J.S. Liesman

Metabolism of long chain fatty acids by ruminant liver

The primary source of fatty acids processed by ruminant liver is nonesterified fatty acids (NEFA) from blood. Uptake is regulated by concentration of NEFA and blood flow. Blood NEFA concentration increases with negative energy balance. Blood flow increases with energy intake. Uptake and secretion of triacylglycerol between blood and the liver is limited. The reason for limited hepatic secretion of triacylglycerol-rich lipoprotein is unclear but probably involves the secretory process, not synthesis of triacylglycerol or apolipoprotein. Oxidation of fatty acids and ketogenesis are inhibited by malonyl-CoA and propionic acid. The stress of late gestation and early lactation increases NEFA supply to the liver, where they cause deposition of fat. Ketogenesis and oxidation in the liver increase but not sufficiently to prevent an accumulation of fat, which may contribute to decreased feed intake in the peripartum period.

Effect of intensified feeding of heifer calves on growth, pubertal age, calving age, milk yield, and economics

The objective of this study was to determine if increasing the energy and protein intake of heifer calves would affect growth rates, age at puberty, age at calving, and first lactation milk yield. A second objective was to perform an economic analysis of this feeding program using feed costs, number of nonproductive days, and milk yield data. Holstein heifer calves born at the Michigan State Dairy Cattle Teaching and Research Center were randomly assigned to 1 of 2 dietary treatments (n=40/treatment) that continued from 2 d of age until weaning at 42 d of age. The conventional diet consisted of a standard milk replacer [21.5% crude protein (CP), 21.5% fat] fed at 1.2% of body weight (BW) on a dry matter basis and starter grain (19.9% CP) to attain 0.45 kg of daily gain. The intensive diet consisted of a high-protein milk replacer (30.6% CP, 16.1% fat) fed at 2.1% of BW on a dry matter basis and starter grain (24.3% CP) to achieve 0.68 kg of daily gain. Calves were gradually weaned from milk replacer by decreasing the amount offered for 5 and 12 d before weaning for the conventional and intensive diets, respectively. All calves were completely weaned at 42 d of age and kept in hutches to monitor individual starter consumption in the early postweaning period. Starting from 8 wk of age, heifers on both treatments were fed and managed similarly for the duration of the study. Body weight and skeletal measurements were taken weekly until 8 wk of age, and once every 4 wk thereafter until calving. Calves consuming the intensive diet were heavier, taller, and wider at weaning. The difference in withers height and hip width was carried over into the early post-weaning period, but a BW difference was no longer evident by 12 wk of age. Calves fed the intensive diet were younger and lighter at the onset of puberty. Heifers fed the high-energy and protein diet were 15 d younger at conception and 14 d younger at calving than heifers fed the conventional diet. Body weight after calving, daily gain during gestation, withers height at calving, body condition score at calving, calving difficulty score, and calf BW were not different. Energy-corrected, age-uncorrected 305-d milk yield was not different, averaging 9,778 kg and 10,069 kg for heifers fed the conventional and intensive diets, respectively. However, removing genetic variation in milk using parent average values as a covariate resulted in a tendency for greater milk from heifers fed the intensive diet. Preweaning costs were higher for heifers fed the intensive diet. However, total costs measured through first lactation were not different. Intensified feeding of calves can be used to decrease age at first calving without negatively affecting milk yield or economics.

Intramammary infusion of leptin decreases proliferation of mammary epithelial cells in prepubertal heifers

High energy intake and excessive body fatness impair mammogenesis in prepubertal ruminants. High energy intake and excessive fatness also increase serum leptin. Our objective was to determine if an infusion of leptin decreases proliferation of mammary epithelial cells of prepubertal heifers in vivo. Ovine leptin at 100 microg/ quarter per d with or without 10 microg of insulin-like growth factor (IGF)-I was infused via the teat canal into mammary glands of prepubertal dairy heifers; contralateral quarters were used as controls. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered approximately 2 h later. Tissue from 3 regions of the mammary parenchyma was collected and immunostained for bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (Ki-67), and caspase-3. Leptin decreased the number of mammary epithelial cells in the S-phase of the cell cycle by 48% in IGF-I-treated quarters and by 19% in saline-treated quarters. Leptin did not alter the number of mammary epithelial cells within the cell cycle, as indicated by Ki-67 labeling. Caspase-3 immunostaining within the mammary parenchyma was very low in these heifers, but leptin significantly increased labeling in saline-treated quarters. Leptin enhanced SOCS-3 expression in IGF-I-treated quarters but did not alter SOCS-1 or SOCS-5 expression. We conclude that a high concentration of leptin in the bovine mammary gland reduces proliferation of mammary epithelial cells. The reduced proliferation is accompanied by an increase in SOCS-3 expression, suggesting a possible mechanism for leptin inhibition of IGF-I action. Whether leptin might be a physiological regulator of mammogenesis remains to be determined.

Effects of Feeding Prepubertal Heifers a High-Energy Diet for Three, Six, or Twelve Weeks on Mammary Growth and Composition

The experimental objective was to determine the effects of feeding prepubertal dairy heifers a high-energy diet for 3, 6, or 12 wk on mammary growth and composition. Holstein heifers (age = 11 wk; body weight = 107 +/- 1 kg) were assigned to 1 of 4 treatments (n = 16/ treatment). The treatment period lasted 12 wk and treatments were H0 (low-energy diet fed for 12 wk, with no weeks on the high-energy diet); H3 (low-energy diet fed for 9 wk, followed by the high-energy diet for 3 wk); H6 (low-energy diet fed for 6 wk, followed by the high-energy diet for 6 wk); and H12 (high-energy diet for all 12 wk). The low- and high-energy diets were formulated to achieve 0.6 and 1.2 kg of average daily gain, respectively. Heifers were slaughtered at 23 wk of age and mammary tissue was collected. A longer duration of feeding the high-energy diet increased total mass of the mammary gland, extraparenchymal fat, and intraparenchymal fat, but did not alter the mass of fat-free parenchymal tissue. When adjusted for carcass weight to reflect differences in physical maturity, the mass of fat-free parenchymal tissue decreased in a linear fashion with a longer duration on the high-energy diet. Total masses of mammary parenchymal DNA and RNA were not different. However, after adjustment for carcass weight, the masses of DNA and RNA decreased as heifers were fed the high-energy diet for a longer duration. The percentages of epithelium, stroma, and lumen, the number of epithelial structures, and the developmental scores of mammary parenchymal tissue were not different among treatments. However, the percentage of proliferating epithelial cells in the terminal ductal units, as indicated by Ki-67 labeling, decreased as heifers were fed the high-energy diet for a longer duration. We concluded that feeding prepubertal heifers a high-energy diet for a longer duration resulted in a linear decrease in both the percentage of mammary epithelial cells that were proliferating and in the mass of fat-free mammary parenchyma per unit of carcass. High-energy feeding hastens puberty and, in this study, decreased mammary epithelial cell proliferation in areas of active ductal expansion. These data are consistent with the idea that feeding heifers a high-energy diet will reduce mammary parenchymal mass at puberty.

Enteric methane emissions and lactational performance of Holstein cows fed different concentrations of coconut oil

To determine if dietary medium-chain fatty acids (FA; C(8) to C(14)) may mitigate enteric methane emissions, 24 cows were blocked by body size (n=2) and randomly assigned to 1 sequence of dietary treatments. Diets were fed for 35 d each in 2 consecutive periods. Diets differed in concentrations of coconut oil (CNO; ~75% medium-chain FA): 0.0 (control) or 1.3, 2.7, or 3.3% CNO, dry matter basis. The control diet contained 50% forage (74% from corn silage), 16.5% crude protein (60% from rumen-degradable protein), 34% neutral detergent fiber (NDF; 71% from forage), and 28% starch, dry matter basis. Data and sample collections were from d 29 to 35 in environmentally controlled rooms to measure methane (CH(4)) production. Methane emitted was computed from the difference in concentrations of inlet and outlet air and flux as measured 8 times per day. Control cows emitted 464 g of CH(4)/d, consumed 22.9 kg of DM/d, and produced 34.8 kg of solids-corrected milk/d and 1.3 kg of milk fat/d. Treatment with 1.3, 2.7, or 3.3% dietary CNO reduced CH(4) (449, 291, and 253 g/d, respectively), but concomitantly depressed dry matter intake (21.4, 17.9, and 16.2 kg/d, respectively), solids-corrected milk yield (36.3, 28.4, and 26.8 kg/d, respectively), and milk fat yield (1.4, 0.9, and 0.9 kg/d, respectively). The amount of NDF digested in the total tract decreased with increased dietary CNO concentrations; thus, CH(4) emitted per unit of NDF digested rose from 118 to 128, 153, and 166 g/kg across CNO treatments. Dietary CNO did not significantly affect apparent digestibility of CP but increased apparent starch digestibility from 92 to 95%. No FA C(10) or shorter were detected in feces, and apparent digestibility decreased with increasing FA chain length. Coconut oil concentrations of 2.7 or 3.3% decreased yields of milk FA C(14). The highest milk fat concentration (3.69%; 1.3% CNO) was due to the greatest yields of C(12) to C(16) milk FA. Milk FA concentrations of C(18:2 trans-10,cis-12) were related to increased dietary CNO concentrations and presumably to depressed ruminal NDF digestion. Moderate dietary CNO concentrations (e.g., 1.3%) may benefit lactational performance; however, CNO concentrations greater than or equal to 2.7% depressed dry matter intake, milk yield, milk fat yield, and NDF utilization. If mitigation of enteric CH(4) emissions is due to decreased digestion of dietary NDF, then this will lessen a major advantage of ruminants compared with nonruminants in food-production systems. Thus, CNO has limited use for enteric CH(4) mitigation in lactating dairy cows.

Effects of Feeding Prepubertal Heifers a High-Energy Diet for Three, Six, or Twelve Weeks on Feed Intake, Body Growth, and Fat Deposition

The objective was to determine the effects of feeding prepubertal dairy heifers a high-energy diet for a duration of 0, 3, 6, or 12 wk on feed intake, growth, and fat deposition. We also used feed composition, daily intake, and body growth data to evaluate the nutritional model of the 2001 National Research Council (NRC) Nutrient Requirements of Dairy Cattle. Holstein heifers (age = 11 wk; body weight = 107 +/- 1 kg) were assigned to 1 of 4 treatments (n = 16/treatment) designated H0, H3, H6, and H12 and fed a low-energy diet for 12, 9, 6, or 0 wk, followed by a high-energy diet for 0, 3, 6, or 12 wk, respectively. Four heifers were killed initially (11 wk of age) and 64 heifers were killed at the end of the treatment period (23 wk of age). The low-energy diet was formulated to achieve 0.6 kg of average daily gain and contained 16% crude protein, and 45% neutral detergent fiber. The high-energy diet was formulated to achieve an average daily gain of 1.2 kg and contained 18% crude protein and 23% neutral detergent fiber. Actual daily gains averaged over the 12-wk treatment period were 0.64, 0.65, 0.83, and 1.09 kg for the H0, H3, H6, and H12 groups, respectively. Body weight, withers height, hip width, carcass weight, liver weight, and perirenal fat increased in heifers fed a high-energy diet for a longer duration. In addition, percentage of fat increased and percentage of protein decreased in rib sections with a longer duration on the high-energy diet. Uterine and ovarian weights adjusted for body weight decreased when heifers were fed the high-energy diet for a longer duration. The 2001 NRC underestimated dry matter intake of the high-energy diet and overestimated dry matter intake of the low-energy diet. On the basis of actual intakes of each diet, the NRC slightly underestimated gain for the low-energy diet and overestimated gain by 40% for the high-energy diet. The likely explanation for this is that the NRC underestimated the proportion of gain that was fat in the heifers fed the high-energy diet and therefore predicted more body gain per unit of energy intake. We concluded that feeding a high-energy diet for a short duration altered body growth and fat deposition in a time-dependent, linear manner consistent with feeding a high-energy diet for a long duration.

Transcriptome Analysis of Epithelial and Stromal Contributions to Mammogenesis in Three Week Prepartum Cows

Transcriptome analysis of bovine mammary development has provided insight into regulation of mammogenesis. However, previous studies primarily examined expression of epithelial and stromal tissues combined, and consequently did not account for tissue specific contribution to mammary development. Our objective was to identify differences in gene expression in epithelial and intralobular stromal compartments. Tissue was biopsied from non-lactating dairy cows 3 weeks prepartum, cut into explants and incubated for 2 hr with insulin and hydrocortisone. Epithelial and intralobular stromal tissues were isolated with laser capture microdissection. Global gene expression was measured with Bovine Affymetrix GeneChips, and data were preprocessed using RMA method. Moderated t-tests from gene-specific linear model analysis with cell type as a fixed effect showed more than 3,000 genes were differentially expressed between tissues (P<0.05; FDR<0.17). Analysis of epithelial and stromal transcriptomes using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathways Analysis (IPA) showed that epithelial and stromal cells contributed distinct molecular signatures. Epithelial signatures were enriched with gene sets for protein synthesis, metabolism and secretion. Stromal signatures were enriched with genes that encoded molecules important to signaling, extracellular matrix composition and remodeling. Transcriptome differences also showed evidence for paracrine interactions between tissues in stimulation of IGF1 signaling pathway, stromal reaction, angiogenesis, neurogenesis, and immune response. Molecular signatures point to the dynamic role the stroma plays in prepartum mammogenesis and highlight the importance of examining the roles of cell types within the mammary gland when targeting therapies and studying mechanisms that affect milk production.

Protein quality and utilization of timothy, oat-supplemented timothy, and alfalfa at differing harvest maturities in exercised Arabian horses

To evaluate the protein quality and postgut N utilization of full-bloom timothy hay, oat-supplemented timothy-hay diets, and alfalfa hay harvested at different maturities, apparent whole tract N digestibility, urinary N excretion, and serum AA profiles were determined in light to moderately exercised Arabian horses. Six Arabian geldings (16.0 ± 0.3 yr; 467 ± 11 kg of BW) were randomly allocated to a 6 × 6 Latin square design. Diets included full-bloom timothy grass hay (G), G + 0.2% BW oat (G1), G + 0.4% BW oat (G2), mid-bloom alfalfa (A1), early-bloom alfalfa (A2), and early-bud alfalfa hay (A3). Forages were fed at 1.6% of the BW of the horse (as-fed). Each period consisted of an 11-d adaptation period followed by total collection of feces and urine for 3 d. Blood samples were taken on d 11 for analysis of serum AA concentrations. During the 3-d collection period, urine and feces were collected every 8 h and measured and weighed, respectively. Approximately 10% of the total urine volume and fecal weight per period was retained for N analyses. Fecal DM output was less (P < 0.05) in A1, A2, or A3 compared with G, G1, or G2. Apparent whole tract N digestibility was greater (P < 0.01) in A1, A2, and A3 compared with G, G1, or G2, and was greater (P < 0.05) in G1 and G2 compared with G. Nitrogen retention was not different from zero, and there were no differences (P > 0.05) in N retention among diets. Urinary N excretion and total N excretion were greater (P < 0.05) in A1, A2, and A3 compared with G, G1, or G2. Plasma concentrations for the majority of AA increased curvilinearly in response to feeding G, A1, A2, and A3 (quadratic, P < 0.05), with values appearing to maximize 2-h postfeeding. Although alfalfa N digestibility increased with decreasing harvest maturity, N retention did not differ and urinary volume and N excretion increased, indicating that postabsorptive N utilization decreased. In contrast, inclusion of oats at either 0.2 or 0.4% of the BW of the horse to timothy hay markedly enhanced N digestibility without increasing N excretion, indicating improvement in postgut N utilization. These findings indicate that feeding oat-supplemented timothy hay is more environmentally sustainable than feeding alfalfa to the horse at maintenance or under light to moderate exercise.

Mammary lipoprotein lipase in plasma of cows after parturition or prolactin infusion

Plasma lipase activity from the mammary vein and a tail blood vessel was measured in periparturient Holstein cows treated in one of three ways: control, CB154 (2-Br-α-ergocryptin) or CB154 plus prolactin. CB154 administration decreased basal serum prolactin concentration by 80% and blocked the normal parturient increase of serum prolactin. In CB154 plus prolactin-treated cows, prolactin was infused continously for six days starting five and eight days prepartum. Plasma lipase activity was not detectable up to 26 hr prepartum in control and CB154-treated cows or before the start of prolactin infusion in CB154 plus prolactin-treated cows. After two hr prepartum, plasma lipase activity was detected in all treatments. In CB154 plus prolactin-treated cows, plasma lipase activity was detected in the presence of high concentrations of serum progesterone four days after the start of prolactin infusion and at least two days before parturition. Plasma lipase activity was four times greater in the mammary vein than in the tail vessel at sampling times at which activity was detected in both vessels. We propose the difference between plasma lipase activity from the mammary vein and tail vessel is due to release of lipoprotein lipase from the mammary gland into blood, and this activity can be induced prepartum by prolactin or at parturition even if the parturient increase in prolactin is suppressed.

A simple analytical and experimental procedure for selection of reference genes for reverse-transcription quantitative PCR normalization data

Variation in cellular activity in a tissue induces changes in RNA concentration, which affects the validity of gene mRNA abundance analyzed by reverse transcription quantitative PCR (RT-qPCR). A common way of accounting for such variation consists of the use of reference genes for normalization. Programs such as geNorm may be used to select suitable reference genes, although a large set of genes that are not co-regulated must be analyzed to obtain accurate results. The objective of this study was to propose an alternative experimental and analytical protocol to assess the invariance of reference genes in porcine mammary tissue using mammary RNA and DNA concentrations as correction factors. Mammary glands were biopsied from 4 sows on d 110 of gestation (prepartum), on d 5 (early) and 17 (peak) of lactation, and on d 5 after weaning (postweaning). Relative expression of 7 potential reference genes, API5, MRPL39, VAPB, ACTB, GAPDH, RPS23, and MTG1, and one candidate gene, SLC7A1, was quantified by RT-qPCR using a relative standard curve approach. Variation in gene expression levels, measured as cycles to threshold at each stage of mammary physiological activity, was tested using a linear mixed model fitting RNA and DNA concentrations as covariates. Results were compared with those obtained with geNorm analysis, and genes selected by each method were used to normalize SLC7A1. Quantified relative mRNA abundance of GAPDH and MRPL39 remained unchanged across stages of mammary physiological activity after accounting for changes in tissue RNA and DNA concentration. In contrast, geNorm analysis selected MTG1, MRPL39, and VAPB as the best reference genes. However, when target gene SLC7A1 was normalized with genes selected either based on our proposed protocol or by geNorm, fold changes in mRNA abundance did not differ. In conclusion, the proposed analytical protocol assesses expression invariance of potential reference genes by accounting for variation in tissue RNA and DNA concentrations and thus represents an alternative method to select suitable reference genes for RT-qPCR analysis.