B. J. Johnson

Daily Injection of Tumor Necrosis Factor-α Increases Hepatic Triglycerides and Alters Transcript Abundance of Metabolic Genes in Lactating Dairy Cattle

To determine whether inflammation can induce bovine fatty liver, we administered recombinant bovine tumor necrosis factor-alpha (rbTNF) to late-lactation Holstein cows. Cows (n = 5/treatment) were blocked by feed intake and parity and randomly assigned within block to control (CON; saline), rbTNF at 2 microg/(kg.d), or pair-fed control (saline, intake matched) treatments. Treatments were administered once daily by subcutaneous injection for 7 d. Plasma samples were collected daily for analysis of glucose and FFA and a liver biopsy was collected on d 7 for triglyceride (TG) and quantitative RT-PCR analyses. Data were analyzed using treatment contrasts to assess effects of tumor necrosis factor-alpha (TNFalpha) and decreased feed intake. By d 7, feed intake of both rbTNF and pair-fed cows was approximately 15% less than CON (P < 0.01). Administration of rbTNF resulted in greater hepatic TNFalpha mRNA and protein abundance and 103% higher liver TG content (P < 0.05) without affecting the plasma FFA concentration. Hepatic carnitine palmitoyltransferase 1 transcript abundance tended to be lower (P = 0.09) and transcript abundance of fatty acid translocase and 1-acyl-glycerol-3-phosphate acyltransferase was higher (both P < 0.05) after rbTNF treatment, consistent with increased FFA uptake and storage as TG. Transcript abundance of glucose-6-phosphatase (P < 0.05) and phosphoenolpyruvate carboxykinase 1 (P = 0.09), genes important for gluconeogenesis, was lower for rbTNF-treated cows. These findings indicate that TNFalpha promotes liver TG accumulation and suggest that inflammatory pathways may also be responsible for decreased glucose production in cows with fatty liver.

Omega-3 fatty acids in the gravid pig uterus as affected by maternal supplementation with omega-3 fatty acids

Two experiments evaluated the ability of maternal fatty acid supplementation to alter conceptus and endometrial fatty acid composition. In Exp. 1, treatments were 1) the control, a corn-soybean meal diet; 2) flax, the control diet plus ground flax (3.75% of diet); and 3) protected fatty acids (PFA), the control plus a protected fish oil source rich in n-3 PUFA (Gromega, JBS United Inc., Sheridan, IN; 1.5% of diet). Supplements replaced equal parts of corn and soybean meal. When gilts reached 170 d of age, PG600 (PMSG and hCG, Intervet USA, Millsboro, DE) was injected to induce puberty, and dietary treatments (n = 8/treatment) were initiated. When detected in estrus, gilts were artificially inseminated. On d 40 to 43 of gestation, 7 gilts in the control treatment, 8 gilts in the PFA treatment, and 5 gilts in the flax treatment were pregnant and were slaughtered. Compared with the control treatment, the flax treatment tended to increase eicosapentaenoic acid (EPA: C20:5n-3) in fetuses (0.14 vs. 0.25 +/- 0.03 mg/g of dry tissue; P = 0.055), whereas gilts receiving PFA had more (P < 0.05) docosahexaenoic acid (DHA: C22:6n-3) in their fetuses (5.23 vs. 4.04 +/- 0.078 mg/g) compared with gilts fed the control diet. Both the flax and PFA diets increased (P < 0.05) DHA (0.60, 0.82, and 0.85 +/- 0.078 mg/g for the control, flax, and PFA diet, respectively) in the chorioallantois. In the endometrium, EPA and docosapentaenoic acid (C22:5n-3) were increased by the flax diet (P < 0.001; P < 0.05), whereas gilts receiving PFA had increased DHA (P < 0.001). The flax diet selectively increased EPA, and the PFA diet selectively increased DHA in the fetus and endometrium. In Exp. 2, gilts were fed diets containing PFA (1.5%) or a control diet beginning at approximately 170 of age (n = 13/treatment). A blood sample was collected after 30 d of treatment, and gilts were artificially inseminated when they were approximately 205 d old. Conceptus and endometrial samples were collected on d 11 to 19 of pregnancy. Plasma samples indicated that PFA increased (P < 0.005) circulating concentrations of EPA and DHA. Endometrial EPA was increased (P < 0.001) for gilts fed the PFA diet. In extraembryonic tissues, PFA more than doubled (P < 0.001) the EPA (0.13 vs. 0.32 +/- 0.013 mg/g) and DHA (0.39 vs. 0.85 +/- 0.05 mg/g). In embryonic tissue on d 19, DHA was increased (P < 0.05) by PFA (0.20 vs. 0.30 +/- 0.023 mg/g). Supplementing n-3 PUFA, beginning 30 d before breeding, affected endometrial, conceptus, and fetal fatty acid composition in early pregnancy. Dynamic day effects in fatty acid composition indicate this may be a critical period for maternal fatty acid resources to affect conceptus development and survival.

Tissue expression of angiopoietin-like protein 4 in cattle

Angiopoietin-like protein 4 (ANGPTL4; also known as fasting-induced adipose factor) is a plasma protein that stimulates oxidation of fatty acids and inhibits fat accumulation. The gastrointestinal tract appears to play an important role in regulating plasma ANGPTL4 concentration in some situations and may be influenced by microbes within the gastrointestinal tract. Our aim was to determine which tissues express ANGPTL4 in the bovine. Rumen, omasum, abomasum, duodenum, jejunum, ileum, colon, pancreas, liver, and subcutaneous adipose tissue samples were collected postmortem from 2 steers. Abundance of ANGPTL4 messenger RNA was quantified by quantitative real-time PCR, and was most abundant in liver and adipose tissue (P < 0.05). We also detected ANGPTL4 messenger RNA throughout the gastrointestinal tract, although its abundance was approximately 10% of that found in liver and adipose tissue. Western blot analysis revealed that ANGPTL4 protein was most abundant in liver and adipose tissue (P < 0.05), but omasal, abomasal, and ileal samples contained at least 60% as much ANGPTL4 protein as the liver and adipose tissue samples, and the protein was detected in all tissues. Finally, cross-sections of the liver, pancreas, and rumen wall were used for indirect immunofluorescent detection of ANGPTL4. Despite the low abundance of ANGPTL4 measured by quantitative real-time PCR and Western blot in ruminal tissue, immunofluorescence demonstrated that expression of ANGPTL4 in ruminal epithelial cells was equivalent to or greater than that in liver hepatocytes. These findings indicate that, as in other species studied, liver and adipose tissue are key sources of ANGPTL4 in cattle. However, the protein was also highly abundant in ruminal epithelium, making it possible that commensal microbes may influence ANGPTL4 synthesis and secretion in the ruminant gastrointestinal tract.

Maternal Dietary l-Carnitine Supplementation Influences Fetal Carnitine Status and Stimulates Carnitine Palmitoyltransferase and Pyruvate Dehydrogenase Complex Activities in Swine

Effects of increasing maternal L-carnitine on carnitine status and energy metabolism in the fetus were evaluated by feeding pregnant swine a corn-soybean-based diet containing either 0 or 50 mg/kg added L-carnitine (n = 10/treatment) during the first 70 d of gestation. Carnitine, carnitine palmitoyltransferase (CPT), and pyruvate dehydrogenase complex (PDHC) activities were analyzed in tissues collected from fetuses on d 55 and 70. Maternal L-carnitine supplementation increased both fetal free and long-chain carnitine concentrations by 45% in liver and free carnitine by 31% in heart tissues but did not affect kidney tissue. Elevations in free and acylcarnitines increased with gestational age from 55 to 70 d in liver but not in heart and kidney. The increased carnitine concentrations resulted in a 45% increase in PDHC activity in heart and liver on d 70 of gestation but did not affect kidney and liver on d 55 of gestation. The increases in carnitine concentrations were accompanied by a 70% increase in hepatic CPT activity in 70-d-old fetuses, but activities in heart and kidney were unaffected. The Michaelis constant (K(m)) of CPT for carnitine in fetal tissues was not influenced by carnitine supplementation (P > 0.1). Notably, the concentrations of carnitine measured on d 70 were only 25-40% of the K(m) values in liver, 60-70% in heart, and 30-40% in kidney (P < 0.001). We conclude that carnitine ingestion during pregnancy increases fetal carnitine concentrations and stimulates heart PDHC and liver CPT activity without altering carnitine K(m).

Effects of feeding L-carnitine to gilts through day 70 of gestation on litter traits and the expression of insulin-like growth factor system components and L-carnitine concentration in foetal tissues.

We investigated the influence of supplemental L-carnitine on foetal blood metabolites, litter characteristics, L-carnitine concentration in skeletal muscle and insulin-like growth factor (IGF) axis components in foetal hepatic and skeletal muscle tissues at day 40, 55 and 70 of gestating gilts. A total of 59 gilts (body weight = 137.7 kg) received a constant feed allowance of 1.75 kg/day and a top-dress containing either 0 or 50 ppm of L-carnitine starting on the first day of breeding through the allotted gestation length. Foetuses from the gilts fed diets with L-carnitine tended to be heavier (p = 0.06) and the circulating IGF-II tended to be lower (p = 0.09) at day 70, compared with the foetuses from the control gilts. Insulin-like growth factor-I messenger RNA (mRNA) was lower (p = 0.05) in hepatic tissue in the foetuses collected from gilts fed L-carnitine. Free and total carnitine concentration increased (p < 0.05) in the skeletal muscle from the foetuses collected from gilts fed supplemental L-carnitine. This study showed that L-carnitine had beneficial effects on the average foetal weight at day 70 of gestation, associated with changes in the foetal IGF system.

Effects of flax supplementation and a Revalor-S implant on circulating insulin-like growth factor 1 (IGF-1) and muscle IGF-1 mRNA levels in finishing cattle

Sixteen crossbred steers weighing 875 lb were used to evaluate the effects of a 5% ground flaxseed supplement and a combined trenbolone acetate/estradiol (TBA/E2) growth promoting implant, Revalor-S, on both circulating insulin-like growth factor-1 (IGF-1) and local muscle IGF-1 mRNA concentrations. Steers were randomly assigned to one of four treatments: . 1) Flax/Implant, 2) No Flax/ Implant, 3) Flax/No Implant, 4) No Flax/No Implant. Serum was harvested from blood collected via jugular venipuncture on day 0 (before implantation or flax addition), 14, and 28. Muscle biopsy samples were obtained from the longissimus muscle on days 0, 14, and 28. Implanted steers had 52 and 84% higher (P<0.05) circulating IGF-1 levels than nonimplanted steers on days 14 and 28, respectively. Cattle fed diets without flax had higher levels of muscle IGF-1 mRNA than cattle fed diets with flax on day 28 (4.4-fold, P<0.01). Our data support that the administration of a combined TBA/E2 growth promotant increases circulating IGF-1 and local muscle IGF-1 mRNA concentrations in finishing cattle. However, this increase in muscle IGF-1 mRNA appears to be attenuated by the addition of a dietary flax supplement.

L-carnitine supplementation to gestating gilts alters the IGF axis in porcine embyronic myoblasts.

We determined the effects of supplemental L-carnitine on the insulin-like growth factor (IGF) system in porcine embryonic myoblasts (PEM) from gilts. Forty gilts (BW = 303.6 lb) were allotted to 1 of 4 treatments that were arranged in a 2 × 2 factorial, with main effects of L-carnitine (0 or 50 ppm) and day of gestation (55 or 70). All gilts were fed 3.86 lb/day and a top-dress containing either 0 or 50 ppm of L-carnitine, starting on the first day of breeding and continuing through the allotted gestation length. At d 55 or 70 of gestation, fetuses were removed for isolation of PEM from the hind-limb muscles. Real-time quantitative PCR was used to determine growth factor messenger RNA (mRNA) expression in cultured PEM at 72-, 96-, 120-, and 144-h after plating. Flow cytometry was used to analyze percentage of myogenic cells with a myoblast/myotube-specific monoclonal antibody 5.1H11, and for determination of cell cycle stage. There was no treatment differences (P>0.10) for the expression of IGF-I, IGF-II, or IGFBP-5 mRNA levels. But PEM isolated from fetuses collected from gilts fed diets with L-carnitine had lower (P = 0.08) IGFBP-3 mRNA levels, compared with levels in the controls. Myoblasts isolated from fetuses from gilts fed diets with added Lcarnitine had greater (P = 0.09; 8.8%) 5.1H11 monoclonal antibody attachment, compared with the controls, after 72 hours in culture (91.8% vs. 87.4%). Although not significant (P = 0.31), the total number of PEM in the S phase of the cell cycle was 4.7% greater in PEM collected from fetuses obtained from gilts fed diets with L-carnitine, compared with numbers from the control-fed gilts (37.5% vs. 34.2%). These data suggest that L-carnitine influences the IGF system, stage of the cell cycle, and recognition of muscle development, resulting in enhanced proliferation and delayed differentiation of PEM.

Influence of L-carnitine on growth and plasma IGF-I from gilts harvested at three gestation lengths.

A total of 59 gilts were used to determine the effects of supplemental L-carnitine on gilt growth and maternal insulin-like growth factor-I (IGF-I). Experimental treatments were arranged in a 2 × 3 factorial with main effects of L-carnitine (0 or 50 ppm) and day of gestation (40, 55, or 70). All gilts received a constant feed allowance of 3.86 lb/day and a topdress containing either 0 or 88 mg of Lcarnitine, starting on the first day of breeding. No differences (P>0.05) between treatments were observed for BW, estimated protein mass, or estimated fat mass at any gestation length. At d 70 of gestation, there was a numeric increase (P>0.10) in BW for the gilts fed L-carnitine, compared with those fed the control diet. At d 40 of gestation, gilts fed Lcarnitine tended to have greater (P = 0.10) backfat, compared with the gilts fed the control diet; but no differences (P>0.05) were observed in backfat on d 0, 55, or 70 of gestation. In addition, no differences (P>0.05) were observed in maternal IGF-I between treatments at any gestation length. Total and free plasma L-carnitine concentrations were similar (P>0.10) at d 0 of gestation, but concentrations were higher (P<0.01) by d 40 of gestation in the gilts fed L-carnitine. These results show that supplemental L-carnitine numerically increases BW of gestating gilts. This data represents the first part of an ongoing study, with the rest of the data being reported in subsequent publications.

Feeding L-carnitine to gestating sows alters the insulin-like growth-factor system in cultured porcine embryonic muscle cells isolated from fetal skeletal muscle

The objective was to determine the effects of L-carnitine on cell proliferation and on messenger RNA (mRNA) concentrations in the insulin-like growth factor (IGF) system. Cultured porcine embryonic myoblasts (PEM) were isolated from fetuses at mid-gestation from sows fed a common gestation diet with a 50-g top dress of 0 (control, n = 6) or 100 mg of L-carnitine (n = 6). Proliferation of PEM was evaluated at 36, 48, 60, and 72 h postplating. Real-time quantitative PCR was used to determine growth factor mRNA concentrations in culture. The number of cells/cm did not differ (P>0.05) from sows fed either diet, but the number of cells/cm increased (P 0.05). There was no treatment difference (P>0.05) for the expression of IGF-I or insulin-like growth factor binding protein 5 (IGFBP-5). But PEM isolated from sows fed L-carnitine had decreased (P<0.05) IGF-II, IGFBP-3, and myogenin (61, 59, and 67%, respectively) mRNA concentrations compared with those of controls. These data suggest that L-carnitine influences the IGF system and myogenin, resulting in enhanced proliferation and delayed differentiation of porcine embryonic myoblasts. These results show that L-carnitine plays a role in regulating proliferation and differentiation of cultured porcine embryonic myogenic cells and that fetal muscle growth and development could be increased by feeding L-carnitine.

Effect of melengestrol acetate (MGA) on cultured bovine muscle satellite cell proliferation and differentiation

Melengestrol acetate (MGA) increases growth rate and inhibits estrus in feedlot heifers. Little is known of MGA’s effect on skeletal muscle growth and differentiation. The purpose of this trial was to investigate the potential direct effects of MGA on cultured bovine muscle satellite cell proliferation and differentiation. Satellite cells isolated from yearling cattle were used to assess the effect of MGA in a dose titration (0, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM) study on [H]-thymidine incorporation. Likewise, satellite cell cultures were allowed to differentiate, and nuclei were stained at 168 hours to determine the effect of MGA (10 nM and 100 μM) addition during the first 48 hours on extent of differentiation and absolute myotube nuclei number. MGA addition resulted in a dose-dependent decrease (P<0.05) in DNA synthesis as measured by [H]-thymidine incorporation. MGA addition (10 nM) did not significantly alter the extent of differentiation or myotube nuclei number at 168 hours in culture even though this concentration reduced DNA synthesis. However, 100 μM MGA addition significantly (P<0.05) reduced both fusion percentage and myotube nuclei number as compared to control cultures. These data suggest MGA addition at concentration between 10 nM and 100 μM affected bovine muscle cell proliferation and differentiation. A better understanding of these effects will increase our knowledge of bovine muscle growth and development. Introduction