In response to negative energy balance, overconditioned cows mobilize more body fat than thin cows and subsequently are prone to develop metabolic disorders. Changes in adipose tissue (AT) metabolism are barely investigated in overconditioned cows. Therefore, the objective was to investigate the effect of increasing body condition on key regulator proteins of fat metabolism in subcutaneous AT and circulation of dairy cows. Nonlactating, nonpregnant dairy cows (n=8) investigated in the current study served as a model to elucidate the changes in the course of overcondition independent from physiological changes related to gestation, parturition, and lactation. Cows were fed diets with increasing portions of concentrate during the first 6wk of the experiment until 60% were reached, which was maintained for 9wk. Biopsy samples from AT of the subcutaneous tailhead region were collected every 8wk, whereas blood was sampled monthly. Within the experimental period cows had an average BW gain of 243±33.3 kg. Leptin and insulin concentrations were increased until wk 12. Based on serum concentrations of glucose, insulin, and nonesterified fatty acids, the surrogate indices for insulin sensitivity were calculated. High-concentrate feeding led to decreased quantitative insulin sensitivity check index and homeostasis model assessment due to high insulin and glucose concentrations indicating decreased insulin sensitivity. Adiponectin, an adipokine-promoting insulin sensitivity, decreased in subcutaneous AT, but remained unchanged in the circulation. The high-concentrate diet affected key enzymes reflecting AT metabolism such as AMP-activated protein kinase and hormone-sensitive lipase, both represented as the proportion of the phosphorylated protein to total protein, as well as fatty acid synthase. The extent of phosphorylation of AMP-activated protein kinase and the protein expression of fatty acid synthase were inversely regulated throughout the experimental period, whereas the extent of phosphorylation of hormone-sensitive lipase was consistently decreasing by the high-concentrate diet. Overcondition in nonpregnant, nonlactating dairy cows changed the expression of key regulator proteins of AT metabolism and circulation accompanied by impaired insulin sensitivity, which might increase the risk for metabolic disorders.

Energy balance in dairy cows changes during the course of lactation due to alterations in voluntary feed intake and energy required for milk synthesis. To adapt to the demands of lactation, energy metabolism needs to be regulated and coordinated in key organs such as adipose tissue (AT), liver, and mammary gland. Mitochondria are the main sites of energy production in mammalian cells and their number varies depending on age, organ, and physiological condition. The copy number of the mitochondrial genome, the mitochondrial DNA (mtDNA), reflects the abundance of mitochondria within a cell and is regulated by transcriptional and translational factors. Environmental, physiological, and energetic conditions change during lactation and we thus hypothesized that these changes may influence the mtDNA copy number and the abundance of genes regulating mitochondrial biogenesis. Therefore, we aimed to provide an overview of mitochondrial biogenesis in liver, subcutaneous (sc)AT, mammary gland, and peripheral blood cells during early and late lactation in dairy cows. German Holstein cows (n=21) were fed according to their requirements, and biopsies from scAT, liver, mammary gland, and blood were collected in early and late lactation and assayed for relative mtDNA copy numbers and the mRNA abundance of genes regulating mitochondrial biogenesis, such as nuclear-respiratory factor 1 and 2 (NRF-1, NRF-2), mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC-1α). The number of mtDNA copies increased from early to late lactation in all tissues, whereas that in peripheral blood cells was greater in early compared with late lactation. Moreover, mitochondrial activity enzymes (i.e., citrate synthase and cytochrome c oxidase) increased from early to late lactation in scAT. Comparing the number of mtDNA copies between tissues and blood in dairy cows, the highest mtDNA content was observed in liver. The mRNA abundance of genes related to mitochondrial biogenesis changed in a tissue-specific manner when comparing early versus late lactation. The mtDNA copy number was associated with transcriptional factors only in AT, suggesting nontranscriptional regulation of mtDNA in the other tissues. We detected strong correlations between peripheral blood mtDNA and tissue mtDNA content in early lactation. Peripheral blood forms an appropriate medium to display the cellular content of mtDNA copy numbers and consequently the cellular energy status of tissues during early lactation.

With the onset of lactation, dairy cows with a body condition score >3.5 are sensitive to oxidative stress and metabolic disorders. Adipose tissue (AT) can adapt to varying metabolic demands and energy requirements by the plasticity of its size during lactation. In AT, angiogenesis is necessary to guarantee sufficient oxygen and nutrient supply for adipocytes. Cellular energy metabolism is reflected mainly by mitochondria, which can be quantified by the mitochondrial DNA copy number per cell. In the present study, we aimed to investigate the effect of overconditioning on angiogenesis and mitochondrial biogenesis in AT of nonlactating cows, irrespective of the physiological influences of lactation and pregnancy. Eight nonpregnant, nonlactating cows received a ration of increasing energy density for 15 wk, during which body weight and body condition increased substantially. Subcutaneous AT was biopsied every 8 wk, and blood was sampled monthly. The blood concentrations of indicators of oxidative stress increased continuously throughout the experimental period, possibly damaging mitochondrial DNA. Concomitantly, HIF-1α, a major marker for hypoxia, increased until wk 8, indicating insufficient angiogenesis in the rapidly expanding AT. Based on the observation that the number of apoptotic cells decreased with increasing hypoxia, the increasing mitochondrial DNA copy numbers might compensate for the hypoxia, reinforcing the production of oxidative stressors. Key transcription factors of mitochondrial biogenesis were largely unaffected. Thus, increased oxidative stress does not impair mitochondrial DNA.

Telomeres create a protective cap on the ends of chromosomes that shorten with cell division and are influenced by stressful conditions. With the onset of lactation, high-yielding dairy cows are exposed to metabolic stress. In the present study, we aimed to analyze telomere length (TL) in key metabolic organs, such as liver, subcutaneous (sc) adipose tissue (AT), and mammary gland, as well as in peripheral blood cells during early and late lactation in German Holstein cows (n=21). Animals were fed according to their requirement, and biopsies from scAT, liver, and mammary gland as well as blood cells were collected in early and late lactation. The relative quantity of telomere products (qT), which is proportional to the average TL, was determined in genomic DNA by multiplex quantitative PCR. In this study, relative qT varied widely in the investigated tissues and blood. In late lactation, slowly proliferating tissues, such as liver and scAT, had the highest qT, whereas peripheral blood cells and in the mammary gland had the lowest qT. Comparing early with late lactation, relative qT attrition was limited to blood and mammary gland. Relationships between relative qT in blood, mammary gland, scAT, and liver suggest that blood qT might serve as a surrogate marker for tissue-specific qT. Cows with high initial qT in tissues and blood in early lactation had greater qT attrition during the course of lactation than cows with lower qT. The determination of qT could be included when phenotyping dairy cattle to test for associations with performance and fitness traits.

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