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Comparison of production-related responses to hyperinsulinemia and hypoglycemia induced by clamp procedures or heat stress of lactating dairy cattle

Open AccessPublished:August 30, 2022DOI:https://doi.org/10.3168/jds.2022-21922

      ABSTRACT

      Hyperinsulinemia concurrent with hypoglycemia is one of a myriad of physiological changes typically experienced by lactating dairy cows exposed to heat stress, the consequences of which are not yet well defined or understood. Therefore, the objective of this experiment was to separate the production-related effects of hyperinsulinemia with hypoglycemia from those of a hyperthermic environment. Multiparous lactating Holstein cows (n = 23; 58 ± 4 d in milk, 3.1 ± 0.3 lactations) were housed in temperature-controlled rooms and all were subjected to 4 experimental periods as follows: (1) thermoneutral (TN; temperature-humidity index of 65.1 ± 0.2; d 1–5), (2) TN + hyperinsulinemic-hypoglycemic clamp (HHC; insulin infused at 0.3 µg/kg of BW per h, glucose infused to maintain 90 ± 10% of baseline blood glucose for 96 h; d 6–10), (3) heat stress (HS; temperature-humidity index of 72.5 ± 0.2; d 16–20), and (4) HS + euglycemic clamp (EC; glucose infused to reach 100 ± 10% of TN baseline blood glucose for 96 h; d 21–25). Cows were fed and milked twice daily. Feed refusals were collected once daily for calculation of daily dry matter intake, and milk samples were collected at the beginning and end of each period for component analyses. Circulating insulin concentrations were measured in daily blood samples, whereas glucose concentrations were measured more frequently and variably in association with clamp procedures. Rectal temperatures and respiration rates were greater during HS than TN, as expected, and states of hyperinsulinemia and hypoglycemia were successfully induced by the HHC and high ambient temperatures (HS and EC). Feed intake differed based upon thermal environment as it was similar during TN and HHC periods, and declined for HS and EC. Milk production was not entirely reflective of feed intake as it was greatest during TN, intermediate during HHC, and lowest during HS and EC. All milk components differed with the experimental period, primarily in response to the thermal environment. Interestingly, TN baseline glucose concentrations were highly correlated with the change in glucose from TN to HS, and were related to glycemic status during HS. Furthermore, although few in number, those cows that failed to become hypoglycemic during HS tended to have a greater reduction in milk yield. The work presented here addresses a critical knowledge gap by broadening our understanding of the physiological response to heat stress and the related changes in glycemic state. This broadened understanding is fundamental for the development of novel, innovative management strategies as the dairy industry is compelled to become increasingly efficient in spite of global warming.

      Key words

      INTRODUCTION

      Environmental heat stress is detrimental to nearly all facets of animal agriculture, and concern over its far-reaching ramifications has increased alongside the growing threats of global warming. Among other consequences, heat stress reduces the efficiency of animal production, and within the US dairy industry, the immediate and long-term effects of heat stress cause severe financial loss. Older estimates place economic losses to the dairy industry at $897 million to $1.5 billion annually (for optimal and minimal heat abatement, respectively;
      • St-Pierre N.R.
      • Cobanov B.
      • Schnitkey G.
      Economic losses from heat stress by US livestock industries.
      ). In the harshest US climates (such as Florida and Texas), producers lose $337 to $383 per cow per year as a result of environmental heat stress (
      • St-Pierre N.R.
      • Cobanov B.
      • Schnitkey G.
      Economic losses from heat stress by US livestock industries.
      ). Actual recent losses are likely much greater as a result of particularly severe weather conditions and the ever-increasing costs of production. Climate change will continue to exacerbate the already-severe consequences of seasonal heat stress and is predicted to cause additional losses in milk production of more than 2% in southern states by 2030 (
      • Key N.
      • Sneeringer S.
      • Marquardt D.
      Climate change, heat stress, and U.S. dairy production. SSRN.
      ).
      Although production losses are greatest in subtropical or tropical regions worldwide, seasonal heat stress is a concern in most any place dairy cattle can be found. Meteorological summaries indicate that even in Western Canada, ambient temperatures reach or exceed the upper critical temperature for lactating dairy cows during 40% of summer days (
      • Ominski K.H.
      • Kennedy A.D.
      • Wittenberg K.M.
      • Moshtaghi Nia S.A.
      Physiological and production responses to feeding schedule in lactating dairy cows exposed to short-term, moderate heat stress.
      ). Bouts of heat stress in temperate climates are comparatively short and intermittent, but they are more than sufficient to cause losses in milk production and may be particularly problematic as the cows in these regions are less likely to be acclimated to heat stress (
      • Ominski K.H.
      • Kennedy A.D.
      • Wittenberg K.M.
      • Moshtaghi Nia S.A.
      Physiological and production responses to feeding schedule in lactating dairy cows exposed to short-term, moderate heat stress.
      ;
      • Rhoads M.L.
      • Rhoads R.P.
      • VanBaale M.J.
      • Collier R.J.
      • Sanders S.R.
      • Weber W.J.
      • Crooker B.A.
      • Baumgard L.H.
      Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin.
      ;
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      • Rhoads R.P.
      • Vanbaale M.J.
      • Sanders S.R.
      • Baumgard L.H.
      Effects of heat stress on energetic metabolism in lactating Holstein cows.
      ).
      Regardless of the region in which dairy cattle reside, when subjected to summertime thermal stress, their feed intake and milk production decline. Interestingly, however, decreased DMI does not fully explain milk production loss in heat-stressed dairy cattle (
      • Baumgard L.H.
      • Rhoads Jr., R.P.
      Effects of heat stress on postabsorptive metabolism and energetics.
      ). This discrepancy was first demonstrated in a previous experiment during which cows were housed in environmentally controlled chambers where they were either fed ad libitum and heat stressed or pair-fed in thermoneutral conditions. In essence, the cows in thermoneutral conditions were being limit-fed to separate the direct effects of reductions in feed intake from those induced by heat stress. This unique experimental design revealed that reduced nutrient intake during heat stress accounts for only a portion (approximately 35%) of milk production loss, as pair-fed cows housed in thermoneutral conditions experienced a 21% decrease in milk yield, whereas those cows subjected to heat stress experienced a 41% decrease in milk yield (
      • Rhoads M.L.
      • Rhoads R.P.
      • VanBaale M.J.
      • Collier R.J.
      • Sanders S.R.
      • Weber W.J.
      • Crooker B.A.
      • Baumgard L.H.
      Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin.
      ).
      In addition to heat-stress-specific limitations of milk synthesis, many other physiological changes have been observed in lactating dairy cattle, including altered circulating nonesterified fatty acids, urea nitrogen, IGF-I, total protein, albumin, methyl histidine, creatinine, immune function markers, and heat shock proteins (
      • Rhoads M.L.
      • Rhoads R.P.
      • VanBaale M.J.
      • Collier R.J.
      • Sanders S.R.
      • Weber W.J.
      • Crooker B.A.
      • Baumgard L.H.
      Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin.
      ;
      • Wheelock J.B.
      • Rhoads R.P.
      • Vanbaale M.J.
      • Sanders S.R.
      • Baumgard L.H.
      Effects of heat stress on energetic metabolism in lactating Holstein cows.
      ;
      • Lamp O.
      • Derno M.
      • Otten W.
      • Mielenz M.
      • Nurnberg G.
      • Kuhla B.
      Metabolic heat stress adaption in transition cows: differences in macronutrient oxidation between late-gestating and early-lactating German Holstein dairy cows.
      ;
      • Min L.
      • Cheng J.B.
      • Shi B.L.
      • Yang H.J.
      • Zheng N.
      • Wang J.Q.
      Effects of heat stress on serum insulin, adipokines, AMP-activated protein kinase, and heat shock signal molecules in dairy cows.
      ,
      • Min L.
      • Cheng J.
      • Zhao S.
      • Tian H.
      • Zhang Y.
      • Li S.
      • Yang H.
      • Zheng N.
      • Wang J.
      Plasma-based proteomics reveals immune response, complement and coagulation cascades pathway shifts in heat-stressed lactating dairy cows.
      ;
      • Kekana T.W.
      • Nherera-Chokuda F.V.
      • Muya M.C.
      • Manyama K.M.
      • Lehloenya K.C.
      Milk production and blood metabolites of dairy cattle as influenced by thermal-humidity index.
      ). Heat stress is known to concomitantly alter digestive tissue integrity and function (
      • Bedford A.
      • Beckett L.
      • Harthan L.
      • Wang C.
      • Jiang N.
      • Schramm H.
      • Guan L.L.
      • Daniels K.M.
      • Hanigan M.D.
      • White R.R.
      Ruminal volatile fatty acid absorption is affected by elevated ambient temperature.
      ;
      • Opgenorth J.
      • Abuajamieh M.
      • Horst E.A.
      • Kvidera S.K.
      • Johnson J.S.
      • Mayorga E.J.
      • Sanz-Fernandez M.V.
      • Al-Qaisi M.A.
      • DeFrain J.M.
      • Kleinschmit D.H.
      • Gorden P.J.
      • Baumgard L.H.
      The effects of zinc amino acid complex on biomarkers of gut integrity, inflammation, and metabolism in heat-stressed ruminants.
      ;
      • Guo Z.
      • Gao S.
      • Ding J.
      • He J.
      • Ma L.
      • Bu D.
      Effects of heat stress on the ruminal epithelial barrier of dairy cows revealed by micromorphological observation and transcriptomic analysis.
      ). Furthermore, adipose responsiveness to an adrenergic stimulus is blunted and exogenous glucose is cleared more quickly (
      • Wheelock J.B.
      • Rhoads R.P.
      • Vanbaale M.J.
      • Sanders S.R.
      • Baumgard L.H.
      Effects of heat stress on energetic metabolism in lactating Holstein cows.
      ;
      • Baumgard L.H.
      • Wheelock J.B.
      • Sanders S.R.
      • Moore C.E.
      • Green H.B.
      • Waldron M.R.
      • Rhoads R.P.
      Postabsorptive carbohydrate adaptations to heat stress and monensin supplementation in lactating Holstein cows.
      ), indicating that cows experiencing a thermal load reprioritize fuel selection in favor of glucose. This is supported by observations of lower circulating glucose concentrations in heat-stressed cows (
      • Rhoads M.L.
      • Rhoads R.P.
      • VanBaale M.J.
      • Collier R.J.
      • Sanders S.R.
      • Weber W.J.
      • Crooker B.A.
      • Baumgard L.H.
      Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin.
      ;
      • Wheelock J.B.
      • Rhoads R.P.
      • Vanbaale M.J.
      • Sanders S.R.
      • Baumgard L.H.
      Effects of heat stress on energetic metabolism in lactating Holstein cows.
      ;
      • Alves B.G.
      • Alves K.A.
      • Martins M.C.
      • Braga L.S.
      • Silva T.H.
      • Alves B.G.
      • Santos R.M.
      • Silva T.V.
      • Viu M.A.
      • Beletti M.E.
      • Jacomini J.O.
      • Gambarini M.L.
      Metabolic profile of serum and follicular fluid from postpartum dairy cows during summer and winter.
      ) despite sustained capacity for whole-body glucose production (
      • Baumgard L.H.
      • Wheelock J.B.
      • Sanders S.R.
      • Moore C.E.
      • Green H.B.
      • Waldron M.R.
      • Rhoads R.P.
      Postabsorptive carbohydrate adaptations to heat stress and monensin supplementation in lactating Holstein cows.
      ). For reasons that are not yet understood, reduced glucose concentrations in heat-stressed lactating dairy cows coexist with greater circulating insulin concentrations (
      • Wheelock J.B.
      • Rhoads R.P.
      • Vanbaale M.J.
      • Sanders S.R.
      • Baumgard L.H.
      Effects of heat stress on energetic metabolism in lactating Holstein cows.
      ). This state of hyperinsulinemia is inconsistent with the substantial reductions in feed intake and the hypoglycemia typical of periods of heat stress, especially in light of sustained insulin responsiveness (unlike underfed dairy cows that typically become insulin insensitive;
      • Wheelock J.B.
      • Rhoads R.P.
      • Vanbaale M.J.
      • Sanders S.R.
      • Baumgard L.H.
      Effects of heat stress on energetic metabolism in lactating Holstein cows.
      ). Heat-stress-induced hyperinsulinemia and hypoglycemia have also been reported in other mammalian species (rodents, pigs, and calves;
      • Torlińska T.
      • Banach R.
      • Paluszak J.
      • Gryczka-Dziadecka A.
      Hyperthermia effect on lipolytic processes in rat blood and adipose tissue.
      ;
      • O'Brien M.D.
      • Rhoads R.P.
      • Sanders S.R.
      • Duff G.C.
      • Baumgard L.H.
      Metabolic adaptations to heat stress in growing cattle.
      ;
      • Pearce S.C.
      • Gabler N.K.
      • Ross J.W.
      • Escobar J.
      • Patience J.F.
      • Rhoads R.P.
      • Baumgard L.H.
      The effects of heat stress and plane of nutrition on metabolism in growing pigs.
      ), suggesting these changes may be important for successful adaptation to heat stress (
      • Baumgard L.H.
      • Rhoads Jr., R.P.
      Effects of heat stress on postabsorptive metabolism and energetics.
      ).
      Elucidating the physiological significance of heat-stress-induced hyperinsulinemia and hypoglycemia in lactating dairy cows is an important step toward broadening our understanding of the whole-body response to elevated ambient temperatures. This knowledge is necessary as it will fuel the development of novel, innovative heat abatement strategies that go beyond cooling technology. Even now, advanced cooling strategies being employed on dairies throughout the United States are unable to fully recover heat-related economic losses (
      • Armstrong D.V.
      Heat stress interaction with shade and cooling.
      ;
      • St-Pierre N.R.
      • Cobanov B.
      • Schnitkey G.
      Economic losses from heat stress by US livestock industries.
      ), and those inadequacies will be exacerbated by temperature increases associated with global warming. To contribute to the current understanding of the whole-body response to heat stress, the objective of this work was to isolate the physiological consequences of altered glycemic state by comparing production-related variables measured during euglycemia or hyperinsulinemia with hypoglycemia in both thermoneutral and heat stress conditions. We hypothesize that the hyperinsulinemia and hypoglycemia experienced during heat stress is directly responsible for some of the associated production-related consequences in lactating dairy cattle. Furthermore, we believe the negative effect of heat stress on the identified variables could be ameliorated through management of the glycemic state during heat stress.

      MATERIALS AND METHODS

      All procedures involving animals were approved by the Virginia Tech Institutional Animal Care and Use Committee. Twenty-three multiparous (3.1 ± 0.3 lactations; second lactation, n = 8; third lactation, n = 9; and fourth lactation or greater, n = 6), lactating Holstein cows (58 ± 4 DIM, 687.1 ± 25.8 kg of BW) with clinically normal periparturient periods were selected from the Virginia Tech Dairy Science Complex. Before clamp procedures (described below), 4 bilateral indwelling jugular catheters were placed in each cow (2 on each side) for delivery of treatments and blood sampling. At minimum, catheters were flushed with saline once daily and locked with heparinized saline. Most catheters remained patent for the duration of the experiment.
      The total length of the trial was 28 d (from d −3 to 25) with 4 experimental periods and 2 acclimation periods. All cows were subjected to the same experimental design, which is depicted in Figure 1. Three to 4 cows were enrolled in each of 6 replicates conducted between mid October and March. The experiment was purposely planned for the fall, winter, and spring to reduce the likelihood that cows would be exposed to heat stress in the weeks before enrollment. Briefly, the experiment began with a 3-d period that allowed the cows to acclimate to the tiestalls and general husbandry procedures (d −3 to −1). Acclimation began in tiestalls at the Dairy Science Complex, after which cows were transported to Litton-Reaves Hall where cows were randomly paired and housed in 1 of 2 environmentally controlled chambers. Acclimation was immediately followed by the first and second experimental periods, consisting of (1) thermoneutral (TN; d 1–5) and (2) TN + hyperinsulinemic-hypoglycemic clamp (HHC; d 6–10). After the HHC, the heat was turned on and cows were allowed 5 d of acclimation to the thermal conditions (described in detail below). The third and fourth experimental periods consisted of (3) heat stress (HS; d 16–20) and (4) HS + euglycemic clamp (EC; d 21–25).
      Figure thumbnail gr1
      Figure 1All cows (n = 23) were subjected to the same experimental design, which consisted of 4 experimental periods: thermoneutral (TN), hyperinsulinemic-hypoglycemic clamp (HHC), heat stress (HS), and euglycemic clamp (EC). Cows acclimated to tiestall housing and experimental procedures from d −3 to −1 (ACC). Days 11 to 15 of the experiment served as a period for acclimation to HS (ACC).

      Daily Care, Samples, and Monitoring

      All cows were fed a TMR formulated to meet or exceed the nutrient requirements of early lactation (Table 1). Feeding took place twice daily (at 0030 and 1230 h), feed refusals were measured once daily (before the 1230 h feeding), and the amount of feed offered was adjusted as needed for ad libitum intake. Cows were milked twice daily (0100 and 1300 h) with yields recorded at each milking. Daily blood samples were collected by coccygeal venipuncture into sterile evacuated sodium heparin and EDTA tubes (1230 h). Blood samples were immediately centrifuged at 4°C at 1,500 × g for 15 min. Plasma was separated and stored at −20°C until analyses. Rectal temperatures (°C) and respiration rates (breaths/min) were measured once daily (1230 h) during thermoneutral periods (TN and HHC) and 3 times daily (0030, 0900, and 1230 h) under heat stress conditions (HS and EC). Milk samples were collected at the beginning and end of each experimental period from a subset of the cows (n = 16). A representative sample of milk was obtained after stirring milk collected in the bucket of the portable milker. Samples were placed in vials containing a preservative (bronopol tablet, D&F Control System) and stored at 4°C until being shipped to Lancaster DHIA (Manheim, PA) for component analyses. The resulting milk protein, fat, and lactose values were used to calculate ECM containing 3.14 MJ/kg (
      • Madsen T.G.
      • Nielsen M.O.
      • Andersen J.B.
      • Ingvartsen K.L.
      Continuous lactation in dairy cows: Effect on milk production and mammary nutrient supply and extraction.
      ).
      Table 1Ingredients and chemical composition of diet
      Values represent an average of samples collected and composited throughout the trial. Diet DM averaged 48.0%.
      ItemValue
      Ingredient, % of DM
       Corn silage36.5
       Milk cow concentrate32.7
       Dry ground corn14.0
       Brewers grain10.0
       Whole cottonseed6.7
      Chemical analysis, % of DM
       TDN73.5
       NDF32.5
       ADF20.2
       CP16.8
      NEL, Mcal/kg1.6
      1 Values represent an average of samples collected and composited throughout the trial. Diet DM averaged 48.0%.

      Thermoneutral and Hyperinsulinemic-Hypoglycemic Clamp

      Both of the first 2 experimental periods (TN and HHC) were conducted in constant ambient conditions that would be considered thermoneutral for lactating dairy cattle, which equates to a temperature-humidity index (THI) of 68 or less (
      • Zimbelman R.B.
      • Rhoads R.P.
      • Rhoads M.L.
      • Duff G.C.
      • Baumgard L.H.
      • Collier R.J.
      A re-evaluation of the impact of temperature humidity index (THI) and Black Globe Humidity Index (BGHI) on milk production in high producing dairy cows.
      ). Actual mean daily THI (± SE) was 65.1 ± 0.2 and 65.3 ± 0.2 for TN and HHC, respectively. The first experimental period, TN, was a period during which samples and data were collected, but no additional treatments were applied (d 1–5; no thermal treatment and no infusions).
      The second experimental period, which included the HHC, commenced in the afternoon on d 6 and continued for 96 h, ending in the afternoon of d 10 of the experiment. Blood glucose concentrations were monitored with handheld glucometers (Contour Next EZ, Ascensia Diabetes Care US Inc.) using blood collected from jugular catheters. Before the initiation of the HHC, baseline glucose values for each individual cow were determined (6 samples over 24 h). The insulin infusate was prepared for each individual cow in the laboratory using sterile technique. Purified bovine insulin (I5500, Sigma-Aldrich Inc.) was dissolved in 0.01 M HCl, which was then added to bagged saline containing 1.25% of each specific cow's plasma (
      • Butler S.T.
      • Pelton S.H.
      • Butler W.R.
      Insulin increases 17 beta-estradiol production by the dominant follicle of the first postpartum follicle wave in dairy cows.
      ). Bovine insulin was continuously infused using syringe pumps (Genie Plus, Kent Scientific) at a rate of 0.3 µg/kg of BW per h (
      • Butler S.T.
      • Pelton S.H.
      • Butler W.R.
      Insulin increases 17 beta-estradiol production by the dominant follicle of the first postpartum follicle wave in dairy cows.
      ). Simultaneously, glucose (50% Dextrose, Nova-Tech Inc.) was infused (Plum A+, Hospira) at variable rates to maintain a hypoglycemic state (90 ± 10% of baseline blood glucose concentrations of each individual cow). Initially, blood glucose was checked every 5 min. Once blood glucose and infusion rates stabilized, intervals between measurements increased to a maximum of 30 min. In some instances, blood glucose concentrations were not affected by the low-dose insulin infusion. In these cases, insulin was slowly increased (maximum of 0.7 µg/kg of BW per h) until blood glucose dropped to the desired range.

      Heat Stress and Euglycemic Clamp

      After the HHC, heat stress conditions were applied and cows were allowed 5 d to acclimate to those conditions. Experimental periods 3 and 4 (HS and EC) were conducted during heat stress that varied diurnally to mimic ambient conditions that would be typical for summertime in the southeastern United States (NOAA; www.noaa.gov). The room temperature was set to gradually reach a maximum of 32.2°C during the afternoon (1400 to 2000 h; maximum 76.8 ± 0.5 THI), after which it gradually cooled to reach the lowest temperature of 21.1°C overnight (2200 to 0700 h; minimum 70.3 ± 0.4 THI). Actual mean daily THI was 72.5 ± 0.2 and 71.9 ± 0.2 for HS and EC, respectively. In instances where individual cows did not acceptably acclimate to heat stress (multiple, recurring interventions when rectal temperature reached or exceeded 40.5°C), room temperature was adjusted so that the maximum temperature duration was shortened. If cows continued to require intervention for high rectal temperature, the maximum temperature was incrementally lowered until rectal temperatures stabilized below 40.5°C.
      The third experimental period (HS) was a period during which samples and data were collected, and heat stress was the only treatment that was applied (d 16–20; thermal treatment with no infusions). The fourth experimental period (EC) commenced in the afternoon on d 21 and continued for 96 h, ending in the afternoon of d 25 of the experiment. General procedures for the EC were similar to the HHC, with blood glucose concentrations monitored with handheld glucometers and baseline blood glucose concentrations determined before the initiation of the infusion. During the EC, glucose was infused (Plum A+, Hospira) at varying rates to bring circulating blood glucose concentrations back to the baseline values observed during thermoneutral (100 ± 10% of thermoneutral baseline of each individual cow). Initially, blood glucose was checked every 5 min. Once blood glucose and infusion rates stabilized, intervals between measurements increased to a maximum of 60 min.

      Insulin Analyses

      Plasma insulin concentrations were determined using commercially available, bovine-specific ELISA kits (Bovine Insulin ELISA 10–1201–01, Mercodia Inc.). The intra- and interassay coefficients of variation were 7.0 and 15.2%, respectively. Circulating insulin and glucose concentrations were used to calculate an indicator of insulin sensitivity, the homeostasis model assessment of insulin resistance (HOMA-IR) where HOMA-IR = [glucose (mg/dL) × insulin (µU/mL)]/405 (
      • Matthews D.R.
      • Hosker J.P.
      • Rudenski A.S.
      • Naylor B.A.
      • Treacher D.F.
      • Turner R.C.
      Homeostasis model assessment: Insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man.
      ;
      • Yanase M.
      • Takatsu F.
      • Tagawa T.
      • Kato T.
      • Arai K.
      • Koyasu M.
      • Horibe H.
      • Nomoto S.
      • Takemoto K.
      • Shimizu S.
      • Watarai M.
      Insulin resistance and fasting hyperinsulinemia are risk factors for new cardiovascular events in patients with prior coronary artery disease and normal glucose tolerance.
      ;
      • Hackbart K.S.
      • Cunha P.M.
      • Meyer R.K.
      • Wiltbank M.C.
      Effect of glucocorticoid-induced insulin resistance on follicle development and ovulation.
      ).

      Net Energy Balance Determination

      Net energy balance (EBAL) was calculated for a single day during each experimental period using the calculated NEL value of the diet as net energy intake in the following equation (
      • NRC
      Nutrient Requirements of Dairy Cattle.
      ;
      • Rhoads M.L.
      • Rhoads R.P.
      • VanBaale M.J.
      • Collier R.J.
      • Sanders S.R.
      • Weber W.J.
      • Crooker B.A.
      • Baumgard L.H.
      Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin.
      ): EBAL = net energy intake − (NEM + NEL). Body weights collected at the beginning and end of the experiment were used in the calculation of NEM (
      • NRC
      Nutrient Requirements of Dairy Cattle.
      ), where NEM = (0.08 × BW0.75). Some evidence suggests that maintenance energy requirements increase as much as 25% during periods of heat stress (
      • NRC
      Nutrient Requirements of Dairy Cattle.
      ). For this reason, EBAL was assessed using 2 separate approaches as follows: (1) with no adjustment in maintenance requirements and (2) with a 25% increase in maintenance requirements during those periods conducted in heat stress (HS and EC). The net energy required for lactation was calculated as NEL = [(0.0929 × fat %) + (0.0547 × protein %) + (0.0395 × lactose %)] × milk yield (
      • NRC
      Nutrient Requirements of Dairy Cattle.
      ). Energy contributed by the dextrose infused during the HHC and EC was added after multiplying the mass infused by its energy value (3.66 Mcal/kg;
      • Butler S.T.
      • Pelton S.H.
      • Butler W.R.
      Insulin increases 17 beta-estradiol production by the dominant follicle of the first postpartum follicle wave in dairy cows.
      ;
      • Rhoads R.P.
      • Kim J.W.
      • Leury B.J.
      • Baumgard L.H.
      • Segoale N.
      • Frank S.J.
      • Bauman D.E.
      • Boisclair Y.R.
      Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows.
      ).

      Statistical Analyses

      Data were analyzed using the MIXED and CORR procedures of SAS (version 9.4, SAS Institute Inc.). Independent variables included the main effects of experimental period and day of period, as well as their interactions. Cow served as the experimental unit as heat stress response (rectal temperatures and respiration rates) was measured on an individual basis. Furthermore, ambient temperature and humidity information was collected by sensors in each individual stall and those values were used in analyses rather than set points. One cow was excluded from all analyses for reasons unrelated to the experiment. Changes in rectal temperatures, respiration rates, feed intake, milk production, and blood glucose were calculated by subtracting the value of each parameter during HS from the value observed during TN. Replicate number was included in the model as the random effect and cow was included as the repeated variable. For each analysis, 8 covariance structures were tested and the most appropriate structure for each variable was selected based upon Akaike's information criterion, Akaike's information criterion with correction, and Bayesian information criterion values. In separate analyses, cows were categorized based upon their glycemic status during HS (either did or did not become hypoglycemic), and changes in rectal temperatures, respiration rates, feed intake, milk production, and blood glucose were analyzed based upon that glycemic status. For all analyses, when effects of day of period or the period × day of period interaction were not significant, they were removed from the statistical model. Results are reported as least squares means ± standard errors of the means. Means were separated using the Tukey procedure of SAS. Statistical significance was declared at P < 0.05 and a tendency for a difference at 0.05 < P < 0.15.

      RESULTS

      Rectal Temperatures and Respiration Rates

      Rectal temperatures were affected by the period of the experiment (P < 0.01; Figure 2A). Mean rectal temperatures were lowest during the TN period (38.64 ± 0.11°C) and increased when cows were subjected to the HHC (39.10 ± 0.14°C; P < 0.02) even though the ambient temperature was the same during these 2 periods. Rectal temperatures increased further during the HS period (39.50 ± 0.07°C; P < 0.02) and remained elevated during the EC (39.50 ± 0.07°C). In addition to a main effect of period, there was an interaction between period and the day of the period (P < 0.02; Figure 2A). Pairwise comparisons of days within periods revealed tendencies for differences during the HHC, with d 4 and 5 tending to be greater than d 1 (P < 0.14 and P < 0.12, respectively).
      Figure thumbnail gr2
      Figure 2Rectal temperatures (A) and respiration rates (B) during each of the 4 experimental periods: thermoneutral (TN), hyperinsulinemic-hypoglycemic clamp (HHC), heat stress (HS), and euglycemic clamp (EC). Rectal temperatures were lowest during the TN period, intermediate during HHC (P < 0.02), and greatest during HS and EC (P < 0.05). Respiration rates were lowest during TN and HHC (P < 0.01), greatest during HS (P < 0.03), and intermediate during EC (P < 0.03). Within experimental periods, pairwise comparisons determined differences (a,b: P < 0.05) and tendencies for differences (y,z: P < 0.14). Results are reported as least squares means ± standard errors of the means.
      Respiration rates were also affected by period of the experiment (P < 0.01; Figure 2B) and were lowest during TN (54.02 ± 4.04 breaths per minute, BPM). Interestingly, although rectal temperatures increased from TN to HHC, respiration rates did not differ (57.30 ± 4.05 BPM; P = 0.36). Respiration rates were greater during the HS period (76.48 ± 3.83 BPM) than during TN (P < 0.01) or HHC (P < 0.01). After reaching their peak during HS, respiration rates during the EC period declined to levels in between TN/HHC and HS (70.15 ± 3.83 BPM; P < 0.03). An interaction between period and day of the period also existed (P < 0.01; Figure 2B), with respiration rates on d 1 of TN being lower than all other days of the period (P < 0.05).
      Change in rectal temperatures and change in respiration rates from TN to HS were also calculated and found to be correlated with each other. As rectal temperatures increased, respiration rates increased as well (r = 0.53; P < 0.02).

      Feed Intake

      Mean feed intake for all full days in all periods is shown in Figure 3A. The first (d 1) and last (d 5) days of each period were excluded from statistical analyses because, as a result of the daily schedule, they truly only represented half days of the respective treatments. For example, the HHC ended on the afternoon of d 5 of the second period, but due to the feeding schedule, refusals were not collected until noon the next day. By the time the refusals were collected, cows had been off the clamp for approximately 18 h, thus potentially skewing the feed intake measurements for that day. The feeding and weigh back schedule remained consistent for the entire experiment, thus requiring the exclusion of intake data from d 1 and 5 of each period. Feed intake was affected by period (P < 0.01), with intake during TN and HHC periods (24.7 ± 0.8 and 24.5 ± 1.1 kg of DMI/d, respectively) exceeding that of HS and EC (21.8 ± 0.7 and 21.3 ± 1.0 kg of DMI/d; P < 0.03). It is worthwhile to emphasize that feed intake did not differ between TN and HHC (P = 0.99), indicating that the low insulin dose and glucose infused during the HHC did not affect the cows' appetites, nor did the observed increase in rectal temperatures during the HHC alter feed intake. There was a tendency for an interaction between period and day of period (P < 0.14), but no day-to-day differences were observed within individual periods (Figure 3A).
      Figure thumbnail gr3
      Figure 3Dry matter intake (A) and milk production (B) during the 4 experimental periods: thermoneutral (TN), hyperinsulinemic-hypoglycemic clamp (HHC), heat stress (HS), and euglycemic clamp (EC). Dry matter intake during TN and HHC periods exceeded that of HS and EC (P < 0.03). Milk production was greatest during TN (P < 0.01), intermediate during HHC (P < 0.01), and lowest during HS and EC (P < 0.01). Despite a significant interaction between period and day of period, there were no day-to-day differences within periods. Results are reported as least squares means ± standard errors of the means.

      Milk Production, Milk Composition, and EBAL

      Milk production for all full days of all experimental periods is presented in Figure 3B. As with feed intake, the first and last day of each period were excluded from statistical analyses. Milk production was affected by period (P < 0.01) and the interaction between period and day of the period (P < 0.03). Most periods differed with each other, with the exception being HS and EC. Milk production was greatest during TN (50.7 ± 2.7 kg/d; P < 0.01), intermediate during HHC (46.6 ± 2.8 kg/d; P < 0.01), and lowest during HS and EC (41.5 ± 2.3 and 41.3 ± 2.5 kg/d, respectively; P < 0.01). Within periods, there were no day-to-day differences in milk yield (Figure 3B). The change in milk production from TN to HS was correlated with the change in feed intake (r = 0.53; P < 0.02), the age of the cows (r = −0.48; P < 0.04), and their parity (r = −0.47; P < 0.05).
      Energy-corrected milk yield also differed between experimental periods (P < 0.01). Similar to unadjusted milk yield, it was greatest during TN (49.9 ± 2.3 kg/d; P < 0.01) and lowest during HS and EC (37.9 ± 1.6 and 39.3 ± 1.5 kg/d, respectively; P < 0.01). During the HHC, ECM was intermediate (42.4 ± 2.2 kg/d) as it was lower than ECM during TN (P < 0.01) and tended to be greater than the ECM during HS (P = 0.10). Energy-corrected milk did not differ between the HHC and EC. Again, similar to unadjusted milk yield, ECM was affected by the interaction between period and day of period (P < 0.05), but pairwise comparisons revealed no day-to-day differences within periods.
      Most milk components, including milk fat, protein, lactose, urea nitrogen, and other solids, were affected by period (Table 2). There was also a difference in milk protein based on the period by sample day interaction (P < 0.02). Pairwise comparisons revealed only one within-period change in milk protein. Milk protein was greater at the beginning of HHC (2.71 ± 0.05%) than the end of HHC (2.52 ± 0.05%, P < 0.01).
      Table 2Effects of treatments on milk components
      Milk componentTN
      Thermoneutral (TN) and hyperinsulinemic-hypoglycemic clamp (HHC) periods, which were conducted in thermoneutral ambient conditions.
      HHC
      Thermoneutral (TN) and hyperinsulinemic-hypoglycemic clamp (HHC) periods, which were conducted in thermoneutral ambient conditions.
      HS
      Heat stress (HS) and euglycemic clamp (EC) periods, which were conducted in heat stress ambient conditions.
      EC
      Heat stress (HS) and euglycemic clamp (EC) periods, which were conducted in heat stress ambient conditions.
      P-value
      Protein, %2.69 ± 0.05
      Within a row, different superscripts differ at P < 0.05.
      2.61 ± 0.05
      Within a row, different superscripts differ at P < 0.05.
      2.59 ± 0.05
      Within a row, different superscripts differ at P < 0.05.
      2.53 ± 0.05
      Within a row, different superscripts differ at P < 0.05.
      <0.01
      Fat, %4.20 ± 0.19
      Within a row, different superscripts differ at P < 0.05.
      4.14 ± 0.18
      Within a row, different superscripts differ at P < 0.05.
      3.61 ± 0.18
      Within a row, different superscripts differ at P < 0.05.
      3.67 ± 0.15
      Within a row, different superscripts differ at P < 0.05.
      <0.01
      Lactose, %5.01 ± 0.04
      Within a row, different superscripts differ at P < 0.05.
      4.97 ± 0.04
      Within a row, different superscripts differ at P < 0.05.
      4.92 ± 0.04
      Within a row, different superscripts differ at P < 0.05.
      4.93 ± 0.04
      Within a row, different superscripts differ at P < 0.05.
      <0.01
      Other solids, %5.92 ± 0.04
      Within a row, different superscripts differ at P < 0.05.
      5.88 ± 0.04
      Within a row, different superscripts differ at P < 0.05.
      5.83 ± 0.04
      Within a row, different superscripts differ at P < 0.05.
      5.84 ± 0.43
      Within a row, different superscripts differ at P < 0.05.
      <0.01
      SCC, × 1,000346.92 ± 230.55264.86 ± 230.34271.63 ± 230.52349.45 ± 245.660.29
      MUN, mg/dL8.34 ± 0.70
      Within a row, different superscripts differ at P < 0.05.
      9.38 ± 0.81
      Within a row, different superscripts differ at P < 0.05.
      10.51 ± 0.77
      Within a row, different superscripts differ at P < 0.05.
      8.96 ± 0.86
      Within a row, different superscripts differ at P < 0.05.
      <0.01
      a–c Within a row, different superscripts differ at P < 0.05.
      1 Thermoneutral (TN) and hyperinsulinemic-hypoglycemic clamp (HHC) periods, which were conducted in thermoneutral ambient conditions.
      2 Heat stress (HS) and euglycemic clamp (EC) periods, which were conducted in heat stress ambient conditions.
      Net EBAL tended to be affected by experimental period when calculated using no change in maintenance energy requirements (−7.01 ± 2.23, 0.56 ± 2.31, −2.23 ± 2.31, and −0.14 ± 2.31 Mcal/d for TN, HHC, HS, and EC, respectively; P < 0.09). Alternatively, when calculated using maintenance energy requirements adjusted for heat stress during the HS and EC periods (+25%), no differences in net EBAL were detected (−7.01 ± 2.40, −1.27 ± 2.40, −4.78 ± 2.49, and −2.69 ± 2.49 Mcal/d for TN, HHC, HS, and EC, respectively; P = 0.37).

      Circulating Insulin, Glucose, and Clamp Responses

      Baseline blood glucose concentrations were measured during TN and HS periods before being altered by the clamps conducted during the HHC and EC periods, respectively. Baseline circulating glucose concentrations were greater during TN (58.5 ± 1.3 mg/dL) than during HS (56.0 ± 0.7 mg/dL; P = 0.05). Baseline blood glucose during TN only tended to be correlated with those concentrations observed during HS (r = 0.37; P < 0.15).
      Interestingly, baseline glucose concentrations during TN were highly correlated with the change in blood glucose from TN to HS (r = −0.86; P < 0.01; Figure 4). Closer examination of the baseline blood glucose concentrations revealed a natural break at 53 mg/dL where, with only one exception, cows whose TN concentrations were >53 mg/dL became hypoglycemic during HS (15 of the 20 cows that completed both periods became hypoglycemic, with hypoglycemia defined as an individual having a mean blood glucose concentration lower than was measured for that individual during TN), whereas those cows with baseline blood glucose concentrations <53 mg/dL did not become hypoglycemic (5 cows out of 20 did not become hypoglycemic). For this reason, production and stress parameters were analyzed based upon TN baseline blood glucose concentrations and glycemic state during HS (i.e., did or did not become hypoglycemic). Cows that failed to become hypoglycemic during HS tended to have a greater reduction in milk yield (−14.9 ± 3.2 kg/d) than those cows that did become hypoglycemic (−9.4 ± 2.2 kg/d; P < 0.10). This relationship persisted when change in milk yield was calculated as a percent reduction from TN to HS, as the cows that failed to become hypoglycemic experienced a 26.3 ± 4.5% reduction in milk yield, whereas for the hypoglycemic cows milk yield was reduced by 17.8 ± 3.1% (P = 0.06) during HS. Feed intake, rectal temperatures, and respiration rates did not differ between cows that did or did not become hypoglycemic.
      Figure thumbnail gr4
      Figure 4Regression of the change in blood glucose from thermoneutral (TN) to heat stress (HS) conditions on baseline blood glucose during TN (r = −0.86; P < 0.01). With only one exception, cows whose TN baseline glucose concentrations were >53 mg/dL became hypoglycemic during HS, whereas those cows with baseline blood glucose concentrations <53 mg/dL did not become hypoglycemic.
      As the HHC began, insulin concentrations increased over pre-infusion values (Figure 5) and remained relatively stable for the duration of the clamp. The dextrose infused during the EC did not affect circulating insulin concentrations (Figure 5). Overall, circulating insulin concentrations were affected by experimental periods as expected. The concentrations were lowest during TN (0.26 ± 0.04 µg/L) and increased with insulin infusion during the HHC (0.52 ± 0.06 µg/L; P < 0.01). Insulin concentrations during the HS and EC (0.47 ± 0.06 and 0.52 ± 0.07 µg/L, respectively) remained elevated (P < 0.01), and were similar to concentrations observed during the HHC.
      Figure thumbnail gr5
      Figure 5Mean circulating insulin and glucose concentrations during the hyperinsulinemic-hypoglycemic clamp (HHC) and euglycemic clamp (EC). Baseline, pre-clamp values of insulin and glucose are presented as time 0, followed by mean values calculated over 24-h intervals of the clamp. Both clamps lasted 96 h. Within lines, points with different letters differ (P < 0.05). Results are reported as least squares means ± standard errors of the means.
      A total of 20 cows completed the HHC. The mean dextrose infusion rate needed to maintain hypoglycemia (90 ± 10% of baseline blood glucose concentrations of each individual cow) during the HHC was 29.4 ± 8.3 mL/h. Of the 20 cows that completed the HHC, 7 failed to respond to the low-dose insulin infusion (0.3 µg/kg of BW per h) with declining blood glucose concentrations after at least 5 h of insulin infusion. Retrospective analyses revealed that baseline blood glucose concentrations, feed intake, milk production, and HOMA-IR were similar between those cows that did or did not respond to the low dose of insulin (data not shown).
      Nearly half of the cows that did not respond to the low-dose insulin infusion also failed to become hypoglycemic during HS (3 of the 7 cows), whereas the majority of the cows that responded to the low dose of insulin became hypoglycemic during HS as expected (11 of the 13 cows). The mean dextrose infusion rate needed to achieve normoglycemia (100 ± 10% of thermoneutral baseline of each individual cow) during the EC was 69.8 ± 17.1 mL/h.

      DISCUSSION

      The HS treatment that cows were subjected to during this experiment was based upon temperatures recorded during summertime in the southeastern United States. This thermal treatment successfully elicited the expected responses to elevated ambient temperatures. Rectal temperatures and respiration rates both increased from TN to HS conditions and were comparable to values that have been previously observed (
      • Rhoads M.L.
      • Rhoads R.P.
      • VanBaale M.J.
      • Collier R.J.
      • Sanders S.R.
      • Weber W.J.
      • Crooker B.A.
      • Baumgard L.H.
      Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin.
      ;
      • Wheelock J.B.
      • Rhoads R.P.
      • Vanbaale M.J.
      • Sanders S.R.
      • Baumgard L.H.
      Effects of heat stress on energetic metabolism in lactating Holstein cows.
      ).
      Interestingly, although the HHC was conducted in thermoneutral conditions, rectal temperatures were greater during the HHC than during the TN period, with a tendency for the rectal temperatures on d 4 and 5 to be greater than d 1. The reason for this increase in rectal temperature is unknown and was not representative of respiration rates, as they did not differ between TN and HHC. Fortunately, there is no reason to believe that the increase in rectal temperature was caused by an immune response. The insulin used in this study is commonly administered intravenously to cattle with no adverse effects (
      • Butler S.T.
      • Marr A.L.
      • Pelton S.H.
      • Radcliff R.P.
      • Lucy M.C.
      • Butler W.R.
      Insulin restores GH responsiveness during lactation-induced negative energy balance in dairy cattle: effects on expression of IGF-I and GH receptor 1A.
      ;
      • Butler S.T.
      • Pelton S.H.
      • Butler W.R.
      Insulin increases 17 beta-estradiol production by the dominant follicle of the first postpartum follicle wave in dairy cows.
      ;
      • Piantoni P.
      • Lock A.L.
      • Allen M.S.
      Palmitic acid increased yields of milk and milk fat and nutrient digestibility across production level of lactating cows.
      ;
      • McCracken V.L.
      • Xie G.
      • Deaver S.E.
      • Baumgard L.H.
      • Rhoads R.P.
      • Rhoads M.L.
      Short communication: Hepatic progesterone-metabolizing enzymes cytochrome P450 2C and 3A in lactating cows during thermoneutral and heat stress conditions.
      ;
      • Xie G.
      • Cole L.C.
      • Zhao L.D.
      • Skrzypek M.V.
      • Sanders S.R.
      • Rhoads M.L.
      • Baumgard L.H.
      • Rhoads R.P.
      Skeletal muscle and hepatic insulin signaling is maintained in heat-stressed lactating Holstein cows.
      ), and the infusate was prepared under sterile conditions using pharmaceutical-grade materials. Furthermore, endotoxin levels are measured by the insulin supplier as part of their quality control assessment and were found to be negligible (0.1 endotoxin units/mg). Most convincing, however, is the lack of difference in feed intake between TN and HHC. Overall feed intake during TN and HHC was nearly identical, and although not significant, it numerically increased over the course of the HHC. This would not have been the case if the immune system was activated (
      • Oh J.
      • Harper M.
      • Giallongo F.
      • Bravo D.M.
      • Wall E.H.
      • Hristov A.N.
      Effects of rumen-protected Capsicum oleoresin on immune responses in dairy cows intravenously challenged with lipopolysaccharide.
      ;
      • Dickson M.J.
      • Kvidera S.K.
      • Horst E.A.
      • Wiley C.E.
      • Mayorga E.J.
      • Ydstie J.
      • Perry G.A.
      • Baumgard L.H.
      • Keating A.F.
      Impacts of chronic and increasing lipopolysaccharide exposure on production and reproductive parameters in lactating Holstein dairy cows.
      ;
      • Kayser W.C.
      • Carstens G.E.
      • Washburn K.E.
      • Welsh Jr., T.H.
      • Lawhon S.D.
      • Reddy S.M.
      • Pinchak W.E.
      • Chevaux E.
      • Skidmore A.L.
      Effects of combined viral-bacterial challenge with or without supplementation of Saccharomyces cerevisiae boulardii strain CNCM I-1079 on immune upregulation and DMI in beef heifers.
      ). Instead, the observed difference in rectal temperature could be related to the unique characteristics of the HHC. During the HHC, hyperinsulinemia and hypoglycemia were maintained over a period of 96 h. Although this is not the first time a clamp procedure was performed for this length of time (
      • Butler S.T.
      • Marr A.L.
      • Pelton S.H.
      • Radcliff R.P.
      • Lucy M.C.
      • Butler W.R.
      Insulin restores GH responsiveness during lactation-induced negative energy balance in dairy cattle: effects on expression of IGF-I and GH receptor 1A.
      ,
      • Butler S.T.
      • Pelton S.H.
      • Butler W.R.
      Insulin increases 17 beta-estradiol production by the dominant follicle of the first postpartum follicle wave in dairy cows.
      ;
      • Rhoads R.P.
      • Kim J.W.
      • Leury B.J.
      • Baumgard L.H.
      • Segoale N.
      • Frank S.J.
      • Bauman D.E.
      • Boisclair Y.R.
      Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows.
      ), it is the first time cows were maintained in hyperinsulinemia with hypoglycemia for 96 h and rectal temperatures were reported. It is possible that the prolonged state of hyperinsulinemia and hypoglycemia altered metabolic heat production causing an increase in rectal temperatures. A direct effect of insulin is also plausible as insulin crosses the blood-brain barrier (
      • Frank H.J.
      • Pardridge W.M.
      • Morris W.L.
      • Rosenfeld R.G.
      • Choi T.B.
      Binding and internalization of insulin and insulin-like growth factors by isolated brain microvessels.
      ;
      • Banks W.A.
      • Jaspan J.B.
      • Huang W.
      • Kastin A.J.
      Transport of insulin across the blood-brain barrier: Saturability at euglycemic doses of insulin.
      ) and is known to affect warm-sensing neurons in the rat hypothalamus, ultimately causing an increase in core body temperature (
      • Sanchez-Alavez M.
      • Tabarean I.V.
      • Osborn O.
      • Mitsukawa K.
      • Schaefer J.
      • Dubins J.
      • Holmberg K.H.
      • Klein I.
      • Klaus J.
      • Gomez L.F.
      • Kolb H.
      • Secrest J.
      • Jochems J.
      • Myashiro K.
      • Buckley P.
      • Hadcock J.R.
      • Eberwine J.
      • Conti B.
      • Bartfai T.
      Insulin causes hyperthermia by direct inhibition of warm-sensitive neurons.
      ). Within the present study, results from the EC partially support the involvement of glycemic state, as the infusion of glucose during heat stress was associated with reduced respiration rates. Rectal temperatures were not significantly affected by the EC, but did numerically decline over the 96-h EC treatment period.
      When considering effects of ambient temperature aside from the effects of the 2 clamps (i.e., TN vs. HS only), feed intake and milk production were both lower during HS than during TN. Reductions in these 2 parameters are well-established responses to heat stress and are often the first to be recognized on farm. In this experiment, feed intake was reduced by approximately 12% and milk production by approximately 18%. These are more modest values than previous experiments where feed intake and milk production were reduced by 30 to 35% and 27 to 40%, respectively (
      • Rhoads M.L.
      • Rhoads R.P.
      • VanBaale M.J.
      • Collier R.J.
      • Sanders S.R.
      • Weber W.J.
      • Crooker B.A.
      • Baumgard L.H.
      Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin.
      ;
      • Wheelock J.B.
      • Rhoads R.P.
      • Vanbaale M.J.
      • Sanders S.R.
      • Baumgard L.H.
      Effects of heat stress on energetic metabolism in lactating Holstein cows.
      ). The differences between experiments are partly explained by the magnitude and duration of the overnight cool period. In previous experiments, overnight temperatures reached a nadir of 29.4°C for 7 h. In contrast, the overnight temperature for this experiment dropped to 21.1°C for 9 h. Lower overnight temperatures and cooling efforts are known to improve production even in the absence of daytime cooling (
      • Spiers D.E.
      • Spain J.N.
      • Ellersieck M.R.
      • Lucy M.C.
      Strategic application of convective cooling to maximize the thermal gradient and reduce heat stress response in dairy cows.
      ). Thus, these differences, in combination with a less extreme maximum temperature (32.2 vs. 38.9°C) could easily explain differences in feed intake and milk production between experiments. Furthermore, differences in mean DIM between studies are noteworthy. The cows used in this experiment were approximately 58 DIM, whereas cows in the other studies were approximately 100 (
      • Wheelock J.B.
      • Rhoads R.P.
      • Vanbaale M.J.
      • Sanders S.R.
      • Baumgard L.H.
      Effects of heat stress on energetic metabolism in lactating Holstein cows.
      ) and 140 DIM (
      • Rhoads M.L.
      • Rhoads R.P.
      • VanBaale M.J.
      • Collier R.J.
      • Sanders S.R.
      • Weber W.J.
      • Crooker B.A.
      • Baumgard L.H.
      Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin.
      ). It is important to distinguish these relative stages of lactation because in comparisons of early-lactation cows to those in mid-lactation, it is actually the early-lactation cows that are best able to sustain milk production during periods of heat stress (
      • Maust L.E.
      • McDowell R.E.
      • Hooven N.W.
      Effect of summer weather on performance of Holstein cows in three stages of lactation.
      ;
      • Perera K.S.
      • Gwazdauskas F.C.
      • Pearson R.E.
      • Brumback Jr., T.B.
      Effect of season and stage of lactation on performance of Holsteins.
      ).
      Over the entirety of the study (i.e., TN, HHC, HS, and EC), patterns of feed intake followed ambient temperature as the 2 periods conducted in thermoneutral conditions (TN and HHC) differed from the 2 periods conducted in heat stress (HS and EC). The similarities in feed intake between TN and HHC periods as well as HS and EC periods indicate that the clamp procedures did not affect the cows' appetites. Sustained feed intake during the clamp periods was a secondary goal and was the reason that a comparatively low insulin infusion dose was selected for the HHC. In previous work, when a more commonly used insulin dose was administered during long-term hyperinsulinemic-euglycemic clamps (1.0 µg/kg of BW per h of insulin infusion), voluntary feed intake declined between 28 and 33% (
      • McGuire M.A.
      • Dwyer D.A.
      • Harrell R.J.
      • Bauman D.E.
      Insulin regulates circulating insulin-like growth factors and some of their binding proteins in lactating cows.
      ;
      • Griinari J.M.
      • McGuire M.A.
      • Dwyer D.A.
      • Bauman D.E.
      • Palmquist D.L.
      Role of insulin in the regulation of milk fat synthesis in dairy cows.
      ;
      • Leury B.J.
      • Baumgard L.H.
      • Block S.S.
      • Segoale N.
      • Ehrhardt R.A.
      • Rhoads R.P.
      • Bauman D.E.
      • Bell A.W.
      • Boisclair Y.R.
      Effect of insulin and growth hormone on plasma leptin in periparturient dairy cows.
      ). In contrast, a previous 96-h hyperinsulinemic-euglycemic clamp utilizing the lower insulin infusion dose (0.3 µg/kg of BW per h of insulin infusion) successfully produced differences in circulating insulin concentrations without affecting DMI (
      • Butler S.T.
      • Pelton S.H.
      • Butler W.R.
      Insulin increases 17 beta-estradiol production by the dominant follicle of the first postpartum follicle wave in dairy cows.
      ). Based upon these published outcomes, the lower insulin infusion dose was chosen for the current study. Ultimately, similar feed intake within ambient temperature categories was advantageous for interpretation of the results from the clamps as they are not confounded by changes in nutrient consumption.
      Interestingly, milk production did not mirror the similarities and differences observed for feed intake. Although milk production was greatest during TN as would be expected, it declined during the HHC even while feed intake remained stable. This decline in milk production is presumably due to the induced state of hypoglycemia and may have contributed to the observed increase in rectal temperatures during the HHC. In another study where mid-lactation cows were subjected to a long-term hyperinsulinemic and hypoglycemic clamp, milk yield also declined (
      • Kreipe L.
      • Vernay M.C.
      • Oppliger A.
      • Wellnitz O.
      • Bruckmaier R.M.
      • van Dorland H.A.
      Induced hypoglycemia for 48 hours indicates differential glucose and insulin effects on liver metabolism in dairy cows.
      ). In fact, the amount of the decline was nearly identical (4.60 vs. 4.07 kg/d), whereas the percent decline was much greater in the work described by
      • Kreipe L.
      • Vernay M.C.
      • Oppliger A.
      • Wellnitz O.
      • Bruckmaier R.M.
      • van Dorland H.A.
      Induced hypoglycemia for 48 hours indicates differential glucose and insulin effects on liver metabolism in dairy cows.
      than for the current experiment (22 vs. 8%, respectively). In contrast, in a separate study, when early-lactation cows were maintained in euglycemia during a 96-h low-dose hyperinsulinemic clamp, milk production did not change over time or differ from control cows (
      • Butler S.T.
      • Pelton S.H.
      • Butler W.R.
      Insulin increases 17 beta-estradiol production by the dominant follicle of the first postpartum follicle wave in dairy cows.
      ). The results of these 3 long-term infusion experiments (current experiment;
      • Butler S.T.
      • Pelton S.H.
      • Butler W.R.
      Insulin increases 17 beta-estradiol production by the dominant follicle of the first postpartum follicle wave in dairy cows.
      ;
      • Kreipe L.
      • Vernay M.C.
      • Oppliger A.
      • Wellnitz O.
      • Bruckmaier R.M.
      • van Dorland H.A.
      Induced hypoglycemia for 48 hours indicates differential glucose and insulin effects on liver metabolism in dairy cows.
      ) are difficult to compare as each experiment targeted a different stage of lactation. Physiological changes over those stages of lactation (such as overall level of milk production, reproductive status, insulin sensitivity, and so on) as well as differences in the experimental designs limit our ability to draw conclusions about the observed reduction in milk production in the current study. The most apparent difference between the studies that did or did not result in a reduction in milk yield is the targeted glycemic state (hypoglycemic or euglycemic, respectively). The plausibility of a relationship between the decline in milk production and the hypoglycemic state can be attributed to the role of glucose in milk production. Glucose is a necessary precursor for lactose production and lactose is the primary osmotic factor driving milk production (
      • Guinard-Flament J.
      • Delamaire E.
      • Lemosquet S.
      • Boutinaud M.
      • David Y.
      Changes in mammary uptake and metabolic fate of glucose with once-daily milking and feed restriction in dairy cows.
      ). When blood glucose is limited, so is the supply to the mammary gland for lactose production, thus limiting milk yield.
      Although the supply of glucose to the lactating mammary gland is clearly prerequisite for milk production, it is only one of many factors that affect overall yield. The EC conducted as part of the current experiment revealed that factors other than circulating blood glucose and hypoglycemia are limiting milk production during heat stress. This was evident as the normoglycemia induced by the dextrose infusion during the EC did not improve milk production. The Michaelis constant (Km) values of glucose transporters found in the mammary gland are quite low (
      • Zhao F.Q.
      • Keating A.F.
      Expression and regulation of glucose transporters in the bovine mammary gland.
      ), further indicating that the amount of glucose available for uptake is likely not a factor limiting milk production, even during heat-stress-induced hypoglycemia. Instead, evidence from the literature suggests that glucose transporters are downregulated and the use of glucose for lactose synthesis is reduced in heat-stressed dairy cattle (
      • Gao S.T.
      • Ma L.
      • Zhou Z.
      • Zhou Z.K.
      • Baumgard L.H.
      • Jiang D.
      • Bionaz M.
      • Bu D.P.
      Heat stress negatively affects the transcriptome related to overall metabolism and milk protein synthesis in mammary tissue of midlactating dairy cows.
      ;
      • Abbas Z.
      • Sammad A.
      • Hu L.
      • Fang H.
      • Xu Q.
      • Wang Y.
      Glucose metabolism and dynamics of facilitative glucose transporters (gluts) under the influence of heat stress in dairy cattle.
      ).
      Regardless of the underlying mechanisms, the lack of improvement in milk production during the EC is indicative of sustained homeorhetic adjustments that could be compared with the shift in metabolic priorities observed during various physiological states, including immune activation. Similar to heat stress, LPS administration to lactating dairy cattle is known to reduce milk production and blood glucose concentrations. Markers of the metabolism of the mammary gland after an LPS challenge during a hyperinsulinemic-hypoglycemia clamp indicate that glucose is preserved for use by the immune system, whereas this is not the case following an LPS challenge during a hyperinsulinemic-euglycemic clamp (when circulating glucose is adequate;
      • Gross J.J.
      • van Dorland H.A.
      • Wellnitz O.
      • Bruckmaier R.M.
      Glucose transport and milk secretion during manipulated plasma insulin and glucose concentrations and during LPS-induced mastitis in dairy cows.
      ). The contrast in priorities of the mammary gland based on glycemic state would suggest that restoration of circulating glucose concentrations would improve milk production. Induction of euglycemia following LPS administration does not rescue milk production, however (
      • Kvidera S.K.
      • Horst E.A.
      • Abuajamieh M.
      • Mayorga E.J.
      • Fernandez M.V.
      • Baumgard L.H.
      Glucose requirements of an activated immune system in lactating Holstein cows.
      ), much like milk production was not restored by dextrose infusion during heat stress in the current experiment. Thus, it is apparent that circulating glucose concentrations during heat stress are not limiting milk production. Additional analyses will be necessary to elucidate all of the mechanisms involved in the control of milk production during exposure to elevated ambient temperatures.
      Although it is well established that both feed intake and milk production decline during heat stress, and those changes are generally predictable and consistent across studies, the same cannot be said for principal milk components. In fact, it would be difficult to identify an overall trend in milk components during heat stress as they vary from study to study (
      • Rhoads M.L.
      • Rhoads R.P.
      • VanBaale M.J.
      • Collier R.J.
      • Sanders S.R.
      • Weber W.J.
      • Crooker B.A.
      • Baumgard L.H.
      Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin.
      ;
      • Wheelock J.B.
      • Rhoads R.P.
      • Vanbaale M.J.
      • Sanders S.R.
      • Baumgard L.H.
      Effects of heat stress on energetic metabolism in lactating Holstein cows.
      ;
      • Baumgard L.H.
      • Wheelock J.B.
      • Sanders S.R.
      • Moore C.E.
      • Green H.B.
      • Waldron M.R.
      • Rhoads R.P.
      Postabsorptive carbohydrate adaptations to heat stress and monensin supplementation in lactating Holstein cows.
      ). For the current work, all measurements of milk composition except SCC were affected by period of the experiment. Milk fat, lactose, and other solids exhibited similar patterns of differences with each of these components being greatest during TN and lowest during HS. The observed reduction in milk fat during HS agrees with the widely reported on-farm phenomenon of milk fat depression during the summer months (
      • Bernabucci U.
      • Basiricò L.
      • Morera P.
      • Dipasquale D.
      • Vitali A.
      • Piccioli Cappelli F.
      • Calamari L.
      Effect of summer season on milk protein fractions in Holstein cows.
      ;
      • Harvatine K.J.
      Managing Milk Fat Depression.
      ). Although milk fat, lactose, and other solids during the clamp periods were statistically similar to their respective environmental counterparts (HHC with TN; EC with HS), each component numerically declined with insulin infusion and hypoglycemia during the HHC and numerically increased with glucose infusion during the EC. For lactose, these differences may be simply a reflection of the amount of glucose available for lactose synthesis (
      • Baumgard L.H.
      • Rhoads Jr., R.P.
      Effects of heat stress on postabsorptive metabolism and energetics.
      ).
      Milk protein percent was also greatest during TN. Unlike milk fat, lactose, and other solids, it decreased during the HHC and remained low for the remainder of the experiment. The factors responsible for the observed changes in milk protein content were not discernible in the current experiment, but others have found evidence of altered casein synthesis (
      • Bernabucci U.
      • Basiricò L.
      • Morera P.
      • Dipasquale D.
      • Vitali A.
      • Piccioli Cappelli F.
      • Calamari L.
      Effect of summer season on milk protein fractions in Holstein cows.
      ), downregulated protein synthetic activity (
      • Cowley F.C.
      • Barber D.G.
      • Houlihan A.V.
      • Poppi D.P.
      Immediate and residual effects of heat stress and restricted intake on milk protein and casein composition and energy metabolism.
      ), and deficiencies in amino acid precursors for milk protein synthesis (
      • Gao S.T.
      • Guo J.
      • Quan S.Y.
      • Nan X.M.
      • Fernandez M.V.S.
      • Baumgard L.H.
      • Bu D.P.
      The effects of heat stress on protein metabolism in lactating Holstein cows.
      ) during periods of heat stress. The reduction in milk protein from TN to HS conditions could also be indicative of casein degradation, which appears to be an important step in the negative regulation of milk production during stressful conditions (
      • Silanikove N.
      • Shapiro F.
      • Shinder D.
      Acute heat stress brings down milk secretion in dairy cows by up-regulating the activity of the milk-borne negative feedback regulatory system.
      ). Interestingly, in the current experiment, reductions in milk protein percent were first observed during the HHC, which was conducted in thermoneutral ambient conditions before any thermal stress. This observation suggests the hyperinsulinemic and hypoglycemic state, which is characteristic of periods of heat stress, is involved in the regulation of milk protein concentrations.
      For reasons that are not yet completely understood, heat stress is generally associated with increased circulating and MUN concentrations (
      • Wheelock J.B.
      • Rhoads R.P.
      • Vanbaale M.J.
      • Sanders S.R.
      • Baumgard L.H.
      Effects of heat stress on energetic metabolism in lactating Holstein cows.
      ;
      • Fatehi F.
      • Zali A.
      • Honarvar M.
      • Dehghan-Banadaky M.
      • Young A.J.
      • Ghiasvand M.
      • Eftekhari M.
      Review of the relationship between milk urea nitrogen and days in milk, parity, and monthly temperature mean in Iranian Holstein cows.
      ;
      • Hou Y.
      • Zhang L.
      • Dong R.Y.
      • Liang M.Y.
      • Lu Y.
      • Sun X.Q.
      • Zhao X.
      Comparing responses of dairy cows to short-term and long-term heat stress in climate-controlled chambers.
      ). In the current experiment, MUN was greater during HS than TN. This phenomenon may be a consequence of increased skeletal muscle breakdown rather than the usual culprit, hepatic detoxification of ammonia. This premise is supported by heat-stress-induced changes in indicators of muscle catabolism. Direct effects of heat stress on muscle breakdown (indicated by 3-methyl-histidine or creatine) have been reported in exercising men (
      • Febbraio M.A.
      Alterations in energy metabolism during exercise and heat stress.
      ), rabbits (
      • Marder J.
      • Eylath U.
      • Moskovitz E.
      • Sharir R.
      The effect of heat exposure on blood chemistry of the hyperthermic rabbit.
      ), poultry (
      • Yunianto V.D.
      • Hayashit K.
      • Kaiwda S.
      • Ohtsuka A.
      • Tomita Y.
      Effect of environmental temperature on muscle protein turnover and heat production in tube-fed broiler chickens.
      ), and lactating cows (
      • Schneider P.L.
      • Beede D.K.
      • Wilcox C.J.
      Nycterohemeral patterns of acid-base status, mineral concentrations and digestive function of lactating cows in natural or chamber heat stress environments.
      ;
      • Kamiya M.
      • Kamiya Y.
      • Tanaka M.
      • Oki T.
      • Nishiba Y.
      • Shioya S.
      Effects of high ambient temperature and restricted feed intake on urinary and plasma 3-methylhistidine in lactating Holstein cows.
      ). Other factors that may contribute to urea nitrogen concentrations are increased digestibility of the diet (due to slower passage rate;
      • West J.W.
      Nutritional strategies for managing the heat-stressed dairy cow.
      ) and decreased blood flow to the kidneys, which ultimately limits urea excretion (
      • Kenney M.J.
      • Musch T.I.
      Senescence alters blood flow responses to acute heat stress.
      ;
      • Lee I.Y.
      • Lee C.C.
      • Chang C.K.
      • Chien C.H.
      • Lin M.T.
      Sheng mai san, a Chinese herbal medicine, protects against renal ischaemic injury during heat stroke in the rat.
      ). Cumulatively, these physiological changes could easily explain the observed increase in MUN concentrations in heat-stressed cows irrespective of dietary formulation or declining DMI.
      Hyperinsulinemic clamps are often used in dairy cattle as a method to assess insulin sensitivity (
      • De Koster J.
      • Hostens M.
      • Hermans K.
      • Van den Broeck W.
      • Opsomer G.
      Validation of different measures of insulin sensitivity of glucose metabolism in dairy cows using the hyperinsulinemic euglycemic clamp test as the gold standard.
      ;
      • Alves-Nores V.
      • Castillo C.
      • Hernandez J.
      • Abuelo A.
      Comparison of surrogate indices for insulin sensitivity with parameters of the intravenous glucose tolerance test in early lactation dairy cattle.
      ;
      • Davis A.N.
      • Rico J.E.
      • Myers W.A.
      • Coleman M.J.
      • Clapham M.E.
      • Haughey N.J.
      • McFadden J.W.
      Circulating low-density lipoprotein ceramide concentrations increase in Holstein dairy cows transitioning from gestation to lactation.
      ). However, this is not their sole application, as many researchers have used them as an experimental treatment to raise circulating insulin concentrations in dairy cattle (
      • Butler S.T.
      • Pelton S.H.
      • Butler W.R.
      Insulin increases 17 beta-estradiol production by the dominant follicle of the first postpartum follicle wave in dairy cows.
      ;
      • Rhoads R.P.
      • Kim J.W.
      • Leury B.J.
      • Baumgard L.H.
      • Segoale N.
      • Frank S.J.
      • Bauman D.E.
      • Boisclair Y.R.
      Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows.
      ;
      • Kreipe L.
      • Vernay M.C.
      • Oppliger A.
      • Wellnitz O.
      • Bruckmaier R.M.
      • van Dorland H.A.
      Induced hypoglycemia for 48 hours indicates differential glucose and insulin effects on liver metabolism in dairy cows.
      ). In the current study, the HHC was used as a treatment to induce hyperinsulinemia and hypoglycemia (comparable to levels observed during HS) in the absence of heat stress. Although the HHC was used as a treatment rather than an assessment, it is notable that some cows failed to respond to the low-dose insulin infusion. This was observed as a lack of change in blood glucose concentrations after at least 5 h of insulin infusion. Closer examination of these results revealed no relationship between the response to insulin infusion and baseline glucose concentrations, feed intake, milk production, or HOMA-IR. Considering the comparatively low insulin infusion dose (approximately one-third of most reported hyperinsulinemic clamp procedures in dairy cattle) and the absence of related variables, it was concluded that the differences in response to insulin infusion were likely the result of cow-to-cow variation.
      Despite the variation in response observed during the HHC, mean baseline blood glucose concentrations decreased as expected when cows were subjected to high ambient temperatures. Blood glucose concentrations typically decline during heat stress, and this response has been observed in dairy cattle (
      • Rhoads M.L.
      • Rhoads R.P.
      • VanBaale M.J.
      • Collier R.J.
      • Sanders S.R.
      • Weber W.J.
      • Crooker B.A.
      • Baumgard L.H.
      Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin.
      ;
      • Wheelock J.B.
      • Rhoads R.P.
      • Vanbaale M.J.
      • Sanders S.R.
      • Baumgard L.H.
      Effects of heat stress on energetic metabolism in lactating Holstein cows.
      ;
      • Xie G.
      • Cole L.C.
      • Zhao L.D.
      • Skrzypek M.V.
      • Sanders S.R.
      • Rhoads M.L.
      • Baumgard L.H.
      • Rhoads R.P.
      Skeletal muscle and hepatic insulin signaling is maintained in heat-stressed lactating Holstein cows.
      ) as well as other species (
      • Lee F.S.
      • Scott E.L.
      The action of temperature and humidity on the working power of muscles and on the sugar of the blood.
      ;
      • Mahjoubi E.
      • Amanlou H.
      • Mirzaei-Alamouti H.R.
      • Aghaziarati N.
      • Yazdi M.H.
      • Noori G.R.
      • Yuan K.
      • Baumgard L.H.
      The effect of cyclical and mild heat stress on productivity and metabolism in Afshari lambs.
      ;
      • Victoria Sanz Fernandez M.
      • Johnson J.S.
      • Abuajamieh M.
      • Stoakes S.K.
      • Seibert J.T.
      • Cox L.
      • Kahl S.
      • Elsasser T.H.
      • Ross J.W.
      • Clay Isom S.
      • Rhoads R.P.
      • Baumgard L.H.
      Effects of heat stress on carbohydrate and lipid metabolism in growing pigs.
      ). Although it is logical to assume that the decline in circulating glucose concentrations during heat stress is a consequence of reduced feed intake, the accompanying collection of metabolic changes suggest that glucose is being preferentially used by extramammary tissues during heat stress (reviewed in
      • Baumgard L.H.
      • Rhoads Jr., R.P.
      Effects of heat stress on postabsorptive metabolism and energetics.
      ). Whole-body consequences of the hypoglycemia originating from heat stress are not completely understood and this knowledge gap was the impetus for inclusion of the EC in the current experiment. We initially hypothesized that, among other benefits, the additional glucose would stimulate milk production. Although circulating glucose concentrations were indeed restored by dextrose infusion during the EC, that glucose was not used in the mammary gland as milk yield remained low and no change in milk lactose concentration was observed. The ultimate fate of the excess glucose supplied during the EC remains to be determined.
      Results from the EC indicate that there is still much to be learned about blood glucose and its significance for dairy production during heat stress. Additional analyses of the characteristic decline in baseline blood glucose concentrations during heat stress revealed an interesting relationship between the blood glucose concentrations in TN conditions and the subsequent response to heat stress. Thermoneutral baseline glucose concentrations were negatively correlated with the change in blood glucose from TN to HS. In fact, for the group of cows included in this study, TN blood glucose concentrations were almost always (only one exception) predictive of whether cows did or did not become hypoglycemic during HS. The physiological significance of hypoglycemia during heat stress has not yet been determined, but results from this study indicate it may actually be beneficial for dairy cattle. Those cows that became hypoglycemic during heat stress tended to better sustain milk production than those cows that did not become hypoglycemic, even though both groups consumed the same amount of feed. These results must be interpreted with caution, however, as only a small proportion of the cows failed to become hypoglycemic (5 of the 20 that completed both TN and HS periods). Nonetheless, this relationship is worthy of further investigation as any variable that could be easily measured during thermoneutral conditions and be predictive of subsequent performance during heat stress would greatly benefit the dairy industry.

      CONCLUSIONS

      The work presented herein broadens our understanding of the physiological response to heat stress and the related changes in glycemic state. Taken together, the results from the HHC indicate that the hyperinsulinemia and hypoglycemia typical of the heat stress response participate in the regulation of milk production irrespective of feed intake. Unfortunately, management of the glycemic state during heat stress conditions through restoration of normoglycemia (EC) did not improve milk production. This observation aligns with previous work suggesting that glucose in the heat-stressed dairy cow is conserved for preferential use in extramammary tissues (reviewed in
      • Baumgard L.H.
      • Rhoads Jr., R.P.
      Effects of heat stress on postabsorptive metabolism and energetics.
      ) and demonstrates that glucose is not limiting milk production during heat stress. Relationships between glycemic state and the heat stress response were evident in the current work, and suggest that thermoneutral glucose concentrations could be predictive of performance during heat stress. Although potential predictive capabilities require additional evaluation due to the small number of cows available for analyses, these relationships suggest that endocrine and metabolic adaptations may, in some ways, help sustain the production of lactating dairy cows during periods of heat stress.

      ACKNOWLEDGMENTS

      The authors gratefully acknowledge funding of this work by the Agriculture and Food Research Initiative: Competitive Grant no. 2016-67015-24897 from the USDA National Institute of Food and Agriculture (Washington, DC). Additional support was provided by the Virginia Agricultural Experiment Station (Blacksburg) and the Hatch Program of the National Institute of Food and Agriculture, USDA. Many thanks to the staff and students from the Virginia Tech Dairy Science Complex, the Department of Animal and Poultry Sciences, and the Department of Dairy Science. The authors have not stated any conflicts of interest.