Effects of a mannan-rich yeast cell wall-derived preparation on cecal concentrations and tissue prevalence of Salmonella Enteritidis in layer chickens.

Salmonella Enteritidis (SE) has been the most common Salmonella serotype associated with foodborne infections in the last several years. Dietary applications of yeast-based preparations in feed have shown to reduce Salmonella colonization in chickens augmenting SE control strategies. This study was conducted to evaluate the effects of a mannan-rich yeast cell wall-derived preparation (Actigen®) administered in feed at a rate of 400 g/ton on SE colonization in the cecum and internal organs of commercial layer chickens. Sixteen week-old layer pullets were orally challenged with a selected nalidixic acid resistant SE strain at a dose of 1.7×10^9 colony forming units (CFU) per bird. SE colonization was assessed by evaluating isolation rates from ovary and pooled liver/spleen samples as well as enumeration of SE in cecal pouches one week post-challenge. Recovery rates of SE from the ovaries of directly challenged birds receiving Actigen® were significantly lower (P <0.02) than those in directly challenged birds fed an unsupplemented control diet. Recovery rates of SE from pooled liver/spleen samples were not significantly different between Actigen®-treated pullets and controls (P = 0.22). Using direct plate count methods, cecal SE concentrations were 1 log10 lower (P <0.001) in challenged pullets in the Actigen®-supplemented group than in the challenged controls. The SE concentration distributions in the ceca were similar in groups testing positive and groups testing negative for SE in the ovaries and liver/spleens tissues. As a result, SE concentrations in the ceca could not be directly related to the occurrence or prevalence of SE in these tissues. In conclusion, Actigen® supplementation appears to decrease the prevalence of SE in ovarian tissue and concentrations of SE in cecal contents and may be useful as a tool for reducing the risk of eggshell contamination and transovarian transmission of SE in eggs.

Can dietary zinc diminish the impact of necrotic enteritis on growth performance of broiler chickens by modulating the intestinal immune-system and microbiota?

The objective of this study was to compare the effects of inorganic and proteinate Zn in chickens challenged with coccidia and Clostridium perfringens. A 3 × 2 factorial design was used, with 3 dietary formulations (0 or 90 mg/kg supplemental Zn from ZnSO4 or Zn proteinate, with or without challenge). On day 14, challenged birds were orally gavaged with approx. 5,000 Eimeria maxima sporulated oocysts, and on day 19 to 21 with C. perfringens (108 CFU/D). Productive performance was assessed at day 21 and 28. At 21 D, necrotic enteritis (NE) lesion severity, intestinal permeability, gene expression, and ileal and cecal microbiota were evaluated. An interaction of Zn source by challenge was observed for lesion score and mortality, wherein Zn supplementation decreased the degree of NE lesions (P = 0.02) and mortality due to NE (P = 0.008). In the jejunum, an interaction of Zn source by challenge was observed for the expression of IL-8 (P = 0.001) and INF-γ (P = 0.03), wherein the NE challenge upregulated their expression, but not in the Zn proteinate supplemented birds. Zn proteinate supplementation downregulated iNOS vs. ZnSO4 supplemented birds (P = 0.0003), and supplemental Zn downregulated TLR-2 (P = 0.05) and ZnT5 (P = 0.04), regardless of the source. In the ileal microbiota, Zn proteinate supplementation decreased the frequency of Lactobacillus (P = 0.01), and the challenge increased Enterobacteriaceae (P = 0.01). Dietary Zn decreased NE lesion severity and mortality due to NE; Zn proteinate led to lower expression of IL-8 and INF-γ in challenged birds which may be an indicative of a lessened inflammatory response.

Zinc source modulates intestinal inflammation and intestinal integrity of broiler chickens challenged with coccidia and Clostridium perfringens.

Two dietary sources of zinc (ZnSO4 or organic Zn) were tested in chickens challenged with coccidiosis (Co) or coccidiosis plus Clostridium perfringens (CoCPF). On day 14, the chickens were orally gavaged with ∼5,000 Eimeria maxima sporulated oocysts. On day 19, 20, and 21 chickens challenged with C. perfringens were given a broth culture containing 108 cfu of this bacterium. Productive performance parameters were determined at d 14, 21, and 28. On day 21, necrotic enteritis (NE) lesions were scored, and intestinal permeability was evaluated. Jejunum and cecal tonsils were collected for morphology and gene expression analysis. On day 21, organic Zn improved BW gain by 18.6% (P = 0.07), and FCR by 12% (P = 0.09) in CoCPF challenged chickens vs. birds fed ZnSO4. From 1 to 28, organic Zn increased BW gain (P = 0.02), and improved FCR (P = 0.03) vs. birds fed ZnSO4. At 21 d, NE lesions were only observed in CoCPF birds (P < 0.001), and mortality due to NE was only observed when CoCPF birds were fed ZnSO4 (P = 0.001). Organic Zn fed birds had increased villus height in the jejunum (P = 0.005) and decreased intestinal permeability (P = 0.01) vs. ZnSO4. In the jejunum, organic Zn fed birds showed a downregulation of expression of IL-8 (P = 0.02), and upregulation of IL-10 (P = 0.05) in CoCPF birds vs. ZnSO4- CoCPF birds. As main effect, birds supplemented with organic Zn had higher mRNA expression of TLR-2 (P = 0.02) and IgA (P = 0.01). In the cecal tonsils, organic Zn fed birds showed upregulation of iNOS (P = 0.008) in CoCPF birds vs. ZnSO4-CoCPF birds. Organic Zn supplementation reduced intestinal permeability and attenuated intestinal inflammation of broilers co-challenged with coccidia and C. perfringens.

Dietary supplementation of weaned piglets with a yeast-derived mannan-rich fraction modulates cecal microbial profiles, jejunal morphology and gene expression.

The development of nutritional strategies to improve microbial homeostasis and gut health of piglets post-weaning is required to mitigate the high prevalence of post-weaning diarrhea and subsequent growth checks typically observed during the weaning transition. Therefore the objective of this study was to determine the effect of supplementing piglet creep and nursery feed with a yeast-derived mannan-rich fraction (MRF) on piglet growth performance, cecal microbial profiles, and jejunal morphology and gene expression. Ten litters of piglets (n=106) were selected on postnatal day (PND) 7 and assigned to diets with or without MRF (800 mg/kg) until weaning (n=5 litters/treatment; initial weight 3.0±0.1 kg). On PND 21, 4 piglets per litter (n=40) were selected and weaned into the nursery where they remained on their respective diets until PND 42. A two-phase feeding program was used to meet nutrient requirements, and pigs were switched from phase 1 to phase 2 on PND 28. Feed intake and piglet weights were recorded on PND 7, 14, 21, 28, 35 and 42. On PND 28 and 42, ten piglets per treatment were euthanized to collect intestinal tissue and digesta. Piglets supplemented with MRF had 21.5% greater (P<0.05) average daily feed intake between PND 14-21. However, MRF supplementation did not affect piglet growth performance compared to control. On PND 28, jejunal villus height was 16.8% greater (P<0.05) in piglets consuming MRF supplemented diets. Overall microbial community structure in cecal digesta on PND 28 tended to differ in pigs supplemented with MRF (P=0.076; analysis of similarities (ANOSIM)) with increased (P<0.05) relative abundance of Paraprevotellaceae genera YRC22 and CF231, and reduced (P<0.05) relative abundance of Sutterella and Prevotella. Campylobacter also tended to reduce (P<0.10) in MRF supplemented piglets. On PND 28 differential gene expression in jejunal tissue signified an overall effect of supplementing MRF to piglets. Downstream analysis of gene expression data revealed piglets supplemented with MRF had enriched biological pathways involved in intestinal development, function and immunity, supporting the observed improvement in jejunal villus architecture on PND 28. On PND 42 there was no effect of MRF supplementation on jejunal morphology or overall cecal microbial community structure. In conclusion, supplementing Actigen™, a MRF, to piglets altered cecal microbial community structure and improved jejunal morphology early post-weaning on PND 28, which is supported by enrichment of intestinal development pathways.

Zinc source influences the gene expression of zinc transporters in jejunum and cecal tonsils during broiler challenge with Eimeria maxima and Clostridium perfringens.

The objective of this experiment was to study the effects of dietary zinc (Zn) source on gene expression of Zn transporters (metallothionein [MT], ZIP 3, 5, 8, 9, 10, 11, 13, and 14, and ZnT 1, 4, 5, 6, 7, 9, and 10) in the jejunum and cecal tonsils of broilers challenged with coccidia or coccidia plus Clostridium perfringens. A 2 × 2 factorial design was used with 2 Zn sources (90 mg Zn/kg from either ZnSO4 or an organic Zn, Bioplex® Zn) and challenged with approximately 5,000 oocysts of Eimeria maxima at 14 d of age with or without C. perfringens (108 CFU/bird) at 18, 19, and 20 d of age (8 pens per treatment and 8 birds per pen) after which 1 bird/pen was sampled at 21 d of age. In the jejunum, co-infection resulted in higher ZnT 5 and 6 gene expression, while organic Zn fed birds had lower ZIP 5 and 11, and higher ZnT1. Additionally, an interaction of challenge by Zn source was noted wherein ZnT10 was unaffected by the C. perfringens in the organic Zn treatment but was 2.7-fold lower in the co-infected ZnSO4 fed birds. S100A9 gene expression, a biomarker of inflammatory response in necrotic enteritis, increased 2 and 2.8-fold in the cecal tonsils and jejunum with the co-infection, respectively. Supplementation with organic Zn lowered S100A9 by 1.9 and 4.4-fold in the cecal tonsils and jejunum, respectively, when birds were supplemented with ZnSO4. Notably, MT, ZIP 3, 8, 9, 10, 13, or 14, and ZnT 4, 7, and 9 were unaffected by Zn source and/or method of challenge. An interaction of challenge by Zn source was also noted for serum Zn concentration, which was reduced when birds were challenged with C. perfringens and fed ZnSO4 but no difference between challenge method when birds were fed organic Zn. Based on the expression of ZnT and ZIP genes, more Zn trafficking due to treatment occured in the jejunum than cecal tonsils, but further studies are needed to ascertain how Zn source regulates intracellular free Zn concentrations and whole-body Zn status during an enteric challenge.

Prepartal Energy Intake Alters Blood Polymorphonuclear Leukocyte Transcriptome During the Peripartal Period in Holstein Cows.

In the dairy industry, cow health and farmer profits depend on the balance between diet (ie, nutrient composition, daily intake) and metabolism. This is especially true during the transition period, where dramatic physiological changes foster vulnerability to immunosuppression, negative energy balance, and clinical and subclinical disorders. Using an Agilent microarray platform, this study examined changes in the transcriptome of bovine polymorphonuclear leukocytes (PMNLs) due to prepartal dietary intake. Holstein cows were fed a high-straw, control-energy diet (CON; NEL = 1.34 Mcal/kg) or overfed a moderate-energy diet (OVE; NEL = 1.62 Mcal/kg) during the dry period. Blood for PMNL isolation and metabolite analysis was collected at -14 and +7 days relative to parturition. At an analysis of variance false discovery rate <0.05, energy intake (OVE vs CON) influenced 1806 genes. Dynamic Impact Approach bioinformatics analysis classified treatment effects on Kyoto Encyclopedia of Genes and Genomes pathways, including activated oxidative phosphorylation and biosynthesis of unsaturated fatty acids and inhibited RNA polymerase, proteasome, and toll-like receptor signaling pathway. This analysis indicates that processes critical for energy metabolism and cellular and immune function were affected with mixed results. However, overall interpretation of the transcriptome data agreed in part with literature documenting a potentially detrimental, chronic activation of PMNL in response to overfeeding. The widespread, transcriptome-level changes captured here confirm the importance of dietary energy adjustments around calving on the immune system.

Prepartal dietary energy level affects peripartal bovine blood neutrophil metabolic, antioxidant, and inflammatory gene expression.

During the dry period, cows can easily overconsume higher-grain diets, a scenario that could impair immune function during the peripartal period. Objectives were to investigate the effects of energy overfeeding on expression profile of genes associated with inflammation, lipid metabolism, and neutrophil function, in 12 multiparous Holstein cows (n=6/dietary group) fed control [CON, 1.34 Mcal/kg of dry matter (DM)] or higher-energy (HE, 1.62 Mcal/kg of DM) diets during the last 45 d of pregnancy. Blood was collected to evaluate 43 genes in polymorphonuclear neutrophil leukocytes (PMNL) isolated at -14, 7, and 14 d relative to parturition. We detected greater expression of inflammatory-related cytokines (IL1B, STAT3, NFKB1) and eicosanoid synthesis (ALOX5AP and PLA2G4A) in HE cows than in CON cows. Around parturition, all cows had a close balance in mRNA expression of the pro-inflammatory IL1B and the anti-inflammatory IL10, with greater expression of both in cows fed HE than CON. The expression of CCL2, LEPR, TLR4, IL6, and LTC4S was undetectable. Cows in the HE group had greater expression of genes involved in PMNL adhesion, motility, migration, and phagocytosis, which was similar to expression of genes related to the pro-inflammatory cytokine. This response suggests that HE cows experienced a chronic state of inflammation. The greater expression of G6PD in HE cows could have been associated with the greater plasma insulin, which would have diverted glucose to other tissues. Cows fed the HE diet also had greater expression of transcription factors involved in metabolism of long-chain fatty acids (PPARD, RXRA), suggesting that immune cells might be predisposed to use endogenous ligands such as nonesterified fatty acids available in the circulation when glucose is in high demand for milk synthesis. The lower overall expression of SLC2A1 postpartum than prepartum supports this suggestion. Targeting interleukin-1ß signaling might be of value in terms of controlling the inflammatory response around calving. The present study revealed that overfeeding cows during late pregnancy results in activation, ahead of parturition, of PMNL responses associated with stress and inflammation. These adaptations observed in PMNL did not seem to be detrimental for production.

Intestinal gene expression profiles of piglets benefit from maternal supplementation with a yeast mannan-rich fraction during gestation and lactation.

The objective was to study the effect of maternal supplementation with a yeast cell wall-based product containing a mannan-rich fraction (MRF) during gestation and lactation on piglet intestinal gene expression. First parity sows were fed experimental gestation and lactation diets with or without MRF (900 mg/kg). After farrowing, piglets were fostered within treatment, as necessary. Sow and litter production performance data were collected until weaning. On day 10 post farrowing, jejunum samples from piglets were collected for gene expression analysis using the Affymetrix Porcine GeneChip array. Most performance parameters did not differ between the treatments. However, protein (P<0.01), total solids less fat (P<0.03) and the concentration of immunoglobulin G (IgG) in milk were greater (P<0.05) in the MRF-supplemented group. Gene expression results using hierarchical clustering revealed an overall dietary effect. Further analysis elucidated activation of pathways involved in tissue development, functioning and immunity, as well as greater cell proliferation and less migration of cells in the jejunum tissue. In conclusion, feeding the sow MRF during pregnancy and lactation was an effective nutritional strategy to bolster colostrum and milk IgG that are essential for development of piglet immune system and gut. In addition, the gene expression patterns affected by the passive immunity transfer showed indicators that could benefit animal performance long term.

Visceral adipose tissue mass in nonlactating dairy cows fed diets differing in energy density(1).

Our objective was to determine dietary energy effects on feed intake, internal fat deposition, body condition score (BCS), visceral organ mass, and blood analytes in Holstein cows. Eighteen nonpregnant, nonlactating cows (BCS = 3.04 ± 0.25) were blocked based on initial BCS and were randomly assigned within each block to 2 treatments. Treatments were either high energy [HE; net energy for lactation (NEL)=1.62 Mcal/kg] or low energy (LE; NEL = 1.35 Mcal/kg) diets fed as total mixed rations for 8 wk. The LE diet consisted of 81.7% forage, including 40.5% wheat straw and 28.3% corn silage, whereas the HE diet contained 73.8% forage with no straw and 49.9% corn silage (dry matter basis). Cows were fed for ad libitum intake once daily at 0800 h. Feed intake was recorded daily, blood was sampled at wk 1, 4, and 7, and BCS was assigned at wk 1, 4, and 7. Cows were killed following the 8-wk period, and visceral organs, mammary gland, and internal adipose tissues were weighed and sampled. The HE group had greater dry matter intake (15.9 vs. 11.2 ± 0.5 kg/d) and energy intakes than cows fed LE, but neutral detergent fiber intake did not differ (5.8 vs. 5.6 ± 0.25 kg/d for HE and LE). Final body weight was greater for cows fed HE (807 vs. 750 kg), but BCS did not differ between groups (3.52 vs. 3.47 for HE and LE). Omental (26.8 vs. 15.2 ± 1.6 kg/d), mesenteric (21.5 vs. 11.2 ± 1.9 kg), and perirenal (8.9 vs. 5.4 ± 0.9 kg) adipose tissue masses were larger in HE cows than in LE cows. Although subcutaneous adipose mass was not measured, carcass weight (including hide and subcutaneous fat) did not differ between HE (511 kg) and LE (496 kg). Liver weight tended to be greater for cows fed HE, but weights of gastrointestinal tract, heart, and kidney did not differ. Serum insulin tended to be greater and the glucose to insulin ratio was lower for cows fed HE. Serum concentrations of ß-hydroxybutyrate and cholesterol were greater for HE cows than for LE cows but concentrations of glucose, nonesterified fatty acids, total protein, and albumin did not differ. Final BCS was correlated with masses of omental (r = 0.57), mesenteric (r = 0.59), and perirenal (r = 0.72) adipose tissue, but mesenteric adipose mass increased more as BCS increased for cows fed HE. The similar final BCS between HE and LE cows demonstrates that BCS may lack sensitivity to detect differences in visceral fat deposition that might increase risk for peripartal diseases and disorders.

Postpartal immunometabolic gene network expression and function in blood neutrophils are altered in response to prepartal energy intake and postpartal intramammary inflammatory challenge.

The effect of over-feeding energy prepartum on blood polymorphonuclear neutrophil (PMN) response remains unclear. Cows fed controlled (CON; 1.34Mcal/kg of dry matter) or excess energy (OVE; 1.62Mcal/kg dry matter) during the dry period (~45d before expected calving date) received an intramammary (IM) challenge with Escherichia coli lipopolysaccharide (LPS) during the postpartal period to determine the effects of IM LPS and prepartal diet on the expression of key genes associated with immunometabolic response in blood PMN. Feed intake and daily milk yield were recorded throughout the study period. At 7d in milk (DIM), all cows received LPS (200µg) into 1 rear mammary quarter. Blood PMN were isolated at 7, 14, and 30 DIM, as well as before (0h) and after (12h) IM LPS challenge for gene expression analysis using quantitative real time PCR. Phagocytosis capabilities in vitro were assessed at 7, 14, and 30 DIM. Data were analyzed using the MIXED procedure of SAS with repeated measures. No differences in feed intake and milk yield were observed between OVE- and CON-fed cows. As expected, IM LPS challenge altered the expression of genes associated with the immune response (e.g., 1.9- and 1.8-fold for SELL and TLR2, respectively), metabolism (e.g., 1.8- and -1.8-fold for LDHA and SLC2A1, respectively), and transcription (e.g., 1.1- and 1.7-fold for NCOR1 and PPARD, respectively). At 12h postchallenge, an upregulation of TLR2 (1.8-fold), HIF1A (1.9-fold), and NFKB1 (1.5-fold) was observed for OVE rather than CON. At 7 DIM, S100A9 tended (2.2-fold) to be upregulated for OVE rather than CON. At 14 DIM, OVE resulted in lower PMN phagocytosis and an upregulation of NCOR2 (1.6-fold) and RXRA (1.9-fold) compared with CON-fed cows. At 30 DIM, an upregulation of MPO (3.5-fold) and PLA2G4A (1.5-fold) and a tendency for RXRA (1.7-fold) was observed for OVE- rather than CON-fed cows. Our results suggest that IM LPS challenge altered gene expression associated with metabolism in PMN and that OVE impaired PMN phagocytosis and increased the expression of immunometabolic genes after IM LPS challenge and during the postpartal period. The current study provides new linkages among prepartal feed energy intake, metabolism, and immune response of blood PMN and risk of disease during early lactation.

Comparison of gene expression profiles of the jejunum of broilers supplemented with a yeast cell wall-derived mannan oligosaccharide versus bacitractin methylene disalicylate.

1. The addition of yeast cell wall (YCW) mannan fractions or low concentrations of antibiotics to the diet of broilers positively affects gut health by improving intestinal cell morphology and improves feed efficiency and performance; however the exact mechanisms are unclear. Based on these production responses, the objective of this study was to compare the effects of supplementing YCW and bacitracin methylene disalicylate (BMD) in the diet on mRNA levels in the jejunum of 6-week-old broilers. 2. Dietary treatments were a maize-soya control diet and the control diet with the addition of YCW or BMD. Birds (n = 7) from each dietary treatment were randomly selected and killed at d 42. Whole jejunum (with serosa) samples were collected for RNA isolation. Gene expression analysis was performed using the AffymetrixGeneChip Chicken Genome Array (Santa Clara, CA, USA). 3. Supplementation with YCW resulted in 928 genes that were significantly changed (456 down-regulated, 472 up-regulated) and supplementation with BMD resulted in 857 genes that significantly changed (408 down-regulated, 449 up-regulated). In addition, 316 genes were significantly changed by both YCW and BMD (146 down-regulated, 170 up-regulated). 4. BMD increased the expression of genes involved in lipid and carbohydrate metabolism and decreased expression of genes associated with T-helper cell pathways. Gene expression profiles from birds fed on diets containing YCW showed changes on a genomic level that correspond to slower gut cell turnover and therefore increased energy preservation for growth. 5. In conclusion, supplementation with BMD or YCW had similar influences on the number of differentially expressed genes in the jejunum. Biological functions common to both YCW and BMD with positive activation scores included antiviral response and antimicrobial response. Genes that were affected by BMD or YCW classified into both different and common biological functions and pathways related to improved metabolism and health in the jejunum.

Liver lipid content and inflammometabolic indices in peripartal dairy cows are altered in response to prepartal energy intake and postpartal intramammary inflammatory challenge.

This study evaluated the effect of feeding a control diet (CON) or a moderate energy diet (overfed, OVE) during the dry period (∼45d) and a postpartum intramammary lipopolysaccharide (LPS) challenge on blood metabolic and inflammatory indices, milk production, and hepatic gene expression. A subset of cows (n=9/diet) in CON (1.34 Mcal/kg of dry matter) and OVE (1.62 Mcal/kg of dry matter) received an intramammary LPS challenge (200 µg; CON-LPS, OVE-LPS, respectively). Liver biopsies were harvested at -14 d from calving, and postpartum at 2.5h post-LPS on d 7, 14, and 30. Prepartum, the OVE group was in more positive energy balance (EB) and had greater body condition score compared with CON. In contrast, during wk 1 postpartum and before the LPS challenge, the OVE group was in greater negative EB than CON. Prepartal diet did not affect postpartal milk production or dry matter intake. At 2h postchallenge on d 7, we observed an increase in serum nonesterified fatty acids (NEFA) and bilirubin and a decrease in hydroxybutyrate, regardless of diet. That was coupled with greater haptoglobin in OVE-LPS compared with CON-LPS. In addition, OVE-LPS cows versus CON nonchallenged, OVE nonchallenged, and CON-LPS had greater liver triacylglycerol (TAG) concentration 2.5h postchallenge on d 7. The concentration of TAG in liver of OVE-LPS remained elevated by 30d postpartum. The liver TAG concentration in OVE-LPS compared with CON-LPS cows was associated with greater serum concentration of NEFA and reactive oxygen metabolites on d 10 and 14 postpartum. Cows in OVE-LPS also had greater concentrations of ceruloplasmin, cholesterol, and vitamin E from d 10 through 21. Among 28 genes associated with fatty acid oxidation, inflammation, oxidative stress, and gluconeogenesis, only SAA3 (which encodes an acute phase protein) was greater in CON-LPS compared with OVE-LPS at 2.5h postchallenge. Expression of HP, which encodes another acute phase protein, was greater in OVE-LPS than in CON-LPS at 14 and 30 d postpartum. Several inflammation-related genes (TNF, IRAK1, NFKB1, ANGPTL4) showed markedly decreased expression between 7 and 14 d, after which expression remained unchanged. No differences were observed in several genes of the growth-hormone/insulin-like growth factor-1 axis, except for SOCS2, expression of which decreased markedly between 7 and 14 d in OVE-LPS but not in CON-LPS. These data suggest that overfeeding a moderate-energy diet prepartum alters the response of the cow to an intramammary challenge after calving and may predispose it to sustained liver lipidosis.

Corium tissue expression of genes associated with inflammation, oxidative stress, and keratin formation in relation to lameness in dairy cows.

Objectives were to (1) determine the feasibility of performing hoof biopsies without impairing locomotion; (2) evaluate the feasibility of using biopsied tissue for quantitative PCR; and (3) compare relative gene expression among claws for several target genes. Biopsies were performed on 6 Holstein cows, yielding 4 tissue specimens per cow from front leg, right limb, and medial claw (claw position 3); rear leg, left limb, and lateral claw (claw position 5); and rear leg, right limb, medial claw (claw position 7). Cows were monitored for lameness daily for 7 d post-biopsy and then weekly for 8 wk. Histopathological analysis confirmed that tissue collected was from between the stratum corneum and dermis. Biopsied tissue was used for RNA extraction, including evaluation of yield and purity. The profile by claw position of 19 genes with key functions in cell differentiation, proliferation, inflammation, and keratin formation was assessed via quantitative reverse transcription-PCR. Other than transient disturbances in locomotion score in some cows during 2 to 4 d post-biopsy, no signs of pain, locomotion impairment, or clinical lameness were observed post-biopsy. Total RNA yields averaged 259.7±100, 447.8±288, and 496.4±118 µg/mg of tissue for claw positions 3, 5, and 7, respectively. The biopsy procedure was successful for obtaining corium for gene expression. Among 5 keratin proteins analyzed, only keratin 5 was expressed. Transcripts related to inflammation and oxidative stress (STAT3, MYD88, SOD2, and TLR4) were among the more abundant in corium tissue, but expression did not differ between claws. Biotinidase (BTD) expression was greater in claw 3 versus claw 5, whereas the ligand-activated nuclear receptor retinoic acid receptor-α (RXRA) was greater in claws 3 + 5 compared with claw 7. Overall, results from this pilot study revealed modest differences at the transcriptome level, suggesting that biotin availability and lipid metabolism differ between claw positions, whereas inflammation and oxidative stress seem to play an important role across claws. More comprehensive studies of the hoof transcriptome are required to improve our understanding of the mechanisms that link environmental and dietary factors to development of lameness.

Blood immunometabolic indices and polymorphonuclear neutrophil function in peripartum dairy cows are altered by level of dietary energy prepartum.

Cows experience some degree of negative energy balance and immunosuppression around parturition, making them vulnerable to metabolic and infectious diseases. The effect of prepartum feeding of diets to meet (control, 1.34 Mcal/kg of dry matter) or exceed (overfed, 1.62 Mcal/kg of dry matter) dietary energy requirements was evaluated during the entire dry period (∼45 d) on blood polymorphonuclear neutrophil function, blood metabolic and inflammatory indices, and milk production in Holstein cows. By design, dry matter intake in the overfed group (n=9) exceeded energy requirements during the prepartum period (-4 to -1 wk relative to parturition), resulting in greater energy balance when compared with the control group (n=10). Overfed cows were in more negative energy balance during wk 1 after calving than controls. No differences were observed in dry matter intake, milk yield, and milk composition between diets. Although nonesterified fatty acid concentration pre- (0.138 mEq/L) and postpartum (0.421 mEq/L) was not different between diets, blood insulin concentration was greater in overfed cows prepartum (16.7 µIU/mL) compared with controls pre- and postpartum (∼3.25 µIU/mL). Among metabolic indicators, concentrations of urea (4.63 vs. 6.38 mmol/L), creatinine (100 vs. 118 µmol/L), and triacylglycerol (4.0 vs. 8.57 mg/dL) in overfed cows were lower prepartum than controls. Glucose was greater pre- (4.24 vs. 4.00 mmol/L) and postpartum (3.49 vs. 3.30 mmol/L) compared with control cows. Among liver function indicators, the concentration of bilirubin increased by 2 to 6 fold postpartum in control and overfed cows. Phagocytosis capacity of polymorphonuclear neutrophils was lower prepartum in overfed cows (32.7% vs. 46.5%); phagocytosis in the control group remained constant postpartum (50%) but it increased at d 7 in the overfed group to levels similar to controls (48.4%). Regardless of prepartum diet, parturition was characterized by an increase in nonesterified fatty acid and liver triacylglycerol, as well as blood indices of inflammation (ceruloplasmin and haptoglobin), oxidative stress (reactive oxygen metabolites), and liver injury (glutamic oxaloacetic transaminase). Concentrations of the antioxidant and anti-inflammatory compounds vitamin A, vitamin E, and ß-carotene decreased after calving. For vitamin A, the decrease was observed in overfed cows (47.3 vs. 27.5 µg/100 mL). Overall, overfeeding energy and higher energy status prepartum led to the surge of insulin and had a transient effect on metabolism postpartum.

Endocannabinoid system and proopiomelanocortin gene expression in peripartal bovine liver in response to prepartal plane of nutrition.

Endocannabinoids are fatty acid amides (FAE; oleoylethanolamide and anandamide) which have orexigenic, anorexigenic or anti-inflammatory properties. We examined mRNA expression via qPCR of endocannabinoid receptors (CNR1 and CNR2), enzymes that synthesize FAE (HRASLS5 and N-acyl phosphatidylethanolamine phospholipase D), enzymes that degrade FAE [fatty acid amide hydrolase (FAAH), N-acylethanolamine acid amidase (NAAA) and monoglyceride lipase (MGLL)], and the hormone precursor proopiomelanocortin (POMC) in liver at -14, 7, 14 and 30 days around parturition from cows fed with a control (CON; NE(L) = 1.34 Mcal/kg) or moderate-energy (OVER; NE(L) = 1.62 Mcal/kg) diet during the dry period. Expression of CNR2 and POMC was greater at 7 days in cows fed with OVER because of a decrease in expression between -14 and 7 days in cows fed with CON. Cows fed with CON had an increase in expression of FAAH, HRASLS5, NAA, MGLL and POMC between 7 and 14 days; for FAAH and HRASLS5, such response led to greater expression at 14 days vs. cows fed with OVER. Cows fed with OVER vs. CON had a approximately twofold increase in expression of MGLL between -14 and 7 days followed by a gradual decrease through 30 days at which point expression was still greater in OVER vs. CON. FAAH, MGLL and HRASLS5 were the most abundant genes measured. Expression of the hepatic endocannabinoid system and POMC was altered by plane of dietary energy prepartum particularly during the first 2-week postpartum. Such responses may play a role in the physiological adaptations to the onset of lactation, including energy balance and feed intake.

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

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

Adipose tissue lipogenic gene networks due to lipid feeding and milk fat depression in lactating cows.

Dietary lipid supplements have been extensively evaluated for their effects on mammary tissue mRNA abundance, including the classical lipogenic genes ACACA, SCD, FASN, and the transcription regulators SREBF1, THRSP, and PPARG. Novel gene isoforms with key regulatory roles in triacylglycerol synthesis have been recently identified including LPIN1 and AGPAT6. Transcriptional networks (i.e., genes whose mRNA expression is regulated by a transcription factor or nuclear receptor) coordinate adipogenesis and lipid filling in nonruminant adipose tissue. To investigate whether long-term milk fat depression affects adipogenic networks in subcutaneous adipose tissue, we characterized mRNA expression via quantitative PCR of 20 genes in cows fed saturated and polyunsaturated lipid for 3 wk. Adipose tissue from cows fed a control diet, control with fish (10 g/kg of dry matter) and soybean oil (25 g/kg of dry matter) (FSO), or control with saturated lipid (35 g/kg, EB100; Energy Booster 100, Milk Specialties, Dundee, IL) was biopsied after 21 d of feeding. Milk production did not differ across treatments (averaged 32 kg +/- 2.8 kg/d during the 21 d) but dry matter intake (DMI) decreased in cows fed FSO versus controls (averaged 18 vs. 22 kg/d during the 21 d). Despite the decrease in DMI, FSO resulted in similar energy intake as EB100 during the last 2 wk of the study. Cows fed FSO had a gradual decline in milk fat and energy yield leading to an overall 25% decrease in milk fat yield during the study (averaged 0.90 vs. 1.2 kg/d) compared with control or EB100. Thus, during the 21-d study, FSO led to a gradual increase in intake energy available for adipose tissue deposition. Relative mRNA expression of LPL and SCD as well as ADFP (coding for a protein involved in lipid droplet formation) and LPIN1 (coding for a protein involved in diacylglycerol synthesis/transcriptional regulation) was upregulated with FSO relative to other diets. Expression of the transcription regulator THRSP tended to be greater in cows fed FSO. Overall, results suggest that long-term milk fat depression caused by feeding FSO provided additional energy as well as long-chain fatty acids that, coupled with upregulation of a subset of adipogenic genes in subcutaneous adipose tissue, might have resulted in greater tissue lipid deposition.

Internal controls for quantitative polymerase chain reaction of swine mammary glands during pregnancy and lactation.

High-throughput microarray analysis is an efficient means of obtaining a genome-wide view of transcript profiles across physiological states. However, quantitative PCR (qPCR) remains the chosen method for high-precision mRNA abundance analysis. Essential for reliability of qPCR data is normalization using appropriate internal control genes (ICG), which is now, more than ever before, a fundamental step for accurate gene expression profiling. We mined mammary tissue microarray data on >13,000 genes at -34, -14, 0, 7, 14, 21, and 28 d relative to parturition in 27 crossbred primiparous gilts to identify suitable ICG. Initial analysis revealed TBK1, PCSK2, PTBP1, API5, VAPB, QTRT1, TRIM41, TMEM24, PPP2R5B, and AP1S1 as the most stable genes (sample/reference = 1 +/- 0.2). We also included 9 genes previously identified as ICG in bovine mammary tissue. Gene network analysis of the 19 genes identified AP1S1, API5, MTG1, VAPB, TRIM41, MRPL39, and RPS15A as having no known co-regulation. In addition, UXT and ACTB were added to this list, and mRNA abundance of these 9 genes was measured by qPCR. Expression of all 9 of these genes was decreased markedly during lactation. In a previous study with bovine mammary tissue, mRNA of stably expressed genes decreased during lactation due to a dilution effect brought about by large increases in expression of highly abundant genes. To verify this effect, highly abundant mammary genes such as CSN1S2, SCD, FABP3, and LTF were evaluated by qPCR. The tested ICG had a negative correlation with these genes, demonstrating a dilution effect in the porcine mammary tissue. Gene stability analysis identified API5, VABP, and MRPL39 as the most stable ICG in porcine mammary tissue and indicated that the use of those 3 genes was most appropriate for calculating a normalization factor. Overall, results underscore the importance of proper validation of internal controls for qPCR and highlight the limitations of using absence of time effects as the criteria for selection of appropriate ICG. Further, we showed that use of the same ICG from one organism might not be suitable for qPCR normalization in other species.