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The aim of this study was to evaluate the effect of sampling technique and milk fraction on bovine milk microbiota data and to compare the microbiota in milk to microbiota on the teat end and in the teat canal. Representative milk samples are highly important for assessment of bacteriological findings and microbiota in milk. Samples were obtained from 5 healthy lactating dairy cows at udder quarter level during 1 milking. Swab samples from the teat end and teat canal, and milk samples collected using different techniques and in different milk fractions were included. Milk was collected by hand stripping and through a teat canal cannula before and after machine milking, through a trans-teat wall needle aspirate after milking, and from udder quarter composite milk. The microbiota of the samples was analyzed with sequencing of the V1-V3 region of the 16S rRNA gene. In addition, somatic cell counts and bacterial cultivability were analyzed in the milk samples. Microbiota data were analyzed using multivariate methods, and differences between samples were tested using analysis of similarity (ANOSIM). Differences between samples were further explored via individual studies of the 10 most abundant genera. The microbiota on the teat end, in the teat canal, and in udder quarter composite milk, collected using a milking machine, differed in composition from the microbiota in milk collected directly from the udder quarter. No differences in milk microbiota composition were detected between hand-stripped milk samples, milk samples taken through a teat canal cannula, or milk samples taken as a trans-teat wall needle aspirate before or after milking. We conclude that for aseptic milk samples collected directly from the lactating udder quarter, sampling technique or milk fraction has minor effect on the microbiota composition., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Staphylococcus aureus is an opportunistic pathogen affecting both human and animal species. An effective vaccine to prevent S. aureus bovine disease and transmission would have positive effects on animal well-being, food production, and human health. The objective of this study was to identify multiple antigens that are immunoreactive during udder colonization and disease for exploration as vaccine antigens to prevent bovine mastitis. Staphylococcus aureus produces several cell wall-anchored and surface-associated virulence factors that play key roles in the pathogenesis of mastitis. Many of these proteins are conserved between different strains of S. aureus and represent promising vaccine candidates. We used an immunoproteomics approach to identify antigenic proteins from the surface of S. aureus. The expression of cell wall and surface proteins from S. aureus was induced under low iron conditions, followed by trypsin extraction and separation by 2-dimensional electrophoresis. The separated proteins were blotted with antibodies from mastitic bovine milk and identified by liquid chromatography-mass spectrometry. Thirty-eight unique proteins were identified, of which 8 were predicted to be surface exposed and involved in S. aureus virulence. Two surface proteins, iron-regulated surface determinant protein C (IsdC) and ESAT-6 secretion system extracellular protein (EsxA), were cloned, expressed, and purified from Escherichia coli for confirmation of immune reactivity by ELISA. A PCR of 37 bovine S. aureus isolates indicated that the presence of esxA and isdC is conserved, and amino acid alignments revealed that IsdC and EsxA sequences are highly conserved. The immunoproteomics technique used in this study generated reproducible results and identified surface exposed and reactive antigens for further characterization., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

During the periparturient period, elevated circulating nonesterified fatty acids (NEFA) from excessive lipid mobilization affect not only the circulating fatty acid (FA) composition, but also that of the peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMNL). However, the changes to specific lipid fractions remain unknown. We hypothesized that elevated lipid mobilization will alter FA profiles and gene expression of selected proinflammatory mediators in PBMC and PMNL. Starting -28 d relative to expected calving (d 0), treatment cows (n = 18) received a dry cow ration plus an additional 10 kg of corn/head per day, while the control cows (n = 16) received the dry cow ration (no additional corn) supplemented with 400 mg of monensin/head per day to minimize lipid mobilization. Postpartum, treatment cows were feed deprived for 8 h on d +3. For FA analysis, serum was collected on d -28 and -7 relative to expected parturition and d +1, +3, +6, +15, and +21 postpartum, in addition to milk samples. Immune cells, PBMC and PMNL, were isolated on d -28, +3, +12, and +21 for FA analysis and gene expression analysis by reverse-transcription PCR. Serum, PBMC, and PMNL lipids were fractionated into NEFA and phospholipids (PL). The FA composition of milk, serum, PBMC, and PMNL was analyzed by gas chromatography. Data were analyzed as repeated measures ANOVA using mixed model procedures in SAS (9.3) with significance declared at P ≤ 0.05. Several FA varied by treatment and across time and parity. Within the serum PL fraction, FA associated with altered immune function, C18:3n-6, C20:4, C20:5, total n-3, and the ratio of n-6 to n-3 varied significantly by a treatment × parity × time interaction. Overall, FA composition of NEFA and PL fractions from PBMC and PMNL did not significantly reflect FA of serum. Gene expression for IL-1β in PBMC was greater for control, whereas ICAM, IL-1β, IL-6, and TNF-α were greater in primiparous than multiparous cows, without a detectable treatment effect. Whereas gene expression of CASP, IL-8R, and SELL in PMNL changed over time, no treatment effect was detected. In summary, high-energy prepartal diets altered FA profile in serum, milk, PBMC, and PMNL lipids; however, gene expression of selected proinflammatory mediators was not significantly affected., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Feeding rations supplemented with fats may provide an opportunity to manipulate the health and performance of dairy cows; however, the relative effects of specific fats, such as trans fatty acids (TFA), are poorly understood. The objective of this study was to investigate the effects of a ration supplemented with TFA on the fatty acid (FA) profile of peripheral blood mononuclear cells (PBMC), plasma lipids, and milk; the gene expression of inflammatory markers; production of acute phase proteins; and production performance in early lactating dairy cows. Trans fat was fed at 0, 1.5, and 3% of dry matter, replacing (1:1 wt:wt) saturated fatty acids (SFA). Multiparous lactating Holstein cows at 7 d in milk (n=12) were randomly assigned to a treatment sequence in a 3 × 3 balanced Latin square design; each period lasted 14 d. Milk and heparinized blood were collected on d 0 (pretreatment) and on d 10 and 14 of each period. Plasma was collected and solid-phase extraction was used to isolate plasma phospholipids and nonesterified fatty acids. Additionally, PBMC were isolated for FA analysis and gene expression analysis by reverse transcription-PCR using bovine RPS9 as the endogenous control. The FA composition of PBMC, plasma lipid fractions, and milk were analyzed by gas chromatography. Data were analyzed using the MIXED procedure (SAS Institute Inc., Cary, NC). As dietary TFA increased, the percentage of some 18:1 trans isomers increased in PBMC, plasma lipids, and milk. Dietary TFA had no detectable effect on mRNA expression of proinflammatory TNFα or IL6. Expression of IL1β and ICAM1 decreased with increasing TFA. In addition, supplementation of TFA did not affect percentages of milk fat, protein, lactose, or solids-not-fat, or somatic cell count. Overall, dietary TFA increased the trans FA present in PBMC, plasma lipids, and milk; however, dietary TFA decreased PBMC expression of some of the proinflammatory markers tested at the mRNA level compared with SFA in early lactating dairy cows. Together, these findings provide evidence that over short period of times, dietary TFA might be slightly less immune-stimulatory than dietary SFA., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Betaine, naturally found in plants and an oxidative product of choline, is converted to acetate in the rumen, which may be used for milk fat synthesis. The objective of this study was to determine the effect of supplemental dietary betaine on milk yield and milk composition. Eighteen Holstein dairy cows (126±5 d in milk; mean ± SD) were randomly assigned to a sequence of treatments of rumen-unprotected betaine at 0, 25, 50, and 100 g/d added to a standard lactation ration in a 4×4 Latin square design. Animals were fed individually with feed intake and milk yield recorded daily. Body condition score and body weight were recorded on the last day of each period that lasted 16 d, with milk sampled on the last 2 d of each period. Milk composition was determined by a Dairy Herd Improvement Association laboratory and milk fatty acids were determined by gas chromatography. Data collected over the last 2 to 3 d were analyzed using the MIXED procedure in SAS (SAS Institute Inc., Cary, NC). Milk yield (mean ± SEM) was increased by betaine when fed at 100g/d (22.4, 22.5, 22.8, 24.1±1.19 kg/d for 0, 25, 50, and 100g of betaine/d, respectively). No effect of dietary betaine was detected on dry matter intake, feed efficiency, body weight, or body condition score. Percentages of milk fat, lactose, solids-not-fat, and somatic cell count were not altered; however, protein concentration was decreased by betaine supplementation as compared with the control (3.35, 3.28, 3.27, and 3.28±0.07% for 0, 25, 50, and 100 g of betaine/d, respectively). Daily yields of milk protein, fat, lactose, energy-corrected milk, and 3.5% fat-corrected milk did not differ with betaine supplementation. Overall, inclusion of dietary betaine at 100 g/d increased milk yield, whereas all levels of betaine supplementation decreased milk protein percent and slightly altered milk fatty acid profile. Further studies are needed to determine the ruminal fermentation characteristics and the optimum rate of supplemental betaine for dairy cows., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Trans fatty acids (tFA) contribute to inflammation. The objective was to investigate the effects of tFA on mRNA expression of proinflammatory markers in cultured bovine mammary epithelial cells (MAC-T cell line). Bovine mammary epithelial cells were grown in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum. Cells were then subcultured in a medium lacking fetal bovine serum, to which incremental concentrations (up to 90 μM) of elaidic acid (trans-9 C18:1) or linoleidic acid (trans-9, trans-12 C18:2) were added. Bovine serum albumin (fatty acid-free) solutions were added and cells were collected at specific time points over 48 h. Then, RNA was extracted and converted to complementary DNA for quantitative real-time PCR analysis of proinflammatory gene expression. Presence of elaidic acid caused increases in mRNA expression of interleukin (IL)-1β (3.4-fold; dose-independently over a 6-h period) and intercellular adhesion molecule (ICAM)-1 (up to 1.4-fold) relative to that for cells treated with no tFA, whereas expression of IL-6 and IL-8 was reduced 0.75- and 0.85-fold, respectively. Presence of linoleidic acid reduced mRNA expression of IL-6 and IL-8 relative to that for control (0.95- and 0.87-fold, respectively). Trans mono- and dienoic fatty acids upregulated mRNA expression of IL-1β and ICAM-1, whereas expression of IL-6 and IL-8 was downregulated in MAC-T cells. Because these genes are ultimately involved in inflammation, elaidic or linoleidic acid, either directly fed or formed in the rumen during biohydrogenation, may alter the risk for mastitis in vivo., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Conjugated linoleic acids (CLA) are potent anticarcinogens in animal and in vitro models as well as inhibitors of fatty acid synthesis in mammary gland, liver, and adipose tissue. Our objective was to evaluate long-term CLA supplementation of lactating dairy cows in tropical pasture on milk production and composition and residual effects posttreatment. Thirty crossbred cows grazing stargrass (Cynodon nlemfuensis Vanderyst var. nlemfüensis) were blocked by parity and received 150 g/d of a dietary fat supplement of either Ca-salts of palm oil fatty acids (control) or a mixture of Ca-salts of CLA (CLA treatment). Supplements of fatty acids were mixed with 4 kg/d of concentrate. Grazing plus supplements were estimated to provide 115% of the estimated metabolizable protein requirements from 28 to 84 d in milk (treatment period). The CLA supplement provided 15 g/d of cis-9,trans-11 and 22g of cis-10,trans-12. Residual effects were evaluated from 85 to 112 d in milk (residual period) when cows were fed an 18% crude protein concentrate without added fat. The CLA treatment increased milk production but reduced milk fat concentration from 2.90 to 2.14% and fat production from 437 to 348 g/d. Milk protein concentration increased by 11.5% (2.79 to 3.11%) and production by 19% (422 to 504 g/d) in the cows fed CLA. The CLA treatment decreased milk energy concentration and increased milk volume, resulting in unchanged energy output. Milk production and protein concentration and production were also greater during the residual period for the CLA-treated cows. The CLA treatment reduced production of fatty acids (FA) of all chain lengths, but the larger effect was on short-chain FA, causing a shift toward a greater content of longer chain FA. The CLA treatment increased total milk CLA content by 30% and content of the trans-10,cis-12 CLA isomer by 88%. The CLA treatment tended to decrease the number of days open, suggesting a possible effect on reproduction. Under tropical grazing conditions, in a nutritionally challenging environment, CLA-treated cows decreased milk fat content and secreted the same amount of milk energy by increasing milk volume and milk protein production.

Recent research has focused on the importance of supplying essential fatty acids to the lactating dairy cow. The addition of essential fatty acids, specifically linoleic and linolenic acid, to dairy cow diets has been investigated as a method to increase reproductive efficiency. Rumen bacteria, however, biohydrogenate polyunsaturated fatty acids (PUFA) to saturated fatty acids. This is an important issue because it can also lead to milk fat depression when unsaturated fatty acids are fed. The formation of Ca salts has previously been shown to partially protect unsaturated fatty acids from rumen biohydrogenation. The objective of this experiment was to evaluate feed intake, milk production, and milk composition of cows fed Ca salts of palm fatty acids (CS) compared with those fed Ca salts of palm fatty acids with an increased content of PUFA (CS+PUFA). Nineteen lactating Holstein cows were used in a switchback experiment to determine any differences between CS and CS+PUFA on milk production and composition. This experiment consisted of 3 consecutive periods of 14 d. Treatments were formulated to provide 450 g/d (dry matter basis) of the Ca salt supplement and were mixed with the same basal ration. Milk weights and feed intakes were recorded daily for each cow. Milk samples were collected the last 2 d of each period and analyzed for milk composition and fatty acids. Dry matter intake [28.0 vs. 27.0 kg/d; standard error of the mean (SEM) = 0.4] and milk production (44.4 vs. 44.0 kg/d; SEM = 0.7) were not different between treatments for CS and CS+PUFA, respectively. Milk fat percentage (3.34 vs. 3.22%; SEM = 0.07) and milk protein percentage (2.78 vs. 2.80%; SEM = 0.01) were not different for CS- and CS+PUFA-fed cows. Feeding CS+PUFA reduced the concentration of palmitic acid in milk fat (28.3 vs. 26.8 wt%; SEM = 0.3). Supplementation of CS+PUFA increased the linoleic acid concentration (3.96 vs. 4.61 wt%; SEM = 0.1) of milk fat, indicating that linoleic acid was partially protected from rumen biohydrogenation. Concentrations of conjugated linoleic acid were also increased (0.44 vs. 0.52 wt%; SEM = 0.02) when cows consumed CS+PUFA, indicating that some biohydrogenation did occur. Supplementing CS+PUFA did not alter milk production, milk fat percentage, or dry matter intake when compared with CS. The CS+PUFA supplement supplied more linoleic acid to the small intestine for milk fat synthesis.

To determine the optimum feeding level of fatty acids of palm oil (PALM; Energizer RP10; 86.6% palmitic acid) on milk production, lactating cows (n = 18) were randomly assigned to a treatment sequence in replicated 4 x 4 Latin squares. Animals were assigned to squares by parity (3 multiparous and 1 primiparous squares with primiparous in the incomplete square). The 4 diets were designed to provide 0, 500, 1,000, and 1,500 g of PALM per day. Cows were fed individually with feed intake measured daily. Each period lasted 16 d with milk production and composition determined the final 2 d. Milk production, milk composition and feed intake data were analyzed using the MIXED procedure of SAS. Milk yields were 30.9, 34.0, 34.2, and 34.2 kg/ d (SEM = 1.9) for the 0, 500, 1,000, and 1,500 g levels, respectively. Milk yield was increased by the addition of PALM; however, there were no differences among the levels of PALM. Milk fat percentage was also increased from 3.44% for 0 g to 3.95% (SEM = 0.17) across all levels of PALM but there were no differences among the PALM treatments. Dry matter intakes were 23.3, 26.4, 24.7, and 23.8 kg/d (SEM = 1.4) for the 0, 500, 1,000 and 1,500 g levels, respectively. The addition of PALM increased milk yield and milk fat percentage, and no adverse effects on dry matter intake were observed.

Objectives were to determine effects of continuous milking (CM) and bovine somatotropin (bST) administration on 1) mammary epithelial cell (MEC) proliferation, apoptosis, and ultrastructure during late gestation and early lactation, 2) expression of genes associated with proliferation, and apoptosis in mammary epithelial cells, and 3) milk yield and composition. Second-gestation, first dry-period cows were randomly assigned to either continuous bST throughout late gestation and early lactation (+bST; n = 4) or no bST (-bST; n = 4) administration. Within each animal, udder halves were randomly assigned to CM or a 60-d dry period (control) treatment. Daily milk yield and weekly milk composition were measured during the last 60 d of gestation in CM halves and from 1 to 30 d postpartum for both halves. Mammary biopsies were obtained at -20 +/- 7, -8 +/- 3, +1 +/- 0, +7 +/- 0, and +20 +/- 0 d (mean +/- standard error) relative to parturition. Prepartum half-udder milk yield was greater in +bST cows than in -bST cows (9.9 vs. 8.2 kg/d) and postpartum half-udder milk yields were dramatically reduced in CM halves compared with control halves (10.6 vs. 22.2 kg/d), regardless of bST treatment. Proliferation of MEC was reduced in CM halves at -8 d (2.7 vs. 5.4%). Apoptosis of MEC was elevated during early lactation for d +1 and +7 in control halves, but was only increased at d +1 in CM halves. Turnover of MEC was not affected by bST. Ultrastructure data indicated complete involution of the control half and lactation maintenance in CM glands (d -20). By d -8, control tissue contained alveoli in an immature secretory state, but CM tissue contained both lactating and immature alveoli. Postpartum ultrastructure parameters were similar between halves until d 20 when control tissue was composed of a homogeneous population of lactating alveoli, but CM tissue contained lactating, engorged, and resting alveoli. Expression of CCAAT/enhancer binding protein-beta (CEBP-beta), cyclin D1, and bcl(2) were up-regulated during late gestation, but did not differ between control and CM halves. Expression of alpha-lactalbumin was increased in CM halves during late gestation, but was not different in CM and control tissue after parturition. Other genes evaluated (bax, insulin-like growth factor binding protein 5, ATP-binding cassette 1, and p27) were not differentially expressed at any timepoints evaluated. Results indicate that CM reduced subsequent half-udder milk yield in primiparous cows through altered MEC turnover and secretory capacity. Negative effects of CM on the subsequent lactation were not alleviated by bST supplementation.

A number of major scientific advances have been realized in the last 25 yr in determining the opportunities and limitations of altering milk composition through nutritional manipulation. Because of the greater sensitivity of milk fat to dietary manipulation than either protein or lactose, nutritional control of milk fat content and fatty acid composition received a great deal of attention. New information emerged linking ruminal production of trans fatty acid isomers with milk fat depression. As a result, research on fatty acid biohydrogenation intensified yielding new insight on the origin of specific trans fatty acid isomers originating from ruminal biohydrogenation and how these isomers were modified by the action of mammary enzymes. The discovery of conjugated linoleic acid (CLA) as a potent anticarcinogen also led to extensive work on enhancing its concentration in milk through nutritional manipulation and discovering the physiological effects of specific CLA isomers. New protected fats were developed in recent years that were designed to resist biohydrogenation and enhance the concentration of unsaturated fatty acids in milk. The nutritional factors receiving the most attention during the last 25 yr for their influence on milk protein content were forage-to-concentrate ratio, the amount and source of dietary protein, and the amount and source of dietary fat. New insights were tested on modes of action whereby fat supplements caused a decline in protein concentration. Changes in milk lactose concentration occur only in extreme and unusual feeding situations, but the basic biology of lactose synthesis and regulation are still being explored using modern molecular techniques. This paper highlights the major advances in controlling milk composition by dietary manipulation and how it influences the entire animal system from practical feeding studies to basic cellular work on mammary tissue metabolism.

The seasonal variation in conjugated linoleic acid (CLA) and vaccenic acid (VA) concentrations in sheep dairy products and the extent of their transfer from milk fat to cheese and ricotta fat were investigated. Samples were collected from 2 sheep milk processing plants in North Sardinia (Italy) every 2 wk from March through June. Concentrations of fatty acids (FA) in fresh cheese and ricotta fat were primarily dependent on the fatty acid content of the unprocessed raw milk. The content of c9,t11-CLA averaged 1.73, 1.69, and 1.75 mg/100 mg of FA methyl esters (FAME), respectively, for milk, cheese, and ricotta, and differed significantly between cheese and ricotta. The content of VA averaged 3.40, 3.33, and 3.43 mg/100 mg of FAME, respectively for milk, cheese, and ricotta. The FA composition of dairy products was markedly affected by period of sampling: the mean c9,t11-CLA and VA concentration decreased from March (2.20 and 4.52 mg/100 mg of FAME) to June (1.14 and 1.76 mg/100 mg of FAME) in all dairy products. No differences in c9,t11-CLA and VA concentration of dairy products were observed between the 2 dairy companies obtaining milk from the same geographical origin. The seasonal changes in CLA and VA in milk fat were probably related to changes in pasture quality.

Dry periods of 40 to 60 d have been an industry standard because dry periods <40 d have resulted in reduced milk yields in the subsequent lactation by 10 to 30%. However, recent research has demonstrated no production losses for cows given a 30-d dry period. The current study evaluated milk production effects of shortened or omitted dry periods for cows at mature-equivalent production >12,000 kg of milk and treated with bovine somatotropin (bST). The study used 2 commercial dairies and one university dairy and included 4 treatments. Five multiparous and 5 primiparous cows from each farm were assigned to each treatment: 1) 60-d dry period, label use of bST (60DD); 2) 30-d dry period, label use of bST (30DD); 3) continuous milking, label use of bST (CMLST); and 4) continuous milking with continuous use of bST (CMCST). Per label, bST use started at 57 to 70 d in milk and ended 14 d before drying (60DD and 30DD) or expected calving date (CMLST). In primiparous cows, average milk yields during the first 17 wk of lactation were reduced for cows on treatments 30DD, CMLST, and CMCST vs. the 60DD treatment. (38.3, 35.1, and 37.5 vs. 44.1 +/- 1.3 kg/d, respectively). For multiparous cows, respective milk yields did not differ (46.6, 43.4, 46.5, and 47.7 +/- 2.1 kg/d). Shortened or omitted dry periods may impede mammary growth in primiparous cows, resulting in reduced milk yield in the subsequent lactation. In contrast, a shortened or omitted dry period with either bST protocol did not alter production in multiparous cows treated with bST. Quality aspects of prepartum milk and colostrum require additional characterization. For multiparous cows, milk income generated for short dry periods or for continuous milking might increase their profitability. At 17 wk of the subsequent lactation, estimates of the cumulative net margins of multiparous cows on the 30DD treatment and continuous milking treatments exceeded those of cows on the 60DD treatment by 40 dollars to 60 dollars per cow.

A crossover design trial with 4 ruminally and duodenally cannulated lactating dairy cows was conducted to study the effect of sodium laurate on ruminal fermentation, nutrient digestibility, and milk yield and composition. The daily dose of sodium laurate (0, control or 240 g/cow, LA) was divided in 2 equal portions and introduced directly into the rumen through the cannula before feedings. Ruminal samples (29 in 114 h) were analyzed for fermentation variables and protozoal counts. Sodium laurate had no effect on ruminal pH and total and individual volatile fatty acids concentrations. Ruminal ammonia concentration, ammonia N pool size, and the irreversible loss of ammonia N were unaffected by treatment. Compared to control, protozoal counts were reduced by 91% by LA. Carboxymethylcellulase and xylanase activities of ruminal fluid were decreased (by 40 and 36%, respectively), and amylase activity was not affected by LA compared with control. Flow of microbial N to the duodenum was reduced by LA. Dry matter intake and apparent total tract digestibility of dry matter, organic matter, crude protein, neutral detergent fiber, and acid detergent fiber were not different between the 2 treatments. Milk yield, fat-corrected milk yield, milk fat and protein concentrations and yields, and milk urea N content were not affected by treatment. Sodium laurate did not affect transfer of ruminal ammonia-15N into bacterial or milk protein. In conclusion, LA at approximately 0.3% of the rumen weight reduced ruminal protozoal population and had a negative effect on fibrolytic activities of ruminal fluid and microbial protein flow to the intestine. Treatment had no other significant effects on ruminal fermentation, total tract digestibility, or transfer of ruminal ammonia-15N into milk protein.

In lactating dairy cows, the onset of negative net energy balance (EB) at parturition causes a reduction in plasma leptin and is also associated with increased concentration of growth hormone (GH) and decreased concentration of insulin. These observations raise the possibility that opposite changes in plasma insulin and GH are partly responsible for reduced plasma leptin. To test this hypothesis, we first examined the effects of undernutrition without the confounding influence of parturition by using late lactating dairy cows fed 120% of their nutrient requirements or restricted to 33% of maintenance requirements. Plasma leptin was reduced within 24 h of feed restriction, and was associated with increased plasma GH and decreased plasma insulin. Complete food deprivation for 48 h caused similar changes in the plasma concentration of leptin. To determine if an elevation in GH is responsible for the fall in plasma leptin, dairy cows were treated with excipient or bovine somatotropin in early lactation or in late lactation. Growth hormone treatment had no significant effect on plasma leptin irrespective of stage of lactation. Finally, the effects of insulin were studied by performing euglycemic hyperinsulinemic clamps in mid-lactating dairy cows. After 96 h of hyperinsulinemia, plasma leptin was increased significantly. These data indicate that insulin regulates plasma leptin in lactating dairy cows. They also suggest that, in undernourished lactating dairy cows, reduced plasma insulin could account for a portion of the decline in plasma leptin but that elevated plasma GH is unlikely to have a major effect.

Intravenous infusion of conjugated linoleic acid (CLA) was evaluated as a simpler method than abomasal infusion and the feeding of calcium salts to examine milk fat depression. The objectives were to determine the dose-dependent response of milk fat and plasma metabolites to intravenous administration of the trans-10, cis-12 isomer of CLA, an isomer identified to possess an inhibitory effect on milk fat synthesis. Four multiparous Holstein cows averaging 123 +/- 30 d in milk were randomly assigned to treatments in a 4 x 4 Latin square design. Catheters were inserted into the jugular vein for infusions and blood sampling. Treatments consisted of intravenous infusions of 0, 2, 4, and 6 g/d CLA (> 95% trans-10, cis-12 CLA). Infusates contained 72 g/d of a parenteral solution, saline, and CLA to 90 ml. Periods were of 5 d duration with a 7 d wash out. Milk was sampled at each milking and analyzed for fat, protein, and fatty acids. Blood samples were obtained on the last day of each period. Dry matter intake (22.4 +/- 2.4 kg/d), milk yield (28.5 +/- 3.3 kg/d), and protein percent (3.26 +/- 0.08%) of cows were not affected by treatment. However, milk fat percentage was reduced linearly with increasing doses of CLA. Milk fat percentage was 4.17, 3.53, 3.29, and 2.92% on d 5 for treatments 0, 2, 4, and 6 g/d CLA, respectively. Concentrations (4.2 mg/g of fat) of cis-9, trans-11 CLA in milk fat were not affected by treatment. However, an increase in the trans-10, cis-12 CLA content of milk fat was observed. Milk fat contained 0.00, 0.02, 0.06, and 0.10 mg of trans-10, cis-12 CLA per g of fat (SEM = 0.065) for treatments 0, 2, 4, and 6 g/d CLA, respectively. Plasma NEFA concentration increased linearly with the dose of the trans-10, cis-12 CLA. Intravenous infusion of the trans-10, cis-12 isomer of CLA depressed milk fat in a linear manner over the range of infusion studied and, therefore, is an alternative to abomasal infusion.

Increasing conjugated linoleic acid (CLA) content of milk fat from lactating dairy cattle has become a research interest due to the possible health benefits afforded humans consuming CLA. Dietary supplementation of CLA to lactating dairy cows is one potential method by which CLA content of milk and dairy products may be enhanced. Feeding CLA in calcium salt form could potentially deliver CLA to the lower digestive tract through prevention of biohydrogenation by rumen microbes. Milk fat depression (MFD) occurs when cows receive CLA-60, a commercially available CLA source containing numerous CLA isomers, abomasally. Our objectives were to determine the quantity of CLA as calcium salts required to elicit maximal MFD and to evaluate the effects of CLA supplementation on fatty acid composition of milk fat. Five Holstein cows at approximately 93 DIM were utilized in a 5 x 5 balanced Latin square crossover design. Periods were 14-d in length with a 5-d treatment phase and 9-d rest phase. Treatments were 5-d supplementation of 0, 12.5, 25, 50, and 100 g of CLA-60 in calcium salt form. Milk samples were collected on d 5 of CLA supplementation and analyzed for composition and fatty acid profile. Regression analysis of milk fat data suggested that MFD was not maximized over the dose levels investigated, despite delivery of 34.5 g of trans-10, cis-12 CLA in the 100-g dose of CLA. Supplementation with 50 and 100 g of CLA per day resulted in a reduction of milk fat percent of 29 and 34%, respectively. Trend analysis indicated a linear decrease in the milk fat content of caprylic, capric, and lauric acids as the dose of CLA increased. Milk fat content of cis-9, trans-11, and trans-10, cis-12 CLA increased at an increasing rate as dose increased.

The primary objective was to determine whether the dietary polyunsaturated fatty acids, eicosapentaenoic (EPA, C20:5, n-3) and docosahexaenoic (DHA, C22:6, n-3), present in fish meal (FM) can attenuate uterine secretion of PGF2alpha in response to a challenge with estradiol and oxytocin in lactating dairy cows. Cycling multiparous cows (n = 32) were fed diets containing 0 (OFM), 2.6 (2.6FM), 5.2 (5.2FM), or 7.8% menhaden FM (7.8FM). The diet consisting of 7.8FM also contained fish oil (0.28% of dietary dry matter) to increase intake of EPA and DHA. Average dry matter intake was 24.9 kg/d and unaffected by diet. Combined intakes of EPA and DHA averaged 0, 12.8, 24.1, and 54.0 g/d from the OFM, 2.6FM, 5.2FM, and 7.8FM diets, respectively. At 30 to 34 d after initiation of dietary treatments, cows received an i.m. injection of 100 microg of GnRH followed by i.m. administration of 25 and 15 mg of PGF2alpha after 7 and 8 d, respectively. Synchronous ovulation was induced by an injection of 3000 IU of human chorionic gonadotropin (hCG) given 24 h later on d 9. Subsequent luteal phase increases in plasma progesterone concentrations did not differ (0.88 ng/ml per day). At 15 d after hCG injection, cows were injected with estradiol-17beta (3 mg, i.v.) at 0900 h and oxytocin (100 IU, i.v.) at 1300 h. Plasma PGF2alpha metabolite concentrations after oxytocin injection were reduced in cows fed diets containing FM compared with those fed OFM. Milk production (39.1 kg/d) and concentrations of fat, protein, or urea nitrogen in milk were not affected by diet. Feeding fish meal and fish oil containing eicosapentaenoic acid and docosahexaenoic acid reduced the proportion of n-6 fatty acids and increased that of n-3 fatty acids in milk in a dose-responsive manner.

A study was conducted to investigate the response to supplemental tallow of lactating cows fed basal diets with different alfalfa silage:corn silage ratios. We postulated that supplemental tallow will have decreasing negative effects on rumen fermentation, dry matter intake (DMI), and milk fat percentage as the dietary ratio of alfalfa silage:corn silage is increased. Eighteen Holstein cows averaging 134 +/- 14 d in milk were used in a replicated 6 x 6 Latin square design with 21-d periods. Treatments were arranged as a 2 x 3 factorial with 0 or 2% tallow (DM basis) and three forage treatments: 1) 50% of diet DM as corn silage, 2) 37.5% corn silage and 12.5% alfalfa silage, and 3) 25% corn silage and 25% alfalfa silage. Cows were allowed ad libitum consumption of a total mixed ration. Diets were formulated to contain 18% crude protein and 32% neutral detergent fiber. No fat x forage treatment interactions were observed. Fat supplemented cows had lower DMI and produced more milk with less milk fat content relative to non-supplemented cows. Concentration of trans-octadecenoic acids was higher in milk fat of tallow-supplemented cows. Tallow supplementation had no effect on ruminal pH and acetate:propionate ratio, but tended to decrease total volatile fatty acid (VFA) concentration in the rumen. Increasing the proportion of alfalfa silage increased DMI, milk fat percentage, and milk fat yield regardless of the fat content of the diet. Total VFA concentration and acetate:propionate ratio in the rumen were increased in response to higher levels of alfalfa in the diets. These results suggest that replacing corn silage with alfalfa silage did not alleviate the negative response of dairy cows to tallow supplementation at 2% of diet DM.

The objective of this study was to investigate the effects of tallow and choice white grease (CWG) fed at 0, 2, and 4% of the diet dry matter (DM) on rumen fermentation and performance of dairy cows when corn silage is the sole forage source. Fifteen midlactation Holstein cows were used in a replicated 5 x 5 Latin square design with 21-d periods. Treatments were 0% fat (control), 2% tallow, 2% CWG, 4% tallow, and 4% CWG (DM basis). The forage:concentrate ratio was 50:50, and diets were formulated to contain 18% crude protein and 32% neutral detergent fiber (DM basis). Cows were allowed ad libitum consumption of diets fed twice daily as total mixed rations. Cows fed supplemental fat had lower DM intake and produced less milk and milk fat than cows fed the control diet. Feeding 4% fat reduced milk production and milk fat yield relative to feeding 2% fat. Treatments had little effect on the concentration of trans-octadecenoic acids in milk fat. Total trans fatty acids were poorly related to changes in milk fat percentage. Ruminal pH and total volatile fatty acids concentration were not affected by supplemental fat. The acetate:propionate ratio, NH3-N, and numbers of protozoa in the rumen were significantly decreased when fat was added to the diets. Source of dietary fat did not affect rumen parameters. There was no treatment effect on in situ corn silage DM and neutral detergent fiber disappearance. Including fat in corn silage-based diets had negative effects on milk production and rumen fermentation regardless of the source or level of supplemental fat.

We examined the role of trans-octadecenoic acids in milk fat depression when low fiber diets were fed. The study consisted of four experimental periods with a 2 x 2 factorial arrangement of treatments to test the effects of dietary fat (saturated vs. unsaturated) and rumen fermentation (high fiber diets vs. low fiber diets) on milk fat depression. Dietary fiber concentration and type of fat had significant effects on milk fat. Effects were most pronounced when unsaturated fat was added to the low fiber diet. When the low fiber diet plus unsaturated fat was fed, milk fat percentage and yield were decreased by 30 and 35%, respectively, compared with the percentage and yield when the high fiber diet plus saturated fat was fed. Alterations in rumen fermentation caused by differences in dietary fiber concentrations had little effect on the amount of trans-octadecenoic acids in milk fat, and the total amount did not correlate with changes in milk fat percentage. Further examination of the isomeric profile of trans-octadecenoic acid revealed substantial differences among the dietary treatments. Although the addition of unsaturated fat resulted in marked increases in the milk fat content of trans-11-octadecenoic acid, regardless of dietary fiber concentration, the low fiber diet plus unsaturated fat increased the content of trans-10-octadecenoic acid. This combination was also associated with a significant decrease in milk fat content and yield. When the low fiber diets were fed, circulating insulin concentrations were elevated, regardless of the type of fat supplement. However, marked milk fat depression occurred only when the low fiber diet was supplemented with unsaturated fat.

The galactopoietic actions of bovine somatotropin are both direct and indirect. Indirect actions are apparently mediated by the insulin-like growth factor (IGF) system. The objective of this study was to compare systemic (plasma) versus local (lymph) concentrations of IGF. Afferent mammary lymph most nearly represents the extracellular fluid that is bathing mammary tissue. Catheters were surgically implanted in the jugular vein and the superficial afferent mammary lymph vessels of four lactating cows. A crossover design was utilized to evaluate the effects of bovine somatotropin (bST). After 2 d of basal sampling, either bST (40 mg/d) or excipient was administered daily for 5 d. After the conclusion of the bST study, a second study was conducted in which cows were deprived of feed for 36 h. Blood and lymph were simultaneously sampled at least every 6 h throughout both studies. Milk yield was increased by bST, and concentrations of IGF-I were increased in plasma and lymph. The relationship between plasma and lymph concentrations for IGF-II, bST, insulin, glucose, urea nitrogen, and nonesterified fatty acids were similar during bST treatment. Milk yield was reduced 76% by 36 h of feed deprivation. Feed deprivation also caused a reduction of IGF-I in plasma, but concentrations of IGF-I in lymph were not altered. In contrast, changes in IGF-II, bST, insulin, glucose, urea nitrogen, and nonesterified fatty acids that were caused by feed deprivation followed similar patterns in plasma and lymph. Clearly, if IGF-I mediates the mammary actions of bST, then concentrations of IGF-I in plasma correlate with milk yield responses as well as, if not better than, concentrations in lymph.

We examined the role of insulin in milk protein synthesis using the hyperinsulinemic-euglycemic clamp approach in combination with abomasal infusion of casein. The two experimental periods consisted of abomasal infusion of water or 0.5 kg/d of casein. An insulin clamp was conducted over the last 4 d of each period. During the insulin clamp, circulating insulin was elevated fourfold, and euglycemia was maintained by the infusion of exogenous glucose. Casein infusion increased milk yield so that milk protein yield was 10% greater than baseline values. Use of the insulin clamp combined with casein infusion increased milk protein yield by 230 g/d (28% greater than baseline values). Milk protein composition was not altered, but content was increased from 3.13% during the baseline period to 3.44% by d 4 of the clamp; calcium concentration in milk increased about 10% to 1.2 g/kg. During the clamp, circulating concentrations of essential amino acids were dramatically reduced. The most pronounced effects were noted for branched-chain amino acids (64% reduction from baseline values). The insulin clamp resulted in alterations in circulating insulin-like growth factor (IGF)-I concentrations (increase) as well as IGF-II and IGF-binding protein-2 concentrations (decreases). Overall, results indicated that the ability of the mammary gland to synthesize milk protein does not function at maximum capacity, and there is a previously unrecognized potential to enhance milk protein percentage and yield.

Five lactating Holstein cows were fitted with rumen fistulas and subjected to a hyperinsulinemic-euglycemic clamp and abomasal casein infusion to examine the effects on milk fat synthesis and the composition of milk fatty acids. The experiment consisted of two periods of abomasal infusions (water or 0.5 kg/d of casein); each period was divided into three 4-d intervals. The initial interval allowed for acclimation, and baseline measurements were established during the second interval. During the third 4-d interval, a hyperinsulinemic-euglycemic clamp was maintained, and insulin was infused continuously at the rate of 1 microgram/kg of body weight per h. Circulating concentrations of insulin were increased more than fourfold, and euglycemia was maintained by infusion of glucose at variable rates. Insulin had no effect on milk fat yield but casein infusion increased milk yield and tended to increase fat yield. A trend toward higher milk yield during the clamp, combined with a slight numerical decrease in milk fat yield, resulted in decreased fat percentage. Calculated net energy balance was positive throughout the study, although feed intake decreased during the insulin clamp, particularly for the water infusion period. Minor changes occurred in the composition of milk fatty acids during the clamp when the balance between de novo and preformed fatty acids shifted slightly toward de novo. Overall, results demonstrated that a relatively constant rate of milk fat synthesis was maintained during chronic hyperinsulinemia. Effects on milk fat yield and composition of fatty acids offered no support for the role of insulin on milk fat depression.

Five lactating Holstein cows were subjected to a hyperinsulinemiceuglycemic clamp to examine the effects of insulin on milk yield and composition. Of special interest was the evaluation of the glucogenic-insulin theory of milk fat depression. Cows were fed every other hour to minimize postprandial effects, and blood samples were obtained via an indwelling jugular catheter every 4 h for 2 d to establish baseline glucose concentrations. For the 4-d clamp, insulin was infused continuously (1 microgram/kg of BW per h) into the contralateral jugular vein, and circulating insulin was increased approximately fivefold. Blood was sampled frequently, and blood glucose was maintained within 10% of baseline concentrations by infusion of exogenous glucose at variable rates (mean = .15 g/kg of BW per h). Dietary intake declined on the 4th d of the insulin clamp (23.0 vs. 16.3 kg/d). Milk yield, however, did not change (32.4 vs. 33.6 kg/d) in support of the lack of sensitivity of the mammary gland to insulin. Milk fat percentage (3.85 vs. 3.66) and yield (1.26 vs. 1.22 kg/d) did not change during the insulin clamp. Milk protein yield increased (.98 vs. 1.05 kg/d), and milk protein percentage tended to increase (3.04 vs. 3.14), during the insulin clamp. This modest increase in milk protein yield may have been constrained by a lack of available amino acids, as indicated by a decrease in circulating concentrations of essential amino acids, urea nitrogen, and plasma proteins. Overall, results offer no support for the glucogenic-insulin theory of milk fat depression but do indicate that the insulin infusion, either directly or indirectly, enhanced secretion of milk protein.

This paper examines whether or not responses of dairy cattle to various dietary macromineral elements differed in hot weather compared with thermoneutral conditions. The consequences of heat stress and the interrelationships of macromineral elements on feed intake, digestive function, use of dietary buffers, mineral element uptake from the portal-drained viscera, perturbation of acid-base physiology and related mineral element nutrition, effects of dietary macromineral element concentrations on lactational performance as affected by season, and influence of mineral elements in drinking water are reviewed, and new research results are presented. Dietary mineral buffers aided in alleviation of the decline in DMI and milk yield induced by heat stress. New research results showed that portal plasma flow declined with heat stress or by restriction of DMI in a thermoneutral environment and that uptake of P from the portal-drained viscera by lactating cows was reduced 50% by heat stress compared with that of cows with the same DMI in a thermoneutral environment. Accelerated respiration rate caused respiratory alkalosis and apparently compensated metabolic acidosis, changing the demand for Na and K during heat stress. There is need to characterize more accurately the nyctohemeral pattern of acid-base physiology in the heat-stressed lactating dairy cow and to relate it to macromineral element needs. A large data set (n = 1444 cow period means) was used to compare milk yield and DMI responses to varying dietary concentrations of P, Na, K, Cl, Ca, Mg, and cation-anion difference in summer compared with those in winter. Interactions of dietary concentrations of Cl, Ca, and Mg with season on DMI and K, Ca, and Mg with season on 4% FCM yield were detected. Interactions of Na by Cl, Na by P, and Cl by P with season on DMI and of Cl by P with season on 4% FCM yield were detected. High concentrations of sulfate and chloride in drinking water jeopardized productivity of cows during hot weather.