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ANIMAL NUTRITION |
* Department of Animal Science, University of Wyoming, Laramie, 82071; and
and
Department of Animal Science, Colorado State University, Ft. Collins, 80523
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: mammary gland • mRNA • milk somatic cell • lipogenesis
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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). Certain isomers of CLA are known to regulate fatty acid metabolism in various tissues including the mammary gland. Baumgard et al. (2002), for example, demonstrated the trans-10, cis-12 CLA isomer altered expression of genes involved in milk lipid synthesis in dairy cows by decreasing mRNA for acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase (SCD). Lipogenic enzymes are regulated, in part, through mRNA transcription (Munday, 2002; Stoeckman and Towle, 2002; Barber et al., 2003); thus, quantifying the respective mRNA should provide insight into their dietary regulation. Mammary tissue from lactating goats was used by Bernard et al. (2005) to measure abundance of mRNA for lipoprotein lipase (LPL), ACC, FAS, and SCD; those researchers determined that supplemental fat was a modifier of gene expression. Our laboratory demonstrated that supplemental linoleic acid fed to beef cows increased CLA (18:2 cis-9, trans-11) in milk fat and decreased milk output of 10:0, 12:0, and 14:0 (Lake et al., 2004). Reduced output of these medium-chain fatty acids may reflect dietary effects on enzymes involved in mammary lipogenesis because these fatty acids are typically synthesized de novo.
Obtaining RNA to study transcripts of enzymes involved in lipid metabolism from the mammary gland of a lactating beef cow is difficult because the process requires either mammary tissue dissection or biopsy; the latter can be costly and potentially injurious to both animal and technician. On the other hand, Boutinaud et al. (2002) used milk somatic cells to study gene expression for casein and 
-lactalbumin in mammary gland of lactating goats. In the current study, we hypothesized that somatic cells obtained from milk of lactating beef cows could provide RNA similar to that extracted from mammary gland tissue, as well as provide valuable information regarding gene expression of enzymes involved in lipid metabolism. Our objectives were 2-fold: to determine the effect of dietary linoleate on milk fat composition and transcript abundance of ACC, FAS, LPL, and SCD mRNA in mammary tissue, and to evaluate milk somatic cell mRNA as a source of mammary tissue mRNA for these enzymes.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Beginning on d 1 postpartum, 18 primiparous, cross-bred beef cows (primarily Angus x Gelbvieh; 411 ± 24 kg of BW; 5.25 BCS on a 1 to 9 scale) were offered Foxtail millet hay at 1.68% of BW daily (DM basis). Cows were assigned randomly to receive either a low-fat control supplement (n = 9; 64.2% cracked corn, 32.1% safflower seed meal, and 3.7% liquid molasses; as-fed basis) fed at 0.35% of BW daily (DM basis) or a cracked high-linoleate safflower seed supplement (n = 9; 94.0% cracked high-linoleate safflower seeds [79% of total fatty acids as 18:2 cis-9,cis-12], and 6% liquid molasses; as-fed basis) at 0.23% of BW daily (DM basis). Diets were formulated to meet the nutrient requirements of a 410-kg, primiparous beef cow producing 9.4 kg of milk at peak lactation (NRC, 2000), as well as to provide similar daily quantities of N and TDN.
Cows were placed in individual stanchions twice daily and allowed 2 h to consume their respective diets. Cows were offered the supplement first to ensure its complete consumption, after which hay was delivered. All supplements were readily consumed; however, in the event that hay was left after each 2-h feeding period, refusals were weighed and sampled for laboratory analysis. Hay intake did not differ (P = 0.42) between treatment groups (control cows, 1.44% of BW daily; linoleate-supplemented cows, 1.40% of BW daily; DM basis). Dietary ingredients were analyzed for CP (Leco FP-528; Leco Corp., St. Joseph, MO) and fatty acids via direct trans-esterification (Whitney et al., 1999) with methanolic HCl (Kucuk et al., 2001). Composition of diets presented in Table 1 was based on actual consumption. The linoleate diet provided 5.4% of DM as total fatty acids. Cows were slaughtered (stunning followed by exsanguination) on d 37 ± 3 postpartum, at which time mammary gland and milk samples for somatic cells were obtained.
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Fatty Acid Methyl Ester Analysis
Approximately 30 mg of each milk fat sample were subjected to fatty acid methyl ester (FAME) preparation using 0.2 M KOH in methanol (Murrieta et al., 2003). A triacylglycerol of tridecanoic acid (13:0, 1.0 mg) was used as the internal standard. Fatty acid methyl esters were separated using an Agilent 6890 GLC (Agilent Technologies, Wilmington, DE) equipped with a flame ionization detector and a 100 m x 0.25 mm (i.d.), fused silica capillary column (SP-2560, 0.2 µm film thickness, Supelco, Bellefonte, PA). Oven temperature was maintained at 175°C for 40 min, and then increased to 240°C at 10°C/min. Injector and flame-ionization detector temperatures were 245°C. Helium was the carrier gas at a split ratio of 50:1 and a constant flow rate of 0.8 mL/min. Fatty acid peaks were recorded and integrated using GC ChemStation software (version A.09.03, Agilent Technologies). Individual fatty acids were identified by comparing retention times with known FAME standards (Nu-Chek Prep, Inc., Elysian, MN, and Matreya Inc., Pleasant Gap, PA). Identification of 18:1 trans-10 was putative and based on the position of a peak observed between peaks identified as 18:1 trans-9 and 18:1 trans-11 (Molkentin and Precht, 1995).
RNA Extraction and Analysis
Approximately 60 mg of frozen mammary tissue, representing right and left halves of the excised mammary gland, was weighed into sterile 12 x 75 mm plastic tubes and immediately homogenized with a Tekmar Tissuemizer (Tekmar Company, Cincinnati, OH) using the buffer provided with the RNA extraction kit. Total RNA was extracted using an RNeasy Lipid Tissue Mini Kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions, suspended in 50 µL of sterile water containing 0.1% diethyl pyrocarbonate, and stored at –80°C.
After milk centrifugation and milk fat removal, the cellular pellets from each tube were washed in PBS (pH 7.4, 4°C) to remove any remaining infranatant. The total somatic cell pellet, obtained from the 200-mL milk sample, was resuspended in 0.75 mL of Tri Reagent LS (Molecular Research, Inc., Cincinnati, OH) for total RNA extraction according to the manufacturer’s instructions. After extraction, the total RNA pellet was suspended in 50 µL of sterile water containing 0.1% diethyl pyrocarbonate and stored at –80°C.
Concentrations of total extracted RNA for all samples were determined using absorbance at 260 nm (A260) and averaged 80 µg of total RNA from mammary tissue and 20 µg of total RNA from milk somatic cells. The integrity of the total RNA was verified by using a denaturing, 6% acrylamide gel containing 7 M urea, adjusted to pH 8.3. Ten micrograms of total RNA, extracted from either mammary tissue or milk somatic cell samples, was used for each ribonuclease protection assay (RPA).
Probes from cDNA for ACC, FAS, SCD, LPL, and 18S RNA were generated from bovine mammary RNA as described by Lee et al. (2002). The RPA was used to quantify mRNA according to Lee et al. (2002) and modified such that samples were placed in a 95°C water bath for 2 min followed by a 15-h hybridization in a 48°C water bath. After hybridization, the samples were loaded onto a 10 cm x 8 cm x 1.5 mm-thick, vertical PAGE apparatus containing a 7 M urea, and 4% acryl-amide:bisacrylamide gel (19:1) mixture, adjusted to pH 8.3. All probes were hybridized to the target mRNA simultaneously. Membrane transfer and autoradiography were conducted according to Lee et al. (2002), except that a 45-min exposure to x-ray film (Sterling RX- B, bioWorld, Dublin, OH) was used. Films were digitized and analyzed using Quantity One Imaging Software with a Gel Doc 1000 camera and light box (BioRad, Hercules, CA). Data for mRNA represented relative abundance of mRNA expressed as optical density units of each band per optical density units of the 18S RNA band. Samples of both mammary and milk were analyzed on the same gel.
Statistical Analyses
Data from FAME analysis were expressed as weight percentage of total fatty acids and analyzed as a completely randomized design using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC). The model included dietary treatment as a fixed effect. Messenger RNA abundance data were analyzed as a split-plot design with the main plot as a completely randomized design and with tissue (mammary vs. milk) as the sub plot using the MIXED procedure of SAS. The main plot error term was the random effect of animal within dietary treatment. Because the mRNA abundance response to dietary treatment seemed to be greater for mammary samples, posthoc statistical analyses (as described for FAME data) were conducted to evaluate dietary treatment effects within tissue type. Pearson correlation coefficients between mammary and milk mRNA relative abundance within dietary treatments were determined using the CORR procedure of SAS.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Fatty Acid Profile of Milk Fat
Weight percentages of 10:0, 12:0, and 14:0 were lower (P = 0.01, 0.01, and 0.02, respectively) in the milk fat of linoleatefed cows than in milk fat of control cows (Table 2). The lower proportions of these fatty acids in the linoleatefed cows suggest that de novo fatty acid synthesis was decreased in the mammary tissue of these cows. Milk fat is composed primarily of triacylglycerols, with the fatty acids arising from de novo synthesis within the mammary tissue and uptake from the general circulation. Griinari et al. (1998) reported that trans-octadecenoic acids, a metabolite of ruminal biohy-drogenation of unsaturated fatty acids, reduced fatty acids produced de novo by 32% and reduced overall milk fat yield by 36%. In our study, milk production was not measured; however, the weight percentages of fatty acids 10:0, 12:0, and 14:0 in milk fat was 33% less for linoleate-fed cows than for control cows. Thus, it is possible that de novo fatty acid synthesis was decreased by dietary linoleate supplementation, which resulted in increased (P = 0.02 to 0.06) trans-octadecenoic acids.
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demonstrated that supplemental high-linoleate safflower seeds, providing 5% of DMI as fat, increased duodenal flow of trans-vaccenic acid and other biohydrogenation intermediates. A cow in lactation will predominantly transport fatty acids to the mammary gland for incorporation into milk lipids (Palmquist et al., 1993), which would explain the increased availability of biohydrogenation intermediates to the mammary gland and subsequent incorporation into milk fat of linoleate-fed cows.
Although total milk fat concentration was not different between treatments (P = 0.30), the milk fat of linole-ate-fed cows tended to have lower (P = 0.06) proportions of 16:0, whereas proportions of 18:0 (P = 0.05) and 18:1 cis-9 (P = 0.04) were greater than those of the control cows. These results were similar to previous studies from our laboratory (Lake et al., 2004), in which a decrease in the amounts of 16:0 and an increase in the amounts of 18:0 and 18:1 cis-9 in milk fat of cows fed a similar dietary fat supplement were reported. Because 16:0 is partially derived from de novo synthesis (Palmquist et al., 1993), the lower proportions of this fatty acid in linoleate-fed cows may have been due to lower proportions of fatty acid biosynthesis intermediates (10:0, 12:0, and 14:0). In early lactation, regardless of dietary treatment, cows are typically in a negative energy balance; thus, fatty acids made available through the hydrolysis of circulating triacylglycerols by mammary LPL and mammary uptake of NEFA mobilized from adipose tissue represent a significant source of milk fatty acids (Bell, 1995). Therefore, in addition to mammary lipogenesis, numerous upstream biochemical events occur to provide the mammary gland with fatty acids. For example, hormone-sensitive lipase catalyzes hydrolysis of stored triacylglycerols resulting in mobilization of fatty acids stored in adipose tissue, which increases to establish and maintain lactation (McNamara et al., 1995). These fatty acids will be transported to the liver where triacylglycerol synthesis occurs, as well as to the mammary gland for uptake. Thus, very low density lipoprotein synthesis and plasma membrane fatty acid transport also contribute significantly to mammary fatty acid supply during lactation (McNamara, 1991). Hyperinsulinemia in the cow during late gestation is coupled with an increase in prolactin levels (Ramos et al., 1999), both of which can contribute to an upregulation in the expression of mammary LPL during lactation (Carrascosa et al., 1998). Thus, an increase in mammary LPL activity coupled with increased adipose tissue fatty acid mobilization could result in increased availability of 18:0 and 18:1 cis-9. In addition, modification of the diet during early lactation may affect the source and supply of fatty acids to the lactating mammary gland.
Effect of Diet on mRNA Abundance
Results of a typical RPA are shown in Figure 1. Probes for the 4 mRNA types analyzed were hybridized simultaneously with resolution at 501 bp (LPL), 436 bp (ACC), 337 bp (SCD), and 218 bp (FAS). The 18S housekeeping RNA resolved at 81 bp, allowing correction for intraassay variation due to differing amounts of total RNA loaded within each lane of the gel.
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illustrates abundance of mRNA transcripts of the 4 enzymes studied as affected by both diet and tissue. Interactions between diet and tissue were not observed (P 
0.20) for mRNA abundances of any of the 4 enzymes. Although the numerical differences between dietary treatments within tissue type seemed to be greater for mRNA abundance within mammary tissue than in milk (Table 3), results of our posthoc statistical analyses indicated that dietary treatment did not affect mRNA abundance for the 4 transcripts in mammary (P = 0.13 to 0.20) or milk (P = 0.41 to 0.94) samples. The SEM for mRNA abundance of ACC (0.0033 vs. 0.0028), FAS (0.28 vs. 0.23), and LPL (0.34 vs. 0.24) transcripts in mammary tissue were slightly greater than in milk samples, which limited detection of statistical differences between treatments when mammary tissue was the source of RNA. The SEM for mRNA abundance of SCD was similar between mammary tissue and milk samples (0.11). Results discussed hereafter are those from the split-plot analyses presented in Table 3.
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in which vegetable oil supplements fed to lactating goats did not affect mRNA abundance in mammary tissue for these enzymes. Acetyl-CoA carboxylase and FAS catalyze the reactions responsible for de novo fatty acid synthesis. In addition to transcriptional regulation of ACC and FAS (Mao et al., 2002), it is possible that posttranscriptional regulation of ACC occurred because each of the 8 phosphorylation sites on ACC- (Hardie, 1989) are involved in covalent modification of the enzyme, which would affect enzyme activity (Boone et al., 1999). Moreover, PUFA were shown to activate phosphorylation cascades in rat liver (Hii et al., 1995), which might suggest that elevated PUFA in lactating bovine mammary gland could decrease ACC activity through similar mechanisms.
Fatty acid synthase catalyzes fatty acid biosynthesis with the ultimate formation and release of 16:0 (Stoops et al., 1975). Polyunsaturated fatty acids are known to inhibit mRNA transcription of the FAS gene, thereby reducing FAS protein (Xu et al., 2002). It is possible, therefore, that our linoleate treatment inhibited mammary FAS mRNA expression rather than abundance. This is supported by a decrease in milk fatty acids likely produced de novo (Table 2
). However, because there was no treatment difference (P = 0.38) for FAS mRNA abundance between control and linoleate-fed cows, other regulatory mechanisms were likely involved. Nakamura et al. (2000), for example, demonstrated in mice that hepatic µ-6-desaturase enzyme activity could be suppressed artificially while mRNA abundance for this enzyme was induced.
Abundance of LPL mRNA transcripts tended (P = 0.09) to be greater for cows receiving the high-linoleate supplement compared with those receiving the low-fat control diet. Greater intake of dietary fat provided a greater influx of fatty acids associated with increased abundance of LPL mRNA and likely an increase in LPL activity by the mammary gland of the linoleate-fed cows. This could have blunted ACC activity compared with the control because fatty acids, and more so fatty acyl-CoA, inhibit ACC activity (Nikawa et al., 1979). Furthermore, dietary fatty acids taken up by the mammary gland of linoleate-fed cows would have diluted medium-chain fatty acids (C10 to C14), effectively decreasing their proportions, and thus reducing measured milk profile of these fatty acids. Thus, the treatment effects noted in milk fatty acid proportions (Table 2) might not have been the result of only variations in mRNA transcription.
Stearoyl-CoA desaturase mRNA abundance was not affected (P = 0.45) by dietary treatment. This enzyme is known to be transcriptionally regulated through the actions of peroxisome proliferator-activated receptor and sterol regulatory element binding proteins (Tabor et al., 1999), so the dietary treatment differences noted for cis and trans C18 MUFA may have been attributed to increased supply provided by upregulated LPL as well as catabolism of stored lipids occurring in adipose tissue (McNamara et al., 1995; Ramos et al., 1999). Our SCD mRNA abundance results were inconsistent with results reported by Bernard et al. (2005) who reported a decrease in mammary SCD mRNA in lactating goats fed supplemental lipid. In the current study, lactating beef cows were fed cracked safflower seed supplements beginning at d 1 postpartum, whereas Bernard et al. (2005) fed goats either formaldehyde-treated linseed oil or high-oleic sunflower oil supplements during mid lactation.
Effect of Tissue Type on mRNA Abundance
Relative abundance of each mRNA was similar (P = 0.15 to 0.89) for mammary tissue and milk somatic cell mRNA (Table 3). These results were similar to those of Boutinaud et al. (2002), in which significant positive correlations between mammary tissue mRNA and milk somatic cell mRNA for -S1 casein, -lactalbumin, and 
-casein were observed. Those authors indicated that exfoliated epithelial cells of milk were biochemically similar to alveolar epithelial cells. In the current study, total somatic cells were used for the RNA extractions. Newman et al. (1991) reported the occurrence of mastitis in beef cows during early lactation to be less than 10% for cows 3 yr of age or younger with somatic cell counts of uninfected beef cows at 20,000 cells/mL of milk. Although somatic cell counts were not obtained for the current study, our unpublished data indicated an average somatic cell count of 22,000 cells/mL in beef cows fed diets similar to those in our study. Therefore, the risk of mastitis negatively influencing subsequent mammary gland analysis for this study was considered small. Nevertheless, RNA extracted from milk somatic cells of cows experiencing advanced mastitis could result in interference or artifacts from excessive leukocyte levels in the somatic cell pool.
Correlations Between Mammary Gland mRNA and Milk Somatic Cell RNA
Positive correlations of 0.68 to 0.90 (P = 0.001 to 0.05) for the 4 mRNA types between mammary tissue and milk somatic cells were observed within both dietary treatments (Table 4). Correlation values from our study were not as high as those reported by Boutinaud et al. (2002), wherein values from 0.924 to 0.999 were reported for -S1 casein, -lactalbumin, and 
-casein between milk epithelial cells and mammary tissue from lactating goats. Boutinaud et al. (2002) reported mRNA abundance of each gene as a proportion of the total signal detected, whereas we reported density of each band relative to the 18S band, which is expressed both in epithelial cells and leukocytes. Our results suggest that milk somatic cells were a reliable source of mRNA for use in analysis of lipogenic enzymes in mammary tissue. Our results further suggest that this approach would be valid for nutritional studies using dietary lipid supplementation, which may alter the fat composition of milk through effects on mammary lipid metabolism in beef cows.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Present address: USDA-ARS, NGPRL, Mandan, ND 58554.
3 Corresponding author: dcrule{at}uwyo.edu
Received for publication November 21, 2005. Accepted for publication April 13, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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gene in mammary gland is associated with recruitment of SREBP-1 to a region of the proximal promoter defined by a DNase I hypersensitive site. Biochem. J. 375:489–501.[CrossRef][Medline]-encoding gene in the mammary gland. J. Mol. Endocrinol. 29:73–88.[Abstract]This article has been cited by other articles:
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  M. D. Thomas, K. C. Olson, J. S. Stevenson, J. R. Jaeger, J. W. Bolte, N. A. Sproul, D. A. Linden, and L. A. Pacheco Effects of Prepartum Whole Fuzzy Cottonseed or Whole Raw Soybean Supplementation on Response to Timed Artificial Insemination by Suckled Mature Beef Cows Following Ovulation Synchronization Professional Animal Scientist, February 1, 2009; 25(1): 41 - 48. [Abstract] [PDF]
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 B. W. Hess, G. E. Moss, and D. C. Rule A decade of developments in the area of fat supplementation research with beef cattle and sheep J Anim Sci, April 1, 2008; 86(14_suppl): E188 - E204. [Abstract] [Full Text] [PDF]
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 M. Boutinaud, M. H. Ben Chedly, E. Delamaire, and J. Guinard-Flament Milking and Feed Restriction Regulate Transcripts of Mammary Epithelial Cells Purified from Milk J Dairy Sci, March 1, 2008; 91(3): 988 - 998. [Abstract] [Full Text] [PDF]
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S. Feng, A. M. Salter, T. Parr, and P. C. Garnsworthy Extraction and Quantitative Analysis of Stearoyl-Coenzyme A Desaturase mRNA from Dairy Cow Milk Somatic Cells J Dairy Sci, September 1, 2007; 90(9): 4128 - 4136. [Abstract] [Full Text] [PDF]
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S. L. Lake, T. R. Weston, E. J. Scholljegerdes, C. M. Murrieta, B. M. Alexander, D. C. Rule, G. E. Moss, and B. W. Hess Effects of postpartum dietary fat and body condition score at parturition on plasma, adipose tissue, and milk fatty acid composition of lactating beef cows J Anim Sci, March 1, 2007; 85(3): 717 - 730. [Abstract] [Full Text] [PDF]
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