Dexamethasone administration restored growth in dairy calves exposed to heat stress

Recent evidence indicates that the heat stress loss on the growth performance of calves is associated with the diversion of nutrients to control enteritis and systemic inflammation. In this study, we investigated the impact of heat stress on markers of inflammation, feed use-efficiency, and growth of dairy calves. We hypothesized that dexamethasone, which is known for its immunosuppressive and anti-inflammatory properties, would reduce inflammation and restore the growth of calves exposed to heat stress. Thirty-two Holstein bull calves (body weight (BW) 68.5 ± 1.37 kg; age 3.5 ± 0.5-week-old; mean ± SD) were housed in individual pens in climate-controlled rooms at constant ambient temperature and allowed to adjust to facilities for 5 d before the start of treatments. Calves were randomly assigned to one of 4 treatments (n = 8/treatment) in a 2 × 2 factorial arrangement of environment (ENV, thermoneutral or heat stress) and intervention (INT, saline or dexamethasone) imposed for 5 d as follow: 1) thermoneutral (constant ambient temperature of 20°C 24 h/d) and administration of saline, 2) thermoneutral (constant ambient temperature of 20°C 24 h/d) and administration of dexamethasone, 3) cyclic heat stress (40°C ambient temperature, from 0800 to 1900 h/d) and administration of saline, 4) cyclic heat stress (40°C ambient temperature, from 0800 to 1900 h/d) and administration of dexamethasone. Dexamethasone (0.05 mg/kg BW), or saline (1.2 mL) was administered intra-muscularly on d 1 and 3. Upon completion of treatments, calves were euthanized on d 5 to obtain jejunum mucosa samples. Commercial milk replacer, starter grain, and water were offered, and intake was monitored daily. Rectal temperature and respiratory rate were monitored 3 times daily. Blood samples were collected on d 1, 3, and 5 to determine serum pro-inflammatory cytokine concentrations. A section of the jejunum was collected and snap-frozen to determine the concentration of pro-inflammatory markers. Statistical analyses included a mixed model, fixed effects of ENV, INT, consecutive measurements taken over time (d, h, or both), replica, and random effects of calf and error (SAS version 9.4, SAS Institute Inc., Cary, NC). The measurements collected immediately before treatment allocation were included as covariates in the model. An ENV effect showed that heat stress increased rectal temperature (38.72 vs. 39.21°C), respiratory rate (36 vs. 108 breaths/min), and water intake (3.2 vs. 6.6 L/d). The treatments did not affect dry matter intake. An ENV × INT interaction showed that heat stress with saline decreased average daily gain (ADG) by 35% and tended to decrease feed use-efficien-cy by 36%, but the use of dexamethasone to treat heat stress restored ADG and feed use-efficiency comparable to their basal levels. An ENV × INT interaction revealed that heat stress with saline increased jejunal interleukin (IL)-6 concentration 2-fold, but dexamethasone treat-ment of heat stress restored jejunal IL-6 concentration to basal levels. The bioenergetic cost of the heat stress-immune pro-inflammatory response ranged between 1.18 and 1.50 Mcal of ME. Overall, the administration of dexamethasone reduced the jejunal concentration of a pro-inflammatory marker and restored the heat stress-associated reduction in growth and feed use-efficiency. The immunomodulation and anti-inflammatory effects of dexamethasone could be part of a homeorhetic change that results in a shift from maintenance functions to support growth on calves exposed to heat stress.


INTRODUCTION
Heat stress is a significant challenge for dairy cattle because of escalating global temperatures (IPCC, 2018) and the promotion of intensive agriculture (Renaudeau et al., 2012).Dairy calves are susceptible to summer heat stress but have received little attention from scientific and management perspectives (Roland et al., 2016).
Environmental stressors (e.g., ambient temperature and relative humidity) imposed in summer occur during critical periods of immune system plasticity and maturation.To this end, heat stress in the early life of calves can have long-lasting effects on the endocrine, immune, and reproductive systems (Dahl et al., 2016).Therefore, it is vital to focus on enhancing our understanding of the effects of heat stress on the physiological functions of growing dairy calves.
Endocrine secretions regulate and prioritize nutrient partitioning and utilization in tissues to support maintenance and growth (Bell and Bauman, 1997).In dairy cattle exposed to heat stress, circulating concentrations of thyroxine presumably increase to direct resources in support of maintenance functions, while circulating growth hormone decreases (McGuire et al., 1991), potentially favoring energy partitioning away from protein synthesis in skeletal muscle and growth.Considering that the immune system is energetically demanding with the most important priority (Lochmiller and Deerenberg, 2000), the activation of immunity and a pro-inflammatory response shift energy and nutrients away from growth toward maintenance functions.The activation of the immune system induced by heat stress may contribute to changes in feed use-efficiency and impair growth performance in dairy calves (Ríus et al., 2022).
The intestinal epithelium is the most regenerative tissue in the body and is regulated by cytokines.Our previous work has shown that heat stress is associated with enteritis, impaired intestinal barrier function, and altered cytokine concentrations (Cantet et al., 2021;Yu et al., 2024).Interleukin-6 (IL-6), a regeneration-promoting or pro-inflammatory cytokine, is crucial for regulating immune responses within the intestinal microenvironment.For example, in rodents, the administration of IL-6 inhibits both constitutive and induced enterocyte cell death, resulting in intestinal hyperplasia under homeostasis (Jin et al., 2010), and promotes the proliferation and survival of intestinal epithelial cells following injury (Kuhn et al., 2014).Others have reported that increasing concentrations of IL-6 linearly undermine the integrity of the intestinal barrier by increasing the trans-epithelial flux of small-sized molecules by 3-4-fold via paracellular pathways without affecting the flux of large-sized molecules (Suzuki et al., 2011).Under pathophysiological conditions, IL-6 signaling is dispensable for intestinal epithelial proliferation and repair (Aden et al., 2016); however, excessive secretion and dysregulation of IL-6 play major roles in the pathogenesis of intestinal diseases (Suzuki et al., 2011).Therefore, it remains unclear whether IL-6 concentrations in the intestine are associated with the pathogenesis of heat stress in dairy cattle.
Glucocorticoids are anti-inflammatory hormones that limit the migration of immune cells and suppress the pro-duction and release of various inflammatory mediators, such as cytokines, chemokines, and prostaglandins (Burton et al., 1995, Yubero et al., 2012).In cattle, dexamethasone has been used to treat various pro-inflammatory and immune-mediated conditions, such as endotoxemia (Walz et al., 2008).However, the administration of glucocorticoids to control the heat stress-induced activation of immune function has not been studied in dairy cattle models.Given that heat stress triggers a sustained proinflammatory response with impaired intestinal function (Kaufman et al., 2020;Kaufman et al., 2021;Ríus et al., 2022), we hypothesized that the administration of a therapeutic dose of dexamethasone would reduce intestinal concentrations of pro-inflammatory cytokines and restore feed use-efficiency and growth in heat-stressed calves.To test this hypothesis, a proof-of-concept study was designed with the following objectives: to determine the effects of heat stress on feed use-efficiency and growth performance and to determine the effect of dexamethasone administration on pro-inflammatory markers, feed use-efficiency, and growth in heat-stressed dairy calves.

On-farm pre-study monitoring
All procedures were approved by the University of Tennessee IACUC (protocol# 2851-0921).Newborn Holstein bull calves gathered at the East Tennessee Ag Research and Education Center (Little River Animal and Environmental Unit, Walland, TN) were raised in individual pens following current commercial management practices in October 2022.Ambient temperature and relative humidity data were collected from the National Weather Service (Walland, TN).Calves were monitored daily to maintain intake and health records, ensure the wellbeing and health of the animals, and identify clinical illnesses (data not shown; calf health scorer, University of Wisconsin School of Veterinary Medicine).Fresh water and a commercial calf starter (Table 1) were provided ad libitum once daily.Milk replacer was fed to each calf using commercial bottles at 0530 and 1600 (0.34 kg milk replacer/feeding).The starter and milk replacer were manufactured to meet nutrient requirements of growing dairy calves following NASEM (2021) recommendations (AgCentral Co-op, Athens, TN, Table 1).Body weight (BW), starter intake, milk replacer intake, and average daily gain (ADG) were monitored to ensure the health of animals for 2 weeks before transportation to the Johnson Research and Teaching Unit (East Tennessee Research and Education Center, Knoxville, TN) to conduct the study.

Animal housing and management
Thirty-two calves (BW 68.5 ± 1.37 kg, age 3.5 ± 0.5-week-old, mean ± SD) were housed in individual pens in climate-controlled rooms (19.8 ± 0.8°C constant ambient temperature).To ensure that the animals fully recovered after being moved to climate-controlled rooms, a 5-d acclimation period was imposed.Each room accommodated 8 pens, and the study was conducted in 2 cohorts of 16 calves each, following our previous protocol (Ríus et al., 2022).Animals were stratified based on BW, DMI and ADG (average 61.3 kg, 1,311 g, 917 g and 62.1 kg, 1,378 g, 860 g) and randomly assigned to treatments environment (ENV) and intervention (INT) in a complete randomized design with a 2 × 2 factorial arrangement of treatments (n = 8 calves/treatment) imposed for 5 d as follow: 1) thermoneutral (TN, constant ambient temperature of 20°C 24 h/d) and administration of saline, 2) thermoneutral (TND, constant ambient temperature of 20°C 24 h/d) and administration of dexamethasone, 3) cyclic heat stress (HS, 40°C ambient temperature from 0800 to 1900 h/d followed by 27°C) and administration of saline, 4) cyclic heat stress (HSD, 40°C ambient temperature from 0800 to 1900 h/d followed by 27°C) and administration of dexamethasone.The thermostat was set at 40°C at 0800 and ambient temperature gradually increased and reached 40°C between 1230 and 1400 h.Dexamethasone (0.05 mg/kg BW), and saline (1.2 mL) were administered intramuscularly at 0700 h on d 1 and 3.The dexamethasone dose was a recommendation to treat pro-inflammatory conditions in cattle (Walz et al., 2008).The circadian heat stress climate imposed approximately 12 h/d of heat stress from d 1-5, following our previous work (Kassube et al., 2017;Ríus et al., 2022).Upon completion of the treatment period, euthanasia was performed on the calves by administration of pentobarbital (Euthasol, Virbac TX).Following confirmation of cardiac arrest, tissue samples were procured within 15 min.A segment of the jejunum approximately 10 m distal to the descending duodenum was excised and mucosal scrapings were collected according to our previous work (Yu et al., 2024).The jejunum was selected because heat stress compromises the jejunal mucosa in dairy calves (Yu et al., 2024), pigs (Pearce et al., 2013), poultry (Mazzoni et al., 2022) and rodents (Cantet et al., 2021).
Thermal load assessment.Climate-controlled rooms temperature and relative humidity records were monitored every 10 min using loggers located in the front and back of the room at the animal level (HOBO U23 Pro v2; Onset Computer Corp., Bourne, MA; accuracy ± 0.21°C and 2.5% relative humidity).Rectal temperature (RT; GLA M700 digital thermometer; accuracy ± 0.1°C) and respiratory rate (RR, breaths per min) were measured at 0630, 1400, and 1800 daily in all calves.Personnel were trained to monitor the RR, utilizing the method of quantifying the movement of the flank area within a 15-s timeframe and multiplying by 4 (Ríus et al., 2022).
Performance measurements.Body weight (Tru-test XR-3000, Datamars, Lamone, Switzerland) was recorded on d 0, 2, 4, and 6 at 0600 before feeding.Body weight records were regressed against time and the slope was used to obtain ADG.Fresh water and the calf starter were provided ad libitum.All calves consumed the total milk replacer offered, and the DMI was calculated by adding the amount of milk replacer and starter consumed on a DM basis.Feed use-efficiency (FE) was calculated using the formula BW gain (kg) / dry matter intake (kg) (Ríus et al., 2022).Daily rectal content samples were collected to determine the fecal water content (Ríus et al., 2022).
NASEM (2021) calf sub-model predicted nutrients and energy requirements.Observed 5-d average ambient temperature (31.1°C for heat stress treatments), values of nutrient composition of milk replacer and starter (Table 1), and LSM of animal variables (i.e., age, BW, ADG, and DMI) were entered in NASEM (2021).These computations were used to predict numerical values of energy supply and requirements (maintenance, thermal, growth and energy balance) for each of the treatment combinations.Predictions of energy and MP allow for growth were also calculated.
Cytokine concentrations.On d −5, 1, 2, and 5, individual blood samples were collected by jugular venipuncture in sodium heparin tubes and separated for serum collection at 1200 × g for 10 min at 4°C within 30 min and stored at − 80°C.Enzyme-linked immunoassay reagent kits (Invitrogen, Waltham, MA; catalog No. ESS0027 and ESS0029) were used to measure IL-6 and IL-1β levels in serum and jejunum lysate, respectively.Jejunal lysates were prepared according to previously published methods (Yu et al., 2024).A 96-well microplate (Corning, Tewksbury, MA, catalog no.9018) was used, and standards or samples were added to each well in triplicate, incubated at room temperature for 1 h, followed by the addition of a detection antibody (detection antibody: reagent diluent = 1:100), and kept for another hour of incubation.After adding the streptavidin-HRP reagent, the plate was incubated for 30-min at room temperature.After adding the substrate solution and incubating for 20 min at room temperature in the dark, the reaction was terminated with stop solution.Spectrophotometer (Biotek, Santa Clara, CA) readings were taken at 450 nm according to the manufacturer's instructions, and cytokine concentrations were calculated based on a standard curve.Intra-assay and inter-assay coefficients of variation showed a range of 2.5-14.1%.

Statistical analyses.
SAS v. 9.4 (SAS Inst.Inc., Cary, NC) was used to analyze results presented herein.Data recorded on farm (d −5; RR, RT, BW, DMI, water intake, fecal water content, and serum IL-6) were used for covariate analysis and included in the model if P < 0.05.Normal distribution of residuals and homoscedasticity criteria were conducted using the univariate and GLM procedures and data were log-transformed when needed.Mixed models were used to analyze the results and repeated measurements were included in the model for consecutive measurements obtained over time (h, d, or both) applied on calf.The statistical model included the overall mean, the fixed effects of ENV, INT, experimental replicate, time, interactions among main effects, and the random effect of calf and the error term.Best-fit models were evaluated manually and the covariance structure with the smallest Akaike information criterion (AIC) and Bayesian (BIC) values was chosen.The effects were tested using the PDIFF option and significance was declared at P < 0.05.The average mean and standard deviation of ambient temperature and humidity were calculated during the on-farm pre-study period and in the climate-controlled rooms to assess environmental conditions.

RESULTS
The ambient temperature and relative humidity were monitored to characterize the environmental conditions throughout the study.The ambient temperature ranged from 19.5 to 22°C during the on-farm period (Table 2, 19.5 ± 4.1 to 22.0 ± 2.5°C).The thermostat in the heat stress room was set at 40°C at 0800 and ambient temperature gradually increased and reached 40°C between 1330 and 1430 h.The ambient temperature in the heat stress room ranged from 19.1 to 30.9°C between 2000 and 0800, from 21.5 to 40.6°C between 0800 and 1900, and the relative humidity percentage ranged from 12.7 to 69.2.The ambient temperature ranged from 19.7 to 25.6°C and the relative humidity percentage ranged from 33.8 to 58.0 in the thermoneutral room (Figure 1 A and B).The ambient temperature increased from 19.7 to 25.6°C in the thermoneutral room on d 4 and 5.We are unsure what caused the increase in ambient temperature, but it could be attributed to warmer climatic conditions during those days.Nonetheless in dairy calves, the thermoneutral zone ranges between 15 and 25°C (NASEM, 2021) and, thus, the animals in the thermoneutral room experienced ambient temperature consistent with their thermoneutral conditions throughout the study.

Dexamethasone increased body temperature in heat stress calves
The RT and RR of the calves did not change before treatment initiation (Table 2).By design, ENV increased the mean RT in heat stress calves compared with that in thermoneutral calves.Unexpectedly, dexamethasone increased RT in heat stress by 0.16°C relative to saline heat stress but the effect of dexamethasone was not observed in thermoneutral treatments (Table 3, Figure 2, ENV × INT, P < 0.05).Compared with thermoneutral, heat stress calves increased RR 2-fold (Table 3, Figure 3, ENV, P < 0.01).

Dexamethasone intervention to treat heat stress restored productivity
Milk replacer intake (data not shown) and DMI was not affected by the treatments (Table 4, P > 0.31).An ENV × INT interaction indicated that dexamethasone restored ADG in heat stress calves but did not affect ADG in thermoneutral calves ( 2 Data collected on d 1 and 5 of the adjustment period. Figure 1.A) Ambient temperature and relative humidity in the thermoneutral room during the treatment period (average 20.3°C and 39.3%, standard deviation 1.5°C and 6.1% respectively).B) Ambient temperature and relative humidity in the heat stress room during the treatment period (average 31.1°C and 27.5%, standard deviation 6.1°C and 10.4% respectively).The NASEM predictions of ME supply were between 6.39 and 6.82 Mcal/d and showed negligible disparity, expected for deterministic models (Table 6).Compared with thermoneutral treatments, the maintenance requirement was greater in heat stress groups because these calves were heavier than their thermoneutral counterparts.The ME of thermoregulation due to heat stress was estimated at 0.

Dexamethasone intervention during heat stress reduced IL-6 concentration in jejunum mucosa
There was an ENV × INT interaction that showed 100% increase IL-6 concentrations in jejunum mucosa of heat stress calves compared with their thermoneutral counterparts.Dexamethasone intervention reduced jejunal IL-6 concentration in the heat stress treatment group compared with that in the saline treatment group.Surdexamethasone intervention increased jejunal IL-6 levels in thermoneutral calves (Table 7, ENV × INT, P < 0.01).

DISCUSSION
Heat stress pathophysiology favors the use of nutrients to support immunity and pro-inflammatory responses at the expense of animal growth.Thus, we hypothesized that anti-inflammatory intervention with glucocorticoids would reduce inflammation and subsequently support feed use-efficiency and growth in dairy calves subjected to heat stress.The feed and equipment used for feeding and housing did not differ between the on-farm monitoring period and the climate-controlled room.This approach minimized the impact of external factors on our research protocol (Ríus et al., 2022).
The ambient temperature before the start of the study was consistent with the thermoneutral range shown for dairy calves (Berman et al., 1985).In line with the objectives of this work, the temperature in the heat stress room mimicked a circadian pattern that is observed during spring and summer in dairy regions worldwide (Berman et al., 1985, Kaufman et al., 2021).Calves in the heat stress room were exposed to warm ambient temperatures for approximately 10 h per d and heat stress ranged from moderate (0900 to noon) to severe (noon to 2000).The changes in ambient temperature observed in the present study were similar to those observed in our previous studies on heat stress in Holstein bull calves and lactating cows (Ríus et al., 2022;Kassube et al., 2017).
Before initiation of the study, the calves showed thermoneutral RR and RT.Body temperature data confirmed that the animals housed under heat-stress conditions  experienced an increased mean RT throughout the study.The physiological response was noticeable within 4 h of the initiation of the heat stress treatment, which agrees with our previous studies on dairy calves and cows (Ríus et al., 2022;Kassube et al., 2017).The magnitude in RT increase ranged from 0.5°C at 1400 h to 1.5°C at 2000 h and agreed with previous circadian heat stress work in pre-weaned dairy calves (Ríus et al., 2022), dairy bulls (O'Brien et al., 2010), and constant heat stress in growing beef calves (Kim et al., 2018).In the present study, heat stress increased water intake.The effect of heat stress on water intake is well-described in the literature and aims to maintain thermal homeostasis by promoting heat loss through evaporative cooling (Berman et al., 1985, Bernabucci et al., 2010, Ríus et al., 2022).Loss of heat by the utilization of the latent heat of water vaporization is an important component of the physiological strategies available to cattle that regulate their body temperature in the face of a heat load.Furthermore, our data showed that heat stress elicited a 2-fold increase in RR, supporting respiratory evaporative heat loss, which agrees with previous research on dairy cattle (Bernabucci et al., 2010, Renaudeau et al., 2012), pigs (Renaudeau et al., 2012), and poultry (Lara and Rostagno, 2013).Thus, our heat stress model elicited the expected changes in thermoregulation and body temperature.
The hyperthermic effect of dexamethasone increased RT on heat stress calves.Surprisingly, in this group, the increase in RT was not accompanied by a subsequent increase in RR, facilitating additional heat loss through evaporative cooling (Berman, et al., 1985).In ectotherms, corticosterone treatment at physiological levels raised the body temperature and oxygen consumption by 50%, indicating a high metabolic rate (Preest and Cree, 2008).In rodents, social stress dramatically activated the hypothalamic-pituitary-adrenal axis, increasing plasma corti-costerone 4-fold and core body temperature from 37.0 to 39.5°C (Pardon et al., 2004).In mammals, glucocorticoid administration elicits an immediate increase in metabolic rate (Haase et al., 2016).Dexamethasone exhibits 20-30 times greater binding affinity for glucocorticoid receptors than endogenous cortisol.This increased affinity may subsequently affect the hypothalamus, leading to an increase in the thermoregulatory set-point (Oka et al., 2001).Collectively, the increase in RT observed on heat stress dexamethasone-treated calves may be associated with a change in the thermoregulatory set-point.
Compared with saline, dexamethasone increased water intake.Studies in rodents have shown that dexamethasone indirectly increases water consumption elicited by angiotensin and sodium ingestion, possibly by targeting glucocorticoid receptors in the brain to increase thirst (Summers et al., 1991).Glucocorticoids exert powerful renal effects that influence water and sodium ingestion by promoting urine excretion.Glucocorticoids released under stressful conditions regulate electrolyte transport and ion channel activation in the gut epithelium (Binder 1978, Ahsan et al., 2020).Glucocorticoids are important regulators of sodium/hydrogen exchanger 3, the major ion transport counterpart to the CFTR channel in enterocytes that regulates the recovery of anions, Na + , and water from the lumen through the apical side of the cells (Ahsan et al., 2020).Together, the increase in water intake in response to dexamethasone intervention may have supported the absorption of electrolytes and water, thus helping to cope with the increased water demand to support evaporative cooling in the heat stress animals.
Compared with thermoneutral, heat stress did not affect DMI.Milk replacer accounted for 40% of the DMI.Calves fed milk replacer containing 20% fat should result in low heat increment of digestion relative to mature animals fed a carbohydrate-based diet.To this end, dietary supplementation with inert fat is a management practice proposed to partially reduce NDF concentrations and overcome the heat stress-associated reduction of DMI in dairy cattle (NRC, 2001).Another factor worth considering is that nutrients in the milk replacer bypass rumen digestion, with the subsequent expected reduction in heat production associated with rumen fermentation of carbohydrates and proteins.Regarding calf starter grain, the intake of a low-fiber diet is associated with low enteric fermentation.Furthermore, rumen development and functional maturity occur later in life, after weaning.In agreement with the present results, our previous work showed that DMI was not affected in pre-weaned 12-week-old bull calves exposed to heat stress (4.10 kg/d) compared with a thermoneutral control (4.15 kg/d, Ríus et al., 2022).In comparison, 18-week-old Holstein bulls consuming a highly fermentable carbohydratebased diet (61% steam-flaked corn and 21% alfalfa hay) dropped 12% DMI when exposed to circadian heat stress (O'Brien et al., 2010).The effect of heat stress on DMI has also been reported in Holstein heifers of 20 weeks of age exposed to a 5-week constant heat stress treatment (Baccari et al., 1983).In agreement with the DMI results, the ME supply predictions showed a narrow range between 6.4 and 6.8 Mcal/d among all treatments and a negligible difference between thermoneutral and heat stress calves (6.7 vs 6.8 Mcal/d).Collectively, the current view that heat stress in pre-weaned calves elicits a decrease in DMI should be revised to highlight the animal and environmental factors that positively or negatively influence heat stress effect on DMI.
Compared with the thermoneutral control, heat stress did not affect body weight gain, in agreement with our previous work on growing calves (Ríus et al., 2022), but in contrast to studies in poultry (Lara and Rostagno, 2013) and swine (Pearce et al., 2013).Factors contributing to these differences include animal-related factors (metabolic heat production, surface-to-mass ratio, genetics, and sex) and non-animal-related factors (mild vs. severe hyperthermia and concentrate vs. forage-based diets).As expected, in agreement with the literature on dairy calves (Baccari et al., 1983, Place et al., 1998, Rauba et al., 2019), heat stress caused a 32% decrease in ADG, and the dexamethasone treatment restored ADG compared with thermoneutral treatments.Our bioenergetic predictions indicated that the heat stress calves allocated 1.94 Mcal/d compared with their dexamethasone counterparts that allocated 3.44 Mcal/d of ME to support body weight gain.Rodent data have shown that glucocorticoid administration enhances glucose availability and mitochondrial biogenesis in metabolically active tissues (Picard et al., 2022).It is possible that glucose availability and energy production increased in metabolically active tissues of dexamethasone-heat-stress-treated calves owing to the ADG results observed herein.Our results showed that dexamethasone-heat-stress-treated calves had a 14% increase in water intake which may or may not have contributed to the increased ADG results.Alternatively, the observed ADG restoration in dexamethasone-heatstress-treated calves may be attributed to a reduction in the resources needed to mount an immune response.Predictions of energetic cost for mounting a heat stressimmune pro-inflammatory response using direct and indirect approach resulted in 1.18 and 1.50 Mcal of ME.Given that an active immune response is energetically costly (Lochmiller and Deerenberg, 2000), a strong anti-inflammatory and immunosuppressive response may shift fuel utilization from maintenance (i.e., the immune system) to growth.In rodents exposed to heat stress, the administration of dexamethasone decreased the mortality rate and multi-organ dysfunction (i.e., cardiovascular, gastrointestinal, immune, and renal functions) in a dose-dependent manner, highlighting the benefits of anti-inflammatory and immunosuppressive effects in treating heat stress (Walter and Gibson, 2020).The reasons for the differences between the models and species are unclear; however, they illustrate the importance of anti-inflammatory and immunosuppressive strategies in the treatment of heat stress illness.
Compared with thermoneutral, heat stress decreased FE by 33%, and as mentioned above, DMI was not reduced by heat stress.The ME balance results indicated that 1.74 Mcal of ME were unaccounted for in the saline heat stress group.This result indicates that ME supply was used to support maintenance functions increased by heat stress, consequently, against ME allocation for growth and FE.Our results agree with the literature on growing dairy cattle (Baccari et al., 1983) and suggest that the requirements for maintenance increased.Nonetheless, considering unchanged energy supply if heat stress increased the cost of thermoregulation in dexamethasone calves, then the energy requirement should have exceeded that observed in calves exposed to thermoneutral conditions, and the heat-stressed calves would have been showing insufficient energy supply and should have blunted growth performance.However, this was not the case suggesting that the maintenance costs of thermoregulation may not have increased.Dexamethasone intervention during heat stress restored the FE to the levels observed in the thermoneutral treatment.This finding agrees with our previous work on bull calves, in which heat stress reduced FE, but supplementation with a postbiotic with anti-inflammatory properties reversed this effect (Ríus et al., 2022).Alternatively, dexamethasone, a potent anti-inflammatory and immunosuppressive synthetic hormone, may shift fuels from immune cells (maintenance) to skeletal muscle synthesis (growth), suggesting a homeorhetic mechanism that partitions fu-Yu et al.: Dexamethasone administration restored… els and nutrients among tissues.In support of this view, heat stress in lactating dairy cows increases nutrient (e.g., glucose and amino acids) partitioning toward maintenance functions to meet the increasing cost of an active heat shock pathway and immunity (Cantet et al., 2021) away from galactopoiesis.Collectively, further research is necessary to elucidate the effects of hyperthermia on energy requirements and feed use-efficiency in growing and lactating dairy cattle.
Cytokine analyses showed that the plasma IL-6 basal concentrations in our calves were comparable to those found in humans (Nikalaus et al., 2018), mice (Bethin et al., 2000), and poultry (Luo et al. 2013) in homeostasis.As expected, heat stress increased jejunal IL-6 concentrations in saline-treated calves.Previous research has demonstrated that heat stress activates the TLR/MyD88/ NF-κB pathway and, in turn, upregulates the expression of pro-inflammatory cytokines (i.e., IL-1, IL-6, TNF-α) in the spleen of broilers (He et al., 2019).In the current study, dexamethasone administration during heat stress treatment restored jejunal IL-6 concentrations comparable to values observed in thermoneutral animals.The fundamental anti-inflammatory and immunomodulatory roles of glucocorticoids have been elucidated in studies using glucocorticoid inhibition or adrenalectomy combined with systemic immune stimulation, which resulted in septic shock and death (Gonzalo et al. 1993, Ruzek et al., 1999).Glucocorticoids may provide negative feedback, which is a critical protective mechanism for preventing inflammatory overreactions (Munck and Náray-Fejes-Tóth, 1994).In our study, the decreased IL-6 concentration in jejunum is likely due to dexamethasone inhibitory effect on NF-κB activity and inflammation (Auphan et al., 1995).These changes might have contributed to the observed improvements in growth and feed efficiency.

CONCLUSIONS
Understanding the mechanisms through which environmentally induced hyperthermia impairs animal health and performance will provide insights for developing strategies to reduce the economic impact of heat stress on dairy cattle.This study confirms our earlier reports on the heat stress immune pro-inflammatory response in cattle and clearly demonstrates that the administration of an anti-inflammatory immunomodulatory drug effectively reduces jejunal production of pro-inflammatory markers in heat-stressed dairy calves.The administration of dexamethasone restored heat stress-associated losses in ADG and feed use-efficiency, supporting a homeorhetic shift in post-absorptive metabolism and nutrient partitioning from immune function to skeletal muscle synthesis and growth in dairy calves.The addition of the heat stress-immune pro-inflammatory response should improve NASEM predictions of nutrients and energy requirements and utilization.

Table 1 .
Yu et al.: Dexamethasone administration restored… Calf starter and milk replacer chemical composition guaranteed by manufacturer (% of DM or stated otherwise)

Table 2 .
Ambient temperature and relative humidity, and rectal temperature data recorded before treatments

Table 3 .
Rectal temperature and respiratory rate in heat stress (HS) or thermoneutral (TN) calves with dexamethasone (D) or saline (S) solution injection xyz Values within the same variable with different superscripts indicate significant differences in E × I interaction, P < 0.05.

Table 4 .
Dry matter (milk replacer plus starter) and water intake in heat stress (HS) or thermoneutral (TN) calves with dexamethasone (D) or saline (S) solution injection of ME lost to the heat stress response that was not linked to thermoregulation.The prediction of MP and ME allow for growth showed that the requirements computed by NASEM were likely less than the observed requirements (i.e., underpredicted) otherwise calves should have displayed an ADG ≥0.78 k/d.

Table 5 .
Growth performance in heat stress (HS) or thermoneutral (TN) calves treated with dexamethasone (D) or saline (S) Chemical composition of starter (18% CP, high starch) and milk replacer (26/20), and LSM of animal variables (i.e., age, BW, ADG, and DMI) were computed in NASEM, (2021) to predict nutrient supply and animal requirements.Non-liquid intake input as LSM of starter intake.Requirements for thermal energy were computed at 31.1°C.Metabolizable energy (ME) and metabolizable protein (MP).