Azolla leaf meal at 5% of the diet improves growth performance, intestinal morphology and p70S6K1 activation, and affects cecal microbiota in broiler chicken.

With growing concern about including unconventional dietary protein sources in poultry diets to substitute the protein sources that are essential for human consumption such as soybean meal, Azolla leaf meal (ALM) has grown in popularity. In our prior experiment, ALM was used at inclusion rates of 5 and 10%. Five per cent inclusion of ALM increased broiler chicken growth performance, the concentration of cecal propionic acid, and activation of skeletal muscle p70S6 Kinase1 (p70S6K1) without having detrimental effects on the meat quality. Those results prompted us to further evaluate the effect of the same inclusion rates of ALM on phase feeding and intestine and liver health of the broiler chicks. The current study hypothesis is that dietary ALM positively affects phase feeding, intestinal morphology and p70S6K1 activation, cecal microbial gene expression, and improves the liver energy status. For this, we enrolled 135 one-day-old broiler chicks and collected growth performance data (starter, grower, and finisher stages) and samples of the gastrointestinal tract to analyse the morphology of the villi, immune-related organs, mucin, and abundance of intestinal p70S6K1. Cecal bacterial species were analysed using qPCR and liver samples were collected to analyse adenosine monophosphate (AMP) and ATP content and selected oxidative stress biomarkers. ALM increased BW and feed intake during the starter and grower phases but did not affect the feed conversion ratio. Liver oxidative stress and AMP: ATP ratio increased in chickens fed on a diet containing 10% ALM (AZ10; P < 0.05). Jejunum villi length and abundance of duodenal neutral mucin increased but villi of the ileum decreased in chickens fed on a diet containing 5% ALM (AZ5), while lymphoid follicle areas of the cecal tonsils decreased with both doses of ALM. Activation of p70S6K1 increased with AZ10 in the duodenum and AZ5 in the jejunum. In the gut, the family of Enterobacteriaceae decreased with both ALM doses. In conclusion, our results indicate an overall positive effect of dietary inclusion of ALM in the broiler chicken diet via its positive effect on intestinal morphology and function; however, a negative effect on the liver was observed with 10% ALM.

Short communication: Molecular markers for epithelial cells across gastrointestinal tissues and fecal RNA in preweaning dairy calves.

The objective of this study was to compare the transcription of gene markers for gastrointestinal (GI) epithelial cells, including fatty acid binding protein 2 (FABP2) and cytokeratin 8 (KRT8), and tight junction complex genes (TJP1, CLDN1, CLDN4) in fecal RNA against several GI tract tissue sections in dairy calves. Eight healthy Jersey calves were euthanized at 5 wk of age, and postmortem samples were collected from rumen, duodenum, jejunum, ileum, large intestine, cecum, and feces for total RNA isolation. Tissues and fecal samples were immediately frozen in liquid nitrogen until RNA isolation. A real-time quantitative PCR analysis was performed using a single standard curve composited of equal amounts of all samples, including cDNA from fecal and GI tract tissues. The mRNA expression of the tight junctions TJP1, CLDN1, and CLDN4 was greater in fecal RNA compared with lower GI tract tissues (i.e., duodenum, jejunum, ileum, large intestine, and cecum). Similar to fecal RNA, rumen tissue had greater expression of tight junctions CLDN1 and CLDN4 than lower GI tract tissues. Similarly, rumen tissue had greater expression of TPJ1 than all lower GI tract tissues except duodenum. The expression of TJP1 and CLDN4 was greater in fecal RNA than in rumen tissue; in contrast, CLDN1 mRNA expression was greater in rumen tissue than in the fecal RNA. The expression of FABP2 was greater in duodenum in comparison to all tissue except ileum. The mRNA expression of FABP2 in fecal samples was similar to jejunum and ileum. The expression of KRT8 in fecal samples was similar to duodenum, large intestine, and cecum. The fecal RNA had a greater expression of KRT8 in comparison to jejunum and ileum. The rumen tissue had the lowest mRNA expression of KRT8. The expression levels of FABP2, KRT8, and tight junction genes observed in fecal transcripts suggest that a considerable amount of RNA derived from GI tract epithelial cells can be detected in fecal RNA, which is in agreement with previous data in neonatal dairy calves and other biological models including humans, rodents, and primates. The greater expression of tight junctions in fecal RNA in comparison to sections of the low GI remains to be understood, and due to the importance of tight junctions in GI physiology, further clarification of this effect is warranted. The similarities in mRNA expression of FABP2 and KRT8 between fecal RNA and intestinal sections add up to the accumulating evidence that fecal RNA can be used to investigate molecular alterations in the GI tract of neonatal dairy calves. Further research in this area should include high-throughput transcriptomic analysis via RNA-seq to uncover novel molecular markers for specific sections of the GI tract of neonates.

Long term conjugated linoleic acid supplementation modestly improved growth performance but induced testicular tissue apoptosis and reduced sperm quality in male rabbit.

Conjugated linoleic acid (CLA) is known for its multiple benefits including improvement of growth, increasing lean mass, and anti-carcinogenic effects. However, when used in long-term supplementations CLA does not improve semen parameters in boar and bull and reduces fertility in Japanese quails. The content of unsaturated fatty acids in dietary lipids plays a significant role in spermatogenesis owning the high proportion of unsaturated fatty acids in plasma membrane of sperms. Whether CLA plays a role in testicular tissue and epididymal fat is still unknown. Therefore, in this study we hypothesize that long-term supplementation of equal proportion of CLA isomer mix (c9,t11-CLA and t10,c12- CLA) in rabbit bucks might alter male reproductive potentials. Twelve V-Line weaned male rabbits were used in 26 weeks trial, rabbits were individually raised and randomly allocated into three dietary groups. Control group (CON) received a basal diet, a group received 0.5% CLA (CLA 0.5%), and a group received 1% CLA (CLA 1%). Rabbits were euthanized at the end of the trial and several parameters were evaluated related to growth, semen quality, and testicular and epididymal tissue histopathology and transcriptome. The long-term supplementation of CLA increased feed intake by 5% and body weight by 2-3%. CLA 1% decreased sperm progressive motility. In testicular tissue L-carnitine and α-tocopherol were decreased by CLA supplementation. In epididymal fat, CLA tended to decrease concentration of polyunsaturated fatty acids, the expression of SCD5 gene was upregulated by CLA 1% and CASP3 gene was upregulated by CLA 0.5%. Transcription of PPARG was downregulated by CLA. Feeding 1% CLA also decreased testicular epithelial thickness. Long-term supplementation of CLA modestly enhanced male rabbit growth, but negatively impacted male reproduction, especially at high dose of CLA.

Heat stress negatively affects the transcriptome related to overall metabolism and milk protein synthesis in mammary tissue of midlactating dairy cows.

Inadequate dry matter intake only partially accounts for the decrease in milk protein synthesis during heat stress (HS) in dairy cows. Our hypothesis is that reduced milk protein synthesis during HS in dairy cows is also caused by biological changes within the mammary gland. The objective of this study was to assess the hypothesis via RNA-Seq analysis of mammary tissue. Herein, four dairy cows were used in a crossover design where HS was induced for 9 days in environmental chambers. There was a 30-day washout between periods. Mammary tissue was collected via biopsy at the end of each environmental period (HS or pair-fed and thermal neutral) for transcriptomic analysis. RNA-Seq analysis revealed HS affected >2,777 genes (false discovery rate-adjusted P value < 0.05) in mammary tissue. Expression of main milk protein-encoding genes and several key genes related to regulation of protein synthesis and amino acid and glucose transport were downregulated by HS. Bioinformatics analysis revealed an overall decrease of mammary tissue metabolic activity by HS (especially carbohydrate and lipid metabolism) and an increase in immune activation and inflammation. Network analysis revealed a major role of TNF, IFNG, S100A8, S100A9, and IGF-1 in inducing/controlling the inflammatory response, with a central role of NF-κB in the process of immunoactivation. The same analysis indicated an overall inhibition of PPARγ. Collectively, these data suggest HS directly controls milk protein synthesis via reducing the transcription of metabolic-related genes and increasing inflammation-related genes.

Activation of liver X receptor promotes fatty acid synthesis in goat mammary epithelial cells via modulation of SREBP1 expression.

In bovine mammary tissue and cells, liver X receptor (LXR) regulates lipid synthesis mainly via transactivation of the transcription factor sterol regulatory element binding protein 1 (SREBP1). In the present work, we investigated the role of LXR in controlling lipid synthesis via transactivation of SREBP1 in goat primary mammary cells (GMEC). The GMEC were treated with a synthetic agonist of LXR, T0901317, and transactivation and transcription of SREBP1, expression of lipogenic genes, and fatty acid profiling and triacylglycerol (TAG) content of the cells were measured. A mild increase in the mRNA expression level of LXRα (NR1H3) was observed following treatment with different concentrations of T0901317, and a dose-dependent increase in mRNA and transactivation of SREBP1 was detected. Activation of LXR resulted in a significant increase in the mRNA expression of most of the measured genes related to de novo synthesis, desaturation, and transport of fatty acids; TAG synthesis; and transcription regulators. Compared with the control, total content of cellular TAG increased by more than 20% with T0901317 treatment. Furthermore, addition of T0901317 increased the proportion of unsaturated fatty acids (e.g., C16:1, C18:1, C20:1, and C22:1), and decreased the proportion of saturated fatty acids (e.g., C16:0, C18:0, C20:0, and C22:0). These results provide evidence that LXR regulates the expression and activity of SREBP1. Our results indicated that LXR participate in regulating the transcription of genes involved in milk fat synthesis in GMEC in an SREBP1-dependent fashion.

Overexpression of SREBP1 (sterol regulatory element binding protein 1) promotes de novo fatty acid synthesis and triacylglycerol accumulation in goat mammary epithelial cells.

Sterol regulatory element binding protein 1 (SREBP1; gene name SREBF1) is known to be the master regulator of lipid homeostasis in mammals, including milk fat synthesis. The major role of SREBP1 in controlling milk fat synthesis has been demonstrated in bovine mammary epithelial cells. Except for a demonstrated role in controlling the expression of FASN, a regulatory role of SREBP1 on milk fat synthesis is very likely, but has not yet been demonstrated in goat mammary epithelial cells (GMEC). To explore the regulatory function of SREBP1 on de novo fatty acids and triacylglycerol synthesis in GMEC, we overexpressed the mature form of SREBP1 (active NH2-terminal fragment) in GMEC using a recombinant adenovirus vector (Ad-nSREBP1), with Ad-GFP (recombinant adenovirus of green fluorescent protein) as control, and infected the GMEC for 48 h. In infected cells, we assessed the expression of 20 genes related to milk fat synthesis using real time-quantitative PCR, the protein abundance of SREBP1 and FASN by Western blot, the production of triacylglycerol, and the fatty acid profile. Expression of SREBF1 was modest in mammary compared with the other tissues in dairy goats but its expression increased approximately 30-fold from pregnancy to lactation. The overexpression of the mature form of SREBP1 was confirmed by >200-fold higher expression of SREBF1 in Ad-nSREBP1 compared with Ad-GFP. We observed no changes in amount of the precursor form of SREBP1 protein but a >10-fold increase of the mature form of SREBP1 protein with Ad-nSREBP1. Compared with Ad-GFP cells (control), Ad-nSREBP1 cells had a significant increase in expression of genes related to long-chain fatty acid activation (ACSL1), transport (FABP3), desaturation (SCD1), de novo synthesis of fatty acids (ACSS2, ACLY, IDH1, ACACA, FASN, and ELOVL6), and transcriptional factors (NR1H3 and PPARG). We observed a >10-fold increase in expression of INSIG1 but SCAP was downregulated by Ad-nSREBP1. Among genes related to milk fat synthesis and lipid droplet formation, only LPIN1 and DGAT1 were upregulated by Ad-nSREBP1. Compared with the Ad-GFP, the cellular triacylglycerol content was higher and the percentage of C16:0 and C18:1 increased, whereas that of C16:1, C18:0, and C18:2 decreased in Ad-nSREBP1 cells. Overall, the data provide strong support for a central role of SREBP1 in the regulation of milk fat synthesis in goat mammary cells.

Strategies for regeneration of the bone using porcine adult adipose-derived mesenchymal stem cells.

Bone is a plastic tissue with a large healing capability. However, extensive bone loss due to disease or trauma requires tissue-engineering applications. Presently, bone grafting is the gold standard for bone repair, but presents serious limitations including donor site morbidity, rejection, and limited tissue regeneration. The use of stem cells appears to be a means to overcome such limitations. Bone marrow mesenchymal stem cells (BMSC) have been the choice, thus far, for stem cell therapy for bone regeneration. However, it has been shown that adipose-derived stem cells (ASC) have similar immunophenotype, morphology, multilineage potential, and transcriptome compared to BMSC. Moreover, ASC are much more abundant, more accessible and have lower donor morbidity, which combined may make ASC a better alternative to BMSC. ASC are also able to migrate to the site of injury and have immunosuppressive abilities similar to BMSC. Further, ASC have demonstrated extensive osteogenic capacity both in vitro and in vivo in several species, greatly enhancing the healing of critical size defects. The use of scaffolds in combination with ASC and growth factors provides a valuable tool for guided bone regeneration, especially for complex anatomic defects. Some critical elements include ASC-scaffold interactions and appropriate three-dimensional design of the porous osteoinductive structures. This review examines data that provides strong support for the clinical translation of ASC for bone regeneration.

Effects of the peroxisome proliferator-activated receptor-alpha agonists clofibrate and fish oil on hepatic fatty acid metabolism in weaned dairy calves.

Peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists increase fatty acid oxidation in liver of nonruminants. If similar effects occur in dairy cattle, enhanced hepatic oxidative capacity could decrease circulating nonesterified fatty acids and hepatic triacylglycerol accumulation in periparturient cows. The objectives of this study were 1) to determine whether partitioning of fatty acid metabolism by liver slices from weaned Holstein calves treated with PPARalpha agonists in vivo is altered compared with partitioning by liver slices from control (untreated) calves, and 2) to measure in vitro metabolism of palmitate and oleate by bovine liver slices and relate these to mRNA abundance for key enzymes. Weaned male Holstein calves (7 wk old; n=15) were assigned to 1 of 3 groups for a 5-d treatment period: control (untreated), clofibrate (62.5 mg/kg of BW), or fish oil (250 mg/kg of BW). Calves treated with clofibrate consumed less dry matter. Body weight, liver weight, liver weight:body weight ratio, blood nonesterified fatty acids, beta-hydroxybutyrate, and liver composition were not significantly different among treatments. Liver slices were incubated for 2, 4, and 8 h to determine in vitro conversion of [1-(14)C] palmitate and [1-(14)C] oleate to CO(2), acid-soluble products, esterified products, and total metabolism. In liver slices incubated for 8 h, conversion of palmitate to CO(2) was greater for calves treated with clofibrate compared with control calves or calves treated with fish oil. Conversion of palmitate to esterified products, total palmitate metabolism, and metabolism of oleate were not different among treatments. Conversion of palmitate to CO(2) was greater than that from oleate for all treatments, but rates of total metabolism did not differ. Clofibrate increased or tended to increase liver expression of several PPARalpha target genes involved in fatty acid oxidation (e.g., ACADVL, ACOX1, CPT1A), whereas fish oil did not significantly affect genes associated with fatty acid oxidation but tended to increase DGAT1. Overall, our data indicated that bovine liver responded to clofibrate treatment but not fish oil, although increases in hepatic lipid metabolism were much less than those reported in rodents treated with clofibrate or fish oil. Applications of PPARalpha agonists may be of interest to increase the rate of hepatic fatty acid oxidation and decrease triacylglycerol accumulation in periparturient dairy cows.

Adipose tissue depots of Holstein cows are immune responsive: inflammatory gene expression in vitro.

The transcriptional response of adipose tissue depots with respect to their immune responsiveness in dairy cows remains largely unknown. Thus, we examined mRNA expression and responsiveness of subcutaneous (SUB) and mesenteric (MES) adipose tissue from nonpregnant dairy cows to a short-term (2 h), in vitro lipopolysaccharide (LPS) challenge (20 microg/mL in physiological saline). Abundance of mRNA for tumor necrosis factor-alpha (TNFA), interleukin-6 (IL6), serum amyloid A3 (SAA3), toll-like receptor 4 (TLR4), monocyte chemoattractant protein-1 (CCL2), and RANTES/chemokine C-C motif ligand 5 (CCL5) were analyzed using quantitative polymerase chain reaction (PCR) from tissue samples collected at slaughter from 5 nonpregnant/nonlactating Holstein cows. Prior to LPS challenge, SAA3 mRNA abundance was greater in MES than SUB tissue. Regardless of depot site, LPS led to greater mRNA abundance of TNFA and IL6 and was more pronounced for IL6 in MES. We also observed a marked increased in expression of CCL2, CCL5, TLR4, IL6, and TNFA in both MES and SUB during the 2-h incubation with saline alone (ie, the control). Because mRNA expression of the apoptotic markers B-cell CLL/lymphoma 2 (BCL2) and tumor protein p53 (TP53) did not differ during the 2-h incubation, it is less likely that the response to saline was a result of increased rate of cell death during incubation. Analysis using semiquantitative PCR of the 16s rRNA gene in cDNA from tissue explants revealed the presence of bacteria likely arising from contamination during sample collection. Furthermore, surfactant medium from about 50% of explant cultures had viable aerobic bacteria without differences between treatments or tissue samples. Thus, the presence of bacteria could partly explain the large increase in inflammatory-related genes after 2-h incubation with saline. The higher SAA3 expression in MES suggests that this acute-phase protein has a role in lipid metabolism and/or transport during an immune challenge. Overall, results provided evidence that adipose depots of dairy cows are capable of synthesizing chemokines and are immune responsive when exposed to inflammatory conditions that can arise from a pathogenic insult or during and soon after parturition.

Peroxisome proliferator-activated receptor-gamma activation and long-chain fatty acids alter lipogenic gene networks in bovine mammary epithelial cells to various extents.

Several long-chain fatty acids (LCFA) are natural ligands of nonruminant peroxisome proliferator-activated receptor-gamma (PPARG), which, along with its lipogenic target genes, is upregulated in bovine mammary tissue during lactation. Thus, PPARG might represent an important control point of bovine milk fat synthesis. We tested lipogenic gene network expression via quantitative PCR of 19 genes in bovine mammary epithelial cells cultured with 16:0, 18:0, cis-9 18:1, trans-10 18:1, trans-10,cis-12 18:2 [t10c12 conjugated linoleic acid (CLA)], 20:5, ethanol (control), and the PPARG agonist rosiglitazone (ROSI). Triplicate cultures were maintained for 12 h with 50 muM ROSI or 100 muM LCFA. Responses common to 16:0 and 18:0 relative to the control included significantly greater expression of INSIG1 (+298%, +92%), AGPAT6 (+137%, +169%), FABP3 (+755%, +338%), and FABP4 (+171%, 157%). These were coupled with greater intracellular lipid droplet formation and mRNA of ACSS2, LPIN1, SCD, and SREBF2 in response to 16:0, and greater DGAT1 and THRSP with 18:0. Trans-10 18:1 and t10c12 CLA reduced expression of FASN (-60%, -31%), SCD (-100%, -357%), and SREBF1 (-49%, -189%). Furthermore, t10c12 CLA downregulated ACSS2, FABP3, INSIG1, SREBF2, and THRSP expression. Expression of SREBF1 was lower with cis-9 18:1 (-140%) and 20:5 (-125%) compared with the control. This latter LCFA also decreased SCD, SREBF2, and LPL expression. No effects of LCFA or ROSI on PPARG were observed, but ROSI upregulated (+39% to +269%) expression of ACACA, FASN, LPIN1, AGPAT6, DGAT1, SREBF1, SREBF2, and INSIG1. Thus, these genes are putative PPARG target genes in bovine mammary cells. This is the first report showing a direct effect of trans-10 18:1 on bovine mammary cell lipogenic gene expression. The coordinated upregulation of lipogenic gene networks in response to ROSI and saturated LCFA offers support for PPARG activation in regulating bovine milk fat synthesis.