Energy Requirements For Lactation Research Articles

Abstract P5-02-01: A FACS-free purification method to study estrogen signaling, organoid formation, and metabolic reprogramming in mammary epithelial cells

Abstract Mammary epithelial cells (MECs) are known to have their metabolism reprogrammed following pregnancy to allow the increased energy requirements for lactation. The estrogen receptor α (ERα) is mainly associated with the regulation of biological pathways linked to mammary gland development but its influence on MECs metabolism is still unknown. Our hypothesis is that ERα reprograms cell metabolism in normal MECs, a phenomenon that would be reprogrammed during breast carcinogenesis. Few in vitro models are used to study MECs, and most of them do not express ERα. Primary MECs can be used to overcome this issue, but methods to purify these cells generally require flow cytometry and fluorescence-activated cell sorting (FACS), which require specialized instruments and expertise. Herein, we present in detail a FACS-free protocol for purification and primary culture of mouse MECs to study ERα metabolic functions using mass spectrometry (MS). Purified MECs from nulliparous mice remain differentiated for up to six days with >85% luminal epithelial cells in two-dimensional culture. When seeded in Matrigel, they form organoids that recapitulate the mammary gland morphology in vivo by developing lumens, contractile cells, and lobular structures. MECs express a functional ERα signaling pathway in both two- and three-dimensional cell culture, as shown at the mRNA and protein levels and by the phenotypic characterization. Extracellular metabolic flux analysis showed that estrogens induce a metabolic switch favouring aerobic glycolysis over mitochondrial respiration in MECs grown in two-dimensions, a phenomenon known as the Warburg effect. We also performed (MS)-based metabolomics in organoids. Estrogens altered the levels of metabolites from various pathways, including aerobic glycolysis, citric acid cycle, urea cycle, and amino acid metabolism, demonstrating that ERα reprograms cell metabolism in mammary organoids. To further understand this reprogramming, stable isotope tracer analysis in primary culture organoids are currently performed. In addition, pregnancy and breast-feeding are known to be protective against breast carcinogenesis. Consequently, we also performed MEC purification and organoid culture using mammary glands from multiparous mice. Intriguingly, organoid phenotypic characterization indicated a difference in organoid structures between MECs from nulliparous and multiparous mice. Furthermore, we observe significant differences in estrogenic response between both conditions, suggesting that pregnancy and/or lactation promotes the establishment of specific epigenetic marks that are preserved even ex vivo. Chromatin immunoprecipitation of specific histone marks and MS-based metabolic studies are ongoing to better understand the different responses of mammary organoids to estrogens between nulliparous and multiparous mice. Overall, we have optimized mouse MEC isolation and purification for two- and three-dimensional cultures and for MS-based metabolomics. We demonstrated that these organoids retain a functional ERα pathway over time and that ERα significantly reprograms multiple metabolic pathways. This model represents a valuable tool to study how estrogens modulate mammary gland biology, and particularly how these hormones reprogram metabolism during lactation and breast carcinogenesis. Citation Format: Aurélie Lacouture, Cynthia Jobin, Alisson Clemenceau, Cindy Weidmann, Line Berthiaume, Dominic Bastien, Isabelle Laverdière, Martin Pelletier, Caroline Diorio, Francine Durocher, Étienne Audet-Walsh. A FACS-free purification method to study estrogen signaling, organoid formation, and metabolic reprogramming in mammary epithelial cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-02-01.

Energy metabolism and partition of lactating Zebu and crossbred Zebu cows in different planes of nutrition.

The aim of this study was to determine the energy metabolism and partition of lactating Gyr and F1 Holstein x Gyr (F1 HxG) cows in different planes of nutrition. Six F1 HxG and six Gyr cows with 130 days in milking (DIM) fed corn silage and concentrate were evaluated. The experiment consisted of four periods with different levels of feeding: 1st ad libitum dry matter intake (DMI) and the others with 5, 10 and 20% restricted DMI, related to the first one. An apparent digestibility assay was performed before measurements in the respiration chamber. Total feces were collected for three days. The cows were confined for 24h in the chamber in each period to determine methane and heat production (HP). F1 HxG had higher gross energy intake (GEI), metabolisable energy intake (MEI) and digestible energy intake (DEI). GE lost in feces was higher in F1 HxG (23.7% GEI) than in Gyr (20.5%) cows. Energy lost as methane and urine was similar between the groups. The metabolisability (q) was 0.67, and the efficiency of converting ME to NE (k) was 0.56. There was no difference in the energy requirements for maintenance between breeds (426.6 MJ/kg BW0,75 average value). The energy requirements for lactation were higher in F1 HxG animals due to the higher volume of milk produced, since there was no difference in energy requirements for production of one kg of milk.

Effect of short term diet restriction on gene expression in the bovine hypothalamus using next generation RNA sequencing technology

BackgroundNegative energy balance (NEB) is an imbalance between energy intake and energy requirements for lactation and body maintenance affecting high-yielding dairy cows and is of considerable economic importance due to its negative impact on fertility and health in dairy herds. It is anticipated that the cow hypothalamus experiences extensive biochemical changes during the early post partum period in an effort to re-establish metabolic homeostasis. However, there is variation in the tolerance to NEB between individual cows. In order to understand the genomic regulation of ovulation in hypothalamic tissue during NEB, mRNA transcriptional patterns between tolerant and sensitive animals were examined. A short term dietary restriction heifer model was developed which induced abrupt onset of anoestrus in some animals (Restricted Anovulatory; RA) while others maintained oestrous cyclicity (Restricted Ovulatory; RO). A third control group (C) received a higher level of normal feeding.ResultsA total of 15,295 genes were expressed in hypothalamic tissue. Between RA and C groups 137 genes were differentially expressed, whereas between RO and C, 32 genes were differentially expressed. Differentially expressed genes were involved in the immune response and cellular motility in RA and RO groups, respectively, compared to C group. The largest difference between groups was observed in the comparison between RA and RO heifers, with 1094 genes shown to be significantly differentially expressed (SDE). Pathway analysis showed that these SDE genes were associated with 6 canonical pathways (P < 0.01), of which neuroactive ligand-receptor interaction was the most significant. Within the comparisons the main over-represented pathway functions were immune response including neuroprotection (CXCL10, Q1KLR3, IFIH1, IL1 and IL8; RA v C and RA v RO); energy homeostasis (AgRP and NPY; RA v RO); cell motility (CADH1, DSP and TSP4; RO v C) and prevention of GnRH release (NTSR1 IL1α, IL1β, NPY and PACA; RA v RO).ConclusionsThis information will assist in understanding the genomic factors regulating the influence of diet restriction on fertility and may assist in optimising nutritional and management systems for the improvement in reproductive performance.

RNA-seq analysis of differential gene expression in liver from lactating dairy cows divergent in negative energy balance

BackgroundThe liver is central to most economically important metabolic processes in cattle. However, the changes in expression of genes that drive these processes remain incompletely characterised. RNA-seq is the new gold standard for whole transcriptome analysis but so far there are no reports of its application to analysis of differential gene expression in cattle liver. We used RNA-seq to study differences in expression profiles of hepatic genes and their associated pathways in individual cattle in either mild negative energy balance (MNEB) or severe negative energy balance (SNEB). NEB is an imbalance between energy intake and energy requirements for lactation and body maintenance. This aberrant metabolic state affects high-yielding dairy cows after calving and is of considerable economic importance because of its negative impact on fertility and health in dairy herds. Analysis of changes in hepatic gene expression in SNEB animals will increase our understanding of NEB and contribute to the development of strategies to circumvent it.ResultsRNA-seq analysis was carried out on total RNA from liver from early post partum Holstein Friesian cows in MNEB (n = 5) and SNEB (n = 6). 12,833 genes were deemed to be expressed (>4 reads per gene per animal), 413 of which were shown to be statistically significantly differentially expressed (SDE) at a false discovery rate (FDR) of 0.1% and 200 of which were SDE (FDR of 0.1%) with a ≥2-fold change between MNEB and SNEB animals. GOseq/KEGG pathway analysis showed that SDE genes with ≥2- fold change were associated (P <0.05) with 9 KEGG pathways. Seven of these pathways were related to fatty acid metabolism and unexpectedly included ‘Steroid hormone biosynthesis’, a process which mainly occurs in the reproductive organs rather than the liver.ConclusionsRNA-seq analysis showed that the major changes at the level of transcription in the liver of SNEB cows were related to fat metabolism. 'Steroid hormone biosynthesis', a process that normally occurs in reproductive tissue, was significantly associated with changes in gene expression in the liver of SNEB cows. Changes in gene expression were found in this pathway that have not been previously been identified in SNEB cows.

Seasonal and Sexual Variation in the Diet and Gastrointestinal Features of the Sika Deer in Western Japan: Implications for the Feeding Strategy

Sexual and seasonal variation in diet quality and the gastrointestine have important implications for forage acquisition and the feeding strategy. We assessed the botanical and chemical compositions of the diet and the gastrointestinal macrostructure of the sika deer (Cervus nippon) in western Japan. The sexual dimorphism-body size hypothesis predicts that smaller females will have a better diet than larger males to meet a higher nutritional demand due to a higher metabolic rate. According to the optimal digestion theory, the gut of larger males will retain a greater quantity of digesta of poor forage to compensate for the reduction in net energy obtained per unit of digesta. The relatively greater omasum content in winter than in summer was the only feature consistent with this prediction. This may suggest that the omasum is the organ that most sensitively reflects diet quality, because the summer diet quality is little better than that in winter. Contrary to predictions, the botanical composition of the diet did not show a sexual difference, but females had greater relative weights of stomach contents, tissues, and hindgut segments (small and large Intestines, and cecum-colon), and a relatively greater small Intestine length than males in summer. Further, females had greater relative weights of abomasum contents, ruminoreticulum tissue, and small Intestine in summer than in winter, but no seasonal variation in gastrointestinal features was detected in males. The sexual variation in diet and in the gastrointestine in summer suggest that lactating females Intake more forage and maintain more digesta in the gut, due to not body size but to higher energy requirements for lactation. The greater gut fill and tissue weight of females in summer than in winter likely resulted from a nutrient demand from lactation greater than that from gestation.

Reproductive Strategies of Female Northern Hairy-Nosed Wombats

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Homeostatic Mechanisms that Regulate Lactation during Energetic Stress ,

Apart from the metabolic differences between species (ruminant vs. nonruminant), there are other important physiologic differences in both the energy requirements for lactation and in the control of milk production between dairy cows and women. Unlike dairy cows, the partitioning of nutrients for lactation in women therefore cannot be generalized to all lactating women but must be related to individual women, taking into account their particular metabolic circumstances. Homeorhetic models may be appropriate for women in developing countries, whereas in developed countries, the flexibility in both homeostatic and homeorhetic adaptations to the substrate demands for milk synthesis means that women can adopt a variety of strategies to support the metabolic demands of lactation. In these women, as in dairy cows, body reserves, dietary intake and milk production vary widely among individuals, and individual differences in capacity for homeorhetic regulation of nutrient partitioning under these conditions require further investigation. J. Nutr. 128: 394S–399S, 1998.

Estimates of energy expenditure and energy consumption of ringed seals (Phoca hispida) throughout the year

We have employed a model for the energy balance of seals to estimate the energy consumption and energy expenditure of ringed seals throughout the year, using biological and physical parameters as input. Data on growth and seasonal changes in body mass and fat content “drives” the seasonal dynamics of the model output. The energy requirements for lactation and activity are based on data from earlier published studies. The analysis suggests that the food intake of ringed seals is highly seasonal. In adult males it is low during the periods of territory defense, mating and moulting from March to June. During this period the seals lose body mass, mainly as fat. The model predicts that lactating females increase their food intake to some extent during the approximately six-week lactation period. After the ice breakup, food intake increases in both sexes, partly as a result of increasing maintenance energy requirements, and partly because the body fat stores are rebuilt in late summer and autumn. The over-all energy requirements of the ringed seal appear to be basically similar to those of terrestrial mammals.

Protein and energy metabolism of lactating Granadina goats

Twelve goats of the Granadina breed in mid- and late lactation were used in two consecutive years to determine their protein and energy requirements for lactation. The animals were individually fed on diets based on pelleted lucerne (Medicago sativa) hay and barley. A total of six balance experiments were carried out. Gas exchange was measured using open-circuit respiration chambers. Milk yield ranged widely from 0.649 to 1.742 kg/d in the first year and from 0.222 to 1.989 kg/d in the second year, a steady decline in milk output being observed as lactation progressed. Milk composition remained rather constant during the midstage of lactation, with an average content (/kg milk) for total solids, total nitrogen, fat and gross energy of 149.7 g, 5.39 g, 58.8 g and 3.59 MJ respectively. Total endogenous N, endogenous urinary N and maintenance requirement for N in lactating goats were estimated to be 244, 218 mg N/kg body-weight (W)0.75 per d and 478 mg total N/kg W0.75 per d respectively from regression equations. A constant efficiency of use of dietary N for milk N plus retained N of 51.0% was found. By regressing milk energy plus apparent body energy retention or loss on metabolizable energy (ME) intake, the maintenance energy requirement was estimated to be 401 kJ ME/kg W0.75 per d. When estimating the corrected milk yield as milk energy + (0.84 x negative energy retentio) + (1.05 x positive energy retention), regression analysis indicated that the overall efficiency of use of ME for lactation was 66.7%. Also, from a plot of apparent body energy retention v. milk energy yield, both expressed as a percentage of ME intake above maintenance, the efficiency with which ME was used to promote energy retention in the body during lactation was calculated to be 0.907 times that for milk secretion.

Energy Requirements for Lactation and Postnatal Growth in Captive Golden-Mantled Ground Squirrels

Pregnant female golden-mantled ground squirrels (Spermophilus saturatus) were captured in the field and maintained in the laboratory at 22° C on a diet of Purina Rodent Chow. ® Three to five young were born (mode 4) in each litter. Growth of young was linear (2.13 g • d⁻¹) for the first 30-35 d, by the end of which the young reached thermal and nutritional independence. From the time of this transition until adult body mass was reached at day 60, the linear growth rate increased more than 2.5-fold (5.67 g • d⁻¹). Pup body mass at birth was independent of litter size (mean 5.97g), but the average mass of young in the smallest litters was significantly greater than that in the largest litters by day 35. Over the course of lactation mothers showed an average 2.1-fold increase in food consumption, with an increase of about 10% in body mass and about 20% in rate of both whole-animal and mass-specific oxygen consumption. Estimated milk production per pup was 115 kJ • d⁻¹ milk energy for litters of three versus ...

Life cycle characteristics of northern Peromyscus maniculatus borealis

Life cycle characteristics of northern Peromyscus maniculatus borealis were examined in the laboratory in order to identify specific adaptations to strongly seasonal environmental conditions. Although northern P. m. borealis are demographically K selected, body weight (19.2 g), gestation time (26.3 days for postpartum litters), litter size (5.0), birth weight (1.87 g), age at weaning (21.4 days), weight at weaning (9.26 g), growth rates (0.35 g/day), and energy requirements for lactation (181% increase attributable to five offspring) are all similar to those of other subspecies of P. maniculatus. General demographic conditions do not appear to have been important in the evolution of the life cycle characteristics of P. maniculatus.