Expression of hepatic genes involved in bile acid metabolism in dairy cows with fatty liver.

Bile acids are cholesterol-derived molecules that are primarily produced in the liver. In nonruminants with fatty liver, overproduction of bile acids is associated with liver injury. During the transition period, fatty liver is a metabolic disorder that can affect up to 50% of high-producing dairy cows. The purpose of this study was to provide a comprehensive evaluation of hepatic bile acid metabolism in dairy cows with fatty liver by assessing the expression changes of genes involved in bile acid synthesis, export, and uptake. The serum activities of aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase and the concentration of total bile acids were all greater, whereas the serum concentration of total cholesterol was lower in cows with fatty liver than in healthy cows. The content of total bile acids was higher, but total cholesterol was slightly lower in liver tissues from fatty liver cows than from healthy cows. The hepatic mRNA abundance of cholesterol 7a-hydroxylase (CYP7A1); hydroxy-delta-5-steroid dehydrogenase, 3 ß- and steroid delta-isomerase 7 (HSD3B7); and sterol 12α-hydroxylase (CYP8B1), enzymes involved in the classic pathway of bile acid synthesis, was higher in fatty liver cows than in healthy cows. Compared with healthy cows, the hepatic mRNA abundance of alternative bile acid synthesis pathway-related genes sterol 27-hydroxylase (CYP27A1) and oxysterol 7α-hydroxylase (CYP7B1) did not differ in cows with fatty liver. The protein and mRNA abundances of bile acid transporter bile salt efflux pump (BSEP) were lower in the liver of dairy cow with fatty liver. Compared with healthy cows, the hepatic mRNA abundance of bile acid transporters solute carrier family 51 subunit α (SLC51A) and ATP binding cassette subfamily C member 1 (ABCC1) and 3 (ABCC3) was greater in cows with fatty liver, whereas the solute carrier family 51 subunit ß (SLC51B) did not differ. The expression of genes involved in bile acid uptake, including solute carrier family 10 member 1 (NTCP), solute carrier organic anion transporter family member 1A2 (SLCO1A2) and 2B1 (SLCO2B1) was upregulated in dairy cows with fatty liver. Furthermore, the hepatic protein and mRNA abundance of bile acid metabolism regulators farnesoid X receptor (FXR) and small heterodimer partner (SHP) were lower in cows with fatty liver than in healthy cows. Overall, these data suggest that inhibition of the FXR signaling pathway may lead to increased bile acid synthesis and uptake and decreased secretion of bile acids from hepatocytes to the bile, which elevates hepatic bile acid content in dairy cows with fatty liver. Because the hepatotoxicity of bile acids has been demonstrated on nonruminant hepatocytes, it is likely that liver injury is induced by increased hepatic bile acid content in dairy cows with fatty liver.

Evaluation of hepatic AMPK, mTORC1, and autophagy-lysosomal pathway in cows with mild or moderate fatty liver.

The aim of the present study was to investigate the activity of AMPK and mTORC1 as well as TFEB transcriptional activity and autophagy-lysosomal function in the liver of dairy cows with mild fatty liver (FL) and cows with moderate FL. Liver and blood samples were collected from healthy dairy cows (n = 10; hepatic triglyceride content <1% wet weight) and cows with mild FL (n = 10; 1% ≤ hepatic triglyceride content < 5% wet weight) or moderate FL (n = 10; 5% ≤ hepatic triglyceride content < 10% wet weight) that had a similar number of lactations (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9). Blood parameters were determined using a Hitachi 3130 autoanalyzer with commercially available kits. Protein and mRNA abundances were determined using western blotting and quantitative real-time PCR, respectively. Activities of calcineurin and ß-N-acetylglucosaminidase were measured with commercial assay kits. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Blood concentrations of glucose were lower in moderate FL cows (3.03 ± 0.21 mM) than in healthy (3.71 ± 0.14 mM) and mild FL cows (3.76 ± 0.14 mM). Blood concentrations of ß-hydroxybutyrate (BHB, 1.37 ± 0.15 mM in mild FL, 1.88 ± 0.17 mM in moderate FL) and free fatty acids (FFA, 0.69 ± 0.05 mM in mild FL, 0.96 ± 0.09 mM in moderate FL) were greater in FL cows than in healthy cows (BHB, 0.76 ± 0.12 mM; FFA, 0.42 ± 0.04 mM). Compared with healthy cows, phosphorylation of AMPK was greater and phosphorylation of its downstream target acetyl-CoA carboxylase 1 was lower in cows with mild and moderate FL. Phosphorylation of mTOR was lower in cows with mild FL compared with healthy cows. In cows with moderate FL, phosphorylation of mTOR and its downstream effectors was greater than in healthy cows and cows with mild FL. The mRNA abundance of TFEB was downregulated in cows with moderate FL compared with healthy cows and mild FL cows. In mild FL cows, the mRNA and protein abundances of TFEB were greater than in healthy cows. Compared with healthy cows, the mRNA abundances of autophagy markers sequestosome-1 and microtubule-associated protein 1 light chain 3-II, and the protein and mRNA abundances of lysosome-associated membrane protein 1 and cathepsin D were increased in mild FL cows but decreased in moderate FL cows. Compared with healthy cows, the mRNA abundance of mucolipin 1 and activities of ß-N-acetylglucosaminidase and calcineurin were higher in cows with mild FL but lower in cows with moderate FL. These data demonstrate that hepatic AMPK signaling pathway, TFEB transcriptional activity, and autophagy-lysosomal function are increased in dairy cows with mild FL; the hepatic mTORC1 signaling pathway is inhibited in mild FL cows but activated in moderate FL cows; and activities of AMPK and TFEB as well as autophagy-lysosomal function are impaired in moderate FL cows.

Nuciferine protects bovine hepatocytes against free fatty acid-induced oxidative damage by activating the transcription factor EB/peroxisome proliferator-activated receptor γ coactivator 1 alpha pathway.

Excessive free fatty acid (FFA) oxidation and related metabolism are the major cause of oxidative stress and liver injury in dairy cows during the early postpartum period. In nonruminants, activation of transcription factor EB (TFEB) can improve cell damage and reduce the overproduction of mitochondrial reactive oxygen species. As a downstream target of TFEB, peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α, gene name PPARGC1A) is a critical regulator of oxidative metabolism. Nuciferine (Nuc), a major bioactive compound isolated from the lotus leaf, has been reported to possess hepatoprotective activity. Therefore, the objective of this study was to investigate whether Nuc could protect bovine hepatocytes from FFA-induced lipotoxicity and the underlying mechanisms. A mixture of FFA was diluted in RPMI-1640 basic medium containing 2% low fatty acid bovine serum albumin to treat hepatocytes. Bovine hepatocytes were isolated from newborn calves and treated with various concentrations of FFA mixture (0, 0.3, 0.6, or 1.2 mM) or Nuc (0, 25, 50, or 100 µM), as well as co-treated with 1.2 mM FFA and different concentrations of Nuc. For the experiments of gene silencing, bovine hepatocytes were transfected with small interfering RNA targeted against TFEB or PPARGC1A for 36 h followed by treatment with 1.2 mM FFA for 12 h in presence or absence of 100 µΜ Nuc. The results revealed that FFA treatment decreased protein abundance of nuclear TFEB, cytosolic TFEB, total (t)-TFEB, lysosome-associated membrane protein 1 (LAMP1) and PGC-1α and mRNA abundance of LAMP1, but increased phosphorylated (p)-TFEB. In addition, FFA treatment increased the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) and decreased the activities of catalase (CAT) and glutathione peroxidase (GSH-Px) in bovine hepatocytes. Moreover, FFA administration enhanced the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactose dehydrogenase (LDH) in the medium of FFA-treated hepatocytes, but reduced the content of urea. In FFA-treated bovine hepatocytes, Nuc administration increased TFEB nuclear localization and the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, decreased the contents of MDA and H2O2 and the protein abundance of p-TFEB, and enhanced the activities of CAT and GSH-Px in a dose-dependent manner. Consistently, Nuc administration reduced the activities of ALT, AST, and LDH and increased the content of urea in the medium of FFA-treated hepatocytes. Importantly, knockdown of TFEB reduced the protein abundance of p-TFEB, t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, and impeded the beneficial effects of Nuc on FFA-induced oxidative damage in bovine hepatocytes. In addition, PPARGC1A silencing did not alter Nuc-induced nuclear translocation of TFEB, increase of the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, or decrease of the protein abundance of p-TFEB, whereas it partially reduced the beneficial effects of Nuc on FFA-caused oxidative injury. Taken together, Nuc exerts protective effects against FFA-induced oxidative damage in bovine hepatocytes through activation of the TFEB/PGC-1α signaling pathway.

Inositol-requiring enzyme 1α and c-Jun N-terminal kinase axis activation contributes to intracellular lipid accumulation in calf hepatocytes.

During the perinatal period, dairy cows undergo negative energy balance, resulting in elevated circulating levels of nonesterified fatty acids (NEFA). Although increased blood NEFA concentrations are a physiological adaptation of early lactation, excessive NEFA in dairy cows is a major cause of fatty liver. Aberrant lipid metabolism leads to hepatic lipid accumulation and subsequently the development of fatty liver. Both inositol-requiring enzyme 1α (IRE1α) and c-Jun N-terminal kinase (JNK) have been validated for their association with hepatic lipid accumulation, including their regulatory functions in calf hepatocyte insulin resistance, oxidative stress, and apoptosis. Meanwhile, both IRE1α and JNK are involved in lipid metabolism in nonruminants. Therefore, the aim of this study was to investigate how IRE1α and JNK regulate lipid metabolism in bovine hepatocytes. An experiment was conducted on randomly selected 10 healthy cows (hepatic triglyceride [TG] content <1%) and 10 cows with fatty liver (hepatic TG content >5%). Liver tissue and blood samples were collected from experimental cows. Serum concentrations of NEFA and ß-hydroxybutyrate (BHB) were greater, whereas serum concentrations of glucose and milk production were lower in cows with fatty liver. The western blot results revealed that dairy cows with fatty liver had higher phosphorylation levels of JNK, c-Jun, and IRE1α in the liver tissue. Three in vitro experiments were conducted using primary calf hepatocytes isolated from 5 healthy calves (body weight: 30-40 kg; 1 d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h, which showed that the phosphorylated levels of JNK, c-Jun, and IRE1α increased in both linear and quadratic effects. In the second experiment, hepatocytes were treated with high concentrations of NEFA (1.2 mM) for 12 h with or without SP600125, a canonical inhibitor of JNK. Western blot results showed that SP600125 treatment could decrease the expression of lipogenesis-associated proteins (PPARγ and SREBP-1c) and increase the expression of fatty acid oxidation (FAO)-associated proteins (CPT1A and PPARα) in NEFA-treated hepatocytes. The perturbed expression of lipogenesis-associated genes (FASN, ACACA, and CD36) and FAO-associated gene ACOX1 were also recovered by JNK inhibition, indicating that JNK reduced excessive NEFA-induced lipogenesis and FAO dysregulation in calf hepatocytes. Third, short hairpin RNA targeting IRE1α (sh-IRE1α) was transfected into calf hepatocytes to silence IRE1α, and KIRA6 was used to inhibit the kinase activity of IRE1α. The blockage of IRE1α could at least partially suppressed NEFA-induced JNK activation. Moreover, the blockage of IRE1α downregulated the expression of lipogenesis genes and upregulated the expression of FAO genes in NEFA-treated hepatocytes. In conclusion, these findings indicate that targeting the IRE1α-JNK axis can reduce NEFA-induced lipid accumulation in bovine hepatocytes by modulating lipogenesis and FAO. This may offer a prospective therapeutic target for fatty liver in dairy cows.

Targeting PHB2-mediated mitophagy alleviates nonesterified fatty acid-induced mitochondrial dysfunction in bovine mammary epithelial cells.

Mitochondrial dysfunction has been reported to occur in the mammary gland of dairy cows suffering from ketosis. Prohibitin 2 (PHB2) plays a crucial role in regulating mitophagy, which clears impaired mitochondria to maintain normal mitochondrial function. Therefore, the current study aimed to investigate how PHB2 mediates mitophagy, thereby influencing mitochondrial function in the immortalized bovine mammary epithelial cell line (MAC-T cells). First, mammary gland tissue and blood samples were collected from healthy cows (n = 15, BHB <0.6 mM) and cows with clinical ketosis (n = 15, BHB >3.0 mM). Compared with healthy cows, cows with clinical ketosis exhibited lower DMI, milk production, milk protein, milk lactose, and serum glucose. In contrast, milk fat, serum nonesterified fatty acids (NEFA) and BHB were greater in cows with clinical ketosis. The protein abundance of PHB2, peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α), mitofusin 2 (MFN2) in whole cell lysates (WCL), as well as PHB2, sequestosome-1 (SQSTM1, also called p62), microtubule-associated protein 1 light chain 3-II (MAP1LC3-II, also called LC3-II), and ubiquitinated proteins in mitochondrial fraction were significantly lower in cows with clinical ketosis. The ATP content of mammary gland tissue in cows with clinical ketosis was lower than that of healthy cows. Second, MAC-T were cultured and treated with NEFA (0, 0.3, 0.6, 1.2 mM). The MAC-T treated with 1.2 mM NEFA displayed decreased protein abundance of PHB2, PGC-1α, and MFN2 in WCL, as well as protein abundance of PHB2, p62, LC3-II, and ubiquitinated proteins in mitochondrial fraction. The content of ATP and JC-1 aggregates in 1.2 mM NEFA group were lower than in the 0 mM NEFA group. Additionally, 1.2 mM NEFA disrupted the fusion between mitochondria and lysosomes. The MAC-T were then pretreated with 100 nM rapamycin, followed by treatment with or without NEFA. Rapamycin alleviated impaired mitophagy and mitochondria dysfunction induced by 1.2 mM NEFA. Third, MAC-T were transfected with small interfering RNA to silence PHB2 or a plasmid for overexpression of PHB2, followed by treatment with or without NEFA. The silencing of PHB2 aggravated 1.2 mM NEFA-induced impaired mitophagy and mitochondrial dysfunction, whereas the overexpression of PHB2 alleviated these effects. Overall, this study provides evidence that PHB2, in regulation of mitophagy, is a mechanism for bovine mammary epithelial cells to counteract NEFA-induced mitochondrial dysfunction.

Thioredoxin-2 suppresses hydrogen peroxide-activated nuclear factor kappa B signaling via alleviating oxidative stress in bovine adipocytes.

During the periparturient period, both oxidative stress, and inflammation of adipose tissue are considered high risk factors for metabolic disorder of dairy cows. Oxidative stress can activate transcription factor nuclear factor kappa B (NF-κB), which lead to the upregulation of genes involved in inflammatory pathways. Thioredoxin-2 (TXN2) is a mitochondrial protein that regulates cellular redox by suppressing mitochondrial reactive oxygen species (ROS) generation in nonruminant, whereas the function of TXN2 in bovine adipocytes was unclear. Thus, the objective of this study was to evaluate how or by which mechanisms TXN2 regulates oxidative stress and NF-κB signaling pathway in bovine adipocytes. Bovine pre-adipocytes isolated from 5 healthy Holstein cows were differentiated and used for (1) treatment with different concentrations of hydrogen peroxide (H2O2; 0, 25, 50, 100, 200, or 400 µM) for 2 h; (2) transfection with or without TXN2 small interfering RNA (si-TXN2) for 48 h and then treated with or without 200 µM H2O2 for 2 h; (3) transfection with scrambled negative control siRNA (si-control) or si-TXN2 for 48 h, and then treatment with or without 10 mM N-acetylcysteine (NAC) for 2 h; (4) transfection with or without TXN2-overexpressing plasmid for 48 h and then treatment with or without 200 µM H2O2 for 2 h. High concentrations of H2O2 (200 and 400 µM) decreased protein and mRNA abundance of TXN2, reduced total antioxidant capacity (T-AOC) and ATP content in adipocytes. Moreover, 200 and 400 µM H2O2 reduced protein abundance of inhibitor of kappa B α (IκBα), increased phosphorylation of NF-κB and upregulated mRNA abundance of tumor necrosis factor-α (TNFA) and interleukin-1B (IL-1B), suggesting that H2O2-induced oxidative stress and activated NF-κB signaling pathway. Silencing of TXN2 increased intracellular ROS content, phosphorylation of NF-κB and mRNA abundance of TNFA and IL-1B, decreased ATP content and protein abundance of IκBα in bovine adipocytes. Knockdown of TXN2 aggravated H2O2-induced oxidative stress and inflammation. In addition, treatment with antioxidant NAC ameliorated oxidative stress and inhibited NF-κB signaling pathway in adipocytes transfected with si-TXN2. In bovine adipocytes treated with H2O2, overexpression of TXN2 reduced the content of ROS and elevated the content of ATP and T-AOC. Overexpression of TXN2 alleviated H2O2-induced inflammatory response in adipocytes, as demonstrated by decreased expression of phosphorylated NF-κB, TNFA, IL-1B, as well as increased expression of IκBα. Furthermore, the protein and mRNA abundance of TXN2 was lower in adipose tissue of dairy cows with clinical ketosis. Overall, our studies contribute to the understanding of the role of TXN2 in adipocyte oxidative stress and inflammatory response.

Caveolin 1 ameliorates nonesterified fatty acid-induced oxidative stress via the autophagy regulator Beclin 1 in bovine mammary gland epithelial cells.

High blood concentrations of nonesterified fatty acids (NEFA) during ketosis enhance uptake by the mammary gland and impair autophagy while causing oxidative stress. Caveolin 1 (CAV1) is closely related to autophagy and plays a role in regulating oxidative stress. The aim of this study was to explore the potential role of CAV1 on oxidative stress and autophagy during a high NEFA challenge in the immortalized bovine mammary epithelial cell line MAC-T. Mammary gland tissue biopsies and blood samples were collected from healthy (n = 15) and clinically ketotic (n = 15) Holstein cows at 3 to 10 (average = 6) days in milk. Compared with healthy cows, ketotic cows had lower dry matter intake (DMI), daily milk yield, serum glucose and greater serum NEFA and BHBA, accompanied by greater milk fat and lower milk protein. Malondialdehyde (MDA) was greater but activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH) were lower in cows with clinical ketosis. A lower protein abundance of CAV1, Beclin 1, autophagy relative gene 5 (ATG5), and microtubule-associated protein 1 light chain 3 (LC3) as well as greater protein abundance of sequestosome-1 (SQSTM1, also called p62) were detected in mammary tissue of cows with clinical ketosis. In vitro, the MAC-T cells were treated with 0, 0.6 and 1.2 mM NEFA for 12 h or treated with 1.2 mM NEFA for various time points (0, 0.5, 1, 2, 4, 8, 12 and 24 h). Compared with 0 mM NEFA, protein abundance of CAV1, Beclin 1, ATG5 and LC3 was greater in the MAC-T challenged with 0.6 mM NEFA, but lower in the 1.2 mM NEFA group. Protein abundance of p62 was lower with 0.6 mM NEFA, but higher with 1.2 mM NEFA. In response to increasing doses of NEFA, mRNA abundance of CAV1, total antioxidant capacity (T-AOC) and SOD activity decreased while the level of reactive oxygen species (ROS) and content of MDA increased. The protein abundance of CAV1, Beclin 1, ATG5 and LC3 peaked at 0.5 h and 1 h, resulting in both linear and quadratic effects. The protein abundance of p62 decreased, reaching a nadir at 4 h in both a linear and quadratic manner. The silencing of CAV1 in MAC-T cells aggravated the 1.2 mM NEFA-induced decrease in Beclin 1 expression, impaired autophagy, and increase in oxidative stress, whereas the overexpression of CAV1 alleviated these effects. Pretreatment of MAC-T cells with Beclin 1 siRNA (si-Beclin 1) and overexpressing CAV1 followed by challenged with 1.2 mM NEFA reversed the CAV1 induced autophagy, thereby enhancing oxidative stress. Overall, these data suggest that CAV1 protects bovine mammary epithelial cells from NEFA-induced oxidative stress through enhancing the expression of Beclin 1 and activating autophagy.

Research Progress on the Role of M6A in Regulating Economic Traits in Livestock.

Reversible regulation of N6-methyladenosine (m6A) methylation of eukaryotic RNA via methyltransferases is an important epigenetic event affecting RNA metabolism. As such, m6A methylation plays crucial roles in regulating animal growth, development, reproduction, and disease progression. Herein, we review the latest research advancements in m6A methylation modifications and discuss regulatory aspects in the context of growth, development, and reproductive traits of livestock. New insights are highlighted and perspectives for the study of m6A methylation modifications in shaping economically important traits are discussed.

Drug Repurposing-Based Brain-Targeting Self-Assembly Nanoplatform Using Enhanced Ferroptosis against Glioblastoma.

Glioblastoma (GBM), the most aggressive and lethal form of malignant brain tumor, is a therapeutic challenge due to the drug filtration capabilities of the blood-brain barrier (BBB). Interestingly, glioblastoma tends to resist apoptosis during chemotherapy, but is susceptible to ferroptosis. Developing therapies that can effectively target glioblastoma by crossing the BBB and evoke ferroptosis are, therefore, crucial for improving treatment outcomes. Herein, a versatile biomimetic nanoplatform, L-D-I/NPs, is designed that self-assembled by loading the antimalarial drug dihydroartemisinin (DHA) and the photosensitizer indocyanine green (ICG) onto lactoferrin (LF). This nanoplatform can selectively target glioblastoma by binding to low-density lipoprotein receptor-related protein-1 (LRP1) and crossing the BBB, thus inducing glioblastoma cell ferroptosis by boosting intracellular reactive oxygen species (ROS) accumulation and iron overload. In addition, L-D-I/NPs have demonstrated the ability to effectively suppress the progression of orthotopic glioblastoma and significantly prolong survival in a mouse glioblastoma model. This nanoplatform has facilitated the application of non-chemotherapeutic drugs in tumor treatment with minimal adverse effects, paving the way for highly efficient ferroptosis-based therapies for glioblastoma.

Continuous-wave terahertz quantum cascade microlaser arrays operating on various bound states in the continuum.

We report on the continuous-wave (CW) operation of 1D terahertz quantum cascade (THz QC) microlaser arrays working on various bound states in the continuum (BICs). We first created a quasi-BIC state by breaking the inversion symmetry of the microlaser array, which enables flexible control of the radiation efficiency. The optimized multi-periods array exhibits single-mode emission with the maximum output power of 21 mW (at 30 K), and the maximum operation temperature (Tcw) of 45 K. To further increase Tcw, we created a hybrid-BIC state by hybridizing a quasi-BIC generated in a few-periods array and a high-Q surface plasmon polariton mode formed in an unbiased array. The hybridization minimizes the pumping area with no obvious degradation of the threshold current density. The reduced pumping area, together with the discrete distribution of microlasers, significantly decreases the device thermal resistance. Such scheme improves the Tcw up to 79 K with a low beam divergence of 17°×17°, and the output power remains 3.4 mW at 20 K. Our work provides a novel solution to control the output power, the operating temperature, and the beam quality of THz QC lasers in CW mode.

Abernethy malformation with unusual cardiac malformation: Case report and literature review.

Abernethy malformation, also known as congenital extrahepatic shunt, is a rare anomaly, which is characterized by partial or complete diversion of the portal blood into the systemic venous circulation. The clinical manifestations of Abernethy malformation during childhood include neonatal cholestasis, failure to thrive, mental retardation, and other congenital defects. We report a case of Abernethy malformation Type II in a 9-year-old boy, whose left ventricle was slightly enlarged because of several major aortopulmonary collateral arteries (MAPCAs) but laboratory examinations were normal 5 years earlier. The characteristics of congenital heart disease in patients with Abernethy malformation are discussed. We propose that physicians should be aware of the possibility of Abernethy malformation in children with enlargement of the left ventricular due to systemic-pulmonary collateral circulation.

Sirtuin 3 mitigates oxidative-stress-induced apoptosis in bovine mammary epithelial cells.

Ketosis is often accompanied by a reduction in milk production in dairy cows, but the molecular mechanism has not been fully elucidated. Ketotic cows possess systemic oxidative stress (OS), which may implicate apoptosis in mammary glands. Sirtuin 3 (SIRT3) is a vital regulator of cellular redox homeostasis and is under the control of AMP-activated protein kinase (AMPK) signaling in nonruminants. Thus, we aimed to investigate (1) the AMPK-SIRT3 and apoptosis status of mammary glands from ketotic cows, (2) the effect of SIRT3 on OS-induced apoptosis in bovine mammary epithelial cells (BMEC), and (3) the role of AMPK signaling on SIRT3-mediated effects on apoptosis. Mammary gland samples were reused from a previous study, which contained healthy and ketotic cows (both n = 15). BMEC were incubated with 0, 0.3, 0.6, or 0.9 mM H2O2 for 6 h with/without a 30 min incubation of an antioxidant MitoQ (1 µM). Then BMEC were incubated with SIRT3 overexpression adenovirus (Ad-SIRT3) for 6 h followed by a 6 h incubation with 0.6 mM H2O2. Finally, BMEC were treated with the AMPK inhibitor Compound C (Cd C,10 µM) for 30 min before the H2O2 challenge, or cells were initially treated with the AMPK agonist MK8722 (10 µM) for 30 min followed by a 30-h culture with/without si-SIRT3 and eventually the H2O2 exposure. Ketotic cows displayed higher levels of Bax, Caspase-3 and Bax/Bcl-2 but lower levels of Bcl-2 in mammary glands. H2O2 incubation displayed similar results, exhibiting a dose-dependent manner between the H2O2 concentration and the apoptosis degree. Mito Q pretreatment reduced cellular reactive oxygen species and rescued cells from apoptosis. Ketotic cows had a lower mammary protein abundance of SIRT3. Similarly, H2O2 incubation downregulated both mRNA and protein levels of SIRT3 in a dose- and time-dependent manner. Ad-SIRT3 infection lowered levels of cellular reactive oxygen species, Bax, Caspase-3 and Bax/Bcl-2 but increased levels of Bcl-2. TUNEL assays confirmed that Ad-SIRT3 infection mitigated H2O2-induced apoptosis. Both ketotic cows and H2O2-induced BMEC had lower levels of p-AMPK and p-AMPK/AMPK. Additionally, Cd C pretreatment decreased SIRT3 and Bcl-2 expression but increased levels of Bax and Caspase-3. Contrary to the inhibitor, MK8722 had opposite effects and reduced the percentage of apoptotic cells. However, these effects of MK8722 were reversed upon SIRT3 silencing. In conclusion, in vivo data confirmed that ketosis is associated with greater apoptosis and restricted AMPK-SIRT3 signaling in mammary glands; in vitro data indicated that SIRT3 mitigates OS-induced apoptosis via AMPK signaling. As such, there may be potential benefits for targeting the AMPK-SIRT3 axis to help counteract the negative effects of mammary glands during ketosis.

Inhibition of cluster antigen 36 protects against fatty acid-induced lipid accumulation, oxidative stress, and inflammation in bovine hepatocytes.

When ketosis occurs, supraphysiological concentrations of nonesterified fatty acids (NEFA) display lipotoxicity and are closely related to the occurrence of hepatic lipid accumulation, oxidative stress, and inflammation, resulting in hepatic damage and exacerbating the progression of ketosis. However, the mechanism of these lipotoxic effects caused by high concentrations of NEFA in ketosis is still unclear. Cluster antigen 36 (CD36), a fatty acid transporter, plays a vital role in the development of hepatic pathological injury in nonruminants. Thus, the aim of this study was to investigate whether CD36 plays a role in NEFA-induced hepatic lipotoxicity in dairy cows with clinical ketosis. Liver tissue and blood samples were collected from healthy (n = 10) and clinically ketotic (n = 10) cows at 3 to 15 d in milk. In addition, hepatocytes isolated from healthy calves were treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 12 h; or infected with CD36 expressing adenovirus or CD36 silencing small interfering RNA for 48 h and then treated with 1.2 mM NEFA for 12 h. Compared with healthy cows, clinically ketotic cows had greater concentrations of serum NEFA and ß-hydroxybutyrate and activities of aspartate aminotransferase and alanine aminotransferase but lower serum glucose. In addition, dairy cows with clinical ketosis displayed excessive hepatic lipid accumulation. More importantly, these alterations were accompanied by an increased abundance of hepatic CD36. In the cell culture model, exogenous NEFA (0, 0.6, 1.2, or 2.4 mM) treatment could dose-dependently increase the abundance of CD36. Meanwhile, NEFA (1.2 mM) increased the content of triacylglycerol, reactive oxygen species and malondialdehyde, and decreased the activities of glutathione peroxidase and superoxide dismutase. Moreover, NEFA upregulated phosphorylation levels of nuclear factor κB (NF-κB) and the inhibitor of NF-κB (IκB) α, along with the upregulation of protein abundance of NLR family pyrin domain containing 3 (NLRP3) and caspase-1, and mRNA abundance of IL1B, IL6, and tumor necrosis factor α (TNFA). These alterations induced by NEFA in bovine hepatocytes were associated with increased lipid accumulation, oxidative stress and inflammation, which could be further aggravated by CD36 overexpression. Conversely, silencing CD36 attenuated these NEFA-induced detriments. Overall, these data suggest that CD36 may be a potential therapeutic target for NEFA-induced hepatic lipid accumulation, oxidative stress, and inflammation in dairy cows.

Liver fibrosis is a common pathological change in the liver of dairy cows with fatty liver.

Fatty liver (i.e., hepatic lipidosis) is a prevalent metabolic disorder in dairy cows during the transition period, characterized by excess hepatic accumulation of triglyceride (TG), tissue dysfunction, and cell death. Detailed pathological changes, particularly hepatic fibrosis, during fatty liver remain to be determined. Liver fibrosis occurs as a consequence of liver damage, resulting from the excessive accumulation of extracellular matrix, which distorts the architecture of the normal liver, compromising its normal synthetic and metabolic functions. Thus, we aimed to investigate liver fibrosis status and its potential causal factors including oxidative stress, hepatocyte apoptosis, and production of inflammatory cytokines in the liver of cows with fatty liver. Forty-five dairy cows (parity, 3-5) were selected, and liver biopsy and blood were collected on the second week postpartum (days in milk, 10-14 d). On the basis of the degree of lipid accumulation in liver, selected cows were categorized into normal (n = 25; TG <1% wet wt), mild fatty liver (n = 15; 1% ≤ TG <5% wet wt), and moderate fatty liver (n = 5; 5% ≤ TG <10% wet wt). Compared with normal cows, blood concentrations of nonesterified fatty acids and ß-hydroxybutyrate, along with alanine aminotransferase and aspartate aminotransferase activities, were greater in the cows with fatty liver (mild and moderate). Hepatic extracellular matrix deposition, as indicated by Picrosirius red staining, was greater in cows with fatty liver than those with normal ones. In addition, we observed an increased proportion of collagen type I fiber in extracellular matrix with increased lipid accumulation in the liver. Compared with normal cows, the area of α-smooth muscle actin (α-SMA)-positive staining along with the mRNA abundance of collagen type I α 1 (COL1A1), ACTA2 (gene encoding α-SMA), and transforming growth factor-ß (TGFB) were greater in cows with fatty liver. Compared with normal cows, hepatic contents of malondialdehyde, glutathione disulfide, and 8-isoprostane were greater, whereas total antioxidant capacity, the hepatic content of glutathione, and activities of antioxidant indicators, including superoxide dismutase, glutathione peroxidase, and catalase, were lower in cows with fatty liver. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells and abundance of apoptosis-related molecules BAX, CASP3, CASP8, and CASP9 were greater in cows with fatty liver. However, mRNA abundance of the anti-apoptotic gene BCL2 did not differ. The mRNA abundance of pro-inflammatory cytokines including tumor necrosis factor-α (TNFA), interleukin-1ß (IL1B), and interleukin-6 (IL6) was greater in the liver of cows with fatty liver. Overall, the present study indicated that fibrosis is a common pathological response to liver damage and is associated with oxidative stress, hepatocyte death, and inflammation.

ß-hydroxybutyrate impairs the directionality of migrating neutrophils through inhibiting the autophagy-dependent degradation of Cdc42 and Rac1 in ketotic cows.

Dairy cows have high incidence of ketosis during perinatal. According to our previous studies, elevated ketone bodies (mainly ß-hydroxybutyrate, BHB) in the peripheral blood are believed to contribute to the impairment of neutrophils mobility and directionality thereby contributing to the immunosuppression and further infectious disease secondary to ketosis. However, the specific effect of BHB on the directionality of bovine neutrophils needs further study and the underlying molecular mechanisms are still unclear. According to the concentration of serum BHB, 40 multiparous cows (within 3 wk postpartum) were selected and divided into the control (n = 20, BHB <0.6 mM) or clinical ketosis (n = 20, BHB >3.0 mM) group. Blood samples were collected for baseline serum characteristics analysis and neutrophil mobility and directionality detection. Platelet activation factor was used as a chemoattractant in cell migration experiments. Our ex-vivo data showed ketotic cows, compared with control cows, were in a negative energy balance state, and their neutrophils had shorter migration distance, lower migration speed, and impaired migration directionality. Neutrophils from control cows were incubated with 3.0 mM BHB for 6 h in vitro. Similarly, BHB stimulation resulted in impaired mobility and directionality of bovine neutrophils. We further specifically studied the underlying molecular mechanism of BHB regulating neutrophil migration directionality in the present study. Cell division control protein 42 homolog (Cdc42) and Ras-related C3 botulinum toxin substrate 1 (Rac1), 2 key markers in the regulation of migration directionality, were found increased after BHB treatment in their total and activated protein levels while decreasing in their transcription level, suggesting that an imbalance of the protein degradation system may be involved. Interestingly, transmission electron microscopy data revealed a decrease in autophagosome number in neutrophils from ketotic cows. Western blotting data showed the accumulation of sequestosome-1 (p62) protein and a decrease in microtubule-associated protein 1 light chain 3-II (LC3-II) protein abundance after BHB treatment, further confirming that the autophagy flux was inhibited in neutrophils from ketotic cows. Additionally, rapamycin (RAPA), a specific autophagy activator, was used with or without BHB treatment in vitro. Accordingly, the BHB-induced impairment of migration directionality but not mobility was relieved by RAPA. Furthermore, as verified by in vivo experiments, compared with the control cows, the protein abundance of total and activated Cdc42 and Rac1 increased and their mRNA abundance decreased in neutrophils from ketotic cows. Overall, the present study revealed that pathological concentration of BHB impairs neutrophil migration directionality through inhibiting the autophagy-mediated degradation of Cdc42 and Rac1. These findings help explain the immunosuppression caused by ketosis.

Low abundance of insulin-induced gene 1 contributes to SREBP-1c processing and hepatic steatosis in dairy cows with severe fatty liver.

Fatty liver is a major metabolic disorder of high-producing dairy cows during the transition period. In nonruminants, it is well established that insulin-induced gene 1 (INSIG1) plays a crucial role in regulating hepatic lipogenesis by controlling the anchoring of sterol regulatory element-binding protein 1 (SREBP-1) on the endoplasmic reticulum along with SREBP cleavage-activating protein (SCAP). Whether the INSIG1-SCAP-SREBP-1c transport axis is affected in cows experiencing fatty liver is unknown. Thus, the aim of this study was to investigate the potential role of INSIG1-SCAP-SREBP-1c axis in the progression of fatty liver in dairy cows. For in vivo experiments, 24 dairy cows at the start of their fourth lactation (median; range 3-5) and 8 d in milk (median; range 4-12 d) were selected into a healthy group [n = 12; triglyceride (TG) content <1%] and a severe fatty liver group (n = 12; TG content >10%) according to their hepatic TG content. Blood samples were collected for detecting serum concentrations of free fatty acids, ß-hydroxybutyrate, and glucose. Compared with healthy cows, cows with severe fatty liver had higher serum concentrations of ß-hydroxybutyrate and free fatty acids and lower concentration of glucose. Liver biopsies were used to detect the status of INSIG1-SCAP-SREBP-1c axis, and the mRNA expression of SREBP-1c-target lipogenic genes acetyl-CoA carboxylase α (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1). Cows with severe fatty liver had lower protein expression of INSIG1 in the hepatocyte endoplasmic reticulum fraction, greater protein expression of SCAP and precursor SREBP-1c in the hepatocyte Golgi fraction, and greater protein expression of mature SREBP-1c in the hepatocyte nuclear fraction. In addition, the mRNA expression of SREBP-1c-target lipogenic genes ACACA, FASN, and DGAT1 was greater in the liver of dairy cows with severe fatty liver. In vitro experiments were conducted on hepatocytes isolated from 5 healthy 1-d-old female Holstein calves, and hepatocytes from each calf were run independently. First, hepatocytes were treated with 0, 200, or 400 µM palmitic acid (PA) for 12 h. Exogenous PA treatment decreased INSIG1 protein abundance, enhanced the endoplasmic reticulum to Golgi export of SCAP-precursor SREBP-1c complex and the nuclear translocation of mature SREBP-1c, all of which was associated with increased transcriptional activation of lipogenic genes and TG synthesis. Second, hepatocytes were transfected with INSIG1-overexpressing adenovirus for 48 h and treated with 400 µM PA 12 h before the end of transfection. Overexpressing INSIG1 inhibited PA-induced SREBP-1c processing, upregulation of lipogenic genes, and TG synthesis in hepatocytes. Overall, the present in vivo and in vitro results indicated that the low abundance of INSIG1 contributed to SREBP-1c processing and hepatic steatosis in dairy cows. Thus, the INSIG1-SCAP-SREBP-1c axis may be a novel target for treatment of fatty liver in dairy cows.

AdipoRon alleviates fatty acid-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes by promoting autophagy.

During the transition period in dairy cows, high circulating concentrations of nonesterified fatty acids (NEFA) increase hepatic lipid deposits and are considered a major pathological factor for liver damage. We investigated whether AdipoRon, a synthetic small-molecule agonist of adiponectin receptors 1 and 2 shown to prevent liver lipid accumulation in nonruminants, could alleviate NEFA-induced lipid accumulation and mitochondrial dysfunction. Bovine hepatocytes were isolated from 5 healthy Holstein female newborn calves (1 d of age, 30-40 kg, fasting), and independently isolated hepatocytes from at least 3 different calves were used for each subsequent experiment. The composition and concentration of NEFA used in this study were selected according to hematological criteria of dairy cows with fatty liver or ketosis. First, hepatocytes were cultured with various concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 12 h. In a second experiment, hepatocytes were treated with AdipoRon at different concentrations (0, 5, 25, or 50 µM for 12 h) and times (25 µM for 0, 6, 12, or 24 h) with or without NEFA (1.2 mM) treatment. In the last experiment, hepatocytes were treated with AdipoRon (25 µM), NEFA (1.2 mM), or both for 12 h after treatment with or without the autophagy inhibitor chloroquine. Hepatocytes treated with NEFA had increased protein abundance of sterol regulatory element-binding protein 1c (SREBP-1c) and mRNA abundance of acetyl-CoA carboxylase 1 (ACACA), and decreased protein abundance of peroxisome proliferator-activated receptor α (PPARA), proliferator-activated receptor gamma coactivator-1 α (PGC-1α), mitofusin 2 (MFN2), cytochrome c oxidase subunit IV (COX IV), and mRNA abundance of carnitine palmitoyltransferase 1A (CPT1A), along with lower ATP concentrations. AdipoRon treatment reversed these effects, suggesting this compound had a positive effect on lipid metabolism and mitochondrial dysfunction during the NEFA challenge. In addition, upregulated expression of microtubule-associated protein 1 light chain 3-II (LC3-II, encoded by MAP1LC3) and downregulated expression of sequestosome-1 (SQSTM1, also called p62) indicated that AdipoRon enhanced autophagic activity in hepatocytes. The fact that chloroquine impeded the beneficial effects of AdipoRon on lipid accumulation and mitochondrial dysfunction suggested a direct role for autophagy during NEFA challenge. Our results suggest that autophagy is an important cellular mechanism to prevent NEFA-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes, which is consistent with other studies. Overall, AdipoRon may represent a promising therapeutic agent to maintain hepatic lipid homeostasis and mitochondrial function in dairy cows during the transition period.

Tumor necrosis factor-α reduces adiponectin production by decreasing transcriptional activity of peroxisome proliferator-activated receptor-γ in calf adipocytes.

Adiponectin (encoded by ADIPOQ) is an adipokine that orchestrates energy homeostasis by modulating glucose and fatty acid metabolism in peripheral tissues. During the periparturient period, dairy cows often develop adipose tissue inflammation and decreased plasma adiponectin levels. Proinflammatory cytokine tumor necrosis factor-α (TNF-α) plays a pivotal role in regulating the endocrine functions of adipocytes, but whether it affects adiponectin production in calf adipocytes remains obscure. Thus, the present study aimed to determine whether TNF-α could affect adiponectin production in calf adipocytes and to identify the underlying mechanism. Adipocytes isolated from Holstein calves were differentiated and used for (1) BODIPY493/503 staining; (2) treatment with 0.1 ng/mL TNF-α for different times (0, 8, 16, 24, or 48 h); (3) transfection with peroxisome proliferator-activated receptor-γ (PPARG) small interfering RNA for 48 h followed by treatment with or without 0.1 ng/mL TNF-α for 24 h; and (4) overexpression of PPARG for 48 h followed by treatment with or without 0.1 ng/mL TNF-α for 24 h. After differentiation, obvious lipid droplets and secretion of adiponectin were observed in adipocytes. Treatment with TNF-α did not alter mRNA abundance of ADIPOQ but reduced the total and high molecular weight (HMW) adiponectin content in the supernatant of adipocytes. Quantification of mRNA abundance of endoplasmic reticulum (ER)/Golgi resident chaperones involved in adiponectin assembly revealed that ER protein 44 (ERP44), ER oxidoreductase 1α (ERO1A), and disulfide bond-forming oxidoreductase A-like protein (GSTK1) were downregulated in TNF-α-treated adipocytes, while 78-kDa glucose-regulated protein and Golgi-localizing γ-adaptin ear homology domain ARF binding protein-1 were unaltered. Moreover, TNF-α diminished nuclear translocation of PPARγ and downregulated mRNA abundance of PPARG and its downstream target gene fatty acid synthase, suggesting that TNF-α suppressed the transcriptional activity of PPARγ. In the absence of TNF-α, overexpression of PPARG enhanced the total and HMW adiponectin content in supernatant and upregulated the mRNA abundance of ADIPOQ, ERP44, ERO1A, and GSTK1 in adipocytes. However, knockdown of PPARG reduced the total and HMW adiponectin content in supernatant and downregulated the mRNA abundance of ADIPOQ, ERP44, ERO1A, and GSTK1 in adipocytes. In the presence of TNF-α, overexpression of PPARG decreased, while knockdown of PPARG further exacerbated TNF-α-induced reductions in total and HMW adiponectin secretion and gene expression of ERP44, ERO1A, and GSTK1. Overall, TNF-α reduces adiponectin assembly in the calf adipocyte, which may be partly mediated by attenuation of PPARγ transcriptional activity. Thus, locally elevated levels of TNF-α in adipose tissue may be one reason for the decrease in circulating adiponectin in periparturient dairy cows.

Histamine promotes adhesion of neutrophils by inhibition of autophagy in dairy cows with subacute ruminal acidosis.

Subacute ruminal acidosis (SARA), a common digestive disease in dairy cows, is accompanied by systemic inflammation and high concentrations of histamine in blood. Histamine-induced neutrophil adhesion may play an important role in the systemic inflammation experienced by cows during SARA. Autophagy, an intracellular degradation system, regulates recycling of membrane-associated integrin and may be involved in histamine-induced adhesion of bovine neutrophils. In the present study, 20 multiparous mid-lactation cows (average body weight 486 ± 24 kg) fitted with ruminal fistula were assigned to a control group (n = 10) or a SARA group (n = 10). We induced SARA by feeding different combinations of wheat-barley pellets and chopped alfalfa hay for 8 wk; SARA was defined as a ruminal pH <5.6 for at least 3 h/d. Blood samples were collected in wk 8. Compared with controls, SARA cows had greater serum concentrations of tumor necrosis factor-α, IL-6, IL-1ß, lipopolysaccharide (LPS)-binding protein, haptoglobin, and serum amyloid A. Serum concentrations of these proinflammatory factors had strong positive correlations with the concentration of serum histamine and LPS. In ex vivo adhesion experiments, the number of adherent neutrophils was greater in the SARA group. Additionally, membrane protein abundance of adhesion molecules such as integrin α-L precursor (CD11a) and integrin α-M precursor (CD11b) was greater in neutrophils of the SARA group, confirming enhanced adhesion ability. Neutrophils of SARA cows had greater number of autophagosomes, greater protein abundance of autophagy substrate sequestosome-1 (p62), and higher ratio of microtubule associated proteins 1A/1B light chain 3 (LC3)-II to LC3-I, indicating congestion during the late phase of autophagy flux. For in vitro experiments, neutrophils isolated from control cows were incubated with 0.4 endotoxin units (EU)/mL LPS or 7 µM histamine for 0, 1, 2, and 4 h, respectively. We detected linear and quadratic effects for the number of adherent neutrophils after histamine treatment with a peak response at 2 h, whereas no significant effect was detected after LPS treatment. Membrane protein abundance of CD11a and CD11b was greater after histamine treatment, suggesting that it may have an inhibitory effect on the degradation of adhesion molecules. The greater abundance of p62, higher ratio of LC3-II to LC3-I, and increased co-localization between CD11b and LC3 after histamine treatment indicated that recycling of adhesion molecules and autophagy flux were blocked. These effects were not aggravated further in the presence of chloroquine, a specific inhibitor of the late phase of autophagy flux. Overall, our results revealed that histamine increases adhesion of neutrophils by inhibiting autophagy in dairy cows with SARA.

ß-Hydroxybutyrate impairs the release of bovine neutrophil extracellular traps through inhibiting phosphoinositide 3-kinase-mediated nicotinamide adenine dinucleotide phosphate oxidase reactive oxygen species production.

Ketosis in dairy cows often occurs in the peripartal period and is accompanied by immune dysfunction. High concentrations of ß-hydroxybutyrate (BHB) in peripheral blood during ketosis inhibits the release of neutrophil extracellular traps (NET) and contributes to immune dysfunction. However, the mechanisms whereby BHB affects NET release remains unclear. In this study, 5 healthy peripartal dairy cows (within 3 wk postpartum) with serum BHB concentrations <0.6 mM and glucose concentrations >3.5 mM were used as blood donors. Blood samples were collected before feeding, and the isolated polymorphonuclear neutrophils were incubated with 3 mM BHB for different times. Inhibition of Cit-H3 (citrullinated histone 3) protein abundance, a marker of NET activation, in response to BHB was used to determine an optimal incubation time for in vitro experiments. Four hours was selected as the optimal duration of BHB treatment. Phorbol-12-myristate-13-acetate (PMA) was used to induce the release of NET in vitro. The BHB treatment with or without PMA treatment decreased protein abundance of Cit-H3 and PAD4 (arginine deiminase 4) and increased neutrophil elastase. Immunofluorescence and scanning electron microscope analyses revealed that BHB treatment inhibited PMA-induced NET release. The BHB treatment also decreased double strain DNA content in the supernatant, further confirming the inhibitory effect of BHB on NET release. Furthermore, BHB treatment decreased the level of intracellular reactive oxygen species (ROS), phosphorylation level of p47, and protein abundance of Rac2, suggesting that BHB-induced NET inhibition may have been caused by decreased NADPH oxidase-derived ROS. The phosphorylation level of phosphoinositide 3-kinase (PI3K), an important upstream regulator of NADPH oxidase, was attenuated by BHB treatment. To confirm the involvement of PI3K signaling pathway in BHB-induced NET inhibition, 740Y-P, a potent activator of PI3K signaling pathway, was used. Data indicated that 740Y-P relieved the inhibitory effects of BHB on ROS production and NADPH oxidase activation. Importantly, as revealed by immunofluorescence and scanning electron microscopy analyses, 740Y-P also dampened the inhibitory effect of BHB on NET release and the protein abundance of Cit-H3 and PAD4. Overall, the present study revealed that high concentration of BHB impairs NET release through inhibiting PI3K-mediated NADPH oxidase ROS production. These findings help partly explain the immune dysfunction in cows experiencing negative energy balance or ketosis in early lactation.