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Steaming Conditions Enhance Hygienic Quality of the Compromised Equine Hay With Minimal Losses of Nonfiber Carbohydrates
Journal of Equine Veterinary Science 74 (2019) 28e35
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Original Research
Steaming Conditions Enhance Hygienic Quality of the Compromised Equine Hay With Minimal Losses of Nonfiber Carbohydrates € gmüller b, Qendrim Zebeli a, * Elke Humer a, Manfred Hollmann a, Gerald Sto a
Department for Farm Animals and Veterinary Public Health, Institute of Animal Nutrition and Functional Plant Compounds, University of Veterinary Medicine Vienna, Vienna, Austria b Feed Laboratory Rosenau, Chamber of Agriculture of Lower Austria, Wieselburg-Land, Austria
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 October 2018 Received in revised form 25 November 2018 Accepted 24 December 2018 Available online 28 December 2018
Compromised hygienic quality of hay often is a serious threat to horse health. The study examined the effect of different steaming conditions on molds, bacteria, and yeast as well as on the content of main macro- and micro-nutrients of either normal-quality or hygienically compromised hays. Hay samples were subjected to the following eight treatments each: unsteamed hays as control or hays steamed at 50 C for 90 minutes, at 80 C for 10, 20, or 30 minutes, and at 100 C for 5, 10, or 20 minutes. To evaluate a possible storage effect of the treated hays, subsamples were stored for 24 hours at room temperature. Data indicated increased bacterial counts, molds, and yeasts in hygienically compromised hay. Steaming did not affect total bacterial counts in this hay, but all steaming conditions were equally effective leading to a strong decline of molds and yeasts to a level comparable to the normal-quality hay. Interestingly, only steaming at 100 C for at least 10 minutes reduced Bacillus spp. significantly. In the normal-quality hay, steaming lowered the already low bacterial counts further but caused leakage of sodium and copper. Hay steamed and left for 24 hours at room temperature retained its microbial and nutrient profiles. Overall, the study showed that steaming represents a useful strategy for reducing mold contamination in hygienically compromised hay, with no differences among steaming conditions. Most importantly, to reduce contamination of Bacillus spp. in hygienically compromised hays, steaming at 100 C for at least 10 minutes is required. © 2018 Elsevier Inc. All rights reserved.
Keywords: Bacteria Hay nutritive value Hay steaming Horse Mold concentration Yeast
1. Introduction Forage is a crucial ingredient of the equine diet, which serves both as energy and nutrient source as well as is needed to maintain normal feeding behavior and promote teeth and gut health. Meadow hay is the most common forage fed to horses and can vary significantly in its hygienic quality, depending on the weather during the conservation process and storage conditions [1,2]. In an effort to avoid the presence of adverse weather conditions, hay is
Animal welfare/ethical statement: Animal experimentation was not conducted in this research. Conflict of interest statement: All the authors declare no conflict of interests related to this article. * Corresponding author at: Qendrim Zebeli, Department for Farm Animals and Veterinary Public Health, Institute of Animal Nutrition and Functional Plant Compounds, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. E-mail address: [email protected] (Q. Zebeli). https://doi.org/10.1016/j.jevs.2018.12.024 0737-0806/© 2018 Elsevier Inc. All rights reserved.
often insufficiently dried and baled before achieving recommended moisture contents, resulting in reduced forage quality and mold development [3]. Moldy hay serves as the major source of mold or dust particles [4], which predispose horses to respiratory disorders, including recurrent airway obstruction and chronic obstructive pulmonary disease [5,6]. As horses are highly sensitive to mold particles, there is a need for effective feed management strategies to reduce the number of airborne mold particles released during feeding. Soaking of hay is commonly applied by horse owners in an attempt to reduce dust levels. Furthermore, soaking commonly causes a significant loss of nutrients, that is, water-soluble carbohydrates, soluble protein, and minerals [7e9]. One major disadvantage of soaking hay is the rapid and extensive proliferation of bacteria, which is highly undesirable if the level of contamination increases the number of bacteria above the recommended safe limit and/or if potentially pathogenic bacteria are present [10]. To avoid this aspect, steaming of hay has become more and popular as a management tool to reduce mold and bacterial concentrations [11].
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Steaming has also been attributed an additional benefit such as the reduction of potentially hazardous post-soak liquor [12]. Nevertheless, steaming might also have undesirable consequences as it might leach essential nutrients, including soluble nonfiber carbohydrates (NFC) and protein as well as minerals and trace elements [11,13]. However, little is known about the impact of different steaming conditions especially the combination of temperature and duration steaming on nutritive value and microbial content. A further aspect that has to be considered is that horse owners often owe only one horse or have to restrict the hay intake due to adiposity risk [14]. Thus, it is often not feasible to feed all the steamed hay immediately after steaming than rather preparing the respective hay for 1e2 days in advance. However, there is no information available on the effect on the microbial and nutrient concentrations in hay left for 24 hours poststeaming. Therefore, the main aim of this study was to evaluate the effect of different steaming conditions on microbial concentrations and nutritive values of the hay of the same origin but of different hygienic traits. Furthermore, a possible effect of an additional 1-day storage period after the hay treatments was evaluated, too. 2. Material and Methods 2.1. Hay Preparation and Treatments A round bale hay provided by a local supplier was identified during the routine hay quality control at the Institute of Animal Nutrition and Functional Plant Compounds for the study. After the official organoleptic (sight, smell, color, foreign substances including poisonous plants contamination) and feeding value evaluation [15], the hay was deemed adequate to feed to horses. The hay was free of any foreign material or unpalatable weeds and musty or moldy smell. Also, no unusual dust or visual signs of rain or heat damage were observed. The hay was field-dried Austrian meadow hay of the first cut harvested at bloom, consisting mainly of common meadow-grass (Poa pratensis L.), orchard grass (Dactylis glomerata), false oat-grass (Arrhenatherum elatius), and meadow foxtail (Alopecurus pratensis L.), harvested in 2017 and conserved for 12 months. The bale weighed approximately 300 kg and was divided into two equal sections. While one section was kept in a dry, nonheated room to serve as normal-quality hay, the other half was moistened with distilled water (about 0.7 L/kg of hay) and allowed to mold for 6 days at room temperature to prepare the hygienically compromised hay. The hygienically compromised hay showed visual signs of mold and moldy smell, starting almost 1 day after the moistening treatment. The experiment was set up in a completely randomized arrangement. Three randomly collected replicates of each hay quality type were individually subjected to one of the following treatments: (1) unsteamed (original hay) where no additional treatment was applied to the hay; (2) steamed at 50 C for 90 minutes; (3) steamed at 80 C for 10 minutes; (4) steamed at 80 C for 20 minutes; (5) steamed at 80 C for 30 minutes; (6) steamed at 100 C for 5 minutes; (7) steamed at 100 C for 10 minutes; and (8) steamed at 100 C for 20 minutes. The steaming was carried out in a specifically designed steamer composed of a preserving boiler (TZS, First Austria, Article-No.: FA-5180) with a total capacity of 27 L and a continuously adjustable thermostat. A total of 2 L of distilled water was placed in the bottom section of the container, and when the targeted temperature was reached, 750 g of hay was put in laundry nets and placed on a plastic insert on the bottom of the steamer. After closing the steamer, the hay was left to steam for either 5, 10, 20, 30, or 90 minutes. The temperature was confirmed with the thermometer (Basetech ET-1; Conrad, ArticleNo.: 1396295) located within the hay sample. Subsequently, the steamer was turned off, opened, and the hay was removed. The
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steamer was washed with water and disinfected with ethanol between each treatment to reduce the chance of contamination. Poststeaming the hay was stored at 4 C until further analysis. All hay samples were kept on ice during transportation before sample admission. In addition, the effect of a 1-day storage at room temperature was evaluated for the following treatments: (1) unsteamed (original hay); (2) steamed for 90 minutes at 50 C; (3) steamed for 30 minutes at 80 C; (4) steamed for 20 minutes at 100 C. Therefore, poststeaming hay samples were stored for 24 hours in a dry, nonheated room before further analysis. 2.2. Microbial and Nutrient Analyses Microbial counts (aerobic, mesophilic bacteria, molds, and yeasts) were determined of all samples. For analysis of microbial counts, 10 g from each sample was placed into a sterile Stomacher bag, and 90 mL of sterile phosphate-buffered saline solution (PBS; sodium hydrogen phosphate dihydrate, sodium chloride, sodium dihydrogen phosphate dihydrate, and neutralized bacteriological peptone) was added resulting in a 1:10 dilution. Bags were tightly closed to exclude air and were agitated for 30 minutes at ambient temperature. Colony-forming units (CFU) were determined from serial dilutions in PBS (102e104), which were made from a 1 mL subsample of the supernatant fluid. From dilutions 102 and 104 aliquots of 100 mL each were spread evenly on agar plates. Nutrient agar in accordance with Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung, two plates per dilution, was used to determine the CFU of aerobic mesophilic bacteria. Dichloran glycerol agar (DG 18 Agar) and Sabouraud dextrose agar, two plates per dilution, were used to determine the CFU of molds and yeasts. Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung agar was incubated at 37 C, whereas DG 18 Agar and Sabouraud agar plates were incubated at 27 C before colonies were counted and reported as CFU per gram. Nutrient analyses of hay samples were conducted in duplicate according to the German Handbook of Agricultural Experimental and Analytical Methods [16]. The dry matter (DM) of the hay samples was determined by oven drying at 105 C for 4 hours (method 3.1). Ash was analyzed by combustion of samples at a temperature of 550 C (method 8.1). The crude protein (CP) content was determined using the Dumas method (method 4.1.2). The content of crude fiber (CF) was determined gravimetrically after chemical digestion with sulfuric acid and was expressed exclusive residual ash following method 6.1.1 of VDLUFA [16]. Ether extract (EE) was analyzed by extraction with petroleum ether (method 5.1.1). The content of NFC was calculated as NFC ¼ DM (Ash þ CP þ EE þ NDF). Sugar was analyzed via the Luff-Schoorl method according to method 7.1.1 of VDLUFA [16]. The contents of phosphorus (P), calcium (Ca), magnesium (Mg), potassium (K), sodium (Na), iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu) were determined by inductively coupled plasma emission spectroscopy following method 10.8.2 of VDLUFA [16]. 2.3. Statistical Analyses Statistical analyses were performed by analysis of variance using the MIXED procedure of SAS (version 9.4; SAS Institute Inc, Cary, NC). For each variable tested, the model included the fixed effects of treatment, hay quality, the interaction between hay quality and treatment, and the random effect of the replicate within the feed. As no interaction between hay quality and treatment was observed, normalquality and hygienically compromised hays were analyzed separately. To evaluate a possible storage effect, further analyses were carried out considering only the respective steaming treatments
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that underwent the postprocessing test later on compared with their counterparts that were immediately stored at 4 C after steaming. As no effect of the time duration within steaming temperature as well as an additional storage for 24 hours was observed, results from all treatments within one temperature were pooled and subjected to further statistical analysis considering the effect of temperature, hay quality, and their interaction. Degrees of freedom were estimated with the method of KenwardeRoger. The PDIFF option was used for each least square mean (LSM) comparison. Differences among LSM with P .05 were considered as representing significant differences, and differences with P > .05 and P .10 were considered to represent a trend. 3. Results 3.1. Effect of Steaming on the Microbial Contamination Table 1 summarizes the microbial analyzes of the normalquality hay subjected to different steaming conditions. Microbial analyses confirmed the organoleptic evaluation showing the presence of only 5.3 105 bacteria and 1.7 103 mold CFU/g hay, whereas no yeast was detected. Steaming caused a reduction of the total bacteria count by an average 96% irrespective of the steaming conditions (P < .01). The overall low mold content in the untreated hay was reduced by 89% when hay was steamed but did not reach statistical significance (P ¼ .29). As summarized in Supplementary Table 1, additional storage of the steamed hays at room temperature for 24 hours did not affect the microbial counts compared with the samples that were immediately analyzed after steaming. The hygienically compromised hay contained 11.9 106 bacterial CFU/g, 5.3 106 mold CFU/g, and 1.3 105 yeast CFU/g (Table 2). Although the total bacterial counts were not affected by steaming (P ¼ .57), a more detailed investigation of the bacterial groups revealed a significant effect of the steaming at 100 C to reduce Bacillus spp. Indeed, multiple comparisons among treatments showed that solely steaming at 100 C for 10 minutes (P ¼ .06) or 20 minutes (P ¼ .05) was effective in reducing Bacillus spp. compared with untreated hay by on average 88%. A strong reduction in mold counts (99.5%) was observed for all steaming treatments (P ¼ .04), showing similar effectiveness among all investigated species (Aspergillus niger, Mucor, Aspergillus fumigatus, and Rhizopus). Yeast counts were reduced by 99.9% when hygienically compromised hay was steamed, showing a statistical tendency when multiple comparisons of the steaming treatments with the original hay were carried out (P .08). Storage of steamed hays showed no effect on the microbial counts (Supplementary Table 2).
3.2. Effect of Steaming on the Nutritive Profile As demonstrated in Table 3, steaming of normal-quality hay caused a 23% reduction in the DM compared with the original hay (P ¼ .08). No differences in the content of crude nutrients were found, whereas among minerals, measured Na was reduced by on average 33% (P < .01), and Cu tended to be reduced by 26% (P ¼ .07) when hay was steamed. However, no differences among the seven different steaming methods were found. As summarized in Supplementary Table 3, no differences between samples analyzed immediately after steaming compared with samples stored for 24 hours at room temperature were found. Steaming of hygienically compromised hay reduced the DM content by on average 32% (P ¼ .03) and caused a tendential decrease in NFC (7%, P ¼ .09, Table 4). Overall, no effect of the different time durations within steaming temperature groups was observed. Supplementary Table 4 shows the effect of an additional storage period on the nutrient content of steamed hygienically compromised hay samples. There was an effect on the DM content, showing an increase by on average 31% with storage (P ¼ .01). However, no differences in nutrient profiles between hay analyzed immediately after steaming and hay stored for 24 hours were found. 3.3. Interactive Effects of Steaming and Hay Quality on the Microbial Contamination Fig. 1 shows the interactive effect of steaming temperature and hay quality on microbial contamination. Compared with the original hay, the total bacterial count (P ¼ .09; Fig. 1A) and the Bacillus spp. were only reduced in hygienically compromised hay when steamed at 100 C (P ¼ .06; Fig. 1B). Among mold (Fig. 1C) and yeast (Fig. 1D), all steaming treatments caused a strong decline to a level comparable to the normal-quality hay. 3.4. Interactive Effects of Steaming and Hay Quality on the Nutritive Profile Several nutrients were affected by the hay quality (Supplementary Table 5 and Fig. 2), showing a decrease in NFC, sugar, and digestible energy when hay was hygienically compromised (P .01), whereas the opposite was observed for CP and CF (P .06). Among minerals, higher contents of P, K, and Mn were measured in hygienically compromised hay compared with normal-quality hay (P .07). Fig. 2 shows the interactive effect of steaming temperature and hay quality on nutrient concentration. An interaction between hay
Table 1 The effect of steaming on bacterial, mold, and yeast concentrations in the normal-quality hay expressed as geometric mean colony-forming units 103 per gram of hay. Item
Bacteria Yellow Pigmented Bacteria Bacillus spp. Molds A. niger Mucor A. glaucus A. fumigatus Rhizopus Yeasts
Treatment Unsteamed
50 C, 90 min
80 C, 10 min
80 C, 20 min
80 C, 30 min
100 C, 5 min
100 C, 10 min
100 C, 20 min
528a 135a
10.3b 5.13b
17.0b 2.80b
19.7b 6.93b
10.7b 6.63b
43.0b 3.78b
15.7b 0.863b
29.0b 10.3b
5.20b 1.00 1.00 0 0 0 0 0
13.6b 0.333 0.333 0 0 0 0 0
12.7b 0 0 0 0 0 0 0
4.03b 0 0 0 0 0 0 0
39.2b 0.667 0.333 0.333 0 0 0 0
14.8b 0 0 0 0 0 0 0
18.7b 0.333 0.333 0 0 0 0 0
392a 1.67 1.47 0 0 0 0.200 0
Abbreviations: A., Aspergillus; SEM, standard error of the mean. a and b indicate differences among treatments at a level of P .05.
SEM
P Value
66,03 20.398
<.01 <.01
47.715 0.553 0.485 0.118
<.01 .29 .26 .47 d d .47 d
d d 0.070 d
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Table 2 The effect of steaming on bacterial, mold, and yeast concentrations in the hygienically compromised hay expressed as geometric mean colony forming units 103 per gram of hay. Item
Treatment
Bacteria Yellow Pigmented Bacteria Bacillus spp. Molds A. niger Mucor A. glaucus A. fumigatus Rhizopus Yeasts
Unsteamed
50 C, 90 min
80 C, 10 min
80 C, 20 min
80 C, 30 min
100 C, 5 min
100 C, 10 min
100 C, 20 min
11,883 2,950
10,500 6,253
11,033 602
13,867 1,269
10,000 6,850
60,233 602
5,275 4,042
1,107 110
10,431a 0b 0b 0b 0 0 0b 0z
12,539a 58b 26b 0.777b 0.035 17 6b 0.973z
8,933ab 5,267a 1,407a 610a 0 2,113 963a 133y
4,247b 3b 0b 0.003b 0 0 0b 0z
3,150b 2b 0.6b 0.133b 0 0.933 0b 0z
5,421bc 3b 0.027b 0b 0.133 0.133 0b 0z
1,233c 6b 0.500b 0b 0.500 3 2b 0z
997c 101b 0.051b 15b 10 10 15b 0.333z
SEM
P Value
4,599 3,332
.57 .65
2,858 1,083 332 134 0.0035 0.573 0.173 47.1
.07 .04 .09 .06 .48 .19 .01 .47
Abbreviations: A., Aspergillus; SEM, standard error of the mean. a, b, and c indicate differences among treatments at a level of P .05. y and z indicate differences among treatments at a level of P > .05 and .10.
quality and steaming temperature (P ¼ .01) revealed that the sugar content was only reduced by steaming hygienically compromised hay (on average 39% compared with the original hay; Fig. 2A). However, the opposite effect was found for the ash content, where steamed normal-quality hay had, on average, 24% less ash compared with the unsteamed hay (P .10; Fig. 2B). In more details, mainly Mg (19%; P .10; Fig. 2D), Na (35%; P < .01; Fig. 2E), and Cu (27%; P < .01; Fig. 2F) concentrations were lowered when normal-quality hay was subjected to steaming, whereas for P (Fig. 2C), a reduction by, on average, 7% was found in good quality hay, and even an increase was observed in hygienically compromised hay (þ7%; P .10). 4. Discussion 4.1. Effect of Steaming on the Microbial Concentration Across all treatments examined, the results showed that steaming was effective to reduce mold and yeasts, especially in hygienically compromised hays. Although no differences among different steaming temperatures and durations were observed for molds and yeast, total viable bacteria numbers, especially spoilage
indicating bacteria (Bacillus spp.), were solely reduced when the highest temperature treatment (i.e., 100 C) was applied for at least 10 minutes. These results agree with those reported by Moore-Colyer et al [13] who found that steaming of hay at 102 C caused a strong reduction in hay bacteria, whereas steaming at 58 C was not effective in reducing bacteria concentrations. Also, Taylor and Moore-Colyer [17] concluded that hay must be steamed at >90 C for a minimum of 10 minutes for most of the bacteria to be killed. Moore-Colyer et al [10] observed a reduction of 98.6% of bacteria when hay was steamed 50 minutes at about 100 C, whereas soaking hay caused a fivefold increase in the average bacterial contamination. Therefore, soaking might cause the number of bacteria ingested to increase above the recommended safe limit of 2,000,000 CFU/g [15] when normal-quality hay is submerged in water, whereas steaming at 100 C might even decrease the number of bacteria below this limit in contaminated hay. Although information regarding the pathogenicity of spoilage-indicating bacteria is lacking [18], poor feed hygiene, in general, has been linked to digestive disorders such as colic [19]. Furthermore, ingestion of feed-associated gram-negative bacteria also causes the ingestion of lipopolysaccharides, which have been reported to play a pivotal
Table 3 The effect of steaming on hay nutritive values (in g/kg on a DM basis, except DM), digestible energy (in MJ/kg DM), macrominerals (in g/kg DM), and trace minerals (in mg/kg DM) in the normal-quality hay. Item
DM CP CF NFC Sugar EE Ash DE Ca P Mg K Na Fe Mn Zn Cu
Treatment Unsteamed
50 C, 90 min
80 C, 10 min
80 C, 20 min
80 C, 30 min
100 C, 5 min
100 C, 10 min
100 C, 20 min
905a 87.3 269 519 148 22.0 103 9.12 6.03 2.13 2.13 21.0 0.43a 1,833 142 29.5 7.73a
692b 83.3 302 522 157 20.7 71.0 9.36 5.73 2.00 1.67 21.6 0.26b 430 102 25.8 5.33b
762b 83.7 304 515 145 20.7 77.3 9.30 6.27 1.97 1.83 21.5 0.32b 821 118 41.2 5.63b
679b 86.7 296 513 142 20.7 83.0 9.26 5.77 2.07 1.87 21.1 0.30b 1,075 123 22.2 6.33b
647b 82.0 311 511 153 20.7 75.7 9.29 5.07 1.97 1.63 21.9 0.26b 677 100 22.3 5.63b
683b 79.7 310 516 156 19.7 73.7 9.30 4.97 1.93 1.63 22.0 0.27b 674 107 22.7 5.70b
689b 84.3 302 511 151 20.3 82.7 9.25 5.43 2.00 1.73 21.7 0.30b 961 114 22.8 5.97b
720b 80.3 321 500 147 20.3 78.7 9.24 5.27 2.00 1.70 21.2 0.27b 826 118 20.2 5.30b
SEM
P Value
53.2 4.82 14.3 11.8 7.0 0.66 8.71 0.077 0.685 0.057 0.146 0.942 0.035 351.8 13.5 7.40 0.565
.08 .93 .15 .86 .78 .47 .33 .60 .85 .33 .27 .71 .01 .26 .30 .56 .07
Abbreviations: Ca, Calcium; CF, crude fiber; CP, crude protein; Cu, Copper; DE, digestible energy; DM, dry matter; EE, ether extract; Fe, Iron; K, Potassium; Mg, Magnesium; Mn, Manganese; Na, Sodium; NFC, nonfiber carbohydrates; P, Phosphorus; SEM, standard error of the mean; Zn, Zinc. a and b indicate differences among treatments at a level of P .05.
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Table 4 The effect of steaming on hay nutritive values (in g/kg on a DM basis, except DM), digestible energy (in MJ/kg DM), macrominerals (in g/kg DM), and trace minerals (in mg/kg DM) in the hygienically compromised hay. Item
DM CP CF NFC Sugar EE Ash DE Ca P Mg K Na Fe Mn Zn Cu
Treatment Unsteamed
50 C, 90 min
80 C, 10 min
80 C, 20 min
80 C, 30 min
100 C, 5 min
100 C, 10 min
100 C, 20 min
749a 80.7 320 494a 119 21.3 84.3 9.18 4.97 1.97 1.70 22.6 0.32 1,065 136 21.6 6.10
509b 92.7 342 452b 65.3 23.0 89.7 9.14 5.67 2.17 1.93 24.3 0.30 1,021 125 26.4 6.43
547b 91.7 341 443b 60.7 21.0 103 8.99 5.50 2.07 2.03 23.8 0.34 1,566 152 28.2 6.40
533b 85.3 321 489a 102 21.7 82.7 9.21 5.43 2.00 1.83 21.9 0.32 980 133 22.9 6.37
440b 94.3 332 437b 61.7 21.0 115 8.90 6.33 2.20 2.27 23.2 0.36 1,967 157 31.3 6.77
519b 87.7 335 471a 86.3 21.3 84.7 9.18 5.80 2.13 1.87 24.3 0.32 954 125 26.4 7.13
519b 92.7 337 462ab 71.3 22.7 84.0 9.20 6.30 2.13 1.90 23.9 0.27 738 130 22.8 6.07
502b 88.0 325 467ab 76.0 19.7 101 9.03 5.60 2.10 1.97 23.5 0.34 1,576 140 29.8 7.47
SEM
P Value
52.6 3.28 14.9 13.5 14.61 1.69 15.86 0.148 0.472 0.067 0.193 0.95 0.047 667 24.2 3.28 0.396
.03 .12 .91 .09 .11 .91 .77 .74 .46 .15 .63 .57 .93 .89 .97 .34 .19
Abbreviations: Ca, Calcium; CF, crude fiber; CP, crude protein; Cu, Copper; DE, digestible energy; DM, dry matter; EE, ether extract; Fe, Iron; K, Potassium; Mg, Magnesium; Mn, Manganese; Na, Sodium; NFC, nonfiber carbohydrates; SEM, standard error of the mean; P, Phosphorus; Zn, Zinc. a and b indicate differences among treatments at a level of P .05.
role in several equine diseases such as laminitis, acute abdominal disease, recurrent airway obstruction and exertion, and are able to elicit strong immune responses in horses [20]. Thus, the reduction in viable bacteria by steaming hay at 100 C for at least 10 minutes might also improve digestive health in horses. In our study, all steaming treatments were equally effective in reducing the mold contamination. This is contrary to the findings by Moore-Colyer et al [13], where a significant decrease in mold
was only found for the higher temperature (102 C vs. 58 C). One possible explanation for the differing results might be the shorter duration (50 minutes) of the steaming treatment at the lower temperature in the respective study compared with our study (90 minutes). This speculation seems to be supported by Earing et al [11], who tested the effect of steaming on low and moderately moldy hay. In their study, steaming for 90 minutes and reaching a final temperature of 46 C caused a reduction in mold count by 99%
Fig. 1. Effect of different steaming temperatures on the counts of (A) total bacteria, (B) Bacillus spp. (C) mold and (D) yeast expressed as geometric mean colony-forming unit (CFU) per gram of normal-quality (gray) or hygienically compromised (black) hay, respectively. a, b, and c indicate differences among treatments at a level of P .05.
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Fig. 2. Effect of different steaming temperatures on the content of sugar (A), ash (B), phosphorus (C), magnesium (D), sodium (E) and copper (F) in normal-quality (gray) or hygienically compromised (black) hay. a, b, c, and d indicate differences among treatments at a level of P .05.
in low moldy and 91% in moderately moldy hays. Also, MooreColyer et al [10] showed a reduction of mold content of 99.7% across the range of low, medium, and highly contaminated hays after steaming at about 100 C for 50 minutes. Overall, it is somewhat unclear as to what an acceptable concentration of mold contamination is with regards to horse feeds. Although it has been suggested that hay containing <1,000,000 CFU mold per gram is considered relatively safe for livestock [15], Martinson et al [3] suggested a threshold of 500,000 CFU/g of mold for hay intended for horse, as horses are known to be more susceptible to mold exposure [21]. Overall, in the present study, both thresholds have been only exceeded in the unsteamed hygienically compromised hay. A further finding was that steaming reduced the number of yeasts below the limiting value for horses in hygienically compromised hay. Although studies investigating the effect of steaming on yeast concentrations are lacking, increased yeast counts have been reported in hay after soaking [22]. Although yeast species present in
forages are generally not considered pathogenic and thus might not pose an immediate health risk to horses, elevated yeast contents might cause excessive gas formation in the stomach and small intestine, thereby paving the way for gas colics [18]. Therefore, it can be suggested that horses affected by respiratory and digestive diseases likely benefit from the reduction in mold contamination when hay is steamed before feeding. 4.2. Effect of Steaming on the Nutritive Profile Overall, the results showed that steaming of normal-quality hays conserved nutrients and energy content while reducing Na and Cu contents. On the opposite, the already decreased content of NFC (mainly sugars) of hygienically compromised hay compared with the normal-quality hay was further reduced by steaming. However, no further decline due to steaming was observed for minerals. Poststeaming DM concentrations were reduced to 69.6% and 51.0% for normal-quality hay and hygienically compromised hay,
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respectively, demonstrating that moisture was absorbed during the steaming process. Previously, it has been speculated that the higher water content, among others, might increase the palatability of steamed hay [10]. In this regard, previous studies demonstrated improved palatability of steamed hay compared with dry or soaked hay [11,23]. A loss of essential nutrients that might occur when hay is submerged in water has been reported to be one of the disadvantages of steaming or soaking hay. In accordance with Earing et al [11], steaming of hay with appropriate hygienic quality showed no effects on forage nutritive values, except a decreasing effect on specific minerals. Regardless of hay quality, no effect on CP and fiber content was observed, which was also observed by Earing et al [11] and Moore-Colyer et al [10,13]. However, inconsistent effects of soaking on CP contents have been reported previously [9,13,24]. In general, a reduction of soluble forage carbohydrates is a claim made by hay steamer manufacturers. Interestingly, steaming showed no effect on sugar in the normal-quality hay but caused a decline by 39% in hygienically compromised hay. A reduction in reducing sugars after steaming alfalfa hay at a temperature ranging from 100 to 110 C for 47 minutes has been observed previously [25]. Furthermore, Martinson et al. [26] observed a reduction in soluble carbohydrates when four different hays were soaked for 15 minutes in water, whereas Moore-Colyer et al [10] found no effect of steaming hays at about 100 C for 50 minutes on watersoluble carbohydrates. Similar to our results, Earing et al [11] found only a reduction in water-soluble and ethanol-soluble carbohydrates when moderately moldy hay was steamed, whereas it remained unaffected in the low moldy hay. The authors speculated that for hay with low mold amounts, higher temperature for a longer duration has to be applied to reduce soluble carbohydrate fractions. However, this is not supported by the present results. Overall, sugar contents were 20% lower in the untreated hygienically compromised hay compared with normal-quality hay. It is likely that the molding process increased the availability of NFC, that is, sugars, rendering these carbohydrates more prone to leaching during the steaming treatment. A disadvantage of steaming or soaking hay might be a loss of essential minerals. Several studies have shown significant losses in minerals (i.e., P, K, and Mg) after soaking hay in water for 15 minutes [8,24,27,28]. Also, losses of Na and Cu have been reported when hay was soaked for 10 or 30 minutes [27]. On the contrary, little effects of steaming on the mineral content have been reported. For instance, Moore-Colyer et al [13] observed that steaming hay at about 100 C for 50 minutes conserved minerals, such as Ca, K, Mg, Na, P, Cu, Mn, and Zn. Also, Blackman and MooreColyer [27] found little effect of steaming at 100 C for 50 minutes on the nutrient content, whereas soaking caused a loss of minerals. Thus, steaming hay may best conserve the total nutrient content. Overall, in the present study, water-insoluble minerals, such as Ca, were not affected by steaming, whereas soluble minerals, that is, Na and Cu, were decreased in the normal-quality hay. The effect of steaming on P content depended on the hay quality, showing a reduction in the normal-quality hay, whereas an increase was observed in hygienically compromised hay. On the contrary, Earing et al [11] observed a decrease in P content when hay was steamed for 90 minutes and reaching a final temperature of 46 C irrespective of the hay quality. However, one has to consider that results are not directly comparable, as the low moldy hay used in their study had about a 10-times higher initial mold concentrations (10,020 CFU/g vs. 1,667 CFU/g in our study), whereas the moderately moldy hay in their study had a much lower mold content (268,102 CFU/g) compared with the hygienically compromised hay used in our study (5,266,670 CFU/g). Furthermore, although P was leached in the normal-quality hay, it is unlikely to be deficient in
horse diets, at least for maintenance and light work. For instance, a horse in light work weighing 500 kg requires 14 g P/d [29]. This requirement would be even exceeded when consuming 10 kg of either steamed or unsteamed hay. Mares and growing horses, however, might experience P deficiencies if fed the steamed hay without adequate supplementation. 4.3. Effect of Storage on Microbial Concentration and Nutritive Profile A consequence of the steaming process was the reduced DM content 76%. As hay with <84% DM has been reported to have an increased probability of mold formation [3,30], the increase in moisture might enhance the susceptibility of steamed hay to mold formation if not consumed soon after steaming. Thus, a possible effect of an additional storage at room temperature for 24 hours was evaluated. However, the additional storage showed no deleterious effects on hay nutritive value. Research investigating the effect of time on the microbial concentration in hay left for up to 24 hours poststeaming is lacking. Similar to our results, Brookes and Lambert [14] observed that the reduction of bacteria and mold concentrations in the hay after steaming at 100 C was maintained for up to 10 hours poststeaming. Furthermore, palatability was not affected when steamed hay was left for up to 24 hours compared with freshly steamed hay. Thus, hay can be steamed and fed up to 24 hours later without compromising its hygienic quality. 5. Conclusions In conclusion, steaming of hygienically compromised hay is a feasible long-term strategy for reducing mold and yeasts and is suitable for providing hygienically clean hay to horses while conserving minerals and most nutrients. Steaming at different temperatures and time durations, as well as a possible additional storage at room temperature after steaming, showed no effect on the mold and yeast counts. However, for the reduction of Bacillus spp. counts, a temperature treatment of 100 C is required. Steaming of normal-quality hay leaches minerals, such as Na and Cu, and therefore does not provide any benefit compared with unsteamed hay. Acknowledgments The authors acknowledge H. von Butler and S. Begic for their help during the experiment. The authors thank A. Dockner and M. Wild (Institute of Animal Nutrition and Functional Plant Compounds, Vetmeduni Vienna) for their assistance with laboratory analyses and experimental material preparation. Financial disclosure This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jevs.2018.12.024. References [1] Longland A, Barfoot C, Harris P. Effects of soaking on the water-soluble carbohydrate and crude protein content of hay. Vet Rec 2011;168:618.
E. Humer et al. / Journal of Equine Veterinary Science 74 (2019) 28e35 [2] Ellis AD. Biological basis of behaviour and feed intake. In: Longland AC, Coenen M, Miraglia N, editors. The impact of nutrition on the health and welfare of horses. Wageningen: EAAP Publications; 2010. p. 53e74. [3] Martinson K, Coblentz W, Sheaffer C. The effect of harvest moisture and bale wrapping on forage quality, temperature, and mold in orchardgrass hay. J Equine Vet Sci 2011;31:711e6. [4] Clarke A, Madelin T. Technique for assessing respiratory health-hazards from hay and other source material. Equine Vet J 1987;19:442e7. [5] McGroum B, Dixon P, Halliwell R. Responses of horses affected with chronic obstructive pulmonary disease to inhalation challenges with mold antigens. Equine Vet J 1993;25:261e7. [6] Beeler-Marfisi J, Clark M, Wen X, Sears W, Huber L, Ackerley C, Viel L, Bienzle D. Experimental induction of recurrent airway obstruction with inhaled fungal spores, lipopolysaccharide, and silica microspheres in horses. Am J Vet Res 2010;71:682e9. [7] Longland A, Barfoot C, Harris P. Effect of period, water temperature and agitation on loss of water-soluble carbohydrates and protein from grass hay: implications for equine feeding management. Vet Rec 2014;174:68. [8] Mack S, Dugdale A, Argo C, Morgan R, McGowan C. Impact of water-soaking on the nutrient composition of UK hays. Vet Rec 2014;174:452. [9] Hansen T, Fowler A, Strasinger L, Harlow B, Hayes S, Lawrence L. Effect of soaking on nitrate concentrations in teff hay. J Equine Vet Sci 2016;45:53e7. [10] Moore-Colyer M, Lumbis K, Longland A, Harris P. The effect of five different wetting treatments on the nutrient content and microbial concentration in hay for horses. PLoS One 2014;9:e114079. [11] Earing J, Hathaway M, Sheaffer C, Hetchler B, Jacobson L, Paulson J, Martinson K. Effect of hay steaming on forage nutritive values and dry matter intake by horses. J Anim Sci 2013;91:5813e20. [12] Warr E, Petch J. Effects of soaking on its nutritional quality. Equine Vet Educ 1992;5:169e71. [13] Moore-Colyer M, Taylor J, James R. The effect of steaming and soaking on the respirable particle, bacteria, mould, and nutrient content in hay for horses. J Equine Vet Sci 2016;39:62e8. [14] Brookes R, Lambert P. Investigations into the bacteria (TVC) and fungi concentrations and palatability of hay steamed in the HG600 and left for up to 24 hours before testing. BSc Thesis. Royal Agricultural College; 2012. [15] Kamphues J, Wolf P, Coenen M, Eder K, Iben C, Kienzle E, Liesegang A, €nner K, Zebeli Q, Zentek J. Supplemente zur Tiererna €hrung für Studium Ma und Praxis. 12th ed. Hannover: Schaper; 2014. p. 203e7.
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[16] Naumann C, Bassler R. Handbuch der Landwirtschaftlichen Versuchs- und Untersuchungsmethodik (VDLUFA-Methodenbuch), Bd. III. Die chemische Untersuchung von Futtermitteln. Darmstadt: VDLUFA-Verlag; 2012. [17] Taylor J, Moore-Colyer MJS. The effect of 5 different wetting treatments on the bacteria and mould concentrations in hay for horses. 2013. 6th European equine health and nutrition Congress, Ghent. [18] Vervuert I, Meyer H. Schadwirkungen durch Futtermittel. In: Meyer H, Coenen M, editors. Pferdefütterung. 5th ed. Stuttgart: Enke; 2014. p. 216e7. [19] Kaya G, Sommerfeld-Stur I, Iben C. Risk factors of colic in horses in Austria. J Anim Physiol Anim Nutr 2009;93:339e49. [20] Werners A, Bull S, Fink-Gremmels J. Endotoxaemia: a review with implications for the horse. Equine Vet J 2005;37:371e83. [21] Smith TK, Girish CK. The effects of feed born mycotoxins on equine performance and metabolism. In: Oswald IP, Taranu I, editors. Mycotoxins in farm animal. Kerala: Transworld Research Network; 2008. p. 48e70. [22] Müller C, Nostell K, Brojer J. Microbial counts in forages for horses - effect of storage time and of water soaking before feeding. J Equine Vet Sci 2015;35: 622e7. [23] Moore-Colyer MJS, Payne V. Palatability and ingestion behaviour of 6 polo ponies offered a choice of dry, soaked and steamed hay for 1 hour on three separate occasions. 3 (Part 1). Adv Anim Biosci Healthy Food Healthy Anim; 2012. p. 127. [24] Martinson K, Hathaway M, Jung H, Sheaffer C. The effect of soaking on protein and mineral loss in archardgrass and alfalfa hay. J Equine Vet Sci 2012;32: 776e82. [25] Broderick G, Yang J, Koegel R. Effect of steam heating alfalfa hay on utilization by lactating dairy cows. J Dairy Sci 1993;76:165e74. [26] Martinson K, Jung H, Hathaway M, Sheaffer C. The effect of soaking on carbohydrate removal and dry matter loss in orchardgrass and alfalfa hays. J Equine Vet Sci 2012;32:332e8. [27] Blackman M, Moore-Colyer M. Hay for horses: the effects of three different wetting treatments on dust and nutrient content. Anim Sci 1998;66:745e50. [28] MooreColyer M. Effects of soaking hay fodder for horses on dust and mineral content. Anim Sci 1996;63:337e42. [29] NRC. Nutrient requirements of horses. 6th ed. Washington: The National Academic Press; 2007. [30] Montgomery M, Tineo A, Bledsoe B, Baxter H. Effect of moisture-content at baling on nutritive value of alfalfa orchardgrass hay in conventional and large round bales. J Dairy Sci 1986;69:1847e53.
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Original Research
Steaming Conditions Enhance Hygienic Quality of the Compromised Equine Hay With Minimal Losses of Nonfiber Carbohydrates € gmüller b, Qendrim Zebeli a, * Elke Humer a, Manfred Hollmann a, Gerald Sto a
Department for Farm Animals and Veterinary Public Health, Institute of Animal Nutrition and Functional Plant Compounds, University of Veterinary Medicine Vienna, Vienna, Austria b Feed Laboratory Rosenau, Chamber of Agriculture of Lower Austria, Wieselburg-Land, Austria
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 October 2018 Received in revised form 25 November 2018 Accepted 24 December 2018 Available online 28 December 2018
Compromised hygienic quality of hay often is a serious threat to horse health. The study examined the effect of different steaming conditions on molds, bacteria, and yeast as well as on the content of main macro- and micro-nutrients of either normal-quality or hygienically compromised hays. Hay samples were subjected to the following eight treatments each: unsteamed hays as control or hays steamed at 50 C for 90 minutes, at 80 C for 10, 20, or 30 minutes, and at 100 C for 5, 10, or 20 minutes. To evaluate a possible storage effect of the treated hays, subsamples were stored for 24 hours at room temperature. Data indicated increased bacterial counts, molds, and yeasts in hygienically compromised hay. Steaming did not affect total bacterial counts in this hay, but all steaming conditions were equally effective leading to a strong decline of molds and yeasts to a level comparable to the normal-quality hay. Interestingly, only steaming at 100 C for at least 10 minutes reduced Bacillus spp. significantly. In the normal-quality hay, steaming lowered the already low bacterial counts further but caused leakage of sodium and copper. Hay steamed and left for 24 hours at room temperature retained its microbial and nutrient profiles. Overall, the study showed that steaming represents a useful strategy for reducing mold contamination in hygienically compromised hay, with no differences among steaming conditions. Most importantly, to reduce contamination of Bacillus spp. in hygienically compromised hays, steaming at 100 C for at least 10 minutes is required. © 2018 Elsevier Inc. All rights reserved.
Keywords: Bacteria Hay nutritive value Hay steaming Horse Mold concentration Yeast
1. Introduction Forage is a crucial ingredient of the equine diet, which serves both as energy and nutrient source as well as is needed to maintain normal feeding behavior and promote teeth and gut health. Meadow hay is the most common forage fed to horses and can vary significantly in its hygienic quality, depending on the weather during the conservation process and storage conditions [1,2]. In an effort to avoid the presence of adverse weather conditions, hay is
Animal welfare/ethical statement: Animal experimentation was not conducted in this research. Conflict of interest statement: All the authors declare no conflict of interests related to this article. * Corresponding author at: Qendrim Zebeli, Department for Farm Animals and Veterinary Public Health, Institute of Animal Nutrition and Functional Plant Compounds, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. E-mail address: [email protected] (Q. Zebeli). https://doi.org/10.1016/j.jevs.2018.12.024 0737-0806/© 2018 Elsevier Inc. All rights reserved.
often insufficiently dried and baled before achieving recommended moisture contents, resulting in reduced forage quality and mold development [3]. Moldy hay serves as the major source of mold or dust particles [4], which predispose horses to respiratory disorders, including recurrent airway obstruction and chronic obstructive pulmonary disease [5,6]. As horses are highly sensitive to mold particles, there is a need for effective feed management strategies to reduce the number of airborne mold particles released during feeding. Soaking of hay is commonly applied by horse owners in an attempt to reduce dust levels. Furthermore, soaking commonly causes a significant loss of nutrients, that is, water-soluble carbohydrates, soluble protein, and minerals [7e9]. One major disadvantage of soaking hay is the rapid and extensive proliferation of bacteria, which is highly undesirable if the level of contamination increases the number of bacteria above the recommended safe limit and/or if potentially pathogenic bacteria are present [10]. To avoid this aspect, steaming of hay has become more and popular as a management tool to reduce mold and bacterial concentrations [11].
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Steaming has also been attributed an additional benefit such as the reduction of potentially hazardous post-soak liquor [12]. Nevertheless, steaming might also have undesirable consequences as it might leach essential nutrients, including soluble nonfiber carbohydrates (NFC) and protein as well as minerals and trace elements [11,13]. However, little is known about the impact of different steaming conditions especially the combination of temperature and duration steaming on nutritive value and microbial content. A further aspect that has to be considered is that horse owners often owe only one horse or have to restrict the hay intake due to adiposity risk [14]. Thus, it is often not feasible to feed all the steamed hay immediately after steaming than rather preparing the respective hay for 1e2 days in advance. However, there is no information available on the effect on the microbial and nutrient concentrations in hay left for 24 hours poststeaming. Therefore, the main aim of this study was to evaluate the effect of different steaming conditions on microbial concentrations and nutritive values of the hay of the same origin but of different hygienic traits. Furthermore, a possible effect of an additional 1-day storage period after the hay treatments was evaluated, too. 2. Material and Methods 2.1. Hay Preparation and Treatments A round bale hay provided by a local supplier was identified during the routine hay quality control at the Institute of Animal Nutrition and Functional Plant Compounds for the study. After the official organoleptic (sight, smell, color, foreign substances including poisonous plants contamination) and feeding value evaluation [15], the hay was deemed adequate to feed to horses. The hay was free of any foreign material or unpalatable weeds and musty or moldy smell. Also, no unusual dust or visual signs of rain or heat damage were observed. The hay was field-dried Austrian meadow hay of the first cut harvested at bloom, consisting mainly of common meadow-grass (Poa pratensis L.), orchard grass (Dactylis glomerata), false oat-grass (Arrhenatherum elatius), and meadow foxtail (Alopecurus pratensis L.), harvested in 2017 and conserved for 12 months. The bale weighed approximately 300 kg and was divided into two equal sections. While one section was kept in a dry, nonheated room to serve as normal-quality hay, the other half was moistened with distilled water (about 0.7 L/kg of hay) and allowed to mold for 6 days at room temperature to prepare the hygienically compromised hay. The hygienically compromised hay showed visual signs of mold and moldy smell, starting almost 1 day after the moistening treatment. The experiment was set up in a completely randomized arrangement. Three randomly collected replicates of each hay quality type were individually subjected to one of the following treatments: (1) unsteamed (original hay) where no additional treatment was applied to the hay; (2) steamed at 50 C for 90 minutes; (3) steamed at 80 C for 10 minutes; (4) steamed at 80 C for 20 minutes; (5) steamed at 80 C for 30 minutes; (6) steamed at 100 C for 5 minutes; (7) steamed at 100 C for 10 minutes; and (8) steamed at 100 C for 20 minutes. The steaming was carried out in a specifically designed steamer composed of a preserving boiler (TZS, First Austria, Article-No.: FA-5180) with a total capacity of 27 L and a continuously adjustable thermostat. A total of 2 L of distilled water was placed in the bottom section of the container, and when the targeted temperature was reached, 750 g of hay was put in laundry nets and placed on a plastic insert on the bottom of the steamer. After closing the steamer, the hay was left to steam for either 5, 10, 20, 30, or 90 minutes. The temperature was confirmed with the thermometer (Basetech ET-1; Conrad, ArticleNo.: 1396295) located within the hay sample. Subsequently, the steamer was turned off, opened, and the hay was removed. The
29
steamer was washed with water and disinfected with ethanol between each treatment to reduce the chance of contamination. Poststeaming the hay was stored at 4 C until further analysis. All hay samples were kept on ice during transportation before sample admission. In addition, the effect of a 1-day storage at room temperature was evaluated for the following treatments: (1) unsteamed (original hay); (2) steamed for 90 minutes at 50 C; (3) steamed for 30 minutes at 80 C; (4) steamed for 20 minutes at 100 C. Therefore, poststeaming hay samples were stored for 24 hours in a dry, nonheated room before further analysis. 2.2. Microbial and Nutrient Analyses Microbial counts (aerobic, mesophilic bacteria, molds, and yeasts) were determined of all samples. For analysis of microbial counts, 10 g from each sample was placed into a sterile Stomacher bag, and 90 mL of sterile phosphate-buffered saline solution (PBS; sodium hydrogen phosphate dihydrate, sodium chloride, sodium dihydrogen phosphate dihydrate, and neutralized bacteriological peptone) was added resulting in a 1:10 dilution. Bags were tightly closed to exclude air and were agitated for 30 minutes at ambient temperature. Colony-forming units (CFU) were determined from serial dilutions in PBS (102e104), which were made from a 1 mL subsample of the supernatant fluid. From dilutions 102 and 104 aliquots of 100 mL each were spread evenly on agar plates. Nutrient agar in accordance with Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung, two plates per dilution, was used to determine the CFU of aerobic mesophilic bacteria. Dichloran glycerol agar (DG 18 Agar) and Sabouraud dextrose agar, two plates per dilution, were used to determine the CFU of molds and yeasts. Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung agar was incubated at 37 C, whereas DG 18 Agar and Sabouraud agar plates were incubated at 27 C before colonies were counted and reported as CFU per gram. Nutrient analyses of hay samples were conducted in duplicate according to the German Handbook of Agricultural Experimental and Analytical Methods [16]. The dry matter (DM) of the hay samples was determined by oven drying at 105 C for 4 hours (method 3.1). Ash was analyzed by combustion of samples at a temperature of 550 C (method 8.1). The crude protein (CP) content was determined using the Dumas method (method 4.1.2). The content of crude fiber (CF) was determined gravimetrically after chemical digestion with sulfuric acid and was expressed exclusive residual ash following method 6.1.1 of VDLUFA [16]. Ether extract (EE) was analyzed by extraction with petroleum ether (method 5.1.1). The content of NFC was calculated as NFC ¼ DM (Ash þ CP þ EE þ NDF). Sugar was analyzed via the Luff-Schoorl method according to method 7.1.1 of VDLUFA [16]. The contents of phosphorus (P), calcium (Ca), magnesium (Mg), potassium (K), sodium (Na), iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu) were determined by inductively coupled plasma emission spectroscopy following method 10.8.2 of VDLUFA [16]. 2.3. Statistical Analyses Statistical analyses were performed by analysis of variance using the MIXED procedure of SAS (version 9.4; SAS Institute Inc, Cary, NC). For each variable tested, the model included the fixed effects of treatment, hay quality, the interaction between hay quality and treatment, and the random effect of the replicate within the feed. As no interaction between hay quality and treatment was observed, normalquality and hygienically compromised hays were analyzed separately. To evaluate a possible storage effect, further analyses were carried out considering only the respective steaming treatments
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E. Humer et al. / Journal of Equine Veterinary Science 74 (2019) 28e35
that underwent the postprocessing test later on compared with their counterparts that were immediately stored at 4 C after steaming. As no effect of the time duration within steaming temperature as well as an additional storage for 24 hours was observed, results from all treatments within one temperature were pooled and subjected to further statistical analysis considering the effect of temperature, hay quality, and their interaction. Degrees of freedom were estimated with the method of KenwardeRoger. The PDIFF option was used for each least square mean (LSM) comparison. Differences among LSM with P .05 were considered as representing significant differences, and differences with P > .05 and P .10 were considered to represent a trend. 3. Results 3.1. Effect of Steaming on the Microbial Contamination Table 1 summarizes the microbial analyzes of the normalquality hay subjected to different steaming conditions. Microbial analyses confirmed the organoleptic evaluation showing the presence of only 5.3 105 bacteria and 1.7 103 mold CFU/g hay, whereas no yeast was detected. Steaming caused a reduction of the total bacteria count by an average 96% irrespective of the steaming conditions (P < .01). The overall low mold content in the untreated hay was reduced by 89% when hay was steamed but did not reach statistical significance (P ¼ .29). As summarized in Supplementary Table 1, additional storage of the steamed hays at room temperature for 24 hours did not affect the microbial counts compared with the samples that were immediately analyzed after steaming. The hygienically compromised hay contained 11.9 106 bacterial CFU/g, 5.3 106 mold CFU/g, and 1.3 105 yeast CFU/g (Table 2). Although the total bacterial counts were not affected by steaming (P ¼ .57), a more detailed investigation of the bacterial groups revealed a significant effect of the steaming at 100 C to reduce Bacillus spp. Indeed, multiple comparisons among treatments showed that solely steaming at 100 C for 10 minutes (P ¼ .06) or 20 minutes (P ¼ .05) was effective in reducing Bacillus spp. compared with untreated hay by on average 88%. A strong reduction in mold counts (99.5%) was observed for all steaming treatments (P ¼ .04), showing similar effectiveness among all investigated species (Aspergillus niger, Mucor, Aspergillus fumigatus, and Rhizopus). Yeast counts were reduced by 99.9% when hygienically compromised hay was steamed, showing a statistical tendency when multiple comparisons of the steaming treatments with the original hay were carried out (P .08). Storage of steamed hays showed no effect on the microbial counts (Supplementary Table 2).
3.2. Effect of Steaming on the Nutritive Profile As demonstrated in Table 3, steaming of normal-quality hay caused a 23% reduction in the DM compared with the original hay (P ¼ .08). No differences in the content of crude nutrients were found, whereas among minerals, measured Na was reduced by on average 33% (P < .01), and Cu tended to be reduced by 26% (P ¼ .07) when hay was steamed. However, no differences among the seven different steaming methods were found. As summarized in Supplementary Table 3, no differences between samples analyzed immediately after steaming compared with samples stored for 24 hours at room temperature were found. Steaming of hygienically compromised hay reduced the DM content by on average 32% (P ¼ .03) and caused a tendential decrease in NFC (7%, P ¼ .09, Table 4). Overall, no effect of the different time durations within steaming temperature groups was observed. Supplementary Table 4 shows the effect of an additional storage period on the nutrient content of steamed hygienically compromised hay samples. There was an effect on the DM content, showing an increase by on average 31% with storage (P ¼ .01). However, no differences in nutrient profiles between hay analyzed immediately after steaming and hay stored for 24 hours were found. 3.3. Interactive Effects of Steaming and Hay Quality on the Microbial Contamination Fig. 1 shows the interactive effect of steaming temperature and hay quality on microbial contamination. Compared with the original hay, the total bacterial count (P ¼ .09; Fig. 1A) and the Bacillus spp. were only reduced in hygienically compromised hay when steamed at 100 C (P ¼ .06; Fig. 1B). Among mold (Fig. 1C) and yeast (Fig. 1D), all steaming treatments caused a strong decline to a level comparable to the normal-quality hay. 3.4. Interactive Effects of Steaming and Hay Quality on the Nutritive Profile Several nutrients were affected by the hay quality (Supplementary Table 5 and Fig. 2), showing a decrease in NFC, sugar, and digestible energy when hay was hygienically compromised (P .01), whereas the opposite was observed for CP and CF (P .06). Among minerals, higher contents of P, K, and Mn were measured in hygienically compromised hay compared with normal-quality hay (P .07). Fig. 2 shows the interactive effect of steaming temperature and hay quality on nutrient concentration. An interaction between hay
Table 1 The effect of steaming on bacterial, mold, and yeast concentrations in the normal-quality hay expressed as geometric mean colony-forming units 103 per gram of hay. Item
Bacteria Yellow Pigmented Bacteria Bacillus spp. Molds A. niger Mucor A. glaucus A. fumigatus Rhizopus Yeasts
Treatment Unsteamed
50 C, 90 min
80 C, 10 min
80 C, 20 min
80 C, 30 min
100 C, 5 min
100 C, 10 min
100 C, 20 min
528a 135a
10.3b 5.13b
17.0b 2.80b
19.7b 6.93b
10.7b 6.63b
43.0b 3.78b
15.7b 0.863b
29.0b 10.3b
5.20b 1.00 1.00 0 0 0 0 0
13.6b 0.333 0.333 0 0 0 0 0
12.7b 0 0 0 0 0 0 0
4.03b 0 0 0 0 0 0 0
39.2b 0.667 0.333 0.333 0 0 0 0
14.8b 0 0 0 0 0 0 0
18.7b 0.333 0.333 0 0 0 0 0
392a 1.67 1.47 0 0 0 0.200 0
Abbreviations: A., Aspergillus; SEM, standard error of the mean. a and b indicate differences among treatments at a level of P .05.
SEM
P Value
66,03 20.398
<.01 <.01
47.715 0.553 0.485 0.118
<.01 .29 .26 .47 d d .47 d
d d 0.070 d
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Table 2 The effect of steaming on bacterial, mold, and yeast concentrations in the hygienically compromised hay expressed as geometric mean colony forming units 103 per gram of hay. Item
Treatment
Bacteria Yellow Pigmented Bacteria Bacillus spp. Molds A. niger Mucor A. glaucus A. fumigatus Rhizopus Yeasts
Unsteamed
50 C, 90 min
80 C, 10 min
80 C, 20 min
80 C, 30 min
100 C, 5 min
100 C, 10 min
100 C, 20 min
11,883 2,950
10,500 6,253
11,033 602
13,867 1,269
10,000 6,850
60,233 602
5,275 4,042
1,107 110
10,431a 0b 0b 0b 0 0 0b 0z
12,539a 58b 26b 0.777b 0.035 17 6b 0.973z
8,933ab 5,267a 1,407a 610a 0 2,113 963a 133y
4,247b 3b 0b 0.003b 0 0 0b 0z
3,150b 2b 0.6b 0.133b 0 0.933 0b 0z
5,421bc 3b 0.027b 0b 0.133 0.133 0b 0z
1,233c 6b 0.500b 0b 0.500 3 2b 0z
997c 101b 0.051b 15b 10 10 15b 0.333z
SEM
P Value
4,599 3,332
.57 .65
2,858 1,083 332 134 0.0035 0.573 0.173 47.1
.07 .04 .09 .06 .48 .19 .01 .47
Abbreviations: A., Aspergillus; SEM, standard error of the mean. a, b, and c indicate differences among treatments at a level of P .05. y and z indicate differences among treatments at a level of P > .05 and .10.
quality and steaming temperature (P ¼ .01) revealed that the sugar content was only reduced by steaming hygienically compromised hay (on average 39% compared with the original hay; Fig. 2A). However, the opposite effect was found for the ash content, where steamed normal-quality hay had, on average, 24% less ash compared with the unsteamed hay (P .10; Fig. 2B). In more details, mainly Mg (19%; P .10; Fig. 2D), Na (35%; P < .01; Fig. 2E), and Cu (27%; P < .01; Fig. 2F) concentrations were lowered when normal-quality hay was subjected to steaming, whereas for P (Fig. 2C), a reduction by, on average, 7% was found in good quality hay, and even an increase was observed in hygienically compromised hay (þ7%; P .10). 4. Discussion 4.1. Effect of Steaming on the Microbial Concentration Across all treatments examined, the results showed that steaming was effective to reduce mold and yeasts, especially in hygienically compromised hays. Although no differences among different steaming temperatures and durations were observed for molds and yeast, total viable bacteria numbers, especially spoilage
indicating bacteria (Bacillus spp.), were solely reduced when the highest temperature treatment (i.e., 100 C) was applied for at least 10 minutes. These results agree with those reported by Moore-Colyer et al [13] who found that steaming of hay at 102 C caused a strong reduction in hay bacteria, whereas steaming at 58 C was not effective in reducing bacteria concentrations. Also, Taylor and Moore-Colyer [17] concluded that hay must be steamed at >90 C for a minimum of 10 minutes for most of the bacteria to be killed. Moore-Colyer et al [10] observed a reduction of 98.6% of bacteria when hay was steamed 50 minutes at about 100 C, whereas soaking hay caused a fivefold increase in the average bacterial contamination. Therefore, soaking might cause the number of bacteria ingested to increase above the recommended safe limit of 2,000,000 CFU/g [15] when normal-quality hay is submerged in water, whereas steaming at 100 C might even decrease the number of bacteria below this limit in contaminated hay. Although information regarding the pathogenicity of spoilage-indicating bacteria is lacking [18], poor feed hygiene, in general, has been linked to digestive disorders such as colic [19]. Furthermore, ingestion of feed-associated gram-negative bacteria also causes the ingestion of lipopolysaccharides, which have been reported to play a pivotal
Table 3 The effect of steaming on hay nutritive values (in g/kg on a DM basis, except DM), digestible energy (in MJ/kg DM), macrominerals (in g/kg DM), and trace minerals (in mg/kg DM) in the normal-quality hay. Item
DM CP CF NFC Sugar EE Ash DE Ca P Mg K Na Fe Mn Zn Cu
Treatment Unsteamed
50 C, 90 min
80 C, 10 min
80 C, 20 min
80 C, 30 min
100 C, 5 min
100 C, 10 min
100 C, 20 min
905a 87.3 269 519 148 22.0 103 9.12 6.03 2.13 2.13 21.0 0.43a 1,833 142 29.5 7.73a
692b 83.3 302 522 157 20.7 71.0 9.36 5.73 2.00 1.67 21.6 0.26b 430 102 25.8 5.33b
762b 83.7 304 515 145 20.7 77.3 9.30 6.27 1.97 1.83 21.5 0.32b 821 118 41.2 5.63b
679b 86.7 296 513 142 20.7 83.0 9.26 5.77 2.07 1.87 21.1 0.30b 1,075 123 22.2 6.33b
647b 82.0 311 511 153 20.7 75.7 9.29 5.07 1.97 1.63 21.9 0.26b 677 100 22.3 5.63b
683b 79.7 310 516 156 19.7 73.7 9.30 4.97 1.93 1.63 22.0 0.27b 674 107 22.7 5.70b
689b 84.3 302 511 151 20.3 82.7 9.25 5.43 2.00 1.73 21.7 0.30b 961 114 22.8 5.97b
720b 80.3 321 500 147 20.3 78.7 9.24 5.27 2.00 1.70 21.2 0.27b 826 118 20.2 5.30b
SEM
P Value
53.2 4.82 14.3 11.8 7.0 0.66 8.71 0.077 0.685 0.057 0.146 0.942 0.035 351.8 13.5 7.40 0.565
.08 .93 .15 .86 .78 .47 .33 .60 .85 .33 .27 .71 .01 .26 .30 .56 .07
Abbreviations: Ca, Calcium; CF, crude fiber; CP, crude protein; Cu, Copper; DE, digestible energy; DM, dry matter; EE, ether extract; Fe, Iron; K, Potassium; Mg, Magnesium; Mn, Manganese; Na, Sodium; NFC, nonfiber carbohydrates; P, Phosphorus; SEM, standard error of the mean; Zn, Zinc. a and b indicate differences among treatments at a level of P .05.
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Table 4 The effect of steaming on hay nutritive values (in g/kg on a DM basis, except DM), digestible energy (in MJ/kg DM), macrominerals (in g/kg DM), and trace minerals (in mg/kg DM) in the hygienically compromised hay. Item
DM CP CF NFC Sugar EE Ash DE Ca P Mg K Na Fe Mn Zn Cu
Treatment Unsteamed
50 C, 90 min
80 C, 10 min
80 C, 20 min
80 C, 30 min
100 C, 5 min
100 C, 10 min
100 C, 20 min
749a 80.7 320 494a 119 21.3 84.3 9.18 4.97 1.97 1.70 22.6 0.32 1,065 136 21.6 6.10
509b 92.7 342 452b 65.3 23.0 89.7 9.14 5.67 2.17 1.93 24.3 0.30 1,021 125 26.4 6.43
547b 91.7 341 443b 60.7 21.0 103 8.99 5.50 2.07 2.03 23.8 0.34 1,566 152 28.2 6.40
533b 85.3 321 489a 102 21.7 82.7 9.21 5.43 2.00 1.83 21.9 0.32 980 133 22.9 6.37
440b 94.3 332 437b 61.7 21.0 115 8.90 6.33 2.20 2.27 23.2 0.36 1,967 157 31.3 6.77
519b 87.7 335 471a 86.3 21.3 84.7 9.18 5.80 2.13 1.87 24.3 0.32 954 125 26.4 7.13
519b 92.7 337 462ab 71.3 22.7 84.0 9.20 6.30 2.13 1.90 23.9 0.27 738 130 22.8 6.07
502b 88.0 325 467ab 76.0 19.7 101 9.03 5.60 2.10 1.97 23.5 0.34 1,576 140 29.8 7.47
SEM
P Value
52.6 3.28 14.9 13.5 14.61 1.69 15.86 0.148 0.472 0.067 0.193 0.95 0.047 667 24.2 3.28 0.396
.03 .12 .91 .09 .11 .91 .77 .74 .46 .15 .63 .57 .93 .89 .97 .34 .19
Abbreviations: Ca, Calcium; CF, crude fiber; CP, crude protein; Cu, Copper; DE, digestible energy; DM, dry matter; EE, ether extract; Fe, Iron; K, Potassium; Mg, Magnesium; Mn, Manganese; Na, Sodium; NFC, nonfiber carbohydrates; SEM, standard error of the mean; P, Phosphorus; Zn, Zinc. a and b indicate differences among treatments at a level of P .05.
role in several equine diseases such as laminitis, acute abdominal disease, recurrent airway obstruction and exertion, and are able to elicit strong immune responses in horses [20]. Thus, the reduction in viable bacteria by steaming hay at 100 C for at least 10 minutes might also improve digestive health in horses. In our study, all steaming treatments were equally effective in reducing the mold contamination. This is contrary to the findings by Moore-Colyer et al [13], where a significant decrease in mold
was only found for the higher temperature (102 C vs. 58 C). One possible explanation for the differing results might be the shorter duration (50 minutes) of the steaming treatment at the lower temperature in the respective study compared with our study (90 minutes). This speculation seems to be supported by Earing et al [11], who tested the effect of steaming on low and moderately moldy hay. In their study, steaming for 90 minutes and reaching a final temperature of 46 C caused a reduction in mold count by 99%
Fig. 1. Effect of different steaming temperatures on the counts of (A) total bacteria, (B) Bacillus spp. (C) mold and (D) yeast expressed as geometric mean colony-forming unit (CFU) per gram of normal-quality (gray) or hygienically compromised (black) hay, respectively. a, b, and c indicate differences among treatments at a level of P .05.
E. Humer et al. / Journal of Equine Veterinary Science 74 (2019) 28e35
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Fig. 2. Effect of different steaming temperatures on the content of sugar (A), ash (B), phosphorus (C), magnesium (D), sodium (E) and copper (F) in normal-quality (gray) or hygienically compromised (black) hay. a, b, c, and d indicate differences among treatments at a level of P .05.
in low moldy and 91% in moderately moldy hays. Also, MooreColyer et al [10] showed a reduction of mold content of 99.7% across the range of low, medium, and highly contaminated hays after steaming at about 100 C for 50 minutes. Overall, it is somewhat unclear as to what an acceptable concentration of mold contamination is with regards to horse feeds. Although it has been suggested that hay containing <1,000,000 CFU mold per gram is considered relatively safe for livestock [15], Martinson et al [3] suggested a threshold of 500,000 CFU/g of mold for hay intended for horse, as horses are known to be more susceptible to mold exposure [21]. Overall, in the present study, both thresholds have been only exceeded in the unsteamed hygienically compromised hay. A further finding was that steaming reduced the number of yeasts below the limiting value for horses in hygienically compromised hay. Although studies investigating the effect of steaming on yeast concentrations are lacking, increased yeast counts have been reported in hay after soaking [22]. Although yeast species present in
forages are generally not considered pathogenic and thus might not pose an immediate health risk to horses, elevated yeast contents might cause excessive gas formation in the stomach and small intestine, thereby paving the way for gas colics [18]. Therefore, it can be suggested that horses affected by respiratory and digestive diseases likely benefit from the reduction in mold contamination when hay is steamed before feeding. 4.2. Effect of Steaming on the Nutritive Profile Overall, the results showed that steaming of normal-quality hays conserved nutrients and energy content while reducing Na and Cu contents. On the opposite, the already decreased content of NFC (mainly sugars) of hygienically compromised hay compared with the normal-quality hay was further reduced by steaming. However, no further decline due to steaming was observed for minerals. Poststeaming DM concentrations were reduced to 69.6% and 51.0% for normal-quality hay and hygienically compromised hay,
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respectively, demonstrating that moisture was absorbed during the steaming process. Previously, it has been speculated that the higher water content, among others, might increase the palatability of steamed hay [10]. In this regard, previous studies demonstrated improved palatability of steamed hay compared with dry or soaked hay [11,23]. A loss of essential nutrients that might occur when hay is submerged in water has been reported to be one of the disadvantages of steaming or soaking hay. In accordance with Earing et al [11], steaming of hay with appropriate hygienic quality showed no effects on forage nutritive values, except a decreasing effect on specific minerals. Regardless of hay quality, no effect on CP and fiber content was observed, which was also observed by Earing et al [11] and Moore-Colyer et al [10,13]. However, inconsistent effects of soaking on CP contents have been reported previously [9,13,24]. In general, a reduction of soluble forage carbohydrates is a claim made by hay steamer manufacturers. Interestingly, steaming showed no effect on sugar in the normal-quality hay but caused a decline by 39% in hygienically compromised hay. A reduction in reducing sugars after steaming alfalfa hay at a temperature ranging from 100 to 110 C for 47 minutes has been observed previously [25]. Furthermore, Martinson et al. [26] observed a reduction in soluble carbohydrates when four different hays were soaked for 15 minutes in water, whereas Moore-Colyer et al [10] found no effect of steaming hays at about 100 C for 50 minutes on watersoluble carbohydrates. Similar to our results, Earing et al [11] found only a reduction in water-soluble and ethanol-soluble carbohydrates when moderately moldy hay was steamed, whereas it remained unaffected in the low moldy hay. The authors speculated that for hay with low mold amounts, higher temperature for a longer duration has to be applied to reduce soluble carbohydrate fractions. However, this is not supported by the present results. Overall, sugar contents were 20% lower in the untreated hygienically compromised hay compared with normal-quality hay. It is likely that the molding process increased the availability of NFC, that is, sugars, rendering these carbohydrates more prone to leaching during the steaming treatment. A disadvantage of steaming or soaking hay might be a loss of essential minerals. Several studies have shown significant losses in minerals (i.e., P, K, and Mg) after soaking hay in water for 15 minutes [8,24,27,28]. Also, losses of Na and Cu have been reported when hay was soaked for 10 or 30 minutes [27]. On the contrary, little effects of steaming on the mineral content have been reported. For instance, Moore-Colyer et al [13] observed that steaming hay at about 100 C for 50 minutes conserved minerals, such as Ca, K, Mg, Na, P, Cu, Mn, and Zn. Also, Blackman and MooreColyer [27] found little effect of steaming at 100 C for 50 minutes on the nutrient content, whereas soaking caused a loss of minerals. Thus, steaming hay may best conserve the total nutrient content. Overall, in the present study, water-insoluble minerals, such as Ca, were not affected by steaming, whereas soluble minerals, that is, Na and Cu, were decreased in the normal-quality hay. The effect of steaming on P content depended on the hay quality, showing a reduction in the normal-quality hay, whereas an increase was observed in hygienically compromised hay. On the contrary, Earing et al [11] observed a decrease in P content when hay was steamed for 90 minutes and reaching a final temperature of 46 C irrespective of the hay quality. However, one has to consider that results are not directly comparable, as the low moldy hay used in their study had about a 10-times higher initial mold concentrations (10,020 CFU/g vs. 1,667 CFU/g in our study), whereas the moderately moldy hay in their study had a much lower mold content (268,102 CFU/g) compared with the hygienically compromised hay used in our study (5,266,670 CFU/g). Furthermore, although P was leached in the normal-quality hay, it is unlikely to be deficient in
horse diets, at least for maintenance and light work. For instance, a horse in light work weighing 500 kg requires 14 g P/d [29]. This requirement would be even exceeded when consuming 10 kg of either steamed or unsteamed hay. Mares and growing horses, however, might experience P deficiencies if fed the steamed hay without adequate supplementation. 4.3. Effect of Storage on Microbial Concentration and Nutritive Profile A consequence of the steaming process was the reduced DM content 76%. As hay with <84% DM has been reported to have an increased probability of mold formation [3,30], the increase in moisture might enhance the susceptibility of steamed hay to mold formation if not consumed soon after steaming. Thus, a possible effect of an additional storage at room temperature for 24 hours was evaluated. However, the additional storage showed no deleterious effects on hay nutritive value. Research investigating the effect of time on the microbial concentration in hay left for up to 24 hours poststeaming is lacking. Similar to our results, Brookes and Lambert [14] observed that the reduction of bacteria and mold concentrations in the hay after steaming at 100 C was maintained for up to 10 hours poststeaming. Furthermore, palatability was not affected when steamed hay was left for up to 24 hours compared with freshly steamed hay. Thus, hay can be steamed and fed up to 24 hours later without compromising its hygienic quality. 5. Conclusions In conclusion, steaming of hygienically compromised hay is a feasible long-term strategy for reducing mold and yeasts and is suitable for providing hygienically clean hay to horses while conserving minerals and most nutrients. Steaming at different temperatures and time durations, as well as a possible additional storage at room temperature after steaming, showed no effect on the mold and yeast counts. However, for the reduction of Bacillus spp. counts, a temperature treatment of 100 C is required. Steaming of normal-quality hay leaches minerals, such as Na and Cu, and therefore does not provide any benefit compared with unsteamed hay. Acknowledgments The authors acknowledge H. von Butler and S. Begic for their help during the experiment. The authors thank A. Dockner and M. Wild (Institute of Animal Nutrition and Functional Plant Compounds, Vetmeduni Vienna) for their assistance with laboratory analyses and experimental material preparation. Financial disclosure This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jevs.2018.12.024. References [1] Longland A, Barfoot C, Harris P. Effects of soaking on the water-soluble carbohydrate and crude protein content of hay. Vet Rec 2011;168:618.
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