Assessment of spent hemp biomass as a potential ingredient in ruminant diet: nutritional quality and effect on performance, meat and carcass quality, and hematological parameters in finishing lambs.

Spent hemp biomass (SHB), a byproduct of cannabinoid extraction from the production of industrial hemp has not been approved by FDA-CVM since its effects on animal health, performance, and product quality are unknown. Our objective was to investigate the effects of feeding two levels of SHB and a 4-wk withdrawal period on performance, carcass characteristic, meat quality, and hematological parameters in finishing lambs. A total of 35 weaned, Polypay male lambs kept in single pens were randomly assigned to five feeding treatments (n = 7) and fed diets containing either no SHB (CON) or SHB at 10% (LH1) or 20% (HH1) for 4 wk with 4 wk of clearing period from SHB, or SHB at 10% (LH2) or 20% (HH2) for 8 wk. Chemical analysis revealed SHB to have a nutritive quality similar to alfalfa with no mycotoxin, terpenes, or organic residuals as a result of the extraction process. Feed intake of lambs was negatively affected by 20% SHB in period 1 but not in period 2 where feed intake was the greatest in HH1 and LH2. In contrast, none of the performance data, including liveweight gains, were different across the groups and periods. In period 1, blood glucose, cholesterol, calcium, paraoxonase, and tocopherol were decreased by the level of SHB fed, while bilirubin and alkaline phosphatase (ALP) were increased. In period 2, the concentration in blood of urea, magnesium, bilirubin, ALP, and ferric reducing ability of the plasma (FRAP) were higher in LH2 and HH2 as compared with CON, while ß-hydroxybutyrate was lower in HH2. Blood parameters related to liver health, kidney function, immune status, and inflammation were unaffected by feeding SHB. Most carcass and meat quality parameters did not differ across feeding groups either. Except carcass purge loss and meat cook loss were larger in lambs that were fed 20% SHB. Although lower feed intake of lambs that were fed 20% SHB initially in period 1 suggested SHB was not palatable to the lambs, increased feed intake at a lower level of inclusion at 10% in period 2 may point to a positive long-term effect of feeding SHB.

The use of hemp by-products in livestock diets holds promise for reducing feed costs and achieving greater resource-use efficiency through integration of livestock production and rapidly growing hemp farming. Spent hemp biomass (SHB), the byproduct of the extraction process of cannabidiol from hemp can potentially be included in the ruminant diets due to its desirable nutritional properties. However, the potential accumulation of tetrahydrocannabinol­a psychotropic compound in animal tissues and its effect on animal health, production, and product quality are still unknown. Therefore, we conducted an indoor feeding study to investigate the effects of varying levels of SHB and a withdrawal period on feed intake, performance, health, and meat quality of lambs at Oregon State University. Our findings indicated that SHB can be included in lamb diets without causing any major detrimental effects on performance, meat quality, or health of the lambs.

Coprophagy Prevention Decreases the Reproductive Performance and Granulosa Cell Apoptosis via Regulation of CTSB Gene in Rabbits.

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.

Long-Term Effects of Dietary Olive Oil and Hydrogenated Vegetable Oil on Expression of Lipogenic Genes in Subcutaneous Adipose Tissue of Dairy Cows.

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.

Effect of Soybean Oil and Fish Oil on Lipid-Related Transcripts in Subcutaneous Adipose Tissue of Dairy Cows.

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.

CRISPR/Cas9-mediated Stearoyl-CoA Desaturase 1 (SCD1) Deficiency Affects Fatty Acid Metabolism in Goat Mammary Epithelial Cells.

Stearoyl-CoA desaturase 1 (SCD1) is a fatty acid desaturase catalyzing cis-double-bond formation in the Δ9 position to produce monounsaturated fatty acids essential for the synthesis of milk fat. Previous studies using RNAi methods have provided support for a role of SCD1 in goat mammary epithelial cells (GMEC); however, RNAi presents several limitations that might preclude a truthful understanding of the biological function of SCD1. To explore the function of SCD1 on fatty acid metabolism in GMEC, we used CRISPR-Cas9-mediated SCD1 knockout through non-homologous end-joining (NHEJ) and homology-directed repair (HDR) pathways in GMEC. We successfully introduced nucleotide deletions and mutations in the SCD1 gene locus through the NHEJ pathway and disrupted its second exon via insertion of an EGFP-PuroR segment using the HDR pathway. In clones derived from the latter, gene- and protein-expression data indicated that we obtained a monoallelic SCD1 knockout. A T7EN1-mediated assay revealed no off-targets in the surveyed sites. The contents of triacylglycerol and cholesterol and the desaturase index were significantly decreased as a consequence of SCD1 knockout. The deletion of SCD1 decreased the expression of other genes involved in de novo fatty acid synthesis, including SREBF1 and FASN, as well the fatty acid transporters FABP3 and FABP4. The downregulation of these genes partly explains the decrease of intracellular triacylglycerols. Our results indicate a successful SCD1 knockout in goat mammary cells using CRISPR-Cas9. The demonstration of the successful use of CRISPR-Cas9 in GMEC is an important step to producing transgenic goats to study mammary biology in vivo.

Peroxisome proliferator-activated receptor ß/δ does not regulate glucose uptake and lactose synthesis in bovine mammary epithelial cells cultivated in vitro.

The hypothesis of the study was that inhibition of PPARß/δ increases glucose uptake and lactose synthesis in bovine mammary epithelial cells by reducing the expression of the glucose transporter mRNA destabiliser calreticulin. Three experiments were conducted to test the hypothesis using immortalised bovine mammary alveolar (MACT) and primary bovine mammary (PBMC) cells. In Experiment 1, the most effective dose to inhibit PPARß/δ activity among two synthetic antagonists (GSK-3787 and PT-s58) was assessed using a gene reporter assay. In Experiment 2, the effect on glucose uptake and lactose synthesis was evaluated by measuring glucose and lactose in the media and expression of related key genes upon modulation of PPARß/δ using GSK-3787, the synthetic PPARß/δ agonist GW-501516, or a combination of the two in cells cultivated in plastic. In Experiment 3, the same treatments were applied to cells cultivated in Matrigel and glucose and lactose in media were measured. In Experiment 1 it was determined that a significant inhibition of PPARß/δ in the presence or absence of fetal bovine serum was achieved with ≥ 1000 nm GSK-3787 but no significant inhibition was observed with PT-s58. In Experiment 2, inhibition of PPARß/δ had no effect on glucose uptake and lactose synthesis but they were both increased by GW-501516 in PBMC. The mRNA abundance of PPARß/δ target gene pyruvate dehydrogenase kinase 4 was increased but transcription of calreticulin was decreased (only in MACT cells) by GW-501516. Treatment with GSK-3787 did not affect the transcription of measured genes. No effects on glucose uptake or lactose synthesis were detected by modulation of PPARß/δ activity on cells cultivated in Matrigel. The above data do not provide support for the original hypothesis and suggest that PPARß/δ does not play a major role in glucose uptake and lactose synthesis in bovine mammary epithelial cells.

Myogenic potential of mesenchymal stem cells isolated from porcine adipose tissue.

Advances in stem cell biology and materials science have provided a basis for developing tissue engineering methods to repair muscle injury. Among stem cell populations with potential to aid muscle repair, adipose-derived mesenchymal stem cells (ASC) hold great promise. To evaluate the possibility of using porcine ASC for muscle regeneration studies, we co-cultured porcine ASC with murine C2C12 myoblasts. These experiments demonstrated that porcine ASC display significant myogenic potential. Co-culture of ASC expressing green fluorescent protein (GFP) with C2C12 cells resulted in GFP+ myotube formation, indicating fusion of ASC with myoblasts to form myotubes. The presence of porcine lamin A/C positive nuclei in myotubes and RTqPCR analysis of porcine myogenin and desmin expression confirmed that myotube nuclei derived from ASC contribute to muscle gene expression. Co-culturing GFP+ASC with porcine satellite cells demonstrated enhanced myogenic capability of ASC, as the percentage of labeled myotubes increased compared to mouse co-cultures. Enhancing myogenic potential of ASC through soluble factor treatment or expansion of ASC with innate myogenic capacity should allow for their therapeutic use to regenerate muscle tissue lost to disease or injury.

Use of Pig as a Model for Mesenchymal Stem Cell Therapies for Bone Regeneration.

Bone is a plastic tissue with a large healing capability. However, extensive bone loss due to disease or trauma requires extreme therapy such as bone grafting or 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, adipose-derived stem cells (ASC) have similar immunophenotype, morphology, multilineage potential, and transcriptome compared to BMSC, and both types have demonstrated extensive osteogenic capacity both in vitro and in vivo in several species. The use of scaffolds in combination with stem cells and growth factors provides a valuable tool for guided bone regeneration, especially for complex anatomic defects. Before translation to human medicine, regenerative strategies must be developed in animal models to improve effectiveness and efficiency. The pig presents as a useful model due to similar macro- and microanatomy and favorable logistics of use. This review examines data that provides strong support for the clinical translation of the pig model for bone regeneration.

Transcription Adaptation during In Vitro Adipogenesis and Osteogenesis of Porcine Mesenchymal Stem Cells: Dynamics of Pathways, Biological Processes, Up-Stream Regulators, and Gene Networks.

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.

Adipose-derived mesenchymal stem cells enhance healing of mandibular defects in the ramus of swine.

PURPOSE: This study investigated the effect of adipose-derived mesenchymal stem cells (ASCs) injected locally or systemically on the bone regeneration of a 10-mm-diameter cylindrical noncritical-size defect in the ramus of the pig mandible. MATERIALS AND METHODS: Fifteen Yorkshire pigs, weighing 60 to 80 kg, received bilateral 10-mm-diameter cylindrical surgical defects in each ramus of the mandible. Pigs received 1) a direct injection into the defect of 2.5 million carboxy-fluorescein diacetate succinimidyl ester-labeled ASCs from 1 of 2 pig donors (n = 6); 2) an ear vein injection of 5 million carboxy-fluorescein diacetate succinimidyl ester-labeled ASCs from 1 of 2 pig donors (n = 6); or 3) an ear vein injection of culture Dulbecco's Modified Eagle's Medium without stem cells (control; n = 3). Pigs from each treatment were sacrificed at 1 hour, 2 weeks, or 4 weeks after surgery. Healing of the defect was evaluated by dual-energy x-ray absorptiometry, micro-computed tomography, fluorescent microscopy, and histology. RESULTS: Bone healing was accelerated in the ASC-injected treatment groups at 2 and 4 weeks after surgery compared with the control pigs. CONCLUSIONS: Results from this animal model provide evidence that the injection of ASC locally into a bone defect or systemically can accelerate the healing of bone.

Transcriptomics comparison between porcine adipose and bone marrow mesenchymal stem cells during in vitro osteogenic and adipogenic differentiation.

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<0.001 between comparisons were used to determine differentially expressed genes (DEG). Ingenuity Pathway Analysis and DAVID performed the functional analysis of the DEG. Functional analysis of highest expressed genes in MSC and genes more expressed in MSC vs. fully differentiated tissues indicated low immunity and high angiogenic capacity. Only 64 genes were differentially expressed between ASC and BMSC before differentiation. The functional analysis uncovered a potential larger angiogenic, osteogenic, migration, and neurogenic capacity in BMSC and myogenic capacity in ASC. Less than 200 DEG were uncovered between ASC and BMSC during differentiation. Functional analysis also revealed an overall greater lipid metabolism in ASC, while BMSC had a greater cell growth and proliferation. The time course transcriptomic comparison between differentiation types uncovered <500 DEG necessary to determine cell fate. The functional analysis indicated that osteogenesis had a larger cell proliferation and cytoskeleton organization with a crucial role of G-proteins. Adipogenesis was driven by PPAR signaling and had greater angiogenesis, lipid metabolism, migration, and tumorigenesis capacity. Overall the data indicated that the transcriptome of the two MSC is relatively similar across the conditions studied. In addition, functional analysis data might indicate differences in therapeutic application.

Functional adaptations of the transcriptome to mastitis-causing pathogens: the mammary gland and beyond.

Application of microarrays to the study of intramammary infections in recent years has provided a wealth of fundamental information on the transcriptomics adaptation of tissue/cells to the disease. Due to its heavy toll on productivity and health of the animal, in vivo and in vitro transcriptomics works involving different mastitis-causing pathogens have been conducted on the mammary gland, primarily on livestock species such as cow and sheep, with few studies in non-ruminants. However, the response to an infectious challenge originating in the mammary gland elicits systemic responses in the animal and encompasses tissues such as liver and immune cells in the circulation, with also potential effects on other tissues such as adipose. The susceptibility of the animal to develop mastitis likely is affected by factors beyond the mammary gland, e.g. negative energy balance as it occurs around parturition. Objectives of this review are to discuss the use of systems biology concepts for the holistic study of animal responses to intramammary infection; providing an update of recent work using transcriptomics to study mammary and peripheral tissue (i.e. liver) as well as neutrophils and macrophage responses to mastitis-causing pathogens; discuss the effect of negative energy balance on mastitis predisposition; and analyze the bovine and murine mammary innate-immune responses during lactation and involution using a novel functional analysis approach to uncover potential predisposing factors to mastitis throughout an animal's productive life.

Selection and reliability of internal reference genes for quantitative PCR verification of transcriptomics during the differentiation process of porcine adult mesenchymal stem cells.

INTRODUCTION: The objective of this study was to find highly reliable internal-control genes (ICGs) for normalization of qPCR data from porcine adult mesenchymal stem cells induced to differentiate toward adipogenic and osteogenic lineages. METHODS: Stem cells were acquired from subcutaneous back fat and bone marrow of three castrated Yorkshire crossbred male pigs. Adipose and bone marrow-derived stem cells (ADSCs and BMSCs) were cultured in vitro with specific osteogenic or adipogenic differentiation medium for 4 weeks. Total RNA was extract for microarray (13,000 oligonucleotides) and qPCR analyses. Microarray data were used to uncover the most stably expressed genes (that is, potential ICGs). Co-regulation among potential ICGs was evaluated with Ingenuity Pathway Analysis. qPCR was performed on the non-coregulated ICGs candidates and on specific osteogenic (COL1A1) and adipogenic (DBI) genes. geNorm was used to uncover the most reliable ICGs by using qPCR data and the optimal number of ICGs to be used to calculate the normalization factor. RESULTS: Microarray data analysis revealed 27 potential ICGs. Among those, 10 genes without known co-regulation were selected to perform qPCR. geNorm performed on qPCR data uncovered high stability in expression ratio among the selected ICGs. However, especially reliable normalization was obtained by geometric mean of NSUN5, TIMM17B, and VPS4A. The effect of normalization, assessed on specific osteogenic (COL1A1) and adipogenic (DBI) genes, was apparent for the adipogenic and less apparent for the osteogenic differentiation. CONCLUSIONS: The combination of microarray data and pairwise gene analysis allowed identification of novel and highly reliable ICGs for qPCR data normalization of adult porcine stem cells induced to differentiate to adipogenic and osteogenic lineages.

Gene expression ratio stability evaluation in prepubertal bovine mammary tissue from calves fed different milk replacers reveals novel internal controls for quantitative polymerase chain reaction.

Prepubertal mammary development can be affected by nutrition partly through alterations in gene network expression. Quantitative PCR (qPCR) remains the most accurate method to measure mRNA expression but is subject to analytical errors that introduce variation. Thus, qPCR data normalization through the use of internal control genes (ICG) is required. The objective of this study was to mine microarray data (> 10,000 genes) from prepubertal mammary parenchyma and stroma to identify the most suitable ICG for normalization of qPCR. Tissue for RNA extraction was obtained from calves ( approximately 63 d old; n = 5/diet) fed a control (200 g/kg crude protein, 210 g/kg crude fat, fed at 441 g/d dry matter) or a high-protein milk replacer (280 g/kg crude protein, 200 g/kg crude fat, fed at 951 g/d dry matter). ICG were selected based on both absence of expression variation across treatment and of coregulation (gene network analysis). Genes evaluated were ubiquitously expressed transcript, protein phosphatase 1 regulatory (inhibitor) subunit 11 (PPP1R11), matrix metallopeptidase 14 (MMP14), ClpB caseinolytic peptidase B, SAPS domain family member 1 (SAPS1), mitochondrial GTPase 1 (MTG1), mitochondrial ribosomal protein L39, ribosomal protein S15a (RPS15A), and actin beta (ACTB). Network analysis demonstrated that MMP14 and ACTB are coregulated by v-myc myelocytomatosis viral oncogene, tumor protein p53, and potentially insulin-like growth factor 1. Pairwise comparison of expression ratios showed that ACTB, MMP14, and SAPS1 had the lowest stability and were unsuitable as ICG. PPP1R11, RPS15A, and MTG1 were the most stable among ICG tested. We conclude that the geometric mean of PPP1R11, RPS15A, and MTG1 is ideal for normalization of qPCR data in prepubertal bovine mammary tissue. This study provides a list of candidate ICG that could be used by researchers working in bovine mammary development and allied fields.