- Email: [email protected]
Effects of corn treated with foliar fungicide on in situ corn silage degradability in Holstein cows
Animal Feed Science and Technology 222 (2016) 149–157
Contents lists available at ScienceDirect
Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci
Research Paper
Effects of corn treated with foliar fungicide on in situ corn silage degradability in Holstein cows K.J. Haerr a , A. Pineda a , N.M. Lopes a,b , J.D. Weems c , C.A. Bradley c , M.N. Pereira b , M.R. Murphy a , G.M. Fellows d , F.C. Cardoso a,∗ a b c d
Department of Animal Sciences, University of Illinois, Urbana, IL 61801, USA Departamento de Zootecnia, Universidade Federal de Lavras, Lavras, MG 37200-000, Brazil Department of Crop Sciences, University of Illinois, Urbana, IL 61801, USA B.A.S.F. Corporation, Research Triangle Park, NC 27709, USA
a r t i c l e
i n f o
Article history: Received 23 February 2016 Received in revised form 5 October 2016 Accepted 18 October 2016 Keywords: In situ Digestibility Corn silage Fungicide
a b s t r a c t With increasing feed prices and decreasing profit margins livestock producers are constantly searching for ways to increase nutritive value of the feed in order to get more production per unit of feedstuff. The objective of this study was to assess the digestibility of corn silage made of corn plants treated with various foliar fungicide applications. Treatments were: control (CON), corn received no foliar fungicide application; 1X, corn received one application of pyraclostrobin foliar fungicide (PYR; Headline; BASF Corp.) at vegetative stage 5 (V5); 2X, corn received 2 applications of foliar fungicides, PYR at stage V5, and a mixture of pyraclostrobin and metconazole (PYR + MET; Headline AMP; BASF Corp.) at reproductive stage 1 (R1); and 3X, corn received 3 applications of foliar fungicides, PYR at stage V5, PYR + MET at stage R1, and PYR + MET at reproductive stage 3. Corn was harvested at the same time and ensiled for 7 months. Dried unground corn silage was put into 288 (3 per time points/treatment/cow) 10 × 20 cm bags and incubated for 8 different time points (0, 2, 4, 8, 12, 48, 72, and 96 h). A sample of unground dried corn silage was also placed into 20 × 40 cm bag and incubated for 48 h. Digestibility of corn silages was estimated using in situ procedure with 3 rumen-cannulated lactating multiparous Holstein cows. The degradable fraction of dry matter (DM) tended to be greater (P = 0.08) for corn silages treated with fungicide when compared with CON. There was no treatment effect (P > 0.05) on neutral detergent fiber (aNDF), acid detergent fiber (ADF), starch, and crude protein (CP). However, the soluble fraction of DM, aNDF, and ADF decreased (P < 0.05) as fungicide applications increased. Effective degradability (ED) was greater (P < 0.05) in CON than corn silages treated with fungicide mainly due to decreased ED in 3X compared with 1X and 2X. In situ digestibility for bigger and smaller bags was different. Degradability of DM, aNDF, and ADF was higher (P < 0.05), while starch and CP degradability was lower (P < 0.01) in the larger bags. Fungicide application to corn for silage lead to higher DM degradable fraction which seems to be the result of increased sugar and starch along with decreased aNDF and ADF. © 2016 Published by Elsevier B.V.
Abbreviations: 1X, treatment 1; 2X, treatment 2; 3X, treatment 3; ADF, acid detergent fiber expressed inclusive of residual ash; aNDF, neutral detergent fiber assayed with a heat stable amylase and expressed inclusive of residual ash; ANOVA, analysis of variance; ATP, adenosine triphosphate; CON, control; CP, crude protein; DM, dry matter; ED, effective digestibility, kd, fractional digestion rate; MET, metconazole; NDF, neutral detergent fiber; NRC, National Research Council; PYR, pyraclostrobin; R1, reproductive stage 1; V5, vegetative stage 5. ∗ Corresponding author at: 290 Animal Sciences Laboratory, Department of Animal Sciences, University of Illinois, 1204 West Gregory Drive, Urbana, IL 61801, USA. E-mail address: [email protected] (F.C. Cardoso). http://dx.doi.org/10.1016/j.anifeedsci.2016.10.010 0377-8401/© 2016 Published by Elsevier B.V.
150
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
1. Introduction As prices of feed increase, particularly corn, and nutritional demands of milk production increase, it is important to evaluate potential ways of increasing feed efficiency in the dairy cow leading to increased profitability for dairy farmers. During the course of a growing season corn plants can be exposed to many environmental stressors including heat, cold, drought, and pathogens (Rivero et al., 2001). Plant stress may lead to decreased quality of the plant when used for animal feed, to decreased potential for digestibility, and altered digestibility characteristics (Fahey et al., 1994). Fungal colonization on the corn plant causes a competition between the plant and the fungus for nutrients. The plant has many mechanisms (e.g., lignification and leaf shedding) to attempt to hinder the growth of the fungal infestation. These mechanisms may potentially decrease the digestibility of the plant. The fungal infestation itself may also change the chemical composition of the plant in the process of competing for nutrients (Venancio et al., 2009). Research also has examined the different effects of the foliar fungicide on the plant at a chemical level (Köhle et al., 2002). The class of foliar fungicides known as strobulins have been studied for their possible increase in the greening effect of plants as well as their ability to handle stressors (Venancio et al., 2009). The aforementioned physiological changes elicited by the foliar fungicide have been shown to increase yield in plants that are not infected with disease, which may cause a possible increase in digestibility and nutritive content of the plant when used as feed for animals (Wise and Mueller, 2011). This increase in quality can be due to decreased lignification, increased grain fill and starch content, and increased nitrate assimilation and consequently increased protein content (Yates et al., 1997; Venancio et al., 2009). Digestion techniques are often used in evaluations of the nutritive value of corn silage (Nocek, 1988). The in vitro technique involves drying and fine grinding of whole plant corn silage for analysis. Grinding may minimize quality differences among whole plant corn silage samples related to physical form such as grain hardness and particle size (Bal et al., 2000). A macro in situ technique using undried, unground whole plant corn silage has been used to evaluate ruminal nutrient disappearance (Doggett et al., 1998). This procedure may provide better estimates of differences in ruminal starch disappearance as it is more influenced by maturity and hybrid than standard in vitro procedures. However, because of the occurrence of particle size reduction during eating and rumination it may tend to overestimate the magnitude of mechanical processing effects on ruminal starch disappearance and likely underestimates ruminal neutral detergent fiber (NDF) disappearance (Bal et al., 2000). In situ studies have been used in ruminant animals for many decades to estimate the potential digestibility of feedstuffs as well as to attempt to understand the complex interactions of the rumen ecology on feedstuffs. Much research has been conducted to create a standard procedure for rumen in situ techniques, which will not only allow research to be sufficiently compared across laboratories but provides the best estimate of what actually happens in the rumen and is most biologically relevant (Vanzant et al., 1998). It is well known that sample size to bag surface area ratio is very important to obtain correct results. From various studies it has been agreed that an appropriate sample size to surface area ratio is 10–30 mg/cm2 (Varga and Hoover, 1983; Vanzant et al., 1998). The objectives of this study were: (1) to evaluate in situ degradability of corn plant treated at various times with foliar fungicide on corn harvested as whole plant silage for lactating Holstein cows and (2) to determine if there were differences in 48 h in situ degradability between samples ruminally incubated in 10 × 20 cm bags when compared with samples in 20 × 40 cm bags. 2. Materials and methods 2.1. Treatments Corn was grown for silage on fields owned by the University of Illinois (Urbana, IL, USA) located at 40.08 latitude, and − 88.22 longitude. The four silages evaluated in this experiment were: control (CON), corn received no foliar fungicide application; treatment 1 (1X), in which corn received 1 application of pyraclostrobin (PYR) foliar fungicide (Headline; BASF Corp., Florham Park, New Jersey, USA) at a rate of 0.11 kg of active ingredient (a.i.)/ha at corn vegetative stage 5 (V5; when 5 visible leaf collars can be seen; Mueller and Pope, 2009); treatment 2 (2X), in which corn received 2 applications of foliar fungicides, PYR at 0.11 kg of a.i./ha at corn stage V5, and a mixture of PYR + metconazole (MET; Headline AMP; BASF Corp., Florham Park, New Jersey, USA) at 0.11 + 0.04 kg of a.i./ha at corn reproductive stage 1 (R1; when silks are visible outside the husks; Mueller and Pope, 2009); and treatment 3 (3X), in which corn received 3 applications of foliar fungicide, PYR at 0.11 kg of a.i./ha at corn stage V5, PYR + MET at 0.11 + 0.04 kg of a.i./ha at corn stage R1, and PYR + MET at 0.11 + 0.04 kg of a.i./ha at corn reproductive stage 3 (when kernel is yellow outside, whereas the inner fluid is now milky white due to accumulating starch; Mueller and Pope, 2009). The dates for fungicide application for first, second, and third applications were: July 7, 2013; July 26, 2013; and August 13, 2013. The corn hybrid planted was ‘LG2636 VT3P RIB’ (LG Seeds; Elmhurst, IL, USA), which is a dual purpose hybrid used for either grain or silage. The hybrid is advertised as 114-day maturity, with high yield potential, strong stalks, and high vigor. This hybrid also is advertised as having a high level of resistance against northern corn leaf blight (caused by the fungus Exserohilum turcicum), southern corn leaf blight (caused by the fungus Bipolaris maydis), and gray leaf spot (caused by the fungus Cercospora zeae-maydis). This hybrid contains transgenic traits that provide protection against corn earworm (Helicoverpa zea). All corn was planted on June 5, 2013 and harvested on September 27, 2013 at a dry matter (DM) of 330, 300, 300, and 325 g/kg for CON, 1X, 2X, and 3X, respectively. Harvester included kernel processing to have the same theoretical length of chop, set at 1.9 cm. Inoculant (Silo-King WS; 1.5 × 105 cfu/g of L. plantarum, P. pentosaceus, and
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
151
Table 1 Mean chemical and nutritive composition (g/kg of DM unless otherwise noted) and standard deviation of nutrient composition, fermentation profile, energy content, and microbial count of corn silage treated without or with applications of foliar fungicide (adapted from Haerr et al., 2015). Item
Treatmenta
SDb
CON
1X
2X
3X
Corn silage composition DM CP aNDF aNDF digestibility, 30 h ADF Fat Lignin (sa) Soluble CP, g/kg of CP Starch Sugar Ash Ca P Mg K S
306.30 86.10 473.20 500.90 292.40 27.10 34.50 561.80 276.10 7.20 51.10 2.20 1.90 1.40 13.40 1.00
309.80 87.20 458.80 496.70 277.20 27.70 36.50 519.80 286.70 11.40 49.20 2.20 1.90 1.30 12.70 1.00
344.10 85.00 454.10 490.00 281.10 27.40 33.60 540.50 292.60 12.30 47.90 2.00 1.80 1.30 12.10 0.90
303.70 89.80 452.80 488.20 273.30 31.60 32.20 590.40 287.00 12.50 47.60 2.20 1.90 1.40 12.40 1.00
16.80 8.10 38.90 62.00 23.40 4.80 11.30 41.70 33.00 2.70 3.90 0.10 0.10 0.10 0.90 0.10
Energy calculationsc TDN NEL , MJ/kg DM NEG , MJ/kg DM NEM , MJ/kg DM
655.50 5.98 4.06 6.61
659.20 5.98 4.14 6.61
665.40 6.07 4.23 6.74
673.70 6.19 4.35 6.90
22.00 0.08 0.17 0.17
Fermentation products pH Lactic acid Acetic acid Ethanol Ammonia N
4.88 49.10 29.70 2.00 0.80
4.99 42.50 27.80 2.10 0.70
4.99 46.30 13.10 1.60 0.60
4.84 46.50 34.70 3.20 0.90
0.30 20.50 9.40 1.70 0.20
Microbial count Yeast, cfu/g Mold, cfu/g
2,663 92,800
1,741 14,300
50,595 51,541
5,153 72,300
94,001 30,750
Particle size distributiond 19 mm 7.8 mm 1.2 mm Pan Mean particle size,e m
87 689 215 21 9134
102 678 218 21 9186
83 672 239 57 8852
96 680 220 36 9401
67 59 67 11 0.36
Abbreviations: 1Xtreatment 1; 2Xtreatment 2; 3Xtreatment 3; ADFacid detergent fiber expressed inclusive of residual ash; aNDFneutral detergent fiber assayed with a heat stable amylase and expressed inclusive of residual ash; Cacalcium; CONcontrol; CPcrude protein; DMdry matter; Kpotassium; Lignin (sa)lignin determined by solubilization of cellulose with sulphuric acid; Mgmagnesium; NEG net energy of growth; NEL net energy of lactation; NEM net energy of maintenance; Pphosphorus; Ssulfur; SDstandard deviation. a Treatment: control (CON), corn received no foliar fungicide application; 1X, corn received one application of pyraclostrobin foliar fungicide (PYR; Headline; BASF Corp.) at vegetative stage 5 (V5; when 5 visible leaf collars can be seen; Mueller and Pope, 2009); 2X, corn received 2 applications of foliar fungicides, PYR at stage V5, and a mixture of pyraclostrobin and metconazole (PYR + MET; Headline AMP; BASF Corp.) at reproductive stage 1 (R1; when silks are visible outside the husks; Mueller and Pope, 2009); and 3X, corn received 3 applications of foliar fungicides, PYR at stage V5, PYR + MET at stage R1, and PYR + MET at reproductive stage 3 (when kernel is yellow outside, whereas the inner fluid is now milky white due to accumulating starch; Mueller and Pope, 2009). b Maximum within treatment SD. c NRC (2001). d Penn State Particle Size Separator (Pennsylvania State University, Pennsylvania, USA) was used for measurements. e Log10 SD.
Enterococcus faecium; Agri-King, Fulton, IL, USA) was applied at a rate of 115 g of inoculant per 1000 kg of corn and then ensiled in silo bags for more than 200 days. Particle size of the corn silages was measured on a weekly basis (3 replicates twice weekly) for 10 weeks using the Penn State Particle Separator [3-sieve model (19, 7.8, and 1.2 mm), Pennsylvania State University, Pennsylvania, USA) as described by Kononoff et al. (2003). Data from the Penn State Particle Separator were then fit to a lognormal distribution to estimate the geometric mean particle size and the log10 standard deviation. Corn silages nutrient profiles are described in Table 1 adapted from Haerr et al. (2015).
152
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
2.2. Animals and housing All experimental procedures were approved by the University of Illinois (Urbana-Champaign, IL, USA) Institutional Animal Care and Use Committee. Three second-lactation lactating Holstein cows (375 ± 21 days in milk; milk yield: 19.7 ± 8 kg/day) fitted with a rumen cannula were used for the experiment. Cows were housed in a tie-stall barn with individual feed access and fed diet to meet the NRC (2001) requirements with ad libitum access to feed. The diet was composed of 500 g/kg concentrate and 500 g/kg forage. The concentrate was a mixture of dry ground corn, soybean meal, soy hulls, blood meal, minerals, and vitamins. The forage consisted of 720 g/kg of corn silage and the rest (280 g/kg) made of alfalfa hay, alfalfa silage, and cottonseed. Cows were fed once daily at 1400 h, and were milked three times daily at 0700, 1400, and 2200 h. Refusals were removed and weighed before new feed was offered. 2.3. Sampling and bag preparation Corn silage was removed from 6 locations along the face of the bunker silo in order to retrieve a representative sample. Once the sample (4 kg per treatment) was obtained, it was dried and then placed on a sheet. The sample was then divided into 24 equal sections to decrease sample error and increase uniformity of the feedstuff placed into each bag. Two sizes of Dacron bags (Ankom Technology, Macedon, NY, USA) were utilized for this study, one 10 × 20 cm and the other 20 × 40 cm, both had 50 m pores. The procedure used was similar to the one described by Vanzant et al. (1998). Briefly, bags of both sizes were labeled, dried, and weighed prior to the addition of the corn silage. Each bag was heat sealed at least twice to ensure no feed particles escaped. Bags were filled to achieve a 20 mg DM/cm2 of bag surface. Small bags were filled with 8 g of DM and large bags were filled with 32 g of DM. For all bags, the sample was unground and particle length was not altered before it was put into the bags. Three replicates were made for each small bag at each time point for a total of 24 bags per treatment per cow. Bags were placed into large mesh garment bags to prevent the loss of bags in the gastrointestinal tract. All bags were soaked in warm (45 ◦ C) water before they were placed into the ventral rumen at the same time. Bags were then removed at the appropriate time point, noting the identification of each bag at the time of removal. Small bags were removed at 0, 2, 4, 8, 12, 48, 72, and 96 h. The same procedure was followed for the large (20 × 40 cm) bags, except that they were removed from the rumen only at 0 and 48 h (3 per time point/treatment/cow). Care was taken to minimize air exposure that could interfere with proper fermentation, and remaining bags were placed back into the ventral rumen. Bags that were removed from the rumen were immediately placed in ice water to stop fermentation. Bags were hand rinsed thoroughly in cold running water until the rinsing water was clear, then bags were immediately frozen (−20 ◦ C) for at least 24 h. After freezing, the bags were thawed and rinsed on a rinse cycle of a washing machine (Roper RTW4641BQ1, Whirlpool Corp., Benton Harbor, MI, USA) 2 times to reduce microbial content. Bags were then oven dried for 24 h at 110 ◦ C and disappearance was calculated. In the instance of torn bags or compromised seals post digestion the data were considered missing (n = 14). 2.4. Chemical analysis The three replicates of each time point from each treatment were combined to make a composite sample per cow that was sent to a commercial laboratory (Rock River Lab, Watertown, WI, USA) for analysis via wet chemistry methods (Schalla et al., 2012). The samples (n = 24 per treatment) were analyzed for neutral detergent fiber (aNDF), acid detergent fiber (ADF), crude protein (CP), starch, and DM contents. The aNDF was analyzed using sulfite and alpha amylase along with a premixed neutral detergent solution (Goering and Van Soest, 1970), and expressed inclusive of residual ash. The ADF was analyzed using the Ankom200 Fiber analyzer (Ankom Technology, Fairport, NY, USA) and expressed inclusive of residual ash. The CP content of the samples was measured using the combustion method (976.06) to determine N content and then multiplying the N content by 6.25 (AOAC, 1995). Starch was measured using alpha amylase, amyloglucosidase, and sodium acetate buffer by the procedure described by Hall and Mertens (2008). 2.5. Statistical analysis Statistical analysis was performed using SAS (v9.4 Institute Inc., Cary, NC, USA). The data were analyzed in two sequential steps. First, a nonlinear model of in situ digestion was analyzed using the NLIN procedure, based on the partitioning of feed such that the fractions of soluble feed (A; washout fraction after rinsing at time point 0), rumen degradable feed (B), and indigestible feed (C) is summed to 1. The disappearance data from the small bags were first fitted to the full model: Y = B (exp−Vkd (t−t1 ) ) + C where B = the proportion of potentially degradable feed, C = the proportion of indigestible feed, t = time point, Y = the proportion of feed remaining at a specified time point (t), kd = the fractional digestion rate constant, and V = 1 when t ≥ lag, and V = 0 when t < lag (McDonald, 1981; Van Milgen et al., 1991). The parameter of lag was restricted so that lag ≥ 0. If the full model failed to converge or lag = 0 (Ørskov and McDonald, 1979) then the reduced model was utilized: Y = B (exp−kd (t) ) + C
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
153
The A (soluble) fraction was then calculated as 1 − (B + C). The effective degradability was estimated as ED = A + B × [kd/(kd + kp)] (McDonald, 1981). Although the rate of passage from the rumen (kp) is affected by many factors [feeding intake, diet composition, feed particle size, and moisture in feeds (Krizsan et al., 2010)], a value of 0.06 was assumed to estimate ED in the present experiment. An ANOVA was then conducted on estimates of soluble feed, rumen degradable feed, indigestible feed, kd , and ED. Secondly, a linear mixed model (MIXED procedure) was created to explore associations among parameters calculated and bag size. Treatment and replicate were treated as fixed effects and cow was considered a random effect. Bag type and treatment as well as the interaction between the two were treated as fixed effects. Three predetermined orthogonal contrasts were used for both models. One contrast compared corn silage with fungicide application, and the other two contrasts examined linear and quadratic effects of fungicide applications. These contrasts were used for all variables analyzed unless otherwise stated. The degrees of freedom method used was Kenward-Rogers (Littell et al., 1998). Residual distribution was evaluated for normality and homoscedasticity using the UNIVARIATE procedure. Statistical significance was declared at P ≤ 0.05 and tendencies when 0.05 < P ≤ 0.10. 3. Results 3.1. Physical characteristics The mean particle size of corn silage for each treatment was 9134, 9186, 8852, and 9401 m for CON, 1X, 2X, and 3X, respectively. Results from the analyses of physical characteristics of corn silage are shown in Table 1. Corn silage had similar physical characteristics among treatments. 3.2. Digestibility kinetics There were no differences among treatments for indigested DM (P = 0.19, Table 2). There was a linear treatment effects for the soluble fraction of DM, with the amount decreasing as the number of foliar fungicide applications increased (P = 0.01). Degradable DM tended to be greater for corn silages treated with foliar fungicide when compared with CON (P = 0.08). There were also tendencies for linear (P = 0.06) and quadratic (P = 0.08) treatment effects for rumen degradable DM in which the proportion of rumen degradable feed increased as the number of foliar fungicide applications increased. The fractional rate of degradation of DM was lower (P = 0.003) for corn silages treated with foliar fungicide when compared with CON. Effective degradability of DM decreased (P = 0.04) with foliar fungicide applications when compared with CON. There were linear (P = 0.001) and quadratic (P = 0.03) treatment effects for ED of DM, it decreased as the number of foliar fungicide applications increased. There were no differences between CON and corn silages treated with foliar fungicide for the soluble fraction of aNDF, rumen degradable aNDF, indigestible aNDF, or kd (P > 0.05, Table 2). However, there was a linear treatment effect for the soluble fraction of aNDF with the amount decreasing as the number of applications of foliar fungicide increased (P = 0.005). There was quadratic treatment effect for the rumen degradable aNDF (P = 0.03), indigestible aNDF (P = 0.05), and kd (P = 0.05). The corn silage treated with 1X and 3X applications of fungicide had greater proportion of degradable aNDF than 2X. The opposite occurred for the indigestible fraction and kd , the mid-level treatment of fungicide application presented greater proportion of indigested aNDF along with greater rate of digestion than 1X and 3X. Effective degradability of aNDF decreased (P = 0.002) with foliar fungicide applications when compared with CON. There were linear (P < 0.001) and quadratic (P = 0.006) treatment effects for ED of aNDF, it decreased as the number of foliar fungicide applications increased. There were no differences between CON and corn silages treated with foliar fungicide for the soluble fraction of ADF, rumen degradable ADF, indigestible ADF, or kd (P > 0.05, Table 2). However, there were linear (P = 0.001) and quadratic (P = 0.03) treatment effects for the soluble fraction of ADF with the amount decreasing as the number of applications of foliar fungicide increased. In addition, there was a quadratic treatment effect for the degradable fraction of ADF with the corn silage treated with the 1X and 3X applications of foliar fungicide having greater degradable ADF fraction. Effective degradability of ADF decreased (P = 0.01) with foliar fungicide applications when compared with CON. There were linear (P < 0.001) and quadratic (P = 0.001) treatment effects for ED of ADF, it decreased as the number of foliar fungicide applications increased. There were no differences between CON and corn silages treated with foliar fungicide for the soluble fraction of starch, rumen degradable starch, indigested starch, kd , and ED (P > 0.05, Table 2). There were no linear or quadratic effects of fungicide applications in the different fractions of starch or in its fractional rate of digestion. In addition, there were no differences between CON and corn silages treated with foliar fungicide for the soluble fraction of CP, rumen degradable CP, indigested CP, kd , or ED (P > 0.05, Table 2). 3.3. Comparison of feed digestion in 10 × 20 cm vs. 20 × 40 cm dacron bags There was a larger (P = 0.01) amount of corn silage DM disappearance for feed in the 20 × 40 cm bags when compared with corn silage in the 10 × 20 cm bags. The DM disappearance at 48 h tended (P = 0.08) to be higher for CON than corn silages
154
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
Table 2 Least squares means and associated standard errors of soluble (washout fraction after rinsing at time point 0) proportion, degradable feed proportion, indigestible feed proportion, fractional rate of digestion (kd ), and effective degradability (ED) of corn silage treated without or with applications of foliar fungicide as determined in 10 × 20 cm Dacron bags. Treatmenta
SEMb
CON
1X
2X
3X
DM Soluble Degradable Indigestible kd, h−1 EDd
0.33 0.39 0.28 0.04 0.48
0.39 0.45 0.16 0.02 0.49
0.35 0.42 0.23 0.03 0.47
0.26 0.58 0.16 0.02 0.37
aNDF Soluble Degradable Indigestible kd, h−1 EDd
0.32 0.49 0.19 0.02 0.42
0.33 0.61 0.06 0.01 0.41
0.30 0.47 0.23 0.02 0.40
ADF Soluble Degradable Indigestible kd, h−1 EDd
0.30 0.53 0.17 0.01 0.39
0.31 0.69 0.00 0.01 0.38
Starch Soluble Degradable Indigestible kd, h−1 EDd
0.36 0.61 0.03 0.16 0.80
CP Soluble Degradable Indigestible kd, h−1 EDd
0.31 0.47 0.22 0.02 0.41
Pc CON vs. FUN
Linear
Quadratic
0.25 0.04 0.06 0.004 0.02
0.95 0.08 0.19 0.003 0.04
0.01 0.06 0.94 0.89 0.001
0.39 0.08 0.34 0.13 0.03
0.25 0.71 0.05 0.01 0.32
0.02 0.06 0.06 0.003 0.02
0.11 0.17 0.35 0.22 0.002
0.005 0.27 0.88 0.68 <0.001
0.70 0.03 0.05 0.05 0.006
0.31 0.46 0.23 0.01 0.40
0.17 0.68 0.15 0.01 0.24
0.02 0.08 0.09 0.003 0.01
0.15 0.41 0.68 0.54 0.01
0.001 0.99 0.27 0.46 <0.001
0.03 0.05 0.18 0.16 0.001
0.35 0.63 0.02 0.14 0.79
0.34 0.64 0.02 0.11 0.75
0.27 0.65 0.07 0.22 0.73
0.04 0.04 0.02 0.06 0.02
0.43 0.50 0.80 0.97 0.14
0.24 0.64 0.11 0.42 0.07
0.60 0.98 0.23 0.38 0.71
0.34 0.51 0.15 0.02 0.45
0.33 0.51 0.17 0.02 0.43
0.28 0.50 0.23 0.07 0.46
0.04 0.10 0.11 0.03 0.03
0.89 0.78 0.75 0.69 0.27
0.32 0.90 0.62 0.21 0.87
0.70 1.00 0.89 0.45 0.54
Abbreviations: 1X, treatment 1; 2X, treatment 2; 3X, treatment 3; ADF, acid detergent fiber expressed inclusive of residual ash; aNDF, neutral detergent fiber assayed with a heat stable amylase and expressed inclusive of residual ash; CON, control; CP, crude protein; DM, dry matter; ED, effective degradability; FUN, fungicide application; kd , fractional digestion rate; SEM, standard error of mean. a Treatment: control (CON), corn received no foliar fungicide application; 1X, corn received one application of pyraclostrobin foliar fungicide (PYR; Headline; BASF Corp.) at vegetative stage 5 (V5; when 5 visible leaf collars can be seen; Mueller and Pope, 2009); 2X, corn received 2 applications of foliar fungicides, PYR at stage V5, and a mixture of pyraclostrobin and metconazole (PYR + MET; Headline AMP; BASF Corp.) at reproductive stage 1 (R1; when silks are visible outside the husks; Mueller and Pope, 2009); and 3X, corn received 3 applications of foliar fungicides, PYR at stage V5, PYR + MET at stage R1, and PYR + MET at reproductive stage 3 (when kernel is yellow outside, whereas the inner fluid is now milky white due to accumulating starch; Mueller and Pope, 2009). b Greatest standard error of mean (SEM). c Contrasts statement for: CON vs. FUN = no fungicide application (CON) versus the average of the three treatments (1X, 2X, and 3X) with fungicide application; linear = linear treatment effect; and quadratic = quadratic treatment effect. d Effective degradability (ED) = A + B × [kd /(kd + kp )]. Rate of passage from the rumen (kp ) assumed to be 0.06.
treated with foliar fungicide. As the number of fungicide applications increased, DM disappearance decreased (P = 0.01). However, there was no interaction between treatment and bag type (P = 0.17) for total DM disappearance (Table 3). Degradability of aNDF was greater in the 10 × 20 cm bags when compared with 20 × 40 cm bags (P = 0.03). No differences (P = 0.15) were observed among CON and corn silages treated with foliar fungicide for aNDF degradability. However, there was a linear treatment effect (P = 0.01) for aNDF degradability with the amount of aNDF after 48 h being decreased as the amount of foliar fungicide application increased. Degradability of aNDF had no interaction between treatment and bag type (P = 0.60) for aNDF disappearance (Table 3). Degradability of ADF was lower (P = 0.01) for 20 × 40 cm bags by 48 h than 10 × 20 cm bags. Lower portion (P = 0.02) of ADF remained in corn silages treated with fungicide after 48 h when compared with CON. There was a linear treatment effect (P = 0.001) for ADF degradability with the amount of ADF after 48 h being decreased as the amount of foliar fungicide application increased. Finally, in regards to ADF, there was no interaction between treatment and type of bag type (P = 0.96) for ADF disappearance (Table 3). Degradability of starch was higher (P < 0.001) in 20 × 40 cm bags when compared with 10 × 20 cm bags. Amount of feed degraded was the same for all treatments (P = 0.32). There was also no interaction between treatment and bag type (P = 0.36) for starch disappearance (Table 3). The CP degradability was greater (P < 0.001) for feed in the 20 × 40 cm bags when compared
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
155
Table 3 Least squares means and associated standard errors for proportion of corn silages disappearance of DM, aNDF, ADF, starch, and CP after 48 h of in situ incubation in 10 × 20 or 20 × 40 cm Dacron bags. Treatmenta CON
DM aNDF ADF Starch CP
1X
SEMb 2X
3X
10 × 20
20 × 40
10 × 20
20 × 40
10 × 20
20 × 40
10 × 20
20 × 40
0.69 0.69 0.66 0.84 0.65
0.72 0.62 0.60 1.00 0.76
0.65 0.68 0.63 0.73 0.62
0.72 0.60 0.56 1.00 0.81
0.69 0.71 0.66 0.79 0.69
0.70 0.58 0.56 1.00 0.81
0.54 0.53 0.48 0.84 0.52
0.67 0.52 0.41 1.00 0.80
0.03 0.05 0.04 0.03 0.06
Pc Bag type
CON vs. FUN
Linear
Quadratic
0.01 0.03 0.01 <0.001 <0.001
0.08 0.15 0.02 0.32 0.98
0.01 0.01 0.001 0.86 0.54
0.14 0.09 0.03 0.11 0.23
Abbreviations: 1X, treatment 1; 2X, treatment 2; 3X, treatment 3; ADF, acid detergent fiber expressed inclusive of residual ash; aNDF, neutral detergent fiber assayed with a heat stable amylase and expressed inclusive of residual ash; CON, control; CP, crude protein; DM, dry matter; FUN, fungicide application; SEM, standard error of mean. a Treatment: control (CON), corn received no foliar fungicide application; 1X, corn received one application of pyraclostrobin foliar fungicide (PYR; Headline; BASF Corp.) at vegetative stage 5 (V5; when 5 visible leaf collars can be seen; Mueller and Pope, 2009); 2X, corn received 2 applications of foliar fungicides, PYR at stage V5, and a mixture of pyraclostrobin and metconazole (PYR + MET; Headline AMP; BASF Corp.) at reproductive stage 1 (R1; when silks are visible outside the husks; Mueller and Pope, 2009); and 3X, corn received 3 applications of foliar fungicides, PYR at stage V5, PYR + MET at stage R1, and PYR + MET at reproductive stage 3 (when kernel is yellow outside, whereas the inner fluid is now milky white due to accumulating starch; Mueller and Pope, 2009). b Greatest standard error of mean (SEM). c Bag type: 10 × 20 or 20 × 40 cm Dacron bags; contrasts statement for: CON vs. FUN = no fungicide application (CON) versus the average of the three treatments (1X, 2X, and 3X) with fungicide application; linear = linear treatment effect; and quadratic = quadratic treatment effect. All treatment × bag type interactions were not significant (P > 0.17).
with feed in the 10 × 20 cm bags. However, there was no difference (P = 0.98) among treatments for degradability of feed. Interaction of treatment and bag type was not significant (P = 0.43) for CP disappearance (Table 3). 4. Discussion The major purpose of this study was to evaluate in situ digestibility of corn plant treated at various times of application with foliar fungicide on corn harvested as whole plant corn silage and fed to lactating dairy cattle. We hypothesized that corn plant treated at various times of application with foliar fungicide would have increased degradability as treated corn plants presented decreased contents aNDF and ADF along with increased contents of sugar and starch (Haerr et al., 2015). According to the product label, the minimum time from application of PYR and PYR + MET to harvest of corn for silage is 7 days. The time from first, second, and third application to harvest was 82, 63, and 45 days which exceeded long the minimum time determined by the manufacturer. 4.1. Effects of foliar fungicide In the present study, the degradable fraction of DM tended to be greater for corn silage treated with foliar fungicide with the proportion of rumen degradable feed increasing as the number of foliar fungicide applications increased. These differences may be associated with the chemical composition of corn silages (Haerr et al., 2015). Starch and sugar contents were numerically higher in corn silages treated with foliar fungicide than CON, whereas aNDF and ADF were numerically higher in CON compared with corn silages treated with foliar fungicide. Yates et al. (1997) proposed that when a corn plant had a fungal infestation of the root, the structural components and rigidity increased, which the authors attributed to the plant attempting to decrease further infestation into the upper portion of the plant by increasing the structurally rigid components of the plant such as lignin. This may also have impacted the amount of aNDF, or the structural composition of the aNDF, and potentially increased the lignification of the aNDF making it less degradable in the present study (Buxton and Redfearn, 1997). Williams et al. (1992) found that severely damaged crops exhibited a higher crude fiber content when compared with non-infected plants. The authors also found that gross energy between infected and non-infected plants did not differ, but found that there was more protein and less fat in mold damaged corn kernels. The difference in nutrient composition was due to the fungi using readily accessible carbohydrates as nutrient source first, and the authors hypothesized that this may affect digestibility of the plant. Similar mechanisms may have occurred in the present study because there was a higher proportion of degradable aNDF in the silage treated with fungicide. The ED of DM, aNDF, and ADF was greater in CON compared to corn silages treated with foliar fungicides. These differences were mainly attributed to the lower ED of treatment 3X compared to 1X and 2X which in turn appeared to have numerically similar ED to the CON treatment. Decreased ED in 3X may be associated with lower soluble fraction compared to 1X, 2X, or CON treatment. There were no differences among corn silages in the degradability of starch or CP, which could be due to the fact that there were no visible signs of infection on the corn ears even from CON, and so corn ears were not damaged. Infection by Fusarium moniliforme as a visible ear rot was shown to decrease the fat content, but increased CP content. This is thought to be due
156
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
to the presence of fungal nitrogen (Williams et al., 1992). When steers were fed increasing amounts of steam-flaked corn infected with fungi, total tract digestibility and ruminal digestibility for organic matter and N as well as overall digestible energy decreased as inclusion of steam-flaked corn infected with fungi increased (Alvarez et al., 2011). Even when plants are not compromised with fungal infections, studies have found that application of pyraclostrobin has increased corn yield. A meta-analysis that looked at data from 172 studies from 2002 to 2009 in 14 different states found a mean increase in yield of corn grain of 255 kg/ha (Paul et al., 2011). This is thought to be due to the fact that pyraclostrobins are from the strobilurin class of fungicide, which work as quinone outside inhibitors. Quinone blocks electron transfer in the cytochrome III (bc1) complex, disrupting the electron transport chain and disrupting ATP production (Bartlett et al., 2002). This can also lead to increased N assimilation in the plant, through increased nitrate reductase that can be especially beneficial when the plant has high N requirements (Venancio et al., 2009). However, in our study we did not see any differences in availability or degradability of CP. There also is some evidence that pyraclostrobin may help to increase water use during drought stress due to increased abcissic acid concentration. Pyraclostrobin works to decrease oxidative stress, due to increased activity of peroxidases that persist weeks after treatment with the fungicide (Venancio et al., 2009). In the present study, the soluble portion of the feed had a linear effect for DM, aNDF, and ADF, with the portion of soluble feed components decreasing as the number of fungicide applications increased. This could have happened due to the difference in corn silage particle size but our evaluations did not show any difference between particle length and physical characteristics of the feed. To further investigate this difference it would have been beneficial to complete a more extensive evaluation of particle size of the feed before ensiling and digestion. It is also important to note that because the samples were not ground before placement in the rumen, degradability may have been underestimated due to the lack of effects of mastication or rumination. However, grinding the samples may lead to an over estimation of true digestibility (Vanzant et al., 1998). Nonetheless, the relatively small sizes of the standard errors calculated in the present study seem to show that a representative sample was obtained in each bag. 4.2. Effects of bag size The DM disappearance was greater for the 20 × 40 cm bags when compared with the 10 × 20 cm bag, which is similar to the results obtained by Varga and Hoover (1983) where two different sample sizes (2.5 and 5 g) were put into two different size bags (13 × 21 and 9 × 17 cm bags). The authors analyzed both concentrates and forages (ground at 4 mm) using a sample size to surface area of 9.2 and 14.6 mg/cm2 , respectively. The authors did not analyze NDF in those samples because the amount remaining at 48 h to complete the analyses was not adequate for all sample sizes. In addition, there was no discussion as to why these results were found (Varga and Hoover, 1983). One possible hypothesis would be that because the 10 × 20 cm bags were subjected to some minimal exposure to air when other bags were being removed at the specified time points feed digestion may have been affected, whereas 20 × 40 cm bags were only exposed to air upon removal. However, studies have shown that complete removal of feed, sampling, and reintroduction of the same feed to the rumen did not affect the digestibility, or rate of digestion (Reid, 1965). Bags were treated the same, therefore, it is not known why degradability differed between 10 × 20 vs. 20 × 40 cm bags. It was also noted that fiber degradability was greater in the 10 × 20 cm bags; however, CP and starch degradability were greater in the 20 × 40 cm bags, so microbial access could differ in the size of the bags. Because there have been limited studies utilizing a bag larger than 10 × 20 cm, we cannot explain why these differed and further research in this area is needed. The 48 h degradability of ADF was higher for CON when compared with silage treated with the fungicide. This could have happened due to a slightly higher kd in the fungicide-treated corn silages when compared with CON, and a tendency for a linear treatment effect for decreasing kd as the number of fungicide applications increased was found in the 10 × 20 cm bags alone. However, this inference to the larger bag should be interpreted cautiously as the degradability of feedstuff differed between 10 × 20 and 20 × 40 cm bags as stated above. There was also less ADF disappearing at 48 h with a linear treatment effect for increasing amount remaining as number of fungicide applications increased. We did not see any differences in degradability when the 10 × 20 cm bags were analyzed alone, but the difference became apparent when 20 × 40 cm bags were analyzed. It may be beneficial to do more replications of the larger bags and 10 × 20 cm bags at the 48 h time point as well as a longer time point to understand which portions of the feed may be indigestible. As well, it may be beneficial to compare ground and unground samples between the two bag sizes to better understand the differences found in this study. 5. Conclusion Fungicide application to corn resulted in higher DM degradable fraction which increased with the number of fungicide applications. It tended to decrease kd and linearly decrease DM solubility. There were no differences on the soluble, degradable, and indigestible fractions or kd of aNDF, ADF, starch, and CP among treatments. However, the soluble fraction of aNDF and ADF linearly decreased with fungicide applications. The ED was greater in CON than corn silages treated with foliar fungicide mainly due to decreased ED in 3X compared with 1X and 2X which in turn appeared to be numerically similar to the ED of the CON treatment. In situ digestibility results for big bags (20 × 40 cm) and small bags (10 × 20 cm) were different in the present study. Degradability of DM, aNDF, and ADF was higher, while starch and CP degradability was lower in the larger bags. Further investigation is needed to clarify why this occurred. Fungicide application on corn for silage seems promising
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
157
as changes in the degradable fraction may be the result of increased sugar and starch contents along with decreased aNDF and ADF. Conflicts of interest We acknowledge the affiliation of one of the co-authors with BASF Corporation. However, there are no conflicts of interest, financial or otherwise, including direct or indirect financial or personal relationships, interests, and affiliations, whether or not directly related to the subject of the paper. References AOAC (Association of Official Analytical Chemists), 1995. AOAC Official Method 972.16. Fat, Lactose, Protein, and Solids in Milk. Mid-Infrared Spectroscopic Method. Official Methods of Analysis of the Association of Official Analytical Chemists, vol. 2., 16th ed. AOAC, Arlington, VA, pp. 23–26, 33. Alvarez, E., Barajas, R., Calderon, F., Montano, M., Salinas-Chavira, J., Zinn, R.A., 2011. Effects of fungal infested steam-flaked corn on characteristics of ruminal and total tract digestion in steers. Anim. Feed Sci. Technol. 168, 145–151. Bal, M.A., Shaver, R.D., Shinners, K.J., Coors, J.G., Lauer, J.G., Straub, R.J., Koegel, R.G., 2000. Stage of maturity, processing, and hybrid effects on ruminal in situ disappearance of whole-plant corn silage. Anim. Feed Sci. Technol. 86, 83–94. Bartlett, D.W., Clough, J.M., Godwin, J.R., Hall, A.A., Hamer, M., Parr-Dobrzanski, B., 2002. The strobilurin fungicides. Pest Manage. Sci. 58, 649–662. Buxton, D.R., Redfearn, D.D., 1997. Plant limitations to fiber digestion and utilization. J. Nutr. 127, 814S–818S. Doggett, C.G., Hunt, C.W., Andrae, J.G., Pritchard, G.T., Kezar, W., Harrison, J.H., 1998. Effect of hybrid and processing on digestive characteristics of corn silage. J. Dairy Sci. 81 (E-Suppl.1):196(Abstr.). Fahey, G.C., Collins, M., Mertens, D.R., Moser, L.E., 1994. Plant factors affecting forage quality. Forage Qual. Eval. Util., 115–154. Goering, H.K., Van Soest, P.J., 1970. Forage fiber analyses (apparatus, reagents, procedures, and some applications). USDA Agric. Handb. 379, 387–598. Haerr, K.J., Lopes, N.M., Pereira, M.N., Fellows, G.M., Cardoso, F.C., 2015. Corn silage from corn treated with foliar fungicide and performance of Holstein cows. J. Dairy Sci. 98, 8962–8972. Hall, M.B., Mertens, D.R., 2008. Technical note: effect of sample processing procedures on measurement of starch in corn silage and corn grain. J. Dairy Sci. 91, 4830–4833. Köhle, H., Grossmann, K., Jabs, T., Gerhard, M., Kaiser, W., Glaab, J., Conrath, U., Seehaus, K., Herms, S., 2002. Physiological Effects of the Strobilurin Fungicide F 500 on Plants. Modern Fungicides and Antifungal Compounds III. Mann GmbH & Co. KG, Bonn, Germany, pp. 61–74. Kononoff, P.J., Heinrichs, A.J., Buckmaster, D.R., 2003. Modification of the penn state forage and total mixed ration particle separator and the effects of moisture content on its measurements. J. Dairy Sci. 86, 1858–1863. Krizsan, S.J., Ahvenjarvi, S., Huhtanen, P., 2010. A meta-analysis of passage rate estimated by rumen evacuation with cattle and evaluation of passage rate prediction models. J. Dairy Sci. 93, 5890–5901. Littell, R.C., Henry, P.R., Ammerman, C.B., 1998. Statistical analysis of repeated measures data using SAS procedures. J. Anim. Sci. 76, 1216–1231. McDonald, I., 1981. A revised model for the estimation of protein degradability in the rumen. J. Agric. Sci. 96, 251–252. Mueller, D., Pope, R., 2009. Corn Field Guide. Iowa State University, Extension and Outreach, Ames, IA. NRC (National Research Council). 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Sci., Washington, DC. Nocek, J.E., 1988. In situ and other methods to estimate ruminal protein and energy digestibility: a review. J. Dairy Sci. 71, 2051–2069. Ørskov, E.R., McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. 92, 499–503. Paul, P.A., Madden, L.V., Bradley, C.A., Robertson, A.E., Munkvold, G.P., Shaner, G., Wise, K.A., Malvick, D.K., Allen, T.W., Grybauskas, A., Vincelli, P., Esker, P., 2011. Meta-analysis of yield response of hybrid field corn to foliar fungicides in the U.S. Corn Belt. Phytopathology 101, 1122–1132. Reid, C., 1965. Quantitative studies of digestion in the reticulo-rumen I. Total removal and return of digesta for quantitative sampling in studies of digestion in the reticulo-rumen of cattle. Proc. N.Z. Soc. Anim. Prod., 65. Rivero, R.M., Ruiz, J.M., Garcı´ıa, P.C., López-Lefebre, L.R., Sánchez, E., Romero, L., 2001. Resistance to cold and heat stress: accumulation of phenolic compounds in tomato and watermelon plants. Plant Sci. 160, 315–321. SAS Institute Inc, 2013. Base SAS® 9.4 Procedures Guide: Statistical Procedures, second edition. SAS Institute Inc., Cary, NC. Schalla, A., Meyer, L., Meyer, Z., Onetti, S., Schultz, A., Goeser, J., 2012. Hot topic: apparent total-tract nutrient digestibilities measured commercially using 120-hour in vitro indigestible neutral detergent fiber as a marker are related to commercial dairy cattle performance. J. Dairy Sci. 95, 5109–5114. Van Milgen, J., Murphy, M.R., Berger, L.L., 1991. A compartmental model to analyze ruminal digestion. J. Dairy Sci. 74, 2515–2529. Vanzant, E.S., Cochran, R.C., Titgemeyer, E.C., 1998. Standardization of in situ techniques for ruminant feedstuff evaluation. J. Anim. Sci. 76, 2717–2729. Varga, G.A., Hoover, W.H., 1983. Rate and extent of neutral detergent fiber degradation of feedstuffs in situ. J. Dairy Sci. 66, 2109–2115. Venancio, W.S., Rodrigues, M.A.T., Begliomini, E., De Souza, N.L., 2009. Physiological effects of strobilurin fungicides on plants. Ci. Exatas e da Terra Ci Agr. Eng. 9, 59–68. Williams, K., Blaney, B., Dodman, R., Palmer, C., 1992. Assessment for animal feed of maize kernels naturally-infected predominantly with Fusarium moniliforme and Diplodia maydis. I. Fungal isolations and changes in chemical composition. Aust J. Agric. Res. 43, 773–782. Wise, K., Mueller, D., 2011. Are fungicides no longer just for fungi? An analysis of foliar fungicide use in corn. APSnet Featur., http://dx.doi.org/10.1094/apsnetfeature-2011-0531. Yates, I.E., Bacon, C.W., Hinton, D.M., 1997. Effects of endophytic infection by Fusarium moniliforme on corn growth and cellular morphology. Plant Dis. 81, 723–728.
Contents lists available at ScienceDirect
Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci
Research Paper
Effects of corn treated with foliar fungicide on in situ corn silage degradability in Holstein cows K.J. Haerr a , A. Pineda a , N.M. Lopes a,b , J.D. Weems c , C.A. Bradley c , M.N. Pereira b , M.R. Murphy a , G.M. Fellows d , F.C. Cardoso a,∗ a b c d
Department of Animal Sciences, University of Illinois, Urbana, IL 61801, USA Departamento de Zootecnia, Universidade Federal de Lavras, Lavras, MG 37200-000, Brazil Department of Crop Sciences, University of Illinois, Urbana, IL 61801, USA B.A.S.F. Corporation, Research Triangle Park, NC 27709, USA
a r t i c l e
i n f o
Article history: Received 23 February 2016 Received in revised form 5 October 2016 Accepted 18 October 2016 Keywords: In situ Digestibility Corn silage Fungicide
a b s t r a c t With increasing feed prices and decreasing profit margins livestock producers are constantly searching for ways to increase nutritive value of the feed in order to get more production per unit of feedstuff. The objective of this study was to assess the digestibility of corn silage made of corn plants treated with various foliar fungicide applications. Treatments were: control (CON), corn received no foliar fungicide application; 1X, corn received one application of pyraclostrobin foliar fungicide (PYR; Headline; BASF Corp.) at vegetative stage 5 (V5); 2X, corn received 2 applications of foliar fungicides, PYR at stage V5, and a mixture of pyraclostrobin and metconazole (PYR + MET; Headline AMP; BASF Corp.) at reproductive stage 1 (R1); and 3X, corn received 3 applications of foliar fungicides, PYR at stage V5, PYR + MET at stage R1, and PYR + MET at reproductive stage 3. Corn was harvested at the same time and ensiled for 7 months. Dried unground corn silage was put into 288 (3 per time points/treatment/cow) 10 × 20 cm bags and incubated for 8 different time points (0, 2, 4, 8, 12, 48, 72, and 96 h). A sample of unground dried corn silage was also placed into 20 × 40 cm bag and incubated for 48 h. Digestibility of corn silages was estimated using in situ procedure with 3 rumen-cannulated lactating multiparous Holstein cows. The degradable fraction of dry matter (DM) tended to be greater (P = 0.08) for corn silages treated with fungicide when compared with CON. There was no treatment effect (P > 0.05) on neutral detergent fiber (aNDF), acid detergent fiber (ADF), starch, and crude protein (CP). However, the soluble fraction of DM, aNDF, and ADF decreased (P < 0.05) as fungicide applications increased. Effective degradability (ED) was greater (P < 0.05) in CON than corn silages treated with fungicide mainly due to decreased ED in 3X compared with 1X and 2X. In situ digestibility for bigger and smaller bags was different. Degradability of DM, aNDF, and ADF was higher (P < 0.05), while starch and CP degradability was lower (P < 0.01) in the larger bags. Fungicide application to corn for silage lead to higher DM degradable fraction which seems to be the result of increased sugar and starch along with decreased aNDF and ADF. © 2016 Published by Elsevier B.V.
Abbreviations: 1X, treatment 1; 2X, treatment 2; 3X, treatment 3; ADF, acid detergent fiber expressed inclusive of residual ash; aNDF, neutral detergent fiber assayed with a heat stable amylase and expressed inclusive of residual ash; ANOVA, analysis of variance; ATP, adenosine triphosphate; CON, control; CP, crude protein; DM, dry matter; ED, effective digestibility, kd, fractional digestion rate; MET, metconazole; NDF, neutral detergent fiber; NRC, National Research Council; PYR, pyraclostrobin; R1, reproductive stage 1; V5, vegetative stage 5. ∗ Corresponding author at: 290 Animal Sciences Laboratory, Department of Animal Sciences, University of Illinois, 1204 West Gregory Drive, Urbana, IL 61801, USA. E-mail address: [email protected] (F.C. Cardoso). http://dx.doi.org/10.1016/j.anifeedsci.2016.10.010 0377-8401/© 2016 Published by Elsevier B.V.
150
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
1. Introduction As prices of feed increase, particularly corn, and nutritional demands of milk production increase, it is important to evaluate potential ways of increasing feed efficiency in the dairy cow leading to increased profitability for dairy farmers. During the course of a growing season corn plants can be exposed to many environmental stressors including heat, cold, drought, and pathogens (Rivero et al., 2001). Plant stress may lead to decreased quality of the plant when used for animal feed, to decreased potential for digestibility, and altered digestibility characteristics (Fahey et al., 1994). Fungal colonization on the corn plant causes a competition between the plant and the fungus for nutrients. The plant has many mechanisms (e.g., lignification and leaf shedding) to attempt to hinder the growth of the fungal infestation. These mechanisms may potentially decrease the digestibility of the plant. The fungal infestation itself may also change the chemical composition of the plant in the process of competing for nutrients (Venancio et al., 2009). Research also has examined the different effects of the foliar fungicide on the plant at a chemical level (Köhle et al., 2002). The class of foliar fungicides known as strobulins have been studied for their possible increase in the greening effect of plants as well as their ability to handle stressors (Venancio et al., 2009). The aforementioned physiological changes elicited by the foliar fungicide have been shown to increase yield in plants that are not infected with disease, which may cause a possible increase in digestibility and nutritive content of the plant when used as feed for animals (Wise and Mueller, 2011). This increase in quality can be due to decreased lignification, increased grain fill and starch content, and increased nitrate assimilation and consequently increased protein content (Yates et al., 1997; Venancio et al., 2009). Digestion techniques are often used in evaluations of the nutritive value of corn silage (Nocek, 1988). The in vitro technique involves drying and fine grinding of whole plant corn silage for analysis. Grinding may minimize quality differences among whole plant corn silage samples related to physical form such as grain hardness and particle size (Bal et al., 2000). A macro in situ technique using undried, unground whole plant corn silage has been used to evaluate ruminal nutrient disappearance (Doggett et al., 1998). This procedure may provide better estimates of differences in ruminal starch disappearance as it is more influenced by maturity and hybrid than standard in vitro procedures. However, because of the occurrence of particle size reduction during eating and rumination it may tend to overestimate the magnitude of mechanical processing effects on ruminal starch disappearance and likely underestimates ruminal neutral detergent fiber (NDF) disappearance (Bal et al., 2000). In situ studies have been used in ruminant animals for many decades to estimate the potential digestibility of feedstuffs as well as to attempt to understand the complex interactions of the rumen ecology on feedstuffs. Much research has been conducted to create a standard procedure for rumen in situ techniques, which will not only allow research to be sufficiently compared across laboratories but provides the best estimate of what actually happens in the rumen and is most biologically relevant (Vanzant et al., 1998). It is well known that sample size to bag surface area ratio is very important to obtain correct results. From various studies it has been agreed that an appropriate sample size to surface area ratio is 10–30 mg/cm2 (Varga and Hoover, 1983; Vanzant et al., 1998). The objectives of this study were: (1) to evaluate in situ degradability of corn plant treated at various times with foliar fungicide on corn harvested as whole plant silage for lactating Holstein cows and (2) to determine if there were differences in 48 h in situ degradability between samples ruminally incubated in 10 × 20 cm bags when compared with samples in 20 × 40 cm bags. 2. Materials and methods 2.1. Treatments Corn was grown for silage on fields owned by the University of Illinois (Urbana, IL, USA) located at 40.08 latitude, and − 88.22 longitude. The four silages evaluated in this experiment were: control (CON), corn received no foliar fungicide application; treatment 1 (1X), in which corn received 1 application of pyraclostrobin (PYR) foliar fungicide (Headline; BASF Corp., Florham Park, New Jersey, USA) at a rate of 0.11 kg of active ingredient (a.i.)/ha at corn vegetative stage 5 (V5; when 5 visible leaf collars can be seen; Mueller and Pope, 2009); treatment 2 (2X), in which corn received 2 applications of foliar fungicides, PYR at 0.11 kg of a.i./ha at corn stage V5, and a mixture of PYR + metconazole (MET; Headline AMP; BASF Corp., Florham Park, New Jersey, USA) at 0.11 + 0.04 kg of a.i./ha at corn reproductive stage 1 (R1; when silks are visible outside the husks; Mueller and Pope, 2009); and treatment 3 (3X), in which corn received 3 applications of foliar fungicide, PYR at 0.11 kg of a.i./ha at corn stage V5, PYR + MET at 0.11 + 0.04 kg of a.i./ha at corn stage R1, and PYR + MET at 0.11 + 0.04 kg of a.i./ha at corn reproductive stage 3 (when kernel is yellow outside, whereas the inner fluid is now milky white due to accumulating starch; Mueller and Pope, 2009). The dates for fungicide application for first, second, and third applications were: July 7, 2013; July 26, 2013; and August 13, 2013. The corn hybrid planted was ‘LG2636 VT3P RIB’ (LG Seeds; Elmhurst, IL, USA), which is a dual purpose hybrid used for either grain or silage. The hybrid is advertised as 114-day maturity, with high yield potential, strong stalks, and high vigor. This hybrid also is advertised as having a high level of resistance against northern corn leaf blight (caused by the fungus Exserohilum turcicum), southern corn leaf blight (caused by the fungus Bipolaris maydis), and gray leaf spot (caused by the fungus Cercospora zeae-maydis). This hybrid contains transgenic traits that provide protection against corn earworm (Helicoverpa zea). All corn was planted on June 5, 2013 and harvested on September 27, 2013 at a dry matter (DM) of 330, 300, 300, and 325 g/kg for CON, 1X, 2X, and 3X, respectively. Harvester included kernel processing to have the same theoretical length of chop, set at 1.9 cm. Inoculant (Silo-King WS; 1.5 × 105 cfu/g of L. plantarum, P. pentosaceus, and
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
151
Table 1 Mean chemical and nutritive composition (g/kg of DM unless otherwise noted) and standard deviation of nutrient composition, fermentation profile, energy content, and microbial count of corn silage treated without or with applications of foliar fungicide (adapted from Haerr et al., 2015). Item
Treatmenta
SDb
CON
1X
2X
3X
Corn silage composition DM CP aNDF aNDF digestibility, 30 h ADF Fat Lignin (sa) Soluble CP, g/kg of CP Starch Sugar Ash Ca P Mg K S
306.30 86.10 473.20 500.90 292.40 27.10 34.50 561.80 276.10 7.20 51.10 2.20 1.90 1.40 13.40 1.00
309.80 87.20 458.80 496.70 277.20 27.70 36.50 519.80 286.70 11.40 49.20 2.20 1.90 1.30 12.70 1.00
344.10 85.00 454.10 490.00 281.10 27.40 33.60 540.50 292.60 12.30 47.90 2.00 1.80 1.30 12.10 0.90
303.70 89.80 452.80 488.20 273.30 31.60 32.20 590.40 287.00 12.50 47.60 2.20 1.90 1.40 12.40 1.00
16.80 8.10 38.90 62.00 23.40 4.80 11.30 41.70 33.00 2.70 3.90 0.10 0.10 0.10 0.90 0.10
Energy calculationsc TDN NEL , MJ/kg DM NEG , MJ/kg DM NEM , MJ/kg DM
655.50 5.98 4.06 6.61
659.20 5.98 4.14 6.61
665.40 6.07 4.23 6.74
673.70 6.19 4.35 6.90
22.00 0.08 0.17 0.17
Fermentation products pH Lactic acid Acetic acid Ethanol Ammonia N
4.88 49.10 29.70 2.00 0.80
4.99 42.50 27.80 2.10 0.70
4.99 46.30 13.10 1.60 0.60
4.84 46.50 34.70 3.20 0.90
0.30 20.50 9.40 1.70 0.20
Microbial count Yeast, cfu/g Mold, cfu/g
2,663 92,800
1,741 14,300
50,595 51,541
5,153 72,300
94,001 30,750
Particle size distributiond 19 mm 7.8 mm 1.2 mm Pan Mean particle size,e m
87 689 215 21 9134
102 678 218 21 9186
83 672 239 57 8852
96 680 220 36 9401
67 59 67 11 0.36
Abbreviations: 1Xtreatment 1; 2Xtreatment 2; 3Xtreatment 3; ADFacid detergent fiber expressed inclusive of residual ash; aNDFneutral detergent fiber assayed with a heat stable amylase and expressed inclusive of residual ash; Cacalcium; CONcontrol; CPcrude protein; DMdry matter; Kpotassium; Lignin (sa)lignin determined by solubilization of cellulose with sulphuric acid; Mgmagnesium; NEG net energy of growth; NEL net energy of lactation; NEM net energy of maintenance; Pphosphorus; Ssulfur; SDstandard deviation. a Treatment: control (CON), corn received no foliar fungicide application; 1X, corn received one application of pyraclostrobin foliar fungicide (PYR; Headline; BASF Corp.) at vegetative stage 5 (V5; when 5 visible leaf collars can be seen; Mueller and Pope, 2009); 2X, corn received 2 applications of foliar fungicides, PYR at stage V5, and a mixture of pyraclostrobin and metconazole (PYR + MET; Headline AMP; BASF Corp.) at reproductive stage 1 (R1; when silks are visible outside the husks; Mueller and Pope, 2009); and 3X, corn received 3 applications of foliar fungicides, PYR at stage V5, PYR + MET at stage R1, and PYR + MET at reproductive stage 3 (when kernel is yellow outside, whereas the inner fluid is now milky white due to accumulating starch; Mueller and Pope, 2009). b Maximum within treatment SD. c NRC (2001). d Penn State Particle Size Separator (Pennsylvania State University, Pennsylvania, USA) was used for measurements. e Log10 SD.
Enterococcus faecium; Agri-King, Fulton, IL, USA) was applied at a rate of 115 g of inoculant per 1000 kg of corn and then ensiled in silo bags for more than 200 days. Particle size of the corn silages was measured on a weekly basis (3 replicates twice weekly) for 10 weeks using the Penn State Particle Separator [3-sieve model (19, 7.8, and 1.2 mm), Pennsylvania State University, Pennsylvania, USA) as described by Kononoff et al. (2003). Data from the Penn State Particle Separator were then fit to a lognormal distribution to estimate the geometric mean particle size and the log10 standard deviation. Corn silages nutrient profiles are described in Table 1 adapted from Haerr et al. (2015).
152
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
2.2. Animals and housing All experimental procedures were approved by the University of Illinois (Urbana-Champaign, IL, USA) Institutional Animal Care and Use Committee. Three second-lactation lactating Holstein cows (375 ± 21 days in milk; milk yield: 19.7 ± 8 kg/day) fitted with a rumen cannula were used for the experiment. Cows were housed in a tie-stall barn with individual feed access and fed diet to meet the NRC (2001) requirements with ad libitum access to feed. The diet was composed of 500 g/kg concentrate and 500 g/kg forage. The concentrate was a mixture of dry ground corn, soybean meal, soy hulls, blood meal, minerals, and vitamins. The forage consisted of 720 g/kg of corn silage and the rest (280 g/kg) made of alfalfa hay, alfalfa silage, and cottonseed. Cows were fed once daily at 1400 h, and were milked three times daily at 0700, 1400, and 2200 h. Refusals were removed and weighed before new feed was offered. 2.3. Sampling and bag preparation Corn silage was removed from 6 locations along the face of the bunker silo in order to retrieve a representative sample. Once the sample (4 kg per treatment) was obtained, it was dried and then placed on a sheet. The sample was then divided into 24 equal sections to decrease sample error and increase uniformity of the feedstuff placed into each bag. Two sizes of Dacron bags (Ankom Technology, Macedon, NY, USA) were utilized for this study, one 10 × 20 cm and the other 20 × 40 cm, both had 50 m pores. The procedure used was similar to the one described by Vanzant et al. (1998). Briefly, bags of both sizes were labeled, dried, and weighed prior to the addition of the corn silage. Each bag was heat sealed at least twice to ensure no feed particles escaped. Bags were filled to achieve a 20 mg DM/cm2 of bag surface. Small bags were filled with 8 g of DM and large bags were filled with 32 g of DM. For all bags, the sample was unground and particle length was not altered before it was put into the bags. Three replicates were made for each small bag at each time point for a total of 24 bags per treatment per cow. Bags were placed into large mesh garment bags to prevent the loss of bags in the gastrointestinal tract. All bags were soaked in warm (45 ◦ C) water before they were placed into the ventral rumen at the same time. Bags were then removed at the appropriate time point, noting the identification of each bag at the time of removal. Small bags were removed at 0, 2, 4, 8, 12, 48, 72, and 96 h. The same procedure was followed for the large (20 × 40 cm) bags, except that they were removed from the rumen only at 0 and 48 h (3 per time point/treatment/cow). Care was taken to minimize air exposure that could interfere with proper fermentation, and remaining bags were placed back into the ventral rumen. Bags that were removed from the rumen were immediately placed in ice water to stop fermentation. Bags were hand rinsed thoroughly in cold running water until the rinsing water was clear, then bags were immediately frozen (−20 ◦ C) for at least 24 h. After freezing, the bags were thawed and rinsed on a rinse cycle of a washing machine (Roper RTW4641BQ1, Whirlpool Corp., Benton Harbor, MI, USA) 2 times to reduce microbial content. Bags were then oven dried for 24 h at 110 ◦ C and disappearance was calculated. In the instance of torn bags or compromised seals post digestion the data were considered missing (n = 14). 2.4. Chemical analysis The three replicates of each time point from each treatment were combined to make a composite sample per cow that was sent to a commercial laboratory (Rock River Lab, Watertown, WI, USA) for analysis via wet chemistry methods (Schalla et al., 2012). The samples (n = 24 per treatment) were analyzed for neutral detergent fiber (aNDF), acid detergent fiber (ADF), crude protein (CP), starch, and DM contents. The aNDF was analyzed using sulfite and alpha amylase along with a premixed neutral detergent solution (Goering and Van Soest, 1970), and expressed inclusive of residual ash. The ADF was analyzed using the Ankom200 Fiber analyzer (Ankom Technology, Fairport, NY, USA) and expressed inclusive of residual ash. The CP content of the samples was measured using the combustion method (976.06) to determine N content and then multiplying the N content by 6.25 (AOAC, 1995). Starch was measured using alpha amylase, amyloglucosidase, and sodium acetate buffer by the procedure described by Hall and Mertens (2008). 2.5. Statistical analysis Statistical analysis was performed using SAS (v9.4 Institute Inc., Cary, NC, USA). The data were analyzed in two sequential steps. First, a nonlinear model of in situ digestion was analyzed using the NLIN procedure, based on the partitioning of feed such that the fractions of soluble feed (A; washout fraction after rinsing at time point 0), rumen degradable feed (B), and indigestible feed (C) is summed to 1. The disappearance data from the small bags were first fitted to the full model: Y = B (exp−Vkd (t−t1 ) ) + C where B = the proportion of potentially degradable feed, C = the proportion of indigestible feed, t = time point, Y = the proportion of feed remaining at a specified time point (t), kd = the fractional digestion rate constant, and V = 1 when t ≥ lag, and V = 0 when t < lag (McDonald, 1981; Van Milgen et al., 1991). The parameter of lag was restricted so that lag ≥ 0. If the full model failed to converge or lag = 0 (Ørskov and McDonald, 1979) then the reduced model was utilized: Y = B (exp−kd (t) ) + C
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
153
The A (soluble) fraction was then calculated as 1 − (B + C). The effective degradability was estimated as ED = A + B × [kd/(kd + kp)] (McDonald, 1981). Although the rate of passage from the rumen (kp) is affected by many factors [feeding intake, diet composition, feed particle size, and moisture in feeds (Krizsan et al., 2010)], a value of 0.06 was assumed to estimate ED in the present experiment. An ANOVA was then conducted on estimates of soluble feed, rumen degradable feed, indigestible feed, kd , and ED. Secondly, a linear mixed model (MIXED procedure) was created to explore associations among parameters calculated and bag size. Treatment and replicate were treated as fixed effects and cow was considered a random effect. Bag type and treatment as well as the interaction between the two were treated as fixed effects. Three predetermined orthogonal contrasts were used for both models. One contrast compared corn silage with fungicide application, and the other two contrasts examined linear and quadratic effects of fungicide applications. These contrasts were used for all variables analyzed unless otherwise stated. The degrees of freedom method used was Kenward-Rogers (Littell et al., 1998). Residual distribution was evaluated for normality and homoscedasticity using the UNIVARIATE procedure. Statistical significance was declared at P ≤ 0.05 and tendencies when 0.05 < P ≤ 0.10. 3. Results 3.1. Physical characteristics The mean particle size of corn silage for each treatment was 9134, 9186, 8852, and 9401 m for CON, 1X, 2X, and 3X, respectively. Results from the analyses of physical characteristics of corn silage are shown in Table 1. Corn silage had similar physical characteristics among treatments. 3.2. Digestibility kinetics There were no differences among treatments for indigested DM (P = 0.19, Table 2). There was a linear treatment effects for the soluble fraction of DM, with the amount decreasing as the number of foliar fungicide applications increased (P = 0.01). Degradable DM tended to be greater for corn silages treated with foliar fungicide when compared with CON (P = 0.08). There were also tendencies for linear (P = 0.06) and quadratic (P = 0.08) treatment effects for rumen degradable DM in which the proportion of rumen degradable feed increased as the number of foliar fungicide applications increased. The fractional rate of degradation of DM was lower (P = 0.003) for corn silages treated with foliar fungicide when compared with CON. Effective degradability of DM decreased (P = 0.04) with foliar fungicide applications when compared with CON. There were linear (P = 0.001) and quadratic (P = 0.03) treatment effects for ED of DM, it decreased as the number of foliar fungicide applications increased. There were no differences between CON and corn silages treated with foliar fungicide for the soluble fraction of aNDF, rumen degradable aNDF, indigestible aNDF, or kd (P > 0.05, Table 2). However, there was a linear treatment effect for the soluble fraction of aNDF with the amount decreasing as the number of applications of foliar fungicide increased (P = 0.005). There was quadratic treatment effect for the rumen degradable aNDF (P = 0.03), indigestible aNDF (P = 0.05), and kd (P = 0.05). The corn silage treated with 1X and 3X applications of fungicide had greater proportion of degradable aNDF than 2X. The opposite occurred for the indigestible fraction and kd , the mid-level treatment of fungicide application presented greater proportion of indigested aNDF along with greater rate of digestion than 1X and 3X. Effective degradability of aNDF decreased (P = 0.002) with foliar fungicide applications when compared with CON. There were linear (P < 0.001) and quadratic (P = 0.006) treatment effects for ED of aNDF, it decreased as the number of foliar fungicide applications increased. There were no differences between CON and corn silages treated with foliar fungicide for the soluble fraction of ADF, rumen degradable ADF, indigestible ADF, or kd (P > 0.05, Table 2). However, there were linear (P = 0.001) and quadratic (P = 0.03) treatment effects for the soluble fraction of ADF with the amount decreasing as the number of applications of foliar fungicide increased. In addition, there was a quadratic treatment effect for the degradable fraction of ADF with the corn silage treated with the 1X and 3X applications of foliar fungicide having greater degradable ADF fraction. Effective degradability of ADF decreased (P = 0.01) with foliar fungicide applications when compared with CON. There were linear (P < 0.001) and quadratic (P = 0.001) treatment effects for ED of ADF, it decreased as the number of foliar fungicide applications increased. There were no differences between CON and corn silages treated with foliar fungicide for the soluble fraction of starch, rumen degradable starch, indigested starch, kd , and ED (P > 0.05, Table 2). There were no linear or quadratic effects of fungicide applications in the different fractions of starch or in its fractional rate of digestion. In addition, there were no differences between CON and corn silages treated with foliar fungicide for the soluble fraction of CP, rumen degradable CP, indigested CP, kd , or ED (P > 0.05, Table 2). 3.3. Comparison of feed digestion in 10 × 20 cm vs. 20 × 40 cm dacron bags There was a larger (P = 0.01) amount of corn silage DM disappearance for feed in the 20 × 40 cm bags when compared with corn silage in the 10 × 20 cm bags. The DM disappearance at 48 h tended (P = 0.08) to be higher for CON than corn silages
154
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
Table 2 Least squares means and associated standard errors of soluble (washout fraction after rinsing at time point 0) proportion, degradable feed proportion, indigestible feed proportion, fractional rate of digestion (kd ), and effective degradability (ED) of corn silage treated without or with applications of foliar fungicide as determined in 10 × 20 cm Dacron bags. Treatmenta
SEMb
CON
1X
2X
3X
DM Soluble Degradable Indigestible kd, h−1 EDd
0.33 0.39 0.28 0.04 0.48
0.39 0.45 0.16 0.02 0.49
0.35 0.42 0.23 0.03 0.47
0.26 0.58 0.16 0.02 0.37
aNDF Soluble Degradable Indigestible kd, h−1 EDd
0.32 0.49 0.19 0.02 0.42
0.33 0.61 0.06 0.01 0.41
0.30 0.47 0.23 0.02 0.40
ADF Soluble Degradable Indigestible kd, h−1 EDd
0.30 0.53 0.17 0.01 0.39
0.31 0.69 0.00 0.01 0.38
Starch Soluble Degradable Indigestible kd, h−1 EDd
0.36 0.61 0.03 0.16 0.80
CP Soluble Degradable Indigestible kd, h−1 EDd
0.31 0.47 0.22 0.02 0.41
Pc CON vs. FUN
Linear
Quadratic
0.25 0.04 0.06 0.004 0.02
0.95 0.08 0.19 0.003 0.04
0.01 0.06 0.94 0.89 0.001
0.39 0.08 0.34 0.13 0.03
0.25 0.71 0.05 0.01 0.32
0.02 0.06 0.06 0.003 0.02
0.11 0.17 0.35 0.22 0.002
0.005 0.27 0.88 0.68 <0.001
0.70 0.03 0.05 0.05 0.006
0.31 0.46 0.23 0.01 0.40
0.17 0.68 0.15 0.01 0.24
0.02 0.08 0.09 0.003 0.01
0.15 0.41 0.68 0.54 0.01
0.001 0.99 0.27 0.46 <0.001
0.03 0.05 0.18 0.16 0.001
0.35 0.63 0.02 0.14 0.79
0.34 0.64 0.02 0.11 0.75
0.27 0.65 0.07 0.22 0.73
0.04 0.04 0.02 0.06 0.02
0.43 0.50 0.80 0.97 0.14
0.24 0.64 0.11 0.42 0.07
0.60 0.98 0.23 0.38 0.71
0.34 0.51 0.15 0.02 0.45
0.33 0.51 0.17 0.02 0.43
0.28 0.50 0.23 0.07 0.46
0.04 0.10 0.11 0.03 0.03
0.89 0.78 0.75 0.69 0.27
0.32 0.90 0.62 0.21 0.87
0.70 1.00 0.89 0.45 0.54
Abbreviations: 1X, treatment 1; 2X, treatment 2; 3X, treatment 3; ADF, acid detergent fiber expressed inclusive of residual ash; aNDF, neutral detergent fiber assayed with a heat stable amylase and expressed inclusive of residual ash; CON, control; CP, crude protein; DM, dry matter; ED, effective degradability; FUN, fungicide application; kd , fractional digestion rate; SEM, standard error of mean. a Treatment: control (CON), corn received no foliar fungicide application; 1X, corn received one application of pyraclostrobin foliar fungicide (PYR; Headline; BASF Corp.) at vegetative stage 5 (V5; when 5 visible leaf collars can be seen; Mueller and Pope, 2009); 2X, corn received 2 applications of foliar fungicides, PYR at stage V5, and a mixture of pyraclostrobin and metconazole (PYR + MET; Headline AMP; BASF Corp.) at reproductive stage 1 (R1; when silks are visible outside the husks; Mueller and Pope, 2009); and 3X, corn received 3 applications of foliar fungicides, PYR at stage V5, PYR + MET at stage R1, and PYR + MET at reproductive stage 3 (when kernel is yellow outside, whereas the inner fluid is now milky white due to accumulating starch; Mueller and Pope, 2009). b Greatest standard error of mean (SEM). c Contrasts statement for: CON vs. FUN = no fungicide application (CON) versus the average of the three treatments (1X, 2X, and 3X) with fungicide application; linear = linear treatment effect; and quadratic = quadratic treatment effect. d Effective degradability (ED) = A + B × [kd /(kd + kp )]. Rate of passage from the rumen (kp ) assumed to be 0.06.
treated with foliar fungicide. As the number of fungicide applications increased, DM disappearance decreased (P = 0.01). However, there was no interaction between treatment and bag type (P = 0.17) for total DM disappearance (Table 3). Degradability of aNDF was greater in the 10 × 20 cm bags when compared with 20 × 40 cm bags (P = 0.03). No differences (P = 0.15) were observed among CON and corn silages treated with foliar fungicide for aNDF degradability. However, there was a linear treatment effect (P = 0.01) for aNDF degradability with the amount of aNDF after 48 h being decreased as the amount of foliar fungicide application increased. Degradability of aNDF had no interaction between treatment and bag type (P = 0.60) for aNDF disappearance (Table 3). Degradability of ADF was lower (P = 0.01) for 20 × 40 cm bags by 48 h than 10 × 20 cm bags. Lower portion (P = 0.02) of ADF remained in corn silages treated with fungicide after 48 h when compared with CON. There was a linear treatment effect (P = 0.001) for ADF degradability with the amount of ADF after 48 h being decreased as the amount of foliar fungicide application increased. Finally, in regards to ADF, there was no interaction between treatment and type of bag type (P = 0.96) for ADF disappearance (Table 3). Degradability of starch was higher (P < 0.001) in 20 × 40 cm bags when compared with 10 × 20 cm bags. Amount of feed degraded was the same for all treatments (P = 0.32). There was also no interaction between treatment and bag type (P = 0.36) for starch disappearance (Table 3). The CP degradability was greater (P < 0.001) for feed in the 20 × 40 cm bags when compared
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
155
Table 3 Least squares means and associated standard errors for proportion of corn silages disappearance of DM, aNDF, ADF, starch, and CP after 48 h of in situ incubation in 10 × 20 or 20 × 40 cm Dacron bags. Treatmenta CON
DM aNDF ADF Starch CP
1X
SEMb 2X
3X
10 × 20
20 × 40
10 × 20
20 × 40
10 × 20
20 × 40
10 × 20
20 × 40
0.69 0.69 0.66 0.84 0.65
0.72 0.62 0.60 1.00 0.76
0.65 0.68 0.63 0.73 0.62
0.72 0.60 0.56 1.00 0.81
0.69 0.71 0.66 0.79 0.69
0.70 0.58 0.56 1.00 0.81
0.54 0.53 0.48 0.84 0.52
0.67 0.52 0.41 1.00 0.80
0.03 0.05 0.04 0.03 0.06
Pc Bag type
CON vs. FUN
Linear
Quadratic
0.01 0.03 0.01 <0.001 <0.001
0.08 0.15 0.02 0.32 0.98
0.01 0.01 0.001 0.86 0.54
0.14 0.09 0.03 0.11 0.23
Abbreviations: 1X, treatment 1; 2X, treatment 2; 3X, treatment 3; ADF, acid detergent fiber expressed inclusive of residual ash; aNDF, neutral detergent fiber assayed with a heat stable amylase and expressed inclusive of residual ash; CON, control; CP, crude protein; DM, dry matter; FUN, fungicide application; SEM, standard error of mean. a Treatment: control (CON), corn received no foliar fungicide application; 1X, corn received one application of pyraclostrobin foliar fungicide (PYR; Headline; BASF Corp.) at vegetative stage 5 (V5; when 5 visible leaf collars can be seen; Mueller and Pope, 2009); 2X, corn received 2 applications of foliar fungicides, PYR at stage V5, and a mixture of pyraclostrobin and metconazole (PYR + MET; Headline AMP; BASF Corp.) at reproductive stage 1 (R1; when silks are visible outside the husks; Mueller and Pope, 2009); and 3X, corn received 3 applications of foliar fungicides, PYR at stage V5, PYR + MET at stage R1, and PYR + MET at reproductive stage 3 (when kernel is yellow outside, whereas the inner fluid is now milky white due to accumulating starch; Mueller and Pope, 2009). b Greatest standard error of mean (SEM). c Bag type: 10 × 20 or 20 × 40 cm Dacron bags; contrasts statement for: CON vs. FUN = no fungicide application (CON) versus the average of the three treatments (1X, 2X, and 3X) with fungicide application; linear = linear treatment effect; and quadratic = quadratic treatment effect. All treatment × bag type interactions were not significant (P > 0.17).
with feed in the 10 × 20 cm bags. However, there was no difference (P = 0.98) among treatments for degradability of feed. Interaction of treatment and bag type was not significant (P = 0.43) for CP disappearance (Table 3). 4. Discussion The major purpose of this study was to evaluate in situ digestibility of corn plant treated at various times of application with foliar fungicide on corn harvested as whole plant corn silage and fed to lactating dairy cattle. We hypothesized that corn plant treated at various times of application with foliar fungicide would have increased degradability as treated corn plants presented decreased contents aNDF and ADF along with increased contents of sugar and starch (Haerr et al., 2015). According to the product label, the minimum time from application of PYR and PYR + MET to harvest of corn for silage is 7 days. The time from first, second, and third application to harvest was 82, 63, and 45 days which exceeded long the minimum time determined by the manufacturer. 4.1. Effects of foliar fungicide In the present study, the degradable fraction of DM tended to be greater for corn silage treated with foliar fungicide with the proportion of rumen degradable feed increasing as the number of foliar fungicide applications increased. These differences may be associated with the chemical composition of corn silages (Haerr et al., 2015). Starch and sugar contents were numerically higher in corn silages treated with foliar fungicide than CON, whereas aNDF and ADF were numerically higher in CON compared with corn silages treated with foliar fungicide. Yates et al. (1997) proposed that when a corn plant had a fungal infestation of the root, the structural components and rigidity increased, which the authors attributed to the plant attempting to decrease further infestation into the upper portion of the plant by increasing the structurally rigid components of the plant such as lignin. This may also have impacted the amount of aNDF, or the structural composition of the aNDF, and potentially increased the lignification of the aNDF making it less degradable in the present study (Buxton and Redfearn, 1997). Williams et al. (1992) found that severely damaged crops exhibited a higher crude fiber content when compared with non-infected plants. The authors also found that gross energy between infected and non-infected plants did not differ, but found that there was more protein and less fat in mold damaged corn kernels. The difference in nutrient composition was due to the fungi using readily accessible carbohydrates as nutrient source first, and the authors hypothesized that this may affect digestibility of the plant. Similar mechanisms may have occurred in the present study because there was a higher proportion of degradable aNDF in the silage treated with fungicide. The ED of DM, aNDF, and ADF was greater in CON compared to corn silages treated with foliar fungicides. These differences were mainly attributed to the lower ED of treatment 3X compared to 1X and 2X which in turn appeared to have numerically similar ED to the CON treatment. Decreased ED in 3X may be associated with lower soluble fraction compared to 1X, 2X, or CON treatment. There were no differences among corn silages in the degradability of starch or CP, which could be due to the fact that there were no visible signs of infection on the corn ears even from CON, and so corn ears were not damaged. Infection by Fusarium moniliforme as a visible ear rot was shown to decrease the fat content, but increased CP content. This is thought to be due
156
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
to the presence of fungal nitrogen (Williams et al., 1992). When steers were fed increasing amounts of steam-flaked corn infected with fungi, total tract digestibility and ruminal digestibility for organic matter and N as well as overall digestible energy decreased as inclusion of steam-flaked corn infected with fungi increased (Alvarez et al., 2011). Even when plants are not compromised with fungal infections, studies have found that application of pyraclostrobin has increased corn yield. A meta-analysis that looked at data from 172 studies from 2002 to 2009 in 14 different states found a mean increase in yield of corn grain of 255 kg/ha (Paul et al., 2011). This is thought to be due to the fact that pyraclostrobins are from the strobilurin class of fungicide, which work as quinone outside inhibitors. Quinone blocks electron transfer in the cytochrome III (bc1) complex, disrupting the electron transport chain and disrupting ATP production (Bartlett et al., 2002). This can also lead to increased N assimilation in the plant, through increased nitrate reductase that can be especially beneficial when the plant has high N requirements (Venancio et al., 2009). However, in our study we did not see any differences in availability or degradability of CP. There also is some evidence that pyraclostrobin may help to increase water use during drought stress due to increased abcissic acid concentration. Pyraclostrobin works to decrease oxidative stress, due to increased activity of peroxidases that persist weeks after treatment with the fungicide (Venancio et al., 2009). In the present study, the soluble portion of the feed had a linear effect for DM, aNDF, and ADF, with the portion of soluble feed components decreasing as the number of fungicide applications increased. This could have happened due to the difference in corn silage particle size but our evaluations did not show any difference between particle length and physical characteristics of the feed. To further investigate this difference it would have been beneficial to complete a more extensive evaluation of particle size of the feed before ensiling and digestion. It is also important to note that because the samples were not ground before placement in the rumen, degradability may have been underestimated due to the lack of effects of mastication or rumination. However, grinding the samples may lead to an over estimation of true digestibility (Vanzant et al., 1998). Nonetheless, the relatively small sizes of the standard errors calculated in the present study seem to show that a representative sample was obtained in each bag. 4.2. Effects of bag size The DM disappearance was greater for the 20 × 40 cm bags when compared with the 10 × 20 cm bag, which is similar to the results obtained by Varga and Hoover (1983) where two different sample sizes (2.5 and 5 g) were put into two different size bags (13 × 21 and 9 × 17 cm bags). The authors analyzed both concentrates and forages (ground at 4 mm) using a sample size to surface area of 9.2 and 14.6 mg/cm2 , respectively. The authors did not analyze NDF in those samples because the amount remaining at 48 h to complete the analyses was not adequate for all sample sizes. In addition, there was no discussion as to why these results were found (Varga and Hoover, 1983). One possible hypothesis would be that because the 10 × 20 cm bags were subjected to some minimal exposure to air when other bags were being removed at the specified time points feed digestion may have been affected, whereas 20 × 40 cm bags were only exposed to air upon removal. However, studies have shown that complete removal of feed, sampling, and reintroduction of the same feed to the rumen did not affect the digestibility, or rate of digestion (Reid, 1965). Bags were treated the same, therefore, it is not known why degradability differed between 10 × 20 vs. 20 × 40 cm bags. It was also noted that fiber degradability was greater in the 10 × 20 cm bags; however, CP and starch degradability were greater in the 20 × 40 cm bags, so microbial access could differ in the size of the bags. Because there have been limited studies utilizing a bag larger than 10 × 20 cm, we cannot explain why these differed and further research in this area is needed. The 48 h degradability of ADF was higher for CON when compared with silage treated with the fungicide. This could have happened due to a slightly higher kd in the fungicide-treated corn silages when compared with CON, and a tendency for a linear treatment effect for decreasing kd as the number of fungicide applications increased was found in the 10 × 20 cm bags alone. However, this inference to the larger bag should be interpreted cautiously as the degradability of feedstuff differed between 10 × 20 and 20 × 40 cm bags as stated above. There was also less ADF disappearing at 48 h with a linear treatment effect for increasing amount remaining as number of fungicide applications increased. We did not see any differences in degradability when the 10 × 20 cm bags were analyzed alone, but the difference became apparent when 20 × 40 cm bags were analyzed. It may be beneficial to do more replications of the larger bags and 10 × 20 cm bags at the 48 h time point as well as a longer time point to understand which portions of the feed may be indigestible. As well, it may be beneficial to compare ground and unground samples between the two bag sizes to better understand the differences found in this study. 5. Conclusion Fungicide application to corn resulted in higher DM degradable fraction which increased with the number of fungicide applications. It tended to decrease kd and linearly decrease DM solubility. There were no differences on the soluble, degradable, and indigestible fractions or kd of aNDF, ADF, starch, and CP among treatments. However, the soluble fraction of aNDF and ADF linearly decreased with fungicide applications. The ED was greater in CON than corn silages treated with foliar fungicide mainly due to decreased ED in 3X compared with 1X and 2X which in turn appeared to be numerically similar to the ED of the CON treatment. In situ digestibility results for big bags (20 × 40 cm) and small bags (10 × 20 cm) were different in the present study. Degradability of DM, aNDF, and ADF was higher, while starch and CP degradability was lower in the larger bags. Further investigation is needed to clarify why this occurred. Fungicide application on corn for silage seems promising
K.J. Haerr et al. / Animal Feed Science and Technology 222 (2016) 149–157
157
as changes in the degradable fraction may be the result of increased sugar and starch contents along with decreased aNDF and ADF. Conflicts of interest We acknowledge the affiliation of one of the co-authors with BASF Corporation. However, there are no conflicts of interest, financial or otherwise, including direct or indirect financial or personal relationships, interests, and affiliations, whether or not directly related to the subject of the paper. References AOAC (Association of Official Analytical Chemists), 1995. AOAC Official Method 972.16. Fat, Lactose, Protein, and Solids in Milk. Mid-Infrared Spectroscopic Method. Official Methods of Analysis of the Association of Official Analytical Chemists, vol. 2., 16th ed. AOAC, Arlington, VA, pp. 23–26, 33. Alvarez, E., Barajas, R., Calderon, F., Montano, M., Salinas-Chavira, J., Zinn, R.A., 2011. Effects of fungal infested steam-flaked corn on characteristics of ruminal and total tract digestion in steers. Anim. Feed Sci. Technol. 168, 145–151. Bal, M.A., Shaver, R.D., Shinners, K.J., Coors, J.G., Lauer, J.G., Straub, R.J., Koegel, R.G., 2000. Stage of maturity, processing, and hybrid effects on ruminal in situ disappearance of whole-plant corn silage. Anim. Feed Sci. Technol. 86, 83–94. Bartlett, D.W., Clough, J.M., Godwin, J.R., Hall, A.A., Hamer, M., Parr-Dobrzanski, B., 2002. The strobilurin fungicides. Pest Manage. Sci. 58, 649–662. Buxton, D.R., Redfearn, D.D., 1997. Plant limitations to fiber digestion and utilization. J. Nutr. 127, 814S–818S. Doggett, C.G., Hunt, C.W., Andrae, J.G., Pritchard, G.T., Kezar, W., Harrison, J.H., 1998. Effect of hybrid and processing on digestive characteristics of corn silage. J. Dairy Sci. 81 (E-Suppl.1):196(Abstr.). Fahey, G.C., Collins, M., Mertens, D.R., Moser, L.E., 1994. Plant factors affecting forage quality. Forage Qual. Eval. Util., 115–154. Goering, H.K., Van Soest, P.J., 1970. Forage fiber analyses (apparatus, reagents, procedures, and some applications). USDA Agric. Handb. 379, 387–598. Haerr, K.J., Lopes, N.M., Pereira, M.N., Fellows, G.M., Cardoso, F.C., 2015. Corn silage from corn treated with foliar fungicide and performance of Holstein cows. J. Dairy Sci. 98, 8962–8972. Hall, M.B., Mertens, D.R., 2008. Technical note: effect of sample processing procedures on measurement of starch in corn silage and corn grain. J. Dairy Sci. 91, 4830–4833. Köhle, H., Grossmann, K., Jabs, T., Gerhard, M., Kaiser, W., Glaab, J., Conrath, U., Seehaus, K., Herms, S., 2002. Physiological Effects of the Strobilurin Fungicide F 500 on Plants. Modern Fungicides and Antifungal Compounds III. Mann GmbH & Co. KG, Bonn, Germany, pp. 61–74. Kononoff, P.J., Heinrichs, A.J., Buckmaster, D.R., 2003. Modification of the penn state forage and total mixed ration particle separator and the effects of moisture content on its measurements. J. Dairy Sci. 86, 1858–1863. Krizsan, S.J., Ahvenjarvi, S., Huhtanen, P., 2010. A meta-analysis of passage rate estimated by rumen evacuation with cattle and evaluation of passage rate prediction models. J. Dairy Sci. 93, 5890–5901. Littell, R.C., Henry, P.R., Ammerman, C.B., 1998. Statistical analysis of repeated measures data using SAS procedures. J. Anim. Sci. 76, 1216–1231. McDonald, I., 1981. A revised model for the estimation of protein degradability in the rumen. J. Agric. Sci. 96, 251–252. Mueller, D., Pope, R., 2009. Corn Field Guide. Iowa State University, Extension and Outreach, Ames, IA. NRC (National Research Council). 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Sci., Washington, DC. Nocek, J.E., 1988. In situ and other methods to estimate ruminal protein and energy digestibility: a review. J. Dairy Sci. 71, 2051–2069. Ørskov, E.R., McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. 92, 499–503. Paul, P.A., Madden, L.V., Bradley, C.A., Robertson, A.E., Munkvold, G.P., Shaner, G., Wise, K.A., Malvick, D.K., Allen, T.W., Grybauskas, A., Vincelli, P., Esker, P., 2011. Meta-analysis of yield response of hybrid field corn to foliar fungicides in the U.S. Corn Belt. Phytopathology 101, 1122–1132. Reid, C., 1965. Quantitative studies of digestion in the reticulo-rumen I. Total removal and return of digesta for quantitative sampling in studies of digestion in the reticulo-rumen of cattle. Proc. N.Z. Soc. Anim. Prod., 65. Rivero, R.M., Ruiz, J.M., Garcı´ıa, P.C., López-Lefebre, L.R., Sánchez, E., Romero, L., 2001. Resistance to cold and heat stress: accumulation of phenolic compounds in tomato and watermelon plants. Plant Sci. 160, 315–321. SAS Institute Inc, 2013. Base SAS® 9.4 Procedures Guide: Statistical Procedures, second edition. SAS Institute Inc., Cary, NC. Schalla, A., Meyer, L., Meyer, Z., Onetti, S., Schultz, A., Goeser, J., 2012. Hot topic: apparent total-tract nutrient digestibilities measured commercially using 120-hour in vitro indigestible neutral detergent fiber as a marker are related to commercial dairy cattle performance. J. Dairy Sci. 95, 5109–5114. Van Milgen, J., Murphy, M.R., Berger, L.L., 1991. A compartmental model to analyze ruminal digestion. J. Dairy Sci. 74, 2515–2529. Vanzant, E.S., Cochran, R.C., Titgemeyer, E.C., 1998. Standardization of in situ techniques for ruminant feedstuff evaluation. J. Anim. Sci. 76, 2717–2729. Varga, G.A., Hoover, W.H., 1983. Rate and extent of neutral detergent fiber degradation of feedstuffs in situ. J. Dairy Sci. 66, 2109–2115. Venancio, W.S., Rodrigues, M.A.T., Begliomini, E., De Souza, N.L., 2009. Physiological effects of strobilurin fungicides on plants. Ci. Exatas e da Terra Ci Agr. Eng. 9, 59–68. Williams, K., Blaney, B., Dodman, R., Palmer, C., 1992. Assessment for animal feed of maize kernels naturally-infected predominantly with Fusarium moniliforme and Diplodia maydis. I. Fungal isolations and changes in chemical composition. Aust J. Agric. Res. 43, 773–782. Wise, K., Mueller, D., 2011. Are fungicides no longer just for fungi? An analysis of foliar fungicide use in corn. APSnet Featur., http://dx.doi.org/10.1094/apsnetfeature-2011-0531. Yates, I.E., Bacon, C.W., Hinton, D.M., 1997. Effects of endophytic infection by Fusarium moniliforme on corn growth and cellular morphology. Plant Dis. 81, 723–728.