Cis-12 Conjugated Linoleic Acid Research Articles

An efficient method for enzymatic purification of cis-9,trans-11 isomer of conjugated linoleic acid

Abstract A method for purification of cis-9,trans-11 isomer of conjugated linoleic acid (CLA) is described. Lipase from Candida cylindracea (CCL) was highly selective towards cis-9,trans-11 isomer and was chosen to catalyze esterification of a mixture of two CLA isomers. The effect of various parameters on the esterification process of an almost equimolar mixture of cis-9,trans-11 (44.0%), trans-10,cis-12 (42.5%) and other CLA isomers (13.5%) was studied. The influence of alcohol substrate, CLA/alcohol molar ratio, lipase amount, reaction temperature, pH and time of the reaction on the total esterification and cis-9,trans-11 isomer purity in ester fraction were examined. The highest isomeric excess of cis-9,trans-11 isomer on trans-10,cis-12 CLA and good esterification degree was observed when the esterification condition were as follows: CLA/2-propanol molar ratio, 1:5; water, 200% wt of CLA; CCL, 24 μg of CCL/mg CLA; temperature 30 C; time of reaction 6 h and when mixture enriched in cis-9,trans-11 CLA was used as a substrate for esterification. A preparative scale purification combining crystallization and one step enzymatic process were made to afford purified cis-9,trans-11 (94.2%) with total recovery of 31%.

48 EFFECTS OF LIPID METABOLIC REGULATORS DURING BOVINE EMBRYO CULTURE ON BLASTOCYST DEVELOPMENT AND CRYOSURVIVAL

The overall objective was to determine the effects of addition of lipid metabolic regulators during embryo culture on blastocyst development and survival following cryopreservation. For Experiment 1, embryos produced in vitro were cultured in 5% (vol/vol) oxygen in SOF-bovine embryo 1 (SOF-BE1) medium supplemented with or without 100 μM trans-10,cis-12 conjugated linoleic acid (CLA) and 0.3 μM phenazine ethosulfate (PES). Treatment with CLA began at the initiation of culture, whereas treatment with PES began at Day 3 after insemination. At Day 7 after insemination, the proportion of oocytes that developed to the blastocyst and advanced blastocyst (expanded, hatching, or hatched) stages was recorded. Blastocysts and expanded blastocyst-stage embryos were harvested and slow frozen in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 medium containing 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. Addition of CLA had no effect on embryo development, whereas PES reduced (P < 0.01) development to the blastocyst (26.0 ± 0.8 v. 22.1 ± 0.8%) and advanced blastocyst (19.2 ± 0.9 v. 14.4 ± 0.9%) stages. Blastocysts cultured in the presence of CLA had higher (P < 0.05) re-expansion rates at 24, 48, and 72 h (50.8 ± 3.7 v. 65.7 ± 3.7%, 57.2 ± 4.0 v. 72.0 ± 4.05%, and 57.2 ± 4.0 v. 72.0 ± 4.0%, respectively). Addition of CLA tended (P < 0.07) to increase the hatching rate at 24 h and did increase (P < 0.05) the hatching rate at 48 h (12.4 ± 1.3 v. 16.2 ± 1.3% and 39.0 ± 3.2 v. 50.0 ± 3.2%, respectively). Treatment with PES had no effect on re-expansion rates but reduced (P < 0.05) hatching rates at 24 and 48 h (18.2 ± 1.3 v. 10.3 ± 1.3 and 50.2 ± 3.2 v. 38.8 ± 3.2%, respectively). There was no interaction between CLA and PES affecting embryo development or cryosurvival. For Experiment 2, embryos were produced in vitro as in Experiment 1 and cultured in SOF-BE1 medium with or without 3.03 mM L-carnitine (LC) and 10 μM forskolin (FK). Treatment with LC began at the initiation of culture and treatment with FK began at Day 6. All other methods were as described for Experiment 1. Addition of LC did not affect development to the blastocyst stage but reduced (P < 0.05) development to the advanced blastocyst stage (21.0 ± 1.2 v. 17.1 ± 1.2%). Treatment with FK had no effect on embryo development to the blastocyst or advanced blastocyst stages. Blastocysts cultured in the presence of LC had increased (P < 0.05) re-expansion rates at 24, 48, and 72 h (60.2 ± 2.0 v. 78.0 ± 2.0%, 62.9 ± 1.2 v. 83.3 ± 1.2%, and 63.0 ± 2.4 v. 82.8 ± 2.4%, respectively) and hatching rates at 48 and 72 h (48.6 ± 4.3 v. 64.1 ± 4.3% and 59.6 ± 3.0 v. 78.5 ± 3.0%, respectively). There was no effect of FK on cryosurvival and no interaction between LC and FK affecting embryo development or cryosurvival. In conclusion, blastocyst yield was not improved by any of the lipid metabolic regulators tested. Cryosurvival was enhanced by addition of CLA and LC but FK reduced survival following freezing. There were no additive effects of either CLA and PES or LC and FK for blastocyst yield or cryosurvival.Support was provided by USDA AFRI Grant 2010-85122-20623.

Conjugated Linoleic Acid (t‐10, c‐12) Reduces Fatty Acid Synthesis de Novo, but not Expression of Genes for Lipid Metabolism in Bovine Adipose Tissue ex Vivo

We hypothesized that exogenous fatty acids, and especially or 18:2 trans-10, cis-12 conjugated linoleic acid (CLA), would decrease adipogenic and lipogenic gene expression and de novo fatty acid biosynthesis in intramuscular (i.m.) and subcutaneous (s.c.) adipose tissues. Fresh i.m. and s.c. adipose tissues were collected from the longissimus thoracis muscle of Angus steers at 12, 14, and 16 months of age (n=4 per time point). Adipose tissue explants were incubated in duplicate for 48h with 40μM α-linolenic (ALA), oleic, stearic, trans-vaccenic, or CLA. Adipocyte size, acetate and glucose incorporation into fatty acids in vitro and mRNA levels for C/EBPβ, CPT1β, GPR43, PPARγ, PRKAA1 (AMPKα) and SCD1 were measured following the incubations. PRKAA1 and SCD1gene expression were greater (P<0.001) in s.c. adipose tissue than in i.m. adipose tissue and acetate incorporation into lipids and C/EBPβ, PPARγ, and SCD1gene expression were greater at 16months of age than at 12months of age in i.m. adipose (P<0.01). C/EBPβ gene expression increased by 16months of age and PRKAA1 gene expression decreased by 16months of age in s.c. adipose tissue. All fatty acids increased s.c. adipocyte volumes whereas CLA decreased acetate incorporation into lipids in s.c. adipose tissue (P<0.05), but none of the fatty acids affected gene expression in i.m. or s.c. adipose tissue (P>0.10). Thus, CLA depressed de novo fatty acid biosynthesis from acetate but neither CLA nor other fatty acids significantly affected adipogenic or lipogenic gene expression.

Preparative separation of conjugated linoleic acids (CLAs) from fermented Camellia oleifera Abel cake by β-cyclodextrin (β-CD) encapsulation using pH-zone-refining countercurrent chromatography

This paper concentrates on the separation of three conjugated linoleic acid (CLA) isomers (trans-9,trans-11 CLA, trans-10,cis-12 CLA and cis-9,trans-11 CLA) by β-cyclodextrin (β-CD) encapsulation using countercurrent chromatography from Camellia oleifera Abel cake fermented by lactic acid bacteria Lactobacillus sp. LL-ZSDS001. The elution sequence of the CLA isomers, the mixing zones and mechanism of separation are discussed. The separation of 305.9mg of the crude sample yielded three isomeric compounds: 91.3mg of trans-9,trans-11 CLA, 84.1mg of trans-10,cis-12 CLA and 79.7mg of cis-9,trans-11 CLA at high purities of 98%, 94% and 96%, respectively.

Changes of rumen pH, fermentation and microbial population as influenced by different ratios of roughage (rice straw) to concentrate in dairy steers

SUMMARYThe current study was designed to determine the effect of roughage to concentrate ratio (R : C) on rumen pH, fermentation and bacterial population in dairy steers. Four rumen fistulated dairy steers (170±20 kg) were randomly assigned according to a 4×4 Latin square design, in which the steers were fed with four dietary treatments with different R : C ratios of 0·8 : 0·2, 0·6 : 0·4, 0·4 : 0·6 and 0·2 : 0·8, respectively. All animals were kept in individual pens and received feed according to the respective R : C ratios at 0·025 body weight (BW)/d; urea-treated rice straw (prepared using 3·5 kg urea+100 kg water sprayed onto 100 kg of rice straw) was used as a roughage source. The experiment was conducted for four periods of 21 days each. During the first 14 days, feed intake was measured and the animals were then moved to metabolism crates for total urine and faecal collection for 7 days. Total dry matter intake (DMI) was similar among treatments. Energy intake increased as the proportion of concentrate in the diet increased. Apparent digestibilities of dry matter (DM), organic matter (OM) and crude protein (CP) were improved, while neutral detergent fibre (NDF) and acid detergent fibre (ADF) were reduced when the levels of concentrate increased. A decreasing ratio of R : C reduced rumen pH linearly, from 6·4 to 5·9 at 0·2 : 0·8. High levels of concentrate impacted on volatile fatty acids (VFA) molar proportions and decreased acetate (C2) linearly, while propionate (C3) was increased, leading to decreased C2 : C3 ratio. Numbers of protozoa, fungi and proteolytic bacteria were not affected by R : C ratio. Cellulolytic bacteria decreased linearly while amylolytic bacteria increased linearly with 0·60 and 0·80 concentrates. Quantitative polymerase chain reaction (qPCR) based on 16S RNA revealed thatFibrobacter succinogenesnumbers were increased when steers were fed with R : C ratio of 0·8 : 0·2. Conjugated linoleic acid (CLA)-producing bacteria, especially those ofButyrivibrio fibrisolvens, increased linearly with R : C ratios of 0·8 : 0·2 and 0·6 : 0·4, whileMegasphaera elsdenii, a lactate-utilizing bacterium and reported producer oftrans-10,cis-12 CLA increased linearly with R : C ratio of 0·8 : 0·2. In addition, microbial CP synthesis increased quadratically when steers were fed high levels of concentrate. However, the efficiency microbial N synthesis (EMNS) based on OM, truly digested in the rumen, was not affected by different R : C ratios. From the current study, it can be concluded that roughage to concentrate ratio of 0·4 : 0·6 had positive effects for the creation of healthy rumen (rumen pH and ecology), and improved energy intake and rumen fermentation, particularly propionic acid and microbial protein synthesis, in dairy steers fed urea-treated rice straw as a roughage source.

Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration

Milk fat depression (MFD) caused by intermediates of ruminal biohydrogenation commonly occurs in dairy cattle. The time course of recovery from MFD is important to mechanistic investigation and management of the condition. Nine cows were used in a repeated design, allowing analysis of recovery from diet-induced MFD. A high-fiber, low-oil diet was fed during the control and recovery periods, and a low-fiber, high-oil (LFHO) diet was fed during the induction period. Milk yield was not affected by treatment. Milk fat percentage and yield decreased progressively during induction and were lower by d 3 and 5, respectively. Milk fat concentration and yield increased progressively when cows were fed the recovery diet and were not different from control on d 19 and 15, respectively. Yield of de novo synthesized fatty acids (FA) decreased progressively during the induction period and was lower than that of controls by d 5. A biphasic response was seen for milk fat trans isomers, where trans-11 C18:1 and cis-9,trans-11 conjugated linoleic acid (CLA) were elevated initially and trans-10 C18:1 and trans-10,cis-12 CLA increased progressively during the induction period. A similar biphasic response was seen during recovery from MFD, with trans-10 C18:1 and trans-10,cis-12 rapidly decreasing initially and trans-11 C18:1 and cis-9,trans-11 CLA increasing slightly above control levels during the second phase. Recovery from diet-induced MFD occurs gradually with a short lag when dietary fiber and oil concentrations are corrected. This time course provides a framework to identify factors causing MFD and set expectations during recovery from MFD.

Induction of apoptosis by c9, t11-CLA in human endometrial cancer RL 95-2 cells via ERα-mediated pathway

Numerous studies have shown that conjugated linoleic acid (CLA) can inhibit cancer cells growth and induce apoptosis in vitro and in vivo. The aim of the present study was to investigate the effects of CLA, including cis9, trans11-conjugated linoleic acid (c9, t11-CLA) and trans10, cis12-conjugated linoleic acid (t10, c12-CLA), on apoptosis of human endometrial cancer RL 95-2 cells and its related mechanisms.The MTT analysis was used to evaluate the effect of CLA isomers on the viability of endometrial cancer RL 95-2 cells. We then estimated the apoptosis by Morphological observation and Annexin V-FITC/PI staining and flow cytometry. We also used Western blot analysis to assess the expression of caspase-3, Bax, Bcl-2 proteins and the activation of Akt/p-Akt and ERα/p-ERα. Propylpyrazole-triol (PPT), a selective ERα agonist was used to confirm the induction of apoptosis by c9, t11 CLA may relate to ERα-mediated pathway.In CLA-treated RL 95-2 cells, we found that c9, t11-CLA inhibited viability and trigged apoptosis, as judged from nuclear morphology and flow cytometric analysis. The expression of caspase-3 and the ratio of Bax/Bcl-2 were significant increased, but no obvious change was observed about Akt and p-Akt in c9, t11-CLA-treated cells. However, the expression of total ERα level in RL 95-2 cells-treated with c9, t11-CLA was unchanged, while in the concentration of 80mM, c9, t11-CLA down-regulated the protein expression level of p-ERα. Then PPT has the antagonistic action on growth inhibitory effect in RL 95-2 cells incubated with c9, t11-CLA.This study demonstrated that c9, t11- CLA could induce apoptosis in RL 95-2 cells, and may involve in ERα-mediated pathway. These results indicated that c9, t11- CLA could induce apoptosis of endometrial cancer cells and may be potential agents for the treatment of endometrial cancer.

Evaluating the performance, carcass traits and conjugated linoleic acid content in muscle and adipose tissues of Black Bengal goats fed soybean oil and sunflower oil

An experiment was conducted to investigate the feeding of soybean oil (SOY) and sunflower oil (SUN) on performance, carcass traits and fatty acid (FA) composition, including conjugated linoleic acids (CLA) in meat of Black Bengal goats. Eighteen growing goats (initial body weight (BW) of 11.1±0.49kg and 9–12 months of age) were equally allotted into 3 groups (control, SOY and SUN) in a randomized complete block design (blocked according to initial BW). Goats were fed concentrate mixtures with similar nitrogen and calculated metabolizable energy content with added vegetable oils (SOY or SUN) at 45g/kg dietary dry matter or without oil (control), and berseem hay (60:40). A metabolic trial was conducted after 105 days of feeding, and carcass traits and fatty acid composition of longissimus dorsi muscle and subcutaneous adipose tissues were determined at 122 days of feeding. Oil supplementation did not affect (P>0.05) average daily gains and feed intake in goats. Digestibilities of nutrients except ether extract (EE), and intake, excretion and balance of nitrogen were also similar (P>0.05) among dietary treatments. Digestibility of EE in goats receiving both plant oils was higher (P<0.01) than control. Feeding of oils did not affect (P>0.05) carcass traits, and percentages of protein, fat and ash in muscle. While supplementing oils did not change (P>0.05) the proportion of total saturated FA, they lowered several medium-chain FA in muscle and adipose tissues. However, the proportion of total polyunsaturated FA (PUFA; P<0.001) and CLA cis-9, trans-11 (P<0.001) in these tissues were highest in SUN, followed by SOY, and control. Oils had no effect (P>0.05) on concentrations of serum glucose, cholesterol and protein, but increased (P<0.05) serum triglyceride concentration compared with control. In conclusion, feeding of SOY and SUN at a concentration of 45g/kg of total diets did not affect digestibility and performance, but increased the PUFA and CLA content in muscle and adipose tissues in Black Bengal goats with the greatest effect noted for SUN.

Effect of pH on conjugated linoleic acid (CLA) formation of linolenic acid biohydrogenation by ruminal microorganisms

Conventional beliefs surrounding the linolenic acid (LNA; cis-9 cis-12 cis-15 C18:3) biohydrogenation (BH) pathway propose that it converts to stearic acid (SA) without the formation of conjugated linoleic acid (CLA) as intermediate isomers. However, an advanced study (Lee and Jenkins, 2011) verified that LNA BH yields multiple CLAs. This study utilized the stable isotope tracer to investigate the BH intermediates of (13)C-LNA with different pH conditions (5.5 and 6.5). The (13)C enrichment was calculated as a (13)C/(12)C ratio of labeled minus unlabeled. After 24 h, eight CLA isomers were significantly enriched on both pH treatment, this result verifies that these CLAs originated from (13)C-LNA BH which supports the results of Lee and Jenkins (2011). The enrichment of cis-cis double bond CLAs (cis-9 cis-11 and cis-10 cis-12 CLA) were significantly higher at low pH conditions. Furthermore, the concentration of cis-10 cis-12 CLA at low pH was four times higher than at high pH conditions after a 3 h incubation. These differences support the LNA BH pathways partial switch under different pH conditions, with a strong influence on the cis-cis CLA at low pH. Several mono-, di-, and tri-enoic fatty acid isomers were enriched during 24 h of incubation, but the enrichment was decreased or restricted at low pH treatment. Based on these results, it is proposed that low pH conditions may cause a changed or limited capacity of the isomerization and reduction steps in BH.

Effect of conjugated linoleic acid and vitamin E on glycemic control, body composition, and inflammatory markers in overweight type2 diabetics

BackgroundThe healthy properties of conjugated linoleic acid (CLA) such as weight loss, reducing cardiovascular risk factors and inflammation have been reported. The trans-10, cis-12 CLA isomer is related to increasing insulin resistance, but the effects of cis-9, trans-11 isomer is not clear. The aim of this study was to investigate the effects of CLA with and without Vitamin E on body weight, body composition, glycemic index, inflammatory and coagulation factors, lipid profile, serum leptin and adiponectin, malondialdehyde (MDA), and blood pressure in type2 diabetes.Methods56 patients with type2 diabetes were included in 8 week double-blind control trial that used metformin. They randomly divided into three groups: CLA + VitE, CLA + VitE placebo, CLA placebo + VitE placebo. All variables, anthropometric measurements, and body composition were evaluated at the beginning and the end of study. Statistical analysis and analysis of dietary data were performed using SPSS and nutritionist IV software, respectively.ResultsThere were not any significant differences in variable changes among three groups. However, there was a trend to increase in MDA and decrease in apoB100 among CLA consumers.ConclusionThe results of this study showed that administration of CLA supplementation for 8 weeks does not affect any indicators of metabolic control in overweight type2 diabetic patients.

Milk production, fatty acid composition and vitamin E content of Payoya goats according to grazing level in summer on Mediterranean shrublands

Dietary intake of certain unsaturated fatty acids (FA), in particular conjugated linoleic acid (CLA) and omega-3 FA (n-3 FA), and fat-soluble antioxidants (e.g. α-tocopherol) has been linked to potential health benefits. Nevertheless, there is little information about how Mediterranean shrublands affect the fat composition and fat-soluble antioxidants of milk of dairy goats. The aim of this study was to evaluate the effect during the summer of different grazing levels on Mediterranean scrublands on milk production, milk FA profile and vitamin E content from Payoya goats. Eight farms were selected and data were collected monthly from June to October of 2010. Indicators for feeding management and productivity were obtained. The Percentage of the milking goat's net energy requirements obtained from grazing (NERG) was calculated. Farms were segregated in two groups according to the grazing level: high (HG, n=3) and medium grazing (MG, n=5). Whole milk was collected monthly from bulk tanks of the farms and analyzed for chemical composition, FA profile, α-tocopherol content and total antioxidant capacity (TAC). NERG differed between treatment groups and months (P<0.001 and P<0.05, respectively). No differences between groups were observed for the most studied chemical parameters. The percentages of C16:1 (P<0.01), C18:1 n-9 trans (P<0.05), C18:1 n-9 cis (P<0.05) and MUFA (P<0.01) were significantly higher and the total SFA significantly lower (P<0.01) in milk from MG farms compared with HG farms. In contrast, percentages of the nutritionally desirable FA (α-linolenic acid, C22:5 n-3, total n-3 PUFA) were significantly higher, while levels of total n-6 PUFA and n6:n3 ratio were significantly lower in milk from HG group. In addition, the percentages of NERG were correlated positively with the contents of several n-3 FA and negatively with the n-6 FA and n-6:n-3 ratio. CLA cis-9, trans-11 and CLA trans-10, cis-12 were not affected by grazing level. The short and long-chain FA showed significant variations during the period of study. HG farms showed significant higher α-tocopherol content in September (P=0.030) and October (P=0.025). The TAC levels did not significantly vary according to the grazing level and the month. In conclusions, the high grazing farms showed significantly higher levels of n-3 FA and α-tocopherol compared with the medium grazing farms, which are widely recognized as having beneficial effects on human health.

A highly selective whole cell biocatalysis method for the production of two major bioactive conjugated linoleic acid isomers

Abstract Conjugated linoleic acid (CLA) is well known potential anticarcinogenic, antiatherogenic and a number of other therapeutic qualities bearing substance. In the present study we have isolated Lactobacillus plantarum from local dairy buffalo milk and used as a whole cell biocatalyst, for selective CLA isomer production from linoleic acid (LA). Quantitative analysis revealed that the cis-9, trans-11 CLA comprised of 51% (w/w), while trans-10, cis-12 CLA accounted for 49% (w/w) of total CLA produced by L. plantarum. The product formation and quantitation was monitored by the spectrophotometric method and the individual isomers separation was done with GC–FID. After optimization of biotransformation conditions such as pH, LA concentration, effect of preincubation, fermentation time; the highest CLA production (19.07% conversion) was achieved at 120 h in the presence of 1.62 mg ml−1 of LA. The results revealed that the bacterium is an efficient biocatalyst for the production of two major bioactive CLA isomers using LA as a substrate.

Effects of conjugated linoleic acids and dietary concentrate proportion on performance, milk composition, milk yield and metabolic parameters of periparturient dairy cows

The study aimed to examine effects of supplemented conjugated linoleic acids (CLA) to periparturient cows receiving different concentrate proportions ante partum (a.p.) to investigate CLA effects on lipid mobilisation and metabolism. Compared to adapted feeding, a high-concentrate diet a.p. should induce a ketogenic metabolic situation post partum (p.p.) to better understand how CLA works. Sixty-four pregnant German Holstein cows had ad libitum access to partial mixed rations 3 weeks prior to calving until day 60 p.p. Ante partum, cows received control fat (CON) or a CLA supplement at 100 g/d, either in a low-concentrate (CON-20, CLA-20) or high-concentrate diet (CON-60, CLA-60). Post partum, concentrate proportion was adjusted, while fat supplementation continued. After day 32 p.p., half of the animals of CLA-groups changed to CON supplementation (CLA-20-CON, CLA-60-CON). A ketogenic metabolic situation p.p. was not achieved and therefore impacts of CLA could not be examined. Live weight, milk yield and composition, blood parameters remained unaffected by the treatments. Only a slightly reduced milk fat yield (not significant) was recorded for Group CLA-20. The proportion of trans-10,cis-12 (t10,c12) CLA in milk fat was significantly increased in CLA-groups compared to CON-groups. With the exception of a reversible CLA effect on milk fat in Group CLA-20, no post-treatment effects occurred. Dry matter intake (DMI) of Group CLA-60 was highest before calving, resulting in a significantly improved estimated energy balance after calving. Ante partum, net energy intakes were significantly increased in high-concentrate groups. Overall, supplemented CLA preparation did not relieve metabolism and lipid mobilisation of early lactating cows. But feeding CLA in a high-concentrate diet a.p. seems to increase DMI and thereby improve the energy balance of cows immediately after calving.

Conjugated linoleic acid-induced milk fat depression in lactating ewes is accompanied by reduced expression of mammary genes involved in lipid synthesis

Conjugated linoleic acids (CLA) are produced during rumen biohydrogenation and exert a range of biological effects. The trans-10,cis-12 CLA isomer is a potent inhibitor of milk fat synthesis in lactating dairy cows and some aspects of the mechanism have been established. Conjugated linoleic acid-induced milk fat depression has also been observed in small ruminants and our objective was to examine the molecular mechanism in lactating ewes. Multiparous lactating ewes were fed a basal ration (0.55:0.45 concentrate-to-forage ratio; dry matter basis) and randomly allocated to 2 dietary CLA levels (n=8 ewes/treatment). Treatments were zero CLA (control) or 15g/d of lipid-encapsulated CLA supplement containing cis-9,trans-11 and trans-10,cis-12 CLA isomers in equal proportions. Treatments were fed for 10wk and the CLA supplement provided 1.5g of trans-10,cis-12/d. No treatment effects were observed on milk yield or milk composition for protein or lactose at wk 10 of the study. In contrast, CLA treatment significantly decreased both milk fat percentage and milk fat yield (g/d) by about 23%. The de novo synthesized fatty acids (FA; <C16) were significantly decreased in proportion (15%) and daily yield (27%), and the proportion of preformed FA (>C16) was increased (10%) for the CLA treatment. In agreement with the reduced de novo FA synthesis, mRNA abundance of acetyl-coenzyme A carboxylase α, FA synthase, stearoyl-CoA desaturase 1, and glycerol-3-phosphate acyltransferase 6 decreased by 25 to 40% in the CLA-treated group. Conjugated linoleic acid treatment did not significantly reduce the mRNA abundance of enzymes involved in NADPH production, but the mRNA abundance for sterol regulatory element-binding factor 1 and insulin-induced gene 1, genes involved in regulation of transcription of lipogenic enzymes, was decreased by almost 30 and 55%, respectively, with CLA treatment. Furthermore, mRNA abundance of lipoprotein lipase decreased by almost 40% due to CLA treatment. In conclusion, the mechanism for CLA-induced milk fat depression in lactating ewes involved the sterol regulatory element-binding protein transcription factor family and a coordinated downregulation in transcript abundance for lipogenic enzymes involved in mammary lipid synthesis.

Conjugated linoleic acid reduces adiposity and increases markers of browning and inflammation in white adipose tissue of mice

The objective of this study was to examine the mechanism by which conjugated linoleic acid (CLA) reduces body fat. Young male mice were fed three combinations of fatty acids at three doses (0.06%, 0.2%, and 0.6%, w/w) incorporated into AIN76 diets for 7 weeks. The types of fatty acids were linoleic acid (control), an equal mixture of trans-10, cis-12 (10,12) CLA plus linoleic acid, and an equal isomer mixture of 10,12 plus cis-9, trans-11 (9,11) CLA. Mice receiving the 0.2% and 0.6% dose of 10,12 CLA plus linoleic acid or the CLA isomer mixture had decreased white adipose tissue (WAT) and brown adipose tissue (BAT) mass and increased incorporation of CLA isomers in epididymal WAT and liver. Notably, in mice receiving 0.2% of both CLA treatments, the mRNA levels of genes associated with browning, including uncoupling protein 1 (UCP1), UCP1 protein levels, and cytochrome c oxidase activity, were increased in epididymal WAT. CLA-induced browning in WAT was accompanied by increases in mRNA levels of markers of inflammation. Muscle cytochrome c oxidase activity and BAT UCP1 protein levels were not affected by CLA treatment. These data suggest a linkage between decreased adiposity, browning in WAT, and low-grade inflammation due to consumption of 10,12 CLA.

Replacing canola meal as the major protein source with wheat dried distillers’ grains alters omasal fatty acid flow and milk fatty acid composition in dairy cows

Chibisa, G. E., Christensen, D. A. and Mutsvangwa, T. 2013. Replacing canola meal as the major protein source with wheat dried distillers’ grains alters omasal fatty acid flow and milk fatty acid composition in dairy cows. Can. J. Anim. Sci. 93: 137–147. Wheat dried distillers’ grains with solubles (W-DDGS) has a high fat content and is now commonly fed to dairy cows in western Canada. The objective was to determine the effects of replacing canola meal (CM) with W-DDGS on omasal fatty acid (FA) flow and milk FA composition. Four ruminally-cannulated lactating dairy cows were used in a 4×4 Latin square design with 28-d periods. Cows were fed either a standard barley silage-based total mixed ration (TMR) (0% W-DDGS, control) or TMR containing 10, 15 and 20% W-DDGS, with W-DDGS replacing CM as the major protein source. The omasal flow of C18:1 trans-10, C18:1 trans-11, C18:2n-6, C18:3n-3, conjugated linoleic acid (CLA) cis-9, trans-11, and total CLA increased linearly with increasing levels of W-DDGS. There were quadratic changes in milk concentrations of total C18:2, C18:3n-3, total n-3 polyunsaturated fatty acid (PUFA), and total n-6 PUFA as the level of W-DDGS increased. Feeding increasing amounts of W-DDGS resulted in a linear increase in milk concentrations of CLA cis-9, trans-11, CLA trans-11, trans-13, and total CLA, whereas milk concentrations of C18:1 trans-11 tended to increase linearly when level of W-DDGS increased. Feeding increasing amounts of W-DDGS resulted in quadratic changes in milk concentrations of C18:2n-6 and C18:3n-3. In summary, feeding increasing amounts of W-DDGS increased milk concentrations of key FA like C18:2n-6, C18:3n-3, CLA cis-9, trans-11, and total CLA, which can potentially improve human health.

Effects of conjugated linoleic acids on prostaglandin secretion by bovine endometrial epithelial cells in vitro

To determine the effects of 2 conjugated linoleic acid (CLA) isomers (cis-9, trans-11 and trans-10, cis-12) on synthesis of prostaglandin (PG) E(2) and F(2α) and expression of prostaglandin H synthase-2 (PGHS-2) of adult and fetal bovine endometrial epithelial cells in vitro. Primary cultures of endometrial epithelial cells obtained from 4 adult cows and 4 fetal bovine carcasses. Cells were exposed to 0, 50, 100, or 200μM cis-9, trans-11 or trans-10, cis-12 CLA isomers for 24 hours. Culture media collected before and after 6 hours of stimulation of cells with phorbol 12-myristate 13-acetate were assayed to detect PGE(2) and PGF(2α) via ELISA. After stimulation, cells were collected for western blot analysis to quantify PGHS-2. Concentrations of PGF(2α) and PGE(2) were significantly lower in culture media of adult and fetal endometrial epithelial cells exposed to any concentration of either CLA than they were in media of cells not exposed to CLAs. The trans-10, cis-12 CLA isomer seemed to decrease PG production more markedly than did the cis-9, trans-11 CLA isomer. Most concentrations of both CLAs significantly reduced culture media PGE(2):PGF(2α) concentration ratios of cells. Exposure of cells to CLAs did not affect expression of PGHS-2 protein. Results of this study indicated CLAs significantly decreased PGF(2α) and PGE(2) concentrations and PGE(2):PGF(2α) concentration ratios for cultures of adult and fetal endometrial epithelial cells with no apparent effect on PGHS-2 expression. Similar effects in cows could have effects on maternal recognition of pregnancy and immune function.

Milk fat responses to butterfat infusion during conjugated linoleic acid-induced milk fat depression in lactating dairy cows

During diet-induced milk fat depression (MFD), the short and medium-chain fatty acids (SMCFA), which are synthesized de novo in the mammary gland, are reduced to a much greater extent than the long-chain fatty acids (LCFA) that originate from the circulation. Our hypothesis was that increased availability of SMCFA might rescue conjugated linoleic acid (CLA)-induced MFD in lactating dairy cows. To test that hypothesis, 4 rumen-fistulated lactating Holstein cows (128±23 d in milk) were used in a 4 × 4 Latin square design with 3-wk experimental periods. Treatments were applied during the last 2 wk of each period and included 3× daily abomasal infusion of a total of (1) 230 g/d of LCFA (blend of 59% cocoa butter, 36% olive oil, and 5% palm oil); (2) 420 g/d of butterfat (BF); (3) 230 g/d of LCFA with 27 g/d of CLA (LC-CLA), containing 10 g/d of trans-10,cis-12 CLA; and (4) 420 g/d of butterfat with 27 g/d of CLA (BF-CLA). Butterfat provided 50% of C16 (115 g/d) and similar amounts of C18 FA as found in LCFA, such that the difference between the BF and LCFA treatments was 190 g/d of SMCFA. No treatment effects were observed for DMI or milk yield. Milk fat content was reduced by 41 and 32%, whereas milk fat yield was reduced by 41 and 38% with LC-CLA and BF-CLA, respectively, compared with their respective controls. Abomasal infusion of CLA reduced de novo synthesized fatty acid (DNFA; SMCFA and 50% C16:0) concentration, whereas DNFA tended to be greater with BF infusion. An interaction was observed between SMCFA and CLA as the increased availability of SMCFA reduced stearoyl-CoA-desaturase-1 gene expression, whereas it tended to reduce lipoprotein lipase (LPL), 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT-6), sterol regulatory element-binding protein cleavage-activating protein (SCAP), and peroxisome proliferator-activated receptor γ (PPAR-γ) gene expression in the presence of CLA. The mRNA expression of genes involved in de novo fatty acid synthesis [acetyl-coenzyme A carboxylase α (ACACA) and fatty acid synthase (FASN)], fatty acid uptake (LPL), and triglyceride synthesis [AGPAT-6 and diacylglycerol O-acyltransferase 1 (DGAT-1)] along with protein abundance of the ACC and FASN were reduced with CLA. However, the increased availability of SMCFA had no effect on lipogenic gene expression except for LPL, whose expression was increased with BF infusion. The nutritional manipulation by increasing the intestinal availability of SMCFA was not sufficient to rescue CLA-induced MFD.

Supplementation of conjugated linoleic acid in dairy cows reduces endogenous glucose production during early lactation

Trans-10,cis-12 conjugated linoleic acid (CLA) supplementation causes milk fat depression in dairy cows, but CLA effects on glucose metabolism are not clear. The objective of the study was to investigate glucose metabolism, especially endogenous glucose production (eGP) and glucose oxidation (GOx), as well as hepatic genes involved in endogenous glucose production in Holstein cows supplemented either with 50g of rumen-protected CLA (9% trans-10,cis-12 and 10% cis-9,trans-11; CLA; n=10) or 50g of control fat (24% C18:2; Ctrl; n=10) from wk 2 before parturition to wk 9 of lactation. Animal performance data were recorded and blood metabolites and hormones were taken weekly from 2wk before to 12wk after parturition. During wk 3 and 9 after parturition, glucose tolerance tests were performed and eGP and GOx were measured by [U-13C] glucose infusion. Liver biopsies were taken at the same time to measure total fat and glycogen concentrations and gene expression of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and carnitine palmitoyl-transferase 1. Conjugated linoleic acid feeding reduced milk fat, but increased milk lactose output; milk yield was higher starting 5wk after parturition in CLA-fed cows than in Ctrl-fed cows. Energy balance was more negative during CLA supplementation, and plasma concentrations of glucose were higher immediately after calving in CLA-fed cows. Conjugated linoleic acid supplementation did not affect insulin release during glucose tolerance tests, but reduced eGP in wk 3, and eGP and GOx increased with time after parturition. Hepatic gene expression of cytosolic phosphoenolpyruvate carboxykinase tended to be lower in CLA-fed cows than in Ctrl-fed cows. In spite of lower eGP in CLA-fed cows, lactose output and plasma glucose concentrations were greater in CLA-fed cows than in Ctrl-fed cows. This suggests a CLA-related glucose sparing effect most likely due to lower glucose utilization for milk fat synthesis and probably because of a more efficient whole-body energy utilization in CLA-fed cows.

Genetic engineering of Yarrowia lipolytica for enhanced production of trans-10, cis-12 conjugated linoleic acid

BackgroundConjugated linoleic acid (CLA) has been extensively studied for decades because of its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and modulation of the immune system. We previously described the production of trans-10, cis-12 CLA in Yarrowia lipolytica by expressing the gene coding for linoleic acid isomerase from Propionibacterium acnes (pai). However the stable strain produced CLA at about 0.08% of dry cell weight (DCW), a level of production which was not high enough for practical applications. The goal of the present study was to enhance production of CLA by genetic engineering of Y. lipolytica strains.ResultsWe have now co-expressed the delta 12-desaturase gene (FADS12, d12) from Mortierella alpina together with the codon-optimized linoleic acid isomerase (opai) gene in Y. lipolytica, expressed under the control of promoter hp16d modified by fusing 12 copies of UAS1B to the original promoter hp4d. A multi-copy integration plasmid was used to further enhance the expression of both genes. Using glucose as the sole carbon source, the genetically-modified Y. lipolytica produced trans-10, cis-12-CLA at a level of up to 10% of total fatty acids and 0.4% of DCW. Furthermore, when the recombinant yeast was grown with soybean oil, trans-10, cis-12-CLA now accumulated at a level of up to 44% of total fatty acids, which represented 30% of DCW after 38.5 h of cultivation. In addition, trans-10, cis-12-CLA was also detected in the growth medium up to 0.9 g/l.ConclusionsWe have successfully produced trans-10, cis-12-CLA with a titre of 4 g/l of culture (3.1 g/l in cells and 0.9 g/l in culture medium). Our results demonstrate the potential use of Y. lipolytica as a promising microbial cell factory for trans-10, cis-12-CLA production.