1. business and industrial
2. agriculture and forestry
3. crops and seed

Research Article
Received: 20 April 2011
Revised: 22 July 2011
Accepted: 23 August 2011
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.4680
How to improve the hygienic quality of forages
for horse feeding
a
´
Virginie Seguin,
David Garon,b∗ Servane Lemauviel-Lavenant,a
´ Bouchart,c Yves Gallard,d Benoˆıt Blanchet,d
Caroline Lanier,b Valerie
a Emmanuelle Personenia and Alain Ourrya
´
Sylvain Diquelou,
Abstract
BACKGROUND: Improving the hygienic quality of forages for horse nutrition seems to be a reasonable target for decreasing the
prevalence of pulmonary diseases. The aim of the experiment was to study the effects of different agricultural practices on the
main aero-allergens contained in forages, including breathable dust, fungi, mycotoxins and pollens.
RESULTS: Results showed that the late harvest of hay, a second crop or a haylage production provides a good alternative to
increase hygienic quality by reducing fungi contamination and breathable dust content. Barn drying of hay, while having no
effect on breathable dust, similarly reduced fungi contamination. In contrast, when hay was harvested at a lower dry mass
content (750 g DM kg−1 versus 850 g DM kg−1 ), both breathable dust and fungi contaminations were increased, which could at
least be reversed by adding propionic acid just before baling. Zearalenone was detected in different hays, and even in one case,
in breathable dust.
CONCLUSION: Overall, our data suggest that different approaches can be used to increase forage hygienic quality for horse
feeding and thus reduce their exposure to factors involved in equine pulmonary disease.
c 2011 Society of Chemical Industry
Keywords: breathable dust; recurrent airway obstruction; grassland; moulds; mycotoxin; pollen
INTRODUCTION
Pulmonary diseases are the prominent cause of a reduction in
horse performance in the northern hemisphere, where horses
can spend most of the time in stalls. Among them, recurrent
airway obstruction (RAO) has become a major concern for horse
owners. RAO, also known as heaves, or chronic obstructive
pulmonary disease, which is similar to asthma in humans, is an
inflammatory obstructive lower airway disease of the adult horse.
It is characterised by variable clinical signs ranging from exercise
intolerance to mucus secretion or chronic cough, to expiratory
dyspnoea.1,2 The aetiology of RAO is not known precisely but it
seems to be associated with chronic and long-term exposure to
dust containing environmental aero-allergens that originate from
hay and straw. According to Clarke3 and Burrell,4 a high concentration of airborne dust increases both the severity and duration
of the disease. Several airborne dust constituents have already
been incriminated in the aetiology of RAO:5 breathable dust as
physical particles,5 fungal spores such as Aspergillus fumigatus
(Fresenius),5 – 7 pollens7 and endotoxins.8 Because moulds are
possibly involved in RAO, mycotoxins could also be incriminated.
The environmental control of breathing zone around horses,
including a decrease in airborne dust concentrations, appears
essential to reduce the prevalence of RAO.9 Horse breeders have
already adapted their management by keeping horses longer in
pasture,10 using forages and straws that are only slightly dusty,
such as haylage and pellets,5,9,11 increasing stall ventilation12 or
J Sci Food Agric (2011)
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soaking hay.13 However, the improvement of forage quality could
be also a reasonable target.
Some studies have been performed to improve the hygienic
quality of hay to prevent a human form of pulmonary disease,
farmer’s lung disease, which is also associated with inhaling dust
from mouldy straw and hay.14 For horses, only a few investigations
have been undertaken on the hygienic quality of forage and
towards reduction in the prevalence of equine pulmonary disease.
Most of them have concerned mould contamination. Both
bad meteorological conditions (rainfall) during harvest and the
harvesting of hay containing more than 20% water increase mould
contamination during storage.15 – 17 Alternatively, barn drying as
∗
Correspondence to: David Garon, GRECAN EA1772-IFR 146 ICORE, Universit´e de
Caen Basse-Normandie et Centre Franc¸ois Baclesse, Avenue du G´en´eral Harris,
14076 Caen Cedex 05, France. E-mail: [email protected]
a UMR INRA 950 Ecophysiologie v´eg´etale, Agronomie et Nutritions N, C, S, IFR 146
ICORE, Esplanade de la Paix, Universit´e de Caen Basse-Normandie, 14032 Caen
Cedex, France
b GRECAN EA1772- IFR 146 ICORE, Universit´e de Caen Basse-Normandie et Centre
Franc¸ois Baclesse, Avenue du G´en´eral Harris, 14076 Caen Cedex 05, France
c Laboratoire D´epartemental Frank Duncombe, Conseil G´en´eral du Calvados,
14053 Caen cedex 4, France
d Unit´e Exp´erimentale INRA du Pin, Domaine du Pin-au-Haras, 61310 Exmes,
France
c 2011 Society of Chemical Industry
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160
140
Rainfall (mm)
120
100
80
60
40
20
Ja
nu
a
Fe ry
br
ua
ry
M
ar
ch
A
pr
il
M
ay
Ju
ne
Ju
l
A y
ug
Se us
pt
t
em
b
e
O
ct r
N obe
ov
r
em
D be
ec
r
em
be
r
0
Months
Figure 1. Monthly rainfall data during 2008 (shaded bars), compared to
the average of the last 20 years, 1987–2008 (––), obtained from the
meteorological data of the INRA experimental unit of Le Pin au Haras.
well as the use of hay preservatives such as propionic acid, allow
a reduction in mould proliferation.18 Besides, hay contaminated
by soil appears to increase the mould concentration and modify
fungal diversity.16,17
The main aim of this study was to evaluate the impact of several
agricultural practices and production processes on forage hygienic
quality, which was defined by all the airborne dust constituents
possibly incriminated in RAO aetiology (breathable dust, mould,
pollen, mycotoxins). A homogeneous grassland was divided in order to apply different treatments: molehill deposition that may increase soil contamination, rainfall simulation before and after cutting, balling at different dry mass content, early or late harvest, field
or barn drying, haylage production and use of hay preservatives.
These experimentally produced forages were then compared so as
to determine which practices improve hygienic qualities of forages.
MATERIALS AND METHODS
Study site and experimental design
The study was carried out in the INRA experimental unit of Pin-auHaras (Normandy, France, 48◦ 77 N, 0◦ 13 W, 205 m). The climate
is temperate with an average annual temperature of 10 ◦ C and
an average annual rainfall of 780 mm. During summer 2008, the
conditions of hay harvest were favourable because in June and
July, rainfall was below the mean of the previous 20 years (Fig. 1).
A permanent grassland of 4.96 ha was chosen for this study
because its flora is typical of Normandy grasslands. Rough blue´ marsh foxtail (Alopecurus geniculatus
grass (Poa trivialis Linne),
´ Yorkshire fog (Holcus lanatus Linne),
´ creeping buttercup
Linne),
´ white clover (Trifolium repens Linne)
´
(Ranunculus repens Linne),
´ are the main
and perennial rye-grass (Lolium perenne Linne)
species with Braun–Blanquet coefficients of 5, 4, 3, 3, 2 and 2,
respectively.19 Other plant species have been identified in this
grassland: sweet vernalgrass, soft-brome, shepherd’s purse, common chickweed, bull thistle, orchard grass, red crane’s bill, meadow
buttercup, curly dock, bitter dock, spiny sowthistle, dandelion, red
clover. This grassland has been subjected for a long time to a lime
application (1.6 t ha−1 ) every 4 years, alternately with organic fertilisation (15 to 20 t ha−1 of compost and 30 m3 ha−1 of cattle liquid
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V Seguin
et al.
manure) every 2 years, to three annual (spring, after the first cut and
the second cut) applications of N fertiliser for a total of 150–180 kg
N ha−1 year−1 , to an annual chemical treatment of weeds (‘ARIANE’
specific weedkiller; 2 L ha−1 ), and to the destruction of molehills.
In April 2008, eighteen plots of 600 m2 were delineated in a
homogeneous area and separated from each other by corridors of
2 m in order to avoid an edge effect between treatments.
Two periods of cutting and hay harvesting were chosen
according to local usual practices and weather conditions: on
7 June 2008 (early harvest), and on 15 July 2008 (late harvest).
In June, the Poaceae were at the beginning of ear emergence
and by July flowering was finished for most of them. For both
early (CONTe,) and late harvest (CONTl), control plots were cut
at 5 cm, tossed once a day until baling, and the hay baled at a
dry mass (DM) content of 850 g kg−1 of fresh matter (FM) in low
density square bales (123 kg m−3 in average) (Table 1). In the
control sub-plots of early harvest, a second cut was performed on
7 September 2008 (CONTs).
In the remaining sub-plots, different agricultural practices were
applied and/or climatic conditions were simulated at early (e) or
late (l) harvests because of an empirical or a supposed effect on
forage quality (Table 1).
Agricultural practices
Agricultural practices consisting of: (1) harvesting and baling at
750 g DM kg−1 FM (HUMIe, HUMIl), (2) harvesting and baling
at 650 g DM kg−1 FM corresponding to haylage, with bales surrounded by 12 layers of plastic film (HAYLe, HAYLl), (3) baling after
barn drying (shoots were harvested in loose at 650 g DM kg−1 FM
and dried loose in a drier at 25 ◦ C with heating ventilation, before
baling at 850 g DM kg−1 FM) (BARNe), (4) a late first toss occurring
48 h after cutting shoots, which is a frequent practice in Norman
stud farms (TOSSl), (5) a greater number of tosses (two tosses per
day versus one toss per day) (HITOe), and (6) a molehill invasion
which can occur when they are not destroyed, simulated by an
application on 25 June 2008. For this, molehills (approximately
2.5 kg) were collected in close grasslands of the INRA experimental
unit of Pin-au-Haras and developing in same kind of soils. Molehills
were applied in the frequency of one molehill every 10 m2 (MOLLl).
Hay preservatives
Two commercial hay preservatives, a solution of propionic acid
buffered with sodium benzoate (CleanGrain liquid, 5 L t−1 of
ˆ rue
hay provided by Biomin, Parc Technologique du Zoopole,
` Joliot-Curie, 22 440 Ploufragan, www.biomin.nat) and lactic
Irene
bacteria at a rate of 20 g t−1 of hay (provided by a society that
wished to remain anonymous), were also tested before baling of a
hay harvested at 850 g DM kg−1 FM (PROPe, LACTe) or harvested
at 750 g DM kg−1 FM (PRHUe, LAHUe). These hay preservatives
were applied to the hay with a vaporiser before baling (Table 1).
Meterorological conditions
Meteorological conditions were modified artificially to increase the
humidity of cut shoots before baling, by simulating two rainfalls of
10 mm, 24 and 48 h before (RAIBe, RAIBl) and after cutting shoots
(RAIAe, RAIAl) (Table 1).
Production and chemical composition
Each plot resulted in the production of about 25 bales and was
sampled by selecting four hay bales at random, and these were
subsequently used for analysis.
c 2011 Society of Chemical Industry
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Improving the quality of forages for horses
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Table 1. Description of experimentally produced forages and their respective treatments
Treatment
Abbreviations
Control
CONTe
Haylage
Barn drying
Higher number of tosses
(2/ day vs. 1/ day)
Rainfall simulation
before cutting
Rainfall simulation after
cutting
Baling at 85% DM with
lactic bacteria
Baling at 85% DM with
propionic acid
Baling at 75% DM
Baling at 75% DM with
lactic bacteria
Baling at 75% DM with
propionic acid
Control
Haylage
First toss 48 h after cut
Baling at 75% DM
Rainfall simulation
before cutting
Rainfall simulation after
cutting
Presence of molehill
Control
CONTs
Period of harvest
Theoretical DM content
(g kg−1 FM)
Conditioning
Particular treatments
Ø∗
850
In the field Square bale
HAYLe
BARNe
HITOe
650
650
850
In the field Haylage
In barn
Square bale
In the field Square bale
Ø
Ø
2 tosses/day vs. 1 toss/day
RAIBe
850
In the field Square bale
10 mm of rainfall before cut
RAIAe
850
In the field Square bale
LACTe
850
In the field Square bale
PROPe
850
In the field Square bale
HUMIe
LAHUe
750
750
In the field Square bale
In the field Square bale
PRHUe
750
In the field Square bale
850
In the field Square bale
2 × 10 mm of rainfall to 24
and 48 h after cut
Addition of lactic bacteria
before conditioning
Addition of propionic acid
before conditioning
Ø
Addition of lactic bacteria
before conditioning
Addition of propionic acid
before conditioning
Ø
HAYLl
TOSSl
HUMIl
RAIBl
650
850
750
850
In the field
In the field
In the field
In the field
Ø
First toss 48h after cut
Ø
10 mm of rainfall before cut
RAIAl
850
In the field Square bale
MOLEl
850
In the field Square bale
850
In the field
CONTl
Early harvest (7 June
2008)
Drying
Late harvest (15 July
2008)
Second crop (7
September 2008)
Haylage
Square bale
Square bale
Square bale
–
2 × 10 mm of rainfall to 24
and 48 h after cut
Application of molehill every
10 m2
Second cut
∗ No particular treatment.
Information in bold type correspond to agricultural practices that were different from control.
Chemical composition of the forages was also evaluated by
crude fibre, crude protein and mineral quantification so as to
complete the description of experimental forages. In this case,
only forages with the treatments that showed significant effects
on the hygienic quality were analysed. The characteristics of
forages including bale density, dry mass content, mineral mass
content, crude fibre and crude protein contents are summarised
in Table 2.
Hygienic quality measurements
For each treatment, four replicate bales were analysed. For each
replicate bale, hygienic quality measurements were performed
on samples randomly selected by manual grabs from open bales.
Thirteen grabs of about 100 g (10 for analysis of dust and moulds,
one for analysis of pollens, one for analysis of mycotoxins, one for
chemical analysis) were sampled randomly in each bale.
Quantification of breathable dust
The quantification of total airborne dust was adapted from Vandenput et al.5 and standardised after preliminary trials to reduce
the variability of measurements. A hermetic glove box (200 L) was
connected to a gas compressor allowing a constant air flow
(200 L min−1 ) on which environmental dusts were previously
J Sci Food Agric (2011)
removed by using a disposable filter capsule with glass microfibre media with polypropylene housing (600 cm2 , Whatman
HEPA-CAP 36; Whatman Ltd, Paris, France). The glove box was
connected to a second hermetic box (80 L) which contained an
aerosol dust counter (Grimm Model 1.108; Grimm Aerosol Technik GmbH & CoKG, Ainring, Germany) with a sample flow rate of
1.2 L min−1 . This system included an optical chamber in which particles of different size categories (from 0.3 to 20 µm) were counted.
For each hay bale, 10 samples of 100 g DM were analysed and hays
from each treatment were submitted to 40 dust analyses. Each
sample was sealed in a hermetic plastic bag, and further introduced into the glove box. Then, dust-free air was flushed through
the glove box for about 15 min, until no dust was detectable.
The hay sample was then released from the plastic bag using
sealed gloves, and mixed for 30 s. Quantification of the airborne
dust was then carried out for 30 min. Particles were collected on
a polytetrafluoroethylene (PTFE) filter (0.2 µm of pore size) for
microbiological analysis. Filters were kept at +4 ◦ C until analysis.
Microbiological analysis
Each PTFE filter was divided in four pieces and suspended in 5 mL
of sterile water containing Tween 80 (0.05%, w/v). After 30 min
of shaking at 420 rpm, three dilutions of the suspension (10−1 ,
c 2011 Society of Chemical Industry
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V Seguin
et al.
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Table 2. Characteristics of experimentally produced forages including their mass, density and their result of chemical composition
Square bale mass Forage density DM content (g kg−1
FM)
(kg)
(kg m−3 )
MM content (g kg−1
DM)
CF content (g kg−1
DM)
CP content (g−1 kg
DM)
harvest
Mean
SEM
Mean
SEM
Mean
SEM
Mean
SEM
Mean
SEM
Mean
SEM
Early
17.4
25.6
12.1
17.2
16.6
18.9
16.9
16.1
15.1
17.4
17.0
12.0
27.4
11.3
13.3
13.4
10.1
10.3
11.1
0.79
1.70
0.78
1.83
1.47
1.13
2.05
0.96
0.62
0.41
0.16
0.09
1.79
0.72
0.58
1.11
0.29
0.36
1.19
133
217
105
142
134
152
142
135
134
147
153
107
236
98
113
113
90
95
92
5.52
13.05
7.61
13.66
6.71
8.76
19.20
9.12
5.05
5.55
2.75
3.31
15.58
4.13
2.20
8.29
5.85
5.61
7.65
874
555
885
868
859
864
846
836
868
854
837
865
663
895
895
882
924
918
892
3.47
72.16
5.79
5.32
4.31
10.13
8.05
24.04
4.17
11.88
8.16
9.30
20.44
7.38
14.99
4.70
12.96
16.12
12.02
69.8
79.6
74.0
1.37
1.20
1.95
328
359
325
92.6
116
91.0
6.48
1.97
1.78
2.93
2.92
0.88
4.63
4.71
359
328
329
340
344
339
331
371
1.91
342
6.15
310
0.72
249
10.76
3.77
8.70
ND
ND
8.77
7.49
10.16
10.34
12.50
5.95
17.93
12.07
ND
11.08
ND
5.19
ND
20.70
4.45
11.56
1.68
ND
ND
4.07
3.20
4.07
3.29
9.76
4.54
1.65
5.37
ND
2.65
ND
2.54
ND
7.77
Period of
Treatment∗
CONTe
HAYLe
BARNe
HITOe
RAIBe
RAIAe
LACTe
PROPe
HUMIe
LAHUe
PRHUe
CONTl
HAYLl
TOSSl
HUMIl
RAIBl
RAIAl
MOLEl
CONTs
Late
ND
ND
84.8
71.6
71.2
81.7
75.4
77.0
59.8
64.2
ND
63.2
ND
66.3
ND
79.8
88.7
91.9
85.9
88.9
88.7
88.3
49.7
72.9
48.2
45.4
80.9
∗ Abbreviations of the treatments are given in Table 1.
DM, dry mass; MM, mineral mass; CF, crude fiber; CP, crude protein; FM, fresh mass; SEM, standard error of the means; ND, not determined.
10−2 and 10−3 ) were made. One millilitre of each dilution (in
triplicate) was deposited in a Petri dish (90 mm diameter) and
the culture medium, containing malt extract (1.5%)/agar (1.5%)
medium (MEA) complemented with chloramphenicol (0.05%, w/v)
was poured over it. The plates were incubated at 25 and 30 ◦ C and
the colony forming units (CFU) of culturable fungi were counted after 3 and 7 days of incubation. Fungal concentration, expressed as
the colony forming units per cubic metre of air (CFU m−3 ), was determined. Colonies were identified after subculturing on MEA. Aspergilli and Penicillia were cultivated and identified on Czapek yeast
autolysate agar (CYA) and 25% glycerol nitrate agar (G25N),20 and
Fusaria were cultured on potato dextrose agar medium (PDA).21
The purity of each strain and its identity were checked through
macro- and microscopic examinations.20,22 – 26 For each treatment,
the mycoflora diversity was determined by the Shannon & Weaver
index.27 The Shannon & Weaver
index (H ) was estimated according
to the expression H = − (pi × log2 pi ), where pi = ni /N, pi is the
relative abundance of each group; ni is the number of individuals
of species, i; and N is the total number of individuals. A value near
zero would indicate that every species in the sample are the same.
Liquid extraction of dust
A liquid extraction of dust was carried out to determine the
pollen concentration and the mineral matter content of dust to
estimate the soil contamination of hay. For each hay bale, 100 g
was aliquoted and shaken for 30 min with 1.5 L of distilled water.
Pollen quantification
Five replicates of 100 µL from the above solution were analysed
by mounting microscope slides according to the Wodehouse
method with glycerine jelly stained with basic fuchsine.28 Then,
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microscope slides were examined under a Nikon inverted
microscope DIAPHOT-TMD (Nippon Kogaku K.K., Tokyo, Japan).
Pollens were then counted.
Mineral mass of dust and soil contamination
One litre of the above solution was filtered on an ashless filter
(90 mm diameter) using a Buchner
funnel connected to a flask.
¨
The filters were then dried at 100 ◦ C, until a constant mass for
dry mass determination, before burning at 550 ◦ C for 48 h in
order to determine the ash content. Previous analysis showed
that the ash content of plant material was close to 6% while the
percentage of ash determined from soil samples was about 90%.
The contamination of dust by soil was therefore estimated by the
dilution of soil ash according to expression: % soil contamination
= [(% ash − 6) × 100]/(90 − 6), where % ash corresponds to the
ash obtained after combustion, and expressed as % of dry mass.
Extraction and purification of mycotoxins from hay
For each bale, 100 g DM of hay were randomly selected and
homogenised in a blender, and then an aliquot of 5 g was
weighed in an Erlenmeyer flask. Sixteen mycotoxins (aflatoxin B1 ,
aflatoxin B2 , aflatoxin G1 , aflatoxin G2 , aflatoxin M1 , alternariol,
citrinin, diacetoxyscirpenol, fumagillin, fumonisin B1 , fumonisin
B2 , gliotoxin, ochratoxin A, T-2 toxin, verruculogen, zearalenone)
were extracted with 100 mL of methanol/water (80 : 20, v/v)
using an Ultra-Turrax basic T25 homogeniser (IKA-Werke, Staufen,
Germany), then shaken on a rotary shaker for 60 min at 100 rpm
and finally centrifuged at 7000 × g for 15 min (10 ◦ C).
The supernatant (15 mL) obtained from the previous centrifugation was diluted in 90 mL of ultrapure water, acidified with
400 µL of acetic acid and was then purified through an Oasis HLB
c 2011 Society of Chemical Industry
J Sci Food Agric (2011)
Improving the quality of forages for horses
www.soci.org
(6 mL, 200 mg) cartridge (Waters, Milford, MA, USA), previously
conditioned with 5 mL of methanol and 5 mL of ultrapure
water. The cartridge was washed with 2 mL of ultrapure water.
Mycotoxins were eluted with 5 mL of methanol followed by 10 mL
of methyl t-butyl ether (MTBE)/methanol (90 : 10, v/v). The eluted
mycotoxins were evaporated in a parallel evaporator (Syncore
polyvap, Buchi
Labotechnik AG, Flawil, Switzerland) and finished
¨
to dryness under a stream of nitrogen. The final residue was dissolved in 1 mL of a mixture of acetonitrile/water (10 : 90, v/v) and
then filtered through a Millex HV 0.45 µm filter before injection for
high-performance liquid chromatography–mass spectrometry
(HPLC-MS). The analytical recoveries and the quantification limits
´
have been previously described by Seguin
et al.17
Extraction and purification of mycotoxins from dust
Fourteen mycotoxins (aflatoxin B1 , aflatoxin B2 , aflatoxin G1 , aflatoxin G2 , aflatoxin M1 , alternariol, deoxynivalenol, diacetoxyscirpenol, fumagillin, gliotoxin, nivalenol, ochratoxin A, T-2 toxin,
zearalenone) were also extracted from dust. Each PTFE filter previously used to collect dust was divided in four pieces and suspended
in 10 mL of acetonitrile acidified with acetic acid (5‰, pH 3) then
put in an ultrasonic bath for 3 min at a maximum power of 100
and shaken during 10 min at 5% of maximum power on a rotary
shaker (VWR VX-2500 Multi-tube Vortexer). This step was carried
out twice. The two supernatants obtained were then evaporated
in a parallel evaporator (Syncore polyvap; Buchi
Labotechnik) and
¨
finished to dryness under a stream of nitrogen. The final residue
was dissolved in 500 µL of a mixture of acetonitrile/water (10 : 90,
v/v) and then filtered through a Millex HV 0.45 µm filter before
injection into the HPLC-MS system. For all mycotoxins, the analytical recoveries were above 55% and the quantification limits were
7.5 ng per filter.
Multi-mycotoxin detection by HPLC-MS
Liquid chromatography was performed using Agilent Technologies series 1100 (Palo Alto, CA, USA) quaternary pump coupled
with an autosampler and an SL model mass spectrometry detector.
The analytes were chromatographed at 40 ◦ C on a 150 × 2.1 mm
i.d., 5 µm, Zorbax SB-C18 column (Agilent Technologies) with a
Securityguard C18 4 × 2 mm cartridge (Phenomenex, Torrance,
CA, USA) allowing the separation of 18 mycotoxins (aflatoxin B1 ,
aflatoxin B2 , aflatoxin G1 , aflatoxin G2 , aflatoxin M1 , alternariol,
citrinin, deoxynivalenol, diacetoxyscirpenol, fumagillin, fumonisin
B1 , fumonisin B2 , gliotoxin, nivalenol, ochratoxin A, T-2 toxin,
verruculogen, zearalenone). Mycotoxins were separated using an
elution gradient with acetonitrile (solvent A) and water acidified
with 0.5% acetic acid (pH 3) (solvent B). The gradient program
was: at time zero, 5% solvent A; linear gradient to 15% solvent A
within 3 min; to 30% solvent A in 11 min; and to 50% solvent A
in 6 min; and finally, to 70% solvent A in 7 min. The flow rate was
400 µL min−1 . The sample injection volume was 10 µL.
Mass spectrometry was performed on a quadrupole analyser
equipped with an electron spray ionisation source and operated
in positive and negative modes. The parameters used for the mass
spectrometer in all experiments were as follows: capillary voltage,
3.0 kV; solvent gas, 720 L h−1 ; evaporation temperature, 350 ◦ C;
pressure of nebulisation, 35 psig.
Statistical analysis
Statistical analyses were carried out with the statistical software
MINITAB (version 13.20, copyright 2000; Minitab Inc., State College,
J Sci Food Agric (2011)
PA, USA). As the data did not fit the parametric test conditions,
we chose to use the non-parametric test of Kruskall–Wallis
to test the effect (1) of harvest time (early, late and second
harvest) for controls, and (2) of treatments for each harvest time.
When treatment effects were significant, signed rank tests were
carried out to determine which forages differed from each other
(P < 0.05).29
RESULTS
Breathable dust content
Breathable dusts of a diameter lower than 5 µm, which is one
of the main factors associated with RAO, represented about 99%
of the total dust. As these two parameters, breathable and total
dust, are highly correlated, only breathable dust data is presented
(Table 3).
Breathable dust in controls varied significantly between harvest
times (H = 19.77, P < 0.001). Hay harvested as a second crop
(CONTs) was less contaminated by dust (51 × 106 particles g−1
of hay) than early harvested hay (69 × 106 particles g−1 of hay)
(CONTe) (Table 3).
For each harvest, the applied treatments modified dust content
(early harvest: H = 172.27, P < 0.001; late harvest: H = 208.06,
P < 0.001). In the early harvest, haylage (HAYLe) was less dusty,
with only 7 × 106 particles g−1 of hay, than other treatments
including the control. The use of lactic bacteria on hay harvested
at 850 g DM kg−1 FM (LACTe) caused a decrease in dust
contamination by about 20% but induced an increase in dust when
hay was harvested at 750 g DM kg−1 FM (LAHUe). The propionic
acid application was not efficient when hay was harvested at
850 g DM kg−1 FM (PROPe), but allowed a reduction in dust
contamination of about 47% (Table 3), when hay was harvested at
750 g DM kg−1 FM (PRHUe) compared to hay harvested at 750 g
DM kg−1 FM without a hay preservative (HUMIe). Forages affected
by rain after cutting (RAIAe) or harvested at 750 g DM kg−1 FM
(HUMie) were the most contaminated by dust with, 80 × 106 and
87 × 106 particles g−1 of hay, respectively.
Between late harvested hays, a significant difference of
breathable dust was also observed (Table 3). As for early harvested
hays, the late harvested haylage (HAYLl) was the least dusty with
13 × 106 particles g−1 of hay, while for control (CONTl), 61 × 106
particles g−1 of hay were measured. At the other extreme, the
rainfall before cutting treatment (RAIBl) was the dustiest with
79 × 106 particles g−1 of hay.
Mould contamination of airborne dust
Significant effects of treatments on fungal contamination were
observed between the controls of different harvests (H = 15.60;
P < 0.001). Indeed, forage harvested in the second crop (CONTs)
was less contaminated by mould than control forage harvested
during the first cut (CONTe) (Table 3).
Fungal contamination varied also significantly between treatments during early (H = 66.20; P < 0.001) and late (H = 30.98;
P < 0.001) harvests. Amongst the forage harvested precociously,
haylage (HAYLe) with 1215 CFU m−3 and hay dried in barns
(BARNe) with 25 104 CFU m−3 (Table 3) were the less contaminated by moulds. Harvesting at 750 g DM kg−1 FM (HUMIe, HUMIl)
provoked an increase of about 61% in mould proliferation compared to control [early (CONTe) and late (CONTl) harvests]. On the
other hand, the use of propionic acid on hay harvested at 750 g DM
kg−1 FM (PRHUe), allowed a reduction in this proliferation by about
c 2011 Society of Chemical Industry
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´
V Seguin
et al.
www.soci.org
Table 3. Breathable dust content (diameter <5 µm, n = 40) and mean counts of colony-forming units of viable spores per cubic metre of air (CFU
m−3 ) (n = 24) in the different experimentally produced forages
Breathable dust (106 particles
g−1 of hay)
Treatment∗
Time of harvest
CONTe
HAYLe
BARNe
HITOe
RAIBe
RAIAe
LACTe
PROPe
HUMIe
LAHUe
PRHUe
Treatment effect
CONTl
HAYLl
TOSSl
HUMIl
RAIBl
RAIAl
MOLEl
Treatment effect
CONTs
Early
69
7
69
62
64
80
55
69
87
109
47
Late
61
13
61
64
79
68
62
Second crop
Time of harvest
51
Mean
DE, B
A
DE
CD
D
EF
C
CDE
F
F
B
H = 172.27; P < 0.001
B, AB
A
B
B
C
BC
B
H = 208.06; P < 0.001
A
H = 19.77; P < 0.001
Viable spores (CFU m−3 )
SEM
Mean
3
0.4
2
3
2
4
2
5
5
9
2
1 302 874
1215
25 104
908 767
660 104
2 800 099
877 715
575 719
3 356 814
33 476 979
417 121
3
1
3
1
4
3
2
113 609
4874
69 229
632 849
124 136
174 503
34 535
2
1390
SEM
BC, B
A
A
BCD
B
CD
BCD
B
D
E
B
H = 66.20; P < 0.001
B, B
A
AB
C
ABC
ABC
AB
H = 30.98; P < 0.001
A
H = 15.60; P < 0.001
356 253
428
2625
425 101
89 689
510 837
394 321
144 338
404 124
6 948 556
95 000
42 408
2012
25 682
123 843
55 929
74 660
14 994
224
∗ Abbreviations of the treatments are given in Table 1.
Mean values with different letters are significantly different (P < 0.05, Kruskall–Wallis test). Treatments were compared between each harvest (bold
capital letters for the early harvest and italics for the late harvest). Then the three controls, early harvest, late harvest and second crop were compared
(normal capital letters).
Values given in bold type correspond to treatment significantly different from control.
88% while this preservative did not modify fungal contamination
when harvested at 850 g DM kg−1 FM. As for early harvested hays,
late harvest haylage (HAYLl) was the least mould-contaminated
forage with 4874 CFU m−3 while hay harvested at 750 g DM kg−1
FM (HUMIl) was the most contaminated by mould with 632 849
CFU m−3 .
Mycoflora analysis revealed the presence of 50 fungal species
distributed in 13 genera (Table 4). Some colonies were not
identified because they did not develop characteristic structures
and were then classified as ‘other’. Profiles of fungal contamination
appeared to vary between the different periods of cutting but
also between treatments of the same harvest. In controls, the
proportion of Aspergillus decreased with the harvest period
and could explain the decrease in total CFU from the early
harvest to the second crop. The Penicillium genus appeared
to develop more during the late harvest in July. Absidia and
Cladosporium genera were identified in 12 different hays. Absidia
varied from 4 CFU m−3 (CONTs) to 130 282 CFU m−3 (LAHUe).
Cladosporium varied from 14 CFU m−3 (MOLEl) to 14 966 CFU
m−3 (LAHUe). Acremonium and Alternaria genera were observed
in six hays with values varying, respectively, between 244 CFU
m−3 (MOLEl) and 28 582 CFU m−3 (LAHUe) for Acremonium, and
46 CFU m−3 (BARNe) to 1484 CFU m−3 (HUMIl) for Alternaria.
Chaetomium, Byssochlamys, Trichoderma, Fusarium, Mucor and
Rhizomucor genera were precisely identified in some hays. The
genus Fusarium, represented by Fusarium culmorum ((W.G. Smith)
wileyonlinelibrary.com/jsfa
Saccardo.), was only observed in hay harvested during the second
crop (CONTs) with 19 CFU m−3 .
Mould diversity of airborne dust
The Shannon & Weaver index was calculated to evaluate fungal
diversity. This index varied with the different periods of cutting
(H = 8.44, P < 0.005), with treatments for early harvest hays
(H = 54.72, P < 0.001) and for late harvested hays (H = 3.53,
P < 0.005) (Fig. 2). Control hay from the early harvest (CONTe)
had a lower fungal diversity than control hay harvested from
the second cut (CONTs). The fungal diversity of the late harvest
control (CONTl) was similar to the two other control hays. Among
early harvested hays, fungal diversity increased with barn drying
(BARNe). The use of preservatives for hay harvested at 750 g DM
kg−1 FM (PRHUe and LAHUe) increased the fungal diversity of hays
(Fig. 2), compared to hay harvested at 750 g DM kg−1 FM without
hay preservatives (HUMIe). When harvested in July, treatments
had no significant effect on the fungal diversity.
Among the identified fungi, Aspergillus and related genera
such as Eurotium and Emericella, were predominant. These genera
represented between 50 and 90% of total CFU for all treatments
(Table 4). Ten species were identified, among which Eurotium
amstelodami (Mangin) and Eurotium repens (de Bary) were the
most frequently represented (Table 5) in the different harvests.
Aspergillus versicolor ((Vuillemin) Tiraboshi) was observed with
values varying from 47 to 72 010 CFU m−3 in all hays but was
c 2011 Society of Chemical Industry
J Sci Food Agric (2011)
Improving the quality of forages for horses
www.soci.org
Table 4. Mean concentrations of different fungal genera (expressed in CFUs m−3 ) obtained after culture at 25 ◦ C from total airborne dust contained
in experimentally produced forages (n = 24)
Fungal genus†
Time of
Treatment∗
CONTe
HAYLe
BARNe
HITOe
RAIBe
RAIAe
LACTe
PROPe
HUMIe
LAHUe
PRHUe
CONTl
HAYLl
TOSSl
HUMIl
RAIBl
RAIAl
MOLEl
CONTs
harvest
A
B
C
D
E
F
Early
G
1 302 874 95 231 10 127
ND
ND 1 197 515
1 215
231
ND
ND
ND
868
25 104
4 480
714
ND
46
18 806
908 767
8 714
2 949
ND
156
896 715
660 104
13 450
ND
1 459 ND
630 541
2 800 099 141 418
ND
ND
ND 2 655 764
877 715
16 274
755
ND
ND
859 655
575 719
16 043
147
1 423 ND
556 012
3 356 814 28 899 27 465
ND
ND 3 194 824
33 476 979 5 875 158 130 282 28 582 ND 27 557 657
417 121
22 593
9 218 1 642 ND
394 370
Late
982 197
15 615
3 913
ND
465
707 234
4 874
378
13
ND
ND
4 446
69 229
4 542
ND
ND
ND
61 999
632 849
44 225
ND
4 167 1 484 584 592
124 136
3 739
ND
ND
ND
117 858
174 503
20 544
ND
ND
ND
154 561
34 535
5 707
173
244
47
27 216
Second crop
1 390
496
4
ND
58
821
H
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND ND
ND 147
ND ND
ND ND
ND ND
ND 91 146
ND 13
ND ND
ND ND
ND ND
ND ND
14
59
8
ND
I
J
K
L
ND
58
93
ND
1 523
ND
1 786
1 599
ND
14 966
158
150
ND
ND
ND
287
1 617
14
15
ND
ND
0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19
ND
ND
290
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
58
513
3 181
13 130
4 377
ND
1 918
1 435
14 966
ND
168 052
63
2 688
5 556
2 395
13 669
1 632
17
M
N
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND 14 23ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
16
ND
ND
18
O
ND
ND
150
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
∗
Abbreviations of the treatments are given in Table 1.
The fungal genera were: A, total; B, other; C, Absidia; D, Acremonium; E, Alternaria; F, Aspergillus; G, Byssochlamys; H, Chaetomium; I, Cladosporium; J,
Fusarium; K, Mucor; L, Penicillium; M, Rhizomucor; N, Trichoderma; O, Trichothecium.
ND, not detected.
†
H′′
2.5 A
AB
B
AB
2
D
A
AB
AB
CD
1.5
B
BC
1 ABCABCD
AB
ABCD
A
A
A
AB
AB
A
0.5
0
Early harvest
Late harvest
Treatments
Second
crop
Figure 2. Shannon & Weaver values estimated for the mycoflora in airborne dust at 25 ◦ C in the different experimentally produced forages. Mean
values (n = 24) with different letters are significantly different (P < 0.05, Kruskall–Wallis and ANOVA). Treatments were compared among each harvest
(bold capital letters for the early harvest and italics for the late harvest). Then the three controls, early harvest, late harvest and second crop were
compared (normal capital letters).
H
absent in haylage for early harvest (HAYle), hay harvested at
850 g DM kg−1 FM with lactic bacteria (LACTe) and at 750 g
DM kg−1 FM with propionic acid (PRHUe). Eurotium herbariorum
(Links) was observed in eight hays and was found particularly in
early harvested hays at 750 g DM kg−1 FM with lactic bacteria
(LAHUe). Aspergillus fumigatus, potentially incriminated in RAO,
was identified in five hays: hay harvested in June with barn drying
J Sci Food Agric (2011)
(BARNe), hay harvested at 850 g DM kg−1 FM with lactic bacteria
(LACTe), control hay of late harvest (CONTl), hay harvested in July
at 750 g DM kg−1 FM (HUMIl) and hay with molehills (MOLEl).
Penicillium was the second most frequently represented genus.
Nevertheless, the Penicillium genus had the highest representation
in terms of species, with 21 different species recorded (Table 6).
The early harvested hays were less contaminated by Penicillium
c 2011 Society of Chemical Industry
wileyonlinelibrary.com/jsfa
´
V Seguin
et al.
www.soci.org
Table 5. Mean concentrations of Aspergillus and Eurotium genera (expressed in CFUs m−3 ) obtained after culture at 25 ◦ C from total airborne dust
contained in experimentally produced forages (n = 24)
Fungal genus†
Time of
Treatment∗
CONTe
HAYLe
BARNe
HITOe
RAIBe
RAIAe
LACTe
PROPe
HUMIe
LAHUe
PRHUe
CONTl
HAYLl
TOSSl
HUMIl
RAIBl
RAIAl
MOLEl
CONTs
harvest
Early
Late
Second crop
A
B
C
D
E
F
G
H
I
J
ND
ND
ND
ND
ND
1 459
ND
ND
ND
ND
ND
ND
ND
ND
ND
143
ND
ND
ND
ND
ND
163
ND
ND
ND
ND
1 447
10 115
217 653
ND
ND
27
ND
ND
ND
146
95
4
ND
ND
ND
ND
ND
ND
ND
ND
4 304
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
139
ND
ND
ND
2 717
ND
ND
ND
ND
159
ND
ND
1 523
ND
ND
28
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
7 441
ND
ND
171
ND
1 447
ND
ND
156
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
143
ND
ND
ND
1 447
ND
163
3 107
18 954
58 345
ND
1 423
25 934
72 010
ND
56 369
65
603
5 880
289
26 908
509
47
176 954
463
11 819
76 884
132 004
1 121 238
396 262
102 046
2 684 411
21 934 741
117 945
308 718
2 831
30 028
496 340
54 102
97 134
17 765
501
ND
ND
12
ND
1 523
ND
4 452
ND
10 056
114 944
ND
ND
ND
11 584
3 007
ND
146
313
ND
1 017 667
405
6 510
816 567
478 060
1 474 722
456 224
451 096
460 003
5 218 310
276 425
341 988
1 524
19 785
70 401
63 180
30 227
8 334
269
∗
Abbreviations of the treatments are given in Table 1.
The fungal genera were: A, Aspergillus alliaceus; B, Aspergillus caespitosus; C, Aspergillus candidus; D, Aspergillus fumigatus; E, Aspergillus parasiticus; F,
Aspergillus sydowii; G, Aspergillus versicolor; H, Eurotium amstelodami; I, Eurotium herbariorum; J, Eurotium repens.
ND, not detected.
†
than late harvested hays. Among forages early harvested hays,
control hay (CONTe), hay harvested at 850 g DM kg−1 FM with
lactic bacteria (LACTe) and hay harvested at 750 g DM kg−1 FM
with propionic acid (PRHUe) were devoided of Penicillium genera.
For the early harvests, Penicillium piceum (Raper & Fennell) was
predominant, while hays harvested in July and from the second
crop (CONTs) were dominated by Penicillium islandicum (Sopp).
Mycotoxins content in forages and in airborne dust
Among the 16 mycotoxins sought in forages, zearalenone was
the only mycotoxin identified from forages. For the early harvest,
haylage (HAYLe), hay harvested at 750 g DM kg−1 FM (HUMIe) and
hay harvested at 750 g DM kg−1 FM with lactic bacteria (LAHUe)
were contaminated by zearalenone (Table 7). For the late harvest,
zearalenone was detected in hay tossed 48 h after cutting (TOSSl),
hay affected by rain after cutting (RAIAl) and hay with molehill
(MOLEl) (Table 7). In the CONTs, zearalenone was detected at
concentrations varying from 25 µg kg−1 to 0.765 mg kg−1 of hay.
Zearalenone was also detected in dust from the hay tossed
48 h after cutting (TOSSl), with a concentration just below the
quantification limits.
Pollens content
Pollen contamination varied, depending on the time of the harvest
(F = 35.28, P < 0.001). The lowest quantity of pollen was detected
in hay harvested from the second crop (CONTs) with 9 × 103 pollen
grains g−1 of hay while the highest contamination was determined
with 83 × 103 pollen grains g−1 of hay in the late harvest control
(CONTl). An effect due to treatments appeared only for the early
harvest (F = 3.88, P < 0.005) (Table 7). The lowest level of pollen
wileyonlinelibrary.com/jsfa
contamination during the early harvest was observed at 750 g DM
kg−1 FM (HUMIe) but was significantly different from only two
treatments (LACTe and PRHUe).
Soil contamination
Evaluation of dust by liquid extraction showed a significant effect
of the harvest time on the contribution of soil particles to the
dust (Table 7) (H = 8, P < 0.05). Hay harvested during the second
crop (CONTs) was characterised by a higher soil contamination
(42.49%) and the late harvested control hay (CONTl) had the
lowest contamination. The effects of treatment on the soil content
in the dust were significant for late harvest hays (H = 22.35,
P < 0.001). Haylage (HAYLl) and hay contaminated by molehill
(MOLEl), and therefore soil, had the highest soil contamination,
while hay harvested at 750 g DM kg−1 FM (HUMIl) contained the
lowest soil contamination.
DISCUSSION
Variation of parameters used for the evaluation of hygienic
quality
All parameters used to evaluate hygienic quality were influenced
by treatments. The most sensitive parameters were dust and
fungal contaminations and they were often correlated. Indeed,
the dustier hay, for example the hay harvested precociously at
750 g DM kg−1 FM (HUMIe), was also the most contaminated by
moulds.
Pollen content did not vary much between treatments, and
depended mostly on the harvest date. As anthesis finished in
c 2011 Society of Chemical Industry
J Sci Food Agric (2011)
Improving the quality of forages for horses
www.soci.org
Table 6. Mean concentrations of the Penicillium genus (expressed in CFU m−3 ) obtained after culture at 25 ◦ C from total airborne dust in
experimentally produced forages (n = 24)
Species in the Penicillium genus†
Time of
Treatment∗
CONTe
HAYLe
BARNe
HITOe
RAIBe
RAIAe
LACTe
PROPe
HUMIe
LAHUe
PRHUe
CONTl
HAYLl
TOSSl
HUMIl
RAIBl
RAIAl
MOLEl
CONTs
harvest
A
B
C
D
E
F
G
H
I
J
K
L
M
Early
ND§
ND
81
ND
5 836
1 459
ND
ND
ND
ND
ND
1 447
ND
ND
ND
ND
5 885
109
3
ND
ND
175
2 097
ND
ND
ND
ND
ND
ND
ND
15 914
ND
ND
ND
140
146
483
ND
ND
ND
140
626
ND
ND
ND
ND
ND
ND
ND
2.894
ND
ND
ND
ND
292
ND
ND
ND
ND
ND
ND
2 918
ND
ND
ND
ND
ND
ND
ND
ND
ND
1 389
ND
ND
156
ND
ND
ND
23
ND
ND
ND
ND
ND
ND
ND
ND
ND
29
ND
4 167
ND
1 021
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
4 854
440
3
ND
ND
ND
ND
ND
ND
ND
ND
1 435
ND
ND
ND
ND
292
ND
ND
ND
17
ND
ND
58
ND
ND
ND
ND
ND
ND
ND
ND
ND
35 552
20
733
ND
575
ND
253
7
ND
ND
58
ND
ND
2 918
ND
1 618
ND
14 966
ND
ND
13
ND
ND
140
ND
173
3
ND
ND
23
146
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
90 775
ND
1 663
ND
700
ND
ND
ND
ND
ND
12
ND
ND
ND
ND
ND
ND
ND
ND
21 471
ND
ND
ND
840
1 471
ND
ND
ND
ND
ND
313
4 377
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Late
Second crop
∗
Abbreviations of the treatments are given in Table 1.
The species in the Penicillium genus were: A, other Penicillium; B, Penicillium brevicompactum; C, P. chrysogenum; D, P. citrinum; E, P. coralligerum;
F, P. fellutanum; G, P. implicatum; H, P. islandicum; I, P. piceum; J, P. roqueforti; K, P. sublateritium; L, P. vellutinum; M, P. verrucosum. The group ‘other
Penicillium’ is constituted of Penicillium species that were identified in one treatment. These species are: P. atramentosum, P. capsulatum, P. expansum,
P. glabrum, P. lividum, P. megasporum, P. spinulosum and P. viridicatum.
§ ND, not detected.
†
September, it was normal that hay produced from the second cut
was weakly contaminated by pollen.
In the same way, the contamination by soil varied slightly
with treatments. Hays harvested late in the first cut were less
contaminated by soil than hays of the early harvest and hays of the
second cut. The conditions were probably more favourable for the
other harvests. Our study also demonstrated that when molehills
are not removed, the soil contamination of hay increased, even
if no effect on breathable dusts or on fungal contamination was
observed. The practice of removing molehills, commonly used by
horse breeders, thus appears important in this respect.
Hygienic quality was influenced by the time of harvest. In this
study, the second cut allowed a reduction in dust and mould
concentrations by about 28% and 99% respectively, compared to
early harvested control hay.
In all hays, the mycoflora were dominated by the genus
Aspergillus and its related genera which represented more than
50% of the total CFU. This result confirms a previous study
´
reported by Seguin
et al.17 Each fungal species is characterised
by its own ecological niche, and each hay harvest time as well
as the different treatments have created particular ecological
conditions that favour particular fungal species. Some differences
in the Aspergillus genus appeared between early and late harvest;
Aspergillus candidus (Link) was only detected in hay harvested
precociously while A. parasiticus (Speare) or A. sydowii ((Bain. &
Sart.) Thom & Church) were only identified in hays harvested from
a late harvest. Some species belonging to the Penicillium genus
were also observed specifically, such as P. atramentosum (Thom)
or P. roqueforti (Thom) that were identified in early harvested hays.
J Sci Food Agric (2011)
Fungal diversity but not overall contamination was amplified with
the use of barn drying or hay preservatives on hay harvested
at 750 g DM kg−1 FM. Allergenic genera such as Alternaria and
Cladosporium were identified in dust from barn dried hay but not
in the control.
Some Aspergilli species are supposed to be incriminated in
pulmonary diseases such as A. fumigatus in equine RAO2,30 and
Eurotium amstelodami in the human pulmonary disease known as
farmers lung disease.31 These two fungi are known to produce toxic
secondary metabolites.32,33 Among toxins of A.fumigatus, gliotoxin
presents immunosuppressive, genotoxic, cytotoxic and apoptotic
effects,34 – 36 verruculogen shows tremorgenic and genotoxic
effects,37,38 fumagillin appeared cytotoxic and genotoxic39,40 and
helvolic acid have a cytotoxic effect.40 Fumagillin, gliotoxin
and verruculogen were not detected in hays produced in
this experiment. Only zearalenone was detected in CONTs,
HAYLe, HUMIe, LAHUe, RAIAl, TOSSl and MOLEl. Zearalenone
has oestrogenic effects on animals and especially pigs41 but has
also shown immunotoxic, hepatotoxic and hematotoxic effects.42
Immune disorders could alter horse performance. One study
showed the development of mycotoxicosis in horses exposed
to maize contaminated with 2.6 mg kg−1 zearalenone.43 Among
the hays in which zearalenone was detected, only CONTs was
contaminated by Fusarium culmorum, a zearalenone producer.
Fusaria are ubiquitous in soil and grow on plants in the field.44
The presence of zearalenone in MOLEl could be explained by the
telluric origin of these zearalenone producing strains.
Other species identified in dust can also be toxigenic (Alternaria
alternata ((Fries) Keissler), P. roqueforti and Trichoderma viridae
c 2011 Society of Chemical Industry
wileyonlinelibrary.com/jsfa
´
V Seguin
et al.
www.soci.org
Table 7. Zearalenone, pollens and soil contamination contained in total dust of the different experimentally produced forages, obtained by liquid
extraction
Pollens (103 pollens
g−1 of hay)
Treatment∗
Time of harvest
CONTe
Early
HAYLe
BARNe
HITOe
RAIBe
RAIAe
LACTe
PROPe
HUMIe
LAHUe
PRHUe
Treatment effect
CONTl
Late
HAYLl
TOSSl
HUMIl
RAIBl
RAIAl
MOLEl
Treatment effect
CONTs
Second crop
Time of harvest effect
Zearalenone (mg kg−1 of hay)
Mean
ND
0.314
ND
ND
ND
ND
ND
ND
1.63
0.232
ND
35
43
43
32
28
32
49
38
26
38
56
ND
ND
2.381
ND
ND
0.260
<QL
<QL to 0.765
Soil contamination of
the total material (%)
SEM
ABC, B
6
ABC
9
ABC
3
AB
4
AB
9
AB
4
BC
7
ABC
4
A
3
ABC
8
BC
2
F = 3.88; P < 0.005
83
C
11
113
24
64
9
60
5
72
10
59
13
63
9
NS
9
A
2
F = 35.28; P < 0.001
Mean
37.66
32.46
31.55
45.21
20.15
26.58
39.04
40.56
25.43
26.35
43.57
SEM
B
7.03
4.25
5.04
3.28
5.36
6.71
2.13
4.91
7.81
12.27
9.81
NS
10.66
B, A
0.58
27.59
C
3.77
10.20
B
1.82
7.23
A
0.34
12.68
B
1.40
13.06
B
2.28
30.64
C
6.14
H = 22.35; P < 0.001
42.49
B
2.59
H = 8; P < 0.05
∗ Abbreviations of the treatments are given in Table 1.
Mean values with different letters are significantly different (P < 0.05, Kruskall–Wallis test and ANOVA test). Treatments were compared between
each harvest (bold capital letters for the early harvest and italics for the late harvest). Then the three controls, early harvest, late harvest and second
crop were compared (normal capital letters).
ND, not detected. NS, not significant. QL, quantification limit (25 µg kg−1 of hay).
Values given in bold type correspond to treatment significantly different from control.
(Persoon)), allergenic (A. alternata, Cladosporium cladosporioides
((Fresenius) de Vries)) or pathogenic, such as Absidia corymbifera
((Cohn) Saccardo & Trotter) which is incriminated in farmer’s lung
disease.31 Another genus, Acremonium, can be an endophyte
and a producer of alkaloids. Although Acremonium bacillisporum
((Onions & Barron) Gams) was not known as a toxinogenic species,
it would be interesting to integrate alkaloids such as lolitrem B in
the multi-mycotoxin method.
Effect of environmental conditions
The decision-making processes around hay production are of
course of great importance, particularly in areas with abundant
rainfall. Producers often need to make a decision between
harvesting hay that is too moist (less than 850 g DM kg−1 FM)
or to take the risk of rainfall after cutting and before harvest.
Harvesting at 750 g DM kg−1 FM during an early or late harvest
resulted in a higher dust content and in mould contamination.
This moisture content probably led to overheating in the bales
and thus, to the proliferation of dust and mould. These results are
in accordance with a previous study,17 and as a consequence, such
hays should be avoided for horse feeding.
The impact of unfavourable meteorological conditions on
hygienic quality was estimated though the simulation of rainfall
before or after cutting. In contrast with the results described by
´
Seguin
et al.,17 rainfall after cutting had no detectable effect on
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contamination by mould. Only rainfall before cutting reduced the
hygienic quality of late harvested hays by increasing hay dustiness.
The previous study17 was undertaken in 2007, a year characterised
by very bad meteorological conditions (heavy rain in June and
July). In this case, hay quality was probably more sensitive to
different environmental events than the hay produced for this
study conducted in 2008, in which drier air during the making
process may have contributed a reduction in the deleterious
effects of simulated rainfalls.
Improvements of the drying step
Some methods have been developed in order to improve hygienic
quality of dry hay and these have been focused mainly on the
drying process. Dalphin et al.45 showed that the use of barn drying
reduced the concentration of thermophilic actinomycetes but
not mould concentrations. In our study, barn drying improved
hay quality by decreasing mould proliferation by about 98%, but
without an effect on the amount of breathable dust. Using such
practices, the mould contamination of breathable dust was similar
for barn dried hay and haylage.
The application of a greater number of tosses is also a practice
used to speed up the drying of hay. On the one hand, this
practice could avoid mould proliferation by providing the best
drying conditions, on the other hand, a higher number of tosses
increases the loss of dry mass,46 and thus could increase dustiness
c 2011 Society of Chemical Industry
J Sci Food Agric (2011)
Improving the quality of forages for horses
www.soci.org
by mechanical damage. These hypotheses were not verified in this
work because an increase of the number of tosses did not modify
hay quality, either by increasing the dustiness or by decreasing
the contamination by moulds.
Effect of storage processes on hygienic quality
Haylage is increasingly used in equine nutrition in order to
reduce equine pulmonary diseases.47 – 49 Accordingly, haylages
produced in the early and late harvests were the least dusty and
contaminated by moulds. These results are in accordance with
´
et al.17 Thus haylage seems
those of Vandenput et al.5 and Seguin
to be an excellent alternative to dry hay because of its low level of
dust and mould and a nutritive quality that is similar or superior
to dry hay.
The use of preservatives could be a relevant alternative
to improve hay quality during the storage process. Different
preservatives such as propionic acid and lactic bacteria are
commonly used in silage and some methods could be adapted to
dry hay. The reduction in the risk of mould contamination during
bad harvest conditions through the use of preservatives has been
observed previously,18,50 and was tested in this experiment using
hay containing only 750 g DM kg−1 FM.
Propionic acid reduced dust and mould contamination, by
approximately 47% and 86%, respectively, compared to hay
harvested at 750 g DM kg−1 FM in June and also reduced
the concentration of Eurotium amstelodami by about 96% in
accordance with the work undertaken by Reboux et al.51 The
lower production of dust from these forages could be explained
by the decrease of mould content and fungal degradation of
forage. The results were not so clear with lactic bacteria. Its
application to hay harvested at 750 g DM kg−1 FM induced a
higher dust and mould contamination. However, the use of lactic
bacteria on hay harvested at 850 g DM kg−1 FM decreased the dust
contamination. These lactic bacteria inoculants are usually used
for silage,52 and some studies revealed their efficiency on hay.53
However, the method of application, and more specifically the
remaining moisture in forage on which the inoculant is sprayed,
must be optimised to allow the growth of bacteria and the
production of lactic acid to repress mould proliferation.
CONCLUSIONS
Even when a good nutritive quality was observed in early harvests,
the experimental production of hay in this study has demonstrated
that the use of a second cut is better adapted for horses as
it leads to good nutritive and health qualities. Health quality
is more dependent on agricultural practices and meteorological
conditions than nutritive quality. This study suggests that hay
quality can be improved by some agricultural practices, for
example, to eliminate molehill; to bale at 850 g kg−1 FM; to use
a barn drying or the application of propionic acid before baling
mostly when enough moisture remains in the hay and to toss
after cutting. Besides, haylage seems to be a good compromise
between nutritive and health values.
ACKNOWLEDGEMENTS
This work was partly funded through a PhD Grant to V. S´eguin
from the Conseil R´egional de Basse-Normandie, while this project
ˆ de Competitivit´
´
` Equine.
has been approved by the Pole
e Filiere
The authors would like to acknowledge the staff of the INRA
J Sci Food Agric (2011)
experimental unit of Borculo (P. Georget, S. Clouard, T. Corbet, M.
Aubry, B. Guibout, J. Levallois and M. Rouillon), from Laboratoire
´
Departemental
Frank Duncombe (M. Houssin and R. Picquet) and
from UMR INRA EVA (D. Ballois, R. Segura and A.F. Ameline) for
´ for
their kind and skillful support, as well as B. de Loynes d’Estree
her efficient help with the analysis of hay dust. We would also
like to acknowledge L. Cantrill for improving the English of the
manuscript.
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