The objective of this study was to evaluate the possible role of the peroxisome proliferator-activated receptors (PPAR: PPAR-α, PPAR-β/δ, and PPAR-γ) in diet and CLA-induced milk fat depression (MFD) in dairy cows. We hypothesized that the expression of PPAR, which regulate lipid metabolism and bind to PUFA, could be modulated by biohydrogenation intermediates that induce MFD, thereby interfering with milk fat synthesis. First, tissue profiling revealed that PPAR-α and PPAR-β/δ had low expression in mammary tissue compared with the liver. A comparison of lactating and nonlactating tissue from the same cows showed that expression of all 3 PPAR isoforms did increase during lactation. Mammary expression of the PPAR family during MFD was then observed in 9 mid-lactation cows in a 3 × 3 Latin square design with MFD induced by a 3-d intravenous infusion of trans-10,cis-12 CLA or feeding a high-oil and low-forage diet. The expression of all 3 PPAR isoforms remained largely unaltered during CLA and diet-induced MFD, except for an increase in PPAR-α target genes CPT1A and ACADVL that are involved in β-oxidation. The interaction of PPAR-γ chemical agonist troglitazone and antagonist T0070907 and CLA was then investigated in bovine mammary epithelial cells. The activation and inhibition of PPAR-γ did not overcome trans-10,cis-12 CLA inhibition of lipogenesis despite the agonist stimulating PPAR-γ expression. Furthermore, PPAR-γ activation did not modify the expression of lipogenic genes. Overall, the results fail to support a functional role of the PPAR family in the inhibition of lipogenesis during MFD in dairy cows.

The conjugated linoleic acid (CLA) isomer cis-9, trans-11 is an anticarcinogen that inhibits cell proliferation and/or induces apoptosis of tumor cells. The objective of this study was to evaluate the expression of genes responsible for cell cycle regulation and apoptosis in tumor explants of mammary anaplastic carcinoma (AC) and mammary tubulopapillary carcinoma (TC) cultured in vitro with the CLA isomers cis-9, trans-11 and trans-10, cis-12. In this study we used mammary explants from two adult female dogs that revealed two types of malignant tumors: (a) anaplastic mammary carcinoma (AC) and (b) mammary tubulopapillary carcinoma (TC). The explants (n = 6 per treatment) had an average weight of 80.0 ± 2.0 mg and were cultured for 24 h in 35 mm culture plates under the following treatments: (a) Control: Culture medium + fatty acid free bovine serum albumin (BSA); (b) Culture medium + cis-9, trans-11 CLA (75 µM) diluted with fatty acid free bovine serum albumin (BSA), and; (c) Culture medium + trans-10, cis-12 CLA (75 µM) diluted with fatty acid free bovine serum albumin (BSA). After that, total RNA was extracted, complementary DNA was synthesized (cDNA), and quantitative analysis by real-time polymerase chain reaction (RT-qPCR) was conducted. Data were analyzed using the MIXED procedure of SAS. Compared with the Control, the CLA trans-10, cis-12 treatment decreased expression of the gene encoding the p53 by 20

The objective was to evaluate the effects of palmitic (PA) and stearic (SA) acids and their combination on gene expression of acetyl-CoA carboxylase alpha (ACACA alpha) PII, fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), fatty acid binding proteins (FABP3 and FABP4), and fatty acid CD36 (CD36) translocator in in vitro cultured lactating mammary tissue. Mammary gland explants of two Lacaune ewes rearing twins at 30 +/- 5 days in milk, and with body weight of 70 +/- 5 kg and body condition score of 3.0 +/- 0.5 were used. Explants were cultured for 24 h in 6-well plates at 37 C with 5% CO2 and saturated humidity. The treatments were a) Control: 3.5 mL of culture medium + 0.1 % bovine serum albumin; b) PA: 3.5 mL of culture medium +200 mu M of palmitic acid; c) SA: 3.5 mL of culture medium +200 mu M of stearic acid, and; d) PASA: 3.5 mL of culture medium +100 mu M palmitic acid +100 mu M stearic acid. There was no effect of treatment on FASN (P = 0.11). Compared to Control, PASA treatment increased ACACA alpha PII and CD36 mRNA abundance by 89 % (P = 0.001) and 44 % (P = 0.02), respectively, whereas, FABP4 mRNA was reduced by 44 % (P = 0.001). Compared to Control, SA increased ACACA alpha PII mRNA by 41 % (P = 0.001), whereas FABP4 mRNA was decreased in 41 %. Compared to PASA, the PA treatment, respectively, reduced the mRNA abundance of ACACA alpha PII, CD36 and FABP3 in 48, 40 and 24 % (P = 0.001) excepting that of FABP4 and SCD, which were increased by 131 and 39 % (P = 0.001), respectively. Compared to Control, all treatments decreased SCD mRNA abundance (P = 0.01). Therefore, PASA in mammary explants of lactating ewes increased the expression of genes involved in milk fat synthesis, FA uptake and cellular transport. The increased mRNA abundance of FA translocator CD36 by the combined FAs supports an important role for this gene in mammary explants.

The objective of this study was to test the hypothesis that stearic acid supplementation increases milk fat content and overcomes the antilipogenic effects of trans-10, cis-12 conjugated linoleic acid (CLA) in lactating ewes. Twenty-eight Lacaune ewes (36 ± 2 DIM; 70.5 ± 9.6 kg BW), producing 1.8 ± 0.4 kg of milk/day were used in a completely randomized design (7 ewes/treatment) for 21 days. The treatments were: 1) Control; 2) CLA (6.4 g/day of trans-10, cis-12 CLA); 3) SA (28 g/day of stearic acid) and; 4) CLASA (6.4 g/day of trans-10, cis-12 CLA plus 28 g/day of stearic acid). All data were analyzed using a mixed model that included the fixed effect of treatment and the random effect of ewe. SA did not alter milk fat content and yield relative to Control (91.9 vs. 91.2 ± 4.1 g/d). SA in association with trans-10, cis-12 CLA (CLASA) was not able to overcome the reduction in fat content and fat yield induced by CLA (75 vs. 82 ± 0.14 g/d). SA increased the relative abundance of CD36, FABP4 and PPAR-γ mRNA by 140%, 112% and 68% compared to CLASA. SA also reduced the relative abundance of ACACAα PII and SCD when compared to Control (45% and 39%). Compared to CLA, CLASA treatment had no effect on the mRNA abundance of FASN, LPL, CD36, SCD, FABP4, AGPAT6, SREBP1 and PPAR-γ. In conclusion, stearic acid supplementation did not increase milk fat synthesis and did not overcome the CLA-induced milk fat depression when associated with trans-10, cis-12 CLA.

The objective of this study was to evaluate stearic acid supplementation (C18:0) on the production, composition, fatty acid profile and the expression of lipogenic genes in the mammary gland of late lactating ewes. We used 30 primiparous and multiparous Lacaune ewes with a mean body weight (BW) of 66.5 +/- 9.4 kg and body condition score (BCS) of 3.0 +/- 0.5 at late lactation (122 +/- 12 DEL), producing 1.0 +/- 0.3 kg of milk/day. The treatments were: Control and C18 (28 g/animal/d of C18:0). When compared with Control, C18:0 reduced silage dry matter intake (DMI) by 13.1 % (P = 0.0003), milk production by 8.1 % (P = 0.05), lactose production by 3.3 % (P = 0.0004), and lactose content by 3.4 % (P = 0.0002). There was no treatment effect on the production and content of fat, protein, and total solids. The gene expression of acetyl-CoA carboxylase alpha promoter 2 (ACACA PII) and fatty acid synthase (FASN) were reduced by 30 % (P = 0.02) and 26.1 % (P = 0.04), respectively. The C18:0 supplementation is not effective in increasing the fat content in late lactating dairy ewes and reduces DMI, milk production, lactose levels, and gene expression involved in fatty acid synthesis.

AbstractTrans-10,cis-12 conjugated linoleic acid (CLA) decreases milk fat synthesis in lactating sows and involves, at least in part, the down-regulation of lipogenic genes. The objective was to evaluate the effect of CLA on milk composition and lipogenic gene expression. Twenty multiparous sows were randomly assigned to one of the two treatments for 18 d (from day 7 to day 25 of lactation): (1) control (no CLA added) and (2) 1 % of CLA mixed into the ration. CLA treatment decreased milk fat and protein content by 20 % (P= 0·004) and 11 % (P= 0·0001), respectively. However, piglet weight did not differ between treatments (P= 0·60). Dietary CLA increased the concentration of SFA in milk fat by 16 % (P< 0·0001) and decreased MUFA by 17·6 % (P< 0·0001). In the mammary gland, CLA reduced gene expression of acetyl-CoA carboxylase-αby 37 % (P= 0·003), fatty acid synthase by 64 % (P= 0·002), stearoyl-CoA desaturase 1 by 52 % (P= 0·003), lipoprotein lipase by 26 % (P= 0·03), acyl glycerol phosphate acyltransferase 6 by 15 % (P= 0·02) and diacylglycerol acyltransferase 1 by 27 % (P= 0·02), whereas the expression of fatty acid binding protein 3 was not altered by CLA treatment (P= 0·09). Mammary expression of casein-βandα-lactalbumin was reduced by CLA by 68 % (P= 0·0004) and 62 % (P= 0·005), respectively. Additionally, CLA had no effect on the expression of lipogenic genes evaluated in adipose tissue. In summary, CLA reduced milk fat content without negatively affecting litter performance and it affected mammary expression of genes involved in all lipogenic pathways studied.

Culture of mammary cells with palmitic acid (PA) has shown contradictory results for gene expression of enzymes involved in de novo synthesis pathway. The objective of this study was to evaluate the effects of PA on the mRNA abundance of enzymes involved in de novo fatty acid (FA) synthesis pathway, acetyl-CoA carboxylase alpha from promoters II and III (ACACA-alpha PII and ACACA-alpha PHI), fatty acid synthase (FASN), fatty acid internalization (CD36 molecule), intracellular transport (fatty acid binding proteins; FABP3 and FABP4) and de saturation (stearoyl-CoA desaturase 1; SCD1) of FA during milk fat synthesis. Mammary explants obtained through biopsy from three Lacaune ewes with 40 days in milk (DIM) were used. The explants were cultured for 24 h using the following treatments: Control (culture medium + bovine serum albumin (BSA, 98%) and Palmitic Acid (culture medium + 75 mu M of C16:0 - 99%). Immediately after incubation, total RNA was extracted, complementary DNA was synthesized, and quantitative real time polymerase chain reaction was performed. The PA treatment upregulated the mRNA expression levels of ACACA-alpha PHI, ACACA-alpha PII, and FASN by 1.5, 1.3, and 1.3 fold, respectively, compared to Control. The gene expression levels of CD36, FABP3, FABP4, and SCD1 were not affected by the PA treatment. Thus, PA treatment upregulated the mRNA expression of genes codifying proteins involved in the de novo synthesis pathway.

Mammary cell cultures have been used to evaluate gene expression under many types of treatments. Additionally, culturing explants of mammary gland of lactating animals could allow the evaluation of the effects of bioactive molecules, considering that the tissue maintains its normal cellular activity as it does in the living animal. The objectives of this study were to evaluate mammary gland explants of lactating ewes as an in vitro model to test the gene expression of transcription factors and lipogenic genes involved in milk synthesis. Explants were cultured for 3 or 24 h and subjected or not to a challenge dose of conjugated linoleic acid (CLA = 290 mu M of trans-10, cis-12) and; No CLA = culture medium. The Comet Assay was performed to evaluate DNA fragmentation. RNA was extracted, cDNA was synthesized, and RT-qPCR was carried out. The Comet Assay showed absence of DNA damage in culture at 24 h. Collectively, our results showed gene expression of lipogenic genes and alpha lactalbumin, supporting that mammary gland explants of lactating ewes can be cultured for 24 h for molecular studies.

This study evaluated the effect of trans-10, cis-12 CLA on the expression of genes involved in decreasing milk fat synthesis in two groups of lactating ewes with different metabolic weights. Twelve Lacaune ewes were alloted, according to metabolic weight (MW), into a Light Metabolic Weight (LMW) and Heavy Metabolic Weigth (HMW) groups, both of which were orally fed 30 g/d of rumen-unprotected CLA supplement containing 29.9% trans-10, cis-12. Milk samples were collected on days 0, 3 and 10 and mammary and adipose tissue biopsies were taken on days 3 and 10. RNA extraction, cDNA synthesis and RT-qPCR were performed and data were analyzed using the MIXED procedure of SAS as repeated measure in a completely randomized design. The trans-10, cis-12 CLA reduced milk fat synthesis between days 3 and 10 (P = 0.001), and the HMW group had a greater expression (P < 0.05) of the genes acetyl-CoA carboxylase alpha - promoter III (ACACA-alpha PIII), fatty acid synthase (FASN), stearoil CoA desaturase 1 (SCD1) and thyroid hormone responsive spotl4 (THRSP) in the mammary gland and of acetyl-CoA carboxylase alpha - promoter I (ACACA-alpha PI) and leptin (P < 0.05) in adipose tissue. Our findings indicate that the response of animals to trans-10, cis-12 CLA appears to be metabolic weight dependent.

The trans-10, cis-12 conjugated linoleic acid (CLA) causes milk fat depression by downregulating expression of genes and transcription factors involved in lipogenesis and it has been proposed that peroxisome proliferator-activated receptor gamma (PPARγ) can be inhibited by trans-10, cis-12 CLA. The PPARγ is a nuclear receptor activated by natural or synthetic ligands and promotes expression of lipogenic genes and its effect on mammary lipogenesis and the interaction with trans-10, cis-12 CLA in lactating ewes was evaluated using thiazolidinedione (TZD), a chemical PPARγ agonist. A total of 24 lactating ewes were randomly assigned to one of the following treatments for 7 days: (1) Control (5 ml/day of saline solution); (2) TZD (4 mg/kg of BW/day in 5 ml of saline solution); (3) CLA (27 g/day with 29.9% of trans-10, cis-12); (4) TZD+CLA. Compared with Control, milk fat content was not changed by TZD, but was decreased 22.3% and 20.5% by CLA and TZD+CLA treatments. In the mammary gland, TZD increased PPARγ gene expression by 174.8% and 207.8% compared with Control and TZD+CLA treatments, respectively. Conjugated linoleic acid reduced sterol regulatory element-binding transcription protein 1 (SREBP1) gene expression 89.2% and 75.3% compared with Control and TZD+CLA, respectively, demonstrating that TZD fails to overcome CLA inhibition of SREBP1 signaling. In adipose tissue, the expression of SREBP1 and stearoyl CoA desaturase 1 (SCD1) genes were increased by the TZD+CLA treatment, compared with the other treatments. Conjugated linoleic acid decreased milk fat concentration and expression of lipogenic genes, while TZD had no effect on milk fat concentration, expression of lipogenic enzymes or regulators in the mammary gland and failed to overcome the inhibition of these by CLA. Therefore, CLA inhibition of milk fat synthesis was independent of the PPARγ pathway in lactating dairy ewes.

A limited number of studies in lactating sheep have compared milk fat responses to calcium salts (Ca-salts) differing in fatty acid (FA) composition and their interactions with different dietary conditions. The objective of this study was to evaluate the effects of Ca-salts of palm or soybean FA on milk fat under Normal and CLA-induced milk fat depression (MFD) scenarios in lactating dairy ewes. Thirty-eight Lacaune and thirty-seven East Friesian multiparous ewes were used in a 2 × 2 factorial design. The main factor consisted of two diets designed to create two distinct scenarios: Normal and CLA-induced MFD (CLA-MFD, 30g/d of CLA 29.9% trans−10, cis−12 CLA as methyl ester). The subfactor was supplementation of Normal or CLA-MFD scenarios with 27g/d of Ca-salts of palm FA, or 30g/d of Ca-salts of soybean FA, resulting in four treatments: 1) Normal + Ca-Palm, 2) Normal + Ca-Soy, 3) CLA-MFD + Ca-Palm, and 4) CLA-MFD + Ca-Soy. Overall, the CLA-MFD scenario decreased milk fat concentration by 1.64% units and decreased milk fat yield by 17.3g/d. Ewes that received Ca-Palm had overall 0.29% units higher milk fat concentration, and 13.4% greater milk fat concentration when fed the CLA-MFD diet. Ca-Soy increased trans−10 C18:1 (131.0%), trans-11 C18:1 (30.4%), and cis-9, trans-11 CLA (21.1%) in the CLA-MFD diet. In conclusion, supplementation with Ca-Palm resulted in overall greater milk fat concentration. In a MFD scenario, supplementation with Ca-Soy increased concentration of milk trans FA linked to altered rumen biohydrogenation and further aggravated MFD.

Feeding trans-10, cis-12 CLA to lactating ewes reduces milk fat by down-regulating expression of enzymes involved in lipid synthesis in the mammary gland and increases adipose tissue lipogenesis. Acetyl-CoA carboxylase α (ACC-α) is a key regulated enzyme in de novo fatty acid synthesis and is decreased by CLA. In the ovine, the ACC-α gene is expressed from three tissue-specific promoters (PI, PII and PIII). This study evaluated promoter-specific ACC-α expression in mammary and adipose tissue of lactating cross-bred Lacaune/Texel ewes during milk fat depression induced by rumen-unprotected trans-10, cis-12 CLA supplement. In all, 12 ewes arranged in a completely randomized design were fed during early, mid and late lactation one of the following treatments for 14 days: Control (forage+0.9 kg of concentrate on a dry matter basis) and CLA (forage+0.9 kg of concentrate+27 g/day of CLA (29.9% trans-10, cis-12)). Mammary gland and adipose tissue biopsies were taken on day 14 for gene expression analysis by real-time PCR. Milk fat yield and concentration were reduced with CLA supplementation by 27%, 21% and 35% and 28%, 26% and 42% during early, mid and late lactation, respectively. Overall, our results suggest that trans-10, cis-12 CLA down-regulates mammary ACC-α gene expression by decreasing expression from PII and PIII in mammary gland and up-regulates adipose ACC-α gene expression by increasing expression from PI.

The current literature shows that there is animal and marker variability in digestibility marker recovery, but does not address the effect of this variability on individual animal DMI predictions. The objective of this study was to test the use of various markers and administration methodologies to predict DMI in grazing systems and determine the main contributors to variability. Eight rumen cannulated Nellore steers were randomly assigned to two 4 × 4 Latin squares. Steers had ad libitum access to pasture of Brachiaria brizantha. Steers were either not supplemented or individually supplemented with a mixture of fine ground corn and sodium monensin at 0.3, 0.6 or 0.9% of body weight. Steers were each administered 3 external markers via rumen cannula: a C32 controlled release capsule (CRC) on d 3; LIPE® (purified lignin), once daily on d 7 to 15; and Cr2O3 once daily on d 1 to 15 of each period. Fecal grab samples were collected twice daily for the last 5 d of the 15-d periods. Intake predictions were calculated using the Large Ruminant Nutrition System (LRNS), C31:C32 ratio, C33:C32 ratio, LIPE®, and Cr2O3. Indigestible DM (iDM), indigestible NDF (iNDF) and indigestible ADF (iADF) were used as internal markers. LIPE® was determined by infrared spectroscopy, Cr2O3 by inductively coupled plasma optical emission spectroscopy and alkanes by gas chromatography. A 240-h rumen incubation was used for the internal markers. A mixed model with the fixed effect of supplementation level and random effects of Latin square, animal(Latin square), period, and animal × period was used to determine treatment effects. LIPE® and Cr2O3 detected a treatment effect on pasture intake (P < 0.10) except for the Cr2O3/iADF combination (P = 0.43). Only LIPE® detected a treatment effect on total intake (P < 0.03). The alkanes provided greater and variable intake predictions. The final 4 d in each period demonstrated decreased CRC release rate compared with the previous 8 (mm/d = -0.012*d2 + 0.091*d + 3.93, R2 = 0.79). A completely random model containing internal and external markers, and their interactions with animal, period, and supplementation level determined which variables contributed to intake prediction variability. Variability was mainly due to internal and external markers (11–66% of variation), residual error (11–24% of variation), and external marker × animal interactions (5–16% of variation). Digestibility markers should be used cautiously to predict individual intakes in a grazing system due to animal by marker interactions, though markers can detect treatment differences.