dose (4 × 10 6 cells per leg; cell-H) groups. Cells or PBS were directly injected into the hamstring muscle 20 min after MI surgery. Four weeks after MI surgery, the cell-M and cell-H groups exhibited significantly better ejection fraction, significantly greater wall thickness, smaller infarct scar sizes, and lower LV expansion index compared to the PBS group. Using in vivo imaging, we showed that the hamstring muscles from animals in the cell-M and cell-H groups had RFP-positive signals. In summary, intramuscular injection of porcine AFPCs reduced scar size, reduced pathological remodeling, and preserved heart function after MI.

Pigs are an optimal animal for conducting biomedical research because of their anatomical and physiological resemblance to humans. In contrast to the abundant resources available in the study of mice, few fluorescent protein-harboring porcine models are available for preclinical studies. In this paper, we report the successful generation and characterization of a transgenic DsRed-Monomer porcine model.The transgene comprised a CMV enhancer/chicken-beta actin promoter and DsRed monomeric cDNA. Transgenic pigs were produced by using pronuclear microinjection. PCR and Southern blot analyses were applied for identification of the transgene. Histology, blood examinations and computed tomography were performed to study the health conditions. The pig amniotic fluid progenitor/stem cells were also isolated to examine the existence of red fluorescence and differentiation ability.Transgenic pigs were successfully generated and transmitted to offspring at a germ-line transmission rate of 43.59% (17/39). Ubiquitous expression of red fluorescence was detected in the brain, eye, tongue, heart, lung, liver, pancreas, spleen, stomach, small intestine, large intestine, kidney, testis, and muscle; this was confirmed by histology and western blot analyses. In addition, we confirmed the differentiation potential of amniotic fluid progenitor stem cells isolated from the transgenic pig.This red fluorescent pig can serve as a host for other fluorescent-labeled cells in order to study cell-microenvironment interactions, and can provide optimal red-fluorescent-labeled cells and tissues for research in developmental biology, regenerative medicine, and xenotransplantation.

Background: Goslings in several Taiwanese farms experienced gosling feather loss disease (GFL) at 21-35 days and goose broke feather disease (GBF) at 42-60 days. The prevalence ranges from a few birds to 500 cases per field. It is estimated that about 12,000 geese have been infected, the morbidity is 70-80% and the mortality is 20-30%. Objectives: This study aims to investigate the pathogens that cause GFL and GBF. Focus on the study of the correlation between goose circovirus (GoCV) and goose parvovirus (GPV) with the goose feather loss in southern Taiwan. Furthermore, a phylogenetic tree was established to align the differences between southern and northern Taiwan and compare with virus strains from China and Europe. Methods: Samples were collected from animal hospitals. Molecular and microscopy diagnostics were used to examine 92 geese. Specific quantitative polymerase chain reaction (Q-PCR) assays are performed to evaluate GPV and GoCV viral loads and simultaneously evaluated the feather loss conditions in geese with the scoring method. Results: High prevalence of GoCV and GPV infection in geese showing signs of GFL and GBF. Inclusion body was detected in the feather follicles and Lieberkühn crypt epithelial cells. The Q-PCR showed the high correlation between feather loss and viruses during 3rd-5th week. However, the infection was not detected using the same test in 60 healthy geese. Conclusions: Thus, GFL and GBF appear to be significantly closely related to GoCV and GPV. The geese feathers showed increasing recovery after being quarantined and disinfected. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to investigate whether goose growth and feather characteristics are influenced by their line and feeding surroundings, inclusive of floor materials and types, since there are no reports regarding these factors. Methods: The 240 White Roman geese which were hatched and sex identified came from 3 commercial goose farms. They were randomly distributed to 24 pens depending on a completely random design. The study continued for 13 weeks and included 3 lines of commercial geese and 2 floor types (cement strip floor [CSF] or cement floor [CF]). Results: The day one gosling weight from A farm was lower than other two farms (96 g vs 107 and 115 g; p<0.001). Afterwards, the body weight, back length, keel length, chest girth and main wing feather length among 3 farms showed no significance difference prior to 12 weeks. The CF group showed heavier body weight, shorter back length, longer keel length, shorter chest girth and shorter main wing feather length than the CSF group prior to 12 weeks. The down weight in the CF was heavier than the CSF group (57.1 g vs 41.8 g; p<0.01) prior to 13 weeks. Conclusion: The body weight showed the positive relations for dry feather weight (r = 0.59), down weight (r = 0.69), percent of the down weight of live body weight prior to 13 weeks (r = 0.61). [ABSTRACT FROM AUTHOR]

The decreasing of yogurt drink quality will occur during storage. Stabilizer that usually used for maintaining the yogurt quality is a chemical compound namely carboxymethyl cellulose (CMC). Canna (Canna edulis Ker) potentially used as a natural stabilizer, which is a local Indonesian tuber with high starch content and antioxidative properties. The purpose of this study is to investigate the capability of Canna starch to replace the used of CMC based on the physicochemical and antioxidant activity of yogurt drink during cold storage. The complete randomized factorial design will be used with two factor. The first factor was Canna starch/CMC levels divided into 5 groups, T0 (0.2%CMC) as a control, T1 (0.15% CMC + 0.025% canna), T2 (0.1% CMC + 0.05% canna), T3 (0.05% CMC + 0.075% canna), and T4 (0.1% Canna), respectively. The second factor was the storage time of yogurt drink divided into four groups, 1, 7, 14 and 21 days, to determine the quality change during stored in the refrigerator at 4°C. Data obtained from the results of subsequent studies analyzed by GLM (General Linear Model) and followed by Duncan's multiple range test (DMRT). This study resulted that Canna starch has the capability as a natural stabilizer for producing functional yogurt drink with potential health benefits related to the high antioxidant activity. The combination of 0.1% CMC and 0.05% Canna starch addition on yogurt drink manufacture showed the best physicochemical quality