Relationship of cow and calf circulating lipidomes with colostrum lipid composition and metabolic status of the cow.

Newborn calves rely on lipids in colostrum for energy and immune function. The lipid concentration in colostrum, however, is highly variable, and little is known about its composition and maternal factors that influence its composition. The first objective was to measure plasma lipid composition of multiparous cows at 35 d before calving (BC; 35 ± 3 d; ± standard deviation) and 7 d BC (7 ± 2 d), their colostrum, and serum lipid composition of calves (24 h after birth) using multiple reaction monitoring profiling, which is an exploratory and highly sensitive lipidomic analysis method that screens lipids based on chemical functionality. Second, data were analyzed to determine if there were relationships between circulating lipids in the cow, colostrum lipids, and calf serum lipids. Third, relationships between markers of metabolic status of the cows and circulating and colostrum lipids were analyzed with correlation analysis. Blood was sampled and plasma prepared from multiparous cows (n = 16) at 35 and 7 d BC. Within 3 h of parturition, colostrum was collected from cows and fed to her calf. Calves received another feeding of colostrum within 12 h after birth and a serum sample was collected from each calf 24 h after the first feeding of colostrum. The metabolic status of cows was evaluated using insulin, glucose, and nonesterified fatty acid area under the curve in response to an intravenous glucose tolerance test performed at 3 wk BC. Lipids were extracted from plasma, colostrum, and calf serum and were analyzed using multiple reaction monitoring profiling. Concentration of lipids were calculated using spiked in standards and expressed as percent of lipids identified. Data were uploaded into MetaboAnalyst 5.0 for multivariate and univariate analysis. Principal component analysis indicated that circulating lipids in the cow and calf were distinct from lipids in colostrum. Phosphatidylglycerol (PG) concentration was greater in colostrum and calf serum than in cow plasma, with 23 of the 24 PG found in colostrum also found in calf serum. In response to intravenous glucose tolerance test in late gestation, nonesterified fatty acid area under the curve was positively related to total triacylglycerols lipids in 7 d BC plasma (r = 0.63) but negatively related to total membrane lipids in colostrum (r = -0.55). Thus, the metabolic status of the dam influences circulating lipids and colostrum lipid content. Moreover, the circulating lipidome of the cow and calf are similar to one another and distinct from the colostrum lipidome, except for PG, where it appears that colostrum serves as the source for PG in the calf's circulation.

Chronic prepartum light-dark phase shifts in cattle disrupt circadian clocks, decrease insulin sensitivity and mammary development, and are associated with lower milk yield through 60 days postpartum.

Circadian and metabolic systems are interlocked and reciprocally regulated. To determine if the circadian system regulates glucose homeostasis and mammary development, the function of the circadian system was disrupted by exposing cattle to chronic light-dark cycle phase shifts from 5 wk before expected calving (BEC) to parturition. Multiparous Holstein cows were exposed to 16 h of light and 8 h of dark (CON, n = 8) or phase shifting (PS, n = 8) the light cycle 6 h every 3 d beginning 35 d BEC. After calving, both treatments were exposed to CON lighting. Mammary biopsies were taken at 21 d BEC and 21 d in milk (DIM), and histological analysis indicated PS treatment decreased the ratio of lumen to alveolar area and percentage of proliferating epithelial cells in the prepartum period. Intravenous glucose tolerance test was performed at 14 d BEC and 7 DIM by administering 50% dextrose. Blood glucose, ß-hydroxybutyrate, insulin, and nonesterified fatty acids were consequently measured over 3 h. At 14 d BEC no treatment differences were observed in baseline glucose or insulin. Treatment had no effect on blood glucose or glucose area under the curve at 14 d BEC and 7 DIM. Insulin area under the curve was higher in PS versus CON at 14 d BEC and 7 DIM. The PS cows produced less milk than CON cows through 60 DIM (40.3 vs. 42.6 kg/d). Exposure to chronic light-dark PS in late gestation decreased mammary development and increased insulin resistance in periparturient cows, which may have caused subsequent lower milk yield.

Global transcriptional differences in myokine and inflammatory genes in muscle of mature steer progeny are related to maternal lactation diet and muscle composition.

Steer progeny suckled by cows fed a dried distillers grains and solubles (DDGS) diet the first 3 mo of lactation were heavier during feedlot finishing and had significantly lower marbling and larger longissimus muscles than steers suckled by cows fed a control diet (CON). These differences were profound in that progeny were managed and fed identically from weaning until finishing, and findings suggest that the suckling period established the developmental program of muscle composition. Here transcriptomes of longissimus muscle were measured by next-generation sequencing to investigate whether there were any developmental clues to the differences in marbling scores and muscle content between steers suckled by DDGS ( n = 5) vs. control (CON; n = 5) diet-fed cows during lactation. There were 809 genes differentially expressed ( P-adj<0.1) between CON and DDGS muscle. Of these 636 were upregulated and 173 downregulated in DDGS relative to CON. Overall the DDGS vs. CON muscle transcriptomic signature was promyogenic and antiadipogenic. In particular, myokines/satellite cell maintenance factors were found among upregulated (LIF, CNTF, FGFB1, EPHB1) genes. The antiadipogenic signature was typified by the upregulation of anti-inflammatory cytokines and receptors (IL1RAP, IL1RL2, IL13RA2, IL1F10), and downregulation of expression of inflammation/inflammatory cytokines and receptor (TNF, IL6R, CXCL9), which suggests a selection of differentiation pathways away from adipogenic line. The upregulation of TGFB, SPP1, and INHBA supports selection of fibroblast lineage of cells. Thus, the lactation phase of production can effect meat quality by affecting transcriptional signatures that favor myogenesis and depress inflammation.

Circadian clocks and their role in lactation competence.

Circadian rhythms are 24 h cycles of behavior, physiology and gene expression that function to synchronize processes across the body and coordinate physiology with the external environment. Circadian clocks are central to maintaining homeostasis and regulating coordinated changes in physiology in response to internal and external cues. Orchestrated changes occur in maternal physiology during the periparturient period to support the growth of the fetus and the energetic and nutritional demands of lactation. Discoveries in our lab made over a decade ago led us to hypothesize that the circadian timing system functions to regulate metabolic and mammary specific changes that occur to support a successful lactation. Findings of studies that ensued are summarized, and point to the importance of circadian clocks in the regulation of lactation competence. Disruption of the circadian timing system can negatively affect mammary gland development and differentiation, alter maternal metabolism and impair milk production.

Changes in sow milk lipidome across lactation occur in fatty acyl residues of triacylglycerol and phosphatidylglycerol lipids, but not in plasma membrane phospholipids.

Milk fats are vital to neonate survival and development, but vary highly by diet, maternal metabolic state and stage of lactation. To gain a better understanding of changes in lipid composition of sow milk across lactation, milk was collected from nine multiparous sows on days 0, 3, 7, and 14, relative to birth and lipids were profiled using multiple reaction monitoring (MRM) profiling. Percent fat was determined by creamatocrit, and found to be different (P < 0.05) between day 0 (12.36 ± 5.90%) and day 3 (16.22 ± 3.65%) but not between day 7 (13.13 ± 2.19%) and day 14 (12.13 ± 2.45%). Fat was extracted from milk (n = 6/day) using the Bligh-Dyer method and profiled using tandem mass spectrometry MRM to determine the abundance of lipids defined by class and fatty acyl residue composition. Lipid species relative concentration was calculated from internal standards, and data analysis was performed using Metaboanalyst 4.0. Concentration of phosphatidyl-choline, -serine, -ethanolamine, -inositol, cholesteryl ester and sphingomyelin did not vary across lactation days, nor did the distribution of associated fatty acyl residues. The total abundance of triacylglycerides (TGs) and phosphatidylglycerols (PGs) increased (P < 0.05) from colostrum (day 0) to transitional (days 3 and 7) and mature milk (day 14). As lactation days increased from day 0 to day 14, the number of carbons and unsaturation within fatty acyl residues decreased (P < 0.05) in both TGs and PGs. The proportion of TGs and PGs increased (P < 0.05) relative to other lipid classes. Changes in composition of milk triglycerides and phosphatidylglycerols likely reflect the metabolic activity of the mammary gland and developmental needs of neonates.

Energetics of caterpillar locomotion: biomechanical constraints of a hydraulic skeleton.

Power input increased linearly with speed and was closely associated with changes in cycle frequency. Minimum cost of transport of gypsy moth caterpillars was 4.5 times as high as predicted for vertebrates and arthropods with jointed framework skeletons. Reduced locomotor economy was associated with stride length only one-third or less than that for animals with solid skeletons.

Caterpillar setae: insulation for an ectotherm.

Gypsy moth caterpillars have long, soft setae distributed along the lateral portions of the body, but only short, stiff setae on the dorsal surface. Setae act as selective insulation for caterpillars by reducing the rates of convective heat exchange without affecting the rates of radiative heat exchange. Changes in posture abolish the effects of the setae by maximizing convection and minimizing radiant heat uptake.

Endothermy during terrestrial activity in large beetles.

The large tropical American beetles Strategus aloeus (Scarabaeidae) and Stenodontes molarium (Cerambycidae) can endogenously maintain metathoracic temperatures 5 degrees to 7 degrees C or more above ambient temperature for many hours. During such periods, their activity is exclusively terrestrial and their oxygen consumption equals that of active mammals of the same size. Before and during flight they elevate metathoracic temperatures by an additional 8 degrees to 10 degrees C.

Peripheral thermoregulation: foot temperature in two Arctic canines.

Arctic foxes and gray wolves maintain their foot temperature just above the tissue freezing point (about -1 degrees C)when standing on extremely cold snow, or when the foot is immersed in a -35 degrees C bath in the laboratory. Proportional thermoregulation stabilized the subcutaneous temperature of the foot pad to a precision of +/- 0.7 degrees C (largest deviations). Selective shunting of blood-borne body heat through a cutaneous vascular plexus in the foot pad accounted for more than 99 percent of measured heat loss from the pad surface. Maximum energetic efficiency is achieved because the unit of heat exchange is located in the pad surface which contacts the cold substrate rather than throughout the pad.

Mammary epithelial cells treated concurrently with TGF-alpha and TGF-beta exhibit enhanced proliferation and death.

Transforming growth factor-alpha (TGF-alpha) stimulates while TGF-beta inhibits mammary epithelial cell growth, suggesting that when cells are treated concurrently with the growth factors their combined effects would result in no net growth. However, combined treatments stimulate proliferation and cellular transformation in several cell lines. The objective of this paper was to describe the effect of long-term (6 days) concurrent TGF-alpha and TGF-beta treatment on normal mammary epithelial cell growth pattern, morphology, and gene expression. Growth curve analysis showed that TGF-alpha enhanced while TGF-beta suppressed growth rate until Day 4, when cells entered lag phase. However, cells treated concurrently with both growth factors exhibited a dichotomous pattern of growth marked by growth and death phases (with no intermittent lag phase). These changes in growth patterns were due to a marked induction of cell death from Day 2 (16.5%) to Day 4 (89.5%), resulting in the transition from growth to death phases, even though the combined treated cultures had significantly more (P < 0.05) cells in S phase on Day 4. TGF-beta stimulated epithelial to mesenchyme transdifferentiation (EMT) in the presence of TGF-alpha, as characterized by increased expression of fibronectin and changes in TGF-beta receptor binding. Expression patterns of genes that regulate the cell cycle showed significant interaction between treatment and days, with TGF-beta overriding TGF-alpha-stimulated effects on gene expression. Overall, the combined treatments were marked by enhanced rates of cellular proliferation, death, and trans-differentiation, behaviors reminiscent of breast tumors, and thus this system may serve as a good model to study breast tumorigenesis.

Hibernation in noncontracting mammalian cardiomyocytes.

BACKGROUND: -The aim of the present study was to establish whether isolated neonatal mammalian cardiomyocytes were capable of downregulating energy-using processes other than contraction while maintaining metabolic stability when oxygen availability was reduced. METHODS AND RESULTS: Metabolic response of cardiomyocytes was investigated under moderate (5 to 6 micromol/L) and severe (2 to 3 micromol/L) forms of hypoxia. Cells exposed to oxygen concentrations of 5 to 6 micromol/L exhibited rates of oxygen consumption, which were decreased to 64% of normoxic rates. Rates of cellular energy usage were decreased because this reduced rate of oxygen consumption was not associated with either decreased intracellular ATP and phosphocreatine concentrations or a compensatory switch to glycolysis. When cells were exposed to oxygen concentrations of 2 to 3 micromol/L, rates of oxygen consumption decreased to 9% of normoxic rates. This decreased rate of oxygen consumption was associated with energetic stress, because a significant switch to glycolysis occurred and intracellular phosphocreatine concentrations were decreased by 40%. Rates of cellular energy usage were further decreased as indicated by stable intracellular ATP concentrations. CONCLUSIONS: -Our results suggest that isolated cardiomyocytes are capable of downregulating energy-consuming processes other than contraction when oxygen supply is decreased. Regions of myocardial tissue are also capable of downregulating metabolic activity during ischemia by shutting down contractile activity (myocardial hibernation). We suggest that metabolic downregulation associated with myocardial hibernation may not be exclusively due to reduced rates of contractile activity. Other energy-using processes (eg, protein synthesis, mRNA synthesis, ion channel activity, and proton leak) may also be shut down.

Monosomy 9p24-->pter and trisomy 5q31-->qter: case report and review of two cases.

Partial deletion of the short arm of chromosome 9 (p24-->pter) and partial duplication of the long arm of chromosome 5 (q32-->qter) were observed in an abnormal boy who died at age 8 weeks of a complex cyanotic cardiac defect. He also had minor anomalies, sagittal craniosynostosis, triphalangeal thumbs, hypospadias, and a bifid scrotum. Two other infants with similar cytogenetic abnormalities were described previously. These patients had severe congenital heart defect, genitourinary anomalies, broad nasal bridge, low hairline, apparently low-set ears, short neck, and triphalangeal thumbs, in common with our patient. We suggest that combined monosomy 9p23,24-->pter and trisomy 5q31,32-->qter may constitute a clinically recognizable syndrome.

Lactation Biology Symposium: circadian clocks as mediators of the homeorhetic response to lactation.

The transition from pregnancy to lactation is the most stressful period in the life of a cow. During this transition, homeorhetic adaptations are coordinated across almost every organ and are marked by changes in hormones and metabolism to accommodate the increased energetic demands of lactation. Recent data from our laboratory showed that changes in circadian clocks occur in multiple tissues during the transition period in rats and indicate that the circadian system coordinates changes in the physiology of the dam needed to support lactation. Circadian rhythms coordinate the timing of physiological processes and synchronize these processes with the environment of the animal. Circadian rhythms are generated by molecular circadian clocks located in the hypothalamus (the master clock) and peripherally in every organ of the body. The master clock receives environmental and physiological cues and, in turn, synchronizes internal physiology by coordinating endocrine rhythms and metabolism through peripheral clocks. The effect of the circadian clock on lactation may be inferred by the photoperiod effect on milk production, which is accompanied by coordinated changes in the endocrine system and metabolic capacity of the dam to respond to changes in day length. We have shown that bovine mammary epithelial cells possess a functional clock that can be synchronized by external stimuli, and the expression of the aryl hydrocarbon receptor nuclear translocator-like gene, a positive limb of the core clock, is responsive to prolactin in bovine mammary explants. Others showed that 7% of genes expressed in breasts of lactating women had circadian patterns of expression, and we report that the diurnal variation of composition of bovine milk is associated with changes in expression of mammary core clock genes. Together these studies indicate that the circadian system coordinates the metabolic and hormonal changes needed to initiate and sustain lactation, and we believe that the capacity of the dam to produce milk and cope with metabolic stresses in early lactation is related to her ability to set circadian rhythms during the transition period.

Flight energetics of sphinx moths: power input during hovering flight.

The energetic cost of hovering flight was measured in sphinx moths from five species. Mean power input per unit mass (Pi/M) varied from 237-2 W kg-1 in Manduca sexta (Subfamily:Sphinginae), mean body mass 1-2 X 10(-3) kg, to 327-9 W kg-1 in Deilephila elpenor (Subfamily: Macroglossinae) mean body mass 7-3 X 10(-4) kg. Mean Pi/M for the five species was inversely proportional to mean body mass and directly proportional to mean wing loading. For any given body mass, Pi/M was greater in Hyles lineata than in M. sexta. This difference is correlated with higher wing loading at any given mass in H. lineata. Energy expenditure per unit mass of thorax was 1018 W kg-1 in H. lineata and 694 W kg-1 in M. sexta. Within each of these species, Pi per unit mass of thorax does not vary with body mass. Power input data are compared with calculated power requirements based on momentum theory and blade-element theory of helicopter aerodynamics. Absolute efficiency, the ratio between calculated power requirements and measured energy expenditure, appears to vary directly with body mass. These data provide an energetic basis for observed correlates between thoracic temperature and flight effort in flying sphinx moths.

Flight energetics in sphinx moths: heat production and heat loss in Hyles lineata during free flight.

1. Mean thoracic temperature of free-flying H. lineata in the field and in the laboratory increased from about 40 degrees C at Ta=16 degrees C to 42-5 degrees C at Ta=32 degrees C. At a given Ta, thoracic temperature was independent of body weight and weakly correlated with wing loading. 2. The difference between abdominal temperature and air temperature increased from 2 degrees C at low Ta to 4-2 degrees C at high Ta. At a given Ta, the difference between Tab and Ta was positively correlated with thoracic temperature. 3. Oxygen consumption per unit weight did not appear to vary with Ta from 15 to 30 degrees C and was inversely proportional to body weight. 4. Thermal conductance of the abdomen (Cab) was greater than thermal conductance of the thorax (Cth) in still air and at wind velocities up to 2-5 m/s. In moving air at speeds approximating flight, Cth was twice as high as in still air. Under the same conditions Cab was 3-4 times as high as in still air. 5. Thoracic and abdominal conductance are inversely proportional to their respective weights. 6. These data are consistent with the hypothesis that thoracic temperature is controlled by regulation of heat loss. However, a heat budget derived from these data suggests that heat dissipation may not be sufficient to offset the decrease in passive cooling of the thorax at high ambient temperatures.

An improved capillary electrophoresis method for measuring tissue metabolites associated with cellular energy state.

An improved method for the measurement of tissue metabolites associated with cellular energetic state by capillary electrophoresis is described. This method allows 17 compounds present in a mixture of standards to be determined simultaneously within 43 min with good reproducibility. ATP, ADP, AMP, UTP, IMP, inosine, hypoxanthine, creatine, phosphocreatine, UDP-galactose, NAD and NADH were detected in samples of either rat heart tissue or rat neonatal cardiomyocytes. This method can detect compounds at concentrations of 5 microm in samples. Recoveries for ATP and phosphocreatine added to cardiomyocyte samples were 99.4 +/- 2.1% and 103.1 +/- 3.3%, respectively (mean +/- SEM, n = 3). Our method has been comprehensively validated and is capable of measuring a wider range of tissue metabolites important in assessing cellular energy status than existing methods.

Involution of mouse mammary glands during whole organ culture occurs via apoptosis of epithelial tissue.

Apoptosis was measured in mammary glands during whole organ culture, to determine whether regression resulting from hormone withdrawal results in epithelial cell death as in vivo involution. Glands were evaluated for morphology and DNA degradation prior to whole organ culture, after lobulo-alveolar development and 2, 4, or 6 days after hormone withdrawal. The data indicated that mammary regression during whole organ culture mimics in vivo involution and results in part from apoptosis of epithelial tissue.

Glucocorticoids maintain the extracellular matrix of differentiated mammary tissue during explant and whole organ culture.

Mouse mammary whole organ culture (WOC) and explant culture of lactating tissue were used to investigate the mechanism by which glucocorticoids maintain secretory epithelium following lobuloalveolar development. The relative number of mammary epithelial cells expressing glucocorticoid receptors did not change with the loss of secretory epithelium during involution as demonstrated with competitive binding assays and immunohistochemistry for the glucocorticoid receptor. Furthermore, glucocorticoids did not inhibit AP-1 binding activity. However, Northern analysis demonstrated that genes associated with the breakdown of the extracellular matrix were not expressed in tissues cultured with glucocorticoids, in contrast to their upregulation during involution of mammary tissue cultured with insulin alone. Tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression was lowest in tissue cultured in the presence of glucocorticoids and increased 2.3-, 3.4-, and 9-fold when tissues were involuted in the presence of insulin (Ins) alone, Ins and hydrocortisone (Hyd) with 0. 005 mg/ml, or 0.01 mg/ml collagenase IV, respectively. These data indicate that glucocorticoids maintain mammary differentiation in part by inhibiting the turnover of basement membrane.

The process of suicide: the sequence of disruptive events in the lives of suicide victims.

Certain disruptions in social relations or loss of social roles tend to precede the act of suicide. In general, this study has attempted to demonstrate the sequential ordering of these social losses and otherdisruptive events. It has been shown that loss of roles irn childhood and early adolescence, parents, home, sibilings, close relatives, and student role tends to be followed by chaotic marriages, loss of occupation role(s), and loss of one's own health either physical or mentalor, in some cases, both. Also parental roles tend to be lost through the death of a child. Take away one's social roles and you take away his humanity (See Palmer, 1970). An individual with nowhere to turn and having lost all, tends to be highly vulnerable to suicide.

The coupling of NAD(P)+-producing reactions to a semiautomated bioluminescent reaction.

Many cellular metabolites can be measured with high sensitivity using bioluminescent techniques. These metabolites are coupled to an appropriate enzyme to produce NAD(P)H, which can then be coupled to the bioluminescent reactions. The sensitivity of bioluminescence cannot be readily applied to methods in which cellular metabolites consume NAD(P)H because of the difficulty in measuring, with sufficient sensitivity, decreases in the concentration of NAD(P)H against a high background NAD(P)H concentration. We have overcome these technical difficulties by developing a bioluminescent reagent to measure the production of NAD(P)+. Assays for creatine/creatine phosphate, pyruvate, and succinate, as well as the kinetic measurement of lactate, are described for a range of biological material. The assays are highly sensitive, quantitative, and reproducible and show no sample-specific inhibition. The range of assays and the diverse biological material tested suggests that NAD(P)+ bioluminescence has a wide potential for application.