Robert P. Rhoads

Effects of heat stress on energetic metabolism in lactating Holstein cows

Heat stress has an enormous economic impact on the global dairy industry, but the mechanisms by which hyperthermia negatively affect systemic physiology and milk synthesis are not clear. Study objectives were to evaluate production parameters and metabolic variables in lactating dairy cows during short-term heat stress or pair-fed conditions coupled with bST administration. Twenty-two multiparous Holstein cows were subjected to 3 experimental periods: 1) thermoneutral conditions with ad libitum intake for 7 d (P1); 2) heat stress (HS) with ad libitum intake (n=10) or pair-fed (PF) in thermoneutral conditions (n=12) for 7 d (P2), and 3) 7 d of HS or PF in conditions as described in P2 with recombinant bovine somatotropin administered on d 1 (P3). All cows received an intravenous glucose tolerance test (GTT) on d 5 of each period. Heat stress conditions were cyclical and temperatures ranged from 29.4 to 38.9 degrees C. Rectal temperatures and respiration rates increased during heat stress (38.6-40.4 degrees C and 44-89 breaths/min, respectively). Heat stress reduced dry matter intake by 30% and by design PF cows had similar intake reductions (28%). During heat stress and pair-feeding, milk yield decreased by 27.6% (9.6kg) and 13.9% (4.8kg), respectively, indicating that reduced feed intake accounted for only 50% of the decreased milk production. Milk yield increased with recombinant bovine somatotropin in both HS (9.7%) and PF (16.1%) cows. Cows in both groups were in positive energy balance (3.95 Mcal/d) during P1 but entered negative energy balance during P2 and P3 (-5.65 Mcal/d). Heat stress and pair-feeding treatments decreased (9.3%) basal glucose concentrations. Heat stress conditions had no effect on basal NEFA levels during P2; however, PF cows (despite a similar calculated energy balance) had a 2-fold increase in basal NEFA concentrations. Both groups had increased plasma urea nitrogen levels during P2 and P3 compared with P1. Basal insulin levels increased (37%) during P2 and P3 in HS cows but did not differ between periods in PF cows. During P2 and compared with P1, PF cows had a decreased rate of glucose disposal, whereas HS cows had a similar disposal rate following the GTT. During P2 and compared with P1, PF cows had a reduced insulin response whereas HS cows had a similar insulin response to the GTT. In summary, reduced nutrient intake accounted for only 50% of heat stress-induced decreases in milk yield, and feed intake-independent shifts in postabsorptive glucose and lipid homeostasis may contribute to the additional reduction in milk yield.

Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin

Heat stress is detrimental to dairy production and affects numerous variables including feed intake and milk production. It is unclear, however, whether decreased milk yield is primarily due to the associated reduction in feed intake or the cumulative effects of heat stress on feed intake, metabolism, and physiology of dairy cattle. To distinguish between direct (not mediated by feed intake) and indirect (mediated by feed intake) effects of heat stress on physiological and metabolic indices, Holstein cows (n = 6) housed in thermal neutral conditions were pair-fed (PF) to match the nutrient intake of heat-stressed cows (HS; n = 6). All cows were subjected to 2 experimental periods: 1) thermal neutral and ad libitum intake for 9 d (P1) and 2) HS or PF for 9 d (P2). Heat-stress conditions were cyclical with daily temperatures ranging from 29.7 to 39.2 degrees C. During P1 and P2 all cows received i.v. challenges of epinephrine (d 6 of each period), and growth hormone releasing factor (GRF; d 7 of each period), and had circulating somatotropin (ST) profiles characterized (every 15 min for 6 h on d 8 of each period). During P2, HS cows were hyperthermic for the entire day and peak differences in rectal temperatures and respiration rates occurred in the afternoon (38.7 to 40.2 degrees C and 46 to 82 breaths/min, respectively). Heat stress decreased dry matter intake by greater than 35% and, by design, PF cows had similar reduced intakes. Heat stress and PF decreased milk yield, although the pattern and magnitude (40 and 21%, respectively) differed between treatments. The reduction in dry matter intake caused by HS accounted for only approximately 35% of the decrease in milk production. Both HS and PF cows entered into negative energy balance, but only PF cows had increased (approximately 120%) basal nonesterified fatty acid (NEFA) concentrations. Both PF and HS cows had decreased (7%) plasma glucose levels. The NEFA response to epinephrine did not differ between treatments but was increased (greater than 50%) in all cows during P2. During P2, HS (but not PF) cows had a modest reduction (16%) in plasma insulin-like growth factor-I. Neither treatment nor period had an effect on the ST response to GRF and there was little or no treatment effect on mean ST levels or pulsatility characteristics, but both HS and PF cows had reduced mean ST concentrations during P2. In summary, reduced nutrient intake accounted for just 35% of the HS-induced decrease in milk yield, and modest changes in the somatotropic axis may have contributed to a portion of the remainder. Differences in basal NEFA between PF and HS cows suggest a shift in postabsorptive metabolism and nutrient partitioning that may explain the additional reduction in milk yield in cows experiencing a thermal load.

Effects of a supplemental yeast culture on heat-stressed lactating Holstein cows.

Multiparous, lactating Holstein cows (n = 23; 120 +/- 30 d in milk, 690 +/- 67 kg of body weight) housed in climatic chambers were randomly assigned to 1 of 2 dietary treatments: a diet containing a novel yeast culture formulation (YC) for heat stress (n = 12, 10 g/d) or a control diet (n = 11). The trial length was 28 d and consisted of a 7-d thermal neutral period (TN; 18 degrees C, 20% humidity) followed by 21 d of heat stress (HS; cyclical daily temperatures ranging from 29.4 to 37.8 degrees C and 20% humidity). Cows were individually fed a total mixed ration consisting primarily of alfalfa hay and steam-flaked corn. During TN, the YC feeding had no effect on production variables or most body temperature indices. During HS, all body temperature indices increased and YC had no effect on rump surface temperature, respiration rate, or sweating rates. Cows fed YC had lower rectal temperatures at 1200 and 1800 h (40.29 vs. 40.02 degrees C and 40.35 vs. 40.12 +/- 0.07 degrees C, respectively) compared with control-fed cows. Cows fed both diets lost body weight (42 kg) during HS, but there were no differences between diets. Control-fed cows had increased dry matter intake (DMI) and milk yield (19.1 vs. 17.9 +/- 0.5 kg/d and 32.15 vs. 29.15 +/- 0.02 kg/d, respectively) compared with YC-fed cows, but intake and milk production were similar between diets when evaluated on a body weight basis. Heat stress progressively decreased DMI (29%) and milk yield, with milk production reaching a nadir (33%) in the third week. Heat stress decreased milk protein (7%) and lactose (5%) levels, but did not alter milk fat content. Heat-stressed cows were in calculated negative energy balance (-1.91 +/- 0.70 Mcal/d) and this was unaffected by diet. Independent of diet, HS decreased plasma glucose (11%), but neither diet nor HS altered basal nonesterified fatty acid levels. Heat stress increased plasma urea N concentrations (11.5 vs. 14.8 +/- 0.4 mg/dL). Despite YC-fed cows having slightly reduced body temperatures indices, feeding YC did not prevent the negative effects of HS.

Metabolic adaptations to heat stress in growing cattle.

To differentiate between the effects of heat stress (HS) and decreased dry matter intake (DMI) on physiological and metabolic variables in growing beef cattle, we conducted an experiment in which a thermoneutral (TN) control group (n=6) was pair fed (PF) to match nutrient intake with heat-stressed Holstein bull calves (n=6). Bulls (4 to 5 mo old, 135 kg body weight [BW]) housed in climate-controlled chambers were subjected to 2 experimental periods (P): (1) TN (18 degrees C to 20 degrees C) and ad libitum intake for 9 d, and (2) HS (cyclical daily temperatures ranging from 29.4 degrees C to 40.0 degrees C) and ad libitum intake or PF (in TN conditions) for 9 d. During each period, blood was collected daily and all calves were subjected to an intravenous insulin tolerance test (ITT) on day 7 and a glucose tolerance test (GTT) on day 8. Heat stress reduced (12%) DMI and by design, PF calves had similar nutrient intake reductions. During P1, BW gain was similar between environments and averaged 1.25 kg/d, and both HS and PF reduced (P<0.01) average daily gain (-0.09 kg/d) during P2. Compared to PF, HS decreased (P<0.05) basal circulating glucose concentrations (7%) and tended (P<0.07) to increase (30%) plasma insulin concentrations, but neither HS nor PF altered plasma nonesterified fatty acid concentrations. Although there were no treatment differences in P2, both HS and PF increased (P<0.05) plasma urea nitrogen concentrations (75%) compared with P1. In contrast to P1, both HS and PF had increased (16%) glucose disposal, but compared with PF, HS calves had a greater (67%; P<0.05) insulin response to the GTT. Neither period nor environment acutely affected insulin action, but during P2, calves in both environments tended (P=0.11) to have a blunted overall glucose response to the ITT. Independent of reduced nutrient intake, HS alters post-absorptive carbohydrate (basal and stimulated) metabolism, characterized primarily by increased basal insulin concentrations and insulin response to a GTT. However, HS-induced reduction in feed intake appears to fully explain decreased average daily gain in Holstein bull calves.

The effects of heat stress and plane of nutrition on metabolism in growing pigs1

Heat stress (HS) jeopardizes pig health, reduces performance variables, and results in a fatter carcass. Whether HS directly or indirectly (via reduced feed intake) is responsible for the suboptimal production is not known. Crossbred gilts (n = 48; 35 ± 4 kg BW) were housed in constantly climate-controlled rooms in individual pens and exposed to 1) thermal-neutral (TN) conditions (20°C; 35% to 50% humidity) with ad libitum intake (n = 18), 2) HS conditions (35°C; 20% to 35% humidity) with ad libitum intake (n = 24), or 3) pair-fed [PF in TN conditions (PFTN), n = 6, to eliminate confounding effects of dissimilar feed intake (FI)]. Pigs in the TN and HS conditions were sacrificed at 1, 3, or 7 d of environmental exposure, whereas the PFTN pigs were sacrificed after 7 d of experimental conditions. Individual rectal temperature (Tr), skin temperature (Ts), respiration rates (RR), and FI were determined daily. Pigs exposed to HS had an increase (P < 0.01) in Tr (39.3°C vs. 40.8°C) and a doubling in RR (54 vs. 107 breaths per minute). Heat-stressed pigs had an immediate (d 1) decrease (47%; P < 0.05) in FI, and this magnitude of reduction continued through d 7; by design the nutrient intake pattern for the PFTN controls mirrored the HS group. By d 7, the TN and HS pigs gained 7.76 and 1.65 kg BW, respectively, whereas the PFTN pigs lost 2.47 kg BW. Plasma insulin was increased (49%; P < 0.05) in d 7 HS pigs compared with PFTN controls. Compared with TN and HS pigs, on d 7 PFTN pigs had increased plasma NEFA concentrations (110%; P < 0.05). Compared with TN and PFTN controls, on d 7 circulating N(τ)-methylhistidine concentrations were increased (31%; P < 0.05) in HS pigs. In summary, despite similar nutrient intake, HS pigs gained more BW and had distinctly different postabsorptive bioenergetic variables compared with PFTN controls. Consequently, these heat-induced metabolic changes may in part explain the altered carcass phenotype observed in heat-stressed pigs.

Heat stress reduces intestinal barrier integrity and favors intestinal glucose transport in growing pigs.

Excessive heat exposure reduces intestinal integrity and post-absorptive energetics that can inhibit wellbeing and be fatal. Therefore, our objectives were to examine how acute heat stress (HS) alters intestinal integrity and metabolism in growing pigs. Animals were exposed to either thermal neutral (TN, 21°C; 35–50% humidity; n = 8) or HS conditions (35°C; 24–43% humidity; n = 8) for 24 h. Compared to TN, rectal temperatures in HS pigs increased by 1.6°C and respiration rates by 2-fold (P<0.05). As expected, HS decreased feed intake by 53% (P<0.05) and body weight (P<0.05) compared to TN pigs. Ileum heat shock protein 70 expression increased (P<0.05), while intestinal integrity was compromised in the HS pigs (ileum and colon TER decreased; P<0.05). Furthermore, HS increased serum endotoxin concentrations (P = 0.05). Intestinal permeability was accompanied by an increase in protein expression of myosin light chain kinase (P<0.05) and casein kinase II-α (P = 0.06). Protein expression of tight junction (TJ) proteins in the ileum revealed claudin 3 and occludin expression to be increased overall due to HS (P<0.05), while there were no differences in claudin 1 expression. Intestinal glucose transport and blood glucose were elevated due to HS (P<0.05). This was supported by increased ileum Na+/K+ ATPase activity in HS pigs. SGLT-1 protein expression was unaltered; however, HS increased ileal GLUT-2 protein expression (P = 0.06). Altogether, these data indicate that HS reduce intestinal integrity and increase intestinal stress and glucose transport.

Satellite cell-mediated angiogenesis in vitro coincides with a functional hypoxia-inducible factor pathway

Muscle regeneration involves the coordination of myogenesis and revascularization to restore proper muscle function. Myogenesis is driven by resident stem cells termed satellite cells (SC), whereas angiogenesis arises from endothelial cells and perivascular cells of preexisting vascular segments and the collateral vasculature. Communication between myogenic and angiogenic cells seems plausible, especially given the number of growth factors produced by SC. To characterize these interactions, we developed an in vitro coculture model composed of rat skeletal muscle SC and microvascular fragments (MVF). In this system, isolated epididymal MVF suspended in collagen gel are cultured over a rat SC monolayer culture. In the presence of SC, MVF exhibit greater indices of angiogenesis than MVF cultured alone. A positive dose-dependent effect of SC conditioned medium (CM) on MVF growth was observed, suggesting that SC secrete soluble-acting growth factor(s). Next, we specifically blocked VEGF action in SC CM, and this was sufficient to abolish satellite cell-induced angiogenesis. Finally, hypoxia-inducible factor-1alpha (HIF-1alpha), a transcriptional regulator of VEGF gene expression, was found to be expressed in cultured SC and in putative SC in sections of in vivo stretch-injured rat muscle. Hypoxic culture conditions increased SC HIF-1alpha activity, which was positively associated with SC VEGF gene expression and protein levels. Collectively, these initial observations suggest that a heretofore unexplored aspect of satellite cell physiology is the initiation of a proangiogenic program.

RUMINANT NUTRITION SYMPOSIUM: Ruminant Production and Metabolic Responses to Heat Stress

Heat stress compromises efficient animal production by marginalizing nutrition, management, and genetic selection efforts to maximize performance endpoints. Modifying farm infrastructure has yielded modest success in mitigating heat stress-related losses, yet poor production during the summer remains arguably the costliest issue facing livestock producers. Reduced output (e.g., milk yield and muscle growth) during heat stress was traditionally thought to result from decreased nutrient intake (i.e., a classic biological response shared by all animals during environmental-induced hyperthermia). Our recent observations have begun to challenge this belief and indicate heat-stressed animals employ novel homeorhetic strategies to direct metabolic and fuel selection priorities independently of nutrient intake or energy balance. Alterations in systemic physiology support a shift in carbohydrate metabolism, evident by increased basal and stimulated circulating insulin concentrations. Perhaps most intriguing given the energetic shortfall of the heat-stressed animal is the apparent lack of basal adipose tissue mobilization coupled with a reduced responsiveness to lipolytic stimuli. Thus, the heat stress response markedly alters postabsorptive carbohydrate, lipid, and protein metabolism independently of reduced feed intake through coordinated changes in fuel supply and utilization by multiple tissues. Interestingly, the systemic, cellular, and molecular changes appear conserved amongst different species and physiological states. Ultimately, these changes result in the reprioritization of fuel selection during heat stress, which appears to be primarily responsible for reduced ruminant animal productivity during the warm summer months.

Nutritional interventions to alleviate the negative consequences of heat stress.

Energy metabolism is a highly coordinated process, and preferred fuel(s) differ among tissues. The hierarchy of substrate use can be affected by physiological status and environmental factors including high ambient temperature. Unabated heat eventually overwhelms homeothermic mechanisms resulting in heat stress, which compromises animal health, farm animal production, and human performance. Various aspects of heat stress physiology have been extensively studied, yet a clear understanding of the metabolic changes occurring at the cellular, tissue, and whole-body levels in response to an environmental heat load remains ill-defined. For reasons not yet clarified, circulating nonesterified fatty acid levels are reduced during heat stress, even in the presence of elevated stress hormones (epinephrine, glucagon, and cortisol), and heat-stressed animals often have a blunted lipolytic response to catabolic signals. Either directly because of or in coordination with this, animals experiencing environmental hyperthermia exhibit a shift toward carbohydrate use. These metabolic alterations occur coincident with increased circulating basal and stimulated plasma insulin concentrations. Limited data indicate that proper insulin action is necessary to effectively mount a response to heat stress and minimize heat-induced damage. Consistent with this idea, nutritional interventions targeting increased insulin action may improve tolerance and productivity during heat stress. Further research is warranted to uncover the effects of heat on parameters associated with energy metabolism so that more appropriate and effective treatment methodologies can be designed.

Heat stress and reduced plane of nutrition decreases intestinal integrity and function in pigs.

Heat stress can compromise intestinal integrity and induce leaky gut in a variety of species. Therefore, the objectives of this study were to determine if heat stress (HS) directly or indirectly (via reduced feed intake) increases intestinal permeability in growing pigs. We hypothesized that an increased heat-load causes physiological alterations to the intestinal epithelium, resulting in compromised barrier integrity and altered intestinal function that contributes to the overall severity of HS-related illness. Crossbred gilts (n=48, 43±4 kg BW) were housed in constant climate controlled rooms in individual pens and exposed to 1) thermal neutral (TN) conditions (20°C, 35-50% humidity) with ad libitum intake, 2) HS conditions (35°C, 20-35% humidity) with ad libitum feed intake, or 3) pair-fed in TN conditions (PFTN) to eliminate confounding effects of dissimilar feed intake. Pigs were sacrificed at 1, 3, or 7 d of environmental exposure and jejunum samples were mounted into modified Ussing chambers for assessment of transepithelial electrical resistance (TER) and intestinal fluorescein isothiocyanate (FITC)-labeled lipopolysaccharide (LPS) permeability (expressed as apparent permeability coefficient, APP). Further, gene and protein markers of intestinal integrity and stress were assessed. Irrespective of d of HS exposure, plasma endotoxin levels increased 45% (P<0.05) in HS compared with TN pigs, while jejunum TER decreased 30% (P<0.05) and LPS APP increased 2-fold (P<0.01). Furthermore, d 7 HS pigs tended (P=0.06) to have increased LPS APP (41%) compared with PFTN controls. Lysozyme and alkaline phosphatase activity decreased (46 and 59%, respectively; P<0.05) over time in HS pigs, while the immune cell marker, myeloperoxidase activity, was increased (P<0.05) in the jejunum at d 3 and 7. These results indicate that both HS and reduced feed intake decrease intestinal integrity and increase endotoxin permeability. We hypothesize that these events may lead to increased inflammation, which might contribute to reduced pig performance during warm summer months.