Effect of diets containing linoleic acid- or oleic acid-rich oils on ruminal fermentation and nutrient digestibility, and performance and fatty acid composition of adipose and muscle tissues of finishing cattle.

Two trials were conducted to determine the effect of linoleic acid- or oleic acid-rich safflower oil on ruminal fermentation, nutrient digestion, feedlot performance, carcass characteristics, and fatty acid composition of adipose and muscle tissues of beef cattle. In both trials, cattle were fed a finishing diet based on barley grain, wheat silage, and alfalfa hay. Oils were fed at 5% of dietary DM. In a metabolism trial, four ruminally and duodenally cannulated Angus crossbred steers were subjected to linoleic acid-rich oil or oleic acid-rich oil in a crossover design with covariate periods (no oil supplementation). In a finishing trial, 16 individually fed Angus crossbred steers and heifers (eight per diet) received linoleic acid- or oleic acid-rich oils during the last 86 d of a 116-d feeding period. Ruminal pH, ammonia concentration, protozoal counts, major VFA concentrations, acetate-to-propionate ratio, polysaccharide-degrading activities, microbial N flow to the duodenum, and the efficiency of microbial N synthesis in the rumen were not affected (P = 0.18 to 0.96) by type of oil. Type of oil had no effect on total-tract apparent digestion of nutrients (P = 0.46 to 0.98). Ruminal true nutrient digestibilities did not differ between oils (P = 0.15 to 0.99), except that the linoleic acid-rich oil decreased (P = 0.05) NDF digestibility. Dry matter intake, ADG, G:F, and carcass characteristics did not differ (P = 0.11 to 0.84) between the two oils. Overall, the difference in dietary fatty acids provided to the cattle produced few changes in tissue fatty acids. Weight percentages of c9t11 CLA were unaltered by the addition of linoleic acid to the diet compared with oleic acid, probably as a result of low vaccenic acid production in the rumen, as the pathway of biohydrogenation was apparently primarily through the t10 pathway.

Effect of cutting height and genetics on composition, intake, and digestibility of corn silage by beef heifers.

This research was conducted to determine the effect of corn genetics and cutting height on the composition and nutritive characteristics of corn silage. An in situ study involving eight commercially available corn hybrids indicated main effects and interactions (P < 0.01) of hybrid and cutting height on NDF, ADF, and starch content and on in situ DM and NDF degradablility. Four ruminally cannulated Angus heifers (initial BW = 378 +/- 3 kg) were used in a 4 x 4 Latin square digestion experiment with a 2 x 2 factorial treatment arrangement. Main effects and interactions of hybrid (Pioneer Hi-Bred Int., Inc., hybrids 3335 and 3223) and cutting height (LO = 20.3 cm, and HI = 61 cm) were evaluated. Dietary treatment consisted of 40% chopped alfalfa hay and 60% corn silage. Although corn silage hybrids used were of equivalent maturity at harvest (60% milkline), 3335 treatments had 37.8% starch and 34.8% NDF, whereas 3223 treatments had 33.7% starch and 38.6% NDF. The LO treatments averaged 3.1 percentage units greater in NDF and 3.45 percentage units less in starch content than the HI treatments. Intake of DM was greater for heifers fed 3335-HI than 3335-LO; however, DMI was greater by heifers fed 3223-LO than 3223-HI (hybrid x cutting height interaction, P < 0.05). Starch intake was greater (P < 0.05) and NDF intake was less (P < 0.05) by heifers fed HI vs. LO and fed 3335 vs. 3223 dietary treatments. Digestibility of DM, starch, and NDF was greater (P < 0.05) by heifers fed 3223 than 3335 dietary treatments, but digestibility differences were not observed (P > 0.10) between cutting heights. Rate of in situ DM and starch degradability was not affected (P > 0.10) by hybrid or cutting height; however DM degradability was greater (P < 0.05) for HI than LO corn silage substrates at 8, 16, and 24 h of incubation. Rate of NDF degradability tended (P = 0.08) to be greater for 3223 than for 3335, and for LO compared with HI corn silage. Degradability of NDF was greater (P < 0.05) for 3223 compared with 3335 substrates at 24, 36, and 48 h of incubation. These data suggest fiber may not be an accurate measure of corn silage quality. Whereas cutting height affected chemical composition, we observed genetics to have a greater effect on corn silage quality.

Effect of hybrid, maturity, and mechanical processing of corn silage on intake and digestibility by beef cattle.

A study involving a 2 x 2 x 2 factorial arrangement of treatments was conducted to evaluate effects of hybrid (Pioneer 3335 and 3489), maturity (half milkline and blacklayer), and mechanical processing (field chopper with and without on-board rollers engaged) on intake and digestibility of corn silage. Forty Angus steers (322 +/- 5.2 kg BW) were assigned to the eight silage treatments (five steers per treatment) and individually fed using electronic gates. Diets consisted of 60% corn silage and 40% chopped alfalfa hay (DM basis). Following a 5-d adaptation period, intake was measured for 7 d and subsequently fecal samples were collected for 5 d. Chromic oxide (5 g/d) was fed beginning 7 d before fecal sample collection and digestibility was determined by the ratio of Cr in the feed and feces. Steers were reallocated to treatments and these procedures were repeated, providing 10 observations per treatment. In addition, all silages were ruminally incubated in six mature cows for 0, 8, 16, 24, 48, and 96 h to determine extent and rate of DM, starch, NDF, and ADF disappearance. Processing increased DMI of hybrid 3489 but did not affect DMI of hybrid 3335 (hybrid x processing; P < 0.06). Total tract digestibility of DM, starch, NDF, and ADF decreased (P < 0.01) as plant maturity increased. Maturity tended to decrease starch digestibility more for hybrid 3489 than for hybrid 3335 (hybrid x maturity; P < 0.10). Processing increased (P < 0.01) starch digestibility but decreased (P < 0.01) NDF and ADF digestibility, resulting in no processing effect on DM digestibility. There was a numerical trend for processing to increase starch digestibility more for latethan for early-maturity corn silage (maturity x processing; P = 0.11). Processing increased in situ rates of DM and starch disappearance and maturity decreased in situ disappearance rates of starch and fiber. These data indicate that hybrid, maturity, and processing all affect corn silage digestibility. Mechanical processing of corn silage increased starch digestibility, which may have been associated with the observed decreased fiber digestibility.

Effect of high-oil corn on growth performance, diet digestibility, and energy content of finishing diets fed to beef cattle.

Sixty crossbred beef steers (initial BW = 412 kg) were used in a 83-d finishing study to determine the effect of feeding dry rolled high-oil corn on performance and total-tract digestibility of finishing diets. Steers were allotted by weight to the following dietary treatments: 1) control corn (C; 82% normal corn, 12% triticale silage), 2) high-oil corn (HO; 82% high-oil corn, 12% silage), and 3) high-oil corn formulated to be isocaloric to C (ISO; 74% high-oil corn, 20% silage). Total lipid content was 4.9% (DM basis) for normal corn and 7.0% for high-oil corn. Steers were individually fed using electronic gates. Quantity of feed offered and refused was recorded daily. Fecal samples were collected on d 63 to 66 of the trial to determine digestibility. Chromic oxide was fed as an indigestible marker for 7 d before fecal collection began. Planned contrasts of HO vs C and ISO vs C were used to assess treatment differences. Dry matter intake was greater for steers fed C vs HO (P < 0.01) or C vs ISO (P < 0.01), but daily gain and feed efficiency were not affected (P > 0.05) by treatments. Digestibility of DM, OM, starch, and GE was greater (P < 0.05) for the HO diet than the C diet, but lipid digestibility did not differ among treatments (P > 0.05). The combined effect of greater GE content and digestibility resulted in greater (P < 0.01) DE content for the HO than for the C diet. Calculated DE of the corn was 8.3% greater (3.74 Mcal/kg; P < 0.01) for the HO diet and 6.5% greater (3.67 Mcal/kg; P < 0.01) for the ISO diet than the corn in the C diet (3.25 Mcal/kg). Dry matter and GE digestibility did not differ (P > 0.05) between the C and ISO diets. Steers consuming ISO had greater (P < 0.05) starch digestibility than steers fed the C diet. Although HO had higher DE, DE intake was similar (P > 0.05) for HO and C due to lower DMI for HO. These results indicate that available energy is greater from high-oil corn than from typical corn, but depressed voluntary feed intake prevented performance improvements and resulted in equal energy intakes between high-oil corn and typical corn diets.

Phospholipase A2 mediates immediate early genes in cultured renal epithelial cells: possible role of lysophospholipid.

BACKGROUND: Exposure to high levels of oxalate induces oxidant stress in renal epithelial cells and produces diverse changes in cell function, ranging from cell death to cellular adaptation, as evidenced by increased DNA synthesis, cellular proliferation, and induction of genes associated with remodeling and repair. These studies focused on cellular adaptation to this oxidant stress, examining the manner by which oxalate exposure leads to increased expression of immediate early genes (IEGs). Specifically, our studies assessed the possibility that oxalate-induced changes in IEG expression are mediated by phospholipase A2 (PLA2), a common pathway in cellular stress responses. METHODS: Madin-Darby canine kidney (MDCK) cells were exposed to oxalate in the presence or absence of PLA2 inhibitors: mepacrine and arachidonyl trifluoromethyl ketone (AACOCF3). Expression of IEG (c-jun, egr-1, and c-myc) mRNA was assessed by Northern blot analysis. PLA2 activity was determined by measuring the release of [3H]arachidonic acid (AA) from prelabeled cells. RESULTS: Oxalate exposure (1 to 1.5 mmol/L) induced time- and concentration-dependent increases in IEG mRNA. Treatment with mepacrine resulted in a 75 to 113% reduction of oxalate-induced c-jun, egr-1, and c-myc mRNA, while AACOCF3 caused a 41 to 46% reduction of oxalate-induced c-jun and egr-1 mRNA. Of the two major byproducts of PLA2, only lysophosphatidylcholine (20 micromol/L) increased c-jun and egr-1 mRNA. In contrast, AA (25 micromol/L) attenuated the oxalate-induced increase in c-jun and egr-1 mRNA, presumably by inhibiting PLA2 activity. CONCLUSIONS: These findings suggest that PLA2 plays a major role in oxalate-induced IEG expression in renal epithelial cells and that lysophospholipids might be a possible lipid mediator in this pathway.

Oxalate toxicity in renal epithelial cells: characteristics of apoptosis and necrosis.

Studies in various tissues, including the kidney, have demonstrated that toxins elicit apoptosis under certain conditions and necrosis under others. The nature of the response has important consequences for the injured tissue in that necrotic cells elicit inflammatory responses, whereas apoptotic cells do not. Thus, there has been considerable interest in defining the mode of cell death elicited by known cytotoxins. The present studies examined the response of renal epithelial cells to oxalate, a metabolite excreted by the kidney that produces oxidant stress and death of renal cells at pathophysiological concentrations. These studies employed LLC-PK1 cells, a renal epithelial cell line from pig kidney and NRK-52E (NRK) cells, a line from normal rat kidney, and compared the effects of oxalate with those of known apoptotic agents. Changes in cellular and nuclear morphology, in nuclear size, in ceramide production, and in DNA integrity were assessed. The ability of bcl-2, an anti-apoptotic gene product, to attenuate oxalate toxicity was also assessed. These studies indicated that oxalate-induced death of renal epithelial cells exhibits several features characteristic of apoptotic cell death, including increased production of ceramide, increased abundance of apoptotic bodies, and marked sensitivity to the level of expression of the anti-apoptotic gene bcl-2. Oxalate-induced cell death also exhibits several characteristics of necrotic cell death in that the majority of the cells exhibited cellular and nuclear swelling after oxalate treatment and showed little evidence of DNA cleavage by TUNEL assay. These results suggest that toxic concentrations of oxalate trigger both forms of cell death in renal epithelial cells.

Oxalate-induced changes in the viability and growth of human renal epithelial cells.

Previous studies on the porcine renal epithelial LLC-PK1 cell line demonstrated that oxalate exposure produces concentration-dependent effects on renal cell growth and viability via process(es) involving free radicals. The present studies were conducted to determine whether these findings could be extended to a renal proximal tubular epithelial cell line derived from the human kidney. These studies examined oxalate-induced changes in membrane integrity after short-term exposure (4 h) and changes in cell survival after longer-term exposure (24 to 72 h). Oxalate-induced changes were also assessed in the expression of two genes: egr-1, a zinc-finger transcription factor, and osteopontin, a protein associated with tissue remodeling. The present studies also determined whether oxalate-induced changes in either cell viability or gene expression depended on free radicals. Oxalate at a concentration > or = 175 microM (free) produced membrane damage within 4 h. This effect was inhibited by Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP), a superoxide dismutase mimetic, but not by N-acetyl cysteine, a glutathione precursor, or by deferoxamine, an iron chelator. Acute oxalate-induced injury was followed by cell loss within 24 h, an effect maintained at 48 and 72 h at high concentrations of oxalate. Oxalate also promoted DNA synthesis. This mitogenic effect offset cell loss at lower oxalate concentrations (88 microM) leading to a small but significant increase in cell number at 72 h. Treatment with oxalate also increased expression of egr-1 mRNA within 1 h, a response that was attenuated by MnTMPyP; oxalate treatment for 8 h also increased abundance of osteopontin mRNA. These studies suggest that oxalate exposure produces changes in human renal cell growth and viability via a process(es) dependent on reactive oxygen intermediates. Such changes may play a role in the development and/or progression of renal disease via generation of reactive oxygen intermediates.

Role of phospholipase A2 in the cytotoxic effects of oxalate in cultured renal epithelial cells.

BACKGROUND: Oxalate, a common constituent of kidney stones, is cytotoxic for renal epithelial cells. Although the exact mechanism of oxalate-induced cell death remains unclear, studies in various cell types, including renal epithelial cells, have implicated phospholipase A2 (PLA2) as a prominent mediator of cellular injury. Thus, these studies examined the role of PLA2 in the cytotoxic effects of oxalate. METHODS: The release of [3H]-arachidonic acid (AA) or [3H]-oleic acid (OA) from prelabeled Madin-Darby canine kidney (MDCK) cells was measured as an index for PLA2 activity. The cell viability was assessed by the exclusion of ethidium homodimer-1. RESULTS: Oxalate exposure (175 to 550 microM free) increased the release of [3H]-AA in MDCK cells but had no effect on the release of [3H]-OA. Oxalate-induced [3H]-AA release was abolished by arachidonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cytosolic PLA2 (cPLA2), but was not affected by selective inhibitors of secretory PLA2 and calcium-independent PLA2. The [3H]-AA release could be demonstrated within 15 minutes after exposure to oxalate, which is considerably earlier than the observed changes in cell viability. Furthermore, AACOCF3 significantly reduced oxalate toxicity in MDCK cells. CONCLUSIONS: Oxalate increases AA release from MDCK cells by a process involving cPLA2. In addition, based on the evidence obtained using a selective inhibitor of this isoform, it would appear that the activity of this enzyme is responsible, at least in part, for the cytotoxic effects of oxalate. The finding that oxalate can trigger a known lipid-signaling pathway may provide new insight into the initial events in the pathogenesis of nephrolithiasis.

Short-term clinical evaluation of post-operative sensitivity with bonded amalgams.

PURPOSE: To compare the in vivo short-term post-operative sensitivity of teeth restored with amalgam using a bonded resin liner vs. teeth restored using a copal varnish liner. MATERIALS AND METHODS: 20 patients received Class I or Class II contralaterally paired restorations which were placed at the same appointment. All restorations were placed by the same operator using an identical technique except that, in each randomized pair, one was lined with an adhesive resin (Scotchbond Multi-Purpose Plus) while the other was lined with copal varnish. (Plastodent) Patients were provided visual analog scale response forms, instructed in their use, and requested to complete and return a form reporting their degree of sensitivity at baseline and on days 1, 3, 7, 14, and 30 post-operatively. Data from the response forms were analyzed for differences using a paired t-test. RESULTS: A response rate of 90% (18/20) was achieved for the complete 30-day assessment. Increases in thermal sensitivity beyond baseline were seen in 13 of the 18 subjects involving 12 restorations lined with copal varnish and 10 lined with adhesive resin. Typically, sensitivity peaked on day 1 or day 3 and diminished to pre-operative levels by day 30. Only three subjects reported greater sensitivity at day 30 than at baseline. No significant difference in post-operative sensitivity was found between the two cavity lining materials at any post-operative interval.

Activation of c-myc gene mediates the mitogenic effects of oxalate in LLC-PK1 cells, a line of renal epithelial cells.

Recent studies on LLC-PK1 cells demonstrated that oxalate, a simple dicarboxylic acid, acts as a mitogen for these renal epithelial cells. Exposure to oxalate initiates DNA synthesis, induces the expression of one of the early growth response genes c-myc and stimulates proliferation of quiescent cultures of LLC-PK1 cells. The present studies examined the possibility that expression of the c-myc protooncogene is obligatory for this mitogenic response. Specifically we determined whether pretreatment with c-myc antisense oligonucleotides would block the proliferative effects of oxalate in LLC-PK1 cells. Quiescent cultures of LLC-PK1 cells were exposed to oxalate in the presence and absence of c-myc antisense and the effects of oxalate on c-myc protein expression (Myc), DNA synthesis and cell growth were assessed. Exposure of cells to oxalate alone increased the expression of Myc within two hours. Pretreatment with c-myc antisense abolished this response. Further, pretreatment of cells with c-myc antisense but not nonsense oligonucleotides blocked the oxalate-induced initiation of DNA synthesis. Increases in cell number in response to oxalate (measured after 72 hr exposure) were also blocked by exposure to c-myc antisense. These findings suggest that c-myc gene expression is critical for the mitogenic effects of oxalate in LLC-PK1 cells.

Oxalate toxicity in LLC-PK1 cells, a line of renal epithelial cells.

PURPOSE: The present studies assessed the possibility that high concentrations of oxalate may be toxic to renal epithelial cells. MATERIALS AND METHODS: Subconfluent cultures of LLC-PK1 cells were exposed to oxalate, and the effects on cell morphology, membrane permeability to vital dyes, DNA integrity and cell density were assessed. RESULTS: Oxalate exposure produced time- and concentration-dependent changes in the light microscopic appearance of LLC-PK1 cells with higher concentrations ( > 140 microM.) inducing marked cytosolic vacuolization and nuclear pyknosis. Exposure to oxalate also increased membrane permeability to vital dyes, promoted DNA fragmentation and, at high concentrations (350 microM. free oxalate), induced a net loss of LLC-PK1 cells. CONCLUSIONS: Since high concentrations of oxalate can be toxic to renal epithelial cells, hyperoxaluria may contribute to several forms of renal disease including both calcium stone disease and end-stage renal disease.

Oxalate toxicity in LLC-PK1 cells: role of free radicals.

Oxalate, the most common constituent of kidney stones, is an end product of metabolism that is excreted by the kidney. During excretion, oxalate is transported by a variety of transport systems and accumulates in renal tubular cells. This process has been considered benign; however, recent studies on LLC-PK1 cells suggested that high concentrations of oxalate are toxic, inducing morphological alterations, increases in membrane permeability to vital dyes and loss of cells from the monolayer cultures. The present studies examined the basis for oxalate toxicity, focusing on the possibility that oxalate exposure might increase the production/availability of free radicals in LLC-PK1 cells. Free radical production was monitored in two ways, by monitoring the reduction of nitroblue tetrazolium to a blue reaction product and by following the conversion of dihydrorhodamine 123 (DHR) to its fluorescent derivative, rhodamine 123. Such studies demonstrated that oxalate induces a concentration-dependent increase in dye conversion by a process that is sensitive to free radical scavengers. Specifically, addition of catalase or superoxide dismutase blocked the oxalate-induced changes in dye fluorescence/absorbance. Addition of these free radical scavengers also prevented the oxalate-induced loss of membrane integrity in LLC-PK1 cells. Thus it seems likely that free radicals are responsible for oxalate toxicity. The levels of oxalate that induced toxicity in LLC-PK1 cells (350 microM) was only slightly higher than would be expected to occur in the renal cortex. These considerations suggest that hyperoxaluria may contribute to the progression of renal injury in several forms of renal disease.

Oxalate-induced damage to renal tubular cells.

Our own studies and those of others have shown that the incidence of calcium oxalate stones and plaques is markedly increased by nephrotoxins. The possible role of oxalate as a nephrotoxin has not been fully appreciated. However, recent studies in experimental animals and in cultured cells support this possibility. The results of these studies led us to hypothesize that hyperoxaluria promotes stone formation in several ways: by providing a substrate for the formation of the most common form of renal stones, calcium oxalate stones, and by inducing damage to renal epithelial cells. Damaged cells in turn would produce an environment favorable for crystal retention and provide membranous debris that promotes crystal nucleation, aggregation and adherence. The present report summarizes evidence for oxalate nephrotoxicity and discusses the potential importance of oxalate toxicity in the pathogenesis of stone disease.

Oxalate-induced initiation of DNA synthesis in LLC-PK1 cells, a line of renal epithelial cells.

These studies examined the effects of oxalate, a constituent of renal stones, on the growth of LLC-PK1 cells. Exposure to oxalate resulted in an initiation of DNA synthesis in serum-starved, growth-arrested cells as measured by 3H-thymidine incorporation. The effects of oxalate were comparable to those observed in response to 10% serum. Moreover, exposure to oxalate plus 10% serum stimulated DNA synthesis to a greater extent than oxalate or serum alone. These studies indicate that oxalate promotes the progression of cells from the G0/G1 to the S phase of the cell cycle. However, the increase in DNA synthesis was not always followed by an increase in cell number since high concentrations of oxalate led to a reduction in cell number.