The purpose of this investigation was to establish the role biological sex plays in circulating factors following heat stress (HS). Barrows and gilts (36.8 +/- 3.7 kg body weight) were kept in either thermoneutral (TN; 20.8 +/- 1.6 degrees C; 62.0% +/- 4.7% relative humidity; n = 8/sex) conditions or exposed to HS (39.4 +/- 0.6 degrees C; 33.7% +/- 6.3% relative humidity) for either 1 (HS1; n = 8/sex) or 7 (HS7; n = 8/sex) d. Circulating glucose decreased as a main effect of the environment (P = 0.03). Circulating non-esterified fatty acid (NEFA) had an environment x sex interaction (P < 0.01) as HS1 barrows had increased NEFA compared to HS1 gilts (P = 0.01) and NEFA from HS7 gilts increased compared to HS1 gilts (P = 0.02) and HS7 barrows (P = 0.04). Cortisol, insulin, glucagon, T3, and T4 were reduced as a main effect of environment (P <= 0.01). Creatinine was increased in HS1 and HS7 animals compared to TN (P <= 0.01), indicative of decreased glomerular filtration rate. White blood cell populations exhibited differential patterns based on sex and time. Neutrophils and lymphocytes had an environment x sex interaction (P <= 0.05) as circulating neutrophils were increased in HS1 barrows compared to TN and HS7 barrows, and HS1 gilts (P <= 0.01) and HS7 barrows had less neutrophils compared to TN barrows (P = 0.01), whereas they remained similar in gilts. In contrast, barrow lymphocyte numbers were similar between groups, but in HS7 gilts they were decreased compared to TN and HS1 gilts (P <= 0.04). In total, these data demonstrate that HS alters a host of circulating factors and that biological sex mediates, at least in part, the physiological response to HS.

Abstract Environmental heat stress (HS) can impair muscle mitochondrial function which may contribute to negative health outcomes, but little is known about potential treatments to rescue mitochondrial function during HS. To test the hypothesis that thyroid hormone (T3) would protect mitochondrial function during HS, samples were collected from the right ventricle (RV) of 3-mo-old, crossbred gilts under 4 different environmental conditions: thermoneutral (TN; 22°C; n = 8); 1 d HS (HS1; 38°C; n = 7); oral supplementation of 1.33 µgּ kg body weight (BW)-1ּ d-1 Cytomel (liothyronine sodium) during the 1 d HS (HS1TH; n = 8); and 7 d HS (HS7; n = 8). The oxidative portion of the semitendinosus (ST) was also collected from all groups plus a 5th group: oral supplementation of Cytomel during 7 d HS (HS7TH, n = 8). Mitochondrial volume density was estimated by citrate synthase (CS) activity, and oxidative phosphorylation (OP) and electron transfer (E) capacities were determined via high resolution respirometry. Data were analyzed using mixed linear models in SAS v9.4 with treatment group as a fixed effect and pig (treatment) as a random effect. Within the RV, the contribution of OP supported by complex I (PCI) to maximal E was less in HS1TH gilts compared with all other groups (P ≤ 0.02), and the contribution of E supported by complex II (ECII) to maximal E was greater in TN than HS1 or HS7 (P ≤ 0.02) and tended to be greater in TN compared with HS1TH gilts (P = 0.07). Within the ST, integrative (relative to tissue wet weight) PCI was greatest in TN gilts (vs. HS7 and HS7TH, P ≤ 0.0002; vs. HS1 and HS1TH, P ≤ 0.06) and least in HS7 and HS7TH (vs. HS1 and HS1TH, P ≤ 0.05). Integrative maximal P (PCI+II) was greatest in TN pigs (compared with HS1TH, HS7, and HS7TH, P ≤ 0.05; compared with HS1, P = 0.09). Integrative maximal E (ECI+II) was also greatest in TN gilts than all other groups (P ≤ 0.05) while ECII was greater in TN pigs compared with HS7 and HS7TH (P ≤ 0.009) and tended to be greater than HS1 and HS1TH (P ≤ 0.09). In ST, intrinsic (relative to CS activity) PCI and PCI+II were greater in TN compared with HS1, HS1TH, and HS7 pigs (P ≤ 0.03) and PCI+II tended to be greater in TN than HS7TH (P ≤ 0.06). Intrinsic ECI+II and ECII were greater in TN compared with all other treatment groups (P ≤ 0.04). These results suggest that T3 supplementation did not protect mitochondrial function from negative impacts of HS in skeletal muscle but may have modified electron transfer through complex I in cardiac muscle which may have implications on oxidative stress.

Oxidative stress contributes to heat stress (HS)-mediated alterations in skeletal muscle; however, the extent to which biological sex mediates oxidative stress during HS remains unknown. We hypothesized muscle from males would be more resistant to oxidative stress caused by HS than muscle from females. To address this, male and female pigs were housed in thermoneutral conditions (TN; 20.8 ± 1.6 °C; 62.0 ± 4.7% relative humidity; n = 8/sex) or subjected to HS (39.4 ± 0.6 °C; 33.7 ± 6.3% relative humidity) for 1 (HS1; n = 8/sex) or 7 days (HS7; n = 8/sex) followed by collection of the oxidative portion of the semitendinosus. While HS increased muscle temperature, by 7 d, muscle from heat-stressed females was cooler than muscle from heat-stressed males (0.3 °C; p <0.05). Relative protein abundance of 4-HNE-modified proteins increased in HS1 females compared to TN (p=0.05). Further, MDA-modified proteins and 8-OHdG concentration, a DNA damage marker, was increased in HS7 females compared to TN females (p=0.05). Enzymatic activities of catalase and SOD remained similar between groups; however, GPX activity decreased in HS7 females compared to TN and HS1 females (p≤0.03) and HS7 males (p=0.02). Notably, HS increased skeletal muscle Ca2+ deposition (p=0.05) and was greater in HS1 females compared to TN females (p<0.05). Heat stress increased SERCA2a protein abundance (p<0.01); however, Ca2+ ATPase activity remained similar between groups. Overall, despite having lower muscle temperature, muscle from heat-stressed females had increased markers of oxidative stress and calcium deposition than muscle from males following identical environmental exposure.

Probiotics for humans and direct-fed microbials for livestock are increasingly popular dietary ingredients for supporting immunity. The aim of this study was to determine the effects of dietary supplementation of Bacillus subtilis MB40 (MB40) on immunity in piglets challenged with the foodborne pathogen Listeria monocytogenes (LM). Three-week-old piglets (n = 32) were randomly assigned to four groups: (1) basal diet, (2) basal diet with LM challenge, (3) MB40-supplemented diet, and (4) MB40-supplemented diet with LM challenge. Experimental diets were provided throughout a 14-day (d) period. On d8, piglets in groups 2 and 4 were intraperitoneally inoculated with LM at 108 CFU/mL per piglet. Blood samples were collected at d1, d8, and d15 for biochemical and immune response profiling. Animals were euthanized and necropsied at d15 for liver and spleen bacterial counts and intestinal morphological analysis. At d15, LM challenge was associated with increased spleen weight (p = 0.017), greater circulating populations of neutrophils (p = 0.001) and monocytes (p = 0.008), and reduced ileal villus height to crypt depth ratio (p = 0.009), compared to non-challenged controls. MB40 supplementation reduced LM bacterial counts in the liver and spleen by 67% (p < 0.001) and 49% (p < 0.001), respectively, following the LM challenge, compared to the basal diet. MB40 supplementation was also associated with decreased circulating concentrations of monocytes (p = 0.007). Altogether, these data suggest that MB40 supplementation is a safe and well-tolerated approach to enhance immunity during systemic Listeria infection.

Consumption of zearalenone (ZEN) detrimentally affects tissues and systems throughout the body, and these deleterious effects are especially pronounced in swine. The objectives of this project were to determine the effects of short-term consumption of ZEN (at concentrations that could be found on-farm) on growth, carcass weight, liver weight, and reproductive tissues of pubertal gilts, and to determine if the effects are transient or persistent. Cross-bred gilts (107.25 +/- 2.69 kg) were randomly assigned to one of three feed treatments: 1) solvent only for 21 d (CON; n = 10), 2) ZEN for 7 d followed by 14 d of solvent (ZEN-7; 6 mg/d; n = 10), and 3) ZEN for 21 d (ZEN-21; 6 mg/d; n = 10). Body weights were collected at the beginning and end of the experiment (189.1 +/- 0.8 and 211.1 +/- 0.8 d of age, respectively). Carcass weights and tissues were collected at harvest. There were no treatment-based differences in growth, carcass, liver, or reproductive tissue weights. Histological analyses revealed differences based on treatment and the interaction between treatment and luteal status. The thickness of the ampullary muscularis declined with ZEN exposure (P < 0.05), while the isthmic epithelial cell height (P < 0.01) and uterine endometrial thickness (P < 0.02) increased. Interestingly, the thickness of the isthmic muscularis, uterine myometrium, and epithelial cell height only differed in the presence of a corpus luteum. Uterine epithelial cell height in the luteal phase was lowest in ZEN-7 pigs (P < 0.01). The isthmic muscularis in the luteal phase was thinner in pigs from both ZEN treatments (P < 0.01). Conversely, the luteal-stage myometrium was thicker in pigs from both ZEN treatments (P < 0.01). The discovery of these tissue-based differences during the luteal phase is particularly concerning since this corresponds with the time when embryos would be affected by the functional competency of the oviduct and uterus. The results of this work demonstrate that short-term consumption of ZEN produces microscopic, but not macroscopic alterations in reproductive organs which are likely to have negative effects on their subsequent function and that these differences persist even after ZEN consumption ceases. Taken together, these results indicate that it is insufficient to rely solely on outwardly visible symptoms as indicators of zearalenone exposure, as detrimental effects on reproductive tissues were found in the absence of phenotypic and morphologic changes. Gilts consuming zearalenone-treated feed exhibited histological changes in their reproductive tissues, even in the absence of gross morphological differences. This observation suggests that reproductive performance is affected by zearalenone consumption at concentrations that do not elicit outwardly visible symptoms. Lay Summary The mycotoxin zearalenone is a common contaminant of livestock feed. The consumption of zearalenone is particularly problematic for pigs as they are very sensitive to its effects. This study evaluated the effects of zearalenone on growth, carcass weight, liver weight, and reproductive tissues in young female pigs. Thirty pigs were split across three treatment groups. The control group was given standard feed (no zearalenone added) for 21 d, the second group received zearalenone-treated feed for 7 d followed by 14 d of standard feed, and the third group received zearalenone-treated feed for the full 21 d. Pigs receiving the treated feed exhibited no visible symptoms associated with zearalenone consumption. There were also no treatment-related differences in growth, carcass weight, liver weight, or reproductive tract weight. Histological analyses of both the oviduct and uterus revealed changes in tissue thickness that could indicate potential impairments in reproductive organ function. Changes in tissue layer thickness were especially prominent in the luteal phase. This interaction between the treatment and the presence of a corpus luteum is noteworthy because tract function during the luteal phase is imperative for fertilization and early embryonic development.

Heat stress (HS) compromises farm animal productivity and it is thus both an economic and food security issue. Reduced output (e.g., milk yield, muscle growth, egg production) during HS was traditionally thought to result from decreased nutrient intake (a classic biological response shared by all animals during environmental-induced hyperthermia). Utilizing pair-feeding experimental designs clearly indicate that reduced feed intake only accounts for a portion of the decreased productivity. The etiological epicenter of many (if not most) of the physiological consequences of HS is gastrointestinal tract hyperpermeability. For reasons not clearly understood, HS compromises the intestinal barrier allowing luminal antigens to infiltrate into local and systemic circulation. The invading antigens are recognized and stimulate the immune system. An activated immune system rearranges the hierarchy of nutrient partitioning, and the entire body participates in ensuring that leukocytes receive the fuel and building blocks necessary to mount an effective response. Central to this is increased insulin secretion coupled with simultaneous decreased adipose tissue and skeletal muscle insulin resistance. Activated leukocytes switch their energetic metabolism away from oxidative phosphorylation to aerobic glycolysis. Utilizing a LPS-Euglycemic clamp we have estimated that an intensely activated immune system can use more than 2 kg of glucose/d in lactating cows. We extended this in growing steers and pigs and the amount of glucose used is approximately 1 g/Kg BW0.75/h regardless of species and physiological state. If the heat load is severe enough, the consequences can become lethal. This is primarily explained by inflammation induced hypercoagulation, endothelial barrier dysfunction and disseminated intravascular coagulation. If the animal survives, these changes result in the reprioritization of fuel selection during HS which appears to be primarily responsible for reduced animal productivity during the warm summer months.

The objective of this work was to evaluate the potential benefits of short-duration, high-dose chromium (Cr) supplementation in early postpartum dairy cows during the summer months. Multiparous, early-lactation cows (20.95 ± 0.21 d in milk) were assigned to 1 of 2 treatment groups: (1) control diet (Con; n = 10) or (2) control diet + Cr propionate (CrPro; 12 mg/head per day Cr; n = 12). Measurements of ovarian structures, respiration rates (RR), rectal temperatures (RT), and blood glucose concentrations were recorded every 3 d. Blood was also collected for analysis of plasma progesterone concentrations. Every 6 d, in conjunction with ultrasonography, endometrial cytology samples were collected via cytobrush from each cow to determine the incidences of subclinical endometritis, as determined by polymorphonuclear leukocyte (PMNL)%. No differences were detected in RR, RT, blood glucose, feed intake, milk yield, or change in body weight. The supplementation did, however, improve some reproductive parameters. At cytology sample 6, the PMNL% increased in Con cows, and was greater than the PMNL% in the CrPro group. Chromium consumption did not affect the number or size of most follicles, with the exception being the 6 to 9 mm category where the CrPro group had a greater average diameter and tended to have greater numbers of follicles in this category. While corpus luteum numbers and size did not differ between treatments, the ratio of progesterone to average corpus luteum volume was greater in the CrPro group compared with the Con group. The results from this study indicate that, whereas the short-term, high-dose supplementation strategy did not affect feed intake or milk yield, this Cr supplementation strategy could benefit reproductive performance during periods of stress.

Substantial economic losses in animal agriculture result from animals experiencing heat stress (HS). Pigs are especially susceptible to HS, resulting in reductions in growth, altered body composition, and compromised substrate metabolism. In this study, an artificial high-intensity sweetener and capsaicin (CAPS-SUC; Pancosma, Switzerland) were supplemented in combination to mitigate the adverse effects of HS on pig performance. Forty cross-bred barrows (16.2 ± 6 kg) were assigned to one of five treatments: thermal neutral controls (TN) (22 ± 1.2 °C; 38%-73% relative humidity) with ad libitum feed, HS conditions with ad libitum feed with (HS+) or without (HS-) supplementation, and pair-fed to HS with (PF+) or without supplementation (PF-). Pigs in heat-stressed treatments were exposed to a cyclical environmental temperature of 12 h at 35 ± 1.2 °C with 27%-45% relative humidity and 12 h at 30 ± 1.1 °C with 24%-35% relative humidity for 21 d. Supplementation (0.1 g/kg feed) began 7 d before and persisted through the duration of environmental or dietary treatments (HS/PF), which lasted for 21 d. Rectal temperatures and respiration rates (RR; breaths/minute) were recorded thrice daily, and feed intake (FI) was recorded daily. Before the start and at the termination of environmental treatments (HS/PF), a muscle biopsy of the longissimus dorsi was taken for metabolic analyses. Blood samples were collected weekly, and animals were weighed every 3 d during treatment. Core temperature (TN 39.2 ± 0.02 °C, HS- 39.6 ± 0.02 °C, and HS+ 39.6 ± 0.02 °C, P < 0.001) and RR (P < 0.001) were increased in both HS- and HS+ groups, but no difference was detected between HS- and HS+. PF- pigs exhibited reduced core temperature (39.1 ± 0.02 °C, P < 0.001), which was restored in PF+ pigs (39.3 ± 0.02 °C) to match TN. Weight gain and feed efficiency were reduced in PF- pigs (P < 0.05) but not in the PF+ or the HS- or HS+ groups. Metabolic flexibility was decreased in the HS- group (-48.4%, P < 0.05) but maintained in the HS+ group. CAPS-SUC did not influence core temperature or weight gain in HS pigs but did restore core temperature, weight gain, and feed efficiency in supplemented PF pigs. In addition, supplementation restored metabolic flexibility during HS and improved weight gain and feed efficiency during PF, highlighting CAPS-SUC's therapeutic metabolic effects.

With continued trends toward more frequent and more severe heat events there is a greater risk of environment‐induced heat stress (HS). How persistently elevated core temperatures impacts body tissues remains largely unknown. Our preliminary evidence indicates HS may negatively impact cardiac tissue and that biological sex may play a role in HS‐mediated pathology. To further test the hypothesis that cardiac muscle mitochondria are more negatively impacted by HS in females than in males, samples were isolated from the left ventricle (LV) of the heart from 3‐month‐old castrated male and prepubertal female pigs that were exposed to thermoneutral (TN) conditions (n = 4 per sex) or HS conditions for 24 h at 37°C (HS; n = 6 per sex). Samples were analyzed for citrate synthase (CS) and cytochrome c oxidase (COX) activities, and for mitochondrial oxidative phosphorylation (P) and electron transfer (E) capacities via high resolution respirometry. Data were analyzed by mixed linear models in SAS v9.4 with treatment, sex, and treatment × sex as fixed effects. A traditional marker of mitochondrial volume density, CS activity, tended to be greater in TN males compared to HS males (P = 0.08) but was unaffected by HS in female pigs. Conversely, intrinsic (relative to CS activity) mitochondrial function, as measured by COX activity, was greater in TN females than HS females (P = 0.04) and within HS, males maintained greater intrinsic mitochondrial function than females (P = 0.02). Both integrative (relative to tissue wet weight) and intrinsic mitochondrial leak respiration were greater in HS than TN pigs (P = 0.03) but the contribution of leak to total E (flux control ratio; FCRLEAK) tended to be greater in males than females (P = 0.07). Conversely, both integrative and intrinsic maximal E (ECI+II) tended to be greater in female than male pigs (P = 0.08) indicating a greater degree of excess electron transfer capacity in females. The contribution of complex I‐supported P (PCI) to total E (FCRPCI) tended to be greater in TN compared to HS pigs regardless of sex (P = 0.09) and the contribution of maximal P to E (FCRPCI+II) was greater in TN females compared to HS females (P = 0.05). This resulted in the FCRPCI+IIbeing greater in males compared to females in the HS condition (P = 0.01), suggesting greater coupling of oxidative phosphorylation and electron transfer in cardiac mitochondria in males during 24 h of HS. These results suggest that HS may negatively impact cardiac muscle mitochondria in female pigs compared to males. Moreover, these data indicate that persistent HS has deleterious consequences for the myocardium and may represent an additional health concern caused by global climate trends. This project was supported by USDA NIFA Award #2020‐68014‐31954.

Heat stress (HS) negatively impacts animal health and impairs growth, but little is known about sex‐specific responses of skeletal muscle mitochondria to HS. To test the hypothesis that HS would have more adverse effects on mitochondria in females than males, samples were collected from the oxidative portion of the semitendinosus muscle of 3‐month‐old female and castrated male pigs under thermoneutral (TN) conditions or after 1 or 7 d of HS at 39.4°C (n=8/sex/group). Mitochondrial volume density and function were determined via citrate synthase (CS) and cytochrome c oxidase activities. Mitochondrial oxidative phosphorylation (P) and electron transfer (E) capacities were evaluated by high resolution respirometry. Data were analyzed using mixed linear models in SAS v9.4 with treatment, sex, and treatment × sex as fixed effects. Overall, integrative (relative to tissue weight) maximal P with complex I and II (PCI+II) and E (ECI+II) were greater after 1 and 7 d of HS compared to TN conditions (P≤0.05). The contribution of PCI+II to maximal E (flux control ratio; FCRPCI+II) was greater in TN pigs compared to either HS group (P≤0.03), suggesting decreased efficiency of mitochondrial energy production following HS. However, there appeared to be some adaptation to heat over time as FCRPCI+IIwas greater in pigs that were heat stressed for 7 d compared to 1 d (P=0.05). As hypothesized, some measures differed by sex. Specifically, in females, intrinsic (relative to CS activity) complex I‐supported P (PCI) tended to be greater following 7 d of HS compared to TN (P=0.07); PCI was unaffected by heat in males, resulting in females having greater intrinsic PCI than males following 7 d of HS (P=0.05). The contribution of PCI to total E (FCRPCI) was lesser with 7 d HS than 1 d or TN conditions in male pigs (P≤0.01) but unchanged in females, resulting in females also having greater FCRPCIthan males following 7 d of HS (P=0.008). Conversely, the contribution of electron transfer supported by complex II to total E (FCRECII) was greater in TN compared to 1 d of HS in males (P=0.004) but then increased at 7 d of HS (P=0.02) so that TN and 7 d HS were similar. In females, FCRECII tended to be lesser following 7 d of HS compared to TN, resulting in a trend for males to have greater FCRECII than females after 7 d of HS (P=0.08). Finally, males tended to have greater FCRPCI+IIthan females (P≤0.08). These results suggest HS increased mitochondrial utilization of complex I in females but reliance on complex II in males, resulting in more efficient mitochondria following HS in males than in females. Therefore, skeletal muscle in females may be more susceptible to negative cellular impacts of HS.

Heat stress can negatively impact pig health and performance but the effects of heat stress on skeletal muscle mitochondrial function are largely unknown. We hypothesized that mitochondrial function and capacity would be impaired in heat stressed (HS) compared to thermoneutral (TN) pigs but mitochondrially-targeted coenzyme Q (MitoQ) supplementation would rescue the impairment. Oxidative portions of the semitendinosus muscle were evaluated from TN and HS gilts receiving no supplementation (CON) or MitoQ for 2 d prior to and during the 24h environmental heat treatment (n = 8 per group). Mitochondrial oxidative phosphorylation (P) and electron transfer (E) capacities were determined via high resolution respirometry and mitochondrial volume density and function were quantified by citrate synthase (CS) and cytochrome c oxidase activities, respectively. Data were analyzed using linear models in SAS v9.4 with fixed effects of heat, MitoQ treatment (trt), and heat×trt interaction. There were trends for the interaction of trt and heat (P≤0.1) on integrative (per mg tissue) and intrinsic (relative to CS) P with complexes I and II (PCI+II), maximum noncoupled E (ECI+II), and E with complex II only (ECII), in which all measures were greater in HS-MitoQ than TN-MitoQ (P≤0.03), but measures did not differ due to HS in CON pigs. The contribution of leak to total E (flux control ratio, FCRLeak) was lesser in HS-MitoQ than HS-CON, TN-CON, and TN-MitoQ (P≤0.02). The FCRPCI was greater (P≤0.05) while the FCRPCI+II was lesser (P=0.01) in TN compared to HS pigs. Finally, the FCRPCI+II was greater (P=0.02) while the FCRECII tended to be lesser (P=0.09) for CON than MitoQ pigs. Neither mitochondrial volume density nor function were affected by HS or MitoQ supplementation. In total, these data indicate improved mitochondrial capacities following heat stress in pigs receiving MitoQ but no difference in mitochondrial capacities in unsupplemented, HS pigs.

Adverse weather conditions are a large constraint to maximizing farm animal productivity. Heat stress, in particular, compromises almost every metric of animal agriculture profitability. Suboptimal production during HS was traditionally thought to result from hypophagia. However, independent of inadequate nutrient consumption, HS affects a plethora of endocrine, physiological, metabolic, circulatory, and immunological variables. Mounting evidence suggest that direct effects of HS originating at the gastrointestinal tract precede the observed effects on the aforementioned systems. Heat stress compromises intestinal barrier integrity causing the appearance of luminal contents, e.g. endotoxin, in circulation. Endotoxin stimulates both a classic immune response with local and systemic inflammatory reactions as well as directly acting on numerous organs and tissues. Once activated, leukocytes switch from oxidative phosphorylation to aerobic glycolysis where the glucose requirement of an intensely triggered immune system can exceed 2 kg/d in a lactating dairy cow. Whole body metabolic adjustments are primarily characterized by increased basal and stimulated circulating insulin, increased hepatic glucose output, decreased adipose tissue mobilization, and decreased skeletal muscle flexibility characterized by a reliance on glucose as a fuel substrate rather than lipid. Ultimately, the metabolic and physiological consequences of heat stress share a similar phenotype with immune challenges. Describing the physiology and mechanisms that underpin how HS jeopardizes animal performance is critical for developing approaches to ameliorate current production issues and requisite for generating future strategies (genetic, managerial, nutritional, and pharmaceutical) aimed at optimizing animal well-being, and improving the sustainable production of high-quality protein for human consumption.

Study objectives were to determine the effects of rapamycin (Rapa) on biomarkers of metabolism and inflammation during acute heat stress (HS) in growing pigs. Crossbred barrows (n = 32; 63.5 ± 7.2 kg body weight [BW]) were blocked by initial BW and randomly assigned to 1 of 4 environmental-therapeutic treatments: 1) thermoneutral (TN) control (n = 8; TNCon), 2) TN and Rapa (n = 8; TNRapa), 3) HS control (n = 8; HSCon), or 4) HS and Rapa (n = 8; HSRapa). Following 6 d of acclimation to individual pens, pigs were enrolled in two experimental periods (P). During P1 (10 d), pigs were fed ad libitum and housed in TN conditions (21.3 ± 0.2°C). During P2 (24 h), HSCon and HSRapa pigs were exposed to constant HS (35.5 ± 0.4°C), while TNCon and TNRapa pigs remained in TN conditions. Rapamycin (0.15 mg/kg BW) was orally administered twice daily (0700 and 1800 hours) during both P1 and P2. HS increased rectal temperature and respiration rate compared to TN treatments (1.3°C and 87 breaths/min, respectively; P < 0.01). Feed intake (FI) markedly decreased in HS relative to TN treatments (64%; P < 0.01). Additionally, pigs exposed to HS lost BW (4 kg; P < 0.01), while TN pigs gained BW (0.7 kg; P < 0.01). Despite marked changes in phenotypic parameters caused by HS, circulating glucose and blood urea nitrogen did not differ among treatments (P > 0.10). However, the insulin:FI increased in HS relative to TN treatments (P = 0.04). Plasma nonesterified fatty acids (NEFA) increased in HS relative to TN treatments; although this difference was driven by increased NEFA in HSCon compared to TN and HSRapa pigs (P < 0.01). Overall, circulating white blood cells, lymphocytes, and monocytes decreased in HS compared to TN pigs (19%, 23%, and 33%, respectively; P ≤ 0.05). However, circulating neutrophils were similar across treatments (P > 0.31). The neutrophil-to-lymphocyte ratio (NLR) was increased in HS relative to TN pigs (P = 0.02); however, a tendency for reduced NLR was observed in HSRapa compared to HSCon pigs (21%; P = 0.06). Plasma C-reactive protein tended to differ across treatments (P = 0.06) and was increased in HSRapa relative to HSCon pigs (46%; P = 0.03). Circulating haptoglobin was similar between groups. In summary, pigs exposed to HS had altered phenotypic, metabolic, and leukocyte responses; however, Rapa administration had limited impact on outcomes measured herein.

The ASAS Public Policy Committee (PPC) provides updates of Grand Challenges (GCs; www.asas.org/about/public-policy/asas-grand-challenges) to clearly articulate research priorities, to provide science-based information for shaping public policy, and to enhance future funding for research and education programs in animal sciences (AS). In this nexus symposium for 2021, PPC examines previous stated priorities and provides a progress report card and offers additional perspectives and recommendations for research needed to address some of the GCs continuing to face animal agriculture. Among the GC topics is growth and development, a rather broad field of inquiry focused on improving the overall growth efficiency of meat producing animals. The genesis of this discipline and its popularity grew mainly in response to heightened efforts by pharmaceutical companies to identify, develop and adopt novel new growth promotants. This included a myriad of work on the highly heralded technologies involving estrogenic and androgenic implants, somatotropin, and beta-adrenergic agonists. Because the potential application of these technologies was so broad, many disciplines within the animal sciences became involved in the process of creating knowledge around these drivers of productivity. In the process, our understanding of how tissues grow in response to these compounds, under a myriad of other conditions, and our fundamental understanding of the molecular and cellular mechanisms regulating growth and development, expanded significantly. Areas of significant expansion included but were not restricted to: satellite cell biology and myogenesis, whole body and tissue-specific protein synthesis and degradation, growth factor biology, adipogenesis, and repartitioning of nutrients throughout the body. In our quest to increase productivity and product quality, coupled with advances in scientific techniques, long-existing and emerging genetic mutations with desirable traits were studied and mechanisms undergirding their biology began to develop. Applying the most innovative tools for the detailed manipulation of cellular processes, great strides were made during this time. However, this eclectic area of investigation is perhaps more important than ever given the inevitable replacement of growth promotant technologies with new and emerging genomic technologies. Many biological research challenges lie ahead such as applications of gene editing, RNA control, and epigenetic regulation through fetal programming. This presentation will review some of the significant advances made in the growth and development area and explore where significant gains may be possible in the future.

Heat stress (HS) poses a major threat to human health and agricultural production. Oxidative stress and mitochondrial dysfunction appear to play key roles in muscle injury caused by HS. We hypothesized that mitoquinol (MitoQ), would alleviate oxidative stress and cellular dysfunction in skeletal muscle during HS. To address this, crossbred barrows (male pigs) were treated with placebo or MitoQ (40 mg/d) and were then exposed to thermoneutral (TN; 20 ?C) or HS (35 ?C) conditions for 24 h. Pigs were euthanized following the environmental challenge and the red portion of the semitendinosus (STR) was collected for analysis. Unexpectedly, malondialdehyde concentration, an oxidative stress marker, was similar between environmental and supplement treatments. Heat stress decreased LC3A/B?I (p < 0.05) and increased the ratio of LC3A/B-II/I (p < 0.05), while p62 was similar among groups suggesting increased degradation of autophagosomes during HS. These outcomes were in disagreement with our previous results in muscle from gilts (female pigs). To probe the impact of biological sex on HS-mediated injury in skeletal muscle, we compared STR from these barrows to archived STR from gilts subjected to a similar environmental intervention. We confirmed our previous findings of HS-mediated dysfunction in muscle from gilts but not barrows. These data also raise the possibility that muscle from gilts is more susceptible to environment-induced hyperthermia than muscle from barrows.

Heat stress (HS) diminishes animal production, reducing muscle growth and increasing adiposity, especially in swine. Excess heat creates a metabolic phenotype with limited lipid oxidation that relies on aerobic and anaerobic glycolysis as a predominant means of energy production, potentially reducing metabolic rate. To evaluate the effects of HS on substrate utilization and energy expenditure, crossbred barrows (15.2 ± 2.4 kg) were acclimatized for 5 days (22 °C), then treated with 5 days of TN (thermal neutral, 22 °C, n = 8) or HS (35 °C, n = 8). Pigs were fed ad libitum and monitored for respiratory rate (RR) and rectal temperature. Daily energy expenditure (DEE) and respiratory exchange ratio (RER, CO2:O2) were evaluated fasted in an enclosed chamber through indirect calorimetry. Muscle biopsies were obtained from the longissimus dorsi pre/post. HS increased temperature (39.2 ± 0.1 vs. 39.6 ± 0.1 °C, p < 0.01) and RER (0.91 ± 0.02 vs. 1.02 ± 0.02 VCO2:VO2, p < 0.01), but decreased DEE/BW (68.8 ± 1.7 vs. 49.7 ± 4.8 kcal/day/kg, p < 0.01) relative to TN. Weight gain (p = 0.80) and feed intake (p = 0.84) did not differ between HS and TN groups. HS decreased muscle metabolic flexibility (~33%, p = 0.01), but increased leucine oxidation (~35%, p = 0.02) compared to baseline values. These data demonstrate that HS disrupts substrate regulation and energy expenditure in growing pigs.

Satellite cells (SC) aid skeletal muscle growth and regeneration. SC-mediated skeletal muscle repair can both be influenced by and exacerbate several diseases linked to a fatty diet, obesity, and aging. The purpose of this study was to evaluate the effects of different lifestyle factors on SC function, including body mass index (BMI), age, and high-fat overfeeding. For this study, SCs were isolated from the vastus lateralis of sedentary young (18–30 years) and sedentary older (60–80 years) men with varying BMIs (18–32 kg/m2), as well as young sedentary men before and after four weeks of overfeeding (OVF) (55% fat/ + 1000 kcal, n = 4). The isolated SCs were then treated in vitro with a control (5 mM glucose, 10% fetal bovine serum (FBS)) or a high substrate growth media (HSM) (10% FBS, 25 mM glucose, and 400 μM 2:1 oleate–palmitate). Cells were assessed on their ability to proliferate, differentiate, and fuel substrate oxidation after differentiation. The effect of HSM was measured as the percentage difference between SCs exposed to HSM compared to control media. In vitro SC function was not affected by donor age. OVF reduced SC proliferation rates (–19% p < 0.05) but did not influence differentiation. Cellular proliferation in response to HSM was correlated to the donor’s body mass index (BMI) (r2 = 0.6121, p < 0.01). When exposed to HSM, SCs from normal weight (BMI 18–25 kg/m2) participants exhibited reduced proliferation and fusion rates with increased fatty-acid oxidation (p < 0.05), while SCs from participants with higher BMIs (BMI 25–32 kg/m2) demonstrated enhanced proliferation in HSM. HSM reduced proliferation and fusion (p < 0.05) in SCs isolated from subjects before OVF, whereas HSM exposure accelerated proliferation and fusion in SCs collected following OVF. These results indicated that diet has a greater influence on SC function than age and BMI. Though age and BMI do not influence in vitro SC function when grown in controlled conditions, both factors influenced the response of SCs to substrate challenges, indicating age and BMI may mediate responses to diet.

Heat stress (HS) alters animal metabolism causing reduced performance (muscle growth) while increasing the incidence of disease and mortality. HS is particularly detrimental in the swine industry where the global economic burden of heat stress is in the billions of dollars annually. Excess environmental heat promotes a HS response increasing the expression of heat shock factors and heat shock proteins which coordinate a shift in metabolic substrate preference. The net result of these changes is a metabolic phenotype with limited lipid oxidation that relies on aerobic and anaerobic glycolysis as a predominant source of energy production. This study was designed to evaluate the effect of HS on substrate utilization and overall animal metabolic rate in growing pigs. Crossbred barrows (15.2±2.4 kg) were exposed to 5 days of TN (thermal neutral, 24 C°) or HS ( 35 C°) (n=8 per treatment), after a 5‐day acclimation period (24 C°). Pigs were fed ad libitum and monitored regularly for respiratory rate (RR) and rectal temperature (3x daily). A metabolic cart was used to assess daily energy expenditure (DEE), and respiratory exchange ratio (RER, CO2:O2), pigs were placed in an enclosed chamber and gas exchange was measured for 1 hour after a 30 min washout. Muscle biopsies were taken from the longissimus dorsi to evaluated palmitate, pyruvate, and leucine oxidation capacity and metabolic flexibility (metflex). Metabolic measures and muscle biopsies were taken after five days of acclimation (pre) and immediately following (post) the 5‐day environmental treatments. Initial DEE was positively correlated to weight (r2=0.55, p<0.01) and feed intake (r2=0.40, p<0.01), initial DEE relative to bodyweight (DEE/BW) correlated to metflex (r2=0.29, p=0.03). HS increased RR (94.3±4.5 vs 55.9±2 BPM, p<0.01) and rectal temperature (39.6±0.1 vs 39.2±0.1 C°, p<0.01) compared to TN controls. Weight gain and feed intake did not differ between HS and TN groups. Seven days of HS increased RER (0.91±0.02 vs 1.02±0.02 VCO2:VO2, p<0.01) and decreased DEE/BW (68.8±1.7 vs 49.7±4.8 kcal/kg, p<0.01) compared to baseline values. Heat stress also decreased muscle palmitate oxidation (−20.1%, p=0.04) and metflex (28.8±3.5 vs 19.2±3.7, P=0.01), while causing an increase in leucine oxidation (14.1±0.9 vs 19.0±2.2 nmol/mg‐pro/hr, p=0.02) compared to baseline values. Growth rates of HS animals were positively correlated to post DEE/BW (r2=0.75, p<0.01), while negatively correlated to changes in rectal temperature (r2=0.54, p=0.04). Previous research has shown a J‐shaped relationship between environmental temperature and energy expenditure, with both cold and hot environments increasing energy expenditure. However, these data demonstrate that chronic HS may decrease energy expenditure at least relative to body weight in growing pigs. This reduction in energy expenditure appears to be mechanistically controlled through substrate regulation as HS increased RER and decreased lipid oxidation and metflex. To overcome limits in lipid oxidation, amino acid use for energy may be upregulated which can further limit potential for muscle growth.

Pigs exposed to elevated ambient temperatures exhibit reduced daily gain, alterations in muscle and fat deposition, and decreased health. Negative aspects of gastrointestinal (GI) function, integrity, and permeability also occur. High-intensity sweeteners can ameliorate the negative effects of heat stress (HS) by increasing GI glucagon-like peptide-2 production while capsicum oleoresin has been shown to reduce inflammatory response. The effects of an artificial high-intensity sweetener and capsicum oleoresin (CAPS-SUC; TakTik X-Hit, Pancosma, Switzerland) on growth performance of pigs were examined. Forty-eight pigs (12 wk of age, 43.2 +/- 4.3 kg) were assigned to six treatments: thermoneutral conditions (21 +/- 1.1 degrees C; 40% to 70% relative humidity) fed ad libitum with (TN+) or without supplement (TN-), heat stress (35 +/- 1 degrees C; 20% to 40% relative humidity) fed ad libitum with (HS+) or without supplement (HS-), and thermoneutral conditions pair-fed to HS intake with (PFTN+) or without supplement (PFTN-). Supplementation (0.1 g/kg feed) began 2 d prior to the 3-d environmental treatment period. Body weights (BWs) and blood samples were collected on days -1 and 3. Rectal temperature (RT) and respiration rate (RR) were measured thrice daily and the feed intake (FI) was recorded daily. Intestinal sections were collected for histology. Pigs in HS conditions exhibited increased RT (similar to 1.2 degrees C) and RR (similar to 2.7-fold) compared with TN and PFTN groups (P < 0.01). HS+ animals had increased RR when compared with HS- animals (P < 0.02). Heat stress decreased FI compared with TN. HS and PFTN decreased (P < 0.05) average daily gain compared with TN. Supplement did not alter the BW gain. HS and PFTN decreased (P < 0.05) Gain:Feed compared with TN during environmental treatment. Supplementation with CAPS-SUC increased Gain:Feed by 0.12 (P < 0.05). Circulating glucose concentrations tended to decrease in CAPS-SUC vs. non-supplemented HS and PFTN animals (P = 0.1). Circulating insulin concentrations as well as monocyte count increased in HS compared with PFTN (P < 0.04) but did not differ from TN and likely linked to altered FI. CAPS-SUC increased basophil count (P < 0.02), irrespective of environment. Ileal villus height tended to decrease during HS and PFTN compared with TN (P < 0.08), indicating an effect of intake. Overall, CAPS-SUC supplementation increased pig feed efficiency and may improve immune response.

BACKGROUND:Dietary calcium and phosphorus are required for bone and muscle development. Deficiencies of these macrominerals reduce bone mineral and muscle accretion potentially via alterations of mesenchymal stem cell (MSC) and satellite cell (SC) activities.OBJECTIVES:With increasing interest in the role of early-life events on lifetime health outcomes, we aimed to elucidate the impact of dietary calcium and phosphorus, from deficiency through excess, on MSC and SC characteristics during neonatal development.METHODS:Neonatal pigs [30 females, 1-d-old, 1.46 ± 0.04 kg body weight (BW)] were fed milk replacers for 16 d that were isonitrogenous and isocaloric with a consistent ratio of calcium to phosphorus, but either 25% deficient (calcium: 0.78%; phosphorus: 0.60%; CaPD), adequate (calcium: 1.08%; phosphorus: 0.84%; CaPA), or 25% in excess (calcium: 1.38%; phosphorus: 1.08%; CaPE) of calcium and phosphorus requirements based on sow-milk composition and extrapolation from NRC requirements for older pigs. BW and feed intake were recorded daily. Blood was collected for serum phosphorus, parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23) determination. Humeri were collected for MSC isolation and radii/ulnae bone were collected for analysis. Longissimus dorsi muscle was collected for SC isolation and analysis.RESULTS:There was 4.6% increase in bone ash percentage in CaPE- versus CaPD-fed pigs (P < 0.05). In vivo proliferation indicated a 41.3% increase in MSCs in CaPA compared with CaPD and a 19% increase in SCs in CaPA compared with both CaPE and CaPD. MSCs from CaPD had 2- to 5-fold greater expression of peroxisome proliferator-activated receptor γ (PPARγ), fatty acid-binding protein 4 (FABP4), and lipoprotein lipase (LPL) but lower osteocalcin (BGLAP) and fibronectin (FN1) expression than CaPA (P < 0.05). SCs from CaPD-fed pigs had 19% lower in vivo proliferation than in CaPA-fed pigs.CONCLUSIONS:These findings demonstrated that feeding a diet marginally deficient in calcium and phosphorus to neonatal pigs had a great impact on bone development, MSC, and SC characteristics. These dietary deficiencies may program future bone health and muscle development by altering MSC and SC activities.