Overfed Cows Research Articles

Responses of body fat mobilization to isoproterenol or epinephrine challenge in adult cows: influence of energy level, breed, and body fatness.

The sustainability of livestock production systems facing climatic or economic changes is linked in part to the potential of the female ruminants to adapt to feeding constraints through metabolic and hormonal regulation, notably responses of body fat mobilization, depending on adipose tissue (AT) lipolysis. Our hypothesis was that these responses could change according to genotype (breed) and body fatness. Six fat, nonpregnant, nonlactating Charolais cows, six fat Holstein cows, and six lean Holstein cows were used in a 2 × 2 crossover design with two treatments (underfeeding or overfeeding, at 62% [low] or 128% [high] of maintenance energy requirements [MER], respectively) and two periods. Isoproterenol (ISO, a nonselective β-adrenergic agonist) or epinephrine (EPI, a β- and α2-adrenergic agonist) was injected (6 nmol/kg of lean mass). Blood samples were collected regularly from -20 to 75 min after the injection and then were analyzed for NEFA, glycerol, glucose, and L-lactate. Underfeeding greatly increased (P < 0.001) basal plasma NEFA concentrations (+467%, +264%, and +600% for fat Charolais, fat Holstein, and lean Holstein cows, respectively). For each drug, underfed cows had higher NEFA or glycerol responses to adrenergic challenges than overfed cows. Fat Charolais cows had higher basal plasma NEFA (P < 0.05) concentrations (+64.9%) than fat Holstein cows. The plasma NEFA or glycerol response at 5 min (P < 0.05) was higher for fat Charolais than for fat Holstein cows, whatever the injected drug. Basal plasma lactate concentration and lactate response to ISO or EPI were higher (P < 0.05) for fat Charolais cows than for fat Holstein cows. Fat Holstein cows had higher (P < 0.01) basal glycerol (+18.4%) than lean Holstein cows. This increase could be linked to the increased AT mass. ISO increased more lipolytic responses in fat than in lean Holstein cows, whereas EPI increased more these responses in lean than in fat Holstein cows (drug × fatness interaction), suggesting an increased antilipolytic effect due to α2-AR stimulation in fat cows. Breed had a significant effect on basal and stimulated fat mobilization as well as lactate concentrations, suggesting that the Charolais breed could be more sensitive to stress.

Supplemental Smartamine M in higher-energy diets during the prepartal period improves hepatic biomarkers of health and oxidative status in Holstein cows

BackgroundFeeding higher-energy prepartum is a common practice in the dairy industry. However, recent data underscore how it could reduce performance, deepen negative energy balance, and augment inflammation and oxidative stress in fresh cows. We tested the effectiveness of rumen-protected methionine in preventing the negative effect of feeding a higher-energy prepartum. Multiparous Holstein cows were fed a control lower-energy diet (CON, 1.24 Mcal/kg DM; high-straw) during the whole dry period (~50 d), or were switched to a higher-energy (OVE, 1.54 Mcal/kg DM), or OVE plus Smartamine M (OVE + SM; Adisseo NA) during the last 21 d before calving. Afterwards cows received the same lactation diet (1.75 Mcal/kg DM). Smartamine M was top-dressed on the OVE diet (0.07% of DM) from -21 through 30 d in milk (DIM). Liver samples were obtained via percutaneous biopsy at -10, 7 and 21 DIM. Expression of genes associated with energy and lipid metabolism, hepatokines, methionine cycle, antioxidant capacity and inflammation was measured.ResultsPostpartal dry matter intake, milk yield, and energy-corrected milk were higher in CON and OVE + SM compared with OVE. Furthermore, milk protein and fat percentages were greater in OVE + SM compared with CON and OVE. Expression of the gluconeogenic gene PCK1 and the lipid-metabolism transcription regulator PPARA was again greater with CON and OVE + SM compared with OVE. Expression of the lipoprotein synthesis enzyme MTTP was lower in OVE + SM than CON or OVE. Similarly, the hepatokine FGF21, which correlates with severity of negative energy balance, was increased postpartum only in OVE compared to the other two groups. These results indicate greater liver metabolism and functions to support a greater production in OVE + SM. At 7 DIM, the enzyme GSR involved in the synthesis of glutathione tended to be upregulated in OVE than CON-fed cows, suggesting a greater antioxidant demand in overfed cows. Feeding OVE + SM resulted in lower similar expression of GSR compared with CON. Expression of the methionine cycle enzymes SAHH and MTR, both of which help synthesize methionine endogenously, was greater prepartum in OVE + SM compared with both CON and OVE, and at 7 DIM for CON and OVE + SM compared with OVE, suggesting greater Met availability. It is noteworthy that DNMT3A, which utilizes S-adenosylmethionine generated in the methionine cycle, was greater in OVE and OVE + SM indicating higher-energy diets might enhance DNA methylation, thus, Met utilization.ConclusionsData indicate that supplemental Smartamine M was able to compensate for the negative effect of prepartal energy-overfeeding by alleviating the demand for intracellular antioxidants, thus, contributing to the increase in production. Moreover Smartamine M improved hepatic lipid and glucose metabolism, leading to greater liver function and better overall health.

Far-off and close-up dry matter intake modulate indicators of immunometabolic adaptations to lactation in subcutaneous adipose tissue of pasture-based transition dairy cows

The common practice of increasing dietary energy density during the close-up dry period (last ∼3 wk prepartum) has been recently associated with a higher incidence of metabolic disorders after calving. Despite these reports, over-feeding of metabolizable energy (ME) during the far-off, nonlactating period is a common management policy aimed at achieving optimum calving body condition score (BCS) in pasture-based systems, as cows are generally thinner than total mixed ration cows at the end of lactation. Our hypothesis was that both far-off and close-up overfeeding influence the peripartum adipose tissue changes associated with energy balance and inflammatory state. Sixty mid-lactation, grazing dairy cows of mixed age and breed were randomly allocated to 1 of 2 groups that were managed through late lactation to achieve a low and high BCS (approximately 4.25 and 5.0 on a 10-point scale) at dry-off. The low BCS cows were then overfed ME to ensure that they achieved the same BCS as the higher BCS group by calving. Within each rate of BCS gain treatment, cows were offered 65, 90, or 120% of their pre-calving ME requirements for 3 wk pre-calving in a 2 × 3 factorial arrangement of treatments (i.e., 10 cows/treatment). Subcutaneous adipose tissue was collected via biopsy at -1, 1, and 4 wk relative to parturition. Quantitative PCR was used to measure mRNA and microRNA expression of targets related to adipogenesis and inflammation. Cows overfed in the far-off period had increased expression of miR-143 and miR-378 prepartum (-1 wk) indicating greater adipogenesis, consistent with their rapid gain in BCS following dry-off. Furthermore, the lower postpartum expression of IL6, TNF, TLR4, TLR9, and miR-145, and a higher abundance of miR-99a indicated lower body fat mobilization in early lactation in the same group. In the close-up period, feeding either 65 or 120% of ME requirements caused changes in FASN, IL1B, IL6R, TLR9, and the microRNA miR-143, miR-155, and miR-378. Their respective expression patterns indicate a tentative negative-feedback mechanism in metabolically compromised, feed-restricted cows, and a possible immune-related stimulation of lipolysis in apparently static adipocytes in overfed cows. Data from cows fed 90% of ME requirements indicate the existence of a balance between lipolytic (inflammatory-related) and anti-lipolytic signals, to prime the mobilization machinery in light of imminent lactation. Overall, results indicate that far-off dry cow nutrition influences peripartum adipose tissue metabolism, with neither strategy negatively affecting the physiological adaptation to lactation. Furthermore, to ensure a favorable transition, cows should be subjected to a small feed restriction in the close-up period, irrespective of far-off nutritional management.

Prepartal dietary energy alters transcriptional adaptations of the liver and subcutaneous adipose tissue of dairy cows during the transition period

Overfeeding during the dry period may predispose cows to increased insulin resistance (IR) with enhanced postpartum lipolysis. We studied gene expression in the liver and subcutaneous adipose tissue (SAT) of 16 Finnish Ayrshire dairy cows fed either a controlled energy diet [Con, 99 MJ/day metabolizable energy (ME)] during the last 6 wk of the dry period or high-energy diet (High, 141 MJ/day ME) for the first 3 wk and then gradually decreasing energy allowance during 3 wk to 99 MJ/day ME before the expected parturition. Tissue biopsies were collected at -10, 1, and 9 days, and blood samples at -10, 1, and 7 days relative to parturition. Overfed cows had greater dry matter, crude protein, and ME intakes and ME balance before parturition. Daily milk yield, live weight, and body condition score were not different between treatments. The High cows tended to have greater plasma insulin and lower glucagon/insulin ratio compared with Con cows. No differences in circulating glucose, glucagon, nonesterified fatty acids and β-hydroxybutyrate concentrations, and hepatic triglyceride contents were observed between treatments. Overfeeding compared with Con resulted in lower CPT1A and PCK1 and a tendency for lower G6PC and PC expression in the liver. The High group tended to have lower RETN expression in SAT than Con. No other effects of overfeeding on the expression of genes related to IR in SAT were observed. In conclusion, overfeeding energy prepartum may have compromised hepatic gluconeogenic capacity and slightly affected IR in SAT based on gene expression.

Overfeeding Dairy Cattle During Late-Pregnancy Alters Hepatic PPARα-Regulated Pathways Including Hepatokines: Impact on Metabolism and Peripheral Insulin Sensitivity.

Hepatic metabolic gene networks were studied in dairy cattle fed control (CON, 1.34 Mcal/kg) or higher energy (overfed (OVE), 1.62 Mcal/kg) diets during the last 45 days of pregnancy. A total of 57 target genes encompassing PPARα-targets/co-regulators, hepatokines, growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis, lipogenesis, and lipoprotein metabolism were evaluated on −14, 7, 14, and 30 days around parturition. OVE versus CON cows were in more negative energy balance (NEB) postpartum and had greater serum non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHBA), and liver triacylglycerol (TAG) concentrations. Milk synthesis rate did not differ. Liver from OVE cows responded to postpartal NEB by up-regulating expression of PPARα-targets in the fatty acid oxidation and ketogenesis pathways, along with gluconeogenic genes. Hepatokines (fibroblast growth factor 21 (FGF21), angiopoietin-like 4 (ANGPTL4)) and apolipoprotein A-V (APOA5) were up-regulated postpartum to a greater extent in OVE than CON. OVE led to greater blood insulin prepartum, lower NEFA:insulin, and greater lipogenic gene expression suggesting insulin sensitivity was not impaired. A lack of change in APOB, MTTP, and PNPLA3 coupled with upregulation of PLIN2 postpartum in cows fed OVE contributed to TAG accumulation. Postpartal responses in NEFA and FGF21 with OVE support a role of this hepatokine in diminishing adipose insulin sensitivity.

Blood immunometabolic indices and polymorphonuclear neutrophil function in peripartum dairy cows are altered by level of dietary energy prepartum

Cows experience some degree of negative energy balance and immunosuppression around parturition, making them vulnerable to metabolic and infectious diseases. The effect of prepartum feeding of diets to meet (control, 1.34Mcal/kg of dry matter) or exceed (overfed, 1.62Mcal/kg of dry matter) dietary energy requirements was evaluated during the entire dry period (∼45 d) on blood polymorphonuclear neutrophil function, blood metabolic and inflammatory indices, and milk production in Holstein cows. By design, dry matter intake in the overfed group (n=9) exceeded energy requirements during the prepartum period (−4 to −1 wk relative to parturition), resulting in greater energy balance when compared with the control group (n=10). Overfed cows were in more negative energy balance during wk 1 after calving than controls. No differences were observed in dry matter intake, milk yield, and milk composition between diets. Although nonesterified fatty acid concentration pre- (0.138mEq/L) and postpartum (0.421mEq/L) was not different between diets, blood insulin concentration was greater in overfed cows prepartum (16.7μIU/mL) compared with controls pre- and postpartum (∼3.25μIU/mL). Among metabolic indicators, concentrations of urea (4.63 vs. 6.38mmol/L), creatinine (100 vs. 118μmol/L), and triacylglycerol (4.0 vs. 8.57mg/dL) in overfed cows were lower prepartum than controls. Glucose was greater pre- (4.24 vs. 4.00mmol/L) and postpartum (3.49 vs. 3.30mmol/L) compared with control cows. Among liver function indicators, the concentration of bilirubin increased by 2 to 6 fold postpartum in control and overfed cows. Phagocytosis capacity of polymorphonuclear neutrophils was lower prepartum in overfed cows (32.7% vs. 46.5%); phagocytosis in the control group remained constant postpartum (50%) but it increased at d 7 in the overfed group to levels similar to controls (48.4%). Regardless of prepartum diet, parturition was characterized by an increase in nonesterified fatty acid and liver triacylglycerol, as well as blood indices of inflammation (ceruloplasmin and haptoglobin), oxidative stress (reactive oxygen metabolites), and liver injury (glutamic oxaloacetic transaminase). Concentrations of the antioxidant and anti-inflammatory compounds vitamin A, vitamin E, and β-carotene decreased after calving. For vitamin A, the decrease was observed in overfed cows (47.3 vs. 27.5μg/100mL). Overall, overfeeding energy and higher energy status prepartum led to the surge of insulin and had a transient effect on metabolism postpartum.

The effect of milk production level on host resistance of dairy cows, as assessed by the severity of experimental Escherichia coli mastitis.

This study investigated the possible effects of milk production level on the host resistance of dairy cows. High (n = 18) and low (n = 18) producing cows on a research farm, which respectively produced 11 443 and 7 727 kg milk in their previous lactation, were compared. To enhance the possible differences in host resistance between high and low producing cows, the animals in both groups were metabolically stressed by overfeeding during the dry period or were fed according to requirements, resulting in four groups of nine cows. The metabolic status was monitored from two weeks pre-partum until 2.5-4.5 weeks post-partum. Host resistance was assessed by measuring the severity of experimentally induced Escherichia coli mastitis. Pre-partum blood glucose levels tended to be higher in overfed cows than in cows fed according to requirements. The post-partum energy balance was significantly more negative in high producing cows than in low producers, and tended to be more negative in overfed cows compared to cows fed according to the requirements. Post-partum plasma glucose, NEFA, beta-OH-butyrate and urea concentrations were similar in the four groups. Plasma glucose concentrations were significantly lower and liver triacylglycerol concentrations were significantly higher in third than in second parity cows. Host resistance was not affected by the production level or feeding regimen. There were no significant correlations between the metabolic status and the severity of experimental E. coli mastitis, except for the relatively more severe mastitis in the cows with beta-OH-butyrate concentrations above 1.4 mmol/L. In conclusion, milk production level did not affect host resistance in dairy cows, as measured by the severity of experimental E. coli mastitis. Even in a situation where cows were metabolically stressed by overfeeding, high producers were as able as low producers to cope with the demands of milk production, without consequences for host resistance.

Interrelation of Fatty Acid Composition in Adipose Tissue, Serum, and Liver of Dairy Cows During the Development of Fatty Liver Postpartum

We investigated the composition of fatty acids in adipose tissue, serum, and liver of cows that were fed at restricted energy intake or were overfed during the dry period. Overfed cows had higher concentrations of serum nonesterified fatty acids and consequently accumulated greater amounts of triacylglycerols in the liver than did cows that were fed at restricted energy intake. The percentages of the different fatty acids present in adipose tissue were similar for both groups and did not change during sampling intervals. Before parturition, concentrations of the individual fatty acids present in serum were similar between groups. After parturition, concentrations of major fatty acids in serum, including palmitic, stearic, oleic, and linoleic acids significantly increased in both groups and were higher in overfed cows than in cows that were fed at restricted energy intake. The shift of concentrations of the different fatty acids present in the liver—as a result of increased lipolysis—was observed in palmitic, oleic, and linoleic acids but not stearic acid, suggesting that stearic acid is used by the liver (i.e., oxidation) or is considerably secreted through the milk, thereby not increasing in accumulation in the liver. In conclusion, different feeding regimens during the dry period do not influence the composition of fatty acids in adipose tissue. More intensive lipolysis results in increased concentrations of palmitic, stearic, oleic, and linoleic acids in the blood; subsequently, these fatty acids, excluding stearic acid, greatly accumulated in the liver.

Effect of Overfeeding During the Dry Period on the Rate of Esterification in Adipose Tissue of Dairy Cows During the Periparturient Period

The in vitro rate of esterification of fatty acids in adipose tissue was compared between cows that were fed at restricted energy intake and cows that were overfed during the dry period. Subcutaneous adipose tissue was biopsied at -1, 0.5, 1, and 3 wk from parturition. The basal in vitro rate of esterification was quantified, as well as the rate of esterification after the addition of glucose or glucose plus insulin. The basal rate in adipose tissue from overfed cows at -1 wk was higher than in adipose tissue from cows that were fed at restricted energy intake and indicated enhanced storage of triacylglycerols in adipose tissue of overfed cows at that time. The rate of esterification after the addition of glucose or glucose plus insulin was increased in both groups at each sampling time, but the mean rates, expressed as a percentage of the basal rates, were lower for overfed cows than for cows that were fed at restricted energy intake at 0.5 and 1 wk. Although the addition of glucose or glucose plus insulin increased esterification rates in adipose tissue from both groups of cows, adipose tissue from overfed cows was less sensitive to the addition of these compounds. In conclusion, overfeeding during the dry period predisposed cows to accumulate fat in adipose tissue during the prepartum period. The smaller increase in esterification rate after the addition of glucose or glucose plus insulin in adipose tissue of overfed cows indicates a lower ability of the adipose tissue to esterify circulating fatty acids or to reesterify mobilized fatty acids, which, combined with higher rates of lipolysis postpartum, contributes to continuously elevated concentrations of circulating nonesterified fatty acids postpartum, leading to a more severe hepatic lipidosis in overfed cows.

Effects of the infusion of non-selective beta-, and selective beta1- or beta2-adrenergic agonists, on body fat mobilisation in underfed or overfed non-pregnant heifers.

The aim of this study was to analyse the effects of the intravenous infusion of a non-selective beta-agonist (beta-A) (isoproterenol, ISO), and selective beta1-A (dobutamine, D) or beta2-A (terbutaline, T) on body fat mobilisation in non-pregnant heifers, in a 2 x 2 crossover design with two treatments (underfeeding or overfeeding) and two periods. The effect of the duration of submission to each energy level on basal and ISO-induced fat mobilisation was also studied. ISO had a high and significant lipolytic effect whatever the energy level. Nevertheless, the response area of non-esterified fatty acids (NEFA) to ISO in underfed cows was 1.7 times greater than that in overfed cows. T had a slight but significant lipolytic effect on NEFA plasma response in the underfed group. D had no lipolytic effect. Basal and ISO-stimulated plasma NEFA levels were similar after 1 or 4 weeks of underfeeding.

Effect of Overfeeding During the Dry Period on Regulation of Adipose Tissue Metabolism in Dairy Cows During the Periparturient Period

During the dry period, cows were either fed restricted amounts or were overfed to study lipolytic rates in adipose tissue. Higher lipolytic rates can result in greater accumulation of triacylglycerols in liver and, subsequently, hepatic lipidosis. Adipose tissue was biopsied at –1, 0.5, 1, 2, and 3 wk from parturition. The basal in vitro lipolytic rate was measured as well as the lipolytic rate as affected by the addition of noradrenaline, 3-hydroxybutyrate, or glucose. Liver was biopsied to quantify triacylglycerol concentrations. Blood was collected to determine insulin and nonesterified fatty acid concentrations. Basal in vitro lipolytic rates at –1 and 0.5 wk were lower in overfed cows. Lipolytic rate was enhanced in both groups of cows when noradrenaline was added, but rates at –1 and 3 wk tended to be higher in overfed cows than in cows that were fed restricted amounts. After the addition of 3-hydroxybutyrate or glucose in vitro, lipolytic rates tended to be higher in overfed cows. Liver triacylglycerol concentration was higher in overfed cows at 0.5 and 1 wk. Plasma insulin concentration tended to be higher in overfed cows at –1 wk. Plasma nonesterified fatty acid concentration was higher in overfed cows at 0.5 and 1 wk. Although overfeeding compared with restricted feeding did not significantly alter the in vitro lipolytic response to 3-hydroxybutyrate or glucose, adipose tissue from overfed cows tended to be less inhibited by these substances, which may contribute to higher lipolytic rates in vivo and a greater triacylglycerol accumulation in the liver after parturition.

Plasma non-esterified fatty acid response to a β-adrenergic challenge before or after feeding in energy underfed or overfed, dry or lactating cows

AbstractThe effects of adrenergic challenge (in order to evaluate the adipose tissue lipolytic potential) and time of challenge relative to feeding on the response curve of plasma non-esterified fatty acids (NEFA) in four underfed or overfed non-lactating non-pregnant cows were studied. Basal NEFA and NEFA response to isoproterenol (ISO; 4 nmol/kg body weight) were higher when challenged before than after feeding and higher in underfed than overfed cows. Interaction between feeding level and time of challenge was significant (P &lt; 0·001) for the rising part of NEFA response. Pooled NEFA response curves from several trials (no. = 63 challenges) were analysed in order to obtain a simplified procedure for the prediction of the NEFA response. High correlations were found between the response area or maximal NEFA response and NEFA response at 15 min (r = 0·95 and 0·98, respectively). This simplified procedure was applied on pooled data from 84 challenges in order to evaluate the effects of energy balance, physiological status, body condition score (BCS) and time of challenge relative to feeding. Energy balance had a significant effect on basal plasma NEFA and NEFA response at 15 min after ISO challenge (–7·6 and –14·2 μmol/l when the daily energy balance increased by 1 MJ, respectively). Time of ISO injection relative to feeding had a greater effect on stimulated NEFA than on basal NEFA (308 v. 239 μmol/l). There was a significant effect of BCS (41 μmol/l per unit of BCS) on the basal plasma NEFA level. The NEFA response at 15 min after ISO challenge was lower (387 μmol/l) in lactating cows than in dry cows. The NEFA response to ISO at 15 min could provide an efficient method of studying the adipose tissue lipolytic potential of cattle in vivo.

Somatotropin Treatment Does Not Affect Nonesterified Fatty Acid Response to Adrenergic Injections in Underfed or Overfed Nonlactating Cows

This experiment was conducted to determine the respective effects of bovine somatotropin (bST) and energy balance on the in vivo responses on plasma nonesterified fatty acids and glucose to isoproterenol (a nonselective β-agonist) or epinephrine injection in nonlactating nonpregnant cows. Two groups of adult Holstein cows were either underfed (n = 4) at 75%, or overfed (n = 5) at 150% of maintenance energy requirement, respectively. Cows received or did not receive a subcutaneous injection of Sometribove (500 mg) during two experimental periods (cross-over design). Adrenergic or placebo injections (4 nmol/kg body weight of epinephrine or isoproterenol or 4 mL of sterile saline) were administered intravenously on d 7–9 after bovine somatotropin injection, 1 h before or 3.5 h after feeding for under- or overfed cows, respectively. Glucose and nonesterified fatty acid responses to each challenge were calculated as area under the response curve and above the base line, from the time of challenge until 60 min postchallenge. Basal plasma nonesterified fatty acids and their response to adrenergic injections were enhanced by underfeeding. Responses of nonesterified fatty acids to isoproterenol injection were higher than they were to epinephrine injection. Basal plasma glucose was enhanced by bovine somatotropin treatment, which increased the glucose response at 5 min after adrenergic injections. Response of plasma glucose was higher after epinephrine than after isoproterenol injection. Treatment with bovine somatotropin did not change plasma nonesterified fatty acid responses to epinephrine or isoproterenol injection in under- or overfed cows, at constant energy intake, whereas underfeeding modified these responses markedly.

Characterization of Metabolic Changes during a Protocol for Inducing Lactation Ketosis in Dairy Cows

During the 5 wk immediately after parturition, five high producing cows that were overfed prepartum completed a protocol for inducement of ketosis. By 12 d postpartum, hepatic glycogen decreased by 75% and hepatic triglyceride, β-hydroxybutyrate, and acetoacetate increased by 6, 4, and 2.5 times. The protocol, initiated at 2 wk postpartum, consisted of a 15 to 20% decrease from ad libitum feed intake plus dietary supplementation with 1,3-butanediol, a ketogenic substrate. Severity of the ketotic state increased gradually, and four cows developed clinical signs of ketosis at an average of 36 d postpartum. Glycogen was 90% depleted, and triglycerides, β-hydroxybutyrate, and acetoacetate concentrations in liver were increased to 10, 10, and 15 times above average prepartum concentrations. In plasma, β-hydroxybutyrate increased and glucose decreased. Plasma insulin exhibited an initial postpartal decrease (40%) but then was stable at that concentration through 36 d. After treatment for ketosis, glucose and insulin concentrations of plasma were greater than prepartal concentrations. Results indicate that a relatively simple protocol of prolonged energy deficit combined with an influx of ketone body precursors can induce experimental lactation ketosis in overfed cows. The protocol should be a valuable tool for ketosis research.