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Effect of diet complexity, multi-enzyme complexes, essential oils, and benzoic acid on weanling pigs
Author’s Accepted Manuscript Effect of diet complexity, multi-enzyme complexes, essential oils, and benzoic acid on weanling pigs Y. Wang, L.I. Chiba, C. Huang, I.M. Torres, L. Wang, E.G. Welles www.elsevier.com/locate/livsci
PII: DOI: Reference:
S1871-1413(17)30378-5 https://doi.org/10.1016/j.livsci.2017.12.007 LIVSCI3369
To appear in: Livestock Science Received date: 27 July 2017 Revised date: 8 December 2017 Accepted date: 9 December 2017 Cite this article as: Y. Wang, L.I. Chiba, C. Huang, I.M. Torres, L. Wang and E.G. Welles, Effect of diet complexity, multi-enzyme complexes, essential oils, and benzoic acid on weanling pigs, Livestock Science, https://doi.org/10.1016/j.livsci.2017.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of diet complexity, multi-enzyme complexes, essential oils, and benzoic acid on weanling pigs
Y. Wang a, L.I. Chiba a,*, C. Huang a, I.M. Torres a, L. Wang a, E.G. Welles b
a
Department of Animal Sciences, College of Agriculture, Auburn University, Auburn, AL 36849,
United States b
Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL
36849, United States
* Corresponding author. Tel.: +1 334 844 1560; fax: +1 334 844 1519. E-mail address: [email protected] (L.I. Chiba).
AB STRACT The study was conducted to investigate the effect of supplementing a simple corn-soybean meal (SBM) diet with multi-enzyme complexes, essential oils, and benzoic acid on growth performance, serum metabolite profile, serum cytokines, and intestinal microbiota in weanling pigs. Forty-eight gilts and 48 castrated males weaned at 3 to 4 wk of age (initial body weight, 7.96 ± 0.89 kg) were randomly assigned to 4 dietary treatments with 3 gilt and 3 castrated male pens per treatment and 4 gilts or 4 castrated males per pen. A complex diet containing palatable and digestible ingredients was formulated (1.30 g standardized ileal digestible Lys/kg) to serve as the positive control (POS) diet. A simple corn-SBM, negative control (NEG) diet was formulated
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to be iso-lysinic to the POS diet, and the NEG diet was supplemented with multi-enzyme complexes (ENZ) or multi-enzyme complexes, essential oils, and benzoic acid (ALL). All diets were formulated to meet or exceed the 2012 NRC nutrient requirements of pigs weighing 7 to 25 kg. During the fourth week, blood samples were collected to determine serum metabolite profile and cytokines, and fecal samples were collected for the enumeration of bacteria. Pigs had ad libitum access to feed and water throughout the 4-wk study. From d 0 to 7 and 7 to 14, pigs fed the POS diet had greater feed and Lys intake (P < 0.05) and weight gain (P < 0.05) than those fed the NEG and ALL diets, but there were no differences in those response criteria between pigs fed the POS and ENZ diets. Weight gain of pigs fed the ENZ diet was 17% greater than those fed the NEG diet during the second week (P < 0.05), but it increased only numerically (16%) during the first week. Overall (d 0 to 28), pigs fed the POS diet consumed more feed, Lys, and digestible energy (DE; P < 0.05) and had greater weight gain (P < 0.05) than those fed the other diets. Dietary treatments had no effect on the efficiency of feed, Lys, or DE utilization for weight gain during the study. Serum total protein in pigs fed the ENZ and ALL diets was greater (P < 0.05) than those fed the POS and NEG diets. Pigs fed the ENZ diet had greater serum albumin (P < 0.05) than those fed the NEG diet. Serum globulin and urea N were lower (P < 0.05) and albumin to globulin ratio, glucose, and cholesterol were greater (P < 0.05) in pigs fed the POS diet than those fed the other diets. Dietary treatments had no clear effect on serum cytokines or fecal microbiota. Pigs fed the POS diet grew faster and had lower serum urea N and globulin and greater serum glucose and cholesterol than those fed the other diets. Although supplementation of the NEG diet with multi-enzyme complexes seemed to have beneficial effect on growth performance during the first 2 wk of the study, supplementation of the NEG diet with various feed additives had no clear effects. Further research is needed to explore further the possibility of
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using a simple corn-SBM diet for weanling pigs by supplementation with multi-enzyme complexes, essential oils, and benzoic acid. Keywords: Weanling pigs, Diet complexity, Multi-enzyme complex, Essential oils, Benzoic acid
1. Introduction Weaning is the most critical period in the pig's life. At the time of weaning, pigs are exposed to various stressors, which can reduce the growth performance of weanling pigs (Le Dividich and Sève, 2000; van Beers-Schreurs et al., 1998). For that reason, weanling pigs are fed highly palatable and digestible diets. Although a mixture of corn and soybean meal (SBM) has been used to satisfy the energy and amino acid requirements of pigs over the years, such a mixture is not appropriate for weanling pigs because of the digestive (Jensen et al., 1997; Lindemann et al., 1986) and other challenges. Weanling pigs need highly palatable and digestible ingredients such as dried whey, plasma protein, oat groats, lipids, fishmeal, and others in their diets. Researchers have been investigating the effect of diets containing such ingredients, i.e., complex diets, on weanling pigs over the years and concluded that complex diets can improve the growth performance of weanling pigs (Whang et al., 2000; Wolter et al., 2003). However, providing such complex diets to weanling pigs can be rather costly. In addition, antibiotics have been included in diets for weanling pigs to enhance their health status and growth. The public concern over the routine use of antibiotics has, however, led to the ban of antibiotics as a feed additive in Sweden in 1986 and Switzerland in 1999 (Wenk, 2003) and the restricted use in 1999 and the complete ban in 2006 by the European Union (Janczyk et al., 2009; Windisch et al., 2008). Considering the ban in those countries and ongoing discussions on the use of antibiotics in other countries, such as in the United States (FDA, 2017),
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it is important to explore alternative ways to protect farm animals and improve their health status (Wenk, 2003; Windisch et al., 2008). With the development of various enzymes or enzyme complexes in recent years, it may be possible for weanling pigs to extract energy and nutrients from corn and SBM meal more efficiently (Kim et al., 2003; Zhang et al., 2014). Similarly, phytogenic compounds (Windisch et al., 2008), such as essential oils (Cho et al., 2005; Kroismayr et al., 2008; Manzanilla et al., 2009), and benzoic acid (Diao et al., 2016; Kluge et al., 2006; Torrallardona et al., 2007) may improve the growth performance of weanling pigs because of their antimicrobial and antioxidative activities. The current study was conducted to assess the possibility of replacing a typical complex diet for weanling pigs with a simple corn-SBM diet by supplementation with various feed additives. Specific objective was to investigate the effect of supplementation of a simple corn-SBM diet with multi-enzyme complexes, essential oils, and benzoic acid on the growth performance, serum metabolite profile, serum cytokines, and fecal microbiota.
2. Materials and methods 2.1. Animals and facilities The protocol for animal care was approved by the Institutional Animal Care and Use Committee of Auburn University (Auburn, AL, US). A total of 96 piglets weaned at 3 to 4 wk of age (initial body weight, 7.96 ± 0.89 kg) were placed in pens (1.5 m2) in an environmentally controlled nursery with slotted floors based on their sex and body weight. Pigs were randomly assigned to 4 dietary treatments with 3 gilt pens and 3 castrated male pens per treatment and 4 gilts or 4 castrated males per pen. Because of the availability of the facility at one time, the study was conducted in 3 trials, and each trial used 16 females and 16 castrated males. Three trials
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were conducted consecutively with approximately 4 wk apart. Pigs were allowed ad libitum access to feed and water throughout each 4-wk trial period, and the weight and feed consumption data were collected weekly. 2.2. Dietary treatments After weaning, pigs were fed a common pre-starter diet for 4 d before beginning of the study, and 1 set of diets was during the 4-wk starter phase (Table 1). A typical complex, positive control (POS) diet was formulated to contain 13.0 g standardized ileal digestible (SID) Lys/kg, which was, essentially, the average SID Lys requirements for pigs weighing 7 to 11 and 11 to 25 kg (NRC, 2012). In addition to corn and SBM, the complex diet contained spray dried whey (Honeyville, Brigham City, UT, US), fishmeal (Seven Springs Farm, Check, VA, US), poultry fat, plasma protein (Appetein; APC Inc., Ankey, IA, US), and antibiotic (Tylan-10 Sulfa-G; Livestock Concepts, Hawarden, IA, US). A simple, corn-SBM negative control (NEG) diet was formulated to be iso-lysinic to the POS diet, and the NEG diet was supplemented with multienzyme complexes (DSM Nutritional Products, Parsippany, NJ, US; ENZ) or multi-enzyme complexes, essential oils (CRENA; DSM Nutritional Products), and benzoic acid (Vevovitall; DSM Nutritional Products; ALL). Feed additives were included in the diets by replacing part of corn. Minerals and vitamins for all diets were provided in amounts calculated to meet or exceed the NRC (2012) recommendations for pigs weighing 7 to 11 and 11 to 25 kg. Feed samples were collected from each batch of feed prepared, and pooled sub-samples were analyzed for crude protein and minerals (AOAC, 2000). The analyzed crude protein content of the NEG diet was slightly lower than intended (Table 1), but the reason is not apparent. 2.3. Collection of blood and fecal samples
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During the fourth week of the study, approximately 5 mL of blood was collected via vena cava puncture using a sterile needle and evacuated tube. Blood samples were allowed to clot and serum samples were separated by centrifugation at 1,500 x g for 15 min at room temperature to obtain clean serum samples. An aliquot was stored at -20°C, and samples were pooled by pen and analyzed for serum metabolites and cytokines. Similarly, fecal samples for bacterial enumeration were collected from, at least, 3 pigs from each pen by rectal stimulation during the fourth week of the study. Fecal samples were immediately chilled with ice, pooled by pen, and an aliquot was used immediately for the assay. 2.4. Analysis of blood and fecal samples Pooled serum samples were analyzed for total protein, albumin, globulin, urea N, glucose, cholesterol, and triglyceride using an automated analyzer at the Auburn University Clinical Pathology Laboratory (Mule et al., 2006). Likewise, pooled serum samples were used for the multiplex cytokine assay (Discovery Assay; Eve Technologies Corp, Calgary, AB, Canada). The multiplex assay consisted of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFNγ), interleukin (IL)-1α, IL-1Ra, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and tumor necrosis factor-α. Bacterial counts in the pooled fecal samples were determined by the plate-count technique (Shen et al., 2009). Each 25 g of fecal sample was homogenized in 225 mL of buffered peptone water with a lab blender (Smasher; AES Chemunex, bioMérieux, France) for 2 min and then serially diluted for the plating. Coliform bacteria concentration was determined by plating the homogenate and dilutions on agar medium (Violet Red Bile Agar; Difco, Detroit, MI, US) and incubated aerobically at 37°C for 24 h. For Lactobacillus spp., samples were plated on MRS agar (Difco) and incubated anaerobically at 37°C for 48 h. To enumerate the total anaerobic and
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aerobic bacteria, fecal samples were plated on plate-count agar (Difco) and incubated anaerobically and aerobically, respectively, for 48 h at 37°C. The numbers of bacteria in each sample were calculated and reported as colony forming units (cfu)/plate. 2.5. Statistical analysis Data were analyzed using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC, US). The pen was considered as the experimental unit. Trial and treatment, along with appropriate body weight as a covariate, were included in the initial statistical model. Covariates considered for the analysis were initial body weight for growth performance data and body weight at third week of the study for cytokine, microbiota, and serum metabolite data. The results of the initial statistical analysis indicated that trial and trial x treatment interactions were not important source of variation, thus, the data for the 3 trials were combined and analyzed accordingly. The PDIFF option of the LSMEANS statement (SAS Inst. Inc.) was used to assess the effect of treatments. The result with P < 0.05 was considered significant, whereas the result with P < 0.10 was considered a trend.
3. Results 3.1. Growth performance From d 0 to 7 and 7 to 14, pigs fed the POS diet had greater feed and SID Lys intake (P < 0.05) and weight gain (P < 0.05) than those fed the NEG and ALL diets (Table 2). However, there were no differences in those response criteria between pigs fed the POS and ENZ diets. Pigs fed the ENZ diet tended to consume more feed and Lys (P = 0.07) than those fed the NEG diet during the first week, whereas the increases in those criteria during the second week were not statistically significant. Weight gain of pigs fed the ENZ diet was 17% greater than those fed
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the NEG diet during the second week (P < 0.05), but it increased only numerically (16%) during the first week. Pigs fed the POS diet consumed more digestible energy (DE; P < 0.05) than those fed the other diets during the first 2 wk of the study. Pigs fed the POS diet had greater feed, Lys, and DE intake (P < 0.05) and weight gain (P < 0.05) from d 14 to 21 and DE intake (P < 0.05) from d 21 to 28 than those fed the other diets. Overall (d 0 to 28), pigs fed the POS diet consumed more feed, Lys, and DE (P < 0.05) and gained more weight (P < 0.05) than those fed the other diets. Dietary treatment had no clear effect on the efficiency of feed, Lys, or DE utilization for weight gain during any weekly period or overall. 3.2. Serum metabolites The effect of supplementation of the corn-SBM diet with feed additives on serum metabolites in weanling pigs during the fourth week of the study is presented in Table 3. Serum total protein in pigs fed the ENZ and ALL diets was greater (P < 0.05) than those fed the POS and NEG diets. Serum albumin in pigs fed the POS diet was greater (P < 0.05) than those fed the NEG and ALL diets, and it was greater (P < 0.05) in pigs fed the ENZ diet than those fed the NEG diet. Pigs fed the POS diet had lower serum globulin and urea N (P < 0.05) and greater albumin to globulin ratio, glucose, and cholesterol (P < 0.05) than those fed the NEG, ENZ, and ALL diets. Serum triglyceride in pigs fed the POS diet seemed to be less than those fed the other 3 diets (P < 0.09), but the overall treatment effect was not statistically significant. 3.3. Serum cytokines and fecal microbiota The effect of feed additives on serum cytokine concentrations and fecal microbiota in weanling pigs is presented in Tables 4 and 5, respectively. The GM-CSF, IFNγ, or IL-6 was either not detected or detected inconsistently, thus, the data for those cytokines were not presented. Weanling pigs fed the ENZ diet seemed to have a greater IL-2 concentration than
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those fed the ALL diet (P = 0.09; Table 4). The IL-8 concentration seemed to be greater in pigs fed the ENZ diet compared with those fed the other diet (P = 0.10). However, the overall treatment effect on IL-2 or IL-8 was not statistically significant. Although pigs fed the ENZ diet seemed to have a greater total fecal anaerobe concentration (P = 0.08) than those fed the POS diet, the overall treatment effect was not statistically significant (Table 5). Dietary treatments had no effect on the total aerobic bacteria, coliforms, or Lactobacillus spp. in the feces of weanling pigs.
4. Discussion The digestive system of weanling pigs is not fully developed to utilize corn and SBM efficiently, and young pigs have been fed complex diets containing many special ingredients. However, using such diets can be rather costly. With the development of multi-enzyme complexes, weanling pigs may be able to utilize a simple corn-SBM diet more efficiently. In addition, because of the public concerns over antibiotic resistance and the possible residue problems, the dietary use of antibiotics has been banned in many countries (Janczyk et al., 2009; Wenk, 2003; Windisch et al., 2008). Considering such bans and ongoing discussions on the restrictive use of antibiotics, finding viable alternatives (Wenk, 2003; Windisch et al., 2008) would be crucial for successful pig production in the future. Although various approaches can be used to protect weanling pigs from disease organisms and enhance their growth (Wenk, 2003; Windisch et al., 2008), the use of essential oils or benzoic acid or both can be a potentially viable alternative. The effect of supplementing weanling pig diets with various enzymes, essential oils, and benzoic acid individually have been reported over the years (Knarreborg et al., 2002; Kroismayr et al., 2008; Zhang et al., 2014).
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However, the effect of combining enzymes, essential oils, and benzoic acid in the weanling pig diet has not been explored fully. Therefore, the current study was conducted to investigate the effect of multi-enzyme complexes, essential oils, and benzoic acid on the growth performance, serum profile and cytokines, and fecal microbiota. During the first 2 wk of the study, pigs fed the NEG diet consumed less feed and SID Lys and grew slower than those fed the POS diet. Similar results have been reported previously (Dritz et al., 1996; Himmelberg et al., 1985; Whang et al., 2000). Pigs fed the ENZ diet seemed to consume more feed and Lys during the first 2 wk of the study than those fed the NEG diet. Similarly, pigs fed the ENZ diet grew faster than those fed the NEG diet, although the increase in weight gain was only numerical during the first week. Furthermore, there were no differences in feed intake, Lys intake, or weight gain between the ENZ and POS diets. The results may indicate that the enzyme supplementation had some positive effects on the growth performance of weanling pigs during the first 2 wk of the study. However, there was no effect of enzyme supplementation during the last 2wk of the study. Our results were, perhaps, a reflection of the enzyme activity in young pigs. Lindemann et al. (1986) reported that the enzyme activity increased throughout the nursing period after birth; however, it decreased sharply at weaning. It is possible that enzyme supplementation compensated the reduced enzyme activity at weaning and soon thereafter, and thus having positive effects on the growth performance of weanling pigs during the first 2 wk of the study. However, pigs fed the ENZ diet had lower feed and Lys intake and weight gain than those fed the POS diet during the third week and overall study period. Similar results have been reported earlier (Kiarie et al., 2007; Omogbenigun et al., 2003).
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The lack of clear response to enzyme supplementation may be related to the type of diets or substrate availability (Bedford, 2000; Emiola et al., 2009; Omogbenigun et al., 2004; Simmons et al., 1990). There have been some reports that supplementation of a corn-SBM diet with carbohydrases can improve the energy and nutrient digestibility in weanling pigs (Kim et al., 2003; Li et al., 2010; Passos et al., 2015). However, other researchers reported that enzyme complexes were not be effective in enhancing the nutritional value of corn and SBM for growing pigs (Cozannet et al., 2012; Jones et al., 2010). Similarly, the effectiveness of supplementing a corn-SBM diet with enzymes has been rather inconsistent in poultry (Olukosi et al., 2007; Rutherfurd et al., 2007; Yang et al., 2010). Essential oils have been shown to improve the growth performance of weanling pigs by enhancing the digestive secretion, nutrient absorption, and immune status because of their antimicrobial or antioxidative property or both (Huang et al., 2010; Maenner et al., 2011; Zeng et al., 2015). Similarly, benzoic acid has been shown to enhance the growth performance of weanling pigs by its effect on the intestinal tract development (Diao et al., 2014, 2016; Halas et al., 2010), nutrient utilization (Kluge et al., 2006), antioxidative property (Diao et al., 2016), and intestinal microbiota (Diao et al., 2014; Torrallardona et al., 2007). The effect of essential oils or benzoic acid on weanling pigs, however, has not been always positive (Muhl and Liebert, 2007; Nemechek et al., 2013b). A combination of dietary essential oils and benzoic acid may improve the growth performance and intestinal microbiota in the weanling pig (Diao et al., 2015; Zhang et al., 2016). In the current study, however, supplementation of the ENZ diet with essential oils and benzoic acid had no effect on the rate and efficiency of growth. It is possible that the efficacy those feed additives can be affected by the nutrient density of the diet. Bühler et al. (2006, 2010) reported
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that the effect of benzoic acid on grower-finisher pigs could be dependent on the dietary protein or P content. However, Nemechek et al. (2013a) indicated that benzoic acid had no effect on growth performance of weanling pig regardless of feeding a simple or complex diet. Similarly, Lan et al. (2016) found no interaction between essential oils and nutrient density in weanling pigs. Thus, the lack of effect of supplementing the ENZ diet with essential oils and benzoic acid observed in the current study may not be related to the diet complexity. Total protein or albumin in blood can be an indicator of protein metabolism in animals (Lowrey et al., 1962). Positive correlations observed between serum albumin and growth performance (Mule at al., 2006) may support their contention. In the current study, serum albumin was slightly greater in pigs fed the ENZ diet than those fed the NEG diet, implying that supplementation of a simple corn-SBM diet with multi-enzyme complexes had some beneficial effect on protein metabolism. Serum globulins, which are involved in, e.g., immune functions, were less in pigs fed the POS diet than those fed the other diets. Supplementation with essential oils or benzoic acid seemed to have no effect on the serum metabolites in the current study. Blood urea N is another important indicator of protein and amino acid adequacy and efficiency of amino acid utilization (Coma et al., 1995; Whang and Easter, 2000). In the current study, pigs fed the POS diet had lower concentration of serum urea N than those fed the NEG, ENZ, and ALL diets, indicating that pigs fed the POS diet utilized dietary protein more efficiently than those fed the other diets. Greater serum glucose concentration in pigs fed the POS diet can be explained by a greater available carbohydrates compared with the other diets. Similarly, the increased available energy content because of the inclusion of 3% poultry fat may be responsible for the greater serum cholesterol in pigs fed the POS diet compared with those fed
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the other diets. Multi-enzyme complexes, essential oils, or benzoic acid seemed to have no effect on serum urea N, glucose, or cholesterol. Cytokines can play an important role in, e.g., immune and inflammatory responses (van der Meide and Schellekens, 1996; Zhang and An, 2007). The IL-1, IL-6, and TNF-α are typical pro-inflammatory cytokines (Dinarello, 1991, 2000), and weaning has been associated with the early and transient response in the expression of genes for inflammatory cytokines (Pié et al., 2004). Li et al. (2012) reported that supplementation of a diet with essential oils reduced plasma IL-6 and increased plasma TNF-α concentrations in weanling pigs. Oh et al. (2012) found that benzoic acid decreased IL-1β, IL-6, and TNF-α concentrations in the small intestine of weanling pigs, however, benzoic acid had no effect on serum and mucosal TNF-α concentrations in weanling pigs (Walsh et al., 2012). In the current study, dietary treatments had no clear effect on serum cytokine concentrations. Supplementation of a weanling pig diet with essential oils (Ahmed et al., 2013; Huang et al., 2010; Janczyk et al., 2009; Li et al., 2012), benzoic acid (Diao et al., 2014; Halas et al., 2010; Kluge et al., 2006; Papatsiros et al., 2011; Torrallardona et al., 2007), or the combination of the two (Diao et al., 2015; Zhang et al., 2016) has been shown to improve intestinal microbiota. On the other hand, other researchers reported no effect of essential oils (Maenner et al., 2011; Muhl and Liebert, 2007) on the intestinal microbiota in weanling pigs. Similarly, Torrallardona et al. (2011) indicated that benzoic acid had no effect on the intestinal microbiota in weanling pigs. In the current study, feeding the diet containing the combination of essential oils and benzoic acid had no effect on the fecal microbiota. In the current study, weanling pigs were kept in a clean, sanitary environment, which may have diminished the possible antimicrobial activity (Lee et al., 2003) of essential oils or benzoic acid or both. In addition, although the antimicrobial effect of
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essential oils and benzoic acid has been demonstrated consistently in in vitro studies, the effect has been rather inconsistent in in vivo studies (Dorman and Deans, 2000; Knarreborg et al., 2002; Si et al., 2006; Wenk, 2003). Therefore, it is possible that, unlike in in vitro studies, some unknown or uncontrollable factors may have been responsible for the lack of response observed in the current study.
5. Conclusion Pigs fed the complex diet consumed more feed, Lys, and DE, grew faster, and had lower serum urea N and globulin and greater serum glucose and cholesterol than those fed the simple corn-SBM diets. Supplementation of a simple corn-SBM diet with multi-enzyme complexes had some beneficial effects on growth performance of weanling pigs during the first 2 wk of the study, however, it had no effects during the last 2 wk of the study or overall. Addition of essential oils and benzoic acid to the simple corn-SBM diet supplemented with multi-enzyme complex had no clear effect on growth performance, serum metabolites and cytokines, or fecal microbiota in weanling pigs. Further research is needed to explore further the possibility of using a simple corn-SBM diet for weanling pigs by supplementation with various feed additives.
Conflict of interest statement The authors certify that there is no financial or personal relationships with other individuals or organizations that can affect the current research project improperly, or no professional or personal interest of any nature or kind in any product, service, or organization that could to construed as influencing the present article.
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Acknowledgements This research project was funded in part by Alabama Agricultural Experiment Station (Auburn University, Auburn, AL, US). Appreciation is extended to Nutra Blend LLC (Neosho, MO, US) and DSM Nutritional Products, LLC (Parsippany, NJ, US) for donating vitamin and trace mineral premix and enzyme complexes, essential oils, and benzoic acid, respectively. Technical assistance of B. Anderson, R. Britton, C.M. Lin, the staff at the Auburn University Swine Research and Education Center, and J. Pate and his staff at the Auburn University Poultry Research Farm is gratefully acknowledged.
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Table 1 Composition of starter pig diets (as-fed basis) 1,2,3. Item POS NEG ENZ ALL Ingredient (g/kg) Corn 413.6 514.9 514.2 511.0 Soybean meal (48% CP) 331.7 457.4 457.4 457.4 Spray dried whey4 150.0 4 Fishmeal 40.0 30.0 Poultry fat 4 Plasma protein 10.0 Dicalcium phosphate 6.6 12.8 12.8 12.8 Limestone 7.1 8.9 8.9 8.9 Salt 3.5 3.5 3.5 3.5 5 0.75 0.75 Enzyme mixture Essential oil6 0.10 6 Benzoic acid 3.0 Antibiotics6 5.0 7 Vitamin-trace mineral premix 2.5 2.5 2.5 2.5 Calculated composition DE (Mcal/kg) 3.61 3.43 3.43 3.42 CP (g/kg) 243 261 261 260 Ca (g/kg) 8 8 8 8 P (g/kg) 7 7 7 7 Ca:P 1.14 1.14 1.14 1.14 SID Lys (g/kg) 13.0 13.0 13.0 13.0 SID Lys:DE (g/Mcal) 3.60 3.79 3.79 3.80 Analyzed composition (g/kg) CP 242 246 274 270 Ca 7.6 8.9 6.7 9.3 P 6.1 6.2 5.8 6.2 1 POS = positive complex diet; NEG = negative simple corn-soybean meal diet; ENZ = NEG diet supplemented with multi-enzyme complexes; and ALL = NEG diet supplemented with multi-enzyme complexes, essential oils, and benzoic acid. 2 Starter diets were fed from 7.96 ± 1.78 to 20.70 ± 3.76 kg. 3 CP = crude protein; DE = digestible energy; and SID = standardized ileal digestible. 4 Spray dried whey: Honeyville (Brigham City, UT, US); fishmeal: Seven Springs Farm (Check, VA, US); and plasma protein: Appetein (APC Inc., Ankeny, IA, US). 5 Enzyme mixtures (Ronozyme; DSM Nutritional Products, LLC, Parsippany, NJ, US): 0.11 g MultiGrain (2,700 fungal xylanase unit xylanase, 700 fungal beta-glucanase unit β-glucanase, and 800 cellulase unit cellulase/g)/kg; 0.09 g VP (50 fungal beta-glucanase unit β-glucanase and 5,000 pectinase unit pectinase/g)/kg; 0.09 g WX (1,000 fungal xylanase unit xylanase/g)/kg; 0.11 g Rumistar (600 kilo novo unit amylase/g)/kg; and 0.35 g HiPhos 2700 (10,000 phytase unit phytase/g)/kg. 6 Essential oil: CRINA (DSM Nutritional Products, LLC); benzoic acid: Vevovitall (DSM Nutritional Products, LLC); and antibiotics: Tylan-10 Sulfa-G (Livestock Concepts, Hawarden, IA, US).
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Provided the following (unit/kg diet; Nutra Blend, Neosho, MO, US): Fe (ferrous sulfate), 150 mg; Zn (zinc oxide), 150 mg; Mn (manganous oxide), 37.5 mg; Cu (copper sulfate), 150 ppm; I (ethylenediamine dihydroiodide), 5 ppm; Se (sodium selenite), 0.3 ppm; vitamin A, 6,614 IU; vitamin D3, 1,102 IU; vitamin E, 26 IU; vitamin B12, 0.03 mg; menadione (menadione Na bisulfite complex), 1 mg; riboflavin, 6 mg; D-pantothenic acid (D-Ca pantothenate), 45 mg; niacin, 28 mg; and choline (choline chloride), 110 mg.
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Table 2 Effect of feed additives on growth performance of weanling pigs 1,2,3 Item Diet POS NEG ENZ ALL d 0 to 7 Feed intake (g/d) 298a 232b 264ab 261b Lys intake (g/d) 3.88a 3.01b 3.44ab 3.39b a b b DE intake (Mcal/d) 1.08 0.79 0.91 0.89b a b ab Weight gain (g/d) 256 182 211 170b Gain:feed (g/kg) 849 782 789 656 Gain:Lys intake (g/g) 65.3 60.2 60.7 50.4 Gain:DE intake (g/Mcal) 235 228 230 192 d 7 to 14 Feed intake (g/d) 647a 552b 587ab 570b a b ab Lys intake (g/d) 8.41 7.18 7.62 7.41b DE intake (Mcal/d) 2.33a 1.90b 2.01b 1.95b a b ac Weight gain (g/d) 537 416 486 456bc Gain:feed (g/kg) 833 752 830 797 Gain:Lys intake (g/g) 64.1 57.8 63.8 61.3 Gain:DE intake (g/Mcal) 231 219 242 233 d 14 to 21 Feed intake (g/d) 874a 737b 789b 745b Lys intake (g/d) 11.35a 9.58b 10.26b 9.68b a b b DE intake (Mcal/d) 3.15 2.53 2.71 2.55b Weight gain (g/d) 648a 528b 542b 529b Gain:feed (g/kg) 745 722 685 709 Gain:Lys intake (g/g) 57.3 55.6 52.7 54.5 Gain:DE intake (g/Mcal) 207 210 200 207 d 21 to 28 Feed intake (g/d) 982 849 871 853 Lys intake (g/d) 12.77 11.04 11.32 11.08 DE intake (Mcal/d) 3.54a 2.91b 2.99b 2.91b Weight gain (g/d) 624 555 561 576 Gain:feed (g/kg) 633 653 637 671 Gain:Lys intake (g/g) 48.7 50.2 49 51.6 Gain:DE intake (g/Mcal) 175 190 186 196 d 0 to 28 Feed intake (g/d) 700a 592b 628b 607b a b b Lys intake (g/d) 9.10 7.70 8.16 7.89b a b b DE intake (Mcal/d) 2.52 2.03 2.15 2.08b Weight gain (g/d) 516a 420b 450b 433b Gain:feed (g/kg) 738 711 715 715 Gain:Lys intake (g/g) 56.8 54.7 55.0 55.0 Gain:DE intake (g/Mcal) 205 207 208 209 a-c Within a row, means without a common superscript differ (P < 0.05).
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SEM
P-value
8 0.11 0.03 12 31 2.4 8
0.013 0.013 0.002 0.010 0.073 0.073 0.148
12 0.16 0.05 15 17 1.3 5
0.041 0.041 0.004 0.004 0.111 0.111 0.173
17 0.22 0.07 15 14 1.1 4
0.005 0.005 < 0.001 0.015 0.504 0.500 0.825
25 0.32 0.09 24 17 1.3 5
0.113 0.113 0.017 0.594 0.801 0.802 0.404
14 0.18 0.05 11 8 0.6 2
0.007 0.007 < 0.001 < 0.001 0.588 0.589 0.889
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POS = positive complex diet; NEG = negative simple corn-soybean meal diet; ENZ = NEG diet supplemented with multi-enzyme complexes; and ALL = NEG diet supplemented with multi-enzyme complexes, essential oils, and benzoic acid. n = 6. 2 Stater diets were fed from 7.96 ± 1.78 to 20.70 ± 3.76 kg. 3 SEM = pooled standard error of the mean; DE = digestible energy; and Lys = standardized ileal digestible Lys.
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Table 3 Effect of feed additives on serum metabolite concentrations in weanling pigs 1,2,3 . Item Diet SEM P-value POS NEG ENZ ALL b b a Total protein (g/dL) 4.63 4.72 4.88 4.87a 0.03 < 0.003 a b ac bc Albumin (g/dL) 3.78 3.49 3.65 3.58 0.05 0.016 Globulin (g/dL) 0.85b 1.22a 1.22a 1.29a 0.05 < 0.001 a b b b Albumin:globulin 4.56 2.94 3.02 2.79 0.20 < 0.001 b a a a Urea N (mg/dL) 14.3 17.6 18.4 17.8 0.5 0.003 Glucose (mg/dL) 134a 116b 115b 120b 2 0.005 a b b b Cholesterol (mg/dL) 81.8 70.4 71.2 75.1 1.4 0.002 Triglyceride (mg/dL) 41.7 50.8 50.6 51.3 2.1 0.230 a-c Within a row, means without a common superscript differ (P < 0.05). 1 POS = positive complex diet; NEG = negative simple corn-soybean meal diet; ENZ = NEG diet supplemented with multi-enzyme complexes; and ALL = NEG diet supplemented with multi-enzyme complexes, essential oils, and benzoic acid. n = 6. 2 SEM = pooled standard error of the mean. 3 Blood samples were collected during the third week of the study.
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Table 4 Effect of feed additives on serum cytokine concentrations (pg/mL) in weanling pigs 1,2,3. Item Diet SEM P-value POS NEG ENZ ALL IL-1α 4.78 2.19 7.51 2.74 1.29 0.438 IL-1β 260 479 307 253 50 0.400 IL-1Ra 242 238 351 308 28 0.536 IL-2 21 22 59 7 10 0.326 IL-4 57 111 114 162 22 0.617 IL-8 275 285 696 361 73 0.129 IL-10 197 154 317 120 43 0.400 IL-12 1,401 1,267 1,523 1,339 62 0.610 IL-18 1,150 668 1,269 1,070 102 0.243 TNFα 8.18 7.99 10.95 6.39 2.22 0.944 1 POS = positive complex diet; NEG = negative simple corn-soybean meal diet; ENZ = NEG diet supplemented with multi-enzyme complexes; and ALL = NEG diet supplemented with multi-enzyme complexes, essential oils, and benzoic acid. n = 6. 2 IL-1α = interleukin-1 α; IL-1β = interleukin-1 β; IL-1Ra = interleukin-1 Ra; IL-2 = interleukin-2; IL-4 = interleukin-4; IL-8 = interleukin-8; IL-10 = interleukin-10; IL-12 = interleukin-12; IL-18 = interleukin-18; and TNFα = tumor necrosis factors α. 3 SEM = pooled standard error of the mean. 3 Blood samples were collected during the third week of the study.
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Table 5 Effect of feed additives on microbiota (log10 cfu/g) in feces of weanling pigs 1,2. Item Diet SEM P-value POS NEG ENZ ALL Total aerobic bacteria 10.71 10.73 10.55 10.51 0.07 0.604 Total anaerobic bacteria 9.90 10.17 10.20 10.17 0.08 0.287 Coliforms 7.39 6.65 7.35 6.92 0.24 0.567 Lactobacillus 10.85 10.79 10.87 10.70 0.05 0.689 1 POS = positive complex diet; NEG = negative simple corn-soybean meal diet; ENZ = NEG diet supplemented with multi-enzyme complexes; and ALL = NEG diet supplemented with multi-enzyme complexes, essential oils, and benzoic acid. n = 6. 2 SEM = pooled standard error of the mean. 3 Fecal samples were collected during the third week of the study.
Highlights:
Weanling pigs fed a complex diet performed better and had different serum metabolite profile than those fed simple corn-soybean meal diets
Supplementation of a simple corn-SBM diet with multi-enzyme complexes had some beneficial effects on growth performance of weanling pigs during the first 2 wk of the study; however, it had no effects during the last 2 wk of the study.
Addition of essential oils and benzoic acid to a simple corn-SBM diet supplemented with multienzyme complex had no clear effect on growth performance, serum metabolites and cytokines, or fecal microbiota in weanling pigs.
Exploring fully the possibility of using a simple corn-soybean meal diet for weanling pigs by supplementation with various feed additives requires further research
PII: DOI: Reference:
S1871-1413(17)30378-5 https://doi.org/10.1016/j.livsci.2017.12.007 LIVSCI3369
To appear in: Livestock Science Received date: 27 July 2017 Revised date: 8 December 2017 Accepted date: 9 December 2017 Cite this article as: Y. Wang, L.I. Chiba, C. Huang, I.M. Torres, L. Wang and E.G. Welles, Effect of diet complexity, multi-enzyme complexes, essential oils, and benzoic acid on weanling pigs, Livestock Science, https://doi.org/10.1016/j.livsci.2017.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of diet complexity, multi-enzyme complexes, essential oils, and benzoic acid on weanling pigs
Y. Wang a, L.I. Chiba a,*, C. Huang a, I.M. Torres a, L. Wang a, E.G. Welles b
a
Department of Animal Sciences, College of Agriculture, Auburn University, Auburn, AL 36849,
United States b
Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL
36849, United States
* Corresponding author. Tel.: +1 334 844 1560; fax: +1 334 844 1519. E-mail address: [email protected] (L.I. Chiba).
AB STRACT The study was conducted to investigate the effect of supplementing a simple corn-soybean meal (SBM) diet with multi-enzyme complexes, essential oils, and benzoic acid on growth performance, serum metabolite profile, serum cytokines, and intestinal microbiota in weanling pigs. Forty-eight gilts and 48 castrated males weaned at 3 to 4 wk of age (initial body weight, 7.96 ± 0.89 kg) were randomly assigned to 4 dietary treatments with 3 gilt and 3 castrated male pens per treatment and 4 gilts or 4 castrated males per pen. A complex diet containing palatable and digestible ingredients was formulated (1.30 g standardized ileal digestible Lys/kg) to serve as the positive control (POS) diet. A simple corn-SBM, negative control (NEG) diet was formulated
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to be iso-lysinic to the POS diet, and the NEG diet was supplemented with multi-enzyme complexes (ENZ) or multi-enzyme complexes, essential oils, and benzoic acid (ALL). All diets were formulated to meet or exceed the 2012 NRC nutrient requirements of pigs weighing 7 to 25 kg. During the fourth week, blood samples were collected to determine serum metabolite profile and cytokines, and fecal samples were collected for the enumeration of bacteria. Pigs had ad libitum access to feed and water throughout the 4-wk study. From d 0 to 7 and 7 to 14, pigs fed the POS diet had greater feed and Lys intake (P < 0.05) and weight gain (P < 0.05) than those fed the NEG and ALL diets, but there were no differences in those response criteria between pigs fed the POS and ENZ diets. Weight gain of pigs fed the ENZ diet was 17% greater than those fed the NEG diet during the second week (P < 0.05), but it increased only numerically (16%) during the first week. Overall (d 0 to 28), pigs fed the POS diet consumed more feed, Lys, and digestible energy (DE; P < 0.05) and had greater weight gain (P < 0.05) than those fed the other diets. Dietary treatments had no effect on the efficiency of feed, Lys, or DE utilization for weight gain during the study. Serum total protein in pigs fed the ENZ and ALL diets was greater (P < 0.05) than those fed the POS and NEG diets. Pigs fed the ENZ diet had greater serum albumin (P < 0.05) than those fed the NEG diet. Serum globulin and urea N were lower (P < 0.05) and albumin to globulin ratio, glucose, and cholesterol were greater (P < 0.05) in pigs fed the POS diet than those fed the other diets. Dietary treatments had no clear effect on serum cytokines or fecal microbiota. Pigs fed the POS diet grew faster and had lower serum urea N and globulin and greater serum glucose and cholesterol than those fed the other diets. Although supplementation of the NEG diet with multi-enzyme complexes seemed to have beneficial effect on growth performance during the first 2 wk of the study, supplementation of the NEG diet with various feed additives had no clear effects. Further research is needed to explore further the possibility of
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using a simple corn-SBM diet for weanling pigs by supplementation with multi-enzyme complexes, essential oils, and benzoic acid. Keywords: Weanling pigs, Diet complexity, Multi-enzyme complex, Essential oils, Benzoic acid
1. Introduction Weaning is the most critical period in the pig's life. At the time of weaning, pigs are exposed to various stressors, which can reduce the growth performance of weanling pigs (Le Dividich and Sève, 2000; van Beers-Schreurs et al., 1998). For that reason, weanling pigs are fed highly palatable and digestible diets. Although a mixture of corn and soybean meal (SBM) has been used to satisfy the energy and amino acid requirements of pigs over the years, such a mixture is not appropriate for weanling pigs because of the digestive (Jensen et al., 1997; Lindemann et al., 1986) and other challenges. Weanling pigs need highly palatable and digestible ingredients such as dried whey, plasma protein, oat groats, lipids, fishmeal, and others in their diets. Researchers have been investigating the effect of diets containing such ingredients, i.e., complex diets, on weanling pigs over the years and concluded that complex diets can improve the growth performance of weanling pigs (Whang et al., 2000; Wolter et al., 2003). However, providing such complex diets to weanling pigs can be rather costly. In addition, antibiotics have been included in diets for weanling pigs to enhance their health status and growth. The public concern over the routine use of antibiotics has, however, led to the ban of antibiotics as a feed additive in Sweden in 1986 and Switzerland in 1999 (Wenk, 2003) and the restricted use in 1999 and the complete ban in 2006 by the European Union (Janczyk et al., 2009; Windisch et al., 2008). Considering the ban in those countries and ongoing discussions on the use of antibiotics in other countries, such as in the United States (FDA, 2017),
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it is important to explore alternative ways to protect farm animals and improve their health status (Wenk, 2003; Windisch et al., 2008). With the development of various enzymes or enzyme complexes in recent years, it may be possible for weanling pigs to extract energy and nutrients from corn and SBM meal more efficiently (Kim et al., 2003; Zhang et al., 2014). Similarly, phytogenic compounds (Windisch et al., 2008), such as essential oils (Cho et al., 2005; Kroismayr et al., 2008; Manzanilla et al., 2009), and benzoic acid (Diao et al., 2016; Kluge et al., 2006; Torrallardona et al., 2007) may improve the growth performance of weanling pigs because of their antimicrobial and antioxidative activities. The current study was conducted to assess the possibility of replacing a typical complex diet for weanling pigs with a simple corn-SBM diet by supplementation with various feed additives. Specific objective was to investigate the effect of supplementation of a simple corn-SBM diet with multi-enzyme complexes, essential oils, and benzoic acid on the growth performance, serum metabolite profile, serum cytokines, and fecal microbiota.
2. Materials and methods 2.1. Animals and facilities The protocol for animal care was approved by the Institutional Animal Care and Use Committee of Auburn University (Auburn, AL, US). A total of 96 piglets weaned at 3 to 4 wk of age (initial body weight, 7.96 ± 0.89 kg) were placed in pens (1.5 m2) in an environmentally controlled nursery with slotted floors based on their sex and body weight. Pigs were randomly assigned to 4 dietary treatments with 3 gilt pens and 3 castrated male pens per treatment and 4 gilts or 4 castrated males per pen. Because of the availability of the facility at one time, the study was conducted in 3 trials, and each trial used 16 females and 16 castrated males. Three trials
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were conducted consecutively with approximately 4 wk apart. Pigs were allowed ad libitum access to feed and water throughout each 4-wk trial period, and the weight and feed consumption data were collected weekly. 2.2. Dietary treatments After weaning, pigs were fed a common pre-starter diet for 4 d before beginning of the study, and 1 set of diets was during the 4-wk starter phase (Table 1). A typical complex, positive control (POS) diet was formulated to contain 13.0 g standardized ileal digestible (SID) Lys/kg, which was, essentially, the average SID Lys requirements for pigs weighing 7 to 11 and 11 to 25 kg (NRC, 2012). In addition to corn and SBM, the complex diet contained spray dried whey (Honeyville, Brigham City, UT, US), fishmeal (Seven Springs Farm, Check, VA, US), poultry fat, plasma protein (Appetein; APC Inc., Ankey, IA, US), and antibiotic (Tylan-10 Sulfa-G; Livestock Concepts, Hawarden, IA, US). A simple, corn-SBM negative control (NEG) diet was formulated to be iso-lysinic to the POS diet, and the NEG diet was supplemented with multienzyme complexes (DSM Nutritional Products, Parsippany, NJ, US; ENZ) or multi-enzyme complexes, essential oils (CRENA; DSM Nutritional Products), and benzoic acid (Vevovitall; DSM Nutritional Products; ALL). Feed additives were included in the diets by replacing part of corn. Minerals and vitamins for all diets were provided in amounts calculated to meet or exceed the NRC (2012) recommendations for pigs weighing 7 to 11 and 11 to 25 kg. Feed samples were collected from each batch of feed prepared, and pooled sub-samples were analyzed for crude protein and minerals (AOAC, 2000). The analyzed crude protein content of the NEG diet was slightly lower than intended (Table 1), but the reason is not apparent. 2.3. Collection of blood and fecal samples
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During the fourth week of the study, approximately 5 mL of blood was collected via vena cava puncture using a sterile needle and evacuated tube. Blood samples were allowed to clot and serum samples were separated by centrifugation at 1,500 x g for 15 min at room temperature to obtain clean serum samples. An aliquot was stored at -20°C, and samples were pooled by pen and analyzed for serum metabolites and cytokines. Similarly, fecal samples for bacterial enumeration were collected from, at least, 3 pigs from each pen by rectal stimulation during the fourth week of the study. Fecal samples were immediately chilled with ice, pooled by pen, and an aliquot was used immediately for the assay. 2.4. Analysis of blood and fecal samples Pooled serum samples were analyzed for total protein, albumin, globulin, urea N, glucose, cholesterol, and triglyceride using an automated analyzer at the Auburn University Clinical Pathology Laboratory (Mule et al., 2006). Likewise, pooled serum samples were used for the multiplex cytokine assay (Discovery Assay; Eve Technologies Corp, Calgary, AB, Canada). The multiplex assay consisted of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFNγ), interleukin (IL)-1α, IL-1Ra, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and tumor necrosis factor-α. Bacterial counts in the pooled fecal samples were determined by the plate-count technique (Shen et al., 2009). Each 25 g of fecal sample was homogenized in 225 mL of buffered peptone water with a lab blender (Smasher; AES Chemunex, bioMérieux, France) for 2 min and then serially diluted for the plating. Coliform bacteria concentration was determined by plating the homogenate and dilutions on agar medium (Violet Red Bile Agar; Difco, Detroit, MI, US) and incubated aerobically at 37°C for 24 h. For Lactobacillus spp., samples were plated on MRS agar (Difco) and incubated anaerobically at 37°C for 48 h. To enumerate the total anaerobic and
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aerobic bacteria, fecal samples were plated on plate-count agar (Difco) and incubated anaerobically and aerobically, respectively, for 48 h at 37°C. The numbers of bacteria in each sample were calculated and reported as colony forming units (cfu)/plate. 2.5. Statistical analysis Data were analyzed using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC, US). The pen was considered as the experimental unit. Trial and treatment, along with appropriate body weight as a covariate, were included in the initial statistical model. Covariates considered for the analysis were initial body weight for growth performance data and body weight at third week of the study for cytokine, microbiota, and serum metabolite data. The results of the initial statistical analysis indicated that trial and trial x treatment interactions were not important source of variation, thus, the data for the 3 trials were combined and analyzed accordingly. The PDIFF option of the LSMEANS statement (SAS Inst. Inc.) was used to assess the effect of treatments. The result with P < 0.05 was considered significant, whereas the result with P < 0.10 was considered a trend.
3. Results 3.1. Growth performance From d 0 to 7 and 7 to 14, pigs fed the POS diet had greater feed and SID Lys intake (P < 0.05) and weight gain (P < 0.05) than those fed the NEG and ALL diets (Table 2). However, there were no differences in those response criteria between pigs fed the POS and ENZ diets. Pigs fed the ENZ diet tended to consume more feed and Lys (P = 0.07) than those fed the NEG diet during the first week, whereas the increases in those criteria during the second week were not statistically significant. Weight gain of pigs fed the ENZ diet was 17% greater than those fed
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the NEG diet during the second week (P < 0.05), but it increased only numerically (16%) during the first week. Pigs fed the POS diet consumed more digestible energy (DE; P < 0.05) than those fed the other diets during the first 2 wk of the study. Pigs fed the POS diet had greater feed, Lys, and DE intake (P < 0.05) and weight gain (P < 0.05) from d 14 to 21 and DE intake (P < 0.05) from d 21 to 28 than those fed the other diets. Overall (d 0 to 28), pigs fed the POS diet consumed more feed, Lys, and DE (P < 0.05) and gained more weight (P < 0.05) than those fed the other diets. Dietary treatment had no clear effect on the efficiency of feed, Lys, or DE utilization for weight gain during any weekly period or overall. 3.2. Serum metabolites The effect of supplementation of the corn-SBM diet with feed additives on serum metabolites in weanling pigs during the fourth week of the study is presented in Table 3. Serum total protein in pigs fed the ENZ and ALL diets was greater (P < 0.05) than those fed the POS and NEG diets. Serum albumin in pigs fed the POS diet was greater (P < 0.05) than those fed the NEG and ALL diets, and it was greater (P < 0.05) in pigs fed the ENZ diet than those fed the NEG diet. Pigs fed the POS diet had lower serum globulin and urea N (P < 0.05) and greater albumin to globulin ratio, glucose, and cholesterol (P < 0.05) than those fed the NEG, ENZ, and ALL diets. Serum triglyceride in pigs fed the POS diet seemed to be less than those fed the other 3 diets (P < 0.09), but the overall treatment effect was not statistically significant. 3.3. Serum cytokines and fecal microbiota The effect of feed additives on serum cytokine concentrations and fecal microbiota in weanling pigs is presented in Tables 4 and 5, respectively. The GM-CSF, IFNγ, or IL-6 was either not detected or detected inconsistently, thus, the data for those cytokines were not presented. Weanling pigs fed the ENZ diet seemed to have a greater IL-2 concentration than
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those fed the ALL diet (P = 0.09; Table 4). The IL-8 concentration seemed to be greater in pigs fed the ENZ diet compared with those fed the other diet (P = 0.10). However, the overall treatment effect on IL-2 or IL-8 was not statistically significant. Although pigs fed the ENZ diet seemed to have a greater total fecal anaerobe concentration (P = 0.08) than those fed the POS diet, the overall treatment effect was not statistically significant (Table 5). Dietary treatments had no effect on the total aerobic bacteria, coliforms, or Lactobacillus spp. in the feces of weanling pigs.
4. Discussion The digestive system of weanling pigs is not fully developed to utilize corn and SBM efficiently, and young pigs have been fed complex diets containing many special ingredients. However, using such diets can be rather costly. With the development of multi-enzyme complexes, weanling pigs may be able to utilize a simple corn-SBM diet more efficiently. In addition, because of the public concerns over antibiotic resistance and the possible residue problems, the dietary use of antibiotics has been banned in many countries (Janczyk et al., 2009; Wenk, 2003; Windisch et al., 2008). Considering such bans and ongoing discussions on the restrictive use of antibiotics, finding viable alternatives (Wenk, 2003; Windisch et al., 2008) would be crucial for successful pig production in the future. Although various approaches can be used to protect weanling pigs from disease organisms and enhance their growth (Wenk, 2003; Windisch et al., 2008), the use of essential oils or benzoic acid or both can be a potentially viable alternative. The effect of supplementing weanling pig diets with various enzymes, essential oils, and benzoic acid individually have been reported over the years (Knarreborg et al., 2002; Kroismayr et al., 2008; Zhang et al., 2014).
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However, the effect of combining enzymes, essential oils, and benzoic acid in the weanling pig diet has not been explored fully. Therefore, the current study was conducted to investigate the effect of multi-enzyme complexes, essential oils, and benzoic acid on the growth performance, serum profile and cytokines, and fecal microbiota. During the first 2 wk of the study, pigs fed the NEG diet consumed less feed and SID Lys and grew slower than those fed the POS diet. Similar results have been reported previously (Dritz et al., 1996; Himmelberg et al., 1985; Whang et al., 2000). Pigs fed the ENZ diet seemed to consume more feed and Lys during the first 2 wk of the study than those fed the NEG diet. Similarly, pigs fed the ENZ diet grew faster than those fed the NEG diet, although the increase in weight gain was only numerical during the first week. Furthermore, there were no differences in feed intake, Lys intake, or weight gain between the ENZ and POS diets. The results may indicate that the enzyme supplementation had some positive effects on the growth performance of weanling pigs during the first 2 wk of the study. However, there was no effect of enzyme supplementation during the last 2wk of the study. Our results were, perhaps, a reflection of the enzyme activity in young pigs. Lindemann et al. (1986) reported that the enzyme activity increased throughout the nursing period after birth; however, it decreased sharply at weaning. It is possible that enzyme supplementation compensated the reduced enzyme activity at weaning and soon thereafter, and thus having positive effects on the growth performance of weanling pigs during the first 2 wk of the study. However, pigs fed the ENZ diet had lower feed and Lys intake and weight gain than those fed the POS diet during the third week and overall study period. Similar results have been reported earlier (Kiarie et al., 2007; Omogbenigun et al., 2003).
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The lack of clear response to enzyme supplementation may be related to the type of diets or substrate availability (Bedford, 2000; Emiola et al., 2009; Omogbenigun et al., 2004; Simmons et al., 1990). There have been some reports that supplementation of a corn-SBM diet with carbohydrases can improve the energy and nutrient digestibility in weanling pigs (Kim et al., 2003; Li et al., 2010; Passos et al., 2015). However, other researchers reported that enzyme complexes were not be effective in enhancing the nutritional value of corn and SBM for growing pigs (Cozannet et al., 2012; Jones et al., 2010). Similarly, the effectiveness of supplementing a corn-SBM diet with enzymes has been rather inconsistent in poultry (Olukosi et al., 2007; Rutherfurd et al., 2007; Yang et al., 2010). Essential oils have been shown to improve the growth performance of weanling pigs by enhancing the digestive secretion, nutrient absorption, and immune status because of their antimicrobial or antioxidative property or both (Huang et al., 2010; Maenner et al., 2011; Zeng et al., 2015). Similarly, benzoic acid has been shown to enhance the growth performance of weanling pigs by its effect on the intestinal tract development (Diao et al., 2014, 2016; Halas et al., 2010), nutrient utilization (Kluge et al., 2006), antioxidative property (Diao et al., 2016), and intestinal microbiota (Diao et al., 2014; Torrallardona et al., 2007). The effect of essential oils or benzoic acid on weanling pigs, however, has not been always positive (Muhl and Liebert, 2007; Nemechek et al., 2013b). A combination of dietary essential oils and benzoic acid may improve the growth performance and intestinal microbiota in the weanling pig (Diao et al., 2015; Zhang et al., 2016). In the current study, however, supplementation of the ENZ diet with essential oils and benzoic acid had no effect on the rate and efficiency of growth. It is possible that the efficacy those feed additives can be affected by the nutrient density of the diet. Bühler et al. (2006, 2010) reported
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that the effect of benzoic acid on grower-finisher pigs could be dependent on the dietary protein or P content. However, Nemechek et al. (2013a) indicated that benzoic acid had no effect on growth performance of weanling pig regardless of feeding a simple or complex diet. Similarly, Lan et al. (2016) found no interaction between essential oils and nutrient density in weanling pigs. Thus, the lack of effect of supplementing the ENZ diet with essential oils and benzoic acid observed in the current study may not be related to the diet complexity. Total protein or albumin in blood can be an indicator of protein metabolism in animals (Lowrey et al., 1962). Positive correlations observed between serum albumin and growth performance (Mule at al., 2006) may support their contention. In the current study, serum albumin was slightly greater in pigs fed the ENZ diet than those fed the NEG diet, implying that supplementation of a simple corn-SBM diet with multi-enzyme complexes had some beneficial effect on protein metabolism. Serum globulins, which are involved in, e.g., immune functions, were less in pigs fed the POS diet than those fed the other diets. Supplementation with essential oils or benzoic acid seemed to have no effect on the serum metabolites in the current study. Blood urea N is another important indicator of protein and amino acid adequacy and efficiency of amino acid utilization (Coma et al., 1995; Whang and Easter, 2000). In the current study, pigs fed the POS diet had lower concentration of serum urea N than those fed the NEG, ENZ, and ALL diets, indicating that pigs fed the POS diet utilized dietary protein more efficiently than those fed the other diets. Greater serum glucose concentration in pigs fed the POS diet can be explained by a greater available carbohydrates compared with the other diets. Similarly, the increased available energy content because of the inclusion of 3% poultry fat may be responsible for the greater serum cholesterol in pigs fed the POS diet compared with those fed
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the other diets. Multi-enzyme complexes, essential oils, or benzoic acid seemed to have no effect on serum urea N, glucose, or cholesterol. Cytokines can play an important role in, e.g., immune and inflammatory responses (van der Meide and Schellekens, 1996; Zhang and An, 2007). The IL-1, IL-6, and TNF-α are typical pro-inflammatory cytokines (Dinarello, 1991, 2000), and weaning has been associated with the early and transient response in the expression of genes for inflammatory cytokines (Pié et al., 2004). Li et al. (2012) reported that supplementation of a diet with essential oils reduced plasma IL-6 and increased plasma TNF-α concentrations in weanling pigs. Oh et al. (2012) found that benzoic acid decreased IL-1β, IL-6, and TNF-α concentrations in the small intestine of weanling pigs, however, benzoic acid had no effect on serum and mucosal TNF-α concentrations in weanling pigs (Walsh et al., 2012). In the current study, dietary treatments had no clear effect on serum cytokine concentrations. Supplementation of a weanling pig diet with essential oils (Ahmed et al., 2013; Huang et al., 2010; Janczyk et al., 2009; Li et al., 2012), benzoic acid (Diao et al., 2014; Halas et al., 2010; Kluge et al., 2006; Papatsiros et al., 2011; Torrallardona et al., 2007), or the combination of the two (Diao et al., 2015; Zhang et al., 2016) has been shown to improve intestinal microbiota. On the other hand, other researchers reported no effect of essential oils (Maenner et al., 2011; Muhl and Liebert, 2007) on the intestinal microbiota in weanling pigs. Similarly, Torrallardona et al. (2011) indicated that benzoic acid had no effect on the intestinal microbiota in weanling pigs. In the current study, feeding the diet containing the combination of essential oils and benzoic acid had no effect on the fecal microbiota. In the current study, weanling pigs were kept in a clean, sanitary environment, which may have diminished the possible antimicrobial activity (Lee et al., 2003) of essential oils or benzoic acid or both. In addition, although the antimicrobial effect of
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essential oils and benzoic acid has been demonstrated consistently in in vitro studies, the effect has been rather inconsistent in in vivo studies (Dorman and Deans, 2000; Knarreborg et al., 2002; Si et al., 2006; Wenk, 2003). Therefore, it is possible that, unlike in in vitro studies, some unknown or uncontrollable factors may have been responsible for the lack of response observed in the current study.
5. Conclusion Pigs fed the complex diet consumed more feed, Lys, and DE, grew faster, and had lower serum urea N and globulin and greater serum glucose and cholesterol than those fed the simple corn-SBM diets. Supplementation of a simple corn-SBM diet with multi-enzyme complexes had some beneficial effects on growth performance of weanling pigs during the first 2 wk of the study, however, it had no effects during the last 2 wk of the study or overall. Addition of essential oils and benzoic acid to the simple corn-SBM diet supplemented with multi-enzyme complex had no clear effect on growth performance, serum metabolites and cytokines, or fecal microbiota in weanling pigs. Further research is needed to explore further the possibility of using a simple corn-SBM diet for weanling pigs by supplementation with various feed additives.
Conflict of interest statement The authors certify that there is no financial or personal relationships with other individuals or organizations that can affect the current research project improperly, or no professional or personal interest of any nature or kind in any product, service, or organization that could to construed as influencing the present article.
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Acknowledgements This research project was funded in part by Alabama Agricultural Experiment Station (Auburn University, Auburn, AL, US). Appreciation is extended to Nutra Blend LLC (Neosho, MO, US) and DSM Nutritional Products, LLC (Parsippany, NJ, US) for donating vitamin and trace mineral premix and enzyme complexes, essential oils, and benzoic acid, respectively. Technical assistance of B. Anderson, R. Britton, C.M. Lin, the staff at the Auburn University Swine Research and Education Center, and J. Pate and his staff at the Auburn University Poultry Research Farm is gratefully acknowledged.
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Table 1 Composition of starter pig diets (as-fed basis) 1,2,3. Item POS NEG ENZ ALL Ingredient (g/kg) Corn 413.6 514.9 514.2 511.0 Soybean meal (48% CP) 331.7 457.4 457.4 457.4 Spray dried whey4 150.0 4 Fishmeal 40.0 30.0 Poultry fat 4 Plasma protein 10.0 Dicalcium phosphate 6.6 12.8 12.8 12.8 Limestone 7.1 8.9 8.9 8.9 Salt 3.5 3.5 3.5 3.5 5 0.75 0.75 Enzyme mixture Essential oil6 0.10 6 Benzoic acid 3.0 Antibiotics6 5.0 7 Vitamin-trace mineral premix 2.5 2.5 2.5 2.5 Calculated composition DE (Mcal/kg) 3.61 3.43 3.43 3.42 CP (g/kg) 243 261 261 260 Ca (g/kg) 8 8 8 8 P (g/kg) 7 7 7 7 Ca:P 1.14 1.14 1.14 1.14 SID Lys (g/kg) 13.0 13.0 13.0 13.0 SID Lys:DE (g/Mcal) 3.60 3.79 3.79 3.80 Analyzed composition (g/kg) CP 242 246 274 270 Ca 7.6 8.9 6.7 9.3 P 6.1 6.2 5.8 6.2 1 POS = positive complex diet; NEG = negative simple corn-soybean meal diet; ENZ = NEG diet supplemented with multi-enzyme complexes; and ALL = NEG diet supplemented with multi-enzyme complexes, essential oils, and benzoic acid. 2 Starter diets were fed from 7.96 ± 1.78 to 20.70 ± 3.76 kg. 3 CP = crude protein; DE = digestible energy; and SID = standardized ileal digestible. 4 Spray dried whey: Honeyville (Brigham City, UT, US); fishmeal: Seven Springs Farm (Check, VA, US); and plasma protein: Appetein (APC Inc., Ankeny, IA, US). 5 Enzyme mixtures (Ronozyme; DSM Nutritional Products, LLC, Parsippany, NJ, US): 0.11 g MultiGrain (2,700 fungal xylanase unit xylanase, 700 fungal beta-glucanase unit β-glucanase, and 800 cellulase unit cellulase/g)/kg; 0.09 g VP (50 fungal beta-glucanase unit β-glucanase and 5,000 pectinase unit pectinase/g)/kg; 0.09 g WX (1,000 fungal xylanase unit xylanase/g)/kg; 0.11 g Rumistar (600 kilo novo unit amylase/g)/kg; and 0.35 g HiPhos 2700 (10,000 phytase unit phytase/g)/kg. 6 Essential oil: CRINA (DSM Nutritional Products, LLC); benzoic acid: Vevovitall (DSM Nutritional Products, LLC); and antibiotics: Tylan-10 Sulfa-G (Livestock Concepts, Hawarden, IA, US).
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Provided the following (unit/kg diet; Nutra Blend, Neosho, MO, US): Fe (ferrous sulfate), 150 mg; Zn (zinc oxide), 150 mg; Mn (manganous oxide), 37.5 mg; Cu (copper sulfate), 150 ppm; I (ethylenediamine dihydroiodide), 5 ppm; Se (sodium selenite), 0.3 ppm; vitamin A, 6,614 IU; vitamin D3, 1,102 IU; vitamin E, 26 IU; vitamin B12, 0.03 mg; menadione (menadione Na bisulfite complex), 1 mg; riboflavin, 6 mg; D-pantothenic acid (D-Ca pantothenate), 45 mg; niacin, 28 mg; and choline (choline chloride), 110 mg.
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Table 2 Effect of feed additives on growth performance of weanling pigs 1,2,3 Item Diet POS NEG ENZ ALL d 0 to 7 Feed intake (g/d) 298a 232b 264ab 261b Lys intake (g/d) 3.88a 3.01b 3.44ab 3.39b a b b DE intake (Mcal/d) 1.08 0.79 0.91 0.89b a b ab Weight gain (g/d) 256 182 211 170b Gain:feed (g/kg) 849 782 789 656 Gain:Lys intake (g/g) 65.3 60.2 60.7 50.4 Gain:DE intake (g/Mcal) 235 228 230 192 d 7 to 14 Feed intake (g/d) 647a 552b 587ab 570b a b ab Lys intake (g/d) 8.41 7.18 7.62 7.41b DE intake (Mcal/d) 2.33a 1.90b 2.01b 1.95b a b ac Weight gain (g/d) 537 416 486 456bc Gain:feed (g/kg) 833 752 830 797 Gain:Lys intake (g/g) 64.1 57.8 63.8 61.3 Gain:DE intake (g/Mcal) 231 219 242 233 d 14 to 21 Feed intake (g/d) 874a 737b 789b 745b Lys intake (g/d) 11.35a 9.58b 10.26b 9.68b a b b DE intake (Mcal/d) 3.15 2.53 2.71 2.55b Weight gain (g/d) 648a 528b 542b 529b Gain:feed (g/kg) 745 722 685 709 Gain:Lys intake (g/g) 57.3 55.6 52.7 54.5 Gain:DE intake (g/Mcal) 207 210 200 207 d 21 to 28 Feed intake (g/d) 982 849 871 853 Lys intake (g/d) 12.77 11.04 11.32 11.08 DE intake (Mcal/d) 3.54a 2.91b 2.99b 2.91b Weight gain (g/d) 624 555 561 576 Gain:feed (g/kg) 633 653 637 671 Gain:Lys intake (g/g) 48.7 50.2 49 51.6 Gain:DE intake (g/Mcal) 175 190 186 196 d 0 to 28 Feed intake (g/d) 700a 592b 628b 607b a b b Lys intake (g/d) 9.10 7.70 8.16 7.89b a b b DE intake (Mcal/d) 2.52 2.03 2.15 2.08b Weight gain (g/d) 516a 420b 450b 433b Gain:feed (g/kg) 738 711 715 715 Gain:Lys intake (g/g) 56.8 54.7 55.0 55.0 Gain:DE intake (g/Mcal) 205 207 208 209 a-c Within a row, means without a common superscript differ (P < 0.05).
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SEM
P-value
8 0.11 0.03 12 31 2.4 8
0.013 0.013 0.002 0.010 0.073 0.073 0.148
12 0.16 0.05 15 17 1.3 5
0.041 0.041 0.004 0.004 0.111 0.111 0.173
17 0.22 0.07 15 14 1.1 4
0.005 0.005 < 0.001 0.015 0.504 0.500 0.825
25 0.32 0.09 24 17 1.3 5
0.113 0.113 0.017 0.594 0.801 0.802 0.404
14 0.18 0.05 11 8 0.6 2
0.007 0.007 < 0.001 < 0.001 0.588 0.589 0.889
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POS = positive complex diet; NEG = negative simple corn-soybean meal diet; ENZ = NEG diet supplemented with multi-enzyme complexes; and ALL = NEG diet supplemented with multi-enzyme complexes, essential oils, and benzoic acid. n = 6. 2 Stater diets were fed from 7.96 ± 1.78 to 20.70 ± 3.76 kg. 3 SEM = pooled standard error of the mean; DE = digestible energy; and Lys = standardized ileal digestible Lys.
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Table 3 Effect of feed additives on serum metabolite concentrations in weanling pigs 1,2,3 . Item Diet SEM P-value POS NEG ENZ ALL b b a Total protein (g/dL) 4.63 4.72 4.88 4.87a 0.03 < 0.003 a b ac bc Albumin (g/dL) 3.78 3.49 3.65 3.58 0.05 0.016 Globulin (g/dL) 0.85b 1.22a 1.22a 1.29a 0.05 < 0.001 a b b b Albumin:globulin 4.56 2.94 3.02 2.79 0.20 < 0.001 b a a a Urea N (mg/dL) 14.3 17.6 18.4 17.8 0.5 0.003 Glucose (mg/dL) 134a 116b 115b 120b 2 0.005 a b b b Cholesterol (mg/dL) 81.8 70.4 71.2 75.1 1.4 0.002 Triglyceride (mg/dL) 41.7 50.8 50.6 51.3 2.1 0.230 a-c Within a row, means without a common superscript differ (P < 0.05). 1 POS = positive complex diet; NEG = negative simple corn-soybean meal diet; ENZ = NEG diet supplemented with multi-enzyme complexes; and ALL = NEG diet supplemented with multi-enzyme complexes, essential oils, and benzoic acid. n = 6. 2 SEM = pooled standard error of the mean. 3 Blood samples were collected during the third week of the study.
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Table 4 Effect of feed additives on serum cytokine concentrations (pg/mL) in weanling pigs 1,2,3. Item Diet SEM P-value POS NEG ENZ ALL IL-1α 4.78 2.19 7.51 2.74 1.29 0.438 IL-1β 260 479 307 253 50 0.400 IL-1Ra 242 238 351 308 28 0.536 IL-2 21 22 59 7 10 0.326 IL-4 57 111 114 162 22 0.617 IL-8 275 285 696 361 73 0.129 IL-10 197 154 317 120 43 0.400 IL-12 1,401 1,267 1,523 1,339 62 0.610 IL-18 1,150 668 1,269 1,070 102 0.243 TNFα 8.18 7.99 10.95 6.39 2.22 0.944 1 POS = positive complex diet; NEG = negative simple corn-soybean meal diet; ENZ = NEG diet supplemented with multi-enzyme complexes; and ALL = NEG diet supplemented with multi-enzyme complexes, essential oils, and benzoic acid. n = 6. 2 IL-1α = interleukin-1 α; IL-1β = interleukin-1 β; IL-1Ra = interleukin-1 Ra; IL-2 = interleukin-2; IL-4 = interleukin-4; IL-8 = interleukin-8; IL-10 = interleukin-10; IL-12 = interleukin-12; IL-18 = interleukin-18; and TNFα = tumor necrosis factors α. 3 SEM = pooled standard error of the mean. 3 Blood samples were collected during the third week of the study.
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Table 5 Effect of feed additives on microbiota (log10 cfu/g) in feces of weanling pigs 1,2. Item Diet SEM P-value POS NEG ENZ ALL Total aerobic bacteria 10.71 10.73 10.55 10.51 0.07 0.604 Total anaerobic bacteria 9.90 10.17 10.20 10.17 0.08 0.287 Coliforms 7.39 6.65 7.35 6.92 0.24 0.567 Lactobacillus 10.85 10.79 10.87 10.70 0.05 0.689 1 POS = positive complex diet; NEG = negative simple corn-soybean meal diet; ENZ = NEG diet supplemented with multi-enzyme complexes; and ALL = NEG diet supplemented with multi-enzyme complexes, essential oils, and benzoic acid. n = 6. 2 SEM = pooled standard error of the mean. 3 Fecal samples were collected during the third week of the study.
Highlights:
Weanling pigs fed a complex diet performed better and had different serum metabolite profile than those fed simple corn-soybean meal diets
Supplementation of a simple corn-SBM diet with multi-enzyme complexes had some beneficial effects on growth performance of weanling pigs during the first 2 wk of the study; however, it had no effects during the last 2 wk of the study.
Addition of essential oils and benzoic acid to a simple corn-SBM diet supplemented with multienzyme complex had no clear effect on growth performance, serum metabolites and cytokines, or fecal microbiota in weanling pigs.
Exploring fully the possibility of using a simple corn-soybean meal diet for weanling pigs by supplementation with various feed additives requires further research