Animal Feed Science and Technology 286 (2022) 115254

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Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Effect of dietary isopropanol on the performance and milk quality

of dairy cows
J.M. Bragatto a, C.S. Parra a, F.A. Piran Filho a, S.M.S. Silva a, J.A.C. Osorio a, S.
C. Buttow a, G.T. Santos a, C.C. Jobim a, L.G. Nussio b, J.L.P. Daniel a, *
a
Department of Animal Science, State University of Maringa, 87020-900 Maringa, Brazil
b
Department of Animal Science, University of Sao Paulo, Luiz de Queiroz College of Agriculture, 13418-900 Piracicaba, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The objective of this study was to examine the effects of isopropanol alone or in combination with
Alcohol ethanol on performance, metabolism, and milk quality of dairy cows. Eight crossbred Jersey ×
Corn silage Holstein cows (527 ± 98 kg body weight; 152 ± 44 days in milk) were allocated in two 4 × 4
Ethyl alcohol test
balanced-Latin squares to receive one of four dietary treatments. Four cows were surgically
2-propanol
adapted with rumen cannula. Diets contained 450 g/kg concentrates and 550 g/kg corn silage
either added with (DM basis) 15 g/kg isopropanol, 15 g/kg ethanol, 15 g/kg isopropanol + 15 g/
kg ethanol, or water (control). Immediately before feeding, corn silage was sprayed with alcohols,
mixed with concentrates, and fed as total mixed rations twice daily. Dry matter intake, total tract
digestibility, ruminal fermentation pattern (pH, volatile fatty acids and NH3), urinary purine
derivatives, milk yield, solids composition and oxidative capacity were not affected by treat­
ments. However, gamma-glutamyl transferase activity increased (P < 0.01) in blood of cows
supplemented with alcohols, indicating that isopropanol and ethanol were absorbed. Moreover,
milk concentration of isopropanol and acetone increased (P < 0.01) in cows receiving iso­
propanol, but not ethanol. Consequently, milk from cows fed corn silage treated with isopropanol
tested false-positive at the ethyl alcohol test in raw milk. In conclusion, dietary isopropanol and
ethanol did not impair the performance of dairy cows, but isopropanol was imparted to milk and
induced false-positive results at the ethyl alcohol test in raw milk.

1. Introduction

Alcohols are common fermentation end-products in silages (Kung et al., 2018). Although ethanol is the main alcohol formed during

Abbreviations: ABTS, 2,2-azinobis-3-ethyl-benzothiazolin-6-sulfonic acid; aNDF, neutral detergent fiber assayed with a heat stable amylase and
expressed inclusive of residual ash; AR1, first-order autoregressive; AST, aspartate aminotransferase; CD, conjugated diene; CP, crude protein; CS,
compound symmetry; DM, dry matter; DMI, dry matter intake; DPPH, 2,2-diphenyl-1-picryl-hydrazyl; ECM, energy-corrected milk; FA, fatty acid;
GC-MS, gas chromatography coupled with mass spectrometry; GGT, gamma-glutamyl transferase; iNDF, indigestible neutral detergent fiber; LCFA,
long-chain fatty acids; MCFA, medium-chain fatty acids; MDA, malondialdehyde; MUFA, monounsaturated fatty acids; PDV, portal drained viscera;
PUFA, polyunsaturated fatty acids; SCFA, short-chain fatty acids; SEM, standard error of the mean; SFA, saturated fatty acids; SCC, somatic cell
count; TBARS, thiobarbituric acid substance; TMR, total mix ration; UN, unstructured; VC, variance components; VFA, volatile fatty acids.
* Corresponding author.
E-mail address: jlpdaniel@uem.br (J.L.P. Daniel).

https://doi.org/10.1016/j.anifeedsci.2022.115254
Received 25 November 2021; Received in revised form 16 February 2022; Accepted 17 February 2022
Available online 22 February 2022
0377-8401/© 2022 Elsevier B.V. All rights reserved.
J.M. Bragatto et al. Animal Feed Science and Technology 286 (2022) 115254

silage fermentation, other alcohols such as 1,2-propanediol, 2,3-butanediol, and 1-propanol have been reported in significant con­
centrations (Kalac, 2011; Hafner et al., 2013). In addition, trace concentrations of methanol, 2-butanol, and isopropanol (i.e., 2-prop­
anol or isopropyl alcohol) may be found in silages (Kalac, 2011; Hafner et al., 2013). When diets containing silages are fed to dairy
cows, alcohols can be metabolized in the rumen fluid, portal drained viscera, and liver, with a small proportion of alcohols passing to
the peripheral blood (Kristensen et al., 2007; Raun and Kristensen, 2009, 2011, 2012). Though, trace amounts of silage alcohols can be
secreted in milk (Shipe et al., 1962; Randby, 2007; Kalac, 2011). Meanwhile, alcohol may be found in raw milk tampered with
extraneous water accompanied of ethanol to mask such adulteration (Wanjala et al., 2018). The presence of ethyl alcohol (i.e., ethanol)
in raw milk samples is by law considered fraud (Brasil, 2018).
Recently we surveyed Brazilian dairy farms where bulk milk samples tested positive using the official method for detecting ethyl
alcohol in raw milk (Brasil, 2018, 2019). In different farms, raw milk collected directly from the udder tested positive, indicating no
fraud and suggesting that alcohol in milk likely originated from corn silage, the sole fermented feed in those herds. Screening alcohol-
positive milk samples by head-space gas chromatography–mass spectrometry (GC-MS) unveiled a higher concentration of isopropanol
but not ethanol, in comparison with negative milk samples from the same farms (J.L.P. Daniel, unpublished data). Moreover, the
analysis of fermentation end-products in corn silage from positive farms revealed a 100-fold increase in isopropanol concentration (~4
g/kg DM) comparatively to values reported in the literature (<50 mg/kg DM; Silva et al., 2017; Gomes et al., 2019; Parra et al., 2019).
Isopropanol is seldomly reported in silage analyses (Kung et al., 2018) and the origin of isopropanol in silage is unknown. Some strains
of Clostridium (e.g., C. beijerinckii syn. C. butylicum) can produce isopropanol, acetone, and butanol (Chen and Hiu, 1986; Xin et al.,
2017), but it is unclear if this occurs in silage. In corn silage, Driehuis et al. (2016) described the occurrence of spores of
lactate-fermenting clostridia (i.e., butyric acid bacteria) and noted that C. beijerinckii represented most of Clostridium population in
poorly-preserved silage containing high counts of butyric acid bacteria (≥4 log MPN/g). Rooke and Hatfield (2003) suggested a
pathway for isopropanol formation via glucose or lactic acid fermentation in silage. The final step is the reduction of acetone to
isopropanol, but other end-products such as butanol and butyric acid were associated to the pathway.
When ingested accidentally by non-ruminant animals, isopropanol is approximately twofold more toxic than ethanol and about as
toxic as methanol, resulting in central nervous system depression, hypotension, vomiting, and abdominal pain (Stice et al., 2018).
However, to the best of our knowledge, the effects of relatively low concentrations of isopropanol in dairy rations and its consequences
on cows’ performance and milk quality are unknown. Therefore, the objective of this study was to examine whether corn silage
enriched with isopropanol, alone or in combination with ethanol, affect the feed intake, ruminal fermentation, urinary purine de­
rivatives, apparent digestibility, blood metabolites, milk yield, milk composition and oxidative capacity. We hypothesize that iso­
propanol can be excreted in milk and that co-supplementation of ethanol may increase the excretion of isopropanol in milk, because of
the co-ingestion of ethanol has been reported to increase the half-life of isopropanol in humans (Pappas et al., 1991). Isopropanol and
ethanol compete for the same metabolic pathway in the liver (i.e., alcohol dehydrogenase enzyme system) (Lieber and Abittan, 1999;
Bruss and Lopez, 2000; Jones and Holmgren, 2015).

2. Material and methods

Animal care and handling procedures were approved by the Ethics Committee for Animal Use of the State University of Maringa
(protocol 4649301019/2019).

2.1. Animals, diets, and treatments

Eight crossbred Jersey × Holstein dairy cows (527 ± 98 kg body weight; 152 ± 44 days in milk) were allocated in two 4 × 4
balanced-Latin squares, with 22-d periods (15 d for adaptation and 7 d for sampling). Four rumen cannulated cows composed one Latin
square and four intact cows composed the other. Cows were housed in a tie-stall barn and milked in a milking parlor at 0700 and 1530
h. During the night, the animals had a period of exercise outside the pens.
Diet was formulated to meet or exceed the nutritional requirements of dairy cows producing 20 kg of milk per day (NRC, 2001). The
experimental diets contained (DM basis) 172 g/kg finely ground dry corn, 154 g/kg soybean meal, 100 g/kg whole cottonseed, 20 g/kg
mineral-vitamin mix, 4 g/kg limestone, and 550 g/kg of corn silage either untreated (CON), added with 15 g/kg of ethanol (ETH),
added with 15 g/kg of isopropanol (ISO), or added with 15 g/kg of ethanol + 15 g/kg of isopropanol (ETH+ISO) (as silage DM basis).
The application rates were defined based on a pre-assay, to reproduce their average contents in corn silage observed in our field survey
(~4.0 g/kg DM). The corn silage used for all treatments was unloaded every morning from a single bunker silo throughout the
experiment. Before each feeding, alcohols were diluted in distilled water (1:2) and sprayed onto the silage. The control silage was
sprayed with the same volume of distilled water to avoid that treatment does not affect silage DM content. After, treated silages were
homogenized with concentrates and fed to the cows as total mixed rations (TMR) at 0800 and 1330 h. The amount of TMR was adjusted
daily to allow 50–100 g/kg as orts.

2.2. Data collection and sampling

Dry matter intake (DMI) was recorded from d 16–22 of each experimental period. Treated silages (including the control), offered
TMR and orts were sampled in those days and composite by animal in each period. Aliquots were dried in an air-forced oven at 55 ◦ C
and ground in a Wiley mill to pass a 1-mm screen (MA340, Marconi, Piracicaba, Brazil). Fresh silage samples were used to prepare an
aqueous extract by homogenizing 25 g of silage into 225 g of distilled water for 1 min in a blender and filtering through two layers of

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J.M. Bragatto et al. Animal Feed Science and Technology 286 (2022) 115254

cheesecloth. The aqueous extract was used to determine silage pH using a pHmeter (Tec5, Tecnal, Piracicaba, Brazil) and aliquots were
frozen at − 20 ◦ C for analyses of fermentation end-products. On d 16 and 17, feeding behavior was monitored during the first 3 h after
morning feeding, as short-term feed intake during conditioned meals is an evidence of diet preference (Gerlach et al., 2019). Eating
time (min/3 h), DMI (kg DM/3 h), and intake rate (g DM/min) were recorded.
Milk yield was recorded from d 16–22. Milk samples were collected from eight consecutive milkings from d 16–19. Milk samples
preserved in bronopol (40 mL) were analyzed for fat, crude protein (CP), lactose, casein, urea, and somatic cell count. Another aliquot
(15 mL) without preservatives was frozen at − 80 ◦ C for determination of fatty acids and antioxidant capacity. A third fresh aliquot
(250 mL) was immediately submitted to the ethyl alcohol test in raw milk, an official method to identify tampered milk in Brazil
(Brasil, 2019).
Fecal grabbed samples were collected every 10 h from d 17–20 (nine subsamples) and stored at − 20 ◦ C. In each period, samples
were pooled to form one composite sample per animal. Afterward, samples were dried in an air-forced oven at 55 ◦ C, and ground to
pass through a 1 mm screen in a Wiley mill before chemical analysis.
Urine samples were collected approximately 3.5 h after morning feeding on d 19 and 20 (two subsamples) for analyses of creat­
inine, allantoin and uric acid. After filtering through 2 layers of cheesecloth, a 10 mL aliquot was diluted in 40 mL of 0.036 N sulfuric
acid and frozen at − 20 ◦ C for determination of allantoin. A second undiluted aliquot was stored at − 20 ◦ C for determination of
creatinine and uric acid. Purine derivative: creatinine ratio was used as an index for microbial protein synthesis (Hristov et al., 2019).
Ruminal fluid was collected from cannulated cows on d 21 at 0, 2, 4, 8 and 12 h after morning feeding. The pH was measured
immediately with a pHmeter (Tec5, Tecnal, Piracicaba, Brazil) and aliquots were stored at − 20 ◦ C for determinations of volatile fatty
acids (VFA) and NH3.
On d 22 of each period, blood samples were collected from the external jugular vein at 4 h after morning feeding in vacuum tubes.
Plasma from tubes with EDTA and serum from tubes without anticoagulant were separated by centrifugation at 2,500g at 4 ◦ C for 15
min and frozen at − 20 ◦ C for analyses of blood metabolites.

2.3. Laboratory analysis

Offered TMR, orts and feces were analyzed for absolute DM (method 930.15), ash (method 924.05), CP (method 984.13) and ether
extract (method 920.29) according to the AOAC (1990). Organic matter was calculated as 1000 - ash. Neutral detergent fiber (aNDF)
was assayed using filter bags (F57, Ankom) and a neutral detergent solution including thermostable amylase and sodium sulfite
(Mertens, 2002). Indigestible NDF (iNDF) was determined by 288 h of in situ ruminal incubation (Huhtanen et al., 1994). Duplicate
bags were incubated in two cows fed a diet containing 550 g/kg of corn silage and 450 g/kg of concentrates (DM basis). Both aNDF and
iNDF were expressed inclusive of residual ash. Nonfiber carbohydrates was calculated as 1000 - CP - aNDF - ether extract - ash (NRC,
2001). Silage concentrations of lactic acid (Pryce, 1969) and ammonia (Chaney and Marbach, 1962) were determined by colorimetry.
Volatile fatty acids, alcohols, esters, and acetone were determined by GC-MS (Lazzari et al., 2021).
The qualitative test of ethyl alcohol in raw milk samples (chromium reduction test) was performance as described by the Brazilian
Ministry of Agriculture, Livestock, and Food Supply (Brasil, 2019). A 100 mL of milk in a Büchner flask was added with 3 mL of
anti-foam solution (Cap-Lab CAS 107-21-1) and closed with a rubber stopper. The end of a silicone tube attached to the Büchner flask
was immersed in a sulfochromic solution (2 mL) placed in a test tube, forming a closed system between the head-space of the flask and
the sulfochromic solution. Afterward, the Büchner flask with the milk sample was heated in a pyroceramic plate (SL 141/A, Solab) and
kept under heating for 5 min after boiling had started. While boiling, the vapor formed in the flask bubbled into the sulfochromic
solution. The test was considered negative if the sulfochromic solution remained orange, but positive if the sulfochromic solution
changed to a greenish color.
Acetone, isopropanol, ethanol, and 2-butanol concentrations in milk were determined by head-space GC-MS. Aliquots of milk (2
mL) were placed in 10-mL glass vials with septum and heated at 60 ◦ C for 30 min under agitation in an autoinjector oven (AOC – 5000
Plus). After, vial head-space was sampled (1 mL) and injected in split mode (1:20) in the GC-MS (as for silage fermentation end-
products).
Milk fat, protein, lactose, casein, and urea were determined by infrared through the spectrometer (Bentley FTS/FCM, Bentley
Instruments Inc., Chaska, USA). Somatic cell count was determined by flow cytometry in Bentley-2000 electronic infrared quantifier
(Bentley Instruments Inc., Chaska, USA). Milk energy secretion (Mcal/d) was calculated as (NRC, 2001): [(0.00929 × fat (g/kg)) +
(0.00547 × protein (g/kg)) + (0.00395 × lactose (g/kg))] × milk production (kg/d). Energy-corrected milk (ECM; kg/d) was
calculated as milk energy secretion/0.70 (i.e., assumes 0.70 Mcal/kg of milk).
Milk antioxidant capacity and reducing power were determined in milk extracts obtained by adding 9 mL of methanol to 1 mL of
milk. Organic radical scavenging was measured using both ABTS and DPPH procedures. The ABTS⋅+ [2,2′ -azino-bis(3-ethyl­
benzothiazoline-6-sulfonic acid)] radical scavenging activity was determined as described by Re et al. (1999). Absorbance reading was
performed in a spectrophotometer (Evolution 300, Thermo Scientific, Madison, USA) at 734 nm after 6 min of reaction, and the
antioxidant capacity was expressed as trolox equivalent (μmol trolox/L). The antioxidant capacity was also determined with the
addition of DPPH radical (2,2-diphenyl-1-picryl-hydrazyl) to the extract. Absorbance reading was performed in a spectrophotometer at
515 nm (Brand-Williams et al., 1995), and the antioxidant capacity was expressed in μmol DPPH/L. The production of hydroperoxides
conjugated diene (CD) was performed according to Kiokias et al. (2006). The absorbance was determined in a UV-Vis spectropho­
tometer at 232 nm and expressed in mmol/kg of fat. The analysis of thiobarbituric acid reactive substances (TBARS) was performed
according to Vyncke (1970) with modifications (Osorio et al., 2021). The absorbance was determined in a spectrophotometer at 538
nm and expressed as mg of malondialdehyde/kg fat.

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J.M. Bragatto et al. Animal Feed Science and Technology 286 (2022) 115254

Table 1
Fermentation profile (mean ± SD) of corn silagea supplemented with isopropanol and ethanol (n = 4).
Treatmentb

Item CON ETH ISO ETH+ISO

pH 3.86 ± 0.09 3.87 ± 0.09 3.82 ± 0.08 3.86 ± 0.08

N-NH3, g/kg N 58.6 ± 18.3 62.0 ± 32.2 68.7 ± 25.4 58.0 ± 7.8
Lactic acid, g/kg DM 30.3 ± 14.0 28.5 ± 18.9 29.7 ± 14.4 28.9 ± 12.9
Acetic acid, g/kg DM 11.1 ± 4.5 11.9 ± 5.1 11.7 ± 3.7 11.8 ± 4.6
Propionic acid, g/kg DM 1.94 ± 0.2 1.92 ± 0.6 2.00 ± 0.2 1.92 ± 0.3
2,3-Butanediol, g/kg DM 1.77 ± 0.3 1.65 ± 0.8 1.62 ± 0.9 1.68 ± 0.3
Ethanol, g/kg DM 1.53 ± 0.4 4.80 ± 0.6 1.89 ± 2.3 4.16 ± 1.0
Isopropanol, g/kg DM 0.10 ± 0.0 0.09 ± 0.1 3.92 ± 0.6 4.15 ± 1.1
n-Propanol, mg/kg DM 325 ± 378 285 ± 547 302 ± 355 362 ± 503
Butyric acid, mg/kg DM 216 ± 229 257 ± 201 295 ± 238 254 ± 179
Valeric acid, mg/kg DM 53.8 ± 0.3 36.7 ± 1.1 60.8 ± 2.7 46.2 ± 0.3
Ethyl lactate, mg/kg DM 47.3 ± 26.3 48.6 ± 40.7 48.0 ± 27.3 50.9 ± 22.8
Methanol, mg/kg DM 42.8 ± 25.9 44.7 ± 36.1 41.1 ± 26.6 44.1 ± 21.4
Acetone, mg/kg DM 23.1 ± 5.4 32.8 ± 3.7 32.5 ± 15.3 34.9 ± 6.5
Ethyl acetate, mg/kg DM 12.5 ± 4.9 17.2 ± 27.2 13.0 ± 11.9 19.5 ± 2.3
Isobutyric acid, mg/kg DM 8.6 ± 12.5 4.5 ± 3.0 11.7 ± 14.4 8.0 ± 5.0
1,2-Propanediol, mg/kg DM 3.4 ± 2.6 2.5 ± 0.8 2.3 ± 1.9 2.8 ± 0.7
Isovaleric acid, mg/kg DM 1.8 ± 0.3 2.5 ± 1.1 3.3 ± 2.7 2.3 ± 0.3
2-Butanol, mg/kg DM 1.5 ± 0.5 2.2 ± 1.2 1.9 ± 0.1 2.8 ± 2.6
Propyl acetate, mg/kg DM 1.5 ± 0.4 1.4 ± 0.8 1.3 ± 0.1 1.5 ± 0.2
a
Untreated corn silage had 320 g DM/kg as fed, and contained on average (DM basis) 45.2 g/kg ash, 61.6 g/kg crude protein, 31.4 g/kg ether
extract, 493 g/kg neutral detergent fiber, and 369 g/kg nonfiber carbohydrates.
b
CON: control; ETH: ethanol; ISO: isopropanol; ETH+ISO: ethanol + isopropanol.

Milk fatty acid (FA) profile was analyzed as described by Murphy et al. (1995). Fatty acids were esterified using KOH/methanol and
n-heptane (ISO method, 5509, 1978. A 100 mg of the extracted fat was transferred to a 10-mL Falcon tube, 2 mL of N-heptane was
added and vortexed until complete solubilization, an additional 2 mL of 2 mol/L solution of KOH in methanol was added and vortexed
for 5 min. After phase separation, the upper phase containing the fatty acid methyl esters was transferred to a vial tube and analyzed.
The fatty acid methyl esters were quantified by gas chromatography (Trace GC 52 Ultra, Thermo Scientific, West Palm Beach, USA)
(Osorio et al., 2021).
Uric acid and creatinine concentrations in urine were determined using commercial kits (Acido urico PP - MS 80022230065,
Creatinina PP - MS 80022230066, respectively; Gold Analisa Diagnostica Ltda, Belo Horinzonte, Brazil). Urine allantoin concentration
was determined by colorimetric method, according to Chen and Gomes (1992).
Ruminal ammonia concentration was analyzed by the phenol-hypochlorite method according to Chaney and Marbach (1962).
Volatile fatty acids in rumen fluid were determined by gas chromatography (GC-2010 Plus, Shimadzu, Kyoto, Japan) using a capillary
column (Stabilwax-DA Restek, Bellefonte, USA, 30 m, 0.25 mm ID, 0.25 lm df), and a flame ionization detector, as described in Del
Valle et al. (2018).
Blood urea and glucose were analyzed in plasma, while aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT)
activities were analyzed in blood serum, using commercial kits (Glicose PP - MS 80022230067, Urea PP - MS 80022230063, AST PP -
MS 80022230083 and Gama GT PP - MS 80022230076, respectively; Gold Analisa Diagnostica Ltda, Belo Horinzonte, Brazil).

2.4. Tests of alcohol in raw milk

Parallel to the lactation trial, we carried-out a laboratory experiment to verify the reactivity of the ethyl alcohol test in raw milk
(Brazilian official method) and Milkscreen strip test to selected alcohols and acetone. The former test is based on the change in color of
a sulfochromic solution when hydroxyls linked to primary or secondary carbon (in alcohols) are oxidized by the chromic acid, with a
concomitant reduction of Cr6+ (orange color) to Cr3+ (greenish color) (Brasil, 2019).
The Milkscreen strip test is based on an enzymatic oxidation reaction with alcohol dehydrogenase, followed by a sequency of
reactions having as a closing point a color change (Christopher and Zeccardi, 1992; Fagnani et al., 2019). The strips are plunged in milk
sample for 10 s, and after 2 min the result was judged observing any color change in the strip, with the assistance of a color scale
supplied by the manufacturer. Color change from white to gray or black indicated positive, whereas no change in color (i.e., white) was
interpreted as negative.
Bulk milk from a neighbor dairy herd fed hay and concentrates (no fermented feeds in TMR) was used in this laboratorial
experiment. Milk sub-samples (100 mL) were contaminated with increased doses of ethanol, isopropanol, 2-butanol, and acetone, from
0 to 200 µg/mL by 4 µg/mL intervals. Tests were performed in duplicate from the lowest to the highest dose. When a compound
induced positive result at a given dose, the sequence was finalized for that compound.

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J.M. Bragatto et al. Animal Feed Science and Technology 286 (2022) 115254

Table 2
Chemical composition (mean ± SD) of experimental diets and concentrations of isopropanol and ethanol in offered rations and orts (g/kg DM, unless
otherwise stated) (n = 4).
Treatmenta

Item CON ETH ISO ETH+ISO

Dry matter, g/kg as fed 427 ± 9.4 427 ± 8.9 424 ± 9.3 422 ± 9.5
Organic matter 941 ± 4.5 943 ± 3.3 943 ± 2.9 940 ± 5.2
Crude protein 155 ± 1.5 153 ± 3.7 155 ± 1.8 155 ± 2.5
Ether extract 46.1 ± 2.4 45.9 ± 2.2 45.3 ± 2.9 46.3 ± 4.2
Neutral detergent fiber 361 ± 6.3 365 ± 25.3 360 ± 2.9 361 ± 9.0
Nonfiber carbohydrates 379 ± 12.8 379 ± 15.5 383 ± 18.7 378 ± 17.6
Offered ration
Isopropanol 0.05 ± 0.01 0.05 ± 0.01 2.15 ± 0.30 2.28 ± 0.60
Ethanol 0.63 ± 0.50 2.64 ± 0.30 1.03 ± 0.45 2.30 ± 0.50
Orts
Isopropanol 0.03 ± 0.01 0.03 ± 0.01 1.25 ± 0.20 1.37 ± 0.32
Ethanol 0.31 ± 0.26 1.19 ± 0.28 0.47 ± 0.25 1.08 ± 0.24
a
CON: control; ETH: ethanol; ISO: isopropanol; ETH+ISO: ethanol + isopropanol.

Table 3
Performance of dairy cows supplemented with isopropanol and ethanol (n = 8).
Treatmenta P-value

Item CON ETH ISO ETH+ISO SEM ETH ISO ISO×ETH

b
DMI , kg/d 16.3 16.3 16.2 16.3 0.78 0.75 0.74 0.65
DMIc, kg/first 3 h 6.62 6.34 6.43 6.43 0.413 0.66 0.88 0.67
Eating timec, min/first 3 h 76.9 77.2 76.5 76.3 6.29 0.99 0.90 0.96
Intake ratec, g DM/min 80.0 84.9 86.3 87.3 6.42 0.66 0.52 0.77
Milk yield, kg/d 17.2 17.1 17.3 17.6 0.69 0.84 0.65 0.74
ECMd, kg/d 17.3 17.0 17.1 17.7 1.42 0.85 0.76 0.52
Fat, g/kg 37.3 37.1 37.4 36.9 1.81 0.70 0.92 0.95
Fat, kg/d 0.642 0.634 0.647 0.649 0.060 0.88 0.80 0.49
Protein, g/kg 33.0 32.9 32.9 33.0 1.65 0.86 0.98 0.70
Protein, kg/d 0.568 0.563 0.569 0.581 0.037 0.80 0.90 0.39
Casein, g/kg 26.5 26.3 26.1 26.4 1.37 0.77 0.51 0.19
Lactose, g/kg 46.2 46.4 46.4 46.3 0.44 0.80 0.87 0.50
Urea-N, mg/dL 13.5 12.7 12.9 12.2 0.93 0.38 0.53 0.92
SCCe, 1000/mL 181 217 192 198 – – – –
Log10 SCC 2.26 2.34 2.28 2.30 0.145 0.67 0.71 0.50
ECM/DMI 1.08 1.08 1.04 1.09 0.088 0.48 0.69 0.51
a
CON: control; ETH: ethanol; ISO: isopropanol; ETH+ISO: ethanol + isopropanol.
b
Dry matter intake.
c
Measured during the first 3 h after morning feeding.
d
Energy-corrected milk.
e
Somatic cell count.

2.5. Statistical analysis

Data were evaluated for normality of residuals (Shapiro-Wilk test), homogeneity of variances (Bartlett test), and considering the
relatively short adaptation periods (15 d), data were also analyzed for carryover effects using the Orthoreg procedure of SAS. As the
variables fulfilled normality of residuals, variance homoscedasticity, and no significant carryover effect was identified for any variable,
data were analyzed using the Mixed procedure of SAS (v. 9.4, SAS Inst. Inc., Cary, NC) with the following model: Yijklm = μ + Si + C(S)ij
+ Pk + El + Im + EIlm + eijklm, where: Yijklm = dependent variable, μ = overall mean, Si = fixed effect of square (i = 1 or 2), C(S)ij =
random effect of cow nested within square (j = 1–8), Pk = fixed effect of period (k = 1–4), El = fixed effect of ethanol (l = ethanol or no
ethanol), Im = fixed effect of isopropanol (m = isopropanol or no isopropanol), EIlm = interaction of ethanol and isopropanol, eijklm =
residual error, assumed independently and identically distributed in a normal distribution with mean zero and variance σ2.
Rumen fermentation outcomes were analyzed as repeated measures. The effect of square was removed while the effects of time and
its interactions with ethanol and isopropanol were added in the model described above. The effect of cow nested within the interaction
ethanol×isopropanol was used as error term. The following covariance structures were tested: variance components (VC), compound
symmetry (CS), first-order autoregressive (AR(1)), and unstructured (UN). For each variable, the covariance structure with the lowest
value for the corrected Akaike information criterion was retained in the final model. Incidence of positive milk samples at the ethyl
alcohol test was compared by Chi-square test using the FREQ procedure of SAS. Significant differences were declared at P ≤ 0.05 and
tendencies to significance at 0.05 < P ≤ 0.10.

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Table 4
Ruminal fermentation pattern in cows supplemented with isopropanol and ethanol (n = 4).
Treatmenta P-valueb

Item CON ETH ISO ETH+ISO SEM ETH ISO ISO×ETH

pH 6.85 6.90 6.94 6.95 0.076 0.68 0.36 0.84

Ammonia, mg/dL 17.5 18.6 18.8 18.7 1.62 0.74 0.66 0.72
Acetate, mM 51.2 49.1 52.3 51.7 4.22 0.75 0.66 0.85
Propionate, mM 23.1 20.3 20.9 20.8 2.51 0.74 0.57 0.61
Butyrate, mM 9.71 8.84 9.99 10.1 0.69 0.60 0.25 0.46
Total VFAc, mM 90.1 85.2 87.8 88.9 6.51 0.77 0.90 0.65
Acetate/Propionate 2.79 2.66 2.85 2.68 0.311 0.63 0.89 0.94
a
CON: control; ETH: ethanol; ISO: isopropanol; ETH+ISO: ethanol + isopropanol.
b
All parameters were affected by sampling time (P < 0.01), but no interactions with the dietary treatments were observed (P > 0.22).
c
Total volatile fatty acids, including isobutyrate, isovalerate, valerate and caproate.

Table 5
Total-tract digestibility, purines in urine, and blood parameters in cows supplemented with isopropanol and ethanol (n = 8).
Treatmenta P-value

Item CON ETH ISO ETH+ISO SEM ETH ISO ISO×ETH

Apparent digestibility
Dry matter 0.660 0.663 0.667 0.663 0.008 0.99 0.58 0.61
Organic matter 0.680 0.681 0.682 0.684 0.007 0.83 0.71 0.93
Neutral detergent fiber 0.516 0.493 0.496 0.494 0.018 0.52 0.61 0.57
Non-fiber carbohydrates 0.886 0.880 0.891 0.876 0.013 0.74 0.99 0.43
Crude protein 0.722 0.726 0.718 0.712 0.012 0.91 0.26 0.52
Ether extract 0.902 0.914 0.900 0.896 0.012 0.66 0.31 0.40
Urine purine derivatives
Allantoin/Creatinine 1.26 1.24 1.10 1.35 0.199 0.54 0.90 0.47
Uric acid/Creatinine 0.885 0.787 0.817 0.866 0.107 0.82 0.96 0.51
(Allantoin+Uric acid)/Creatinine 2.08 1.96 1.91 2.04 0.138 0.97 0.78 0.39
Blood metabolites
Glucose. mg/dL 54.9 52.7 53.7 53.1 2.99 0.55 0.98 0.68
Urea. mg/dL 29.0 24.6 25.7 25.4 4.47 0.16 0.46 0.21
ASTb, U/L 54.0 57.8 57.8 61.8 6.72 0.43 0.42 0.98
GGTc, U/L 4.96 6.34 6.78 8.36 0.998 0.01 < 0.01 0.84
a
CON: control; ETH: ethanol; ISO: isopropanol; ETH+ISO: ethanol + isopropanol.
b
Aspartate aminotransferase.
c
Gamma-glutamyl transferase.

Table 6
Fatty acids (g/100 g of fat) in milk of cows supplemented with isopropanol and ethanol (n = 8).
Treatmenta P-value

Item CON ETH ISO ETH+ISO SEM ETH ISO ISO×ETH

10:0 3.01 2.84 2.78 3.19 0.339 0.61 0.79 0.23

12:0 4.38 3.93 4.20 4.48 0.378 0.76 0.54 0.23
14:0 15.5 14.7 15.4 15.9 0.68 0.99 0.52 0.20
16:0 36.9 37.2 37.9 37.2 0.80 0.62 0.61 0.41
18:0 10.7 11.5 10.5 10.2 0.73 0.69 0.25 0.39
18:1 n9t 5.11 4.95 4.56 4.88 0.411 0.77 0.28 0.41
18:1 n9c 15.0 15.7 15.2 14.8 0.99 0.99 0.41 0.39
18:2 n6t 0.252 0.249 0.231 0.242 0.024 0.80 0.29 0.59
18:2 n6c 1.26 1.39 1.27 1.20 0.101 0.75 0.38 0.29
18:3 n6 0.011 0.015 0.014 0.014 0.006 0.74 0.88 0.78
18:3 n3 0.021 0.020 0.028 0.021 0.011 0.58 0.64 0.68
CLA 18:2 c9 t11 0.130 0.178 0.133 0.121 0.059 0.78 0.66 0.63
SCFAb 4.28 4.07 3.85 4.45 0.454 0.52 0.92 0.20
MCFAc 62.0 60.6 62.3 62.5 1.34 0.65 0.42 0.57
LCFAd 33.6 35.3 33.2 32.4 1.36 0.80 0.12 0.23
a
CON: control; ETH: ethanol; ISO: isopropanol; ETH+ISO: ethanol + isopropanol.
b
Short-chain fatty acids (6:0 a 10:0).
c
Medium-chain fatty acids (11:0 a 16:1).
d
Long-chain fatty acids (17:0 a 22:0).

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J.M. Bragatto et al. Animal Feed Science and Technology 286 (2022) 115254

Table 7
Oxidative profile, acetone and alcohols concentrations in milk of cows supplemented with isopropanol and ethanol (n = 8).
Treatmenta P-value

Item CON ETH ISO ETH+ISO SEM ETH ISO ISO×ETH

Oxidative profile
ABTSb, μmol trolox/L 153 154 160 153 7.6 0.55 0.67 0.48
DPPHc, μmol DPPH/L 116 112 116 114 24.7 0.33 0.64 0.57
CDd, mmol/kg fat 0.432 0.450 0.476 0.423 0.0453 0.67 0.84 0.40
TBARSe, mg MDAf/kg fat 1.33 1.46 1.62 1.52 0.191 0.95 0.39 0.56
Acetone and alcohols
Acetone, µg/mL 21.9 20.6 101 104 4.54 0.88 < 0.01 0.66
Isopropanol, µg/mL 4.61 3.96 97.3 98.0 4.55 0.99 < 0.01 0.88
Ethanol, µg/mL 1.63 1.78 0.951 2.87 0.741 0.16 0.77 0.24
2-Butanol, µg/mL 0.085 0.091 0.091 0.090 0.0036 0.47 0.62 0.37
a
CON: control; ETH: ethanol; ISO: isopropanol; ETH+ISO: ethanol + isopropanol.
b
ABTS: 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid).
c
DPPH: 2,2-diphenyl-1-picryl-hydrazyl.
d
Conjugated diene hydroperoxides.
e
Thiobarbituric reacting substances.
f
Malondialdehyde.

Fig. 1. Qualitative test of ethyl alcohol in raw milk from cows supplemented with ethanol and isopropanol. CON: control; ETH: ethanol; ISO:
isopropanol; ETH+ISO: ethanol + isopropanol. Chi-square test. χ2 < 0.01 for treatment effect.

3. Results

3.1. Performance, metabolism, and milk quality

The profile of fermentation products in treated corn silages collected immediately before feeding is shown in Table 1. Addition of
isopropanol and ethanol was conducive to increase their concentrations in treated silages. Ethanol concentration increased from ~1.7
to ~4.5 g/kg DM, whereas isopropanol increased from < 0.1 to ~4.0 g/kg DM. Consequently, diets containing treated silages had
proportionally greater numerical values of their respective supplemented alcohols (Table 2). Concentrations of ethanol and iso­
propanol were numerically lower in orts than that in offered TMR, but orts from treated TMR had numerically greater values of their
respective alcohols.
There was no interaction between isopropanol and ethanol on the feed intake, ruminal fermentation, urinary purine derivatives,
total-tract apparent digestibility, blood metabolites, milk yield, milk composition and oxidative capacity. Alcohol supplementation did
not alter DMI, eating time and intake rate after feeding (Table 3). Milk yield, solids concentration and yield, milk urea nitrogen,
somatic cell count and feed efficiency were not affected by treatments. Ruminal pH and concentrations of ammonia, acetate, propi­
onate, butyrate, and total volatile fatty acids were similar among treatments (Table 4).
Supplementation with isopropanol and ethanol did not affect total-tract apparent digestibility of DM, organic matter, CP, ether
extract, aNDF and NFC (Table 5). Urinary allantoin/creatinine and uric acid/creatinine ratios were similar among treatments.
However, the enzyme GGT increased in the blood of cows fed silage supplemented with ethanol (P = 0.01) and isopropanol (P < 0.01),
alone or in combination. Treatments did not affect blood concentrations of glucose, urea, and AST.
Milk FA profile (Table 6) and oxidative capacity (Table 7) were not altered by alcohol supplementation. However, acetone and
isopropanol concentrations were increased in milk of cows supplemented with isopropanol, alone or associated with ethanol (P <
0.01). All milk samples from cows receiving isopropanol alone or combined with ethanol were positive at the ethyl alcohol test,
whereas all milk samples from cows fed silage supplemented with ethanol were negative (χ2 < 0.01; Fig. 1).

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J.M. Bragatto et al. Animal Feed Science and Technology 286 (2022) 115254

3.2. Alcohol tests in raw milk

The Brazilian official method for detecting ethyl alcohol in raw milk reacted positively when milk samples contained ethanol
(≥80 µg/mL), 2-butanol (≥40 µg/mL) and isopropanol (≥24 µg/mL), but not reacted to acetone. Milkscreen strips only reacted to
ethanol (≥120 µg/mL).

4. Discussion

4.1. Isopropanol and ethanol in rations

Corn silage is a predominant forage used in dairy cattle diets worldwide (Ferraretto et al., 2018). As a fermented feed, corn silage
carries several fermentation end-products to the TMR, including isopropanol (Kalac, 2011; Hafner et al., 2013). To our best knowledge,
we demonstrated for the first time the effects of isopropanol in TMR for dairy cows.
In the current study, supplementation with pure isopropanol and ethanol was conducive to reproduce their average content in corn
silage observed in our field survey (~4.0 g/kg DM). Approximately 30% of sprayed alcohols (15 g/kg DM) were recovered in treated
silage at feeding. Previous studies have also reported a significant loss of volatile organic compounds when sprayed onto feed in­
gredients prior to feeding (Daniel et al., 2013; Gerlach et al., 2019). Most of those losses likely occurred by volatilization during
spraying and silage homogenization. In addition, alcohols were also lost during TMR exposure in the feedbunk but at a lower rate. The
loss of supplementary compounds during spraying is often higher than the volatilization of compounds originated in silage (Daniel
et al., 2013; Robinson et al., 2016). In the current study, the concentrations of isopropanol and ethanol in orts were on average 60%
and 47% of that in offered TMR, respectively. Assuming that a large proportion of TMR mass is ingested during conditioned meals after
each feeding (Dado and Allen, 1993), most of alcohols delivered in the feedbunk were likely consumed by the cows (Daniel et al.,
2013).

4.2. Performance, metabolism, and milk quality

Isopropanol and ethanol supplementation did not alter diet preference, despite the pungent odor and bitter taste of isopropanol to
the human sense of smell. Hence, physical, chemical, and sensory signals that trigger eating control were not altered by the presence of
alcohols in TMR (Forbes, 2007). Moreover, previous studies with alcohol supplementation in dairy diets, including ethanol (Randby
et al., 1999; Raun and Kristensen, 2011; Daniel et al., 2013), 1-propanol (Raun and Kristensen, 2011, 2012; Silva et al., 2017), and 1,
2-propanediol (Kristensen et al., 2002; Kristensen and Raun, 2007) reported no intake depression or in some cases an increase in DMI
for treatments with alcohol supplementation.
In lactating cows, rumen fermentation is an important component of alcohol metabolism (Pradhan and Hemken, 1970). Ruminal
digesta and mucosa account for more than half of ethanol metabolism, resulting in a little proportion of dietary ethanol reaching the
liver in dairy cows (Raun and Kristensen, 2011). However, isopropanol can be formed in the rumen fluid by acetone reduction (Bruss
and Lopez, 2000). Furthermore, alcohols are potentially toxic for ruminal microorganisms by affecting microbial cell wall fluidity and
transport of metabolites (Hui and Barton, 1973). Hence, theoretically the alcohols would alter the microbial protein synthesis, fiber
degradation, and fatty acid biohydrogenation. Also, isopropanol may reduce the transcription of acetyl-CoA synthetase and the
methanogenesis in anaerobic bioreactors (Ince et al., 2011). In this trial, however, no effect of alcohols was observed on ruminal
fermentation pattern, ruminal microbial yield, fatty acid biohydrogenation pattern, and nutrient digestibility, as assessed directly or
indirectly by the ruminal VFA, purine derivative: creatinine ratio in urine, milk urea nitrogen and daily secretion of milk protein
(Hristov et al., 2019), milk fatty acids composition (Dewanckele et al., 2020), and total-tract apparent digestibility.
Milk yield, solids and oxidative capacity were also unchanged by alcohols supplementation, suggesting that the amount of alcohols
consumed was likely less than that capable of challenging cow metabolism. In the liver, both ethanol and isopropanol are metabolized
by the enzyme alcohol dehydrogenase (Lieber and Abittan, 1999; Bruss and Lopez, 2000; Jones and Holmgren, 2015). Ethanol is
oxidized to acetaldehyde and then acetyl-coA, which is used in the cell metabolism (Lieber and Abittan, 1999). On the other hand,
isopropanol is oxidized to acetone, which is released into the peripheral blood and partially recycled into the rumen fluid. In the
rumen, acetone is reduced to isopropanol again. Isopropanol is absorbed, enters the liver, and is oxidized to acetone (Bruss and Lopez,
2000). The carbon chain of a single acetone/isopropanol molecule can cycle several times before being excreted in milk, urine,
expiration, or, in small amounts, converted to glucose (Black et al., 1972). Therefore, the actual concentration of isopropanol in body
fluids might be greater than expected from the daily intake and its first-order pharmacokinetics. In this study, higher levels of GGT
enzyme in cows supplemented with alcohols confirm that ethanol and isopropanol were at least partially absorbed by portal drained
viscera (PDV) and reached the liver (Tennant and Center, 2008; Sato, 2009). Notably, GGT activity was higher for ETH+ISO compared
to the treatments with alcohols alone, suggesting metabolism of both alcohols by the same pathway in hepatocytes. Despite the in­
crease in GGT activity caused by alcohol supplementation, the values were within the normal range for cattle (<17 U/L; Tennant and
Center, 2008).
Circulation of isopropanol and acetone in the peripheral blood resulted in the excretion of these compounds in milk. Co-ingestion of
ethanol has been reported to increase the half-life of isopropanol in humans (Pappas et al., 1991), but contrary to our hypothesis, in the
current study the concentration of ethanol in milk was not altered by ethanol supplementation alone or in combination with iso­
propanol. It suggests an efficient ethanol metabolism in the cow body compartments, including rumen fluid, PDV, and liver (Kristensen
et al., 2007; Raun and Kristensen, 2009). As a result, all milk samples from cows supplemented with ethanol alone were negative at the

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J.M. Bragatto et al. Animal Feed Science and Technology 286 (2022) 115254

Table 8
Reactivity of raw milk added with increased doses of ethanol, isopropanol, 2-butanol, and acetone.
Item Brazilian official test Milkscreen strip test

Ethanol Reactive at ≥ 80 µg/mL Reactive at ≥ 120 µg/mL

Isopropanol Reactive at ≥ 24 µg/mL No reaction
2-Butanol Reactive at ≥ 40 µg/mL No reaction
Acetone No reaction No reaction

ethyl alcohol test. In contrast, supplementation with isopropanol induced 100% of milk positive at the ethyl alcohol test in raw milk. It
demonstrates that not only primary alcohols, but also secondary alcohols such as isopropanol are able to donate electrons to reduce
Cr6+ to Cr3+ and change the color of the sulfochromic solution from orange (negative) to greenish (positive). Although this official
method is intended to detect ‘ethyl alcohol’, other alcohols may lead to false-positive results.
Although the isopropanol-acetone cycle has been reported in ketotic cows (Sato, 2009), it is unlikely that few ketotic fresh cows
within the herd would lead to bulky milk positive at the ethyl alcohol test, as previously demonstrated by Halfen (2017). Moreover, in
the current study, our cows were in mid-lactation and likely in positive energy balance, but isopropanol was imparted to milk when it
was supplied in the TMR.

4.3. Alcohol tests in raw milk and their implications for the dairy chain

The Brazilian official method for detecting ethyl alcohol in raw milk reacted positively not only with milk samples containing
ethanol, but also 2-butanol and isopropanol, with a 2- to 4-fold greater sensibility for the latter. It confirms that secondary alcohols (e.
g., 2-butanol and isopropanol) oxidize faster than primary alcohols (e.g., ethanol) in the sulfochromic solution, and that isopropanol is
even more reactive than 2-butanol. Contrarily, Milkscreen strips specifically reacted to ethanol (≥120 µg/mL), and with a slightly
lower sensibility than the official method (≥80 µg/mL), as previously observed by Fagnani et al. (2019). As expected, no qualitative
test reacted to acetone, because it is already a carbonyl derivative. Acetone (i.e., propanone) is actually the final product of isopropanol
oxidation in the sulfochromic solution.
By law, the presence of ethyl alcohol in raw milk is considered fraud (Brasil, 2018). However, false-positive results at the official
test may unfairly condemn normal milk erroneously labeled as tampered milk, and then cause economic and social loss for the dairy
chain. Based on our findings, the Brazilian official test to detect ethyl alcohol in raw milk should not be adopted as an ultimate method
to identify tampered milk. Milkscreen strips are an alternative to differentiate milk samples added with water and ethanol and confirm
the occurrence of fraud. Yet, other fast colorimetric methods (e.g., Schiff’s reagent) have potential to discern milk samples containing
ethanol (i.e., primary alcohol) from normal milk containing traces of isopropanol (i.e., secondary alcohol) and can be validated in the
future, as demonstrated for Milkscreen strips in this study.

5. Conclusions

Supplementation with isopropanol and ethanol did not alter feed intake and performance of lactating cows. However, silage
isopropanol, but not ethanol, was imparted to milk. Consequently, milk from cows fed corn silage supplemented with isopropanol
tested false-positive at the ethyl alcohol test in raw milk. Hence, the ethyl alcohol test should not be adopted as an ultimate method for
detecting fraud in tampered milk. Milkscreen strips can be used as a fast complementary test to differentiate milk added with water and
ethanol Table 8.

CRediT authorship contribution statement

Janaina M. Bragatto: Investigation, Formal analysis, Data curation, Writing – original draft, Visualization. Camila S. Parra:
Investigation, Formal analysis, Data curation. Francisco A. Piran Filho: Investigation. Sillas M. S. Silva: Investigation. Jesus A. C.
Osorio: Formal analysis, Data curation. Sara C. Buttow: Investigation. Geraldo T. Santos: Methodology, Resources, Writing – review
& editing, Visualization. Cloves C. Jobim: Methodology, Resources, Writing – review & editing, Visualization. Luiz G. Nussio:
Methodology, Resources, Writing – review & editing, Visualization. Joao L. P. Daniel: Conceptualization, Project administration,
Methodology, Resources, Formal analysis, Writing – review & editing, Visualization.

Acknowledgements

We are grateful to all students of the GESF team and Iguatemi Experimental Farm crew - State University of Maringá for their
support with animal care, sample collections, and analyses. Gratitude is expressed to the Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior (CAPES) - Finance Code 001, Brazil, and the Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq), Brazil, for the scholarships provided to the authors. The authors also acknowledge financial support from the Instituto
Nacional de Ciência e Tecnologia para a Cadeia Produtiva do Leite (INCT-LEITE/UEL), Londrina-PR, Brazil (CNPq/Fundação Arau­
cária – INCT-Leite, Grant 465.725/2014-7).

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