Effects of Chromium (Cr) and Zinc (Zn) Supplementation on Metabolism, Inflammation, and Production Parameters in Heat-Stressed and Nutrient-Restricted Pigs Edith J

Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations

2017 Effects of chromium (Cr) and zinc (Zn) supplementation on metabolism, inflammation, and production parameters in heat-stressed and nutrient-restricted pigs Edith J. Mayorga Lozano Iowa State University

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Recommended Citation Mayorga Lozano, Edith J., "Effects of chromium (Cr) and zinc (Zn) supplementation on metabolism, inflammation, and production parameters in heat-stressed and nutrient-restricted pigs" (2017). Graduate Theses and Dissertations. 15573. https://lib.dr.iastate.edu/etd/15573

This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Effects of chromium (Cr) and zinc (Zn) supplementation on metabolism, inflammation, and production parameters in heat-stressed and nutrient-restricted pigs

Edith J. Mayorga Lozano

A thesis submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Major: Animal Science

Program of Study Committee: Lance H. Baumgard, Major Professor John F. Patience Jason W. Ross

Iowa State University

Ames, Iowa

2017

DEDICATION

To my dear parents, Gonzalo and Teresa, for their infinite love and support over the years. I am extremely grateful for everything you have done for me, for your education, hard work, and perseverance. Thanks for encourage me to better myself every day. Everything I am now is just because of you.

Love you and miss you.

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TABLE OF CONTENTS

DEDICATION ...... ii TABLE OF CONTENTS ...... iii LIST OF FIGURES ...... v LIST OF TABLES ...... vii ACKNOWLEDGMENTS ...... viii ABSTRACT ...... x CHAPTER 1: LITERATURE REVIEW ...... 1

Heat Stress ...... 1 Effects of HS on Pigs ...... 2 Thermoregulation during HS ...... 4 Effects of HS on Metabolism ...... 5 Effects of HS on Voluntary Feed Intake ...... 10 Effects of HS on the Gastrointestinal Tract ...... 12 Inflammation and immune system ...... 14 Dietary Options ...... 16 Chromium supplementation ...... 17 Zinc supplementation ...... 21 Conclusion ...... 24 Literature Cited ...... 25

CHAPTER 2: EFFECTS OF DIETARY CHROMIUM PROPIONATE ON GROWTH PERFORMANCE AND METABOLISM DURING HEAT STRESS ON FINISHING PIGS ....47

Abstract ...... 47 Introduction ...... 48 Materials and Methods ...... 50 Animals, diets, and experimental design ...... 50 Production parameters ...... 52 Body temperature measurements ...... 52 Blood sampling and analysis ...... 53 Statistical analysis ...... 53 Results ...... 54 Growth Performance ...... 54 Body temperature indices ...... 55 Blood metabolites ...... 55 Discussion ...... 58 Literature Cited ...... 66 iv

CHAPTER 3: EFFECTS OF ZINC AMINO ACID COMPLEX ON BIOMARKERS OF GUT INTEGRITY AND METABOLISM DURING HEAT STRESS AND A FOLLOWING RECOVERY PERIOD IN GROWING PIGS ...... 82

Abstract ...... 82 Introduction ...... 83 Materials and Methods ...... 85 Animals, diets, and experimental design ...... 85 Production parameters ...... 87 Body temperature measurements ...... 87 Blood sampling and analysis ...... 88 Statistical analysis ...... 89 Results ...... 89 Growth performance ...... 89 Body temperature indices ...... 91 Blood metabolites ...... 92 Discussion ...... 94 Literature Cited ...... 101

CHAPTER 4: SUMMARY AND CONCLUSIONS ...... 117

Literature Cited ...... 120

APPENDIX: EFFECTS OF LIVE YEAST SUPPLEMENTATION ON GROWTH PERFORMANCE AND METABOLISM IN HEAT-STRESSED AND NUTRIENT- RESTRICTED PIGS ...... 121

LIST OF FIGURES

Figure 1. Relation between heat production or heat losses and body temperature as affected by ambient temperature...... 4

Figure 2. Heat stress and growth performance depend upon the feed intake response ...... 11

Figure 3. Effects of chromium propionate on average daily feed intake and body weight...... 78

Figure 4. Effects of chromium propionate on average daily gain, final body weight, and feed efficiency...... 79

Figure 5. Effects of chromium propionate on rectal temperature, skin temperature, and respiration rate ...... 80

Figure 6. Effects of chromium propionate on circulating insulin, glucose, non-esterified fatty acids, and lipopolysaccharide binding protein ...... 81

Figure 7. Effects of zinc amino acid complex on average daily feed intake and average daily gain ...... 111

Figure 8. Effects of zinc amino acid complex on rectal temperature, skin temperature, and respiration rate ...... 113

Figure 9. Effects of zinc amino acid complex on circulating levels of glucose, non-esterified fatty acids, blood urea nitrogen, and lipopolysaccharide binding protein ...... 114

Figure 10. Effects of zinc amino acid complex on circulating levels of insulin during period 2 and recovery, and among environmental treatments ...... 115

Figure 11. Effects of zinc amino acid complex on circulating levels of tumor necrosis factor alpha during period 1, period 2, and the recovery period ...... 116

Figure 12. Effects of live yeast supplementation on rectal temperature, skin temperature, and respiration rate...... 127

Figure 13. Effects of live yeast supplementation on average daily feed intake and average daily gain...... 128

Figure 14. Effects of live yeast supplementation on body weight during P2 and final body weight...... 129 vi

Figure 15. Effects of live yeast supplementation on circulating glucose, insulin, non- esterified fatty acids, and blood urea nitrogen ...... 130

Figure 16. Effects of live yeast supplementation on circulating triiodothyronine, thyroxine, triiodothyronine to thyroxine ratio, and tumor necrosis factor alpha...... 131

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LIST OF TABLES

Table 1. Effects of heat stress on plasma energetic parameters ...... 9

Table 2. Effects of chromium supplementation on production parameters and metabolism ...... 20

Table 3. Effects of zinc supplementation on intestinal barrier function ...... 23

Table 4. Ingredient composition and chemical and nutritional composition of experimental diets (as-fed basis) ...... 71

Table 5. Effects of chromium propionate supplementation on growth performance and carcass characteristics in heat-stressed and nutrient-restricted pigs ...... 73

Table 6. Effects of chromium propionate supplementation on body temperature indices in heat-stressed and nutrient-restricted pigs ...... 74

Table 7. Effects of chromium propionate supplementation on blood metabolites in heat-stressed and nutrient-restricted pigs ...... 75

Table 8. Effects of chromium propionate supplementation on blood cell count in heat-stressed and nutrient-restricted pigs ...... 77

Table 9. Ingredient composition and chemical and nutritional composition of experimental diets ...... 106

Table 10. Effects of zinc amino acid complex on growth performance in heat-stressed and restricted-fed pigs during P2 and recovery ...... 108

Table 11. Effects of zinc amino acid complex on body temperature indices in heat-stressed and restricted-fed pigs during P2 and recovery ...... 109

Table 12. Effects of zinc amino acid complex on blood metabolites and inflammatory biomarkers in heat-stressed and restricted-fed pigs during P2 and recovery ...... 110

Table 13. Effects of live yeast supplementation on body temperature indices in heat-stressed and nutrient-restricted pigs ...... 123

Table 14. Effects of live yeast supplementation on growth performance in heat-stressed and nutrient restricted pigs ...... 124

Table 15. Effects of live yeast supplementation on blood metabolites and biomarkers of inflammation in heat-stressed and nutrient restricted pigs ...... 125

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ACKNOWLEDGMENTS

I would like to express my most sincere gratitude to my major professor, Dr. Lance

Baumgard, for being an inspiring mentor and for his valuable and unconditional support. I am grateful for believing in me, for all your guidance, encouragement, and patience during the last years; everything has contributed to my academic growth. I would also like to extend my appreciation to the members of my graduate committee, Dr. Jason Ross and Dr. John Patience, for their significant contributions to this research.

I would also like to thank the current and former members of the Baumgard lab, especially to Dr. Victoria Sanz-Fernandez for her friendship, support, and for encouraging me to join the group. To Dr. Mohannad Abuajamieh, Dr. Sara Kvidera, Erin Horst, Mohmmad Al-Qaisi, Carrie

Shouse, Mackenzie Dickson, Jake Seibert, and Samantha Lei, thanks a lot for helping me during my experiments or my laboratory work, and for making my staying in the group a very good experience. I really appreciate all your support and encouragement. It has not been easy living abroad, but I have been very fortunate for having your friendship, company, and help whenever I need it.

I would also like to extend my appreciation to all the undergrads that were involved in my experiments and helped me to complete my research successfully. I would also like to acknowledge the staff at Iowa State University, especially to Donna Nelson and Joan Aiton, for their help and assistance. Also, I would like to thank the staff at the Swine Nutrition Farm, especially to Trey Faaborg and Tim Hicks for all the assistance I received during my experiments, for the animal care, and for making sure everything was working properly at the farm. ix

To my sisters, Yamile and Carolina, thank you for your support, help and encouragement from abroad, and for inspiring me to achieve my dreams; and to my nephew Nicolas for bringing happiness to my life. Love you and miss you!

Finally, I would like to thank my husband, Jesus Acosta. I will never regret the day I decided to join you in this adventure. Life has changed a lot for us and has not been easy to live away from home, but I am grateful for having your love, your company, and your support, and I am willing to continue this journey with you.

ABSTRACT

Heat stress (HS) adversely impacts all aspects of global agriculture, and it particularly constrains domestic animal productivity and compromises animal welfare. Heat-stressed animals employ physiologic and metabolic adjustments to ameliorate the heat insult; consequently, efficiency is compromised because nutrients are partly diverted away from production purposes to maintain euthermia. Reduced animal productivity during HS can also be attributed to the direct effects of HS (independent of nutrient intake) on metabolism, physiology, reproduction, and health. Therefore, identifying nutritional alternatives with the potential to ameliorate the detrimental effects of HS on economically important performance and health is of particular interest. The overall thesis objectives were to investigate the dietary effects of chromium (Cr) and zinc (Zn) supplementation in heat-stressed and nutrient-restricted pigs.

In the first study (Chapter 2), finishing pigs were used in a replicated experiment to evaluate the effects of Cr propionate supplementation on growth performance and metabolism during HS.

As expected, pigs exposed to HS had increased thermal indices and decreased growth performance.

However, Cr supplementation tended to increase average daily gain in chronically heat-stressed pigs. Regardless of environmental treatments, pigs supplemented with Cr had numerically increased feed intake. Further, adding Cr to the HS pig diet increased circulating neutrophils and monocytes.

The second study (Chapter 3) evaluated the effects of Zn amino acid complex on metabolism, leaky gut biomarkers, and inflammation during and following HS. Pigs exposed to

HS had increased body temperature and respiration rates as well as reduced production metrics.

Despite marked reductions in feed intake, circulating insulin increased during HS and remained increased during thermal neutral recovery. Interestingly, supplemental Zn tended to decrease xi plasma tumor necrosis factor alpha (TNFα) levels before and after HS exposure. However, no effects of dietary Zn were observed on production parameters or other blood metabolites.

In conclusion, both experiments demonstrated that HS adversely impacts animal productivity and health. Results suggest that Cr supplementation might be beneficial on growth performance and health during HS. Additionally, Zn supplementation might be advantageous at reducing basal inflammation. Altogether, these findings suggest that dietary interventions aimed at alleviating the negative consequences of HS are plausible. However, additional research is needed to better understand the biology and mode of action of both Cr and Zn supplementation during HS.

CHAPTER 1: LITERATURE REVIEW

Heat Stress

Heat stress (HS) is a common issue in animal agriculture and it negatively impacts welfare and production efficiency (Baumgard and Rhoads, 2013). Production losses due to high ambient temperatures occur not only in tropical areas, but also in temperate countries during the warm summer months (Renaudeau et al., 2012a). In fact, the estimated annual losses due to HS in the

US livestock industry averaged $897 million for dairy, $369 million for beef, $1 billion for swine, and $128 million for poultry industries (St. Pierre, 2003; Pollmann, 2010). These economic constraints are mainly explained by the negative consequences of HS on productive parameters including milk yield and composition, growth, reproduction and carcass traits (Baumgard and

Rhoads, 2013). Further, global animal production systems will be increasingly affected by HS if global temperature continues to rise as predicted (IPCC, 2007). Additionally, with the continuous rise in human population, particularly in the tropical and subtropical areas of the globe, animal agriculture will need to expand to keep pace with the increasing demand for animal protein.

Furthermore, increased genetic selection for production traits (i.e., milk yield, lean tissue accretion, fecundity) results in reduced tolerance to HS as these traits are accompanied with increased basal and metabolic heat production (Brown-Brandl et al., 2004). Therefore, understanding how HS reduces animal productivity and jeopardizes health is crucial to developing strategies aimed at enhancing animal well-being and performance, and to improving global agriculture economics.

Effects of HS on Pigs

In the pig industry, the negative consequences of HS are mainly expressed by reduced and inconsistent growth, decreased efficiency, decreased carcass quality (increased lipid and decreased protein), poor sow performance, and increased mortality (specially in sows and market hogs) and morbidity (Baumgard and Rhoads, 2013; Ross et al., 2015). Unlike other domesticated species, pigs are particularly sensitive to HS due to their low capacity to dissipate heat (they are unable to appreciably sweat); consequently, they rely more on reducing metabolic heat production (HP), via reduced feed intake and physical activity, in an attempt to maintain euthermia during hot conditions (Quiniou et al., 2000; Collin et al., 2001). These adjustments result in physiological and metabolic changes that have direct consequences on energy intake and therefore productivity.

Additionally, decreased performance during HS can also be attributed to the direct effects of HS

(independent of feed intake) on reproduction, health, and energy metabolism (Renaudeau et al.,

2012a,b; Baumgard and Rhoads, 2013).

The effects of HS on carcass composition have been well-documented. Pigs exposed to HS have increased lipid deposition and decreased carcass lean content (Close et al., 1971; Verstegen et al., 1973; Heath, 1983; Collin et al., 2001). This phenomenon is biologically interesting as pigs reared in thermoneutral conditions on a restricted diet will prioritize protein deposition at the expense of lipids (van Milgen and Noblet, 2003; Oresanya et al., 2008); therefore, the relationship between reduced feed intake and body composition is opposite in pigs reared during HS and is independent of nutritional plane (Baumgard and Rhoads, 2013). In addition, pigs exposed to HS have a decreased size of the entire splanchnic bed, particularly the liver, empty digestive tract, and heart (Sugahara et al., 1970; Rinaldo and Le Dividich, 1991, Johnson et al., 2015a,b). These 3 decreased viscera weight, along with reduced physical activity during HS, reduces the fasting heat production and may have an important influence on maintenance costs (Koong et al., 1983).

The reproductive consequences of HS have been related with a certain degree of seasonality, which is referred to as “seasonal infertility”. Reduced fertility in breeding pigs is particularly observed during the hottest months of the year (i.e., summer and early fall). Heat stress decreases fertility in both sows and gilts, mainly by delaying the onset of puberty, extending the weaning-to-estrus interval, and by decreasing farrowing rates (Bertoldo et al., 2009; Ross et al., 2015). Similarly, poor semen production and quality occur in boars exposed to HS (Wettemann et al., 1976; Suriyasomboon et al., 2004). Furthermore, previous studies have demonstrated that gestational exposure to HS has detrimental effects on offspring (Johnson et al., 2013; Boddicker et al., 2014; Johnson et al., 2015a,b). Of particular interest, piglets exposed to HS in utero tended to have increased back fat depth and increased levels of circulating insulin (Boddicker et al., 2014).

In addition, the thermoregulatory response of postnatal pigs exposed to HS in utero was also affected as they had increased body temperature (~0.3°C) when exposed to thermoneutral and HS conditions (Johnson et al., 2013; Johnson et al., 2015a,b). Thus, the increased carcass fat and body temperature of piglets derived from heat-stressed sows has profound implications on maintenance costs, rational formulation, and facility efficiency. In conclusion, HS compromises a variety of production parameters in the swine industry including growth, carcass composition, and reproduction. Furthermore, evidence suggests that maternal exposure to HS has long-lasting effects on postnatal offspring performance. Therefore, it is clear HS has direct and indirect (via reduced nutrient intake) effects on multiple physiological systems that ultimately compromise animal health and productivity. 4

Thermoregulation during HS

The goal of thermoregulation is to maintain a safe body temperature within narrow limits, through a dynamic balance of HP and heat loss. The range of ambient temperature over which energy expended to facilitate heat loss is minimal and energy retention is maximal is called the thermoneutral zone (TNZ; Le Dividich et al., 1998). The lower and upper limits of the TNZ are called the lower (LCT) and the upper (UCT) critical temperature, respectively (Renaudeau et al.,

2012a). Ambient temperature on either side of the TNZ causes activation of thermoregulatory mechanisms that have an energetic cost and thus ultimately affects productivity.

Figure 1. Relation between heat production or heat losses and body temperature as affected by ambient temperature (Th=threshold for hypothermia, LCT=lower critical temperature,

ECT=evaporative critical temperature, UCT=upper critical temperature) (Renaudeau et al.,

2012a).

Animals loss heat in the form of sensible and latent (evaporative) heat. Conduction, convection and radiation are the main ways by which sensible heat loss occurs. Each of these strategies require a gradient or temperature difference between the animal and its surrounding environment (Collier and Gebremedhin, 2015). Therefore, as ambient temperature increases, animals adjust their blood flow towards the skin to maximize heat dissipation to the periphery in an attempt to increase radiant heat loss. With a further increase in ambient temperature (the temperature gradient between the environment and animal becomes smaller), the transfer of heat by conductive, convective and radiative means becomes less efficient, and sensible heat loss decreases. Consequently, thermoregulation can be achieved only by increasing evaporative heat loss from the lungs and the skin (Black et al., 1998). Sweating and panting are two of the main responses exhibited by animals exposed to HS. In fact, when ambient temperature increases above the UCT, evaporation is the only route of heat loss preventing lethal hyperthermia. Cows and horses are considered sweating animals; however, pigs and poultry depend more on the respiratory route for heat dissipation (Collier and Gebremedhin, 2015).

Effects of HS on Metabolism

The dramatic decrease in feed intake during HS was traditionally thought to be the main culprit for the negative effects of HS on animal performance (Fuquay, 1981). However, the effects of HS on some key phenotypes (i.e., carcass fat content, etc.) suggest that animals exposed to HS alter their metabolism independently of nutrient intake or energy balance (Baumgard and Rhoads,

2013). Previous studies using the pair-feeding model (to evaluate the effects of thermal stress while eliminating the confounding effect of dissimilar nutrient intake), demonstrated that reduced 6 feed intake only accounts for ~50% of the milk yield decrease in HS dairy cows (Rhoads et al.,

2009; Wheelock et al., 2010; Baumgard et al., 2011). Likewise, decreased feed intake only accounts for up to 50% of the reduced growth of chickens and lambs exposed to HS (Geraert et al., 1996; Mahjoubi et al., 2014; Zuo et al., 2014). On the contrary, pigs exposed to thermal stress had increased body weight and increased circulating insulin levels when compared to their pair- fed counterparts (Pearce et al., 2013). This indicates that HS has direct effects on metabolism and performance that are independent of nutrient intake or energy balance. Reasons for the species differences in response to HS is not known but are of obvious practical and academic importance.

These findings suggest that metabolic adjustments during HS involve a shift in carbohydrate metabolism, including changes in basal and stimulated circulating insulin levels.

Heat stress induces increased circulating glucose in chickens (Bobek et al., 1997; Garriga et al.,

2006), rabbits (Marder et al., 1990), sows (Prunier et al., 1997), and exercising men (Fink et al.,

1975; Febbraio, 2001). Accordingly, when humans exercise under high ambient temperature there is an increase in hepatic glucose output and increased glucose utilization at the expense of non- esterified fatty acid (NEFA) oxidation (Fink et al., 1975; Febbraio, 2001). Evidence suggests that increased hepatic glucose output is likely due to both increased glycogenolysis and gluconeogenesis (Collins et al., 1980; Febbraio, 2001). Further, increased gene expression of pyruvate carboxylase, a key regulator of the gluconeogenesis pathway, occurs during HS in different animal models (Wheelock et al., 2008; O’Brien et al., 2008; Rhoads et al., 2011).

Additionally, increased intestinal glucose uptake and improved renal glucose reabsorptive capacity have been observed during exposure to HS (Ikari et al., 2005; Garriga et al., 2006; Pearce et al.,

2013b). Altogether, these studies indicate that animals exposed to HS have an increased reliance 7 on glucose as a substrate; however, further research is warranted to better understand the biology and mechanisms underlying this altered metabolism during HS.

Regardless of severely reduced nutrient intake, severe body weight loss and negative energy balance, previous studies have demonstrated that HS animals are hyperinsulinemic

(O’Brien et al., 2010; Wheelock et al., 2010; Pearce et al., 2013a; Sanz-Fernandez et al., 2015a).

It is not well-understood why insulin, a potent anabolic hormone, increases during HS, a condition that induces a catabolic state. O’Brien et al. (2010) and Wheelock et al. (2010), observed that cows and calves exposed to a thermal heat load had an increased insulin response to a glucose tolerance test, relative to their pair-fed counterparts. Similarly, using the hyperinsulinemic- euglycemic clamp technique, growing calves and pigs exposed to HS had increased whole-body insulin sensitivity as they required more glucose to maintain euglycemia (Rhoads, et al., 2009;

Sanz-Fernandez et al., 2015a). Increased circulating insulin likely plays a key role as a HS adaptation mechanism. For instance, decreased survival time was observed in diabetic rats exposed to an acute heat load, and the incidence of heatstroke was markedly reduced after exogenous insulin treatment (Niu et al., 2003). Similarly, previous reports have indicated diabetic patients are more susceptible to heat related-illness and death (Schuman et al., 1972; Semenza et al., 1999). Moreover, studies in humans and rodents reported thermal therapy improved insulin signaling in diseases like obesity (Gupte et al., 2009), diabetes (Hooper, 1999; Kokura et al., 2007), and aging (Gupte et al., 2008; Gupte et al., 2011). Consequently, the aforementioned literature suggests improved circulating insulin coupled with proper insulin action are crucial in regulating the HS response.

Lipid mobilization is attenuated during HS as levels of circulating NEFA are typically reduced in rodents (Sanders et al., 2009), growing pigs (Becker et al., 1992; Pearce et al., 2013a, 8

Sanz-Fernandez et al., 2015a,b), chickens (Geraert et al., 1996), and ruminants (Rhoads et al.,

2009; Wheelock et al., 2010; O’Brien et al., 2010; Baumgard et al., 2011) despite a marked decrease in feed intake. Accordingly, increased carcass lipid retention has been reported in rodents

(Schmidt and Widdowson, 1967), chickens (Geraert et al., 1996), and pigs (Close et al., 1971;

Verstegen et al., 1978; Heath, 1983, Collin et al., 2001) exposed to HS. The lack of lipid mobilization from adipose tissue is coupled with reduced sensitivity to lipolytic stimuli, as demonstrated by decreased NEFA response to an epinephrine challenge in heat-stressed pigs

(Sanz-Fernandez et al., 2015a) and cows (Baumgard et al., 2011). Sanders et al. (2009) reported that adipose tissue lipoprotein lipase is increased during HS, suggesting an increased ability to uptake and store dietary and hepatic-derived triglycerides.

Protein metabolism is also affected during HS as there is decreased protein deposition reported in previous studies (Close et al., 1971; Stahly et al., 1979; Lu et al., 2007). Although the mechanism for reduced protein accretion has not been yet elucidated, previous studies reported decreased DNA and RNA synthesis during HS (Henle and Leeper, 1979; Streffer, 1982). In addition, HS exposure increases markers of protein catabolism, including plasma urea nitrogen, creatine, creatinine, and 3-methylhistidine in different species (Yunianto et al., 1997; Ronchi et al., 1999; Shwartz et al., 2009; Pearce et al., 2013a). A summary of the effects of HS on metabolic parameters is presented in Table 1.

Table 1. Effects of heat stress on plasma energetic parametersa Metabolite Species Response Reference BUNb Chickens (uric acid) =/↑ 32/62 Cows ↓/↑ 55, 58/ 1, 20, 27, 42, 43, 48, 53, 56, 57, 61 Humans ↑ 19, 25 Pigs ↑ 44 Rats ↑ 16, 31 Rabbits ↑ 35 Sheep = 19 NEFAc Chickens ↓/↓↑ 22/ 8 Cows ↓ / = / ↑ 1, 26, 47, 48, 49, 57, 61/ 10/ 42, 55, 58, 59 Pigs ↓ 24, 38, 44, 51 Rats ↓ 12, 17, 18, 40 Sheep ↓ / = / ↑ 49/ 4/ 50, 54 Glucose Cats ↓ 1, 30, 51 Chickens ↓ / = / ↑ 18, 22 / 32, 46 / 6, 21, 23 Cows ↓ / = / ↑ 1, 10, 11, 26, 42, 47, 48, 53, 56, 57, 48, 61, 63/ 55/ 20 Dogs ↓ 28 Humans ↑ 3, 5, 14, 15, 39 Pigs ↑ 24, 44, 45 Rabbits ↑ 35, 41 Rats ↓ / ↑ 37, 38, 40 / 18 Sheep ↓ / = / ↑ 2, 34, 52/ 4, 42, 50/ 7, 13, 33 Insulin Cows =/↑ 10, 36 / 43, 61 Humans = 29 Pigs ↑ 9, 24, 44 Rats ↑ 40, 60 Sheep ↑ 34, 50 aAdapted from (Sanders, 2010; Johnson, 2014; Pearce, 2014; Sanz-Fernandez, 2014; Abuajamieh, 2015) bBlood urea nitrogen cNon-esterified fatty acids 1Abeni et al., 2007 22Geraert et al., 1996 43O’Brien et al., 2010 2Achmadi et al., 1990 23Habibian et al., 2014 44Pearce et al., 2013a 3Al-Harthi et al., 2012 24Hall et al., 1980 45Prunier et al., 1997 4Alhidary et al., 2012 25Hart et al., 1980 46Rahimi, 2005 5Angus et al., 2007 26Itoh et al., 1998 47Rhoads et al., 2009 6Arad et al., 1983 27Kamiya et al., 2006 48Ronchi et al., 1999 7Bell et al., 1989 28Kanter, 1954 49Sano et al., 1983 8Bobek et al., 1997 29Kappel et al., 1997 50Sano et al., 1999 9Boddicker et al., 2014 30Lee and Scott, 1916 51Sanz-Fernandez et al., 2015a,b 10Burdick Sanchez et al., 2013 31Li et al., 2014 52Sejian et al., 2014 11Burge et al., 1972 32Lin et al., 2006 53Settivari et al., 2007 12Burger et al., 1972 33Liu et al., 2015 54Sevi et al., 2002 13Caroprese et al., 2010 34Mahjoubi et al., 2014 55Shahzad et al., 2015 14Febbraio, 2001 35Marder et al., 1990 56Shehab-El-Deen et al., 2010 15Febbraio et al., 1994 36Min et al., 2015 57Shwartz et al., 2009 16Francesconi and Hubbard, 1986 37Miova et al., 2013 58Srikandakumar and Johnson, 2004 17Frankel, 1968 38Mitev et al., 2005 59Tian et al., 2015 18Frascella et al., 1977 39Monteleone and Keefe, 1969 60Torlinska et al., 1987 19Fukumoto et al., 1988 40Morera et al., 2012 61Wheelock et al., 2010 20Garcia et al., 2015 41Nakyinsige et al., 2013 62Yalcin et al., 2009 21Garriga et al., 2006 42Nardone et al., 1997 63Zhang et al., 2014

Effects of HS on Voluntary Feed Intake

In ad libitum feeding settings, pigs adjust their feed intake to compensate for changes in ambient temperature (Le Dividich et al., 1998). When pigs are housed in an ideal stress-free environment within the TNZ, voluntary feed intake is closely related to their capacity to utilize nutrients and varies with genetic and physiological state. Once ambient temperature increases, body temperature maintenance is achieved by increasing heat loss and reducing HP (Collin et al.,

2001). Different methods to reduce HP involve the decrease in voluntary feed intake and the associated thermic effect of feeding (TEF; Quiniou et al., 2001), decreased physical activity or decreased basal metabolic rate (Collin et al., 2001). Considering that the TEF represents the additional heat associated with processes like nutrient digestion, assimilation, fermentation, and metabolism, reduced voluntary feed intake is an efficient strategy used by the animal to cope with high ambient temperatures.

The decrease in voluntary feed intake can be severe during HS. Evidence suggests that feed intake can be reduced by ~40 g for every 1°C above the TNZ (Nyachoti et al., 2004).

Additionally, a quantitative analysis from previous studies has demonstrated a curvilinear decrease in feed intake with increasing ambient temperature (Renaudeau et al., 2001). Consequently, for a

50-kg pig, feed intake decreases at a rate of 8 g/d/°C from 16 to 24°C, and then at a rate of 46 g/d/°C from 24 to 32°C. Across the same ambient temperature range, for a 70-kg pig, feed intake decreases by 30 and 70 g/d/°C (Reneaudeau et al., 2012a). Le Dividich et al. (1998) reported the reduction in feed intake due to HS varies from 40 to 80 g°C-1d-1 depending on factors like pig genotype, diet composition, body weight, and ambient temperature. 11

Figure 2. Heat stress and growth performance depend upon the feed intake response (Baumgard et al., 2015).

Similarly, ADG decreases as a result of both the severe decrease in feed intake and the direct effects of HS. For instance, the estimated reduction in ADG for a 50-kg pig is about 18 g/d-

1/°C-1 when temperature is increased from 20 to 30°C. Interestingly, under these same temperature conditions the decrease in ADG is 2-fold larger in a 75 relative to a 25-kg pig (25 vs. 11 g/d-1/°C-

1); indicating that heavier pigs are more susceptible to HS than younger pigs (Renaudeau et al.,

2011). According to Holmes and Close (1977), the UCT decreases with increasing body weight, and this might be explained by the fact that heavier pigs have lower surface area-to-mass ratio and increased fat deposition on subcutaneous tissue, which negatively affects their capacity to dissipate heat. Thermal stress causes a shift in the feeding behavior, as Nienaber et al. (1996) and Quiniou et al. (2001) reported that HS reduced meal duration and size, but not total number of daily meals in growing pigs. Furthermore, pigs eat more during cooler periods of the day to reduce the detrimental effects of high ambient temperature (Feddes and Deshazer, 1998; Giles, 1992). Taken 12 together, reduced feed intake during HS represents an adaptive strategy to decrease metabolic HP and it partially explains the reduced growth performance during high ambient temperatures.

Effects of HS on the Gastrointestinal Tract

A main role of the gastrointestinal (GI) tract is to digest and absorb nutrients. However, the GI tract also needs to simultaneously preserve a barrier between the luminal content and the internal environment. In other words, the GI tract needs to selectively absorb nutrients, solutes and water, while excluding noxious antigens and bacteria (Ma and Anderson, 2006). Luminal nutrients are transported from apical through basolateral epithelial cells by two main pathways: the transcellular (across cells) and the paracellular (between cells) routes. Transcellular transport is accomplished by active transport and endocytosis, while paracellular transport allows the passage of water solutes and minerals by passive diffusion (de Punder and Pruimboom, 2015). To maintain this selective barrier, the epithelium relies on the formation of protein-protein complexes that mechanically link adjacent cells and seal the intercellular space. Tight junction (TJ) proteins connect adjacent epithelial cells and are associated with cytoplasmic actin and myosin networks that partially regulate intestinal permeability (Groschwitz and Hogan, 2009). Four different transmembrane proteins have been identified within the TJ: claudins, occludins, junctional adhesion molecule, and tricellulin (Jin and Blikslager, 2015). The intracellular domains of these transmembrane proteins interact with scaffold intracellular proteins zona occluden (ZO-1, ZO-2,

ZO-3), which in turn anchor them to the actin cytoskeleton (Farre and Vicario, 2017). Alterations in any TJ structure components can result in increased intestinal permeability, which involves the diffusion of molecules including food antigens, bile, hydrolytic enzymes, and endotoxin (i.e., 13 lipopolysaccharide, LPS) from the GI lumen to the blood, resulting in local and systemic inflammatory reactions (Lambert et al., 2002; Lambert, 2008).

It has been demonstrated that the GI tract is very susceptible to a thermal load. According to previous studies, increased intestinal permeability has been reported in rodents (Hall et al., 2001;

Lambert et al., 2002; Prosser et al., 2004, Oliver et al., 2012), pigs (Pearce et al., 2013c, Sanz-

Fernandez et al., 2014), and humans (Lambert, 2004) exposed to HS. Additionally, HS alters intestinal architecture, reducing the villi height and causing epithelial sloughing (Lambert et al.,

2002; Yu et al., 2010; Oliver, et al., 2012; Pearce et al., 2013c, Abuajamieh et al., 2016). The mechanisms for intestinal barrier dysfunction during HS are likely multifactorial, but they might be related with thermoregulatory responses to high ambient temperature. During HS, blood flow is diverted away from the splanchnic bed to the periphery in a coordinated attempt to maximize heat dissipation (Hall et al., 2001). This circulatory adjustment reduces blood flow to the GI tract, resulting in local hypoxia, free radical production, ATP depletion, acidosis, enterocyte membrane damage and TJ dysfunction (Lambert, 2009). Consequently, the defective intestinal barrier allows the paracellular translocation of microbial and dietary antigens across the gut barrier, leading to intestinal and systemic inflammation (Al-Sadi et al., 2007). In fact, circulating LPS increases in

HS pigs (Pearce et al., 2013c), chickens (Cronje 2007), rodents (Hall et al., 2001; Lim et al., 2007), monkeys (Gathiram et al., 1988), and humans (Broke-Utne et al., 1988; Bouchama et al., 1991).

Consequently, intestinal barrier dysfunction during HS can be attributed to both the direct influence of hyperthermia and the subsequent intestinal ischemia. Furthermore, intestinal epithelium damage and loss of TJ integrity have been associated as important mechanism underlying increased intestinal permeability during HS. 14

Inflammation and immune system

The intestinal mucosal epithelium exhibits tolerance to commensal bacteria due to its ability to differentiate between commensal bacteria and pathogenic microorganisms (Kim and Ho,

2010). Bacteria recognition by the host is mediated by several mechanisms; however, the most important depends on the expression of specific receptors that identify conserved bacterial structures that cannot be found in the host. These structures are known as pathogen-associated molecular patterns (PAMPs), and the receptors that recognize them are referred to as pattern recognition receptors (PRRs), such as cell surface toll-like receptors (TLRs). Lipopolysaccharide, a major cell wall component of gram negative bacteria, is the most widely studied PAMP; while an important mammalian TLR is TLR4, which is actually required for LPS recognition (Eckmann,

2006). The pattern and tissue distribution of the TLR4 receptor is highly diverse across species and involves immune cells, epithelial cells, adipocytes, skeletal muscle, brain, and the reproductive tract, among others (Santaolalla et al., 2011; Vaure and Liu, 2014; de Punder and Pruimboom,

2015). Interestingly, intestinal epithelial cells in normal conditions express low levels of TLR4 and are hyporesponsive to the stimuli caused by LPS (Kim and Ho, 2010).

Translocation of LPS from the lumen to the basolateral membrane elicits a cascade of events that activates the immune response and stimulates pro-inflammatory cytokine production

(Lambert, 2009). Initiating the immune response cascade begins with LPS recognition by TLR4.

Lipopolysaccharide binding protein (LBP) transports and delivers circulating LPS to cluster of differentiation 14 (CD14), leading o TLR4 activation; or delivers LPS to lipoproteins, resulting in hepatic clearance (de Punder and Pruimboom, 2015). Activation of TLR4 induces a complex intracellular signaling pathway, resulting in the activation of different transcription factors involved in inflammation, adaptive immunity, and cellular metabolism. Among these transcription 15 factors, the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κΒ) has an important role in inducing the expression of different cytokines associated with inflammation such as tumor necrosis factor alpha (TNFα), interleukins (IL) IL-1, IL-12, IL-18, and type-I interferons (Tak and

Firestein, 2001). In agreement with this, increased levels of pro-inflammatory cytokines including

TNFα, IL-1α, IL-1β, IL-6, and interferon-γ were observed in heat stroke patients; ostensibly linked to high concentrations of circulating LPS (Bouchama et al., 1991, 1993). Similarly,

Lambert and colleagues (2002) reported increased circulating TNFα in rats exposed to HS, a model that also exhibited increased intestinal permeability. The increased recruitment and expansion of immune cells producing pro-inflammatory cytokines might be interpreted as a vicious cycle of events resulting in further inflammation and epithelial damage (Ma et al., 2005; Lambert, 2009;

Barreau and Hugot, 2014). A better understanding of the inflammatory response and its role during

HS is foundational to developing strategies aimed at improving animal productivity during the warm summer months.

In conclusion, HS induces intestinal barrier dysfunction likely by altering intestinal morphology and increasing barrier permeability. Increased bacteria and bacteria components translocation from the lumen to the basolateral membrane activates the immune system and stimulates an inflammatory response, a consequence that ultimately compromises animal health and productivity. Better understanding the mechanisms by which hyperthermia impairs intestinal permeability is warranted to develop future strategies aimed at enhancing intestinal integrity and health during HS.

Dietary Options

There is growing interest in the use of nutritional strategies with potential to ameliorate the negative effects of HS and improve animal productivity. One of the most common approaches has been balancing diets with the ability to reduce heat increment (i.e., low-protein or low-fiber diets) or increase nutrient density (i.e., high-energy diets). Utilizing high protein and fiber diets is not recommended during the warm summer months as they will increase the heat production associated with TEF (Patience et al., 2015). The increased HP of dietary protein is related to the deamination of excess amino acids for urea synthesis and protein turnover. Similarly, heat increment of fiber is associated with production of gases and heat losses during fermentation processes. Although it can be assumed that feeding a low-protein or low-fiber diet can attenuate the decreased feed intake during HS, there is little or no scientific evidence supporting this dogma.

Additionally, increasing the inclusion levels of fat in the diet has been reported to improve

ADG and G:F (Stahly and Cromwell, 1979; Spencer et al., 2005) in HS pigs. Energy derived from fat sources is thought to be more efficiently used for production purposes than starch. Besides, high energy diets are associated with a decreased TEF and result in increased energy intake, which may ameliorate the reduced feed intake observed during HS (Renaudeau et al., 2012b).

A different nutritional approach is the use of supplements aimed to improve insulin sensitivity. As stated previously, HS alters basal metabolism causing a shift towards carbohydrate utilization and a clear decrease in lipid catabolism (despite marked reductions in feed intake).

These changes are characterized by an unexplainable increase in circulating insulin and whole- body insulin sensitivity, a condition that has been well-described in different species (Baumgard and Rhoads, 2013). Since proper insulin action is crucial in regulating the response to a thermal 17 load, supplementing feed additives to enhance insulin sensitivity might be a good strategy to improve animal performance during HS. Nutritional and pharmaceutical strategies to improve insulin sensitivity include the use of Cr, lipoic acid, and thiazolidinediones (Rhoads et al., 2013).

These supplements have been proven to increase glucose utilization in several species under thermoneutral conditions; however, only Cr supplementation has been shown to improve production parameters in heat-stressed animals (Rhoads et al., 2013).

The GI tract is another important target that can benefit from dietary interventions. During

HS, blood flow is redistributed away from the splanchnic bed in an attempt to dissipate heat; reducing blood flow to other organs and tissues. This predisposes the GI tract to hypoxia, resulting in increased intestinal permeability, inflammation, and villus damage. Previous studies have demonstrated the use of dietary supplements including Zn (Alam et al., 1994; Sanz-Fernandez et al., 2014; Pearce et al., 2015; Abuajamieh et al., 2016), glutamine (Lima et al., 2015), or betaine to improve intestinal integrity. A thorough description of nutritional alternatives is not within the scope of this article. Rather, this section concentrates on Cr and Zn supplementation, as well as on their role in alleviating the negative consequences of HS.

Chromium supplementation

Chromium is a micronutrient that plays an important role in carbohydrate, lipid, and protein metabolism (Anderson, 1992; Mertz, 1993; Pechova and Pavlata, 2007). Chromium is thought to potentiate insulin action by stimulating the insulin-receptor at cell surfaces, therefore enhancing insulin sensitivity and/or responsiveness in peripheral tissues (Davis and Vincent, 1997). Trivalent

Cr (Cr+3), the most stable oxidation state of this mineral, is the form proposed to be biologically active (Vincent and Stallings, 2007). However, only a small percentage of dietary Cr+3 is absorbed 18

(0.5-2% and 10-25% for inorganic and organic forms, respectively), while the remainder is excreted mainly in the feces (Mertz and Roginski, 1969; Underwood, 1977). Currently, different forms of organic Cr are commercially available and thought to be over ten times more available than inorganic forms of Cr. The main forms of dietary Cr allowed to be fed to farm livestock include Cr picolinate, Cr propionate, Cr yeast, and Cr methionine. (Lindeman, 2007).

The mode of Cr action was first elucidated when Yamamoto and collaborators (1984) isolated and characterized a Cr-binding oligopeptide termed low molecular weight Cr binding substance or chromodulin. This oligopeptide consists of four amino acid residues (glycine, cysteine, aspartate and glutamate; Yamamoto et al., 1987) and is able to bind four equivalents of chromic ions (Sun et al., 2000). In response to increasing circulating insulin (e.g., after a meal), insulin binds to the transmembrane insulin receptor triggering a conformational change.

Increasing levels of insulin elicit the movement of Cr into insulin-dependent cells. Following the

Cr ion flux inside the cell, apochromodulin (metal free form of chromodulin) sequesters Cr and becomes activated. Once activated, chromodulin binds to the insulin receptor, probably maintaining the receptor in its active conformation and amplifying its kinase activity. Decreased insulin levels cause chromodulin to be released from the cell and finally excreted in the urine

(Vincent et al., 2015).

The use of Cr as a supplement in animal feed started in the 1990s. However, responses to dietary Cr supplementation across species are inconsistent among studies (NRC, 1997). Evidence suggests that Cr supplementation improved glucose clearance rates in cattle (Bunting et al., 1994;

Summer et al., 2007; Spears et al., 2012) and swine (Amoikon et al., 1995; Guan et al., 2000) following an intravenous glucose tolerance test, suggesting improved tissue insulin sensitivity.

Additionally, supplemental Cr benefits production parameters and growth performance as it 19 improves feed intake, milk production, average daily gain, and carcass traits in different species

(Page et al., 1993, Lindemann et al., 1995; Hayirli et al., 2001; Smith et al., 2005). A summary of the effects of Cr supplementation on production and metabolism across species is presented in

Table 2.

During HS, the use of Cr as a supplement is under-explored. According to Soltan (2010),

Cr supplementation may offer a protective effect during HS as it improved milk production, reduced blood NEFA and cortisol concentrations, and improved reproductive performance in early lactating dairy cows. Similarly, lactating cows supplemented with Cr during the summer had increased feed intake and milk production, and decreased circulating insulin and NEFA levels (Al-

Saiady et al., 2004; Mizraei et al., 2011). In heat-stressed broilers, Cr supplementation increased feed intake, body weight gain, feed efficiency, and carcass composition traits (Sahin et al., 2002;

Sahin et al., 2003; Samantha et al., 2008; Toghyani et al., 2012; Jahanian and Rasouli, 2015). On the contrary, Cr use in swine during HS is more equivocal. For instance, Hung et al. (2014) reported that Cr supplementation in finishing gilts during the summer resulted in increased feed intake and tended to increase ADG; in addition, dietary Cr tended to decrease blood cortisol levels.

However, Kimm et al. (2009), did not observe differences in growth performance or blood metabolites in pigs supplemented with Cr and exposed to HS. It is not clear what is responsible for the inter-experimental variation; therefore, additional research is needed to better understand the ability of Cr supplementation to alleviate the adverse effects of HS.

Table 2. Effects of chromium supplementation on production parameters and metabolism Variable Form of Cr Species Response Reference Feed intake CrCl Poultry ↓ 29 CrMet Pigs = 27 Ruminants = 9 CrPic Pigs =/↓/↑ 1, 4, 12, 16, 30/ 13, 18 /7, 17 Poultry =/ ↑ 20 / 21, 28 Ruminants = 2 CrProp Pigs =/↓ 8, 14 / 13 Ruminants =/↑ 23 / 15 CrYst Pigs = 5 Average daily gain CrCl Poultry = 28 Turkeys ↑ 24 CrMet Pigs = 27 Ruminants = 9 CrPic Pigs =/↑/↓ 1, 3, 4, 10, 30/ 6, 12, 16, 17, 18, 30/ 13, 18 Poultry ↑ 19, 20, 22, 28 Ruminants = 2 CrProp Pigs =/ ↓ 14 / 13 CrYst Pigs = 5 G:F CrCl Poultry ↑ 29 CrPic Pigs =/↑ 1, 3, 10, 12, 13/ 11 Poultry ↑ 19, 20, 21, 22 Ruminants = 2 CrProp Pigs = 13, 14 Loin eye area CrPic Pigs =/↑ 16, 17 / 4, 18 CrProp Pigs = 14 Back fat CrPic Pigs = / ↓ 16, 17, 30/ 4, 8, 11, 18 CrProp Pigs =/↓ 14 Insulin sensitivity Synthetic Cr* Pigs ↑ 25 CrMet Ruminants ↑ 9 CrPic Pigs ↑ 1, 12, 13 Ruminants ↑ 2 CrProp Pigs ↑ 13 Ruminants ↑ 23, 26 Basal insulin CrPic Pigs ↓/= 1, 3 / 7, 13 Poultry ↑ 19, 20, 21 Ruminants = 2 CrProp Pigs = 13 Ruminants =/ ↓ 23/ 26 Glucose CrCl Poultry ↓ 29 CrMet Pigs ↓ 27 CrPic Pigs =/↓ 1, 7, 13/ 3 Poultry ↓ 20, 21 Ruminants = 2 CrProp Pigs = 13 Ruminants =/ ↑ 23/ 26 CrYst Pigs = 5 Cholesterol CrCl Poultry = 29 CrPic Pigs =/↑/↓ 13 / 1 / 18 Poultry ↓ 20, 21 Ruminants ↓ 2 CrProp Pigs = 13 NEFA CrPic Pigs ↓/= 1, 13/ 7 Ruminants = / ↓ 2 CrProp Pigs = 13 Ruminants ↓ 26 1Amoikon et al., 1995 11Lindemann et al., 1995 21Sahin et al., 2003 2Bunting et al., 1994 12Liu et al., 2017 22Sands and Smith, 1999 3Evock-Clover et al., 1993 13Mathews et al., 2001 27Tian et al., 2014 4Gang et al., 2000 14Mathews et al., 2005 28Toghyani et al., 2006 5Guan et al., 2000 15McNamara and Valdez, 2005 23Spears et al., 2012 6Harper et al., 1995 16Mooney and Cromwell, 1995 24Steele and Rosbrough, 1979 7Hung et al., 2014 17Mooney and Cromwell, 1997 25Steele et al., 1977 8Jackson et al., 2009 18Page et al., 1993 26Sumner et al., 2007 9Kegley et al., 2000 19Sahin et al., 2002a 29Uyanik et al., 2002 10Kim et al., 2009 20Sahin et al., 2002b 30Zhang et al., 2011 *Synthetic source of Cr+3 containing glucose tolerance factor

Zinc supplementation

Zinc is a trace element indispensable for all living organisms. Zinc was first discovered to be essential for the normal growth of rats (Todd et al., 1934) and birds (O’Dell et al., 1958).

However, the essentiality of Zn for humans was not identified until 1961, with symptoms of Zn deficiency including severe anemia, impaired growth, persistent diarrhea, chronic inflammation, liver disease, and impaired immune function, leading to a higher susceptibility to infections

(Prasad, 1985; Hambridge, 2000). Zinc plays a key role as a structural, catalytic, and signaling component. It is involved in DNA transcription to RNA (via Zn-finger transcription factors), metalloproteinase catalysis, protection from oxidative stress (when bound to metallothionein), regulation of apoptosis, cell homeostasis, and immune function (Kambe et al., 2015). Due to its important role in many biological processes, Zn homeostasis is tightly controlled by the gastrointestinal system via absorption of exogenous Zn and gastrointestinal secretion and excretion of endogenous Zn (Krebs, 2000). Zinc absorption occurs mainly in the duodenum and jejunum by passive diffusion and carrier-mediated processes (King et al., 2016). Approximately 60% of Zn is stored in skeletal muscle, ~30% in bone, ~5% in liver and skin, and the remainder 2-3% in other tissues (brain, kidney and pancreas; Jackson, 1989). Generally, causes for Zn deficiency include inadequate dietary intake, increased requirements, malabsorption, increased losses, and impaired utilization (Roohani et al., 2012).

The use of dietary Zn impacts intestinal barrier in different disease states. For instance,

Lambert et al. (2003) reported that Zn supplementation reduced alcohol-induced intestinal permeability and liver damage in a mouse model. Similarly, a protective effect of Zn on the mucosa barrier was observed in experimental colitis in rats, as the paracellular permeability to Na+ and mannitol in the distal colon were decreased in Zn-fed rats compared to non-supplemented rats 22

(Rohaweder et al., 1998; Sturniolo et al., 2001;). Dietary Zn oxide in guinea pigs lessened small intestinal permeability and TJ degradation associated with malnutrition (Rodriguez et al., 1996).

Additionally, the use of different forms of dietary Zn has demonstrated improved intestinal barrier function, increased expression of TJ proteins and down-regulation of pro-inflammatory cytokines in weaning pigs (Zhang and Guo, 2009; Hu et al., 2014). The role of Zn in membrane barrier function has also been demonstrated in in vitro studies. For instance, Rosselli et al. (2003) observed that Zn oxide had a protective effect on intestinal Caco-2 cells by reducing TJ permeability induced by enterotoxigenic E. coli. Moreover, this study showed that treatment with

Zn reduced the adhesion and internalization of bacteria, as well as the cytokine-induced inflammatory response in enterotoxigenic E. coli-infected cells. Similarly, Zn oxide supplementation modulated the inflammatory response to enterotoxigenic E. coli infection in porcine epithelial cells, suggesting an important role of Zn in maintaining normal gut function and health via reduced inflammation (Sargeant et al., 2011). The effects of Zn supplementation on the gastrointestinal tract in different species is summarized in Table 3.

The beneficial effects of Zn supplementation during a thermal load have also been reported in previous studies. Accordingly, Sahin and Kucuk (2003) observed dietary Zn sulfate increased feed intake, feed efficiency, nutrient digestibility, and egg production and quality in laying

Japanese quails exposed to HS. Similarly, broiler chickens exposed to HS had increased weight gain and feed efficiency, and decreased oxidative stress when supplemented with Zn sulfate

(Kucuk et al., 2003). Furthermore, organic Zn supplementation improved intestinal barrier integrity in acute and chronically HS pigs and ruminants as previously reported by our group. For instance, dietary Zn increased ileal transepithelial resistance in pigs exposed to an acute and chronic heat load (Sanz-Fernandez et al., 2014). Similarly, Pearce et al. (2015) observed increased 23 epithelial resistance, decreased circulating endotoxin and increased acute phase response (LBP and lysozyme) in acute-HS pigs fed with Zn. Additionally, Abuajamieh et al. (2016) reported Zn supplementation improved intestinal architecture characteristics and decreased rectal temperature in chronically-HS growing steers. Altogether, Zn supplementation not only improves intestinal integrity and health but, reciprocally, enhances animal growth and performance. Although the mechanisms by which Zn enhances intestinal integrity are not fully understood, evidence suggests that Zn supplementation might be beneficial at alleviating the detrimental effects of HS on animal health and productivity.

Table 3. Effects of zinc supplementation on intestinal barrier functiona Species Effect References Human ↑ 2, 3, 13, 17, 19, 24, 26, 29, 33 Cattle =/↑ 5/1 Pigs ↑ 6, 7, 8, 11, 16, 18, 25, 28, 34 Sheep ↑ 10 Rat ↑ 9, 12, 14, 20, 22, 30 In-vitro ↑ 4, 15, 21, 23, 27, 31, 32 aAdapted from: Abuajamieh, 2015 1Abuajamieh et al., 2016 18Pearce et al., 2015 2Alam et al., 1994 19Penney et al., 1999 3Faruque et al., 1999 20Peterson et al., 2008 4Finamore et al., 2008 21Ranaldi et al., 2013 5Glover et al., 2013 22Rodriguez et al., 1996 6Hedeman et al., 2006 23Roselli et al., 2003 7Hojber et al., 2005 24Roy et al., 2008 8Hu et al., 2013 25Sanz-Fernandez et al., 2014 9Ineu et al., 2013 26Sharieff et al., 2006 10Jafarpour et al., 2015 27Shao et al., 2017 11Katouli et al., 1999 28Song et al., 2015 12Lambert et al., 2003 29Sturniolo et al., 2001 13Lazzerini and Ronfani, 2008 30Sturniolo et al., 2002 14Mahmood et al., 2009 31Waeytens et al., 2009 15Mao et al., 2013 32Wang et al., 2013 16Owusu-Asiedu et al., 2003 33Young et al., 2014 17Patel et al., 2010 34Zhang and Gue, 2009

Conclusion

Heat stress negatively impacts animal productivity and health and represents a financial burden for global agriculture. Economic losses due to HS are mainly explained by decreased productivity and product quality, infertility, and increased mortality and health care costs. During

HS, feed intake decreases as a survival strategy to reduce metabolic HP and preserve euthermia.

Reduced nutrient intake was usually assumed to be the main culprit of decreased animal productivity during HS. However, recent studies have indicated that HS has “direct effects” on physiology, metabolism, and health that are independent of feed intake. Consequently, identifying strategies to improve animal performance and welfare during HS is of great interest. Nutritional alternatives to ameliorate the negative consequences of HS represent a practical approach to improve the animal response to a thermal load. Evidence suggests that Cr and Zn supplementation in animal diets improves productivity and intestinal health in different species. Therefore, their use during HS conditions may represent a good strategy to improve animal performance and health under these circumstances.

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CHAPTER 2: EFFECTS OF DIETARY CHROMIUM PROPIONATE ON GROWTH

PERFORMANCE AND METABOLISM DURING HEAT STRESS ON FINISHING PIGS

E. J. Mayorga*, S. K. Kvidera*, J. T. Seibert*, E. A. Horst*, M. Abuajamieh*, M. A. Al-Qaisi*,

S. Lei*, L. Ochoa†, B. Kremer‡, J. W. Ross*, and L. H. Baumgard*

*Iowa State University, Department of Animal Sciences, Ames, IA, 50011

†Carthage Veterinary Service, Ltd., Carthage, IL, 62321

‡Kemin Industries, Des Moines, IA, 50317

Abstract

Study objectives were to determine the effects of chromium (Cr) propionate (KemTRACE

Cr propionate 0.04%, Kemin Industries Inc., Des Moines, IA) on growth performance and biomarkers of metabolism and health in heat stress (HS) and nutrient-restricted pigs. Crossbred barrows (n = 96; 105 ± 1 kg BW) were enlisted in a 2x3 factorial experiment with two replicates, blocked by initial BW, and randomly assigned to one of six dietary-environmental treatments: 1) thermoneutral (TN) conditions and fed ad-libitum a control diet (TNCtl), 2) TN conditions and fed ad-libitum a Cr supplemented diet (TNCr), 3) TN and pair-fed a control diet (PFCtl), 4) TN and pair-fed a Cr supplemented diet (PFCr), 5) HS and ad-libitum fed a control diet (HSCtl), and 6)

HS and ad-libitum fed a Cr supplemented diet (HSCr). Pair-fed animals were provided feed to mirror their HS counterparts to eliminate the confounding effects of dissimilar feed intake. The treatment diet contained 0.5g/kg of feed to deliver 200 ppb Cr/d. The study consisted of three 48 experimental periods (P). During P0 (5 d), all pigs were housed in TN conditions (21.3 ± 0.1 ºC,

56.8 ± 0.3% RH) and fed the control diet ad libitum. During P1 (5 d), pigs were fed their respective dietary treatments ad-libitum and kept in TN conditions. During P2 (35 d), HSCtl and HSCr treated pigs were fed ad-libitum and exposed to progressive cyclical HS conditions (27 to 31ºC,

50 ± 0.3% RH) while TNCtl, TNCr, PFCtl and PFCr pigs remained in TN conditions and were fed ad-libitum or pair-fed to their HSCtl and HSCr counterparts. Overall, pigs exposed to HS had increased (P<0.01) rectal temperature, skin temperature, and respiration rate (0.3ºC, 3.8ºC, and 32 bpm, respectively) relative to TN pigs. Overall, HS decreased (P<0.01) ADFI (20%) and ADG

(21%) compared to TN controls. Final BW tended to be increased in HSCr (2.7 kg, P=0.06) compared to HSCtl pigs. Similarly, ADG tended to be increased during P2 in HSCr relative to

HSCtl treatment (0.77 vs. 0.72 kg/d; P=0.10). There were no effects of Cr on most production parameters, but ADFI tended to be increased in Cr relative to Ctl-fed pigs (3.19 vs. 3.09 kg/d;

P=0.08). No effects of Cr supplementation were detected on circulating glucose, insulin, NEFA, cholesterol, triglycerides, or lipopolysaccharide binding protein. However, blood neutrophils were increased in HSCr (37%; P<0.01) relative to HSCtl pigs. In summary, these findings suggest Cr supplementation may be beneficial to growth performance and health during HS.

Key words: chromium, growth performance, heat stress, pig

Introduction

Heat stress (HS) is a common environmental issue in animal agriculture and is a major limitation to efficient and profitable pig production. The economic losses in the US industry alone are thought be nearly $1 billion annually (St. Pierre et al., 2003; Pollmann, 2010). This lost revenue 49 is mainly explained by the negative effects of HS on animal health (in severe conditions animals can die from HS), decreased and inconsistent growth, inefficient feed utilization, and poor reproduction (Baumgard and Rhoads, 2013; Ross et al., 2015).

Heat-stressed animals employ physiologic and metabolic adjustments to ameliorate the heat insult; however, these thermoregulatory responses have adverse consequences on animal productivity and health. Swine performance during HS varies depending on the age. For example, growing-finishing pigs are more susceptible than young pigs due to their higher energy intake relative to maintenance requirements and their decreased ability to dissipate heat (i.e., a lower surface area-to-mass ratio and thicker subcutaneous fat tissue). Unlike other domesticated species which have the capacity to sweat, pigs rely more on reduced metabolic heat production (HP) to maintain a constant body temperature in hot conditions (Renaudeau et al., 2012). Voluntarily reducing feed intake is the main adaptation pigs employ to decrease HP during HS. Further, HS markedly alters post-absorptive carbohydrate, lipid and protein metabolism (independently of reduced feed intake), a homeorhetic adaptation primarily characterized by an unexplainable increase in circulating insulin (Baumgard and Rhoads, 2013).

Chromium (Cr) is thought to primarily improve productivity via increasing insulin action in insulin sensitive tissues (i.e., adipose and muscle; Davis and Vincent, 1997). Additionally, previous research has demonstrated dietary Cr supplementation improves feed intake in pigs

(Hung et al., 2014) and ruminants (Hayirli et al., 2001; Sadri et al., 2009; Soltan, 2010; Yasui et al., 2014), as well as growth rate, carcass characteristics, and reproductive performance of swine

(Lindemann et al., 1995; Hung et al., 2010; Sales and Jancik, 2011). Feeding supplemental Cr may augment an animal’s ability to upregulate insulin action during HS and improve growth performance. Therefore, our objectives were to evaluate the ability of Cr to ameliorate the negative 50 consequences of HS in a finishing pig model. If Cr helps to maintain productivity during HS, it would be of great benefit to agriculture animal welfare and economics.

Materials and Methods

Animals, diets, and experimental design

Ninety-six crossbred barrows (105 ± 1 kg BW) were utilized in a replicated (48 pigs per replicate) experiment. Pigs were blocked by initial BW and randomly assigned to one of six treatments: 1) thermoneutral (TN) ad-libitum control diet (TNCtl; n = 12), 2) TN ad-libitum Cr supplemented diet (TNCr; n = 12), 3) TN pair-fed control diet (PFCtl; n = 12), 4) TN pair-fed Cr supplemented diet (PFCr; n = 12), 5) HS ad-libitum control diet (HSCtl; n = 24), and 6) HS ad- libitum Cr supplemented diet (HSCr; n = 24). Pigs were allotted to one of two environmentally- controlled rooms. Each room had 24 crates (57 x 221 cm) where pigs were housed individually.

Each crate was equipped with a stainless-steel feeder and a nipple drinker. Water was provided ad-libitum during the entire experiment. All procedures were approved by the Iowa State

University Institutional Animal Care and Use Committee and the experiment was conducted in the

Discovery Wing at the Iowa State University Swine Nutrition Farm research facility (Ames, IA).

One pig from the HSCr treatment was removed from the experiment due to health issues and its data was not included in the final data set.

Two diets were formulated and mixed according to the following specifications: 1) a control diet based on corn-soybean meal and 2) a Cr diet formulated based on corn-soybean meal and supplemented with Cr (KemTRACE Cr propionate 0.04%, Kemin Industries Inc., Des Moines,

IA) at the rate of 0.5 g/kg of feed, to deliver 200 parts per billion Cr. Both Ctl and Cr diets were treated with an antioxidant (ENDOX, Kemin Industries Inc., Des Moines, IA) at the rate of 0.125 51 g/kg of feed. Diets were processed in meal form and mixed at the Iowa State University Swine

Nutrition Center Mill. Diets were formulated to meet or exceed the predicted requirements for finishing pigs for energy, essential amino acids, protein, minerals, and vitamins (NRC, 2012; Table

4).

The study was divided into three experimental periods (P): P0, P1 and P2. Period 0 (5 d) was an acclimation period where all pigs were housed in individual crates, subjected to TN conditions (21.3 ± 0.1°C, 56.8 ± 0.3 % RH) with a 12h:12h light-dark cycle, and fed the control diet ad-libitum. During P1 (5 d), pigs were fed their respective dietary treatments ad-libitum and kept in TN conditions for collection of baseline body temperature indices and production parameters. During P2 (35 d), HSCtl and HSCr animals were fed ad-libitum and exposed to progressive cyclical HS conditions with temperatures ranging from 27 to 31°C (day 1: 26.5 ±

0.3°C, 58.7 ± 1.6% RH from 0800 to 1800 h, and 27.1 ± 0.1°C, 56.2 ± 1.6% RH from 1800 to

0800 h; day 2: 28.5 ± 0.2°C, 55.1 ± 1.1% RH from 0800 to 1800 h, and 26.9 ± 0.1°C, 61.9 ± 0.9%

RH from 1800 to 0800 h; day 3 to day 7: 29.6 ± 0.2°C, 55.3 ± 1.0% RH from 0800 to 1800 h, and

27.1 ± 0.1°C, 55.3 ± 1.4% RH from 1800 to 0800 h; day 8 to day 35: 31.4 ± 0.1°C, 49.2 ± 0.3%

RH from 0800 to 1800 h, and 27.3 ± 0.1°C, 47.5 ± 0.4% RH from 1800 to 0800 h). Both temperature and humidity were monitored and recorded every 5 min by data loggers (Lascar EL-

USB-2-LCD, Erie, PA). One data logger was placed in each quadrant of each room (8 data loggers in total) and data were condensed into room averages by each time point. During P2, pigs assigned to the TNCtl and TNCr treatments remained in TN conditions and were fed ad-libitum. Pigs in

PFCtl and PFCr treatments also remained in TN conditions but were pair-fed to their HS counterparts to evaluate the direct effect of HS while eliminating the confounding effects of dissimilar nutrient intake. Average daily feed intake in P1 was averaged for each pig and used as 52 a baseline. During P2, the decrease in ADFI in HS pigs was calculated everyday as a percentage of ADFI reduction relative to P1. The percentage of ADFI reduction was averaged for all pigs in

HS treatments per day of heat exposure and applied individually to the baseline of each pig in the

PF treatments. The daily amount of feed provided was divided into two equal portions during P2

(~0800 and 1800 h) to minimize large metabolic changes associated with gorging. In order to accomplish the pair-feeding, pigs assigned to the PF treatments spent an additional 2 d in P0, so they were delayed 2 d from the TN and HS treatments during the experimental period (P1 and P2).

This technique of pair-feeding has been extensively utilized by our research program (Pearce et al., 2013a,b; Sanz-Fernandez et al., 2015).

Production parameters

Feed intake was measured daily during P1 and P2 as feed disappearance. Body weights were obtained at the beginning of P0 and P1, at the end of P1, and on d 7, 14, 21, 28 and 35 of P2.

In addition, 10th-rib back fat thickness (BF) and loin eye area (LEA) were measured via ultrasound scan (Cai et al., 2008) on d -10 of P0 and 35 of P2.

Body temperature measurements

During both P1 and P2, body temperature indices (rectal temperature [TR], skin temperature [TS] and respiration rates [RR]) were obtained twice daily (~0700 and 1800 h). Rectal temperatures were measured using an electronic thermometer (SureTemp® Plus 690, WelchAllyn,

Skaneateles Falls, NY, USA). Skin temperature was measured using an infrared thermometer

(IRT207: The Heat Seeker 8:1 Mid-Range Infrared Thermometer, General Tools, New York, NY) and RR were determined by counting flank movements in 15 s intervals and multiplying by four to obtain breaths per minute (BPM). All indices recorded were condensed into AM and PM averages. 53

Blood sampling and analysis

Blood samples were obtained by jugular venipuncture (plasma and serum; K2EDTA and

BD Vacutainers, Franklin Lakes, NJ) on d -5 and -1 of P1, and d 7 and 35 of P2. Samples of serum and plasma were stored at 4ºC and sent later to the Iowa State Department of Veterinary Pathology for hematology and blood chemistry analysis. A second set of plasma samples were harvested by centrifugation at 4ºC and 1500 x g, aliquoted, and stored at -20ºC until analysis. Plasma non- esterified fatty acids (NEFA; Wako Pure Chemical Industries, Ltd., Osaka, Japan), insulin

(Mercodia Porcine Insulin ELISA; Mercodia AB; Uppsala, Sweden), and lipopolysaccharide- binding protein (LBP; Hycult Biotech, Uden, Netherlands) were measured following the manufacturer’s instructions. The intra and inter-assay coefficients of variation for NEFA, insulin, and LBP were 8.4 and 8.8%, 6.2 and 1.6%, and 10.5 and 14.3%, respectively.

Statistical analysis

All data from P2 were statistically analyzed using SAS version 9.4 (SAS Institute Inc.,

Cary, NC). Daily measurements were condensed into weekly means. Body temperature indices, blood metabolites, and production parameters (i.e., ADFI, BW, ADG, and G:F) were analyzed using the PROC MIXED procedure of SAS with an autoregressive covariance structure and week of experiment as the repeated effect. The model included treatment, week, treatment by week interaction, block and replicate as fixed effects. Period 1 values for each variable were used as a covariate. Final BW, BF thickness, and LEA were analyzed using the PROC MIXED procedure with a diagonal covariance structure and the initial value of the parameter of interest as a covariate.

Results are reported as least square means and considered significant if P ≤ 0.05 and a tendency if

0.05 < P ≤ 0.10. 54

Results

Growth Performance

Average daily feed intake decreased during P2 (20%, 0.70 kg; P < 0.01; Table 5; Figure

3A) in HS pigs, relative to TN controls. During HS exposure, BW was reduced in HS pigs (3, 4, and 4% on weeks 2, 3, and 5, respectively; P < 0.01; Figure 3B) relative to TN treatments. Average daily gain was also decreased in HS pigs relative to ad-libitum TN controls (21%, 0.20 kg; P <

0.01; Table 5; Figure 4A). Final BW was decreased in HS treatments (6.5 kg, P < 0.01; Table 5;

Figure 4B) compared to TN treatments. Interestingly, during P2, G:F did not differ between HS and TN environments (P = 0.34; Table 5; Figure 4C). By experimental design, PFCtl and PFCr pigs had a similar pattern and extent in ADFI reduction and their BW and ADG variables were similar to the HS pigs (P > 0.05; Table 5). Similarly, G:F was decreased in PF treatments when compared to TN (8%; P = 0.02) and HS treatments (9%; P < 0.01). There were no differences in

ADFI between HSCtl and HSCr treatments (2.80 vs. 2.92 kg/d, respectively; P = 0.12; Table 5).

Final BW tended to be increased in HSCr pigs (2.7 kg; P = 0.06; Table 5) compared to HSCtl pigs.

Similarly, a tendency for increased ADG during P2 was observed in HSCr (P = 0.10; Table 5) relative to HSCtl treatment, but there were no overall treatment effects on G:F between HSCtl and

HSCr treatments (P = 0.61). When comparing Ctl vs. Cr-fed pigs, no differences were detected for

BW, ADG, final BW, or G:F; however, ADFI tended to be increased (3.19 vs. 3.09 kg/d; P =

0.08; Table 5) in Cr relative to Ctl-fed pigs.

There were no differences in BF between HS and TN treatments (P = 0.18); but, BF was decreased in the PF treatments (12%; P < 0.01; Table 5) when compared to their TN counterparts.

There was no treatment effect on BF when comparing HSCtl and HSCr treatments (P = 0.95).

Overall, no differences in BF were detected when comparing Ctl vs. Cr-fed pigs (P = 0.82). 55

Compared to TN treatments, LEA was decreased in HS and PF treatments (3 and 4%, respectively;

P < 0.05; Table 5). There was no treatment effect on LEA (P = 0.59) between HSCtl and HSCr treatments. No overall effects of dietary Cr were detected in LEA (P = 0.76).

Body temperature indices

During P2, all pigs exposed to HS conditions had an overall increase in TR relative to TN and PF treatments (0.40°C; P < 0.01; Table 6). Skin temperature was also increased during P2

(13%, 4.6°C; P < 0.01; Table 6) in the HS environment when compared to TN and PF treatments.

Respiration rate was increased in the HS pigs relative to TN and PF controls (71%, 33 bpm; P <

0.01; Table 6). Interestingly, TR and TS were decreased (0.16 and 1.69°C, respectively; P < 0.01) in PF treatments relative to TN controls. There were no differences in TS or RR between HSCtl and HSCr treatments, but TR at 1800 h was increased in HSCr (0.11°C; P = 0.01; Table 6; Figure

5A) relative to HSCtl pigs. Overall, irrespective of environment, there were no differences in any body temperature indices comparing Ctl vs. Cr-fed treatments (P > 0.10; Table 6).

Blood metabolites

No treatment differences were observed for circulating insulin during P2 (P = 0.17; Table

7; Figure 6A). However, PF pigs had increased insulin over time (1.6 fold; P = 0.03) relative to

HS pigs. There was no treatment effect on circulating insulin between HSCtl and HSCr-fed pigs

(P = 0.52). Overall, no treatment differences in circulating insulin were observed when comparing

Ctl vs. Cr-fed animals (P = 0.35). The insulin:ADFI did not differ between HS and TN treatments

(P = 0.86). However, the insulin:ADFI tended to be increased in PF treatments, relative to TN controls (P = 0.06). Pair-fed pigs had markedly increased insulin:ADFI compared to HS pigs

(64%; P = 0.04). No differences in the insulin:ADFI were detected between Ctl and Cr-fed pigs

(P = 0.19). Blood glucose content did not differ between treatments (P = 0.69; Table 7; Figure 56

7B) and no differences were detected between HSCtl and HSCr treatments (P = 0.17) or Ctl and

Cr-fed pigs (P = 0.95). The insulin:glucose did not differ among treatments (P = 0.21). However, insulin:glucose was increased in the PF pigs (~65%; P = 0.04) relative to HS pigs. No differences were detected in insulin:glucose between HSCtl and HSCr treatments (P = 0.69) or Ctl and Cr-fed pigs (P = 0.29). No dietary treatment effects were observed for plasma NEFA levels (P = 0.92;

Table 7; Figure 6C). Similarly, there was no difference in NEFA when comparing HSCtl vs. HSCr treatments (P = 0.82). Overall, no differences in NEFA levels were detected between Ctl and Cr- fed pigs (P = 0.65).

Compared to TN treatments, HS treatments tended to have increased LBP levels (36%; P

= 0.07; Table 7; Figure 6D). Similarly, LBP was increased in HS pigs relative to PF pigs (42%; P

= 0.05). No differences in LBP levels were observed between HSCtl and HSCr pigs (P = 0.11).

Additionally, there were no overall treatment effects on LBP levels between Ctl and Cr-fed pigs

(P = 0.15).

No treatment differences were observed in circulating sodium (P = 0.70; Table 7).

Potassium levels did not differ between HS and TN treatments (P = 0.17; Table 7), but tended to be increased in HS pigs (5%; P = 0.10) in comparison to PF controls. Additionally, potassium levels were increased in HSCr relative to their HSCtl counterparts (P = 0.03; Table 4). Chloride levels were increased in HS pigs when compared to TN and PF controls, however these differences were small (1.3 and 1.5% for TN and PF treatments, respectively; P < 0.01). Similarly, bicarbonate levels were decreased (7%; P < 0.01; Table 7) in HS pigs compared to TN and PF controls. Blood calcium tended to be decreased in HS animals compared to the TN controls (10.6 vs. 10.7 mg/dL;

P = 0.06). Additionally, blood phosphorous concentration was decreased in HS relative to TN and

PF treatments (6 and 5%, respectively; P < 0.01). Blood magnesium levels did not differ between 57

HS and TN treatments (P = 0.51), but tended to be increased in PF compared to TN treatments

(3%; P = 0.09). Interestingly, magnesium levels were decreased in Cr compared to Ctl-fed pigs

(3%; P = 0.01). No dietary treatment effects on sodium (P = 0.35), chloride (P = 0.88), bicarbonate

(P = 0.21), calcium (P = 0.71), phosphorous (P = 0.71), or magnesium (P = 0.78) were observed when comparing HSCtl vs. HSCr treatments (Table 7).

Blood urea nitrogen (BUN) was decreased in HS and PF treatments when compared to TN treatments (14 and 18%, respectively; P < 0.01; Table 7). In contrast, creatinine was increased in

HS pigs relative to TN and PF controls (10%; P < 0.01). Similarly, total circulating protein was increased in HS and PF treatments (4 and 3%, respectively; P < 0.05; Table 7) when compared to

TN controls. Additionally, albumin levels in blood tended to be increased in the PF animals relative to their TN counterparts (P = 0.08) and were increased in the HSCr pigs when compared to the

HSCtl treatment (P = 0.05; Table 7). Creatine kinase levels were increased in HS treatments relative to both TN and PF pigs (P < 0.05; Table 4). Alkaline phosphatase in blood was decreased in HS treatments when compared to TN and PF treatments (16 and 21%, respectively; P < 0.01;

Table 7). No dietary treatment effects were observed for blood aspartate aminotransferase (P =

0.67), gamma-glutamyl transferase (P = 0.30), bilirubin (P = 0.80), or anion gap (P = 0.36).

No dietary treatment effects were observed in blood cholesterol (P = 0.20; Table 7), but cholesterol levels in HS pigs tended to be decreased (3%; P = 0.09) and were decreased (4%; P =

0.03) relative to TN and PF pigs, respectively. When comparing HSCtl vs. HSCr, no differences were observed in plasma cholesterol (P = 0.80). Circulating triacylglycerides (TAG) did not differ among treatments (P = 0.23), but were increased in PF relative to TN treatments (15%; P = 0.03).

Overall, no differences were observed in cholesterol or TAG between Ctl and Cr-fed pigs (P =

0.22 and P = 0.65, respectively). 58

Blood neutrophils were increased in HSCr-fed pigs relative to HSCtl treatment (37%; P <

0.01; Table 8). There were no dietary treatment effects on circulating lymphocytes (P = 0.20; Table

8). However, a tendency for decreased lymphocytes were observed in HS relative to TN treatments

(7%; P = 0.08). In addition, PF pigs had increased circulating lymphocytes when compared to their

HS counterparts (9%; P = 0.03). Monocytes were increased in HSCr pigs when compared to HSCtl pigs (16%; P = 0.05; Table 8). However, no dietary treatment effects were detected on other white blood cells including eosinophils and basophils (P > 0.10). A tendency for increased RBC was observed in PF treatments when compared to TN controls (3%; P = 0.07). Additionally, no differences were observed in RBC between HSCtl and HSCr treatments (P = 0.75; Table 8).

Plasma hemoglobin tended to be increased in PF animals (3%; P = 0.06), relative to TN animals.

When comparing within PF treatments, PFCtl pigs had increased hemoglobin relative to PFCr pigs

(5%). No differences in hemoglobin were detected between HSCtl and HSCr treatments (P = 0.55).

Plasma hemoglobin tended to be reduced in Cr compared to Ctl-fed pigs (2%; P = 0.09).

Hematocrit did not differ between HS and TN treatments (P = 0.88), but PF treatments tended to have an increased hematocrit level compared to TN controls (3%; P = 0.07). Similarly, hematocrit was increased in PF pigs in comparison to HS pigs (3%; P = 0.05). Overall, hematocrit was reduced in Cr compared to Ctl-fed pigs (2.5%; P = 0.04). No treatment differences were detected on circulating platelets (P = 0.57).

Discussion

When pigs are exposed to environmental conditions that exceed their thermoneutral zone, changes in metabolic HP are essential to maintain euthermia (Collin et al., 2001). The main 59 adaptation to reduce HP is by decreasing voluntary ADFI, a very conserved response to HS across species. Furthermore, animals alter their carbohydrate, lipid and protein metabolism independently of nutrient intake. This homeorhetic process involves changes in fuel supply and use by different tissues, an energetic strategy mediated in large part by increased basal and stimulated circulating insulin levels (Baumgard and Rhoads, 2013). Consequently, physiological and metabolic adjustments to a thermal load result in decreased animal performance and welfare, which negatively impacts global agriculture. In the pig industry, economic losses caused by increased ambient temperature are mainly expressed by decreased and inconsistent growth, decreased carcass quality, increased health care costs, reduced fecundity, and increased mortality

(Baumgard and Rhoads, 2013; Ross et al., 2015). Thus, identifying protocols to alleviate the negative consequences of HS is critical to enhance the animal response to a thermal challenge.

Nutritional interventions represent a practical and adaptable avenue to improve the animal response to a heat load and to alleviate some of the negative consequences of HS on performance.

Accordingly, dietary Cr as a supplement in animal diets has recently gained much interest. Dietary

Cr is thought to potentiate insulin action by facilitating the binding of insulin to its extracellular receptor (NRC, 1997; Vincent, 2000), subsequently enhancing cellular glucose uptake (Chen et al., 2006). Additionally, previous studies have reported the benefits of Cr supplementation as it improves ADFI, growth performance, reproductive parameters, and the immune response in multiple species (Burton et al., 1993; Lindemann et al., 1995; Hayirli et al., 2001; Sadri et al.,

2009; Sales and Jancik, 2011; Hung et al., 2014; Yasui et al., 2014). We hypothesized supplementing Cr during HS may enhance animal productivity and health. Herein, we evaluated potential effects of Cr supplementation in heat-stressed and nutrient-restricted pigs. 60

Heat stress was successfully implemented during the current study as evident by increased

TR and TS (0.3 and 3.8°C, respectively) and RR (32 bpm) in HS relative to TN pigs. Interestingly,

HSCr pigs had a slightly increased TR (0.1°C) at 1800 h, relative to HSCtl pigs. Presumably, increased BW gain and numerically increased ADFI in HSCr fed pigs relative to their HSCtl counterparts contributed to greater metabolic HP and thus increased body temperature indices.

Conversely, decreased TR and TS (0.2 and 1.7°C, respectively) were observed in the PF treatments when compared to their TN counterparts, suggesting decreased basal HP associated with reduced nutrient intake as previously reported by our group (Pearce et al., 2013a).

As expected, thermal stress considerably decreased ADFI, ADG, and final BW (~20, 21 and 4%, respectively), relative to TN pigs. By experimental design, PF treatments followed the same pattern and extent of decreased ADFI. The pattern and magnitude of reduced ADFI is comparable to that observed in our previous experiments (Johnson et al., 2015a,b). Reduced nutrient intake is a hallmark characteristic of HS and it represents an effort to minimize metabolic

HP associated with digestive processes and nutrient metabolism (Collin et al., 2001; Baumgard and Rhoads, 2013). Decreased ADFI during HS reduces the energy available for tissue synthesis and partly explains the decrease in growth performance observed in animals exposed to high ambient temperatures (Le Bellego et al., 2002; Kerr et al., 2003; Johnson et al., 2015a,b).

Interestingly, regardless of environment, pigs fed Cr had a tendency for increased ADFI

(~3%) compared to Ctl-fed pigs. Additionally, pigs fed Cr during HS had a tendency for increased

ADG and final BW. Improved BW gain and ADFI in Cr-fed pigs during this study agrees with previous reports where Cr supplementation during HS enhanced growth performance in different species. For instance, K. Sahin (2002) and N. Sahin (2005) reported increased ADFI, BW gain, and egg production in heat-stressed laying quails supplemented with Cr. Similarly, dietary Cr 61 increased ADFI, BW gain and carcass composition traits in heat-stressed broilers (Lien et al., 1999;

Toghyani et al., 2012). In heat-stressed lactating dairy cows, dietary Cr improved ADFI, milk production, and reproductive performance (Soltan et al., 2010; Mirzaei et al., 2011). Nevertheless, studies on Cr supplementation during HS in swine are scarce and its effects have been more equivocal. Accordingly, Kim et al. (2009) did not observe any differences in growth performance of heat-stressed growing pigs supplemented with Cr. In contrast, Hung et al. (2014) reported dietary Cr increased ADFI and tended to increase ADG in finisher gilts during the summer.

Results from the current study corroborate with reports of improved productivity in heat-stressed pigs and other animals. However, variations of the phenotypic responses to Cr supplementation during HS might be due to dissimilar experimental models, species, Cr source, supplementation doses, and type of HS insult. Regardless, in the current study, Cr supplementation improved some of the economically important phenotypes during HS.

Intriguingly, G:F in HS pigs was not different when compared to the TN pigs, but was increased in HS pigs relative to their PF counterparts (0.27 vs. 0.24 kg gain/kg feed, respectively).

Traditionally, the dogma is that G:F is compromised during HS because of the ostensible increase in maintenance costs. Increased energy expenditure during HS is attributed to the energy costs for heat dissipation (i.e., panting, sweating) as previously observed in pigs (Campos et al., 2014), poultry (Yahav, 2007), cattle (Mcdowell et al., 1969; Beede and Collier, 1986), and rodents

(Collins et al., 1980) and increased chemical reaction rates as predicted by the Van’t Hoff Law

(Kleiber, 1961; Fuquay, 1981). However, effects of thermal load on G:F are inconsistent as some studies have reported HS either decreases (Kerr et al., 2003; Renaudeau et al., 2008; Pearce et al.,

2013a; Johnson et al., 2015b), increases (Lefaucheur et al., 1991), or has no effect (Collin et al.,

2001; Johnson et al., 2015a) on G:F. Furthermore, reduced levels of circulating thyroid hormones 62 have been reported in animals exposed to HS (Prunier et al., 1997; Garriga et al., 2006; Johnson et al., 2015c; Sanz-Fernandez et al., 2015) which, coupled with reduced activity and reduced voluntary ADFI suggests HP is in fact attenuated during HS (Collin et al., 2001; Huynh, et al.,

2005; Johnson et al., 2015a,b). Consequently, differences in G:F between HS and PF pigs, despite similar ADFI, are of particular interest. Presumably, reduced maintenance costs and HP in pigs exposed to HS allows improved energy partitioning towards growth (Johnson et al., 2015a).

Additionally, as further discussed below, large differences in body composition may partially explain why a simple ratio of gross BW to ADFI was not affected during HS.

Animals exposed to HS have increased adipose tissue accretion, and decreased accumulation of skeletal muscle based upon their nutrient intake (as reviewed by Ross et al., 2015).

This phenomenon is biologically interesting as feed-restricted pigs reared in TN conditions prioritize lean tissue deposition at the expense of fat (van Milgen and Noblet, 2003; Oresanya et al., 2008); and this suggests HS has a direct biological effect on the hierarchy of nutrient partitioning independent of reduced ADFI. Similar results were observed in the current study as subcutaneous fat deposition (as measured by BF) did not differ between HS and TN treatments, despite a 20% reduction in ADFI of HS animals. Furthermore, as expected BF was decreased in the PF treatments when compared to their TN counterparts. This data corroborates previous reports that fat deposition during HS is not explained by reduced ADFI, and these observations are likely due to differences in lipid metabolism and the inability to mobilize adipose tissue as demonstrated in a variety of species (as reviewed by Baumgard and Rhoads, 2013). In contrast to adipose tissue,

LEA (a proxy of muscle accretion) was reduced in HS and PF pigs (~3 and 4%, respectively) relative to their TN counterparts and thus it appears this can be explained by decreased ADFI.

Consequently, our observations validate the fact that HS altered body composition presumably by 63 limiting maximum lean tissue accretion or by altering nutrient partitioning between protein and lipid deposition (Le Bellego et al., 2002; Johnson et al., 2015a,b).

Despite HS-induced reduced ADFI and growth performance, no differences were observed on blood metabolites (i.e., insulin, glucose, NEFA) across treatments. This is surprising as HS is thought to alter post-absorptive carbohydrate, lipid, and protein metabolism, a mechanism characterized by an unexplainable increase in circulating insulin levels and decreased NEFA concentrations, relative to PF animals in TN conditions (Baumgard and Rhoads, 2013). Lack of differences in blood metabolites in this study may reflect the fact that just two blood samples (d 7 and d 35 of P2) were obtained over the 5-wk thermal load period. Additionally, differences in basal metabolism as a response to the environmental treatments could have occurred during the first days of HS exposure, and presumably our design did not allow for identifying those changes.

Reduced intestinal integrity is commonly observed in HS studies (Lambert et al., 2002;

Pearce et al., 2013b; Sanz-Fernandez et al., 2014) and can lead to subsequent inflammatory response. This inflammatory response often includes increased circulating LBP, an acute phase protein produced in the liver in response to lipopolysaccharide, and immunogenic bacterial component (Lu et al., 2008). In the current study, circulating LBP levels in HS pigs tended to or were increased relative to TN and PF pigs. Increased levels of LBP in HS pigs suggest an increased acute phase protein and inflammatory response likely due to intestinal barrier dysfunction. Our results disagree with previous reports in our group where circulating LBP was decreased in acute heat-stressed pigs (Pearce et al., 2014, 2015). A possible explanation for this inconsistency may relate to the chronic duration of the heat load as HS exposure in those studies did not exceed 12 hours, while in the current study pigs were exposed to a chronic long-term HS exposure. 64

Inflammatory conditions during HS warrant further investigation into the potential immunomodulatory role of dietary Cr supplementation, which has been previously reported in beef calves and dairy cows. For instance, Chang and Mowat (1992) and Moonsier-Shageer and Mowat

(1993) observed decreased circulating cortisol and increased immunoglobulin and antibody concentrations in transportation-stressed calves fed Cr. Additionally, Burton et al. (1993) reported increased cell-mediated immune response in periparturient and early lactating dairy cows supplemented with Cr. Furthermore, we and others have observed improved neutrophil count and activity with Cr supplementation (Lien et al., 2004; Kafilzadeh et al., 2012; Yasui et al., 2014;

Horst et al., unpublished data). In agreement with the aforementioned literature, circulating neutrophils were increased in HSCr pigs by 37% relative to their HSCtl counterparts.

Additionally, monocytes were increased in HSCr (16%) compared to the HSCtl pigs. Both increased neutrophil and monocyte levels suggest Cr supplementation during HS may support the requirements of proliferating innate immune cells. Upon activation, immune cells become sensitive to insulin (Maratou et al., 2007) and the insulin-sensitizing properties of Cr may improve immune function in that manner. Moreover, since no dietary differences in neutrophil counts were observed in TN or PF treatments, this indicates that Cr’s beneficial role at improving leukocyte proliferation may be more pronounced during an immune activation scenario.

In conclusion, chronic cyclical HS increased all body temperature indices, markedly reduced ADFI, and decreased productive parameters including ADG, BW, and carcass traits.

Herein we demonstrate Cr supplementation improved BW and ADG in chronically heat-stressed pigs. In addition, ADFI tended to be increased in Cr relative to Ctl-fed pigs, regardless of environment. Interestingly, dietary Cr supplementation increased circulating neutrophil counts as well as circulating monocytes in pigs exposed to HS. No other differences were observed in 65 regards to carcass characteristics, body temperature indices, or blood metabolites when pigs were supplemented with Cr. Altogether, Cr supplementation might be a valuable dietary strategy to ameliorate the negative consequences of HS on growth performance and health. However, additional research is warranted to better understand the biology and mode of action of dietary Cr in pigs under these circumstances.

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Table 4. Ingredient composition and chemical and nutritional composition of experimental diets (as-fed basis) Item Control diet Chromium diet Ingredient composition Corn, % 86.10 86.05 Soybean meal, % 11.43 11.44 L-Lys HCl, % 0.19 0.19 L-Thr, % 0.03 0.03 Monocalcium phosphate, % 0.55 0.55 Limestone, % 0.87 0.87 NaCl, % 0.50 0.50 Vitamin Premix1, % 0.16 0.16 Trace Mineral Premix2, % 0.15 0.15 Endox3, % 0.01 0.01 Chromium propionate4, % 0 0.05 Diet composition Dry matter5, % 86.74 86.93 NE, Kcal/kg 2547 2545 ME, Kcal/kg 3309 3308 NE:ME ratio 0.76 0.76 CP5, % 12.13 11.82 ADF, % 3.1 3.1 NDF, % 8.8 8.8 Ether extract5, % 2.55 2.03 SID6 AA, % Lys 0.61 0.61 Thr 0.40 0.40 Met + Cys 0.39 0.39 Trp 0.11 0.11 Ca, % 0.48 0.48 Total P, % 0.42 0.42 STTD7 P, % 0.22 0.22 1Vitamin premix provided the following (per kg diet): 4,900 IU of vitamin A, 560 IU of vitamin D3, 40 IU of vitamin E, 2.4 mg of menadione (to provide vitamin K), 39 μg of vitamin B12, 9 mg of riboflavin, 22 mg of d-pantothenic acid, and 45 mg of niacin 2Mineral premix provided the following (per kg diet): 165 mg of Fe (ferrous sulfate), 165 mg of Zn (zinc sulfate), 39 mg of Mn (manganese sulfate), 2 mg of Cu (cooper sulfate), 0.3 mg/kg of I (calcium iodate), and 0.3 mg/kg of Se (sodium selenite). 3Endox (Kemin Industries Inc., Des Moines, IA) 4Chromium propionate, 0.5 g/kg of feed to deliver 200 parts per billion (KemTRACE Cr propionate 0.04%, Kemin Industries Inc., Des Moines, IA) 72

5Assayed values 6Standardized ileal digestibility 7Standardized total tract digestibility

Table 5. Effects of chromium propionate supplementation on growth performance and carcass characteristics in heat-stressed and nutrient-restricted pigs Treatment1 P-value Contrasts Trt TN TN PF HSCtl Ctl Parameter TNCtl TNCr PFCtl PFCr HSCtl HSCr SEM Trt2 Wk3 x Wk4 vs. PF vs. HS vs. HS vs. HSCr vs. Cr ADFI, kg 3.52a 3.61a 2.95bc 3.04b 2.80c 2.92bc 0.08 <0.01 <0.01 <0.01 <0.01 <0.01 0.07 0.12 0.08 ADG, kg 0.92a 0.97a 0.72b 0.70b 0.72b 0.77b 0.03 <0.01 <0.01 <0.01 <0.01 <0.01 0.24 0.10 0.19 IBW5, kg 104.0 104.1 104.6 103.8 104.4 103.9 0.7 0.95 - - 0.82 0.91 0.91 0.46 0.45 FBW6, kg 144.2a 146.5a 138.4bc 136.6c 137.5bc 140.2b 1.4 <0.01 - - <0.01 <0.01 0.34 0.06 0.32 G:F7 0.25bc 0.27ab 0.24c 0.24c 0.26ab 0.27ab 0.01 0.02 <0.01 <0.01 0.02 0.34 <0.01 0.61 0.39 BF8, cm 2.04x 2.12x 1.90xy 1.77y 1.97xy 1.97xy 0.08 0.08 - - <0.01 0.18 0.11 0.95 0.82 LEA9, cm2 53.6xy 55.1x 52.2y 51.8y 52.8y 52.3y 0.9 0.09 - - <0.01 0.04 0.53 0.59 0.76 1TNCtl=Thermoneutral control; TNCr=Thermoneutral chromium; PFCtl=Pair-fed control; PFCr= Pair-fed chromium; HSCtl= Heat-stress control; HSCr= Heat-stress chromium 2Treatment

3Week 73

4Treatment by week interactions 5Initial body weight 6Final body weight 7Feed efficiency 8Back fat 9Loin eye area a-cMeans with different superscripts significantly differ (P ≤ 0.05) x-yMeans with different superscripts tended to differ (P ≤ 0.10)

Table 6. Effects of chromium propionate supplementation on body temperature indices in heat-stressed and nutrient-restricted pigs Treatment1 P-value Contrasts Trt TN TN PF HSCtl Ctl Parameter TNCtl TNCr PFCtl PFCr HSCtl HSCr SEM Trt2 Wk3 x Wk4 vs. PF vs. HS vs. HS vs. HSCr vs. Cr 5 TR , °C 0700 h 38.81b 38.78b 38.64c 38.62c 38.98a 38.99a 0.04 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.76 0.71 1800 h 39.19c 39.15c 39.00d 39.02d 39.57b 39.68a 0.04 <0.01 <0.01 0.21 <0.01 <0.01 <0.01 0.01 0.36 6 TS , ° C 0700 h 35.11b 35.30b 33.64c 33.05d 38.57a 38.72a 0.16 <0.01 <0.01 0.02 <0.01 <0.01 <0.01 0.38 0.49 1800 h 36.96b 36.92b 35.37c 35.46c 41.11a 41.19a 0.15 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.58 0.69 RR7, bpm 0700 h 44.5b 45.9b 42.6b 41.6b 64.5a 65.3a 1.6 <0.01 <0.01 <0.01 0.05 <0.01 <0.01 0.62 0.73 1800 h 48.1b 53.2b 48.8b 47.8b 92.9a 96.1a 2.3 <0.01 <0.01 0.02 0.31 <0.01 <0.01 0.18 0.16 1TNCtl=Thermoneutral control; TNCr=Thermoneutral chromium; PFCtl=Pair-fed control; PFCr= Pair-fed chromium; HSCtl= Heat-stress control; HSCr= Heat-stress chromium 74 2 Treatment 3Week 4Treatment by week interactions 5Rectal temperature 6Skin temperature 7Respiration rate a-cMeans with different superscripts significantly differ (P ≤ 0.05)

Table 7. Effects of chromium propionate supplementation on blood metabolites in heat-stressed and nutrient-restricted pigs Treatment1 P-value Contrasts Trt TN TN PF HSCtl Ctl Parameter TNCtl TNCr PFCtl PFCr HSCtl HSCr SEM Trt2 Wk3 x Wk4 vs. PF vs. HS vs. HS vs. HSCr vs. Cr

Insulin, µg/L 0.17 0.23 0.20 0.27 0.16 0.13 0.04 0.17 <0.01 0.03 0.41 0.17 0.03 0.52 0.35

Insulin:ADFI5 0.04 0.07 0.07 0.11 0.06 0.05 0.05 0.12 <0.01 0.01 0.06 0.85 0.04 0.72 0.19

Glucose, mg/dL 75.3 72.2 74.3 73.9 71.6 74.8 2.2 0.69 <0.01 0.56 0.88 0.80 0.68 0.17 0.95

Ins:Glu6 1.99 3.00 2.93 4.08 2.31 1.95 0.67 0.21 <0.01 0.01 0.15 0.57 0.04 0.69 0.29

NEFA7, mEq/L 70.9 62.3 69.5 69.6 69.8 71.3 6.6 0.92 0.66 0.87 0.67 0.56 0.87 0.82 0.65

LBP8, µg/mL 5.74xy 4.91y 3.94yz 6.30xy 6.14xy 8.39x 1.03 0.08 0.21 0.70 0.85 0.07 0.05 0.11 0.15

Na9, mEq/L 146.6 145.9 146.2 146.1 145.0 145.6 0.5 0.70 <0.01 <0.01 0.88 0.34 0.42 0.35 0.24

K10, mEq/L 5.18xy 4.93y 4.90y 5.11xy 5.09y 5.45x 0.16 0.06 0.07 0.10 0.76 0.17 0.10 0.03 0.38

Cl11, mEq/L 99.5b 99.5b 99.4b 99.4b 100.9a 100.8a 0.4 0.01 0.02 0.22 0.70 <0.01 <0.01 0.88 0.92 12 a a a a b b HCO3 , mEq/L 35.0 34.3 34.1 35.4 32.7 32.0 0.5 <0.01 0.11 0.80 0.90 <0.01 <0.01 0.21 0.95

Ca13, mg/dL 10.9a 10.6b 10.6b 10.7a 10.6b 10.6b 0.1 0.01 0.59 0.46 0.51 0.06 0.22 0.71 0.10 75 P14, mg/dL 8.53a 8.63a 8.51a 8.45a 8.05b 8.01b 0.11 <0.01 <0.01 <0.01 0.35 <0.01 <0.01 0.71 1.00

Mg15, mg/dL 1.93b 1.88b 2.01a 1.90b 1.93b 1.92b 0.03 0.02 0.68 0.90 0.09 0.51 0.30 0.78 0.01

BUN16, mg/dL 11.5a 11.6a 9.96bc 8.97c 9.55bc 10.2b 0.4 <0.01 0.88 0.25 <0.01 <0.01 0.35 0.16 0.81

Creat.17, mg/dL 1.94b 1.89b 1.96b 1.90b 2.12a 2.11a 0.05 <0.01 <0.01 <0.01 0.76 <0.01 <0.01 0.78 0.28

Tot. Prot.18, g/dL 6.78bc 6.65c 7.04a 6.80bc 6.88ab 7.06a 0.09 <0.01 0.36 0.06 0.02 <0.01 0.57 0.06 0.30

Albumin, g/dL 3.73xy 3.67y 3.86x 3.72xy 3.68y 3.79x 0.05 0.06 0.20 0.19 0.08 0.48 0.30 0.05 0.55

AST19, IU/L 36.5 32.4 37.9 30.2 31.9 36.9 4.3 0.67 0.03 0.64 0.92 0.99 0.94 0.28 0.49

CK20, IU/L 1365xy 1054y 1358y 1035y 1977x 2619x 463 0.06 0.71 0.04 0.98 0.03 0.02 0.16 1.00

Alk. Phos.21, U/L 120.3a 118.5a 124.3a 128.3a 100.9b 99.9b 4.1 <0.01 0.02 0.29 0.10 <0.01 <0.01 0.81 0.90

GGT22, IU/L 34.7 38.1 41.0 38.8 39.5 42.6 2.5 0.30 <0.01 0.09 0.18 0.07 0.63 0.22 0.47

Bil.23, mg/dL 0.12 0.12 0.12 0.14 0.12 0.13 0.01 0.80 0.59 0.33 0.67 0.88 0.78 0.40 0.23

A.G.24, mEq/L 17.4 16.9 17.6 16.3 17.6 18.2 0.7 0.36 <0.01 <0.01 0.71 0.28 0.16 0.36 0.41

Chol.25, mg/dL 87.6 86.4 89.4 86.4 84.4 84.0 1.6 0.20 0.04 0.54 0.60 0.09 0.03 0.80 0.22

TAG26, mg/dL 35.0 31.4 38.3 38.2 35.4 36.8 2.2 0.23 0.04 0.83 0.03 0.19 0.33 0.51 0.65 1TNCtl=Thermoneutral control; TNCr=Thermoneutral chromium; PFCtl=Pair-fed control; PFCr= Pair-fed chromium; HSCtl= Heat-stress control; HSCr= Heat-stress chromium 2Treatment 3Week 4Treatment by week interactions 76

5Insulin to average daily feed intake ratio 6Insulin to glucose ratio 7Non-esterified fatty acids 8Lipopolysaccharide binding protein 9Sodium 10Potasium 11Chloride 12Bicarbonate 13Calcium 14Phosporus 15Magnesium 16Blood urea nitrogen 17Creatinine 18Total protein 19Aspartate aminotransferase 20Creatine kinase

21Alkaline phosphatase 76

22Gamma-glutamyl transferase 23Bilirrubin 24Anion gap 25Cholesterol 26Triacylglicerol a-c Means with different superscripts significantly differ (P ≤ 0.05) x-z Means with different superscripts tended to differ (P ≤ 0.10)

Table 8. Effects of chromium propionate supplementation on blood cell count in heat-stressed and nutrient-restricted pigs Treatment1 P-value Contrasts Trt TN TN PF HSCtl Ctl Parameter TNCtl TNCr PFCtl PFCr HSCtl HSCr SEM Trt2 Wk3 x Wk4 vs. PF vs. HS vs. HS vs. HSCr vs. Cr

WBC5, x 103/ul 21.7 21.4 20.6 21.1 19.4 21.3 0.9 0.32 <0.01 0.05 0.46 0.16 0.53 0.11 0.36 Neutrop.6, x 103/ul 5.89abc 6.21ab 5.22abc 4.99bc 4.76c 6.54a 0.52 0.05 0.39 0.49 0.10 0.43 0.28 0.02 0.15 Lymp.7, x 103/ul 13.3 13.4 13.1 14.1 12.4 12.4 0.5 0.20 <0.01 0.01 0.71 0.08 0.03 0.83 0.39 Mono.8, x 103/ul 1.25 1.19 1.17 1.23 1.20 1.39 0.09 0.34 0.55 0.51 0.81 0.37 0.24 0.08 0.37 Eosinop.9, x 103/ul 0.56 0.53 0.58 0.68 0.57 0.64 0.07 0.74 0.79 0.15 0.27 0.39 0.71 0.28 0.46 Basophils, x 103/ul 0.12 0.12 0.11 0.09 0.11 0.10 0.01 0.85 0.22 0.81 0.34 0.50 0.66 0.91 0.54 RBC10, x 106/ul 7.59xy 7.47y 7.91x 7.57y 7.60y 7.64y 0.10 0.10 <0.01 0.29 0.07 0.41 0.21 0.75 0.10 Hb.11, gm/dl 13.6b 13.4b 14.2a 13.5b 13.5b 13.6b 0.2 0.05 <0.01 0.27 0.06 0.59 0.11 0.46 0.09 Hct.12, % 43.3ab 42.2b 45.1a 42.9b 42.8b 42.9b 0.6 0.04 <0.01 0.28 0.07 0.88 0.05 0.85 0.04 Platelets x 103/ul 301 620 303 349 287 319 25 0.57 0.14 0.49 0.58 0.74 0.34 0.26 0.12 1TNCtl=Thermoneutral control; TNCr=Thermoneutral chromium; PFCtl=Pair-fed control; PFCr= Pair-fed chromium; HSCtl= Heat-stress control; HSCr= Heat-stress chromium 77 2

Treatment 3Week 4Treatment by week interactions 5White blood cells 6Neutrophils 7Lymphocytes 8Monocytes 9Eosinophils 10Red blood cells 11Hemoglobin 12Hematocrit a-c Means with different superscripts significantly differ (P ≤ 0.05) x-z Means with different superscripts tended to differ (P ≤ 0.10)

5.00 (A) Trt: P<0.01 Wk: P<0.01 4.50 Trt x Wk: P<0.01 4.00

3.50

3.00

ADFI (kg) ADFI 2.50

2.00

1.50 P1 1 2 3 4 5 Week

160 (B) Trt: P<0.01 Wk: P<0.01 150 Trt x Wk: P<0.01

140

130

BW (kg) BW 120

110

100 P1 1 2 3 4 5

Week

TNCtl TNCr PFCtl PFCr HSCtl HSCr

Figure 3. Effects of chromium propionate on (A) average daily feed intake (ADFI) and (B) body weight (BW). Treatments: TNCtl = thermoneutral control diet, TNCr = Thermoneutral chromium diet, PFCtl = pair-fed control diet, PFCr = pair-fed chromium diet, HSCtl = heat- stress control diet, and HSCr = heat-stress chromium diet. 79

1.5 (A) Trt: P<0.01 a a 1.0 b b b b

(kg) ADG 0.5

0.0 TNCtl TNCr PFCtl PFCr HSCtl HSCr

160 (B) Trt: P<0.01 150 a a bc b 140 c bc

(kg) BW Final 130

120 TNCtl TNCr PFCTl PFCr HSCtl HSCr

0.40 (C) Trt: P=0.02 0.35 0.30 ab ab ab bc c c 0.25 G:F 0.20 0.15

0.10 TNCtl TNCr PFCtl PFCr HSCtl HSCr

Figure 4. Effects of chromium propionate on (A) average daily gain (ADG), (B) final body weight (BW), and (C) feed efficiency (G:F). Treatments: TNCtl = thermoneutral control diet,

TNCr = Thermoneutral chromium diet, PFCtl = pair-fed control diet, PFCr = pair-fed chromium diet, HSCtl = heat-stress control diet, and HSCr = heat-stress chromium diet. a-e

Values with differing superscripts denote differences (P ≤ 0.05) between treatments. 80

40.0 (A) Trt: P<0.01 39.8 Wk: P<0.01 Trt x Wk: P=0.21 39.6 39.4

C) ° ( 39.2 R

T 39.0 38.8 38.6

38.4 P1 1 2 3 4 5 Week

Trt: P<0.01 (B) 45.0 Wk: P<0.01 43.5 Trt x Wk: P<0.01 42.0

C) 40.5 °

(

S 39.0

T 37.5 36.0 34.5 33.0 P1 1 2 3 4 5

Week 120 Trt: P<0.01 (C) 100 Wk: P<0.01 Trt x Wk: P=0.02 80 60

RR(pm) 40

20 0 P1 1 2 3 4 5 Week

TNCtl TNCr PFCtl PFCr HSCtl HSCr

Figure 5. Effects of chromium propionate on (A) rectal temperature (TR), (B) skin temperature

(TS) and, (C) respiration rate (RR) at 1800 h. Treatments: TNCtl = thermoneutral control diet,

TNCr = Thermoneutral chromium diet, PFCtl = pair-fed control diet, PFCr = pair-fed chromium diet, HSCtl = heat-stress control diet, and HSCr = heat-stress chromium diet. 81

90 0.5 (A) (B) Trt: P=0.17 Trt: P=0.69

0.4 80 g/L) ! 0.3 70 0.2 Insulin ( Insulin 60 0.1 (mg/dL) Glucose

0 50 TNCtl TNCr PFCtl PFCr HSCtl HSCr TNCtl TNCr PFCtl PFCr HSCtl HSCr

120 (C) 15 (D) Trt: P=0.92 Trt: P=0.92 Trt: P=0.08 100 xy 80 10 Eq/L) y xy xy ! 60 y y 40 5 LBP(µg/mL) NEFA( 20 0 0 TNCtl TNCr PFCtl PFCr HSCtl HSCr TNCtl TNCr PFCtl PFCr HSCtl HSCr

Figure 6. Effects of chromium propionate on circulating (A) insulin, (B) glucose, (C) non- esterified fatty acids (NEFA), and (D) lipopolysaccharide binding protein (LBP). Treatments:

TNCtl = thermoneutral control diet, TNCr = Thermoneutral chromium diet, PFCtl = pair-fed control diet, PFCr = pair-fed chromium diet, HSCtl = heat-stress control diet, and HSCr = heat- stress chromium diet. x-y Values with differing superscripts denote differences (P ≤ 0.05) between treatments.

CHAPTER 3: EFFECTS OF ZINC AMINO ACID COMPLEX ON BIOMARKERS OF GUT

INTEGRITY AND METABOLISM DURING HEAT STRESS AND A FOLLOWING

RECOVERY PERIOD IN GROWING PIGS

E. J. Mayorga*, S. K. Kvidera*, E. A. Horst*, M. A. Al-Qaisi*, M.J. Dickson*, J. T. Seibert*, S.

Lei*, Z. Rambo†, M. Wilson† and L. H. Baumgard*

*Iowa State University, Department of Animal Sciences, Ames, IA, 50011

†Zinpro Corporation, Eden Prairie, MN, 55344

Abstract

Study objectives were to determine the effects of zinc (Zn) amino acid complex (Availa®Zn,

Zinpro Corporation, Eden Prairie, MN) on metabolism, biomarkers of leaky gut and inflammation in heat-stressed and nutrient-restricted pigs. Crossbred gilts (n = 50; 50 ± 2 kg BW) were blocked by initial BW and randomly assigned to one of five treatments: 1) thermoneutral (TN) conditions and ad-libitum consumed control diet (TNCtl), 2) TN and pair-fed the control diet (PFCtl), 3) TN and pair-fed a Zn supplemented diet (PFZn), 4) heat stress (HS) and ad-libitum consumed control diet (HSCtl) and 5) HS and ad-libitum fed a Zn supplemented diet (HSZn). The study consisted of three experimental periods (P): during P1 (7 d), all pigs were fed their respective diets ad-libitum and housed in TN conditions (20.84 ± 0.03˚C, 47.11 ± 0.42% RH). During P2 (7 d), HSCtl and

HSZn fed pigs were exposed to progressive cyclical heat stress conditions (27 to 30˚C, 41.9 ±

0.5% RH), while the TNCtl, PFCtl and PFZn pigs remained in TN conditions and were fed ad- 83 libitum or pair-fed to their HSCtl and HSZn counterparts. During P3 (5 d; the “recovery phase”), all pigs were housed in TN conditions and fed ad-libitum. Pigs exposed to HS had an overall increased rectal temperature, skin temperature and respiration rate (0.33°C, 3.76°C and 27 bpm, respectively; P < 0.01). Relative to TN controls, HS decreased ADFI and ADG (1.79 vs. 2.48 kg/d and 0.60 vs. 0.92 kg, respectively, P < 0.05) but these variables were unaffected by dietary treatment. Additionally, circulating insulin levels were decreased in the PF relative to TN and HS pigs (P = 0.03). During recovery, no differences were observed in rectal temperature or respiration rate across dietary or environmental treatments, but HSZn pigs had decreased skin temperature relative to TN, PF and HSCtl pigs (P < 0.01). During P3, no effects of Zn were observed in production parameters; however, PF pigs had increased ADFI and ADG relative to TN and HS treatments (P < 0.01). During P3, circulating insulin was increased in pigs that were in HS compared to TN and PF pigs in P2 (75%, P < 0.05); furthermore, blood glucose tended to be increased in Zn relative to Ctl-fed pigs (P = 0.07). Interestingly, plasma TNFα levels tended to be decreased during P1 (P = 0.08) and were decreased during P3 (P = 0.04) overall in Zn relative to

Ctl-fed pigs. Circulating lipopolysaccharide binding protein was not different among periods (P

> 0.10). In summary, Zn appeared to reduce TNFα (regardless of HS) and the stimulatory effect of HS on insulin secretion is amplified during HS recovery.

Key words: heat stress, intestinal integrity, recovery, pigs, zinc

Introduction

Heat stress (HS) is an environmental issue in animal agriculture and is a major limitation to efficient and profitable pig production. Environmental hyperthermia causes substantial 84 economic loses to global animal agriculture and this is mainly due to the adverse effects of HS on animal health and productivity (St-Pierre et al., 2003; Pollmann, 2010; Baumgard and Rhoads,

2013). Although certainly multifactorial, many of the negative consequences of HS on animal health and productivity may mediated by reduced intestinal barrier integrity (Baumgard and

Rhoads, 2013).

Heat-stressed animals redistribute blood flow to the periphery (skin) in an attempt to increase radiant heat loss, and the gastrointestinal tract vasoconstricts in a coordinated effort to support the altered cardiac event. The reduced splanchnic blood flow causes ischemic damage and subsequently creates intestinal barrier dysfunction (Hall et al, 1999; Lambert, 2009; Pearce et al.,

2013a). Furthermore, reduced feed intake also compromises intestinal barrier integrity (Rodriguez et al., 1996; Pearce et al., 2013c; Kvidera et al., 2017) and thus HS causes leaky gut by multiple mechanisms. Zinc (Zn) plays an important role at maintaining the physiological function of the gastrointestinal tract (Alam et al., 1994). Dietary Zn decreased intestinal permeability in a variety of laboratory animal models (Rodriguez et al., 1996; Sturniolo et al., 2001; Lambert et al., 2003).

Furthermore, we have demonstrated supplementing organic Zn improves intestinal barrier function and villi architecture in chronic and acute heat-stressed growing pigs and ruminants (Sanz-

Fernandez et al., 2014; Pearce et al., 2015; Abuajamieh et al., 2016). However, whether Zn can improve “recovery” from HS is not clear. Therefore, it was of interest to evaluate whether supplemental Zn can ameliorate the detrimental effects of HS on temporal metabolism and biomarkers of intestinal integrity in a porcine model during and recovery from both HS and nutrient restriction. Evaluating the recovery period provides an indication of how long the two insults (HS and nutrient restriction) negatively affect gastrointestinal integrity and offers an opportunity to assess the role of supplemental Zn in this response. Identifying dietary strategies 85 that can potentially accelerate “recovery” following HS exposure would have practical and immediate implications.

Materials and Methods

Animals, diets, and experimental design

Iowa State University Institutional Animal Care and Use committee approved all procedures involving animals. Fifty crossbred gilts (50 ± 2 kg BW) were utilized in a replicated

(25 pigs per replicate) experiment conducted at the Iowa State University Swine Nutrition Farm

Research Facility (Ames, IA). Pigs were blocked by initial BW and randomly assigned to one of five environmental-dietary treatments: 1) thermoneutral (TN) conditions and fed ad-libitum the control diet (TNCtl; n = 10), 2) TN pair-fed control diet (PFCtl; n = 10), 3) TN pair-fed Zn diet

(PFZn; n = 10), 4) HS conditions and fed ad-libitum the control diet (HSCtl; n = 10), HS ad- libitum Zn diet (HSZn, n = 10). During the experimental period, pigs were allotted to one of two environmentally-controlled rooms. Each room had 24 crates (57 x 221 cm) where pigs were housed individually. Each crate was equipped with a stainless-steel feeder and a nipple drinker.

Water was provided ad-libitum during the entire experiment. A total of two pigs were culled from the study due to health issues and their data were not included in the final analysis.

Two diets were formulated and mixed according to the following specifications: 1) a control (Ctl) diet containing 120 mg/kg of inorganic Zn in the form of Zn sulfate (Sigma-Aldrich,

St. Louis, MO) and 2) a Zn diet containing 60 mg of inorganic Zn in the form of Zn sulfate and 60 mg/kg of organic Zn as Zn amino acid complex (Availa®Zn; Zinpro Corporation, Eden Prairie,

MN, USA). Diets were processed in meal form and mixed at the Iowa State University Swine 86

Nutrition Center Mill. Other than the Zn source, all diets were similar in ingredient and were formulated to meet or exceed the predicted requirements of finishing pigs (NRC, 2012) for energy, essential amino acids, protein, minerals and vitamins (Table 9).

The study was divided into three experimental periods (P): P1, P2 and P3. Prior to the start of the study (i.e., before pigs were moved into the environmental rooms), pigs were fed their respective diets for ~21 d. During the first 14 d, all animals were group-housed according to their dietary treatment. After this initial feeding phase, pigs were moved to individual crates and allowed 7 d to acclimate to their pens before the initiation of the experimental periods. During P1

(7 d), all pigs were fed their respective diets ad-libitum and housed in TN conditions (20.84 ±

0.03˚C; 47.11 ± 0.42% relative humidity [RH]) for collection of baseline body temperature indices and production parameters. During P2 (7 d), HSCtl and HSZn animals were fed ad-libitum and exposed to progressive cyclical HS conditions with temperatures ranging from 27 to 30˚C (30.23

± 0.30˚C, 34.70 ± 0.16% RH from 0800 to 1800 h, and 26.98 ± 0.06˚C, 49.13 ± 0.34% RH from

1800 to 0800 h). During P2, pigs assigned to the TNCtl treatment remained in TN conditions and were fed ad-libitum; pigs assigned to the PFCtl and PFZn treatments also remained in TN conditions, but were pair-fed to their HS and dietary counterparts (to evaluate the direct effect of

HS while eliminating the confounding effects of dissimilar nutrient intake). Daily feed intake during P1 was averaged for each pig and used as a baseline. During P2, the decrease in ADFI in

HS pigs was calculated daily as a percentage of ADFI reduction relative to P1. The percentage of

ADFI reduction was averaged for all pigs in HS treatments per day of heat exposure and applied individually to the baseline of each pig in the PF treatments. The daily amount of feed provided to the PF treatments was divided into three equal portions during P2 (~0800, 1200 and 1800 h) to minimize large metabolic changes associated with gorging. In order to accomplish the pair- 87 feeding, pigs assigned to the PF treatments spent an additional day in acclimation so they were one calendar day behind the TN and HS treatments during the entire experimental period. This technique of pair-feeding has been extensively utilized (Pearce et al, 2013; Sanz-Fernandez et al,

2015).

During P3 (recovery period; 5 d), pigs from the HS treatments (HSCtl and HSZn) returned to TN conditions (21.00 ± 0.08˚C, 45.79 ± 0.66% RH) and were fed ad-libitum; likewise, pigs in the TNCtl, PFCtl and PFZn treatments remained in TN conditions and were fed ad-libitum.

Ambient temperature and humidity were monitored and recorded every 5 minutes by a data logger

(Lascar EL-USB-2-LCD, Erie, PA). One data logger was placed in each quadrant of each room (8 data loggers in total) and data were condensed into room averages by each time point.

Production parameters

Daily feed intake was measured during P1, P2, and P3 as feed disappearance. Body weights were obtained at the beginning and at the end of P1 and at the end of P2 and P3. Average daily gain (ADG) and feed efficiency (G:F) were calculated by period.

Body temperature measurements

During P1, P2, and P3, body temperature indices (rectal temperature [TR], skin temperature

[TS] and respiration rate [RR]) were obtained thrice daily (0700, 1200 and 1800 h). Rectal temperature was measured using an electronic thermometer (SureTemp Plus 590, WelchAllyn,

Skaneateles Falls, NY, USA). Skin temperature was measured at the rump area using an infrared thermometer (IRT207: The Heat Seeker 8:1 Mid-Range Infrared Thermometer, General Tools,

New York, NY) which was ~12 cm from the skin. Respiration rate was determined by counting flank movements in 15 s intervals and multiplied by four to obtain breaths per minute (bpm). All indices recorded were condensed into daily averages. 88

Blood sampling and analysis

An indwelling jugular catheter was surgically inserted on d 1 of P1 using a percutaneous technique as previously described (Sanz-Fernandez et al., 2015a). All pigs received antibiotics

(Ceftiofur, Excede, Pfizer Animal Health, New York, NY) and non-steroidal anti-inflammatory drugs (Flunixin Meglumine, Banamine-S, Schering-Plough Animal Health Corp., Whitehouse

Station, NJ) during the surgery. Intravenous antibiotic therapy continued daily for the rest of the study (Ampicilin, Polyflex, Fort Dodge Animal Health, Fort Dodge, IA) and catheters were flushed once a day with heparinized saline (100 IU/ml). Blood samples were obtained from the jugular

catheter daily at 1200 h into disposable tubes (plasma, K2EDTA tube, BD vacutainers, Franklin

Lakes, NJ; serum, culture glass tubes; Fisher Scientific, Waltham, MA). Samples of plasma and serum were harvested by centrifugation at 1500 x g for 15 min at 4˚C, aliquoted into 2.0 ml microcentrifuge tubes, and stored at -20˚C until analysis. Plasma glucose, non-esterified fatty acids

(NEFA) and blood urea nitrogen (BUN) concentrations were measured enzymatically (glucose,

Wako Chemicals USA, Inc, Richmond, VA; NEFA, Wako Chemicals USA, Inc, Richmond, VA;

BUN, Teco Diagnostics, Anaheim, CA). The intra and inter-assay coefficients of variation for glucose, NEFA, and BUN were 2.3 and 13.5%, 7.3 and 14.9%, and 5.1 and 5.8%, respectively.

An ELISA kit was used to determine plasma insulin (Mercodia Porcine Insulin ELISA; Mercodia

AB, Uppsala, Sweden), lipopolysaccharide (LPS) binding protein (LBP) (Hycult Biotech, Uden,

The Netherlands) and tumor necrosis factor alpha (TNFα) concentrations (R&D Systems, Inc.,

Minneapolis, MN), following the manufacturer’s instructions. The intra- and inter-assay coefficients of variation for insulin, LBP and TNFα were 11.2 and 7.2%, 9.1 and 15.5%, and 10.9 and 10.8%, respectively.

Statistical analysis

Data were analyzed using SAS version 9.4 (SAS Inst. Inc., Cary, NC). Daily measurements (i.e., ADFI, body temperature indices, and blood metabolites) were analyzed by repeated measures, using the MIXED procedure of SAS with an autoregressive covariance structure and day of the experiment as the repeated effect. The model included treatment, day, treatment by day interaction, block and replicate as fixed effects; initial BW was used as a covariate. Production parameters (i.e., BW, ADG and G:F) were analyzed using PROC MIXED with a diagonal structure and initial BW as a covariate. The model included treatment, block and replicate as fixed effects. A third analysis for daily measurements and production parameters was assessed to test the differences across experimental periods (i.e. P1, P2 and P3). Data were analyzed using PROC MIXED with a diagonal covariance structure and initial BW as a covariate; the model included treatment, period and their interaction. Pre-planned contrasts were assessed to evaluate the effects of the environmental treatment and the effects of the diet. Data are reported as least squares means and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.

Results

Growth performance

When comparing across experimental periods, there was a treatment by period interaction on ADFI, as it decreased in HS pigs during P2 (32%; P < 0.01) relative to P1 and then increased during P3 (24% relative to P2; P < 0.01). Interestingly, ADFI in HS pigs during P3 remained decreased compared to its P1values (P < 0.01). By experimental design, PF treatments had a similar pattern and magnitude of reduced ADFI during P2 than that of their HS counterparts. 90

However, during P3, ADFI was increased and tended to be increased in PFCtl and PFZn pigs when compared to P1 (10%; P < 0.01 and 6%; P = 0.08, respectively). There was a treatment by period interaction on ADG (P < 0.01), as it decreased in HS and PF treatments during P2 relative to P1

(30% decrease). During P3, ADG in HS animals returned to its baseline levels, while PF pigs had increased (43%; P < 0.01) ADG relative to P1. In contrast, no treatment by period interactions were observed on BW (P = 0.89) or G:F (P = 0.11).

During P1, ADFI, BW, ADG and G:F were not different across treatments (P > 0.10).

Similarly, no differences were observed for BW or ADG when comparing Ctl vs. Zn-fed pigs during P1; however, FI was decreased in Zn compared to Ctl-fed pigs (2.57 vs. 2.73 kg/d, P =

0.02); thus, G:F tended to be increased in Zn relative to Ctl-fed pigs during P1 (0.36 vs. 0.32; P =

0.09).

During P2, there was an overall decrease in ADFI in HS pigs when compared to TN controls (28%; 0.69 kg/d; P < 0.01; Table 10; Figure 7A). Average daily gain was also decreased in HS pigs compared to TN controls (35%; 0.32 kg/d; P < 0.01; Table 10; Figure 7B). Feed efficiency did not differ between HS and TN environments (P = 0.45; Table 10) during P2; however, HSZn pigs tended to have increased G:F compared to HSCtl counterparts (26%; P =

0.07; Table 10). By experimental design, PFCtl and PFZn pigs followed the same pattern in reduced ADFI, and their BW, ADG and G:F variables were similar to their HS counterparts (P =

0.38; 0.20, and 0.31 for BW, ADG, and G:F, respectively; Table 10).

During P3 (pigs in all treatments were housed in TN conditions), ADFI was similar between HS and TN treatments (P = 0.46; Table 10; Figure 7A); however, PF pigs had increased

ADFI by 21 and 28% when compared to TN and HS animals, respectively (P < 0.01; Table 10;

Figure 7A). Body weight was not different between TN and PF pigs at the end of the recovery 91 period (P = 0.24), but was decreased by 5% in HS pigs compared to their TN counterparts (4.70 kg; P = 0.01; Table 10). Average daily gain did not differ between HS and TN treatments (P =

0.95; Table 10; Figure 7B); however, it was increased by 32% in PF pigs relative to TN and HS animals (P < 0.01; Figure 7B). No treatment differences were observed for G:F during P3 (P =

0.14), but PFZn pigs had increased G:F when compared to PFCtl animals (26%; P = 0.02).

Body temperature indices

When comparing across experimental periods, there were treatment x period interactions

on TR, TS and RR, as they increased in HS pigs during P2 relative to P1 (0.2°C, 4.0°C and 26 bpm, respectively), but returned to baseline values during P3 (P < 0.01; Table 12). Interestingly, TR and

TS in PF treatments decreased during P2 (P < 0.01; 0.2 and 0.5°C, respectively) relative to P1.

However, during P3 TR remained stable in PF treatments, while TS increased relative to P1 and P2

(0.3 and 0.6°C, respectively).

During P1, TR and RR were similar across treatments (P > 0.10; Table 13). In contrast, TS tended to be decreased (0.6°C) in HSZn pigs when compared to TNCtl and PFCtl animals (P =

0.10), while PFZn and HSCtl pigs had intermediate TS values. When comparing Ctl vs. Zn-fed

pigs during P1, no differences were observed in TR; however, TS was decreased in Zn relative to

Ctl-fed pigs (0.4°C; P = 0.02). Similarly, RR tended to be decreased by 5% in Zn compared to

Ctl-fed pigs (P = 0.06).

During P2, all pigs exposed to HS had an overall increase in TR when compared to TN and

PF pigs (0.33°C; P < 0.01; Table 11; Figure 8A). Skin temperature in the HS environment was increased by 8 and 11% when compared to TN and PF treatments, respectively (P < 0.01; Table

11, Figure 8B). HSZn fed pigs tended to have decreased TS during P2 compared to the HSCtl animals (0.40°C; P = 0.06; Table 11). Interestingly, TS during P2 was decreased by 0.64°C in PF 92 relative to TN pigs (P < 0.01). During P2, RR was also increased by 75% in HS pigs relative to their TN and PF counterparts (27 bpm increase; P < 0.01; Table 11; Figure 8C).

During P3 (recovery period), no differences between treatments were observed in TR or RR

(P > 0.10; Table 11; Figure 8A-C). However, HSZn pigs had decreased skin temperature when compared to TN, PF and HSCtl treatments (0.84°C decrease; P < 0.01; Table 11).

Blood metabolites

No treatment by period interaction on circulating glucose was detected among experimental periods (P = 0.56). During P1, circulating glucose levels were similar between treatments (P = 0.30) and no differences where observed between Ctl and Zn-fed pigs (P = 0.38).

During HS, circulating glucose did not differ among treatments (P = 0.71; Table 12; Figure 9A).

During P3, blood glucose tended to be increased (6%) in HS pigs when compared to PF controls

(P = 0.10; Table 12). Similarly, plasma glucose levels were increased during P3 in PFZn pigs compared to their PFCtl counterparts (12%; P = 0.02). When comparing Ctl vs. Zn-fed pigs during

P3, a tendency for increased glucose levels was observed in the Zn-fed pigs compared to the Ctl- fed pigs (6%, P = 0.07; Table 12).

When comparing across experimental periods, no treatment by period interactions were detected in insulin levels nor the insulin:ADFI (P > 0.05). During P1, no treatment differences were detected for circulating insulin or the insulin:ADFI (P = 0.58 and 0.32, respectively; Table

12). Similarly, when comparing Ctl vs. Zn-fed pigs, no differences in blood insulin or insulin:ADFI were detected (P = 0.39 and 0.24, respectively). During HS exposure and despite marked differences in ADFI, insulin levels did not differ between HS and TN treatments (P =

0.41); however, insulin was decreased in the PF pigs relative to TN and HS pigs by 58 and 50%, respectively (P < 0.01; Table 12; Figure 10A-B). Likewise, the insulin:ADFI was increased during 93

P2 in HS pigs when compared to their PF counterparts (100%; P < 0.01; Table 12). However, no differences in the insulin:ADFI were observed between TN and HS pigs (P = 0.52; Table 12).

Also, no differences in insulin levels or the insulin:ADFI were detected between Ctl and Zn-fed pigs (P > 0.10). During P3 (recovery period), circulating insulin was increased in HS treatments by 75% relative to TN and PF pigs (P < 0.05; Table 12; Figure 10A-C). Similarly, the insulin:ADFI was increased by 113 and 143% in HS pigs relative to TN and PF controls, respectively (P < 0.05). When comparing Ctl vs. Zn-fed pigs no differences in insulin levels or the insulin:ADFI were detected during P3 (P > 0.10; Table 12).

There was no treatment by period interaction (P = 0.76) on NEFA levels when comparing across experimental periods. During P1, no treatment differences in plasma NEFA levels were observed (P = 0.89). Similarly, NEFA did not differ between Ctl and Zn-fed pigs (P = 0.47).

During P2 and despite marked differences in ADFI, NEFA levels were similar across treatments

(P = 0.17; table 12; Figure 9B). However, when comparing Ctl vs. Zn-fed pigs, NEFA levels were increased in Zn relative to Ctl-fed pigs (28%; P = 0.03; Table 12). During P3 (recovery period), circulating NEFA levels were similar across treatments (P = 0.43) and no differences in NEFA levels were observed between Ctl and Zn-fed pigs (P = 0.41; Table 12).

When comparing across experimental periods, no treatment x period interaction was observed on BUN levels (P = 0.45). No dietary treatment effects were observed for BUN during

P1 (P = 0.33). Likewise, BUN levels did not differ between Ctl and Zn-fed pigs during P1 (P =

0.67). During P2, BUN did not differ across treatments (P = 0.41; Table 12; Figure 9C); however, there was a tendency for decreased BUN levels in HS and PF pigs relative to TN controls (P =

0.07 and 0.09, respectively). No differences in BUN were detected between Ctl and Zn-fed pigs 94 during P2 (P = 0.44; Table 12). During P3, BUN levels were similar across treatments (P = 0.83;

Table 12; Figure 9C) and did not differ between Ctl and Zn-fed pigs (P = 0.95; Table 12).

No treatment by period interaction was observed between experimental periods on circulating LPB (P = 0.95). During P1, LBP levels did not differ by treatment (P = 0.61) and no differences were observed when comparing LPB levels between Ctl and Zn-fed pigs (P = 0.69).

There were no treatment differences in LBP levels during P2 or P3 (P = 0.68 and 0.84, respectively;

Table 12; Figure 9D). Likewise, LBP levels did not differ between Ctl and Zn-fed pigs during P2 or P3 (P = 0.52 and 0.42, respectively).

When comparing across experimental periods, no treatment x period interaction was detected on plasma TNFα levels (P = 0.96). Similarly, TNFα levels did not differ among treatments during P1 (P = 0.55; Figure 11A); however, overall when comparing Ctl vs. Zn-fed pigs, there was a tendency for decreased circulating TNFα in Zn relative to Ctl-fed pigs (23%; P

= 0.08; Figure 11B). During P2, TNFα levels were similar across treatments (P = 0.60; Table 12;

Figure 11A) and no differences were detected between Ctl and Zn-fed pigs (P = 0.15). No overall treatment effect was observed in circulating TNFα during recovery period (P = 0.31; Table 12;

Figure 11A); however, overall TNFα levels were decreased by 25% in Zn relative to Ctl-fed pigs

(P = 0.04; Figure 11C).

Discussion

Heat stress negatively impacts a variety of animal agriculture parameters including milk yield and composition, growth, reproduction, and carcass traits (St-Pierre et al., 2003; Baumgard and Rhoads, 2013). The detrimental effects of HS on animal productivity may be mediated in part 95 by its effects on intestinal health and function (Baumgard and Rhoads, 2013). During hyperthermia, blood is redistributed away from the splanchnic bed to the periphery in an attempt to maximize radiant heat dissipation (Lambert et al., 2002). This survival strategy results in increased reactive oxygen species production, membrane damage, and disruption of tight junction complexes in the intestinal epithelium (Hall et al., 2001; Lambert, 2009). Moreover, the loss of intestinal barrier function increases permeability to luminal content (e.g., LPS from bacterial origin), resulting in LPS appearance in portal and systemic circulation (Pearce et al., 2013c).

Endotoxin stimulates an immune response which is associated with increased circulating inflammatory biomarkers (Bouchama et al., 1991, 1993; Hall et al., 2001; Lambert, 2001).

Additionally, LPS inexplicably increases circulating insulin levels in a variety of farm-animal models (Baumgard et al., 2016). Consequently, HS-induced endotoxemia and inflammation alter metabolism and nutrient partitioning and this partially explains why HS decreases productivity

(Baumgard and Rhoads, 2013). Furthermore, while the negative effects of HS have been intensively studied during the heat load, little is known regarding the biology of a recovery period following HS exposure. Therefore, studying HS recovery provides insight into how long HS and nutrient-restriction adversely affect intestinal integrity. Identifying effective dietary strategies aimed at alleviating the adverse effects of HS on intestinal health and/or accelerating the recovery following HS exposure would have enormous impact on animal health and agriculture economics.

Zinc is essential for normal barrier function as it improves intestinal integrity during different disease states including malnutrition (Rodriguez et al., 1996), ethanol-induced intestinal damage (Lambert et al., 2003), chronic inflammatory bowel diseases (Sturniolo et al., 2001), and infectious diarrhea (Alam et al., 1994). Furthermore, our group has reported beneficial effects of organic Zn supplementation during a thermal load as it decreased circulating endotoxin levels and 96 improved intestinal epithelial resistance, intestinal architecture, and the acute phase response in heat-stressed pigs and ruminants (Sanz-Fernandez et al., 2014; Pearce et al., 2015; Abuajamieh et al., 2016). Therefore, we hypothesized supplemental Zn would reduce the temporal pattern of inflammatory biomarkers and subsequent altered metabolism in a HS and feed restriction porcine model and would improve the recovery response after HS exposure.

In the current study, pigs in the HS environment experienced marked hyperthermia as

demonstrated by increased TR, TS and RR (0.3°C, 2.8°C, and 27 bpm, respectively) relative to TN pigs. Conversely, decreased TS (0.64°C) was observed in PF pigs during P2 relative to their TN counterparts, indicating decreased heat increment of feeding as reported previously by our group

(Pearce et al., 2013b). During the recovery period, all body temperature indices returned to

baseline values and no differences in TR or RR were observed across treatments. However, despite being in similar environmental conditions, HSZn pigs had a slight decrease in TS (0.84°C) relative

to TN, PF and HSCtl pigs. Understanding why HSZn pigs had decreased TS during recovery and whether this is biologically significant is of interest.

As expected, HS exposure markedly decreased ADFI (28%) and ADG (35%) relative to

TN ad libitum fed pigs. By experimental design, PF treatments mirrored this pattern of reduced

ADFI during P2. The magnitude and pattern of nutrient intake reduction was comparable to our previous studies (Johnson et al., 2015; Sanz-Fernandez et al., 2015b). The negative effects of HS on voluntary ADFI have been well-documented within the literature (as reviewed by Nyachoti et al., 2004). Reduced ADFI during a thermal load is a highly-conserved response across species and is obviously partially responsible for decreased growth performance observed in heat-stressed animals (Baumgard and Rhoads, 2013). During the recovery period, ADFI and ADG in pigs that were exposed to HS returned to TN levels; however, pigs that were PF had an increase in ADFI 97 and ADG (21 and 32%, respectively) above TN levels. Reasons why appetite was blunted in HS relative to PF pigs during recovery are not entirely unclear. Our group previously observed that

ADFI in pigs exposed to 3 d of HS gradually increased during the recovery period but remained less than the TN controls for the remainder of the experiment (7 d; Abuajamieh et al., 2015).

Additionally, Johnson et al. (2016) observed that pigs exposed to a 3 h heat load had a progressively increased feeding behavior to that of the TN pigs during a 3 h recovery period.

Mechanisms underlying compensatory growth are difficult to understand as factors influencing an animal’s ability to recover may be associated with the nature, severity, and/or duration of nutrient restriction (Wilson and Osbourn, 1960; Lovatto et al., 2006). Additionally, compensatory growth can also reflect changes in metabolism as circulating levels of insulin, thyroid, and growth hormones are known to play a key role in the initiation of this response (Prince et al., 1983; Hornick et al., 2000). Insulin has been hypothesized to be an important regulator of feed intake and energy balance (Woods et al., 2006; Perry and Wang, 2012); furthermore, exogenous insulin administration reduces feed consumption in different rodent models (Schwartz et al., 1992; Woods et al., 1995; Air et al., 2002). Interestingly, in the current study, pigs that were previously exposed to HS during P2 had increased circulating insulin relative to TN and PF pigs during the recovery period (as described below) and this may have contributed to the blunted appetite response.

Regardless, the absence of full recovery in nutrient intake and growth in HS pigs is of great interest and suggests the negative consequences of HS have long-lasting effects on animal productivity.

Animals exposed to HS have altered post-absorptive carbohydrate, lipid, and protein metabolism independent of nutrient intake. This energetic phenotype is primarily characterized by increased basal and stimulated circulating insulin levels and blunted adipose tissue mobilization

(Baumgard and Rhoads, 2013). In the current study, no differences in blood glucose, NEFA, or 98

BUN were observed during P2 or recovery among treatments. Despite marked reductions in ADFI during P2, circulating insulin from HS pigs was similar to TN controls, but was decreased in PF pigs when compared to their TN counterparts. The insulin response to the heat load observed in this study is remarkable, but not unusual, as similar observations have been reported in heat- stressed rodents, pigs, and ruminants (Torlinska et al., 1987; O’Brien et al., 2010; Wheelock et al.,

2010, Pearce et al., 2013b; Sanz-Fernandez et al., 2015). Reasons for hyperinsulinemia during HS remain unclear, but likely involves insulin’s role in activating the cellular stress response (Li et al.,

2006) and immunoactivation (as reviewed by Baumgard et al., 2016). In fact, previous reports indicate diabetic rodents and humans are more susceptible to heat related-illness and death

(Schuman et al., 1972; Semenza et al., 1999; Niu et al., 2003). Interestingly, during the recovery period, PF pigs returned to euinsulinemia; while pigs previously exposed to HS had a large increase in circulating insulin relative to both TN and PF pigs (~75%). This is surprising because insulin secretion is very sensitive to changes in nutrient intake, yet ADFI in pigs that were exposed to HS remained below that of PF pigs during the recovery period. Reasons why circulating insulin levels were increased during recovery in pigs that were exposed to HS are not clear but likely suggest the stimulatory effect of LPS on insulin secretion is amplified during HS recovery.

Previous studies suggest the pathophysiological responses to a thermal load might not be due to the direct effects of heat exposure, but the result of a systemic and uncontrolled inflammatory response following thermal injury (Bouchama and Knochel, 2002; Leon, 2007). The increased inflammatory response after the cessation of HS is thought to stem from increased LPS leakage into circulation, which in turn elicits an immune response and stimulates the production of pro-inflammatory cytokines (Lambert, 2008). Accordingly, Johnson and colleagues (2016) observed increased circulating and tissue cytokines levels in pigs after a 3 h recovery period 99 following a 3 h acute heat load. Additionally, our group has recently reported a progressive increase in circulating LBP on d 3 and 7 of a recovery period following 3 days of cyclical HS exposure in growing pigs (Abuajamieh et al., 2015); suggesting the inflammatory and acute phase response is constrained or blunted during the heat load but is expressed during HS recovery and remains robust for at least one week following an intense heat load. Contrarily, in the current study no differences were observed in circulating LBP among treatments during P2 or recovery. Reasons why are not clear, but it could be due to differences in the intensity of heat load utilized between experiments. Interestingly, although no inflammatory differences were observed during HS, dietary Zn supplementation reduced the inflammatory response during the recovery period, as

TNFα levels were decreased (25%) in Zn compared to Ctl-fed pigs. Additionally, circulating

TNFα tended to be decreased during P1 and were numerically decreased during P2 in Zn-fed relative to Ctl-fed animals. Reasons why TNFα was decreased by Zn supplementation are not well- understood, but suggest the importance of Zn’s role in modulating the immune and inflammatory responses (as reviewed by Hasse and Rink, 2007, 2013). A likely explanation is that Zn decreased intestinal permeability and thus reduced passage of antigens from the lumen across the intestinal barrier, consequently reducing the extent of immune activation. A second possible mechanism by which Zn ameliorates the inflammatory response is by regulating the nuclear factor kappa-light- chain-enhancer of activated B-cells (NF-κB) pathway (Prasad, 2008; Foster and Samman, 2012;

Jarosz et al., 2017). The NF-κB signaling pathway plays a key role in the expression of proinflammatory cytokines that regulate the immune response (TNFα, IL-1, IL-6, and IL-8); as well as in different cellular processes including cell proliferation and apoptosis (as reviewed by

Hoesel and Schmid, 2013). Previous studies in vitro suggest Zn may inhibit the activation of NF-

κB pathway via A20 and PPAR-α, two Zn-finger proteins known to have anti-inflammatory and 100 anti-apoptotic properties (Lademann et al., 2001; Prasad, 2008; Foster and Samman, 2012; Jarosz, et al., 2017). Additionally, Hu et al. (2013a,b) and Liu et al. (2014), observed decreased mRNA expression of TNFα, IL-6, IL-8, and interferon-γ along different sections of the gastrointestinal tract with increasing concentrations of dietary Zn in weaning pigs. Decreased circulating TNFα levels observed in this study indicate dietary Zn may be able to ameliorate the inflammatory response and enhance the progression of recovery in pigs that were exposed to HS or were nutrient- restricted.

In conclusion, chronic HS exposure significantly increased all body temperature indices, caused a marked and constant reduction in ADFI and ADG. Additionally, our data suggest HS has long-lasting effects on phenotypic and metabolic responses, and thus on animal health and productivity. Herein, we also demonstrated Zn supplementation might be beneficial at reducing biomarkers of inflammation during stress and maintaining this response during the recovery period. Overall, Zn supplementation may be a plausible dietary strategy to ameliorate some of the negative effects of HS on animal health, as well as to accelerate the recovery response during HS or nutrient restriction scenarios. However, additional research is warranted to better understand whether the benefits of dietary Zn supplementation can be translated in improved animal productivity.

101

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Table 9. Ingredient composition and chemical and nutritional composition of experimental diets (as-fed basis) Item Control Zinc Ingredient Corn, % 63.11 63.11 DDGS1, % 15.00 15.00 Soybean meal, % 18.00 18.00 Soybean oil, % 1.50 1.50 Limestone, % 1.25 1.25 L-Lysine HCl, % 0.27 0.27 L-Threonine, % 0.01 0.01 Quantum Blue (5,000 FTU/g) 2, % 0.005 0.005 Vitamin premix3, % 0.200 0.200 Trace mineral premix4, % 0.150 0.150 Availa zinc5 - 0.05 Zinc sulfate 0.05 0.03 Copper sulfate 0.002 0.002 Iron sulfate 0.06 0.06 Limestone 0.03 0.01 Manganese 0.002 0.002 Potassium iodate 0.00003 0.00003 Selenium 0.00005 0.00005 NaCl 0.500 0.500 Calculated nutrient levels, % ME, kcal/kg 3,342 3,342 NE, kcal/kg 2,477 2,477 CP 17.97 17.97 Ether extract 4.43 4.43 ADF 5.30 5.30 NDF 12.29 12.29 SID6 AA, % Lys 0.86 0.86 Met 0.26 0.26 Thr 0.53 0.53 Trp 0.16 0.16 Ca 0.54 0.54 Total P 0.41 0.41 STTD7 P 0.27 0.27 1Corn distillers dried grains with solubles 2Assume releases 0.08% STTD P 3Vitamin premix provided the following (per kg diet): 4,900 IU of vitamin A, 560 IU of vitamin D3, 40 IU of vitamin E, 2.4 mg of menadione (to provide vitamin K), 39 μg of vitamin B12, 9 mg of riboflavin, 22 mg of d-pantothenic acid, and 45 mg of niacin 107

4Compostion per kg of feed: Ca, 129 mg as limestone; Cu, 7 mg as copper sulfate; Fe, 120 mg as iron sulfate; I, 0.28 mg as potassium iodate; Mn, 5 mg manganese sulfate; Zn, 120 mg as zinc sulfate or 50% zinc sulfate + 50% Availa-Zn. 5Availa zinc (Availa®Zn; Zinpro Corporation, Eden Prairie, MN, USA 6Standardized ileal digestibility 7Standardized total tract digestibility

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Table 10. Effects of zinc amino acid complex on growth performance in heat-stressed and restricted-fed pigs during P2 and recovery Treatment1 P-value Contrasts Trt TN TN PF HSCtl Ctl Parameter TNCtl PFCtl PFZn HSCtl HSZn SEM Trt2 Day x Day3 vs. PF vs. HS vs. HS vs. HSZn vs. Zn Period 2 ADFI4, kg 2.48a 1.79b 1.75b 1.79b 1.79b 0.06 <0.01 0.01 0.03 <0.01 <0.01 0.68 0.99 <0.01 FBW5, kg 84.4x 82.9xy 79.8y 79.7y 80.6y 1.4 0.07 - - 0.07 0.02 0.38 0.64 0.09 ADG6, kg 0.92a 0.53b 0.53b 0.53b 0.67b 0.07 <0.01 - - <0.01 <0.01 0.31 0.18 0.27 G:F7 0.37 0.30 0.30 0.30 0.38 0.03 0.14 - - 0.07 0.45 0.21 0.07 0.57

Recovery ADFI4, kg 2.36bc 3.04a 2.69ab 2.25c 2.22c 0.14 <0.01 <0.01 <0.01 <0.01 0.46 <0.01 0.88 0.46 FBW5, kg 89.7x 89.1xy 86.1y 85.1y 84.9y 1.7 0.08 - - 0.24 0.01 0.10 0.94 0.07 ADG6, kg 0.94b 1.23a 1.24a 0.94b 0.92b 0.13 0.02 - - <0.01 0.95 <0.01 0.83 0.58 G:F7 0.40 0.39 0.49 0.42 0.42 0.04 0.14 - - 0.26 0.61 0.48 0.88 0.07 1 TNCtl=Thermoneutral control; PFCtl=Pair-fed control; PFZn= Pair-fed zinc; HSCtl= Heat-stress control; HSZn= Heat-stress zinc 2Treatment 3 Treatment by day interactions 108 4Average daily feed intake 5Final body weight 6Average daily gain 7Feed efficiency a-cMeans with different superscripts significantly differ (P ≤ 0.05) x-zMeans with different superscripts tended to differ (P ≤ 0.10)

109

Table 11. Effects of zinc amino acid complex on body temperature indices in heat-stressed and restricted-fed pigs during P2 and recovery Treatment1 P-value Contrast Trt TN TN PF HSCtl Ctl Parameter TNCtl PFCtl PFZn HSCtl HSZn SEM Trt2 Day x Day3 vs. PF vs. HS vs. HS vs. HSZn vs. Zn Period 2 4 b b b a a TR , °C 39.01 38.97 38.96 39.32 39.30 0.06 <0.01 <0.01 0.01 0.57 <0.01 <0.01 0.79 0.57 5 b c c a a TS , °C 36.82 36.25 36.11 40.29 39.89 0.16 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.06 0.11 RR6, bpm 36.5b 36.0b 35.0b 61.7a 63.5a 1.6 <0.01 <0.01 0.09 0.61 <0.01 <0.01 0.44 <0.01

Recovery 4 TR , °C 39.02 39.09 39.02 39.06 38.96 0.08 0.78 0.33 0.24 0.67 0.87 0.49 0.34 0.34 5 b a ab b c TS , °C 36.73 37.27 37.14 36.78 36.14 0.16 <0.01 <0.01 <0.01 0.01 0.17 <0.01 0.01 0.05 RR6, bpm 35.0 38.4 36.5 36.1 37.7 1.3 0.35 <0.01 <0.01 0.12 0.24 0.68 0.36 0.60 1TNCtl=Thermoneutral control; PFCtl=Pair-fed control; PFZn= Pair-fed zinc; HSCtl= Heat-stress control; HSZn= Heat-stress zinc 2Treatment 3Treatment by day interactions 4

Rectal temperature 109 5Skin temperature 6Respiration rate a-cMeans with different superscripts significantly differ (P ≤ 0.05) x-zMeans with different superscripts tended to differ (P ≤ 0.10)

110

Table 12. Effects of zinc amino acid complex on blood metabolites and inflammatory biomarkers in heat-stressed and restricted-fed pigs during P2 and recovery Treatment1 P-value Contrasts Trt TN TN PF HSCtl Ctl Parameter TNCtl PFCtl PFZn HSCtl HSZn SEM Trt2 Day x Day3 vs. PF vs. HS vs. HS vs. HSZn vs. Zn Period 2 Glucose, mg/dL 98.1 94.8 99.0 99.6 94.4 3.3 0.71 <0.01 0.65 0.77 0.79 0.98 0.31 0.79 Insulin, !g/L 0.13a 0.05c 0.06b 0.11ab 0.11ab 0.02 0.03 0.69 0.16 <0.01 0.41 0.01 0.93 0.51 Insulin: ADFI4 0.05xy 0.03y 0.03y 0.06x 0.06x 0.01 0.07 0.58 0.13 0.08 0.52 <0.01 0.94 0.87 NEFA5, mEq/L 84.5 83.5 110.0 72.5 95.0 10.9 0.17 0.17 0.48 0.36 0.96 0.25 0.19 0.03 BUN6, mg/dL 8.46 7.13 7.34 7.09 6.97 0.58 0.41 0.72 0.31 0.09 0.07 0.73 0.90 0.44 LBP7, µg/mL 9.68 12.95 9.80 9.19 8.80 4.03 0.68 0.39 0.22 0.54 0.80 0.29 0.90 0.52 TNFα8, pg/L 58.7 59.9 44.6 60.0 53.6 8.0 0.60 0.01 0.07 0.49 0.83 0.57 0.56 0.15

Recovery Glucose, mg/dL 94.2xy 87.8y 98.4x 97.9x 100.2x 4.0 0.09 0.60 0.09 0.79 0.31 0.10 0.67 0.07 Insulin, !g/L 0.10y 0.09y 0.11xy 0.16xy 0.19x 0.03 0.09 0.90 0.11 0.84 0.03 0.02 0.51 0.22 Insulin: ADFI4 0.04bc 0.03c 0.04bc 0.08ab 0.09a 0.02 0.03 0.82 0.11 0.64 0.03 <0.01 0.56 0.37 5 NEFA , mEq/L 77.8 74.5 94.3 71.2 70.9 11.8 0.43 0.38 0.83 0.61 0.63 0.21 0.99 0.41 110 BUN6, mg/dL 8.61 8.62 8.53 7.73 8.05 0.72 0.83 <0.01 0.18 0.96 0.38 0.28 0.75 0.95

LBP7, µg/mL 12.72 13.73 11.59 11.38 9.99 3.08 0.84 <0.01 0.04 0.98 0.50 0.43 0.69 0.42 TNFα8, pg/L 53.4 54.8 39.7 48.4 38.5 8.2 0.31 0.32 0.23 0.45 0.23 0.57 0.31 0.04 1TNCtl=Thermoneutral control; PFCtl=Pair-fed control; PFZn= Pair-fed zinc; HSCtl= Heat-stress control; HSZn= Heat-stress zinc 2Treatment 3Treatment by day interactions 4Insulin to average daily feed intake ratio 5Non-esterified fatty acids 6Blood urea nitrogen 7Lipopolysacharide binding protein 8Tumor necrosis factor alpha a-cMeans with different superscripts significantly differ (P ≤ 0.05) x-zMeans with different superscripts tended to differ (P ≤ 0.10)

111

4.0 (A)

3.5

3.0

2.5 TNCtl

PFCtl 2.0 PFZn

ADFI (kg) ADFI 1.5 HSCtl

1.0 HSZn Trt: P<0.01 Trt: P<0.01 0.5 Day: P=0.01 Day: P<0.01 Trt*Day: P=0.03 Trt*Day: P<0.01 0.0 P1 1 2 3 4 5 6 7 1 2 3 4 5 Day

2.0 (B) Trt: P<0.01 Trt: P=0.02

1.8 1.6 a a 1.4

TNCtl 1.2 a b b b 1.0 PFCtl b PFZn

ADG (kg) ADG 0.8 b b b HSCtl 0.6 0.4 HSZn

0.2 0.0 P2 Recovery

Figure 7. Effects of zinc amino acid complex on (A) average daily feed intake (ADFI) and (B) average daily gain (ADG) during P2 and the recovery period. Treatments: TNCtl = thermoneutral control diet, PFCtl = pair-fed control diet, PFZn = pair-fed zinc diet, HSCtl = heat-stress control diet, and HSZn = heat-stress zinc diet. The dashed line divides period 2 from the recovery period. a-e Values with differing superscripts denote differences (P ≤ 0.05) between treatments. 112

39.9 (A) Trt: P=0.78

39.7 Day: P=0.33 Trt*Day: P=0.24 39.5 C)

( 39.3

R T 39.1

38.9 Trt: P<0.01 38.7 Day: P<0.01 Trt*Day: P=0.01 38.5

P1 1 2 3 4 5 6 7 1 2 3 4 5

Day

44.0 (B) Trt: P<0.01 42.5 Day: P<0.01

41.0 Trt*Day: P<0.01

39.5 C) ° ( 38.0 S T 36.5 35.0 Trt: P<0.01 33.5 Day: P<0.01 Trt*Day: P<0.01 32.0 P1 1 2 3 4 5 6 7 1 2 3 4 5 Day

100 (C) 90 Trt: P=0.35 Day: P<0.01 80 Trt*Day: P<0.01 70 60 50 40 RR(bpm) 30 20 Trt: P<0.01 Day: P<0.01 10 Trt*Day: P=0.09 0 P1 1 2 3 4 5 6 7 1 2 3 4 5 Day

TNCtl PFCtl PFZn HSCtl HSZn

113

Figure 8. Effects of zinc amino acid complex on (A) rectal temperature (TR), (B) skin temperature

(TS), and (C) respiration rate (RR) during period 2 and the recovery period. Treatments: TNCtl = thermoneutral control diet, PFCtl = pair-fed control diet, PFZn = pair-fed zinc diet, HSCtl = heat- stress control diet, and HSZn = heat-stress zinc diet. The dashed line divides period 2 from the recovery period.

114

150 (A) (B) Trt: P=0.71 Trt: P=0.09 220 Trt: P=0.17 Trt: P=0.43 130 170 110 xy x x x Eq/L) y ! 90 120 70 70 ( NEFA Glucose (mg/dl)Glucose 50 20 P2 Recovery P2 Recovery

10 20 g/mL)

! 15 5 10 BUN (mg/dl)BUN LBP ( LBP 5 0 0 P2 Recovery P2 Recovery

Figure 9. Effects of zinc amino acid complex on circulating levels of (A) glucose, (B) non- esterified fatty acids (NEFA), (C) blood urea nitrogen (BUN), and (D) lipopolysaccharide binding protein (LBP) during period 2 and recovery. Treatments: TNCtl = thermoneutral control diet,

PFCtl = pair-fed control diet, PFZn = pair-fed zinc diet, HSCtl = heat-stress control diet, and HSZn

= heat-stress zinc diet. The dashed line divides period 2 from the recovery period. x-y Values with different superscripts denote tendencies (P ≤ 0.10) between treatments.

115

(A)

0.30 Trt: P=0.03 Trt: P=0.09 0.25 x ) xy 0.20 g/L a μ 0.15 ab ab y y xy

bc 0.10 c

( Insulin 0.05 0.00 P2 Recovery

TNCtl PFCtl PFZn HSCtl HSZn

(B) (C)

0.30 TN vs. PF: P<0.01 0.30 TN vs. PF: P=0.84 TN vs. HS: P=0.41 TN vs. HS: P=0.03

0.25 0.25 ) a ) PF vs. HS: P=0.01 PF vs. HS: P=0.02

g/L 0.20 g/L 0.20 μ μ a b b 0.15 a 0.15

0.10 0.10 b Insulin ( Insulin Insulin ( Insulin 0.05 0.05 0.00 0.00 TN PF HS TN PF HS

Figure 10. Effects of zinc amino acid complex on circulating levels of insulin during (A) period 2 and recovery. Treatments: TNCtl = thermoneutral control diet, PFCtl = pair-fed control diet, PFZn

= pair-fed zinc diet, HSCtl = heat stress control diet, and HSZn = heat stress zinc diet. The dashed line divides period 2 from recovery period. Circulating insulin levels during (B) period 2 and (C) the recovery period when comparing among thermoneutral (TN), pair-fed (PF), and heat stress

(HS) treatments. a-e Values with differing superscripts denote differences (P ≤ 0.05) between treatments. x-y Values with different superscripts denote tendencies (P ≤ 0.10) between treatments. 116

100 (A) Trt: P=0.55 Trt: P=0.60 Trt: P=0.31

(pg/mL) α 40

TNF 20

0 P1 P2 Recovery TNCtl PFCtl PFZn HSCtl HSZn

100 (B) 100 (C) Trt: P=0.08 Trt: P=0.04

80 80 /mL) /mL) pg (pg 60 ( 60 α α 40 40 TNF TNF

20 20 Ctl Zn Ctl Zn

Figure 11. Effects of zinc amino acid complex on circulating levels of tumor necrosis factor alpha during (A) period 1, period 2, and the recovery period. Treatments: TNCtl = thermoneutral control diet, PFCtl = pair-fed control diet, PFZn = pair-fed zinc diet, HSCtl = heat-stress control diet, and

HSZn = heat-stress zinc diet. The dashed line divides period 2 from recovery period. Circulating tumor necrosis factor alpha levels during (B) period 1 and (C) recovery period when comparing between dietary control (Ctl) and zinc (Zn) treatments.

117

CHAPTER 4: SUMMARY AND CONCLUSIONS

Heat stress undermines animal welfare and productivity. When animals are exposed to high ambient temperatures, physiological and metabolic changes are essential to ameliorate the heat insult; consequently, efficiency is deemphasized as nutrients are partitioned away from production purposes to maintain euthermia. Economic losses due to HS are mainly explained by decreased growth and efficiency, reduced carcass quality, poor reproduction, and high morbidity and mortality (St. Pierre et al., 2003; Pollmann, 2010; Baumgard and Rhoads, 2013). Hence, identifying mitigation strategies aimed at improving animal welfare and productivity during HS is imperative. Nutritional interventions represent a feasible and practical strategy to improve the animal response to a heat load and to ameliorate some of the negative consequences of HS on animal health and productivity. Therefore, the approach of this thesis was to evaluate the role of two different mineral supplements at ameliorating the adverse effects of HS in growing and finishing pigs.

Objectives of Chapter 2 were to determine the effects of dietary Cr supplementation on performance and metabolism in finishing pigs exposed to a diurnal pattern of HS. Chromium has gained a lot of attention recently as it is thought to improve insulin action by stimulating cell surface insulin receptors, enhancing insulin sensitivity and/or responsiveness in peripheral tissues

(Davis and Vincent, 1997) and potentially improving cellular glucose uptake (Chen et al., 2006).

We hypothesized dietary Cr supplementation during HS may enhance an animal’s ability to upregulate insulin action and therefore improve growth performance. Our results demonstrated HS exposure significantly increased all body temperature indices and caused a marked and constant reduction in ADFI and ADG. Chromium supplementation improved final BW, ADG and ADFI 118 variables in chronically heat-stressed pigs. Additionally, we observed increased leukocytes in pigs fed Cr and exposed to HS, while no dietary differences were observed in TN conditions, suggesting an important role of Cr during stress scenarios. Although studies looking at the effects of supplemental Cr during HS in pigs are scarce, its ability to improve feed intake, production parameters, and the immune response in a variety of species have been consistently reported within the literature. However, the exact mechanism by which Cr exerts these effects is still not fully understood and warrants further investigation.

The adverse effects of HS on animal health can be also mediated by reduced intestinal integrity. During hyperthermia, blood flow is redistributed to the periphery in an attempt to maximize radiant heat dissipation. Reduced blood flow to the splanchnic bed results in oxidative damage and subsequent intestinal dysfunction. Zinc is essential for the development and function of the gastrointestinal tract (Alam et al., 1994). Previous studies in our group demonstrated organic

Zn supplementation improved intestinal integrity in chronic and acute heat-stressed pigs and ruminants (Sanz-Fernandez et al., 2014; Pearce et al., 2015; Abuajamieh et al., 2016). Therefore, the objectives of Chapter 3 were to assess the role of organic Zn supplementation at ameliorating the detrimental effects of HS on metabolism and biomarkers of intestinal health in heat-stressed and nutrient-restricted pigs. Furthermore, the study evaluated the metabolic and physiological responses of a thermal neutral recovery period after heat removal, and dietary Zn’s ability to influence the recovery response. Our results demonstrated environmental hyperthermia resulted in a substantial increase in all body temperature indices, reduced ADFI, and decreased ADG. In agreement with previous studies, we observed pigs exposed to HS had an altered insulin homeostasis that continued, and actually became more pronounced even after the cessation of the thermal load. During recovery, appetite and growth were blunted in pigs that were exposed to HS 119 relative to their PF counterparts, suggesting HS has long-lasting effects on animal health and productivity. We also demonstrated Zn supplementation may be beneficial at reducing biomarkers of inflammation during stress and maintaining this response during the recovery period.

In conclusion, both studies demonstrated dietary Cr and Zn supplementation are two plausible nutritional strategies with potential to ameliorate the negative consequences of HS on growth performance, and animal health and productivity. Further research should focus on the mechanisms underlying recovery after an episode of HS exposure. As observed in Chapter 3, the absence of complete recovery in pigs that were exposed to a thermal load suggests HS has long- term effects on animal health and productivity, which may negatively impact agriculture economics. Understanding the biology of the recovery following a heat load allows for the development of strategies aimed to accelerate this process and help the animal to overcome the adverse effects on HS.

120

Literature Cited

Baumgard, L. H., and R. P. Rhoads. 2013. Effects of heat stress on postabsorbtive metabolism and energetics. Annu. Rev. Anim. Biosc. 1:311-337. doi:10.1146/annurev-animal- 031412-103644

Davis, C. M., and J. B. Vincent. 1997. Chromium oligopeptide activates insulin receptor tyrosine kinase activity. Biochemistry 36:4382-4385.

Pearce, S. C., M. V. Sanz-Fernandez, J. Torrison, M.E. Wilson, L. H. Baumgard, and N. K. Gabler. 2015. Dietary organic zinc attenuates heat stress-induced changes in pig intestinal integrity and metabolism. J. Anim. Sci. 93:4702-4713

Pollmann, D. S. Seasonal effects on sow herds: industry experience and management strategies. 2010. J. Anim. Sci. 88 (Suppl. 3) (Abstr.)

St. Pierre, N. R., B. Cobanov, G. Schnitkey. 2003. Economic losses form heat stress by US livestock industries. J. Dairy Sci. 86:E52-77

121

APPENDIX: EFFECTS OF LIVE YEAST SUPPLEMENTATION ON GROWTH

PERFORMANCE AND METABOLISM IN HEAT-STRESSED AND NUTRIENT-

RESTRICTED PIGS

E. J. Mayorga*, S. K. Kvidera*, E. A. Horst*, M. A. Al-Qaisi*, C. S. Shouse*, S. Lei*, M. A.

Abeyta*, J. Corley†, T. Gobreyohannes†, and L. H. Baumgard*

*Iowa State University, Department of Animal Sciences, Ames, IA, 50011

† Phileo-Lesaffre, Milwaukee, WI, 53214

Abstract

Study objectives were to determine the effects of live yeast supplementation (ActisafHR+;

0.25g/kg of feed; Phileo-Lesaffre, Milwaukee, WI) on growth performance and biomarkers of metabolism and inflammation in heat-stressed and nutrient-restricted pigs. Crossbred barrows (n

= 96; 80 ± 1 kg BW) were enrolled in a replicated experiment, blocked by initial BW, and randomly assigned to one of six dietary-environmental treatments: 1) thermoneutral (TN) ad-libitum control diet (TNCtl), 2) TN ad-libitum yeast diet (TNYst), 3) TN pair-fed (PF) control diet (PFCtl), 4) TN

PF yeast diet (PFYst), 5) heat stress (HS) ad-libitum control diet (HSCtl), and 6) HS ad-libitum yeast diet (HSYst). The study consisted of three experimental periods (P): during P0 (5 d), all pigs were housed in TN conditions (20.23 ± 0.01°C, 57.15 ± 0.30% RH) and fed the control diet ad- libitum. During P1 (7 d), pigs were fed their respective dietary treatments and kept in TN conditions for collection of baseline body temperature indices and production parameters. During

P2 (28 d) HSCtl and HSYst pigs were exposed to progressive cyclical heat stress conditions (28 122 to 33˚C, 37.79 ± 0.20% relative humidity), while TNCtl, TNYst, PFCtl, and PFYst pigs remained in TN conditions and were fed ad-libitum or pair-fed to their HSCtl and HSYst counterparts to eliminate the confounding effects of dissimilar nutrient intake. Pigs exposed to HS had an overall increase in rectal temperature, skin temperature, and respiration rate (0.3˚C, 5.5˚C, and 23 bpm, respectively; P < 0.01) compared to TN pigs. Average daily feed intake was dramatically decreased in HS compared to TN pigs (~1 kg, 30%; P < 0.01). Similarly, ADG and final BW decreased in HS relative to TN pigs (26 and 7%, respectively; P < 0.01); however, no differences in G:F were observed between HS and TN treatments (P = 0.16). Despite marked differences in

ADFI, circulating insulin levels were similar between HS and TN pigs (P = 0.42). Additionally,

HS pigs tended to have decrease plasma non-esterified fatty acids relative to their TN counterparts

(~22%; P = 0.07). Circulating triiodothyronine (T3) levels were decreased in HS compared to TN

treatments (~20%; P = 0.03). Similarly, decreased thyroxine (T4) levels were observed in HS treatments relative to both TN and PF pigs (~20 and 15%, respectively; P < 0.05). Circulating

TNFα did not differ among treatments (P = 0.66). No other differences were observed in glucose, blood urea nitrogen, or the triiodothyronine to thyroxine ratio among treatments. In conclusion, live yeast supplementation did not affect body temperature indices, or growth performance, and had minimal effects on biomarkers of inflammation. Therefore, additional research is warranted to better understand the effects of live yeast supplementation in pigs during HS or nutrient restriction scenarios.

123

Table 13. Effects of live yeast supplementation on body temperature indices in heat-stressed and nutrient-restricted pigs Treatment1 P-value Contrasts Trt Ctl TN TN PF HSCtl Parameter TNCtl TNYst PFCtl PFYst HSCtl HSYst SEM Trt2 Wk3 x Wk4 vs. Yst vs. PF vs. HS vs. HS vs. HSYst 5 b b c c a a TR , C° 39.13 39.12 38.82 38.83 39.46 39.45 0.05 <0.01 <0.01 0.18 0.90 <0.01 <0.01 <0.01 0.78 0700 h 39.05a 39.03a 38.72b 38.75b 39.05a 39.12a 0.04 <0.01 <0.01 <0.01 0.38 <0.01 0.28 <0.01 0.12 1800 h 39.22b 39.21b 38.92c 38.91c 39.87a 39.77a 0.07 <0.01 <0.01 0.25 0.43 <0.01 <0.01 <0.01 0.14 6 b b c c a a TS , C° 32.51 33.02 31.00 31.01 38.23 38.32 0.18 <0.01 <0.01 <0.01 0.15 <0.01 <0.01 <0.01 0.63 0700 h 31.82c 32.52b 30.21d 30.46d 37.14a 37.25a 0.20 <0.01 <0.01 <0.01 0.02 <0.01 <0.01 <0.01 0.58 1800 h 33.20b 33.51b 31.79c 31.56c 39.32a 39.39a 0.17 <0.01 <0.01 <0.01 0.72 <0.01 <0.01 <0.01 0.70 RR7, bpm 49b 48b 43bc 41c 71a 72a 2 <0.01 <0.01 0.24 0.70 0.01 <0.01 <0.01 0.80 0700 h 44b 42bc 38bc 37c 58a 60a 2 <0.01 <0.01 0.03 0.99 0.02 <0.01 <0.01 0.30 1800 h 53b 53b 48bc 45c 84a 83a 3 <0.01 <0.01 0.01 0.54 0.03 <0.01 <0.01 0.74 1TNCtl=Thermoneutral control; TNYst=Thermoneutral yeast; PFCtl=Pair-fed control; PFYst= Pair-fed yeast; HSCtl= Heat-stress control; HSYst= Heat-stress yeast

123 2Treatment 3

Week

4Treatment by week interactions

5Rectal temperature averaged by day

6Skin temperature averaged by day

7Respiration rate averaged by day a-dMeans with different superscripts significantly differ (P ≤ 0.05)

124

Table 14. Effects of live yeast supplementation on growth performance in heat-stressed and nutrient-restricted pigs Treatment1 P-value Contrasts Trt Ctl TN TN PF HSCtl Parameter TNCtl TNYst PFCtl PFYst HSCtl HSYst SEM Trt2 Wk3 x Wk4 vs. Yst vs. PF vs. HS vs. HS vs. HSYst ADFI5, kg 3.81a 3.55a 2.71b 2.62b 2.56b 2.60b 0.10 <0.01 0.57 <0.01 0.18 <0.01 <0.01 0.41 0.64 ADG6. kg 1.04a 1.08a 0.71bc 0.69c 0.77bc 0.79b 0.03 <0.01 <0.01 <0.01 0.63 <0.01 <0.01 0.02 0.57 IBW7, kg 78.1 78.6 79.1 79.6 79.9 79.5 1.0 0.76 - - 0.82 0.30 0.17 0.73 0.63 FBW8, kg 122.8a 123.8a 113.2b 113.3b 114.8b 115.7b 1.2 <0.01 - - 0.47 <0.01 <0.01 0.12 0.44 G:F9 0.27bc 0.30ab 0.26c 0.26c 0.30ab 0.31a 0.01 <0.01 <0.01 <0.01 0.13 0.03 0.16 <0.01 0.62 1TNCtl=Thermoneutral control; TNYst=Thermoneutral yeast; PFCtl=Pair-fed control; PFYst= Pair-fed yeast; HSCtl= Heat-stress control; HSYst= Heat-stress yeast 2Treatment 3Week 4Treatment by week interactions 5Average daily feed intake 6Average daily gain 7Initial body weight 8Final body weight 9Feed efficiency 124 a-cMeans with different superscripts significantly differ (P ≤ 0.05)

125

Table 15. Effects of live yeast supplementation on blood metabolites and biomarkers of inflammation in heat-stressed and nutrient-restricted pigs Treatment1 P-value Contrasts Trt Ctl TN TN PF HSCtl Parameter TNCtl TNYst PFCtl PFYst HSCtl HSYst SEM Trt2 Wk3 x Wk4 vs. Yst vs. PF vs. HS vs. HS vs. HSYst Glucose, mg/dL 91.3 93.7 98.0 94.5 84.9 88.3 3.5 0.11 0.16 0.46 0.77 0.32 0.10 0.01 0.34 Insulin, μg/L 0.12xy 0.12x 0.06y 0.06y 0.12x 0.09xy 0.02 0.09 0.30 0.13 0.54 0.01 0.42 0.03 0.15 NEFA5, mEq/L 106.6b 106.4b 167.0a 172.3a 87.7b 77.8b 10.3 <0.01 0.05 0.24 0.87 <0.01 0.07 <0.01 0.44 BUN6, mg/dL 11.5 10.3 9.1 11.4 10.2 10.5 0.79 0.32 0.01 0.05 0.44 0.45 0.41 0.95 0.72 7 T3 , ng/mL 0.45 0.51 0.45 0.43 0.38 0.40 0.04 0.27 <0.01 0.99 0.45 0.39 0.03 0.24 0.58 8 a a a ab b b T4 , ng/mL 36.0 39.7 36.8 34.3 29.6 31.0 2.1 0.01 <0.01 0.18 0.63 0.32 <0.01 0.02 0.57 9 2 T3:T4 , x10 1.58 1.58 1.50 1.55 1.58 1.54 0.17 0.99 0.29 0.41 0.98 0.78 0.90 0.85 0.84 TNFα10, pg/mL 59.1 61.3 60.8 51.7 61.6 54.7 4.8 0.57 0.15 0.05 0.25 0.45 0.65 0.69 0.20 1TNCtl=Thermoneutral control; TNYst=Thermoneutral yeast; PFCtl=Pair-fed control; PFYst= Pair-fed yeast; HSCtl= Heat-stress control; HSYst= Heat-stress yeast 2Treatment 3Week 4

Treatment by week interactions 125 5Non esterified fatty acids 6 Blood urea nitrogen 7Triiodothyronine 8Thyroxine 9Triiodothyronine to thyroxine ratio (Molar ratio) 10Tumor necrosis factor alpha a-cMeans with different superscripts significantly differ (P ≤ 0.05) x-yMeans with different superscripts tended to differ (P ≤ 0.10)

126

40.5 Trt: P<0.01 (A) Week: P<0.01 40.0 Trt*Week: P=0.18

39.5 (ºC)

R T 39.0

38.5

38.0 P1 1 2 3 4

Week

41.0 Trt: P<0.01 (B) 39.5 Week: P<0.01 38.0 Trt*Week: P<0.01 36.5

(ºC) 35.0 S

T 33.5 32.0 30.5 29.0 P1 1 2 3 4 Week

90 Trt: P<0.01 (C) 80 Week: P<0.01 Trt*Week: P=0.24 70 60

RR(bpm) 50 40

P1 1 2 3 4

Week TNCtl TNYst PFCtl PFYst HSCtl HSYst

127

Figure 12. Effects of live yeast supplementation on (A) rectal temperature (TR), (B) skin temperature (TS) and, (C) respiration rate (RR). Treatments: TNCtl = thermoneutral control diet, TNYst = Thermoneutral yeast diet, PFCtl = pair-fed control diet, PFYst = pair-fed yeast diet, HSCtl = heat-stress control diet, and HSYst = heat-stress yeast diet.

128

5.0 Trt: P<0.01 Week: P=0.57 (A) 4.5 Trt*Week: P<0.01 4.0

3.5 3.0

(kg) ADFI 2.5 2.0

1.5 1.0 P1 1 2 3 4 Week

2.0 Trt: P<0.01 1.8 (B) Week: P<0.01 1.6 Trt*Week: P<0.01 1.4 1.2

1.0

ADG (kg) ADG 0.8 0.6

0.4

0.2 0.0 P1 1 2 3 4 Week

TNCtl TNYst PFCtl PFYst HSCtl HSYst

Figure 13. Effects of live yeast supplementation on (A) average daily feed intake (ADFI) and

(B) average daily gain (ADG). Treatments: TNCtl = thermoneutral control diet, TNYst =

Thermoneutral yeast diet, PFCtl = pair-fed control diet, PFYst = pair-fed yeast diet, HSCtl = heat-stress control diet, and HSYst = heat-stress yeast diet.

129

140 Trt: P<0.01 (A) 130 Week: P<0.01 Trt*Week: P<0.01 TNCtl 120 TNYst 110 PFCtl

(kg) BW 100 PFYst HSCtl 90 HSYst

80 P1 1 2 3 4 Week

140 Trt: P<0.01 (B)

130 a a

120 b b b b 110

(kg) BW Final 100

80 TNCtl TNYst PFCtl PFYst HSCtl HSYst

Figure 14. Effects of live yeast supplementation on (A) body weight (BW) during P2 and (B) final body weight (BW). Treatments: TNCtl = thermoneutral control diet, TNYst =

Thermoneutral yeast diet, PFCtl = pair-fed control diet, PFYst = pair-fed yeast diet, HSCtl = heat-stress control diet, and HSYst = heat-stress yeast diet. a-b Values with different superscripts denote differences (P ≤ 0.05) between treatments.

130

140 0.4 (A) Trt: P=0.11 (B) Trt: P=0.09 120 0.3 g/L) μ 100 0.2 xy x x xy y y 80 ( Insulin 0.1 Glucose (mg/dL) Glucose

60 0.0 TNCtl TNYst PFCtl PFYst HSCtl HSYst TNCtl TNYst PFCtl PFYst HSCtl HSYst

220 20 (C) a a Trt: P<0.01 (D) Trt: P=0.32

170 15 b b 120 b b 10 BUN (mg/dL) BUN

NEFA (mEq/L) NEFA 70 5

20 0 TNCtl TNYst PFCtl PFYst HSCtl HSYst TNCtl TNYst PFCtl PFYst HSCtl HSYst

Figure 15. Effects of live yeast supplementation on circulating (A) glucose, (B) insulin, (C) non-esterified fatty acids (NEFA), and (D) blood urea nitrogen (BUN). Treatments: TNCtl = thermoneutral control diet, TNYst = Thermoneutral yeast diet, PFCtl = pair-fed control diet,

PFYst = pair-fed yeast diet, HSCtl = heat-stress control diet, and HSYst = heat-stress yeast diet. a-b Values with different superscripts denote differences (P ≤ 0.05) between treatments. x-y Values with different superscripts denote tendencies (P ≤ 0.10) between treatments.

131

1.0 (A) 60 (B) Trt: P=0.27 Trt: P=0.01 0.8 50 a a a 40 ab 0.6 b b 30 (ng/mL) (ng/mL) 3 0.4 4 T T 20

0.2 10

0.0 0 TNCtl TNYst PFCtl PFYst HSCtl HSYst TNCtl TNYst PFCtl PFYst HSCtl HSYst

2.5 100 (C) ) Trt: P=0.99 (D) 2 90 Trt: P=0.66 2.0 80 1.5 70 pg/mL) (

1.0 α 60 Molar Ratio (x10 Ratio Molar TNF 4 50

:T 0.5 3

T 40 0.0 30 TNCtl TNYst PFCtl PFYst HSCtl HSYst TNCtl TNYst PFCtl PFYst HSCtl HSYst

Figure 16. Effects of live yeast supplementation on circulating (A) triiodothyronine (T3), (B) thyroxine (T4), (C) triiodothyronine to thyroxine ratio (T3:T4), and (D) tumor necrosis factor alpha (TNFα). Treatments: TNCtl = thermoneutral control diet, TNYst = Thermoneutral yeast diet, PFCtl = pair-fed control diet, PFYst = pair-fed yeast diet, HSCtl = heat-stress control diet, and HSYst = heat-stress yeast diet. a-b Values with different superscripts denote differences (P

≤ 0.05) between treatments.