Physiological responses of Holstein calves to prolonged heat stress and dietary supplementation with a postbiotic from Aspergillus oryzae

Increased ambient temperature causes heat stress in mammals, which affects physiological and 20 molecular functions. We have recently reported that the dietary administration of a postbiotic 21 from Aspergillus oryzae (AO) improves tolerance to heat stress in fruit flies and cattle. 22 Furthermore, heat-induced gut dysfunction and systemic inflammation have been ameliorated in 23 part by nutritional interventions. The objective of this study was to characterize the phenotypic 24 response of growing calves to long-term heat stress compared to thermoneutral ad libitum fed 25 and thermoneutral feed-restricted counterparts and examining the physiologic alterations 26 associated with the administration of the AO postbiotic to heat-stressed calves with emphasis on 27 intestinal permeability. In this report, we expand previous work by first demonstrating that heat 28 stress reduced partial energetic efficiency of growth in control (45%) but not in AO-fed calves 29 (62%) compared to thermoneutral animals (66%). While heat stress increased 20% the 30 permeability of the intestine, AO postbiotic and thermoneutral treatments did not affect this 31 variable. In addition, AO postbiotic reduced fecal water content relative to thermoneutral and 32 heat stress treatments. Heat stress increased plasma concentrations of serum amyloid A, 33 haptoglobin and lipocalin-2, and administration of AO postbiotic did not ameliorate this effect. 34 In summary, our findings indicated that heat stress led to reduced nutrient-use efficiency and 35 increased systemic inflammation. Results suggest that the AO postbiotic improved energy-use 36 efficiency, water absorption, and the intestinal permeability in heat stress-mediated increase in 37 gut permeability but did not reduce heat stress-mediated rise in markers of systemic 38 inflammation. Mean-body confirmed that the heat load increased from d 1 to 7. Our results showed that the mean body temperature peaked earlier (1500 h) than the rectal temperature (1900 h) indicating that the maximum thermal load was reached at 1500 h, about 7 h after the initiation of thermal stress. Metabolic heat production is relatively small in dairy bull calves which are typically consuming low-fiber diets relative to lactating dairy cows. The large surface area to mass ratio of calves may lead to increased absorption of heat from the environment, and this probably influenced body temperature in our study.

Furthermore, heat-induced gut dysfunction and systemic inflammation have been ameliorated in 23 part by nutritional interventions. The objective of this study was to characterize the phenotypic 24 response of growing calves to long-term heat stress compared to thermoneutral ad libitum fed 25 and thermoneutral feed-restricted counterparts and examining the physiologic alterations 26 associated with the administration of the AO postbiotic to heat-stressed calves with emphasis on 27 intestinal permeability. In this report, we expand previous work by first demonstrating that heat 28 stress reduced partial energetic efficiency of growth in control (45%) but not in AO-fed calves 29 (62%) compared to thermoneutral animals (66%). While heat stress increased 20% the 30 permeability of the intestine, AO postbiotic and thermoneutral treatments did not affect this 31 variable. In addition, AO postbiotic reduced fecal water content relative to thermoneutral and 32 heat stress treatments. Heat stress increased plasma concentrations of serum amyloid A, 33 haptoglobin and lipocalin-2, and administration of AO postbiotic did not ameliorate this effect. 34 In summary, our findings indicated that heat stress led to reduced nutrient-use efficiency and 35 increased systemic inflammation. Results suggest that the AO postbiotic improved energy-use Introduction protective response, in both species the AO postbiotic induced alterations in biomarkers of 63 immune function and inflammation reminiscent of reduced gut permeability and entry into 64 circulation of luminal toxins and antigens [11]. This possibility, however, remains unproven 65 because we did not examine intestinal permeability. 66 Therefore, the objective of this study was to expand previous work by characterizing the 67 phenotypic response of growing Holstein calves to long-term heat stress in comparison with 68 thermoneutral feed-restricted and ad-libitum fed controls and examining the physiologic 69 alterations associated with the administration of the AO postbiotic to heat-stressed calves with 70 emphasis on intestinal permeability.

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Heat stress reduced energetic efficiency of growth in control but not in AO-fed calves. Feed 73 consumption was affected by treatments (P < 0.01). Intake of the HSP and TNR calves was 74 similar, and less than for HS and TN treated calves (P < 0.05, Table 1). Contrary to our 75 expectations, feed intake did not differ between HS and TN animals. At first glance, this was 76 surprising because the immediate response of an animal to heat stress is reduced nutrient 77 consumption as an attempt to match heat production from digestion and metabolism with its heat 78 dissipation capabilities [12]. For this reason, the TNR treatment was included to account for the 79 = 0.02) water intake on average by 3.7 L/d, but only HSP reduced (P = 0.04) fecal water content 86 (73.3 vs 72.1, Table 1).

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Heat stress increased body temperature and respiration rate. Surface and core body 88 temperature and respiration rate of all calves were similar before treatment initiation (data not 89 shown). The mean ( Figure 1A) and maximum ( Figure 1B) rectal temperature increased in HS 90 and HSP groups compared with TN and TNR animals (treatment by h interaction; P < 0.01). By 91 design, HS and HSP treatments increased mean rectal temperature on average 0.7, 1.1, and 92 1.2°C, respectively at 1100, 1500, and 1700 (treatment by h interaction; P < 0.01; Figure 1A). At 93 1900, HSP treatment decreased (P = 0.05) rectal temperature of calves by 0.1°C compared with 94 HS treatment, and TNR treatment decreased (P = 0.03) rectal temperature by 0.2°C compared 95 with TN treatment. By design, HS and HSP treatments increased maximum rectal temperature on 96 d 1 through 7 (treatment by d interaction; P < 0.001; Figure 1B).

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The mean respiration rate increased on d 1 and continued this pattern thereafter in HS and HSP 98 compared with TN and TNR calves (treatment by d interaction; P < 0.001; Figure 1C). Mean 99 respiration rate increased more in HS compared with HSP animals at d 4 (P ≤ 0.01). Compared 100 with TN and TNR, HS and HSP treatments increased mean respiration rate at 1100, 1500, and temperature, the respiration rate increased 4.2 and 4.1 bpm in HS and HSP calves. The increase 109 in rectal temperatures explained 52.0 and 59.0% of the variation in respiration rates in HS and 110 HSP animals (P < 0.001; Figure 2C) Figure 3B) because HS and HSP increased circulating NEFA on d 6 134 and 7 compared with TN and TNR treatments. The HS, HSP, and TN treatments registered 135 higher (P = 0.007) PUN concentrations than TNR ( Figure 3C); whereas plasma levels of L-136 lactate remained unaffected ( Figure 3D). Even though statistical differences in the entry rate of 137 amino acids were not observed, HS, HSP and TNR treatments showed numerical differences 138 relative to TN (Table 4). Furthermore, entry rates of several essential amino acids, most notably 139 lysine, were numerically greater in HSP calves compared with HS counterparts (Table 4).

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Indeed, analysis of this difference showed that HS tended (P = 0.07) to decrease lysine entry rate 141 by 167% compared with HSP. Our exploratory research suggests that a greater number of 142 replications would likely increase the statistical power to declare such differences as significant.

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In line with our experimental objectives, the temperature in the heat stress room mimicked a 146 long-term change that is typically observed in spring and summer dairy regions of the world [3, 147 11]. Under this setting, and contrary to our projections, we found that HS calves showed similar 148 feed intake compared to TN counterparts. Body temperature data confirm that animals housed 149 under heat stress conditions experienced increased diurnal mean body, rectal, and skin 150 temperature throughout the study. Furthermore, feed efficiency, total and partial energy  Compared with TN and TNR, HS and HSP calves showed elevated skin rump temperature at 167 0700 h suggesting that these animals maintained elevated skin temperature at nighttime. Heat 168 stress triggers dynamic adaptive physiologic responses associated with a substantial flow of heat 169 from the core to peripheral tissues. Typically, the temperature of the peripheral thermal 170 compartment shows 0.5-6.0°C [14,15] less than core temperature. However, this thermal 171 gradient can range from nearly zero to 6.0°C or more depending on the severity of the thermal 172 stress and the consequent vasomotor responses [16]. For example, heat stress triggered a 300% 173 increase in blood in peripheral tissues in mammals [6]. Thus, in our model, heat stress is 174 associated with substantial changes in the core-to-peripheral tissue temperature gradient and 175 distribution of body heat. Body heat distribution is mainly the result of two thermal mass-weighted average of core and skin temperatures, is thus a fundamental characterization of 178 an animal's thermal status. Mean-body temperature data confirmed that the heat load in heat 179 stressed-calves increased from d 1 to 7. Our results showed that the mean body temperature 180 peaked earlier (1500 h) than the rectal temperature (1900 h) indicating that the maximum thermal 181 load was reached at 1500 h, about 7 h after the initiation of thermal stress. Metabolic heat 182 production is relatively small in dairy bull calves which are typically consuming low-fiber diets 183 relative to lactating dairy cows. The large surface area to mass ratio of calves may lead to 184 increased absorption of heat from the environment, and this probably influenced body 185 temperature in our study.

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These long-lasting responses deplete storage of glycogen and, in turn, stimulate lipolysis of 194 adipose to release NEFA and provide substrates for ATP production. Collectively, data presented 195 here suggest that HS and HSP treatments increased availability and metabolism of carbohydrate.

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Upon utilization of glucose in circulation and stored, lipolysis and NEFA mobilization increased.

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As predicted, exposing calves to heat stress increased small intestine permeability. This response 198 was probably mediated by a reduction of the mucosal surface area, increased leak pathway of 199 paracellular movement of water and nutrients, or both. These changes appeared to be located at 200 the small but not the large intestine, as suggested by the results obtained from gut markers 201 analysis. In agreement with our results, increased intestinal permeability has been also observed 202 in other heat stress animal models [5,6]. HSP calves had similar intestinal permeability relative 203 to TN and TNR calves suggesting that the postbiotic may have improved the barrier function of 204 the intestine in these calves. Further to this point, HSP calves had lower fecal water content 205 suggesting that this treatment may have improved water absorption, hence, gut functionality. The 206 precise mechanism has not been addressed by the experimental design of this study, but future 207 work should be designed to identify a mechanism of action to increase our understanding of the 208 intestinal barrier function.  Treatments. Calves were randomly assigned to 1 of 4 treatment groups (n = 8 calves/treatment).

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Performance measurements. Body weight was measured on d -1 and 7, and water and pelleted 268 starter intake was determined daily. Feed to gain ratio was calculated as kg of total intake on DM 269 basis/kg of BW gain. Total energetic efficiency was calculated as the gross energy gain / 270 metabolizable energy intake [26], and partial energetic efficiency was calculated as gross energy 271 gain / the difference between metabolizable energy intake and net energy maintenance [27].

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The biochemical technique is based on Lipocalin-2 antibody-Lipocalin-2 antigen interactions 283 (immunosorbency) and a colorimetric detection system to detect Lipocalin-2 antigen targets in 284 samples. Bovine zonulin was detected using enzyme-linked immunosorbent assay kit according   Amino acid model descriptions and parameter estimation. Briefly, a 4-pool dynamic model 312 was constructed and used to estimate plasma amino acid (AA) entry rates and AA turnover rates 313 between the fast and slow pool as previously described [30]. The fast pool represents blood, 314 interstitial, and cytoplasmic free AA, which was assumed as 14.9% of BW. The slow pool 315 represents protein-bound AA and was calculated using the assumption that body protein is 18.8%

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Thermoregulatory responses related to changes in ambient temperature and time were 334 characterized using treatment replica, time effects, and all interactions in the model. Significant 335 differences were declared at P ≤ 0.05, and trends were declared at 0.05 < P ≤ 0.10. All results are 336 reported as least squares means or slopes ± standard error of the mean. Models to characterize 337 thermoregulatory responses with treatments were also tested to determine if ambient and rectal 338 temperatures captured the information in both variables. Rectal temperature was characterized 339 using treatment and ambient temperature regression effects. Respiration rate was characterized 340 using treatment, ambient temperature, and rectal temperature regression effects.         in Holstein heifers fed either alfalfa or corn silage diets to produce two daily gains. J. 413 Dairy Sci. 80, 1674-1684 (1997). Sci. 95(4), 1983-1991; 10.3168/jds.2011-4688 (2012).                3 Plasma AA entry rates in TNR, HS, and HSP were expressed relative to TN treatment. 518 4 Plasma Lys entry rate tended (P = 0.07) to decrease more in HS compared with HSP calves. 519 520