Feeding spent hemp biomass to lactating dairy cows: Effects on performance, milk components and quality, blood parameters, and nitrogen metabolism.

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 ß-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.

Compositionality, sparsity, spurious heterogeneity, and other data-driven challenges for machine learning algorithms within plant microbiome studies.

The plant-associated microbiome is a key component of plant systems, contributing to their health, growth, and productivity. The application of machine learning (ML) in this field promises to help untangle the relationships involved. However, measurements of microbial communities by high-throughput sequencing pose challenges for ML. Noise from low sample sizes, soil heterogeneity, and technical factors can impact the performance of ML. Additionally, the compositional and sparse nature of these datasets can impact the predictive accuracy of ML. We review recent literature from plant studies to illustrate that these properties often go unmentioned. We expand our analysis to other fields to quantify the degree to which mitigation approaches improve the performance of ML and describe the mathematical basis for this. With the advent of accessible analytical packages for microbiome data including learning models, researchers must be familiar with the nature of their datasets.

Peroxisome Proliferator-Activated Receptor Activation in Precision-Cut Bovine Liver Slices Reveals Novel Putative PPAR Targets in Periparturient Dairy Cows.

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.

When Two plus Two Is More than Four: Evidence for a Synergistic Effect of Fatty Acids on Peroxisome Proliferator-Activated Receptor Activity in a Bovine Hepatic Model.

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.

Advances in fatty acids nutrition in dairy cows: from gut to cells and effects on performance.

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.

The interplay between non-esterified fatty acids and bovine peroxisome proliferator-activated receptors: results of an in vitro hybrid approach.

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.

What's the norm in normalization? A frightening note on the use of RT-qPCR in the livestock science.

Reverse-Transcription quantitative PCR (RT-qPCR) provides a valuable tool to study gene expression with exquisite sensitivity. To retain its inferential power, user-introduced technical variability must be reduced and accounted for. Selecting a set of stably expressed internal control genes (ICG), validated for each experimental condition/sample set, is widely accepted as a reliable way to normalize RT-qPCR data and account for said variability. Despite significant efforts in establishing standardized and resource-efficient normalization approaches, numerous recent reports have underlined deficiencies in the state of RT-qPCR normalization. Livestock science has benefitted tremendously from the use of RT-qPCR; however, the issue of lack of proper normalization likely affects this discipline as well. We thus decided to determine whether this is true, and to which extent. We conducted an in-depth analysis of all (225) RT-qPCR articles published in the six most prominent livestock journals in the field from 2013 to 2017. A quantitative scale was constructed, and values were assigned to each article based on the number of ICG used, the use of a publicly available algorithm to assess the reliability of ICG, and the reporting of pertinent information related to ICG (ranges from 0 = total noncompliance - to 100 = total compliance). Out of the surveyed group, only 10.7% of the publications obtained a score of 100, while the largest group (n = 158) was represented by articles that scored 0. Subdividing articles based on whether an algorithm to validate ICG was used (YAL) or not (NAL) revealed the use of a larger number of ICG to normalize RT-qPCR in the YAL group compared to NAL (1.4-fold more, 95% C.I.: 1.11-1.84) and was closer to the "gold standard" of three ICG. Using an algorithm also increased the diversity of ICG and significantly reduced the use of RNA18S, whose suitability as ICG has been thoroughly debated. These remarkably low normalization standards are likely to generate questionable results that can severely hinder the advance of transcriptomic studies in livestock science and related fields.

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.

Transcriptional changes detected in fecal RNA of neonatal dairy calves undergoing a mild diarrhea are associated with inflammatory biomarkers.

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.