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ABSTRACT: The legalization of industrial hemp by the 2018 Farm Bill in the United States has driven a sharp increase in its cultivation, including for cannabinoid extraction. Spent hemp biomass (SHB), produced from the extraction of cannabinoids, can potentially be used as feed for dairy cows; however, it is still illegal to do so in the United States, according to the US Food and Drug Administration Center for Veterinary Medicine, due to the presence of cannabinoids and the lack of data on the effect on animals. To assess the safety of this byproduct as feed for dairy cows, late-lactation Jersey cows (245 ± 37 d in milk; 483 ± 38 kg body weight; 10 multiparous and 8 primiparous) received a basal total mixed ration (TMR) diet plus 13% alfalfa pellet (CON) or 13% pelleted SHB for 4 wk (intervention period [IP]) followed by 4 wk of withdrawal period (WP), where all cows received only the basal TMR during WP. The dry matter intake (DMI), body weight, body condition score, milk yield, milk components, and fatty acid profile, blood parameters, N metabolism, methane emission, and activity were measured. Results indicated that feeding SHB decreased DMI mainly due to the low palatability of the SHB pellet, as the cows consumed only 7.4% of the total TMR with 13.0% SHB pellet offered in the ration. However, milk yield was not affected during the IP and was higher than CON during the WP, leading to higher milk yield/DMI. Milk components were not affected, except for a tendency in decreased fat percentage. Milk fat produced by cows fed SHB had a higher proportion of oleate and bacteria-derived fatty acids than CON. The activity of the cows was not affected, except for a shorter overall lying time in SHB versus CON cows during the IP. Blood parameters related to immune function were not affected. Compared with CON, cows fed SHB had a lower cholesterol concentration during the whole experiment and higher β-hydroxybutyric acid during the WP, while a likely low-grade inflammation during the IP was indicated by higher ceruloplasmin and reactive oxidative metabolites. Other parameters related to liver health and inflammatory response were unaffected, except for a tendency for higher activity of alkaline phosphatase during IP and a lower activity of gamma-glutamyl transferase during WP in the SHB group versus CON. The bilirubin concentration was increased in cows fed SHB, suggesting a possible decrease in the clearance ability of the liver. Digestibility of the dry matter and protein and methane emission were not affected by feeding SHB. The urea, purine derivatives, and creatinine concentration in urine was unaffected, but cows fed SHB had higher N use efficiency and lower urine volume. Altogether, our data revealed a relatively low palatability of SHB affecting DMI with minimal biological effects, except for a likely low-grade inflammation, a higher N use efficiency, and a possible decrease in liver clearance. Overall, the data support the use of SHB as a safe feed ingredient for lactating dairy cows.

The inclusion of spent hemp biomass (SHB), an extracted byproduct from industrial cannabidiol (CBD) production, in the diets of dairy cows and lambs appears to be safe with minor effects on the metabolism, including a decrease in circulating cholesterol and increase bilirubinemia, both associated with liver metabolism. Those effects could be consequence of the presence of cannabinoids, particularly Δ9-tetrahydrocannabinol (THC) and CBD in the SHB. This study aimed to study the transcriptional profile of the liver of dairy cows and lambs fed SHB. Dairy cows received SHB or alfalfa pellet for four weeks of intervention (IP) and four weeks of withdrawal periods (WP). Finishing lambs were fed a control diet (CON), 10% (LH2), or 20% (HH2) SHB for 2 months or 1 month followed by 1-month SHB withdrawal (LH1 and HH1, respectively). RNA sequencing was performed, and the mRNA was annotated using the latest reference genomes. The RNAseq data were filtered, normalized for library size and composition, and statistically analyzed by DESeq2. The bioinformatic analysis was performed by using DAVID, Gene Set Enrichment Analysis (GSEA), and the Dynamic Impact Approach. Using a 0.2 FDR cut-off, we identified only ≤24 differentially expressed genes (DEG) in the liver by feeding SHB in dairy cows and a larger number of DEGs in lambs (from 71 in HH1 vs. CON to 552 in LH1 vs. CON). The KEGG analysis demonstrated that feeding SHB in dairy cows and lambs had relatively minor to moderate metabolic alterations in dairy cows and lambs mainly associated with amino acids and lipid metabolism whereas cholesterol synthesis was overall activated in lambs. GSEA identified activation of the PPAR signaling pathway only in dairy cows. We found an opposite effect on activation of metabolism of drug and xenobiotics by cytochrome P450 enzymes in dairy cows and lambs receiving less SHB but an inhibition in HH2 lambs. Immune system-related pathways were inhibited by feeding SHB in lambs, but the impact was minor. Cumulatively, inclusion of SHB containing cannabinoids in dairy and lambs demonstrate very little effects on the alteration of transcriptomic profile of the liver.

Changes during the production cycle of dairy cattle can leave these animals susceptible to oxidative stress and reduced antioxidant health. In particular, the periparturient period, when dairy cows must rapidly adapt to the sudden metabolic demands of lactation, is a period when the production of damaging free radicals can overwhelm the natural antioxidant systems, potentially leading to tissue damage and reduced milk production. Central to the protection against free radical damage and antioxidant defense is the transcription factor NRF2, which activates an array of genes associated with antioxidant functions and cell survival. The objective of this study was to evaluate the effect that two natural NRF2 modulators, the NRF2 agonist sulforaphane (SFN) and the antagonist brusatol (BRU), have on the transcriptome of immortalized bovine mammary alveolar cells (MACT) using both the RT-qPCR of putative NRF2 target genes, as well as RNA sequencing approaches. The treatment of cells with SFN resulted in the activation of many putative NRF2 target genes and the upregulation of genes associated with pathways involved in cell survival, metabolism, and antioxidant function while suppressing the expression of genes related to cellular senescence and DNA repair. In contrast, the treatment of cells with BRU resulted in the upregulation of genes associated with inflammation, cellular stress, and apoptosis while suppressing the transcription of genes involved in various metabolic processes. The analysis also revealed several novel putative NRF2 target genes in bovine. In conclusion, these data indicate that the treatment of cells with SFN and BRU may be effective at modulating the NRF2 transcriptional network, but additional effects associated with cellular stress and metabolism may complicate the effectiveness of these compounds to improve antioxidant health in dairy cattle via nutrigenomic approaches.

ABSTRACT: Flaxseed is a rich source of α-linolenic acid (ALA, 18:3 n-3) and can be used to enrich chicken tissues with n-3 fatty acids (FA). However, antinutritional factors in flaxseed compromise the live performance of birds coupled with increased oxidative stress. Chromium (Cr) is a trace element with antioxidant properties. It is hypothesized that Cr supplementation will affect the hepatic total lipid profile, phospholipid n-3 and n-6 FA molecular species, lipid oxidation products, and transcription of genes associated with lipid metabolism in broiler chickens fed flaxseed. Ninety (n = 90), day-old Cornish cross chicks were fed a corn-soybean meal-based diet containing 0% flaxseed (CTR), 10% flaxseed (FLAX), and FLAX + 0.05% organic Cr (FLAXCr) for 42 d. The chicks were kept in 18 pens with 5 chicks per pen. For all response variables, the effect of dietary treatments were compared separately using SAS 9.4. P values were considered significant at ≤0.05. Total lipids, saturated FA, long-chain (≥20C) n-6 FA were reduced while total n-3 FA and long-chain n-3 FA were higher in the liver of FLAX and FLAXCr than CTR (P < 0.05). Hepatic phosphatidylcholine (PC) and phosphatidylethnolamine (PE) n-3 species (36:5, 38:6) were higher in FLAX and FLAXCr compared to CTR (P < 0.05). On the contrary, n-6 species in PC (36:4, 38:4) and PE (38:4) were lower in FLAX and FLAXCr compared to CTR (P < 0.05). Addition of Cr to a flaxseed-containing diet led to an increase in PE 36:4 (P < 0.05). A decrease in the transcription of ELOVL6 gene involved in de novo lipid synthesis was observed in FLAXCr (P = 0.01). An increase in the transcription of genes involved in FA oxidation (ACAA2, ACOX1) was observed in FLAX compared to FLAXCr (P = 0. 05; P = 0.02). A trend for a decrease in the transcription of FADS2 and HMGCS1 was observed in FLAXCr than CTR and FLAX (P = 0.06; 0.08). Transcription of other genes involved in de novo lipid synthesis (FASN, PPARA), FA oxidation (CPT1A, CPT2, ACAA1), and oxidative stress response (GPX1, NQO11, GSTA2, SLC40A1, NFE2L2) were not affected by the diets (P > 0.05). Lipid peroxidation products measured as thiobarbituric acid reactive substances (TBARS) in liver was reduced in FLAXCr than CTR (P < 0.05) and was not different from FLAX (P > 0.05). Serum cholesterol and aspartic aminotransferase were reduced in FLAX and FLAXCr compared to CTR (P < 0.05). The serum glucose level was decreased in FLAX compared to CTR (P < 0.05) and a trend in decrease was noticed in FLAXCr vs. CTR (P = 0.10). Serum TBARS were higher in CTR and FLAXCr compared to FLAX (P < 0.05). In conclusion, flaxseed supplementation enhances total and long-chain n-3 FA while reducing total lipids, saturated, and n-6 FA in the liver. Supplementing Cr along with flaxseed increased n-6 FA species in the hepatic PE and decreased the transcription of genes involved in FA oxidation and lipid synthesis.

Metabolic challenges experienced by dairy cows during the transition between pregnancy and lactation (also known as peripartum), are of considerable interest from a nutrigenomic perspective. The mobilization of large amounts of non-esterified fatty acids (NEFA) leads to an increase in NEFA uptake in the liver, the excess of which can cause hepatic accumulation of lipids and ultimately fatty liver. Interestingly, peripartum NEFA activate the Peroxisome Proliferator-activated Receptor (PPAR), a transcriptional regulator with known nutrigenomic properties. The study of PPAR activation in the liver of periparturient dairy cows is thus crucial; however, current in vitro models of the bovine liver are inadequate, and the isolation of primary hepatocytes is time consuming, resource intensive, and prone to errors, with the resulting cells losing characteristic phenotypical traits within hours. The objective of the current study was to evaluate the use of precision-cut liver slices (PCLS) from liver biopsies as a model for PPAR activation in periparturient dairy cows. Three primiparous Jersey cows were enrolled in the experiment, and PCLS from each were prepared prepartum (−8.0 ± 3.6 DIM) and postpartum (+7.7± 1.2 DIM) and treated independently with a variety of PPAR agonists and antagonists: the PPARα agonist WY-14643 and antagonist GW-6471; the PPARδ agonist GW-50156 and antagonist GSK-3787; and the PPARγ agonist rosiglitazone and antagonist GW-9662. Gene expression was assayed through RT-qPCR and RNAseq, and intracellular triacylglycerol (TAG) concentration was measured. PCLS obtained from postpartum cows and treated with a PPARγ agonist displayed upregulation of ACADVL and LIPC while those treated with PPARδ agonist had increased expression of LIPC, PPARD, and PDK4. In PCLS from prepartum cows, transcription of LIPC was increased by all PPAR agonists and NEFA. TAG concentration tended to be larger in tissue slices treated with PPARδ agonist compared to CTR. Use of PPAR isotype-specific antagonists in PCLS cultivated in autologous blood serum failed to decrease expression of PPAR targets, except for PDK4, which was confirmed to be a PPARδ target. Transcriptome sequencing revealed considerable differences in response to PPAR agonists at a false discovery rate-adjusted p-value of 0.2, with the most notable effects exerted by the PPARδ and PPARγ agonists. Differentially expressed genes were mainly related to pathways involved with lipid metabolism and the immune response. Among differentially expressed genes, a subset of 91 genes were identified as novel putative PPAR targets in the bovine liver, by cross-referencing our results with a publicly available dataset of predicted PPAR target genes, and supplementing our findings with prior literature. Our results provide important insights on the use of PCLS as a model for assaying PPAR activation in the periparturient dairy cow.

Coprophagy is an instinctive behavior in rabbit with important effects on growth and reproductive performance. The underlying mechanism of this effect in rabbit is unknown. Here, we used Elizabeth circle as a coprophagy preventing model in female rabbits and assess feed intake, growth, and reproductive performance. We found that preventing coprophagy did not affect feed intake but decreased body weight and weight of several organs and tissues and resulted in complete reproductive failure during the late pregnancy period, accompanied by reduced levels of plasma progesterone. RNA-seq analysis of rabbit ovarian tissues revealed that preventing coprophagy affected significantly 241 genes (DEGs), with the large majority being downregulated. Bioinformatic analyses revealed that those DEGs are mostly involved in apoptosis, immune response, and metabolic pathways. Among DEGs, the lysosomal cysteine protease cathepsin B (CTSB) was significantly downregulated in the coprophagy prevention group. Further studies using siRNA and adenovirus overexpression systems revealed that CTSB promotes the proliferation of rabbit granulosa cells (GCS) and prevents apoptosis. Measurement of transcripts coding for proteins related to apoptosis revealed a minor transcriptomic effect of CTSB, indicating that its effect is likely post-transcriptional. Overexpression of CTSB increased secretion of progesterone and estradiol, partly via upregulation of CYP19A1 while inhibition of CTSB decreased progesterone secretion partly via downregulation of the StAR gene. In conclusion, our study demonstrated the detrimental effect on reproduction by preventing coprophagy with a main role for this response played by CTSB on the granulosa cells of the ovary.

The objective of this study was to assess the transcriptome of the mammary tissue of four yaks during the whole lactation cycle. For this purpose, biopsies of the mammary gland were performed at −30, −15, 1, 15, 30, 60, 120, 180, and 240 days relative to parturition (d). The transcriptome analysis was performed using a commercial bovine microarray platform and the results were analyzed using several bioinformatic tools. The statistical analysis using an overall false discovery rate ≤ 0.05 for the effect of whole lactation and p < 0.05 for each comparison identified >6000 differentially expressed genes (DEGs) throughout lactation, with a large number of DEGs observed at the onset (1 d vs. −15 d) and at the end of lactation (240 d vs. 180 d). Bioinformatics analysis revealed a major role of genes associated with BTA3, BTA4, BTA6, BTA9, BTA14, and BTA28 in lactation. Functional analysis of DEG underlined an overall induction of lipid metabolism, suggesting an increase in triglycerides synthesis, likely regulated by PPAR signaling. The same analysis revealed an induction of amino acid metabolism and secretion of protein, with a concomitant decrease in proteasome, indicating a major role of amino acid handling and reduced protein degradation in the synthesis and secretion of milk proteins. Glycan biosynthesis was induced for both N-glycan and O-glycan, suggesting increased glycan content in the milk. The cell cycle and immune response, especially antigen processing and presentation, were strongly inhibited during lactation, suggesting that morphological changes are minimized during lactation, while the mammary gland prevents immune hyper-response. Transcripts associated with response to radiation and low oxygen were enriched in the down-regulated DEG affected by the stage of lactation. Except for this last finding, the functions affected by the transcriptomic adaptation to lactation in mammary tissue of yak are very similar to those observed in dairy cows.

Milk consumption has long been associated with better bone development in children, contrary to sugar-sweetened beverages (SSB); however, stronger evidence are needed. Therefore, this study aimed to use a pig model for growing children to evaluate the effect of milk vs. SBB consumption on the developing skeleton. For this, we used 24 pre-pubertal pigs (12 in Experiment 1, 8 males and 4 females, and 12 in Experiment 2, all males) randomly split into two groups to receive up to 750 mL of whole milk supplementation or an isocaloric maltodextrin solution (as SSB) for 13 or 11 weeks. Ultrasonography was used throughout the trial to record in vivo bone growth. The pigs were euthanised, and bones of the appendicular skeleton and the mandible were collected and measured with Dual X-Ray Absorptiometry (DEXA). A three-point bending test was used for biomechanical testing. After fracture, the cortical bone thickness was measured at three regions of the femur. Of the DEXA measurements taken, only the bone mineral density of the mandible was lower in pigs receiving milk vs. SSB. Compared to SSB, pigs consuming milk had also a larger occlusal surface thickness indicating lower teeth grinding, suggesting a calming effect of the milk. There was no difference in any biomechanical tests. In femur, the medial cortical was thicker in the SSB group, while the lateral cortical was thicker in the group receiving milk. Overall, there appears to be no effect of short-term milk supplementation on bone growth, mineral density and strength.Highlights Larger bone density and size was observed in male vs. female before puberty. Three cups of whole milk per day for 3 months did not improve bone density and strength compared to isoenergetic sugar-sweetened beverage in prepubertal pigs used as model for growing children despite higher amount of calcium in the diet. Lower bone density in the ramus of the mandible and less teeth grinding indicated a reduced stress in the animals feed with milk.

Abstract High producing dairy cows generally receive in the diet up to 5–6% of fat. This is a relatively low amount of fat in the diet compared to diets in monogastrics; however, dietary fat is important for dairy cows as demonstrated by the benefits of supplementing cows with various fatty acids (FA). Several FA are highly bioactive, especially by affecting the transcriptome; thus, they have nutrigenomic effects. In the present review, we provide an up-to-date understanding of the utilization of FA by dairy cows including the main processes affecting FA in the rumen, molecular aspects of the absorption of FA by the gut, synthesis, secretion, and utilization of chylomicrons; uptake and metabolism of FA by peripheral tissues, with a main emphasis on the liver, and main transcription factors regulated by FA. Most of the advances in FA utilization by rumen microorganisms and intestinal absorption of FA in dairy cows were made before the end of the last century with little information generated afterwards. However, large advances on the molecular aspects of intestinal absorption and cellular uptake of FA were made on monogastric species in the last 20 years. We provide a model of FA utilization in dairy cows by using information generated in monogastrics and enriching it with data produced in dairy cows. We also reviewed the latest studies on the effects of dietary FA on milk yield, milk fatty acid composition, reproduction, and health in dairy cows. The reviewed data revealed a complex picture with the FA being active in each step of the way, starting from influencing rumen microbiota, regulating intestinal absorption, and affecting cellular uptake and utilization by peripheral tissues, making prediction on in vivo nutrigenomic effects of FA challenging.

Abstract Background In dairy cows circulating non-esterified fatty acids (NEFA) increase early post-partum while liver and other tissues undergo adaptation to greater lipid metabolism, mainly regulated by peroxisome proliferator-activated receptors (PPAR). PPAR are activated by fatty acids (FA), but it remains to be demonstrated that circulating NEFA or dietary FA activate bovine PPAR. We hypothesized that circulating NEFA and dietary FA activate PPAR in dairy cows. Methods The dose-response activation of PPAR by NEFA or dietary FA was assessed using HP300e digital dispenser and luciferase reporter in several bovine cell types. Cells were treated with blood plasma isolated from Jersey cows before and after parturition, NEFA isolated from the blood plasma, FA released from lipoproteins using milk lipoprotein lipase (LPL), and palmitic acid (C16:0). Effect on each PPAR isotype was assessed using specific synthetic inhibitors. Results NEFA isolated from blood serum activate PPAR linearly up to ~ 4-fold at 400 μmol/L in MAC-T cells but had cytotoxic effect. Addition of albumin to the culture media decreases cytotoxic effects of NEFA but also PPAR activation by ~ 2-fold. Treating cells with serum from peripartum cows reveals that much of the PPAR activation can be explained by the amount of NEFA in the serum (R2 = 0.91) and that the response to serum NEFA follows a quadratic tendency, with peak activation around 1.4 mmol/L. Analysis of PPAR activation by serum in MAC-T, BFH-12 and BPAEC cells revealed that most of the activation is explained by the activity of PPARδ and PPARγ, but not PPARα. Palmitic acid activated PPAR when added in culture media or blood serum but the activation was limited to PPARδ and PPARα and the response was nil in serum from post-partum cows. The addition of LPL to the serum increased > 1.5-fold PPAR activation. Conclusion Our results support dose-dependent activation of PPAR by circulating NEFA in bovine, specifically δ and γ isotypes. Data also support the possibility of increasing PPAR activation by dietary FA; however, this nutrigenomics approach maybe only effective in pre-partum but not post-partum cows.

Abstract Background The aim of the study was to investigate the effect of energy overfeeding during the dry period on adipose tissue transcriptome profiles during the periparturient period in dairy cows. Methods Fourteen primiparous Holstein cows from a larger cohort receiving a higher-energy diet (1.62 Mcal of net energy for lactation/kg of dry matter; 15% crude protein) for ad libitum intake to supply 150% (OVR) or 100% (CTR) of energy requirements from dry off until parturition were used. After calving, all cows received the same lactation diet. Subcutaneous adipose tissue (SAT) biopsies were collected at − 14, 1, and 14 d from parturition (d) and used for transcriptome profiling using a bovine oligonucleotide microarray. Data mining of differentially expressed genes (DEG) between treatments and due to sampling time was performed using the Dynamic Impact Approach (DIA) and Ingenuity Pathway Analysis (IPA). Results There was a strong effect of over-feeding energy on DEG with 2434 (False discovery rate-corrected P

Some pasture species are rich in phytochemicals, able to improve milk yield and quality and to reduce the environmental impacts of livestock farming. The phytochemicals interact with the different gene networks within the animal, such as nuclear factor erythroid 2-related factor 2 (NRF2), but their overall impact on animal health remains to be fully understood. The objective of this study was to identify the effects of pasture Legumes and non-leguminous Forbs containing high bioactive compounds on metabolism and activity of the liver, antioxidant response, kidney function, and inflammation of dairy cows using a large array of blood parameters associated with metabolism and the innate immune system. For this purpose, 26 parameters and the concentration of certain bioactive compounds were assessed in blood plasma, collected from the Jersey cows grazing either Grass, Legume, or Forb-based pastures. In addition, serum collected from all the cows was utilized to detect the changes in NRF2 activation in bovine mammary alveolar cells (MACT) and hepatocytes. Compared with Grass, the cows that grazed both Forb and Legume pastures had lower β-hydroxybutyric acid (BHB) and creatinine and larger vitamin E and the ferric reducing ability of the plasma, supporting an improved antioxidative status for these animals. Compared with both Grass and Legume, the cows that graze Forb pasture had lower urea and urea to creatinine ratio, and lower creatinine, indicating a better kidney function. The cows grazing Legume pasture had greater hematocrit, bilirubin, cholesterol, albumin, β-carotene, retinol, and thiol groups but lower ceruloplasmin, paraoxonase, and myeloperoxidase (MPO) than those grazed Grass and Forb pastures, indicating a positive effect of Legume pasture on the liver, oxidative stress, and red blood cells. The plasma of cows in the various pastures was enriched with various isoflavonoids, especially the cows grazed on Forb and Legume pastures, which likely contributed to improving the antioxidative status of those cows. However, this effect was likely not due to the higher activation of NRF2. Overall, these results indicate that Forb and Legume pastures rich in secondary metabolites do not strongly affect the metabolism but can improve the status of the liver and the kidney and improve the efficiency of N utilization and antioxidant response, compared with the Grass pasture.

Nuclear factor erythroid 2-related factor 2 (NRF2) plays a key role in the response to oxidative stress. Diets containing known NRF2 modulators could be used to minimize oxidative stress in dairy cows. Currently, studies evaluating the activity of NRF2 in bovine have used the classical in vitro approach using synthetic media, which is very different than in vivo conditions. Furthermore, studies carried out in vivo cannot capture the short-term and dynamic response of NRF2. Thus, there is a need to develop new approaches to study NRF2 modulation. The aim of the present study was to establish an in vitro–in vivo hybrid system to investigate activation of NRF2 in bovine cells that can serve as an intermediate model with results closer to what is expected in vivo. To accomplish the aim, we used a combination of a gene reporter assay in immortalized bovine mammary cells, synthetic NRF2 modulators, and blood serum from periparturient cows. Synthetic agonist tert-butylhydroquinone and sulforaphane confirmed to be effective activators of bovine NRF2 with acute and large effect at 30 and 5 μM, respectively, with null response after the above doses due to cytotoxicity. When the agonists were added to blood serum the response was more linear with maximum activation of NRF2 at 100 and 30 μM, respectively, and the cytotoxicity was prevented. High concentration of albumin in blood serum plays an important role in such an effect. Brusatol (100 nM) was observed to be an effective NRF2 inhibitor while also displaying general protein synthesis inhibition and cytotoxicity when added to synthetic media. A consistent inhibition of NRF2 was observed when brusatol was added to the blood serum but the cytotoxicity was reduced. The synthetic inhibitor ML385 had no effect on modulation of bovine NRF2. Hydrogen peroxide activates NRF2 in bovine mammary cells starting from 100 μM; however, strong cytotoxicity was detected starting at 250 μM when cells were cultivated in the synthetic media, while blood serum prevented cytotoxicity. Overall, our data indicated that the use of synthetic media can be misleading in the study of NRF2 in bovine and the use of blood serum appears necessary.

The consequences of a cattle crossbreeding programme on the genes expression, signalling and metabolic pathways, molecular networks, and biological functions is still indistinctive. In this study differences of five cattle populations in a crossbreeding programme were studied using high throughput sequencing technology to characterise their transcriptome. Holstein (Bos Taurus origin) and Taleshi (Bos indicus origin) as purebred cattle were compared with their crossbreds including 50% sire Taleshi and dam Holstein (50CSN), 50% sire Holstein and dam Taleshi (50CSH) and 75% Holstein (75C). Differentially expressed genes (DEGs) and their related enriched metabolic pathways, transcription up-stream regulators and biological functions were studied by bioinformatics tools of transcriptome analysis. Total expressed transcripts in all populations were 72,812 with 22,627 annotated genes. We found 2262 DEGs in which 251 genes were uniquely expressed in purebreds or crossbreds. Functional analysis of DEGs showed that the immune and inflammatory responses were the most highly-impacted pathways and functions. Among all significantly enriched pathways, eukaryotic translation initiation factor-2 signalling had the highest activation Z-score (5.3) in crossbred compared to purebred cattle. The majority of up-stream regulators of genes including transcription regulators and cytokines were differentially expressed among populations in which their activation Z-score in purebred was more than crossbred cattle. Crossing of Holstein with Taleshi breed resulted lower activity of pathways related to immunity and inflammatory responses. The analysis revealed that the most important differences between studied genotypes, especially between purebred and crossbred cattle, were related to immune function.

A greater demand for food animal production without antibiotics has created the common practice of feeding food animals dietary immunomodulatory feed additives (IFA) throughout their life cycle. However, little is known about the impact of IFA on cytokine and chemokine signaling in non-stressed, non-pathogen-challenged food animals during the early feeding period. We evaluated the expression of 82 genes related to cytokine and chemokine signaling in the whole blood of growing Angus heifers to determine the effect of IFA supplementation on cytokine and chemokine signaling during the first 28 d of feeding. One gene ( CCL1 ) was significantly up-regulated and 14 genes (17%) were significantly down-regulated by IFA feeding during the entire early feeding period including 5 of 21 (24%) evaluated chemokine and IL receptors (CCR1 , CCR2 , IL1R1 , IL10RA , IL10RB ). These data when taken together suggest providing an IFA in the diet of growing beef cattle during the early feeding period may suppress the inflammatory response through cytokine–cytokine receptor signaling.

MicroRNAs are conserved, endogenous small RNAs with critical post-transcriptional regulatory functions throughout eukaryota, including prominent roles in development and disease. Despite much effort, microRNA annotations still contain errors and are incomplete due especially to challenges related to identifying valid miRs that have small numbers of reads, to properly locating hairpin precursors and to balancing precision and recall. Here, we present miRWoods, which solves these challenges using a duplex-focused precursor detection method and stacked random forests with specialized layers to detect mature and precursor microRNAs, and has been tuned to optimize the harmonic mean of precision and recall. We trained and tuned our discovery pipeline on data sets from the well-annotated human genome, and evaluated its performance on data from mouse. Compared to existing approaches, miRWoods better identifies precursor spans, and can balance sensitivity and specificity for an overall greater prediction accuracy, recalling an average of 10% more annotated microRNAs, and correctly predicts substantially more microRNAs with only one read. We apply this method to the under-annotated genomes of Felis catus (domestic cat) and Bos taurus (cow). We identified hundreds of novel microRNAs in small RNA sequencing data sets from muscle and skin from cat, from 10 tissues from cow and also from human and mouse cells. Our novel predictions include a microRNA in an intron of tyrosine kinase 2 (TYK2) that is present in both cat and cow, as well as a family of mirtrons with two instances in the human genome. Our predictions support a more expanded miR-2284 family in the bovine genome, a larger mir-548 family in the human genome, and a larger let-7 family in the feline genome.

We evaluated the effects of a treatment diet contaminated with 1.7 mg deoxynivalenol and 3.5 mg fumonisins (B1, B2 and B3) per kg ration on immune status and peripheral blood gene expression profiles in finishing-stage Angus steers. The mycotoxin treatment diet was fed for a period of 21 days followed by a two-week washout period during which time all animals consumed the control diet. Whole-blood leukocyte differentials were performed weekly throughout the experimental and washout period. Comparative profiles of CD4+ and CD8+ T cells, along with bactericidal capacity of circulating neutrophils and monocytes were evaluated at 0, 7, 14, 21 and 35 days. Peripheral blood gene expression was measured at 0, 7, 21 and 35 days via RNA sequencing. Significant increases in the percentage of CD4−CD8+ T cells were observed in treatment-fed steers after two weeks of treatment and were associated with decreased CD4:CD8 T-cell ratios at this same timepoint (p ≤ 0.10). No significant differences were observed as an effect of treatment in terms of bactericidal capacity at any timepoint. Dietary treatments induced major changes in transcripts associated with endocrine, metabolic and infectious diseases; protein digestion and absorption; and environmental information processing (inhibition of signaling and processing), as evaluated by dynamic impact analysis. DAVID analysis also suggested treatment effects on oxygen transport, extra-cellular signaling, cell membrane structure and immune system function. These results indicate that finishing-stage beef cattle are susceptible to the immunotoxic and transcript-inhibitory effects of deoxynivalenol and fumonisins at levels which may be realistically encountered in feedlot situations.

The inclusion of fat in livestock diets represents a valuable and cost-effective way to increase the animal’s caloric intake. Beyond their caloric value, fatty acids can be understood in terms of their bioactivity, via the modulation of the ligand-dependent nuclear peroxisome proliferator-activated receptors (PPAR). Isotypes of PPAR regulate important metabolic processes in both monogastric and ruminant animals, including the metabolism of fatty acids (FA), the production of milk fat, and the immune response; however, information on the modulation of bovine PPAR by fatty acids is limited. The objective of this study was to expand our understanding on modulation of bovine PPAR by FA, both when used individually and in combination, in an immortalized cell culture model of bovine liver. Of the 10 FA included in the study, the greatest activation of the PPAR reporter was detected with saturated FA C12:0, C16:0, and C18:0, as well as phytanic acid, and the unsaturated FA C16:1 and C18:1. When supplemented in mixtures of 2 FA, the most effective combination was C12:0 + C16:0, while in mixtures of 3 FA, the greatest activation was caused by combinations of C12:0 with C16:0 and either C18:0, C16:1, or C18:1. Some mixtures display a synergistic effect that leads to PPAR activation greater than the sum of their parts, which may be explained by structural dynamics within the PPAR ligand-binding pocket. Our results provide fundamental information for the development of tailored dietary plans that focus on the use of FA mixtures for nutrigenomic purposes.

Conjugated linoleic acid was detected in rabbit caecotrophs, due to the presence of microbial lipid activity in rabbit cecum. However, the effect of CLA as a functional food in growing rabbit is not well established. Therefore, this study was conducted to determine the effect of CLA on production, meat quality, and its nutrigenomic effect on edible parts of rabbit carcass including skeletal muscle, liver, and adipose tissue. Therefore, seventy five weaned V-Line male rabbits, 30 days old, were randomly allocated into three dietary treatments receiving either basal control diet, diet supplemented with 0.5% (CLAL), or 1% CLA (CLAH). Total experimental period (63 d) was segmented into 7 days adaptation and 56 days experimental period. Dietary supplementation of CLA did not alter growth performance, however, the fat percentage of longissimus lumborum muscle was decreased, with an increase in protein and polyunsaturated fatty acids (PUFA) percentage. Saturated fatty acids (SFA) and mono unsaturated fatty acids (MUFA) were not increased in CLA treated groups. There was tissue specific sensing of CLA, since subcutaneous adipose tissue gene expression of PPARA was downregulated, however, CPT1A tended to be upregulated in liver of CLAL group only (P = 0.09). In skeletal muscle, FASN and PPARG were upregulated in CLAH group only (P ≤0.01). Marked cytoplasmic vacuolation was noticed in liver of CLAH group without altering hepatocyte structure. Adipocyte size was decreased in CLA fed groups, in a dose dependent manner (P

The objective of the present study was to evaluate the overall adaptations of liver and mammary tissue to a corn stover (CS) compared to a mixed forage (MF) diet in mid-lactation primiparous dairy cows. Twenty-four primiparous lactating Holstein cows were randomly allocated to 2 groups receiving either an alfalfa forage diet (MF, F:C = 60:40) with Chinese wildrye, alfalfa hay and corn silage as forage source or a corn stover forage diet (CS, F:C = 40:60). A subgroup of cows (n = 5/diet) was used for analysis of liver and mammary transcriptome using a 4 × 44K Bovine Agilent microarray chip. The results of functional annotation analysis showed that in liver CS vs. MF inhibited pathways related to lipid metabolism while induced the activity of the potassium channel. In mammary tissue, fatty acid metabolism was activated in CS vs. MF. In conclusion, the analysis of genes affected by CS vs. MF indicated mammary gland responding to lower level of linoleate from the diet (lower in CS vs. MF) by activating the associated biosynthesis metabolic pathway while the liver adaptively activated potassium transport to compensate for a lower K ingestion.

Increasing pasture diversity and spatially separated sowing arrangements can potentially increase the dry matter intake of high-quality forages leading to improved animal production. This study investigated the effects of simple (two-species) and diverse (six-species) pastures planted either in mixed or spatially separated adjacent pasture strips on performance, N partitioning, and methane emission of dairy cows. Thirty-six mid-lactation Jersey cows grazed either (1) simple mixed, (2) simple spatially separated, (3) diverse mixed, or (4) diverse spatially separated pastures planted in a complete randomized block design with three replicates. Compared to simple pasture, diverse pasture had lower CP content but higher condensed tannins and total phenolic compounds with an overall positive effect on yield of milk solids, nitrogen utilization, including a reduction of N output from urine, and methane yields per dry matter eaten. The spatial separation increased legume and CP content in simple pasture but decreased NDF in both diverse and simple pastures. In conclusion, increasing diversity using pasture species with higher nutritive value and secondary compounds can help improving the production while decreasing the environmental effect of dairy farming, while spatial separation had a minor effect on feed intake and yield, possibly due to overall high-quality pastures in early spring.

The anti-hyperlipidemic and anti-inflammatory effects exerted by Aloe on monogastric mammals suggest it as a potential strategy to address the tremendous metabolic alterations that affect dairy cows during their transition to calving. A group of 20 multiparous Italian Holstein dairy cows were housed in freestalls and allocated into two homogeneous groups to receive either 200 g/d of water (CTR) or 200 g/day of Aloe arborescens Mill. whole plant homogenate through a rumen tube (AAM) between −14 and 14 days from calving (DFC). From −14 to 35 DFC, the BCS, and milk yield were measured, and blood samples were collected to assess the hematochemical profile. Data underwent ANOVA testing using a mixed model for repeated measurements, including the treatment and time and their interactions as fixed effects. Compared to CTR cows, AAM cows had a less pronounced BCS loss in early lactation (p < 0.01), indicating less mobilization of body reserves. Compared to CTR cows, AAM cows had a lower plasma concentration of nonesterified fatty acids and beta hydroxybutyrate (p < 0.01 and = 0.01 respectively) that, paired with the lower butterfat content and fat/protein ratio in their milk (p = 0.03 and < 0.01 respectively), indicates that Aloe reduced the mobilization of body fats. AAM cows had a reduced concentration of myeloperoxidase in plasma and a lower SCC in milk compared to CTR cows (p = 0.02 for both), indicating an anti-inflammatory effect of Aloe. Furthermore, AAM cows had a lower plasma concentration of ceruloplasmin (p < 0.05) and higher plasma concentration of cholesterol, retinol, and paraoxonase compared to CTR cows (p < 0.01, < 0.01 and < 0.05 respectively), indicating Aloe was effective in mitigating the acute phase response in early lactation. Finally, AAM cows had lower plasma creatinine concentrations around calving (p < 0.05), a lower concentration of plasma bilirubin, and a higher concentration of plasma tocopherol compared to CTR cows (p = 0.01 for both). These data suggest Aloe has anti-hyperlipidemic and anti-inflammatory effects on transition dairy cows that could have ameliorated liver and kidney function disruption and increased the availability of body antioxidants in early lactation.

The objective of this study was to determine the effect of long-term supplementation of unsaturated oil on lipid metabolism and transcription of genes involved in lipid metabolism in subcutaneous adipose tissue (SAT) of mid-lactating dairy cows. The objective was achieved by supplementing dairy cows with soybean oil (SO; high in linoleic acid) or fish oil (FO; high in EPA and DHA) for 63 days (nine weeks). Cows were fed a control diet with no added lipid, or diets containing SO or FO (n = 5 cows/group). At the onset of the experiment (day 0) and on days 21, 42, and 63 of supplementation, blood and SAT samples were collected from each animal. Oil supplementation increased cholesterol and NEFA in plasma, with a greater effect of SO compared to FO. Concentration of BUN was lower in SO compared to control and FO at the end of the trial. Transcription of few genes was affected by dietary lipids: FABP4 had lowest expression in FO followed by SO and control. ACACA and FASN had higher expression in FO. Transcription of SCAP was higher but expression of INSIG1 was lower in SO. Overall, results revealed that compared to control, SO and FO had lipogenic effect in SAT.

After birth, a newborn calf has to adapt to an extrauterine life characterized by several physiological changes. In particular, maturation of the gastrointestinal tract in a new environment loaded with potential pathogens, which can predispose neonatal calves to develop diarrhea, and is a major cause of morbidity and mortality during the first 4 wks of life. We aimed to investigate the inflammatory adaptations at a transcriptomic level in the gastrointestinal (GI) tract to a mild diarrhea in neonatal dairy calves using RNA isolated from fresh fecal samples. Eight newborn Jersey male calves were used from birth to 5 wks of age and housed in individual pens. After birth, calves received 1.9 L of colostrum from their respective dams. Calves had ad-libitum access to water and starter grain (22% CP) and were fed twice daily a total of 5.6 L pasteurized whole milk. Starter intake, body weight (BW), fecal score, withers height (WH), and rectal temperature (RT) were recorded throughout the experiment. Blood samples were collected weekly for metabolic and inflammatory profiling from wk 0 to wk 5. Fresh fecal samples were collected weekly and immediately flash frozen until RNA was extracted using a Trizol-based method, and subsequently, an RT-qPCR analysis was performed. Orthogonal contrasts were used to evaluate linear or quadratic effects over time. Starter intake, BW, and WH increased over time. Fecal score was greatest (2.6 ± 0.3) during wk 2. The concentrations of IL-6, ceruloplasmin, and haptoglobin had a positive quadratic effect with maximal concentrations during wk 2, which corresponded to the maximal fecal score observed during the same time. The concentration of serum amyloid A decreased over time. The mRNA expression of the proinflammatory related genes TLR4, TNFA, IL8, and IL1B had a positive quadratic effect of time. A time effect was observed for the cell membrane sodium-dependent glucose transporter SLC5A1, for the major carbohydrate facilitated transporter SLC2A2, and water transport function AQP3, where SLC5A1 and AQP3 had a negative quadratic effect over time. Our data support the use of the fecal RNA as a noninvasive tool to investigate intestinal transcriptomic profiling of dairy calves experiencing diarrhea, which would be advantageous for future research including nutritional effects and health conditions.

Liver and mammary gland are among the most important organs during lactation in dairy cows. With the purpose of understanding both the different and the complementary roles and the crosstalk of those two organs during lactation, a transcriptome analysis was performed on liver and mammary tissues of 10 primiparous dairy cows in mid-lactation. The analysis was performed using a 4×44K Bovine Agilent microarray chip. The transcriptome difference between the two tissues was analyzed using SAS JMP Genomics using ANOVA with a false discovery rate correction (FDR). The analysis uncovered >9,000 genes differentially expressed (DEG) between the two tissues with a FDR

Mastitis is a major disease in dairy cows resulting in significant economic losses. In vitro works suggest that ruminants peroxisome proliferator-activated receptor gamma (PPARγ) can aid in improving the response to mastitis and can control milk fat synthesis. The objectives of the present experiment were to test if treatment with the putative PPARγ agonist 2,4-thiazolidinedione (TZD) improves (1) the response to subclinical mastitis and (2) milk fat production. Lactating goats received daily injections of 8 mg/kg BW of TZD or saline for 3 weeks. After one week of TZD injection, half of the goats in each group received intramammary infusion of Strep. uberis or saline in both halves for a total of 4 groups (n=6/group). TZD treatment did not affect milk fat but had positive effect on milk somatic cells count, blood nonesterified fatty acids, inflammatory markers, and liver function. TZD significantly increased myeloperoxidase but did not affect leukocytes phagocytosis or insulin. TZD increased adipocytes size and had minor effect on expression of PPARγ target genes in mammary epithelial cells but not in adipose tissue. Overall, TZD ameliorated the response to intramammary infection but the effect on milk fat synthesis and expression of related transcripts was less than expected.

The objective of this study was to characterize the long-term transcriptomic effects of lipogenic genes in subcutaneous adipose tissue (SAT) of dairy cows supplemented with unsaturated (olive oil; OO) and saturated (hydrogenated vegetable oil; HVO) lipids. Cows were fed a control diet with no added lipid, or diets containing OO or HVO (n = 5 cows/group) for 63 days. SAT was obtained from the tail-head area at the onset of the study and after 21, 42, and 63 days of supplementation. Treatments had minor effects on expression of measured genes. Both fat supplements reduced expression of PPARG, HVO decreased transcription of the desaturase FADS2 and lipid droplet formation PLIN2, and OO increased transcription of FABP3. Both lipid treatments decreased expression of the transcription regulator SREBF1 and its chaperone (SCAP) during the first 21 days of treatment. Our data indicated that long-term feeding of OO and HVO have a relatively mild effect on expression of lipogenic genes in SAT of mid-lactating cows.

In a prior experiment, treatment of goats with the putative PPARγ agonist 2,4-thiazolidinedione (2,4-TZD) ameliorated the response to intramammary infection without evidence of PPARγ activation. The lack of PPARγ activation was possibly due to deficiency of vitamin A and/or a poor body condition of the animals. Therefore, the present study hypothesized that activation of PPARγ by 2,4-TZD in goats supplemented with adequate amounts of vitamin A can improve the response to sub-clinical mastitis. Lactating goats receiving a diet that met National Research Council requirements, including vitamin A, were injected with 8 mg/kg BW of 2,4-TZD (n = 6) or saline (n = 6; control (CTR)) daily. Two weeks into treatment, all goats received Streptococcus uberis (IMI) in the right mammary gland. Blood biomarkers of metabolism, inflammation, and oxidative status plus leukocytes phagocytosis were measured. Mammary epithelial cells (MEC) and macrophages were isolated from milk and liver tissue collected for gene expression analysis. Milk fat was maintained by treatment with 2,4-TZD, but decreased in CTR, after IMI. Haptoglobin was increased after IMI only in 2,4-TZD without any effect on negative acute phase proteins, indicating an improved liver function. 2,4-TZD vs. CTR had a greater amount of globulin. The expression of inflammation-related genes was increased by IMI in both macrophages and MEC. Except for decreasing expression of SCD1 in MEC, 2,4-TZD did not affect the expression of measured genes. Results confirmed the successful induction of sub-clinical mastitis but did not confirm the positive effect of 2,4-TZD on the response to IMI in well-fed goats.

A prior study in bovine mammary (MACT) cells indicated that long-chain fatty acids (LCFA) C16:0 and C18:0, but not unsaturated LCFA, control transcription of milk fat-related genes partly via the activation of peroxisome proliferator-activated receptor gamma (PPARγ). However, in that study, the activation of PPARγ by LCFA was not demonstrated but only inferred. Prior data support a lower response of PPARγ to agonists in goat mammary cells compared to bovine mammary cells. The present study aimed to examine the hypothesis that LCFA alter the mRNA abundance of lipogenic genes in goat mammary epithelial cells (GMEC) at least in part via PPARγ. Triplicate cultures of GMEC were treated with a PPARγ agonist (rosiglitazone), a PPARγ inhibitor (GW9662), several LCFA (C16:0, C18:0, t10,c12-CLA, DHA, and EPA), or a combination of GW9662 with each LCFA. Transcription of 28 genes involved in milk fat synthesis was measured using RT-qPCR. The data indicated that a few measured genes were targets of PPARγ in GMEC (SCD1, FASN, and NR1H3) while more genes required a basal activation of PPARγ to be transcribed (e.g., LPIN1, FABP3, LPL, and PPARG). Among the tested LCFA, C16:0 had the strongest effect on upregulating transcription of measured genes followed by C18:0; however, for the latter most of the effect was via the activation of PPARγ. Unsaturated LCFA downregulated transcription of measured genes, with a lesser effect by t10,c12-CLA and a stronger effect by DHA and EPA; however, a basal activation of PPARγ was essential for the effect of t10,c12-CLA while the activation of PPARγ blocked the effect of DHA. The transcriptomic effect of EPA was independent from the activation of PPARγ. Data from the present study suggest that saturated LCFA, especially C18:0, can modulate milk fat synthesis partly via PPARγ in goats. The nutrigenomic effect of C16:0 is not via PPARγ but likely via unknown transcription factor(s) while PPARγ plays an indirect role on the nutrigenomic effect of polyunsaturated LCFA (PUFA) on milk fat related genes, particularly for CLA (permitting effect) and DHA (blocking effect).

Background: In a prior experiment, treatment of goats with the putative PPARγ agonist 2,4-thiazolidinedione (2,4-TZD) did not affect milk fat or expression of milk-fat related genes. The lack of response was possibly due to deficiency of vitamin A and/or a poor body condition of the animals. In the present experiment, we tested the hypothesis that PPARγ activation affects milk fat synthesis in goats with a good body condition and receiving adequate levels of vitamin A. Methods: Lactating goats receiving a diet that met NRC requirements, including vitamin A, were injected with 8 mg/kg BW of 2,4-TZD (n = 6) or saline (n = 6; CTR) daily for 26 days. Blood metabolic profiling and milk yield and components were measured including fatty acid profile. Expression of genes related to glucose and lipid metabolism was measured in adipose tissue and in mammary epithelial cells (MEC). Size of adipocytes was assessed by histological analysis. Results: NEFA, BHBA, and fatty acids available in plasma decreased while glucose increased in 2,4-TZD vs. CTR. Size of cells and expression of insulin signaling and glucose metabolism-related genes were larger in 2,4-TZD vs. CTR in adipose tissue. In MEC, expression of SCD1 and desaturation of stearate was lower in 2,4-TZD vs. CTR. Conclusions: Overall data revealed a lack of PPARγ activation by 2,4-TZD and no effect on milk fat synthesis despite a strong anti-lipolysis effect on adipose tissue.

Flaxseed is rich in α-linolenic acid and is used in broiler chicken diets to enrich tissues with n-3 fatty acids (FA). However, non-starch polysaccharides (NSP) in flaxseed decreases nutrient digestibility and limits the availability of n-3 FA. Addition of carbohydrase enzymes to flaxseed-based diets can decrease the anti-nutritive effects of NSP. We hypothesized that flaxseed and enzyme supplementation affect lipid content and alter expression of genes related to lipid metabolism in broiler liver. Five day-old broiler chicks were fed a corn-soybean basal diet with 0% flaxseed, a basal diet with 10% of flaxseed, or 10% flaxseed + 0.05% enzyme diet up to day 42 of growth. Total lipids, including long-chain (≥20C) n-3 FA and monounsaturated FA, were increased in flax-fed broiler livers. Enzyme addition reduced arachidonic acid and total long chain n-6 FA. These changes were similarly reflected in phosphatidylcholine lipid species. Dietary flax and enzyme treatments up-regulated PPARα target genes CPT1A and ACOX1 while reducing expression of de novo FA synthesis-related genes. This study concludes that flaxseed and enzyme supplementation in broiler diets enhances LC n-3 FA species, while reducing n-6 FA species in hepatic phospholipids (PL). Flaxseed-based diets changes the expression of genes involved in FA lipid metabolism without affecting growth or production performance in broilers.

The yak is primarily found throughout the Tibetan high plateau and the surrounding mountainous area of south central Asia; among its others attributes, its milk is very important for the local population. A key concern in the field of yak research is the better understanding of which genes control the production and composition of milk. The most accurate and sensitive method for gene expression analysis is quantitative reverse transcription polymerase chain reaction (RT-qPCR). It is essential for reliable RT-qPCR to be able to the normalize the data using internal control genes (ICGs). However, it is critical to assess the reliability of the normalization by testing multiple ICGs. Our objective was to uncover a reliable normalization for RT-qPCR data obtained from yak mammary tissue during the lactation cycle. We assessed the reliability of 10 ICGs (ACTB, EIF6, GAPDH, LRP10, MRPL39, MRPS15, MTG1, RPS8, RPS23, and UXT) using geNorm. The analysis revealed that all of the tested ICGs can be considered to be reliable, but the use of the 6 most stable ICGs should be applied to yield a reliable normalization factor (NF). We compared the results of 3 target genes (CSN1S1, ESR1, and MYC) normalized using 6, 3, or 1 of the best ICGs. We did not observe overall differences between the 3 normalization strategies with the exception of 1 time point in MYC. The use of only a single ICG is not recommended; thus, we concluded that the calculation of the NF using the 3 best ICGs, MRPS15, RPS23, and UXT, is a reliable normalization strategy for RT-qPCR data obtained from yak mammary tissue during pregnancy and lactation. A dilution effect of the ICGs due to a large increase in the mRNA of abundantly expressed genes in bovine and porcine mammary tissue during the lactation cycle was previously observed. To test for the presence of a dilution effect in our study, we evaluated the pattern of non-normalized RT-qPCR data of ICGs from pregnancy to lactation and compared them with the total RNA concentration, milk yield, and non-normalized RT-qPCR data of 3 target genes. With a few exceptions, the non- normalized RT-qPCR data for the tested ICGs was significantly increased by lactation and had a positive correlation with total RNA and the non-normalized RT-qPCR data of CSN1S1. These data clearly indicated the presence of a "concentration effect" of single mRNA that remains unexplained but needs to be accounted for during the normalization of RT-qPCR data. Based on our findings, we recommend that the NF of the MRPS15, RPS23, and UXT genes should be used in the normalization of RT-qPCR data obtained from mammary tissue of lactating yaks during pregnancy and lactation.

Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators.

The importance of mesenchymal stem cells (MSC) for bone regeneration is growing. Among MSC the bone marrow-derived stem cells (BMSC) are considered the gold standard in tissue engineering and regenerative medicine; however, the adipose-derived stem cells (ASC) have very similar properties and some advantages to be considered a good alternative to BMSC. The molecular mechanisms driving adipogenesis are relatively well-known but mechanisms driving osteogenesis are poorly known, particularly in pig. In the present study we have used transcriptome analysis to unravel pathways and biological functions driving in vitro adipogenesis and osteogenesis in BMSC and ASC. The analysis was performed using the novel Dynamic Impact Approach and functional enrichment analysis. In addition, a k-mean cluster analysis in association with enrichment analysis, networks reconstruction, and transcription factors overlapping analysis were performed in order to uncover the coordination of biological functions underlining differentiations. Analysis indicated a larger and more coordinated transcriptomic adaptation during adipogenesis compared to osteogenesis, with a larger induction of metabolism, particularly lipid synthesis (mostly triglycerides), and a larger use of amino acids for synthesis of feed-forward adipogenic compounds, larger cell signaling, lower cell-to-cell interactions, particularly for the cytoskeleton organization and cell junctions, and lower cell proliferation. The coordination of adipogenesis was mostly driven by Peroxisome Proliferator-activated Receptors together with other known adipogenic transcription factors. Only a few pathways and functions were more induced during osteogenesis compared to adipogenesis and some were more inhibited during osteogenesis, such as cholesterol and protein synthesis. Up-stream transcription factor analysis indicated activation of several lipid-related transcription regulators (e.g., PPARs and CEBPα) during adipogenesis but osteogenesis was driven by inhibition of several up-stream regulators, such as MYC. Between MSCs the data indicated an 'adipocyte memory' in ASC with also an apparent lower immunogenicity compared to BMSC during differentiations. Overall the analysis allowed proposing a dynamic model for the adipogenic and osteogenic differentiation in porcine ASC and BMSC.

Using published dairy cattle liver transcriptomics dataset along with novel blood biomarkers of liver function, metabolism, and inflammation we have attempted an integrative systems biology approach applying the classical functional enrichment analysis using DAVID, a newly-developed Dynamic Impact Approach (DIA), and an upstream gene network analysis using Ingenuity Pathway Analysis (IPA). Transcriptome data was generated from experiments evaluating the impact of prepartal plane of energy intake [overfed (OF) or restricted (RE)] on liver of dairy cows during the peripartal period. Blood biomarkers uncovered that RE vs. OF led to greater prepartal liver distress accompanied by a low-grade inflammation and larger proteolysis (i.e., higher haptoglobin, bilirubin, and creatinine). Post-partum the greater bilirubinaemia and lipid accumulation in OF vs. RE indicated a large degree of liver distress. The re-analysis of microarray data revealed that expression of >4,000 genes was affected by diet × time. The bioinformatics analysis indicated that RE vs. OF cows had a liver with a greater lipid and amino acid catabolic capacity both pre- and post-partum while OF vs. RE cows had a greater activation of pathways/functions related to triglyceride synthesis. Furthermore, RE vs. OF cows had a larger (or higher capacity to cope with) ER stress likely associated with greater protein synthesis/processing, and a higher activation of inflammatory-related functions. Liver in OF vs. RE cows had a larger cell proliferation and cell-to-cell communication likely as a response to the greater lipid accumulation. Analysis of upstream regulators indicated a pivotal role of several lipid-related transcription factors (e.g., PPARs, SREBPs, and NFE2L2) in priming the liver of RE cows to better face the early postpartal metabolic and inflammatory challenges. An all-encompassing dynamic model was proposed based on the findings.

Characterization and biological roles of the peroxisome proliferator-activated receptor (PPAR) isotypes are well known in monogastrics, but not in ruminants. However, a wealth of information has accumulated in little more than a decade on ruminant PPARs including isotype tissue distribution, response to synthetic and natural agonists, gene targets, and factors affecting their expression. Functional characterization demonstrated that, as in monogastrics, the PPAR isotypes control expression of genes involved in lipid metabolism, anti-inflammatory response, development, and growth. Contrary to mouse, however, the PPAR gene network appears to controls milk fat synthesis in lactating ruminants. As in monogastrics, PPAR isotypes in ruminants are activated by long-chain fatty acids, therefore, making them ideal candidates for fine-tuning metabolism in this species via nutrients. In this regard, using information accumulated in ruminants and monogastrics, we propose a model of PPAR isotype-driven biological functions encompassing key tissues during the peripartal period in dairy cattle.

The overrepresented approach (ORA) is the most widely-accepted method for functional analysis of microarray datasets. The ORA is computationally-efficient and robust; however, it suffers from the inability of comparing results from multiple gene lists particularly with time-course experiments or those involving multiple treatments. To overcome such limitation a novel method termed Dynamic Impact Approach (DIA) is proposed. The DIA provides an estimate of the biological impact of the experimental conditions and the direction of the impact. The impact is obtained by combining the proportion of differentially expressed genes (DEG) with the log2 mean fold change and mean -log P-value of genes associated with the biological term. The direction of the impact is calculated as the difference of the impact of up-regulated DEG and down-regulated DEG associated with the biological term. The DIA was validated using microarray data from a time-course experiment of bovine mammary gland across the lactation cycle. Several annotation databases were analyzed with DIA and compared to the same analysis performed by the ORA. The DIA highlighted that during lactation both BTA6 and BTA14 were the most impacted chromosomes; among Uniprot tissues those related with lactating mammary gland were the most positively-impacted; within KEGG pathways 'Galactose metabolism' and several metabolism categories related to lipid synthesis were among the most impacted and induced; within Gene Ontology "lactose biosynthesis" among Biological processes and "Lactose synthase activity" and "Stearoyl-CoA 9-desaturase activity" among Molecular processes were the most impacted and induced. With the exception of the terms 'Milk', 'Milk protein' and 'Mammary gland' among Uniprot tissues and SP_PIR_Keyword, the use of ORA failed to capture as significantly-enriched (i.e., biologically relevant) any term known to be associated with lactating mammary gland. Results indicate the DIA is a biologically-sound approach for analysis of time-course experiments. This tool represents an alternative to ORA for functional analysis.

Bone-marrow mesenchymal stem cells (BMSC) are considered the gold standard for use in tissue regeneration among mesenchymal stem cells (MSC). The abundance and ease of harvest make the adipose-derived stem cells (ASC) an attractive alternative to BMSC. The aim of the present study was to compare the transcriptome of ASC and BMSC, respectively isolated from subcutaneous adipose tissue and femur of 3 adult pigs, during in vitro osteogenic and adipogenic differentiation for up to four weeks. At 0, 2, 7, and 21 days of differentiation RNA was extracted for microarray analysis. A False Discovery Rate ≤0.05 for overall interactions effect and P

The cow mammary transcriptome was explored at -30, -15, 1, 15, 30, 60, 120, 240, and 300 d relative to parturition. A total of 6,382 differentially expressed genes (DEG) at a false discovery rate ≤ 0.001 were found throughout lactation. The greatest number of DEG (>3,500 DEG) was observed at 60 and 120 d vs. -30 d with the largest change between consecutive time points observed at -15 vs. 1 d and 120 vs. 240 d. Functional analysis of microarray data was performed using the Dynamic Impact Approach (DIA). The DIA analysis of KEGG pathways uncovered as the most impacted and induced 'Galactose metabolism', 'Glycosylphosphatidylinositol (GPI)-anchor biosynthesis', and 'PPAR signaling'; whereas, 'Antigen processing and presentation' was among the most inhibited. The integrated interpretation of the results suggested an overall increase in metabolism during lactation, particularly synthesis of carbohydrates and lipid. A marked degree of utilization of amino acids as energy source, an increase of protein export, and a decrease of the protein synthesis machinery as well cell cycle also were suggested by the DIA analysis. The DIA analysis of Gene Ontology and other databases uncovered an induction of Golgi apparatus and angiogenesis, and the inhibition of both immune cell activity/migration and chromosome modifications during lactation. All of the highly-impacted and activated functions during lactation were evidently activated at the onset of lactation and inhibited when milk production declined. The overall analysis indicated that the bovine mammary gland relies heavily on a coordinated transcriptional regulation to begin and end lactation. The functional analysis using DIA underscored the importance of genes associated with lactose synthesis, lipid metabolism, protein synthesis, Golgi, transport, cell cycle/death, epigenetic regulation, angiogenesis, and immune function during lactation.