INTRODUCTION
There is growing concern for mycotoxins on swine production as they can cause reduced growth and feed intake, organ damage, and altered immune responses of pigs (Chaytor et al., 2011). Among various mycotoxins, Fusarium mycotoxins are secondary toxic metabolites produced by various species of the genus Fusarium such as F. graminearum and F. culmorum and frequently found in various feedstuffs for swine diet thus have received considerable attention (CAST, 2003; Chaytor et al., 2011). Deoxynivalenol (DON) and zearalenone (ZON) are the most important Fusarium mycotoxins in terms of frequency of occurrence as well as concentration of toxins relative to guidance level (CEC, 2006; Rodrigues and Naehrer, 2012a).
Because pigs are known to be very susceptible to DON (Prelusky et al., 1994), several studies were conducted to find feed additives ameliorating the detrimental effects of DON but their effects are still equivocal (Papaioannou et al., 2002; Weaver et al., 2013; Xiao et al., 2013; Kong et al., 2014). The most apparent signs of DON intoxication are reduced feed intake and consequent body weight (BW) loss of pigs. The DON also modulates immune responses as well as affects blood chemistry in pigs (Bergsjo et al., 1993; Swamy et al., 2002; Dänicke et al., 2004b). The ZON known as a mycoestrogen and estrogenic properties of its metabolites have been reported to cause reproductive disorders by hyperestrogenism (Takemura et al., 2007; Kanora and Maes, 2009; Jiang et al., 2010).
Barley ranks in fifth in global grain production (USDA, 2014). Although energy content in barley is about 10% to 15% less than that in corn, barley is still a very attractive ingredient for swine diet because of higher contents of crude protein (CP), lysine, and digestible phosphorus compared with corn (NRC, 2012; Woyengo et al., 2014). In addition, feeding diets containing barley up to 60% at the expense of corn did not show any detrimental effects on animal performances (Kim et al., 2014). A global survey for the incidence of mycotoxins in various feedstuffs indicated that the positive occurrences of DON and ZON in barley were 42% and 38%, respectively, and the average concentrations of DON and ZON were 1.677 and 0.323 mg/kg which are greater than the suggested guidance levels of mycotoxins for swine diets at 0.9 and 0.1 mg/kg, respectively (CEC, 2006; Rodrigues and Naehrer, 2012b). Moreover, it was reported that the effect of Fusarium mycotoxins on growth performance of pigs was dependent on the source of mycotoxin (i.e. purified vs naturally contaminated) in ingredient (Prelusky et al., 1994). However, little data from study feeding contaminated barley with Fusarium mycotoxins to pigs is available. Therefore, the objectives of the present study were to investigate the effects of feeding barley naturally contaminated with Fusarium mycotoxins on growth performance, vulva swelling, and digestibility of dry matter (DM), organic matter (OM), and CP for gilts and the recovery of gilts fed normal diets immediately after the exposure to the contaminated diets by measuring growth performance and vulva size.
MATERIALS AND METHODS
All the experimental procedures used in the current study were approved by the Institutional Animal Care and Use Committee at Konkuk University.
Exp. 1
Animals and experimental design
In a 14-d experiment, 16 crossbred gilts with an average initial BW of 33.3 kg (standard deviation = 3.0) were individually housed in metabolism crates (0.48×1.49 m) in an environmentally controlled room. The pigs were allotted to 4 dietary treatments in a randomized complete block design with 4 blocks based on BW.
Ingredients, diets, feeding, and sample collection
The concentration of DON in the corn, soybean meal (SBM), control barley, or naturally contaminated barley used were analyzed to be 0.6, 1.0, 0.3, or 25.7 mg/kg, respectively, and the concentrations of ZON were less than the detection limit (25.0 μg/kg) except for contaminated barley which was analyzed to be 26.0 μg/kg (Table 1).
A corn-SBM based diet containing 45% of control barley was prepared and 3 additional diets were also formulated to contain 15%, 30%, or 45% of the contaminated barley at the expense of the control barley (Table 2). Individual BW and feed consumption were measured on d 0, 7, and 14 and at the completion of experiment on d 14 average daily gain (ADG), average daily feed intake (ADFI), and gain:feed (G:F) were calculated. Individual vertical and horizontal lengths of vulva were measured on d 0, 3, 6, 9, and 12. From d 10 to 14, fecal samples were collected quantitatively by the marker-to-marker method as described by Kong and Adeola (2014). Blood samples were collected from all the pigs at the completion of study on d 14.
Mycotoxins and chemical analyses
The concentration of DON and ZON in the ingredients and diets were determined by using enzyme-linked immunosorbent assay kits (AgraQuant, Romer Labs Inc., Singapore, Republic of Singapore) which had quantification ranges for analysis on DON and ZON from 250 to 5,000 and 25 to 1,000 ng/mL, respectively.
All the fecal samples and orts collected for each gilt were dried in a forced-air oven at 55°C to constant weight and ground using a coffee grinder. Dry matter analysis of samples was performed by drying the samples in a forced-air oven at 135°C for 2 h (method 930.15; AOAC, 2005). Nitrogen content of samples was determined by the Kjeldahl method (method 984.13; AOAC, 2005) using a Buchi K-424 digestion and B-324 distillation apparatus (Buchi, Flawil, Switzerland) and gross energy was determined by adiabatic bomb calorimeter (Parr 1261 bomb calorimeter; Parr Instruments Co., Moline, IL, USA) using benzoic acid as a calibration standard. Serum concentration of immunoglobulin subsets (IgA, IgG, and IgM) was determined on a Cobas Integra 800 analyzer (Roche, Mannheim, Germany). An automated blood chemistry analyzer (Roche Cobas c702; Roche, Germany) was used to measure the amounts of albumin, globulin, alkaline phosphatase (ALP), Ala transaminase (ALT), Asp transaminase (AST), γ-glutamyl transferase (GGT), blood urea nitrogen (BUN), calcium, glucose, phosphorus, and total protein in the serum samples.
Exp. 2
Diets and feeding
A 14-d feeding trial was conducted to evaluate the recovery of pigs that previously consumed diets contaminated with mycotoxin in Exp. 1. Immediately after ending of Exp. 1, the pigs were fed a corn-SBM-based standard diet (Table 3) which met or exceed the nutrient requirement estimates of pigs (NRC, 1998). Individual pig BW and feed consumption were measured on d 0, 7, and 14 and the vertical and horizontal lengths of vulva were measured every 3 days from d 0 to 14.
Calculations and statistical analyses
For each of the experimental diets, the apparent total tract digestibility of DM, OM, and CP was determined according to the total collection method as described by Kong and Adeola (2014).
The experimental data were statistically analyzed using MIXED procedures of SAS (SAS Inst. Inc., Cary, NC, USA). The independent variables in the model included diet as a fixed effect and block as a random effect. Growth performance including ADG, ADFI, and G:F, immune responses and blood characteristics were response variables and each animal was considered as an experimental unit. Orthogonal polynomial contrasts were used to show linear or quadratic responses to the level of contaminated barley in the experimental diets and specific orthogonal contrasts were also used to compare the recovery results between pigs previously fed diet without and with contaminated barley. The alpha level used for the determination of statistical differences was set at 0.05. Probability values between 0.05 and 0.10 were considered tendencies.
RESULTS
Exp. 1
Mycotoxin analysis
The concentrations of DON and ZON in diets containing 0%, 15%, 30%, or 45% contaminated barley were analyzed to be 0.6, 6.1, 7.7, or 14.6 mg/kg and 23.8, 35.4, 47.8, or 33.7 μg/kg, respectively (Table 2).
Growth performance and vulva size
From d 0 to 7, the ADFI for pigs fed increasing level of the contaminated barley in the diets showed linearly decreasing trend (p = 0.056) and the ADG and G:F decreased linearly (p = 0.011) as the contaminated barley levels increased (Table 4). From d 7 to 14, the ADG and ADFI of pigs linearly decreased (p<0.01) with increasing level of the contaminated barley in the diets whereas the G:F of pigs showed linearly decreasing trend (p = 0.063). On d 14, the final BW of pigs fed diets with increasing level of the contaminated barley linearly decreased (p = 0.017). During the overall period, all growth performance measurements linearly decreased (p<0.01) as the level of contaminated barley in the diets fed to pigs increased.
The vulva size of pigs fed the diets with increasing level of contaminated barley was not affected throughout the 12-d period (Table 5).
Digestibility
The daily feed DM, OM, and CP intake as well as the respective feces components output linearly decreased (p<0.01) as the level of the contaminated barley in the diets increased (Table 6). However, the CP digestibility linearly increased (p = 0.011), and the digestibility of DM (p = 0.065) and OM (p = 0.060) showed increasing trend as the contaminated barley in the diets increased.
Serum immunoglobulin and blood characteristics
The concentrations of serum IgA, IgG, and IgM were not influenced by the increase of the contaminated barley in diets fed to pigs (Table 7). Feeding of increasing level of the contaminated barley to pigs linearly increased BUN (p = 0.016) whereas other blood chemistry measurements were not affected.
Exp. 2
Growth performance
From d 0 to 7, the ADG and G:F of pigs previously fed 15% or 45% of the contaminated barley in diets was greater (p<0.05) compared with pigs previously fed the control barley in diet (Table 8). No difference was observed in the growth performance among the pigs previously fed the diets containing 0%, 15%, 30%, or 45% of contaminated barley from d 7 to 14. For the overall period, the ADG of pigs previously fed 15% or 45% of the contaminated barley in diets was greater (p<0.05) compared with the pigs previously fed the control barley diet.
Vulva size
During the recovery period, the vulva size measured was not influenced by the levels of the contaminated barley in diets fed previously (Table 9).
DISCUSSION
Exp. 1
The difference between the analyzed and calculated concentrations of mycotoxins in the experimental diets based on the analyzed values of the individual ingredient was observed in the present study. This is in agreement with Swamy et al. (2003) who also observed much higher analyzed values of DON compared with the intended values in diets. This might be attributed to uneven distribution of mycotoxins in the ingredients used. The analyzed mycotoxin also showed that the contaminated barley used in the present study was primarily contaminated with DON rather than ZON.
Increasing level of contaminated barley with Fusarium mycotoxins negatively affected growth performance of growing pigs. Feed intake depression and consequent decrease of BW gain were commonly observed and are considered as the major adverse effects of feeding DON-contaminated diets to pigs (Swamy et al., 2003; Goyarts et al., 2005; Mok et al., 2013) and these effects were also observed in the present study. Moreover, the reductions in ADG of pigs fed diet containing 45% contaminated barley compared with diet containing 0% contaminated barley from d 0 to 7, 7 to 14, and the overall period, were 90.3%, 70.3%, and 80.4%, respectively, which were close to the value (93.8%) predicted by the equation derived from literature data on growth performance of pigs exposed to various levels of DON (Mok et al., 2013). Considering the guidance levels of DON and ZON for swine diets at 0.9 and 0.1 mg/kg, respectively (CEC, 2006), the concentrations of DON in the contaminated diets were above the guidance level while those of ZON were not. In addition, the vulva swelling which is one of common clinical symptoms of pigs exposed to ZON was not observed in the present study. Therefore, it might be speculated that the poor growth performance was primarily attributed to the presence of DON rather than ZON in contaminated diets fed to pigs. However, other unanalyzed Fusarium toxins which might interact with DON could not be excluded as a contributor for the poor growth performance. There was a linear decrease in the G:F of pigs fed diets with increasing levels of contaminated barley with Fusarium mycotoxins from d 0 to 7 during the first week as well as the overall period and this is in accordance with Young et al. (1983) and Smith et al. (1997) who also reported a linear reduction in the G:F as dietary DON increased in the diets. On the contrary, other studies observed a significant depression of feed intake and BW gain but no effect (Swamy et al., 2002) or an increase (Rotter et al., 1994; Swamy et al., 2003) in feed efficiency. This conflict of results among experiments may be, in part, attributed to different physiological stage of pigs (age, sex, or BW), source of contaminations (type of grains), and concentration of mycotoxins (Goyarts et al., 2005).
In contrast to the adverse effects of feeding diets contaminated with Fusarium mycotoxins on the growth performance, the positive effect was shown for the digestibility of DM, OM, and CP. This was in agreement with Dänicke et al. (2004a) who also found a DON-related improvement in nutrient digestibility. Several explanations could be possible for this positive effect. Matthäus et al. (2004) found that there were increased activities of protease, amylase, and non-starch polysaccharide-degrading enzymes in the wheat inoculated with Fusarium culmorum compared with the control wheat and were alterations of the cell penetrating properties by fungus. Reduced feed intake due to the presence of mycotoxin might also be attributed to the increase in nutrient utilization because Haydon et al. (1984) observed that the apparent total tract digestibility of DM and energy tended to increase as the feed intake of 25-kg pigs fed sorghum-SBM based diet decreased. However, Dänicke et al. (2004a) and Goyarts et al. (2005) reported that significant increases in nutrient digestibility for pigs fed diets contaminated with DON regardless of the feeding methods (i.e. ad libitum vs restricted) indicating that levels of feed intake did not affect nutrient digestibility.
Blood urea nitrogen is an indication of renal health and a linear increase of BUN was observed but the values were within the reference range of BUN in pigs (8.2 to 25 mg/dL; Latimer et al., 2003). Signs of liver damage (ALP, ALT, AST, and GGT) were not altered as the concentration of mycotoxin in the diets increased. Similarly, no signs of liver damage were detected in previous studies reported (Dänicke et al., 2004a; Accensi et al., 2006) whereas other study reported that there was a dose-response related decrease in blood measurements for the sign of liver damage (Döll et al., 2003). Two major groups of blood total proteins are albumin and globulin, and their alterations are indicatives of liver damage and abnormal immune functions, respectively (Busher, 1990). In some experiments, increases of serum albumin:globulin ratio were observed in pigs fed diets containing Fusarium mycotoxins (Rotter et al., 1994; Swamy et al., 2003). However, there were no changes in albumin:globulin ratio as well as total protein with increasing level of contaminated barley in experimental diets used in the present study. Rotter et al. (1994) suggested that DON and other Fusarium mycotoxins might directly affect globulin synthesis in the liver and compromise the immune response of pigs exposed to Fusarium mycotoxins. This discrepancy in results might be attributed to differences in age of pigs, types of grain as mycotoxin sources, and composition of Fusarium mycotoxins in grains as well as volume of blood sample collected (Dänicke et al., 2004b).
Exp. 2
A rapid compensation in feed intake and BW gain of the pigs fed the corn-SBM-based standard diet for 14 days was in accordance with Dänicke et al. (2004b) who observed quick and complete growth compensatory effects. During the first week of the recovery period, feed intake of pigs previously fed contaminated diets already returned to that of pigs fed diet without contamination and this remained similar following 7 days. In addition, the difference in ADG between the pigs previously fed diets including 0 and 15% or 45% contaminated barley was significant for the period during d 0 to 7 and the overall period, which resulted in the greater G:F in the pigs previously fed diet containing 15% or 45% contaminated barley for the respective periods.
In conclusion, the present study showed that feeding of barley contaminated with Fusarium mycotoxin negatively affects the growth performance of pigs primarily by feed intake depression but these adverse effects were quickly recovered in 7 days after the diets were replaced to the standard corn-SBM diet. The serum immunoglobulin and blood chemical components were not altered by feeding of contaminated barley diets whereas improvement in the digestibility of DM, OM, and CP was observed and further research is needed to verify underlying mechanisms on this improvement.
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