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Background and Objectives: As cannabis use increases among reproductive-aged women, there is a growing need to better understand the presence of cannabinoids in milk produced by women using cannabis. It is unclear how concentrations of cannabinoids such as delta-9-tetrahydrocannabinol (Δ 9 -THC) persist in milk after cannabis use and what factors contribute to variation in milk Δ 9 -THC concentrations. Our objectives were to measure cannabinoids in human milk following cannabis abstention, after single and repeated instances of cannabis use, and identify factors contributing to concentration variation. Methods: The Lactation and Cannabis (LAC) Study prospectively observed 20 breastfeeding participants who frequently used cannabis (≥1/week), had enrolled <6 months postpartum, were feeding their infant their milk ≥5 times/day, and were not using any illicit drugs. Participants collected a baseline milk sample after ≥12 hours of abstaining from cannabis and five milk samples at set intervals over 8-12 hours after initial cannabis use. Participants completed surveys and recorded self-directed cannabis use during the study period. Results: Δ 9 -THC peaked 120 minutes after a single instance of cannabis use (median, n = 9). More instances of cannabis use during the study period were associated with greater Δ 9 -THC area-under-the-curve concentrations (ρ = 0.65, p = 0.002), indicating Δ 9 -THC bioaccumulation in most participants. Baseline Δ 9 -THC logged concentration was positively associated with self-reported frequency of cannabis use ( b = 0.57, p = 0.01). Conclusions: Cannabinoids are measurable in human milk following cannabis use, and concentrations remain elevated with repeated cannabis use over a day. Substantial variation in Δ 9 -THC milk concentrations reflects individual differences in characteristics and behavior, including average postpartum frequency of cannabis use.

The goal of Working Group 1 in the Breastmilk Ecology: Genesis of Infant Nutrition (BEGIN) Project was to outline factors influencing biological processes governing human milk secretion and to evaluate our current knowledge of these processes. Many factors regulate mammary gland development in utero, during puberty, in pregnancy, through secretory activation, and at weaning. These factors include breast anatomy, breast vasculature, diet, and the lactating parent's hormonal milieu including estrogen, progesterone, placental lactogen, cortisol, prolactin, and growth hormone. We examine the effects of time of day and postpartum interval on milk secretion, along with the role and mechanisms of lactating parent-infant interactions on milk secretion and bonding, with particular attention to the actions of oxytocin on the mammary gland and the pleasure systems in the brain. We then consider the potential effects of clinical conditions including infection, pre-eclampsia, preterm birth, cardiovascular health, inflammatory states, mastitis, and particularly, gestational diabetes and obesity. Although we know a great deal about the transporter systems by which zinc and calcium pass from the blood stream into milk, the interactions and cellular localization of transporters that carry substrates such as glucose, amino acids, copper, and the many other trace metals present in human milk across plasma and intracellular membranes require more research. We pose the question of how cultured mammary alveolar cells and animal models can help answer lingering questions about the mechanisms and regulation of human milk secretion. We raise questions about the role of the lactating parent and the infant microbiome and the immune system during breast development, secretion of immune molecules into milk, and protection of the breast from pathogens. Finally, we consider the effect of medications, recreational and illicit drugs, pesticides, and endocrine-disrupting chemicals on milk secretion and composition, emphasizing that this area needs much more research attention., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The human milk microbiome has been somewhat characterized, but little is known about changes over time and relations with maternal factors such as nutrient intake. Objective: We sought to characterize the human milk microbiome and described associations with maternal nutrient intake, time postpartum, delivery mode, and body mass index (BMI; in kg/m 2 ). Methods: Milk samples ( n = 104) and 24-h diet recalls were collected 9 times from 21 healthy lactating women from day 2 to 6 mo postpartum. Women were classified by BMI as healthy weight (<25) or overweight or obese (≥25). Bacterial taxa were characterized with the use of the high-throughput sequencing of the 16S ribosomal RNA gene. Results: The milk microbiome was relatively constant over time, although there were small changes in some of the lesser-abundant genera. Relative abundances of several taxa were associated with BMI, delivery mode, and infant sex. For instance, overweight and obese mothers produced milk with a higher relative abundance of Granulicatella than did healthy-weight women (1.8% ± 0.6% compared with 0.4% ± 0.2%, respectively; P < 0.05). Relative abundances of several bacterial taxa were also associated with variations in maternal dietary intake. For example, intakes of saturated fatty acids ( r s = -0.59; P = 0.005) and monounsaturated fatty acids ( r s = -0.46; P = 0.036) were inversely associated with the relative abundance of Corynebacterium ; total carbohydrates ( r s = -0.54; P = 0.011), disaccharides ( r s = -0.47; P = 0.031), and lactose ( r s = -0.51; P = 0.018) were negatively associated with Firmicutes; and protein consumption was positively correlated with the relative abundance of Gemella ( r s = 0.46; P = 0.037). Conclusions: Factors associated with variations in the human milk microbiome are complex and may include maternal nutrient intake, maternal BMI, delivery mode, and infant sex. Future studies designed to investigate the relation between maternal nutrient intake and the milk microbiome should strive to also evaluate dietary supplement usage and analyze the collected milk for its nutrient content., Competing Interests: Author disclosures: JEW, JMC, KAL, NFB, MAY, SLB, BS, WJP, MLS, MAM, and MKM, no conflicts of interest.

Background: Human milk is a complex fluid comprised of myriad substances, with one of the most abundant substances being a group of complex carbohydrates referred to as human milk oligosaccharides (HMOs). There has been some evidence that HMO profiles differ in populations, but few studies have rigorously explored this variability. Objectives: We tested the hypothesis that HMO profiles differ in diverse populations of healthy women. Next, we examined relations between HMO and maternal anthropometric and reproductive indexes and indirectly examined whether differences were likely related to genetic or environmental variations. Design: In this cross-sectional, observational study, milk was collected from a total of 410 healthy, breastfeeding women in 11 international cohorts and analyzed for HMOs by using high-performance liquid chromatography. Results: There was an effect of the cohort ( P < 0.05) on concentrations of almost all HMOs. For instance, the mean 3-fucosyllactose concentration was >4 times higher in milk collected in Sweden than in milk collected in rural Gambia (mean ± SEM: 473 ± 55 compared with 103 ± 16 nmol/mL, respectively; P < 0.05), and disialyllacto- N -tetraose (DSLNT) concentrations ranged from 216 ± 14 nmol/mL (in Sweden) to 870 ± 68 nmol/mL (in rural Gambia) ( P < 0.05). Maternal age, time postpartum, weight, and body mass index were all correlated with several HMOs, and multiple differences in HMOs [e.g., lacto- N -neotetrose and DSLNT] were shown between ethnically similar (and likely genetically similar) populations who were living in different locations, which suggests that the environment may play a role in regulating the synthesis of HMOs. Conclusions: The results of this study support our hypothesis that normal HMO concentrations and profiles vary geographically, even in healthy women. Targeted genomic analyses are required to determine whether these differences are due at least in part to genetic variation. A careful examination of sociocultural, behavioral, and environmental factors is needed to determine their roles in this regard. This study was registered at clinicaltrials.gov as NCT02670278.

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.)

Background: The fecal microbiota has been characterized in some adult populations, but little is known about its community structure during lactation., Objectives: We characterized the maternal fecal microbiome during lactation and explored possible mediating factors such as nutrition., Methods: Fecal samples were collected from 20 lactating women from 2 d to 6 mo postpartum, and bacterial taxa were characterized with the use of high-throughput sequencing. Bacterial community structure (at each taxonomic level) and relations between bacterial taxa and environmental and dietary variables were visualized and analyzed with the use of stacked bar charts, principal component analysis, and multivariate analyses such as nonmetric multidimensional scaling and canonical correlation analysis., Results: Complex bacterial community structure was somewhat similar to those previously published for other adult populations (although there were some notable differences), and there were no clear associations with time postpartum or anthropometric or environmental variables. However, Spearman rank correlations suggested that increased intake of pantothenic acid, riboflavin, vitamin B-6, and vitamin B-12 were related to increased relative abundance of Prevotella (r = 0.45, 0.39, 0.34, and 0.24, respectively; P ≤ 0.01) and decreased relative abundance of Bacteroides (r = -0.55, -0.46, -0.32, and -0.35, respectively; P ≤ 0.01). Intakes of copper, magnesium, manganese, and molybdenum were positively associated with Firmicutes (r = 0.33, 0.38, 0.44, and 0.51, respectively; P ≤ 0.01) and negatively associated with Bacteroidetes (r = -0.38, -0.44, -0.48, and -0.53, respectively; P ≤ 0.01). Overall, data consistently suggest that increased consumption of a more nutrient- and calorie-rich diet was positively associated with relative abundance of Firmicutes., Conclusions: The fecal microbiome of lactating women is relatively stable in the postpartum period and somewhat similar to that of other adult populations. Variation in dietary constituents may be related to that of relative abundance of individual bacterial taxa. Controlled dietary intervention studies will be required to determine whether these associations are causal in nature., (© 2015 American Society for Nutrition.)

Previous work has demonstrated that elevated maternal lipid intake (particularly from dairy products) is associated with increased lipids and altered fatty acid profile in milk produced by healthy lactating women. To investigate our primary hypothesis that a maternal diet rich in full-fat dairy products would simultaneously increase milk lipid percent and expression of genes related to the uptake and/or de novo biosynthesis of milk lipids, we provided 15 lactating women with diets enriched in full-fat or nonfat dairy products for 14 days each in a randomized, crossover study with a 2-week washout period. Milk fat (%) was lower when women consumed the low-fat compared with the full-fat dairy diet (2.41% ± 0.31% vs 3.35% ± 0.28%, respectively; P < .05); concentrations of more than 20 fatty acids also differed. However, neither conservatively evaluated microarray data nor quantitative real-time polymerase chain reaction analysis uncovered any treatment effects on expression of genes related to lipid synthesis or uptake. These data suggest that alteration in gene expression in the lactating human mammary gland is likely not the primary mechanism by which consumption of a high-fat diet affects milk fat percent in healthy, lactating women., (Copyright © 2015 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.)

Somatic cell count (SCC) is a widely used marker of udder health and a predictor of inflammation caused by an immune response. The objective of this study was to determine whether selected measures of mammary gland health as well as milk fatty acid profile were altered by an increase in milking frequency using a unilateral frequent milking (UFM) model. Holstein cows at parturition were assigned to UFM, in which the left udder half of each cow was milked four-times daily (4X) and the right udder half was milked twice daily (2X) for the first 21 days in milk (DIM). Milk yields from each udder half were measured from 1-21 DIM and samples were collected on days 3, 7, 10, 14 and 21 for determination of SCC and milk composition. Flow cytometric analysis with bovine monoclonal antibodies was used to identify milk immune cell populations and milk fatty acid (FA) composition was determined using gas chromatography. Gene expression analysis was used to determine whether there was an alteration in mRNA expression of genes involved in milk fat production including lipoprotein lipase (LPL) and FA-binding protein 3 (FABP3) with ribosomal protein S9 (RPS9) as a house-keeping gene. No difference was detected for milk SCC or cell populations between the udder halves milked 4X as compared with the udder halves milked 2X. In addition, no difference was detected for any FA in milk from the udder half milked 4X as compared with the udder half milked 2X. Overall, using a UFM model, increased milking frequency for the first 21 DIM did not affect selected measures of mammary gland health or milk FA, but was associated with greater milk yield, milk fat percent and yield, and milk protein and lactose yields.

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.

Substantial research suggests that the t10,c12-18:2, but not the c9,t11-18:2, isomer of conjugated linoleic acid (CLA) reduces milk fat synthesis in lactating bovine and rodent species. Because fat is the major energy-yielding component in human milk, we were interested in whether this is true for women as well. Thus, the effects of c9,t11-18:2 and t10,c12-18:2 on milk fat were examined in breast-feeding women (n = 12) in a double-blind, placebo-controlled, crossover study with latin-square design. The study was divided into six periods: baseline (3 days), three intervention periods (5 days each), and two washout periods (9 days each). During each intervention period, women consumed 750 mg/day of a supplement containing predominantly c9,t11-18:2, t10,c12-18:2, or 18:1 (olive oil placebo). Milk was collected by complete breast expression on the final day of each period. Infant milk consumption was estimated by 24 h weighing on the penultimate day of each intervention and washout period, and maternal adiposity (% body fat) was determined at baseline using dual energy X-ray absorptiometry. Milk c9,t11-18:2 and t10,c12-18:2 concentrations were greater (P < 0.05) during the corresponding CLA treatment periods as compared to the placebo period, providing strong evidence of subject compliance. Both CLA isomers were transferred into milk fat at relatively high efficiency; average transfer efficiency was estimated to be 23.3%. Compared to the placebo treatment, milk fat content was not reduced during either CLA treatment. Data indicate that body fatness did not modify any putative effect of isomeric CLA consumption on milk fat concentration. The evidence from this study suggests that the sensitivity of lactating women's mammary tissue to an anti-lipogenic effect of the t10,c12-18:2 isoform of CLA may be less than previously hypothesized.

The term "conjugated linoleic acid" (CLA) refers to a group of positional and geometric isomers that are derived from linoleic acid and are found primarily in meat and milk products from ruminant animals. Due to the array of putative benefits associated with various forms of CLA, there has been recent interest in supplementing human diets with these fatty acids especially when weight loss is desired. However, in many animal models, CLA has been shown to decrease milk fat production. There is some concern, therefore, that maternal CLA supplementation during lactation might inadvertently decrease nutrient supply to the nursing infant. However, there is only limited research on the effect of CLA consumption on milk fat content in women. Based on previously published work from our laboratory, we hypothesized that CLA supplementation would reduce the milk fat percentage in lactating women in a dose-dependent manner. Breastfeeding women (n = 12) were assigned randomly to treatments of 4 g/day safflower oil (SFO), 2 g/day CLA plus 2 g/day SFO, or 4 g/day CLA in a double blind, 3 x 3 Latin square design. Conjugated linoleic acid supplements contained approximately equal amounts of cis9,trans11-18:2 and trans10,cis12-18:2; the two most common isoforms of CLA. Milk was collected by complete breast expression on the last day (day 5) of each intervention period and analyzed for macronutrient and fatty acid composition. On day 4 of each intervention period, infant milk consumption was estimated by 24 h weighing of the infant. Washout periods were 9 days in length. We observed a dose-dependent increase in the concentrations of cis9,trans11-18:2 and trans10,cis12-18:2 in the milk fat. However, we detected neither a change in overall macronutrient composition nor infant milk consumption. These data do not support those obtained from animal models or our previous human work suggesting that consumption of CLA mixtures necessarily reduces milk fat. It is possible that either (1) the interpretation of our previously published data should be reevaluated, and/or (2) there are important intra- and inter-species differences in this regard.

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.

We studied the incorporation of the trans-11 vaccenic-1-(13)C acid ((13)C-VA) into milk and endogenous synthesis of cis-9, trans-11 conjugated linoleic acid (CLA) in lactating women. Subjects (n = 4) were 247 +/- 30 d postpartum, weighed 70.8 +/- 3.7 kg, breast-fed at least 6 times/d and consumed self-selected diets. After an overnight fast, they consumed the (13)C-VA (2.5 mg/kg body wt). Milk samples were obtained by complete breast expression at 0, 2, 4, 8, 12, 18, 24, and 48 h post-(13)C-VA ingestion. Lipid was extracted using chloroform:methanol. Fatty acids were methylated and converted to dimethyl disulfide and Diels-Alder derivatives before analysis by gas chromatography mass spectrometry. The mean (13)C-enrichment of milk VA was 3.1% at 8 h and reached maximal enrichment of 7.6% at 18 h. The (13)C enrichment of milk cis-9, trans-11 CLA reached a maximum of 0.4% at 18 h, confirming its conversion of VA to the Delta9-desaturase enzyme product. In the subjects examined, a portion (<10%) of the cis-9, trans-11 CLA present in milk was endogenously synthesized from VA.

The utilization of (13)C-labeled vaccenic acid (VA) by lactating dairy cows to synthesize cis-9, trans-11 conjugated linoleic acid (CLA) was investigated. Primiparous ruminally cannulated Holstein cows (n = 3) were abomasally infused with 1.5 g of VA-1-(13)C. Blood and milk samples were taken frequently before and after VA infusion. Milk and plasma lipid were extracted using chloroform:methanol. Plasma lipid was separated into triacylglycerol (TG), cholesterol ester (CE), phospholipid (PL), nonesterified fatty acid (NEFA), and mono- and diacylglycerol (MDG) fractions. Lipid was methylated, converted to dimethyl disulfide and Diels-Alder adducts, and analyzed by GC-MS. Increased enrichment of (13)C was determined using a 2-sample t test for each sample time compared with -24 h, with significance declared at P < 0.05. Enrichment in milk fat VA was detected at 4 (3.0%), 8 (8.3%), 12 (4.1%), 16 (2.2%), and 20 h (0.8%). Enrichment in VA was also detected in plasma TG, NEFA, PL, and MDG. Enrichment in milk fat cis-9, trans-11 CLA, the Delta9-desaturase product of VA, was detected at 4 (2.6%), 8 (6.6%), 12 (3.4%), 16 (1.7%), and 24 h (0.7%). Enrichment was not detected in cis-9, trans-11 CLA for any plasma lipid fraction. Modeling of the data showed the exponential decay in (13)C enrichment over time for both VA and cis-9, trans-11 CLA in milk fat. Conversion of dietary VA to cis-9, trans-11 CLA endogenously was confirmed with the mammary gland being the primary site of Delta9-desaturase activity; approximately 80% of milk fat cis-9, trans-11 CLA originated from VA.

Trans FA (TFA) have at least one trans double bond and comprise several isomers and types, including many of the CLA (e.g., c9,t11-18:2 CLA). Some TFA may have adverse effects (e.g., cardiovascular disease), whereas some are thought to have beneficial effects (e.g., anticarcinogenicity). The presence of TFA in human tissues and fluids is related to dietary intake, although this relationship is not completely understood--especially in regard to serum lipid fractions. This study was conducted as part of an investigation designed to test the influence of butter (B), "low TFA" margarine (LT), and regular margarine (RM) on milk fat content. Here we tested the secondary hypothesis that consumption of B, LT, and RM by lactating women would result in differential distribution of TFA and CLA in major serum lipid classes. Breast-feeding women (n = 11) participated in this randomized Latin-square study consisting of five periods: intervention I (5 d), washout I (7 d), intervention II (5 d), washout II (7 d), and intervention III (5 d). Extracted serum lipid was separated into cholesterol ester (CE), TAG, and phospholipid (PL) fractions and analyzed for total and isomeric TFA and CLA concentrations. Data indicate that TAG consistently contained the highest concentration of total t-18:1. No interaction between treatment and fraction was found for any of the t-18:1 isomers identified. Absolute concentration of each t-18:1 isomer was greatest during the RM period, regardless of fraction. On a relative basis, concentrations of t10-18:1 and t12-18:1 were most responsive to treatment in the CE fraction. The concentration of c9,t11-18:2 CLA was highest in the TAG fraction and lowest in the PL fraction, regardless of treatment. In summary, these results indicate (i) that there is a differential distribution of some isomeric TFA and CLA among human serum lipid fractions and (ii) that dietary TFA intake influences absolute and relative concentrations of some of the isomers in selected fractions.

Background: trans Fatty acids (FAs) have been identified as negatively affecting human health. The trans FA composition of human milk fat must be examined to establish its influence on the nutritional quality of milk consumed by infants., Objective: We sought to ascertain the individual and total trans FA isomers (sum of FAs containing at least one trans double bond) in human milk and to identify relations between individual FAs and milk fat concentration (% by wt)., Design: The FA composition of milk samples (n = 81) from women living in the southwestern United States was ascertained. The individual 18:1t isomers were separated. Correlations between each FA, total trans FAs, groups of similar FAs, and milk fat concentrations were examined., Results: The mean total trans FA concentration was 7.0 +/- 2.3% (range: 2.5-13.8%). The concentration of total 18:1t was 5.1 +/- 2.0% (range: 1.5-11.6%), and Delta10t (range: Delta9-12t) was the most abundant isomer., Conclusions: Milk fat from women living in the United States contains concentrations of trans FAs similar to those in milk from Canadian women but greater than those reported in milk from women in other countries. In decreasing order of concentration, the Delta10t, Delta11t, Delta9t, and Delta12t isomers represented 78.9% of the total 18:1t. These FAs generally originate from partially hydrogenated vegetable oils and ruminant fat in the diet. No relation was found between the concentration of total trans FAs and milk fat concentration.

Isomers of conjugated linoleic acid (CLA) decreased milk fat, altered immunity, and reduced the risk for cardiovascular disease (CVD) in some animals. The major form of CLA in the human diet is c9,t11-18:2 (rumenic acid; RA). We studied the effects of high RA consumption on plasma and milk RA concentration, milk composition, immunity, and CVD risk factors in lactating women (n = 36) assigned to 1 of 3 treatments: control, low CLA cheese (LCLA; 160 mg RA/d), or high CLA cheese (HCLA; 346 mg RA/d). The increase in plasma RA concentration between baseline and 8 wk in women consuming HCLA cheese was significantly greater than that of controls. At study completion (8 wk), milk RA concentration among women consuming HCLA cheese was greater (P < 0.05) than that of controls (0.37 vs. 0.26% of fatty acids). Treatment did not affect milk fat, protein, or lactose concentrations, immune indices (e.g., plasma T-helper cells and interleukin-2), or measured risk factors for CVD (e.g., plasma triacylglyceride and cholesterol). In summary, consumption of a RA-enriched cheese modestly increased plasma and milk RA concentrations without affecting total milk fat, plasma and milk indices of immunity, or selected risk factors for CVD.

Trans fatty acids (TFA) are found naturally in some foods (e.g., dairy products) as well as many processed foods made with partially hydrogenated vegetable oils (PHVO). Data from a growing literature suggest that some TFA decrease milk fat in lactating animals. Because the physiologic effects of TFA in lactating women are unknown, this study was designed to investigate the effects of TFA consumption on human milk fat. A randomized, crossover design (n = 12) was used to study the effect of 3 dietary treatments: high PHVO (regular margarine), low PHVO (low TFA margarine), or low PHVO but high in naturally occurring TFA (butter) on milk fat. Treatments were administered for 5 d, with 7-d washout periods. Maternal adiposity was estimated by dual-energy X-ray absorptiometry. Milk and blood were collected on d 5 of each intervention period. In general, milk and serum fatty acid concentrations mirrored those of the dietary treatments. There were significant interactions between treatment and maternal adiposity on milk fat and infant milk consumption, as well as on serum glucose and nonesterified fatty acid (NEFA) concentrations. Consumption of regular margarine, compared with low TFA margarine, resulted in lower milk fat in leaner, but not in more obese women. Consumption of either regular or low TFA margarine, compared with butter, elevated serum NEFA concentrations in the more obese women. In summary, consumption of regular margarine, compared with low TFA margarine, decreased milk fat in lean women. Further studies are required to determine whether infant milk consumption might compensate for this potentially important change in milk composition.

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.

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.

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.

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.

Four lactating Holstein cows were subjected to a hyperinsulinemic-euglycemic clamp to evaluate the effects of insulin on circulating concentrations of insulin-like growth factors (IGF) and their binding proteins (IGFBP). Baseline blood samples were taken every 4 h for 2 days. For the 4-day clamp, insulin was infused (1 microgram.kg body wt-1.h-1) into the jugular vein and exogenous glucose was infused to maintain euglycemia. Circulating insulin was increased approximately fivefold, while glucose was maintained within 10% of baseline concentrations by infusion of 0.15 g.kg body wt-1.h-1 glucose. Hyperinsulinemia-euglycemia approximately doubled IGF-I (145 vs. 286 ng/ml, SE = 20) while decreasing circulating IGF-II (285 vs. 180 ng/ml, SE = 32). Densitometry of Western blots demonstrated no change in IGFBP-3 or a 30,000 relative molecular weight (M(r)) band during the clamp. However, IGFBP-2 decreased 73% and a 26,000 M(r) band decreased 58% by the end of the clamp. Therefore, insulin, directly or via secondary changes, increased circulating concentrations of IGF-I while decreasing concentrations of IGF-II, IGFBP-2, and a 26,000 M(r) IGFBP in lactating cows.

Mid-lactation Holstein cows (n = 4) were used to evaluate the effects of complete feed deprivation on circulating insulin-like growth factors (IGF) and their binding proteins (IGFBP). Comparisons were made between cows in fed and unfed conditions and included response to a single injection of bovine somatotropin (bST). Two days of feed deprivation decreased milk yield 66%. Concentrations of bST nearly doubled during feed deprivation, whereas glucose and insulin steadily decreased. Circulating concentrations of IGF-I decreased nearly 50%, IGF-II decreased 28% and IGFBP-2 increased 49% during the 2 d of feed deprivation. In contrast, IGFBP-3 was not affected. Eighteen to 24 h after the bST challenge, circulating concentrations of IGF-I rose 100% during the fed period, but there was no response during the unfed period. Blood concentrations of IGF-II decreased 17% after bST challenge in the fed period but were unchanged in the unfed period. More moderate undernutrition (80% of requirements) did not affect circulating IGFBP-2 but did alter the response of IGFBP-2 to bST. A conceptual model was developed that illustrates that nutritional regulation of the ST/IGF system represents a key component signaling the appropriate use of nutrients. Overall, results demonstrate that nutritional modulation of the ST/IGF system plays a key role in animal performance and well-being over a wide range of physiological situations.

The insulin-like growth factors (IGF) circulate bound to specific high affinity binding proteins (IGFBPs) that modulate physiological responses to both IGF-I and IGF-II. Administration of bovine somatotropin (bST) to lactating cows has been shown to increase total serum levels of IGF-I; however, its effects on IGFBPs are unknown. Therefore, we determined the effects of bST on specific IGFBPs that are present in bovine serum and lymph. The results show that bovine serum contains IGFBPs with mol wt (Mr) estimates of 43,000, 39,000, 34,000, 29,000, and 24,000, as determined by ligand blotting. Using specific antisera, immunoblotting showed that the 43,000 and 39,000 Mr bands were IGFBP-3 and the 34,000 Mr band was IGFBP-2. All five forms of IGFBP were also present in afferent mammary lymph. To determine the effect of bST, four cows were treated with bST (40 mg/day) or vehicle for 12-day periods using a cross-over design. The intensity of the IGFBP-3 band increased 3.3 +/- 0.1-fold (mean +/- SE; P less than 0.01) by day 5 of bST treatment compared to that in controls. The intensity of the IGFBP-2 band decreased 3-fold. Serum levels of IGFBP-2, determined by RIA, decreased from 581 +/- 62 ng/ml during the control period to 156 +/- 52 ng/ml by day 5 of bST treatment (P less than 0.01). IGFBP-2 levels remained low for the entire 12-day treatment interval and rose to control levels within 4 days after cessation of bST. Results of a second study demonstrated that the decrease in IGFBP-2 concentrations in plasma observed during bST treatment (578 +/- 60 vs. 335 +/- 57 ng/ml) was paralleled by a decrease in IGFBP-2 levels in afferent mammary lymph (274 +/- 24 vs. 147 +/- 22 ng/ml). In contrast, the increase in IGFBP-3 levels observed in plasma during bST treatment by ligand blot analysis was not observed in lymph. In summary, bST increased serum levels of IGFBP-3 and decreased serum concentrations of IGFBP-2, while only IGFBP-2 levels were decreased in mammary lymph. Further studies are needed to determine whether these differences reflect differences in transport across capillaries or local production of specific forms of IGFBP.

Mid-lactation Holstein cows (n = 4) were assigned to four dietary sequences in a 4 x 4 Latin square to determine energy and protein effects on somatomedins. Diets were designed so that intakes were either high or low for net energy (NE) or crude protein (CP) with the range being representative for a lactation cycle. Each dietary treatment lasted 16 d and consisted of an adjustment period (d 1 to 7), a basal period (d 8 to 12) and a period of bovine somatotropin (bST) (40 mg/d) administration (d 13 to 16). Blood was obtained via jugular catheters every 4 h on d 11 to 16. Basal milk yield was decreased by NE or CP restriction. Milk yield was increased for cows fed all diets with bST, but response was greatest for those fed the high NE/high CP diet (31%, 7.7 kg/d). Plasma insulin-like growth factor (IGF)-I and IGF-II concentrations were not affected by diet. For all diets, bST caused an increase in plasma IGF-I (125%) and IGF-II (21%), with the increase being substantially greater for cows fed the high NE/high CP diet. Basal insulin levels differed among diets and increased with exogenous bST in cows fed the high NE/high CP diet. Results are consistent with a role of somatomedins in the mechanism by which exogenous bST increases milk yield, and variations in somatomedin response due to nutritional status may explain part of the differences in milk yield response to exogenous bST.