Effect of short-term feed restriction on temporal changes in milk components and mammary lipogenic gene expression in mid-lactation Holstein dairy cows.

Investigations of the temporal changes in mammary gene expression that occur during sudden diet change have been limited by the use of mammary tissue as the source of RNA because of the invasive nature of mammary biopsy procedures. However, the cytosolic crescent, present in 1% of the largest milk fat globules, contains mammary epithelial cell RNA that has become trapped between the inner and outer milk fat globule membranes during final formation and secretion of milk fat into the lumen of the mammary alveoli. We hypothesized that cytosolic crescent RNA extracted from milk fat could be used as an alternative source of mammary epithelial cell RNA to measure the immediate temporal changes in gene expression as a result of changes in diet. In this experiment, feed restriction was used to mimic the state of negative energy balance observed in early lactation and induce a rapid change in milk fat yield and lipogenic gene expression. Ten multiparous Holstein dairy were fed a basal diet ad libitum during a 14-d preliminary period followed by a 4-d experimental period where 5 cows remained on ad libitum feeding and 5 cows were fed at 60% of their d 8-14 intakes (restricted) on d 15 to 18 and then returned to ad libitum feeding on d 19 to 21. Milk samples were collected from each milking on d 13 to 20 and the milk fat was immediately isolated, mixed with Trizol LS, and stored at -80°C for subsequent extraction of RNA that was used for measurement of gene expression. Feed restriction tended to increase milk fat percentage. However, total milk and milk fat production were reduced by 21 and 18%, respectively. Consistent with increased use of body fat for milk synthesis, serum nonesterified fatty acids increased 6-fold (0.78 mEq/L in the feed restriction vs. 0.13 mEq/L ad libitum group), whereas the milk fatty acids

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.

Milk fat responses to dietary supplementation of short- and medium-chain fatty acids in lactating dairy cows.

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 during diet-induced milk fat depression. Our hypothesis was that SMCFA are limiting for milk fat synthesis even under conditions when milk fat is not depressed. Our objective was to test the potential limitation of SMCFA on milk fat synthesis via dietary supplementation. Sixteen lactating Holstein cows (107±18 d in milk) were fed a corn silage-based total mixed ration. Cows were randomly assigned to groups of 4 per pen and supplemented with 1 of 4 dietary fat supplements (600 g/d) supplied in a 4×4 Latin square design with 21-d experimental periods. Treatments consisted of fat supplements containing mixtures of calcium salts of long-chain fatty acids (Megalac; Church & Dwight Co. Inc., Princeton, NJ) and an SMCFA mixture (S; 3.3% C8, 7.6% C10, 9.85% C12, 32.12% C14, and 47.11% C16) that contained 0, 200, 400, and 600 g/d of S substituted for Megalac (S0, S200, S400, and S600, respectively). No treatment effects were observed for dry matter and fat-corrected milk. However, milk yield was decreased with S600. Milk fat increased linearly by 0.17, 0.25, and 0.33 percentage units for the respective S treatments. However, fat yield peaked at S200 and milk protein concentration and yield was significantly decreased at the higher S levels because of a linear trend toward decreased milk yield in the S600 treatment. In conclusion, SMCFA supplementation linearly increased milk fat concentration but decreased milk production at the higher levels of supplementation. The dietary inclusion of SMCFA had no effects on total milk fat yield.

Transfer rate of α-linolenic acid from abomasally infused flaxseed oil into milk fat and the effects on milk fatty acid composition in dairy cows.

The objectives of the present study were to evaluate the transfer efficiency of α-linolenic acid (ALA) from the abomasum into milk fat, its interaction with milk fat content and yield, and the relationship between ALA and C16:0 in milk fat. Three rumen-fistulated multiparous Holstein cows at midlactation were used in a 3×3 Latin square design. Treatments consisted of abomasal infusion of (1) 110 mL of water/d (control), (2) 110 mL of flaxseed oil/d (low flaxseed oil, LFO), and (3) 220 mL of flaxseed oil/d (high flaxseed oil, HFO). Experimental periods were continued for 2 wk and fat supplements were infused abomasally during the last 7 d of each period. Average dry matter intake and milk yield were not affected by oil infusion. Milk fat and lactose content tended to be greater with flaxseed infusion compared with the control. Plasma ALA was 2.9- and 4.0-fold greater with LFO and HFO, respectively. The apparent transfer efficiency of ALA to milk was 44.8 and 45.7% with LFO and HFO, respectively. The C16:0 content in milk fat was decreased by 3.59 and 5.25 percentage units, whereas the ALA content was increased by 1.68 and 3.09 percentage units with LFO and HFO, respectively. Similarly, C18:2n-6 was increased by 0.95 and 1.31 percentage units with LFA and HFO, respectively, without changes in other fatty acids (FA). Total polyunsaturated FA was 4.4 and 2.7% lower in the HFO and LFO, respectively, than in the control. Furthermore, C16:0 content in the milk fat was reduced to a greater extent than the increase in ALA content, as a 1.68 and 3.09 percentage unit increase occurred in ALA compared with a 3.6 and 5.25 percentage unit decrease in C16:0 for LFO and HFO, respectively, such that a negative correlation existed between ALA and C16:0 (r=-0.72). In conclusion, abomasal infusion of flaxseed oil dramatically increased the ALA content in plasma and milk fat. Because the replacement of C16:0 with ALA and C18:2n-6 occurred without changes in other FA presumed to be synthesized de novo in the mammary gland, this suggests that the preformed C16:0 was replaced, rather than being caused, by an overall suppression of de novo FA synthesis in the mammary gland.

Changes in milk fat in response to dietary supplementation with calcium salts of trans-18:1 or conjugated linoleic fatty acids in lactating dairy cows.

Milk fat was investigated in lactating dairy cows fed diets supplemented with Ca salts of trans fatty acids (Ca-tFA) or Ca salts of conjugated linoleic acids (Ca-CLA). Forty-five Holstein cows (115 days in milk) were fed a control diet (51% forage; dry matter basis) supplemented with 400 g of EnerG II (Ca salts of palm oil fatty acids) for 2 wk; subsequently, 5 groups of 9 cows each were assigned for 4 wk to the control diet or diets containing 100 g of Ca-CLA or 100, 200, or 400 g of Ca-tFA in a randomized block design. Treatments had no effect on dry matter intake, milk production, protein, lactose, or somatic cell count. Milk fat percentage was reduced from 3.39% in controls to 3.30, 3.04, and 2.98%, respectively, by the Ca-tFA diets and to 2.54% by the Ca-CLA diet. Milk fat yield (1.24 kg/d in controls) was decreased by 60, 130, and 190 g/d with increasing dose of Ca-tFA and by 290 g/d with the Ca-CLA supplement. Consistent with increased endogenous synthesis of cis-9-containing CLA from precursors provided by the Ca-tFA diets, total CLA were similar in milk of cows fed Ca-CLA or Ca-tFA. Compared with controls, the Ca-CLA diet increased trans-10, cis-12-18:2 yield in milk, without altering levels of trans-18:1 isomers. In contrast, yields of most trans-18:1 isomers were elevated in milk of cows fed Ca-tFA diets, whereas yields of trans-10, cis-12-18:2 remained similar to control values. We conclude that milk fat depression can occur without an increase in trans-10, cis-12-18:2 in milk and that other components, perhaps the trans-10-18:1 isomer, may be involved.

Milk composition and apparent digestibilities of dietary fatty acids in lactating dairy cows abomasally infused with Cis or Trans fatty acids.

Fat supplementation of diets for dairy cows produces changes in nutrient supply and milk composition. The effect of abomasal infusion of either cis-C18:1 or trans-C18:1 fatty acid isomers on the digestibility of fatty acids and milk composition was determined in lactating dairy cows. Six multiparous midlactation Holstein cows were used and fed a control diet containing 50% forage and 50% concentrate. Treatments were (per day): no infusion, infusion of a 630-g fat mixture high in cis-C18:1 isomers, and infusion of a 623-g fat mixture high in trans-C18:1 isomers using two 3 x 3 Latin squares with 4-wk experimental periods. Fat infusion did not affect total dry matter intake and increased apparent digestibilities of total fatty acids. Apparent digestibilities of C18 fatty acids were directly related to the number of double bonds within isomers, and cis-C18:1 isomers were slightly more digestible than trans-C18:1 isomers. The lower yield of C12:0, C14:0, and C16:0 fatty acids in milk fat and higher milk citrate observed when cows were infused with trans-C18:1 suggests a depressed de novo milk fatty acid synthesis. Effects of trans infusion on milk fat were independent of ruminal fermentation, fatty acid apparent absorption, and fatty acid plasma concentrations. Lower milk protein yield in cows infused with fat may have been caused by a decrease in milk protein synthesis.

Mammary lipogenic enzyme activity, trans fatty acids and conjugated linoleic acids are altered in lactating dairy cows fed a milk fat-depressing diet.

The objectives of the present study were to examine the effect of a milk fat-depressing (MFD) diet on: 1) the activity of mammary acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), 2) ACC mRNA relative abundance and 3) distributions of conjugated linoleic acids (CLA) and trans-18:1 fatty acids (tFA) in milk fat. Twelve lactating Holstein cows were used in a single reversal design. Two diets were fed: a control diet (60:40% forage/concentrate) and an MFD diet (25:70% forage/concentrate, supplemented with 5% soybean oil). The MFD diet decreased (P: < 0 0.001) milk fat by 43% and ACC and FAS activity by 61 and 44%, respectively. A reduced ACC mRNA relative abundance (P: < 0.001) corresponded with the lower ACC activity. The fatty acids synthesized de novo were decreased (P: < 0. 002), whereas tFA were increased from 1.9 to 15.6% due predominantly to a change in trans-10-18:1 isomer (P: < 0.001). With the MFD diet, the trans-7, cis-9 and trans-10, cis-12 CLA isomers were elevated (P: < 0.001), in contrast to the decrease in trans-11-18:1 (P: < 0. 001) and cis-9, trans-11-18:2. The data were consistent with a dietary effect on mammary de novo FA synthesis mediated through a reduction in ACC and FAS activity and in ACC mRNA abundance. The results were compatible with a role of trans-10, cis-12 CLA in milk fat depression, but alterations noted in tFA and other CLA isomers suggest that they also may be important during diet-induced milk fat depression.

Effect of dietary forage concentration and buffer addition on duodenal flow of trans-C18:1 fatty acids and milk fat production in dairy cows.

Milk fat depression in cows fed high grain diets has been shown to be related to increased trans-C18:1 fatty acids in milk. Trans-C18:1 fatty acids are produced as a result of incomplete biohydrogenation of dietary polyunsaturated fatty acids in the rumen. The objective of this study was to determine the effects of varying amounts of dietary concentrate and buffer addition on duodenal flow, apparent absorption, and incorporation of trans-C18:1 fatty acids into milk fat. Four ruminally and duodenally cannulated multiparous Holstein cows in midlactation were fed diets consisting of 60% (high) or 25% (low) forage with or without buffer (1.5% NaHCO3 and 0.5% MgO). Treatments were arranged in a 2 x 2 factorial within a 4 x 4 Latin square design. The diet containing low forage and no buffer increased the flow of trans-C18:1 fatty acids to the duodenum compared with the effects of other diets (120 vs. 57 to 66 g/d). Ruminal pH was lower for cows fed the low forage diets. The addition of buffer increased ruminal pH by 0.19 and 0.02 units for cows fed the low forage and high forage diets, respectively. Cows fed the diet containing low forage and no buffer produced milk with a lower fat percentage. The addition of buffer to the low forage diet partially corrected milk fat depression. Milk trans-C18:1 fatty acids were higher for cows fed the low forage diet without buffer than for cows fed the other diets (5.8% vs. 3.0%). Altered ruminal function resulting from low ruminal pH for cows fed the low forage diet and no buffer may play a role in the increased production of trans-C18:1 fatty acids in the rumen.

Effect of fat source on duodenal flow of trans-C18:1 fatty acids and milk fat production in dairy cows.

The objective of the study was to determine the effects of dietary fat source on duodenal flow, apparent absorption, and milk fat incorporation of trans-C18:1 fatty acids. Four ruminally and duodenally cannulated multiparous Holsteins cows in mid to late lactation were fed a basal diet containing 36% corn silage, 24% alfalfa haylage, and 40% concentrate (dry matter basis). Diets contained 0% supplemental fat (control diet), 3.7% high oleic sunflower oil, 3.7% high linoleic sunflower oil, or 3.7% partially hydrogenated vegetable shortening; treatments were administered in a 4 x 4 Latin square design with 3-wk experimental periods. The flow of trans-C18:1 to the duodenum was higher for cows fed diets supplemented with fat than for cows fed the control diet (283 vs. 64 g/d). Incomplete biohydrogenation accounted for the increased flow of trans-C18:1 to the duodenum in cows fed diets containing high oleic and high linoleic sunflower oil. Increased flow of trans-C18:1 in cows fed the diet containing partially hydrogenated vegetable shortening most likely originated from the trans-C18:1 in the diet. Milk fat percentages were 3.48, 3.07, 3.18, and 3.38% for cows fed the control diet and diets containing high oleic sunflower oil, high linoleic sunflower oil, and vegetable shortening, respectively. Milk trans-C18:1 increased from 2.9 to 11.2% of the total fatty acids for cows fed the control diet and the diets supplemented with fat, respectively. Milk trans-C18:1 were equal across all diets supplemented with fat. Ruminal and total tract digestion of organic matter, neutral detergent fiber, and N did not change with fat supplementation. Results showed that trans-C18:1 is increased in the milk of cows with reduced milk fat; however, excessive amounts of trans-C18:1 in milk do not necessarily correspond directly to milk fat depression.

Abomasal infusion of cis or trans fatty acid isomers and energy metabolism of lactating dairy cows.

Diets for dairy cows that provide or induce formation of trans isomers of polyunsaturated fatty acids result in reduced percentages of milk fat. The effect of abomasal infusion of trans-C18:1 fatty acid isomers on energy utilization by mature cows was determined. Six multiparous Holstein cows in midlactation had ad libitum access to a basal diet containing 50% forage and 50% concentrate. Treatments were 1) no infusion, 2) infusion of 630 g/d of a fat mixture high in cis-C18:1 isomers (64% cis-C18:1; 68% high oleic sunflower oil and 32% cocoa butter), and 3) infusion of 623 g/d of a fat mixture high in trans-C18:1 isomers (42% trans-C18:1; 90% partially hydrogenated soybean oil and 10% high linoleic safflower oil). The experiment was a replicated 3 x 3 Latin square design with 4-wk periods. Measurements of energy balance were made in open circuit respiration chambers during wk 4 of each period. Fat infusion increased milk production by 2.5 kg/d; apparent digestibility of DM, OM, energy, ADF, and ash by 1 to 4 percentage units; metabolizable energy by 11%; and NEL of the diet by 15%. Milk fat percentage and yield were higher when cows were infused with cis-C18:1 than when they were infused with trans-C18:1 (4.12% and 1.41 kg/d vs. 3.15% and 1.06 kg/d, respectively). Infusion of fat increased milk production, but trans-C18:1 reduced milk fat and energy output.

Glucose and norepinephrine challenges during abomasal infusion of cis or trans octadecenoates in Holstein cows.

This experiment determined the effects of infusion of mixtures of fat containing predominantly cis-C18:1 or trans-C18:1 fatty acids into the abomasum on responses of cows to glucose and norepinephrine challenges administered i.v. Six lactating Holstein cows, each with a rumen cannula, were arranged in two Latin squares with 21-d periods. The common basal diet contained 40% forage and 60% concentrate. Treatments were the uninfused control, 750 g/d of a cis fat mixture (65% high oleic sunflower oil and 35% cocoa butter), and 750 g/d of a trans fat mixture (93% shortening and 7% corn oil) infused into the abomasum via a tube that passed through the rumen cannula. Glucose challenges (0.4 mg/kg of BW, administered i.v.) were conducted on d 18, and norepinephrine challenges (0.7 microgram/kg of BW, administered i.v.) were conducted on d 19 of each experimental period. Despite a lower percentage of fat in milk for trans than for cis treatment, disappearance rates of glucose, secretion of insulin after glucose challenge, and appearance rates of NEFA and triglycerides after norepinephrine challenge were similar between treatments. Thus, these data support the hypothesis that trans-C18:1 fatty acids affect the synthesis of milk fat in the mammary gland of lactating cows.

Milk fat depression, the glucogenic theory, and trans-C18:1 fatty acids.

Metabolic and endocrinological characteristics were compared for cows that differed in the extent of milk fat depression. Forty-one multiparous Holstein cows were fed control (40% concentrate and 60% forage) and high concentrate (80% concentrate and 20% forage) diets in a doubale-reversal design. Cows showing one or more percentage units of depression in milk fat were arbitrarily classified as responders (n = 26); those remaining were classified as nonresponders (n = 15). Compared with nonresponders, responders had greater increases in DMI, estimated NEL intake and balance, BW, milk yield, protein and lactose yields in milk, weight percentage of trans-C18:1 fatty acids in milk, and concentrations of triiodothyronine and thyroxine in serum when switched from the control diet to the 80% concentrate diet. Lack of an increase in concentrations of glucose and insulin in serum of cows with the greatest decline in percentage of milk fat casts doubt on the ability of the glucogenic theory to explain milk fat depression completely.

Milk fat yield and composition during abomasal infusion of cis or trans octadecenoates in Holstein cows.

The role of trans-C18:1 fatty acids in milk fat depression was examined. Six rumen-cannulated Holstein cows were assigned to two Latin squares with 21-d periods. The common basal diet contained 40% forage and 60% concentrate. Treatments were the uninfused control, 750 g/d of a mixture of cis fat (65% high oleic sunflower oil and 35% cocoa butter), and 750 g/d of a mixture of trans fat (93% shortening and 7% corn oil) infused into the abomasum via a tube that passed through the rumen cannula. Milk yield was similar among treatments. Milk fat percentage and yield were lower, and milk citrate concentration was higher, for the trans than the cis treatment. Changes in the fatty acid composition of milk were similar for the cis and trans treatments compared with the control except for trans-C18:1. The concentration of trans-C18:1 was greater for the cis and trans treatments than for the control and was greater for the trans than for the cis treatment. These data clearly demonstrated that infusion of trans-C18:1 fatty acids into the abomasum depressed milk fat percentage and yield. We speculate that reduced synthesis of fatty acids and reduced activity of acyl transferase in mammary tissue contributed to depressed milk fat percentage for the trans treatment.

Lactation curves and effect of pup removal on milk fat of C57Bl/6J mice fed different diet fats.

Groups of C57Bl/6J mice, fed either a cis (C-Diet) or trans diet (T-Diet) were milked without preconditioning at 6, 8, 10 and 12 days postpartum. On day 10, groups of mice were also milked 4, 6 and 18 h after separation of the pups. Except for the 18-h separation, all T-Diet fed animals produced milk of lower fat content than did the C-Diet animals (P < 0.001) throughout the lactation period measured. In the C-Diet mice, the 6-h separation period resulted in a decrease (P < or = 0.03) in fat, but the diet-depressed milk fat of T-Diet animals was not decreased further until the 18-h separation period. Milk volume increased as lactation progressed and was greatly increased as a result of preconditioning (P < or = 0.001), even at 4 h of separation when fat was not reduced, and was always greater for T-Diet animals. Within diet groups, fatty acid composition was similar throughout the lactation period studied and was not affected by preconditioning except in the 18-h separation period, when de novo fatty acids were significantly reduced (P < or = 0.05). The data are consistent with the hypothesis that preconditioning results in lowered milk fat values and that preconditioning techniques can explain discrepancies in literature values for murine milk fat.

Milk fat depression in C57Bl/6J mice consuming partially hydrogenated fat.

Mice of the C57Bl/6J strain were maintained on diets in which the unsaturated fatty acids were all cis fatty acids (CFA) or a mixture of CFA and trans fatty acids (TFA). The fats used were mixtures of corn oil, olive oil, cocoa butter, margarine and shortening blended to yield similar fatty acid compositions, except for the ratio of the CFA to TFA and the percentage of linoleic acid (EFA). Regardless of the level of fat (20 or 40 energy %) or the level of EFA (2 to 12 energy %), diets with TFA decreased the percentage of fat in mouse milk. When lactating females raised on the CFA diets were crossed to the TFA diets, TFA appeared in the milk at 12 h postcross, and within 4 d postcross the percent of milk fat was decreased to levels similar to that of nursing females raised continuously on the TFA diets. Conversely, lactating females crossed from TFA to CFA diets produced milk with percentage fat values and fatty acid compositions that approached those seen in nursing females fed the CFA diets continuously. The possible involvement of TFA in the classical milk fat depression phenomenon in ruminants and its potential relevance in human lactation are discussed.