Animal Feed Science and Technology 261 (2020) 114406

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Animal Feed Science and Technology

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Effects of organic acid-based products added to total mixed ration

T
on performance and ruminal fermentation of dairy cows
Larissa S. Ghellera, Lucas G. Ghizzia, Júlia A. Marquesa, Caio S. Takiyab,
Nathália T.S. Grigolettoa, Mauro S.S. Diasa, Tássia B.P. Silvaa, Alanne T. Nunesa,
Guilherme G. da Silvaa, Luis G.X. Fernandesa, Luciana N. Rennóc,
Francisco P. Rennóa,*
a
Department of Animal Nutrition and Animal Production, University of São Paulo, Pirassununga, 13635-900, Brazil
b
Department of Animal Sciences and Industry, Kansas State University, Manhattan, KS, 66506, United States
c
Animal Science Department, Universidade Federal De Viçosa, Viçosa, Minas Gerais, Brazil

A R T IC LE I N F O ABS TRA CT

Keywords: An increase in total mixed ration (TMR) temperature is associated with a growth of undesirable
Feed heating microorganisms and decreases in feed intake in dairy cows. We evaluated the effects preservative
Formic acid products containing organic acids (OA) added to TMR on intake and digestibility of nutrients,
Preservative rumen fermentation, milk yield and composition, nitrogen utilization, blood parameters, in-
Propionic acid
gestive behavior of cows. Twenty-five Holstein cows (147.4 ± 87.9 days in milk,
TMR
668.7 ± 78.5 kg of body weight and 34.4 ± 4.2 kg/d milk yield), 10 of which were rumen-
cannulated, were assigned to a 5 × 5 Latin square experiment containing the following treat-
ments: control (CON), no OA added to TMR; and four preservatives composed mainly of either
formic acid (FLE), sodium formate (SOL), propionic acid (SPE) in liquid form, or propionic acid
(PER) in solid form added to TMR. The OA products used in this study were manufactured by
Perstorp Waspik BV (Waspik, The Netherlands). Products were added daily to the TMR at 4 ml/kg
natural matter (NM) for the liquid products (FLE, SPE, PER), and at 4 g/kg for the solid product
(SOL). The experimental barn temperature, relative humidity, and temperature-humidity index
during the experiment were 24.4 ± 4.29 °C, 75.0 ± 16.39%, and 72.7 ± 5.31 (mean ± SD),
respectively. Each experimental period consisted of 14 days for adaptation to treatments and five
days for sampling. Differences among treatments were studied using orthogonal contrasts, as
follows: the effect of OA [control versus OA (SPE + SOL + PER + FLE)], the effect of OA com-
position [formic acid (FLE + SOL) versus propionic acid (PER + SPE)], the effect of different
formic acid products (FLE versus SOL); and the effect of different propionic acid products (SPE
versus PER). Organic acids increased feed intake (23.6 and 24.4 kg/d for CON and OA, respec-
tively) and reduced sorting of feed particles with 8–19 mm size and shorter than 4 mm. Organic
acids, however, decreased the total VFA concentration and increased pH of rumen fluid. Cows fed
TMR with OA had greater fat-corrected milk production and milk protein content compared with
CON. Organic acids avoided an increase in TMR temperature within 24 h air exposure. Organic

Abbreviations: ADF, acid detergent fiber; aNDF, neutral detergent fiber; BW, body weight; CP, crude protein; DM, dry matter; DMI, dry matter
intake; EE, ether extract; FCM, fat-corrected milk; iNDF, indigestible neutral detergent fiber; N, nitrogen; NFC, non-fiber carbohydrate; NH3-N,
ammonia nitrogen; NM, natural matter; OA, organic acid; OM, organic matter; PD, purine derivatives; SCFA, short-chain fatty acids; SD, standard
deviation; TMR, total mixed ration; VFA, volatile fatty acids
⁎
Corresponding author at: Av. Duque de Caxias Norte, 225 – Campus da USP, 13635-900, Pirassununga, SP, Brazil.
E-mail address: [email protected] (F.P. Rennó).

https://doi.org/10.1016/j.anifeedsci.2020.114406
Received 7 June 2019; Received in revised form 10 January 2020; Accepted 14 January 2020
0377-8401/ © 2020 Elsevier B.V. All rights reserved.
L.S. Gheller, et al. Animal Feed Science and Technology 261 (2020) 114406

acids, regardless of their composition, can maintain TMR temperature stable for longer periods
and improve the performance of dairy cows.

1. Introduction

Feeding TMR allows a more consistent and homogenous flow of nutrients in the rumen throughout the day avoiding sharp
decreases in ruminal pH and synchronizes the utilization of available energy and protein. (DeVries et al., 2005). Since the 1960s, the
supply of forage and concentrate in the form of TMR has contributed to an increase of milk production, dry matter intake (DMI), and
milk fat content in dairy cows (McCoy et al., 1966;National Research Council (NRC), 2001; Schingoethe, 2017).
Rises in feed temperature are unavoidable in countries with a tropical climate and are positively associated with microbial
multiplication along with feed deterioration (Ashbell et al., 2002). Undesirable microorganisms (i.e., mold and yeast) rapidly mul-
tiply after the silage air exposure and TMR preparation leading to decreases in feed nutritive value and animal performance (Kung,
2010). Organic acids (OA) have been used to mitigate the feed deterioration by bacteria and fungi as well as to improve the con-
servation of fermented feed (e.g., silages; Goering and Gordon, 1973; Jongbloed et al., 2000; Haque et al., 2012; Moriel et al., 2016;
Sousa et al., 2019). Improvements in conservation and aerobic stability of feeds have been reported after OA treatments, presumably
due to OA inhibitory activity on undesirable microorganisms (Muck et al., 2018; Santos et al., 2019a, b). Organic acids, in the
undissociated form, can cross the cell membrane hence releasing hydrogen into the cytoplasm. The reduction in cytoplasmic pH and
the use of ATP to maintain cell homeostasis cause microorganism inhibition (Lambert and Stratford, 1999). Refusals of TMR mixed
with propionic acid exhibited lower pH and temperature after 24 h, suggesting less feed deterioration (Kung et al., 1998). Although
there is evidence that OA improve TMR nutritive value, studies evaluating the effects of OA added to TMR on digestion and

Table 1
Ingredients and chemical composition of total mixed ration
(TMR).
Item TMRa

Ingredients (g/kg DM)

Corn silageb 480
Ground corn 218
Soybean meal, 48% CP 146
Whole raw soybeans 47.0
Citrus pulp 62.1
Bypass soybean mealc 18.0
Mineral premixd 12.9
Sodium bicarbonate 7.00
Limestone 7.00
Salt 2.00
Chemical composition (g/kg DM)
Dry matter, g/kg as-fed 455
Organic matter 926
Non-fiber carbohydratee 427
Neutral detergent fiber 337
Starch 249
Acid detergent fiber 212
Crude protein 156
Ether extract 33.2
Lignin 33.2
Metabolizable energyf, MJ/kg 11.3
NEl3xf, MJ/kg DM 6.74

a
The experimental diet was formulated according to
National Research Council (NRC) (2001) to meet the nutrient
requirements for cows with 620 kg body weight, 140 days in
milk, 35 kg/d of milk yield, and 3.5% milk fat.
b
Chemical composition (DM basis): 300 g/kg as-fed, 72.0 g/
kg CP, 511 g/kg NDF, and 215 g/kg starch.
c
Soypass BR® (Cargill, Uberlândia, Brazil).
d
Each kg contained: 235 g Ca, 60 g P, 20 g Mg, 70 g Na, 20 g
S, 15 mg Co, 700 mg Cu, 10 mg Cr, 40 mg I, 600 mg Fl, 1,600 mg
Mn, 20 mg Se, 2,500 mg Zn, 200,000.00 IU vitamin A,
50,000.00 IU vitamin D3, and 1,500 IU vitamin E.
e
Estimated according to Hall (2000).
f
Estimated according to NRC (2001), net energy of lactation
(NEl) estimated at three times the maintenance level.

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L.S. Gheller, et al. Animal Feed Science and Technology 261 (2020) 114406

performance of dairy cows are scarce.

The objective of this study was to evaluate the intake and digestibility of nutrients, rumen fermentation, milk yield and com-
position, nitrogen utilization, blood parameters, ingestive behavior of lactating cows fed a TMR treated with OA-based products. We
also evaluated TMR temperature after OA treatment. Our hypothesis was that the addition of different OA-based products to the TMR
would increase DMI and milk production of cows and maintain the TMR temperature stable for longer periods.

2. Materials and methods

The experiment was conducted at the Dairy Cattle Research Laboratory of Department of Animal Production and Animal Nutrition
from the School of Veterinary Medicine and Animal Science, Pirassununga, São Paulo, Brazil. Experimental procedures were carried
out under approval of the Ethics Committee from the School of Veterinary Medicine and Animal Sciences of University of São Paulo
(protocol #7654230518).

2.1. Animals and treatments

Twenty-five Holstein cows (twenty multiparous and five primiparous), ten of which were cannulated in the rumen, were used in
this study. Cows [147.4 ± 87.9 days in milk, 668.7 ± 78.5 kg body weight (BW) and 34.4 ± 4.2 kg/d milk yield (mean ± SD) at
the beginning of the experiment were assigned to a 5 × 5 Latin square experiment, blocked by milk yield, days in milk, parity, BW,
and presence of ruminal cannula. The experiment consisted of 19-d experimental periods, whereas the first 14 days were used for
adaptation to treatments and the last 5 days for sampling.
All cows received the same TMR (Table 1) formulated according to National Research Council (NRC) (2001) to meet or exceed the
nutrient requirements of cows with 620 kg BW, 140 days in milk, 35 kg/d milk yield, and 3.5% milk fat. The TMR was prepared
immediately prior to feed delivery with a forage to concentrate ratio of 48:52 with corn silage being the single forage source. Cows
were randomly assigned to treatment sequences consisting of the following: (1) control (CON), no OA added to TMR; (2) ProMyr TMR
Special® (SPE); (3) ProMyr TMR Solid® (SOL); (4) ProMyr TMR Performance® (PER); and (5) ProMyr TMR Flexible® (FLE). All OA
products used in this study were manufactured by Perstorp Waspik BV (Waspik, The Netherlands; Table 2). For OA products in the
liquid form (SPE, PER, and FLE), 4 ml/kg NM was sprayed onto the TMR and hand-mixed with the other portions, whereas the OA
product (SOL) was top-dressed (4 g/kg NM) and hand-mixed in TMR prior feeding. Cows were housed in a barn containing individual
pens (17.5 m²), sand bed, fans, and ad libitum access to water and feed. Cows were fed individually during the experimental period.
The barn temperature, relative humidity, and temperature-humidity index (Mader et al., 2006) during the experiment were
24.4 ± 4.29 °C, 75.0 ± 16.39 % and 72.7 ± 5.31 (mean ± SD), respectively.

2.2. TMR temperature

The 24 h-temperature of TMR was evaluated over 6 days using data loggers (TagTemp Stick, Novus, Canoas, Brazil). The TMR fed
to cows with different treatments was placed in feed bunks (n = 2 per treatment per day, 4 kg DM each time) at the same time of the
morning and afternoon feedings. The TMR and barn (26.1 ± 4.8 °C) temperatures were recorded every 60 min. The tested TMR were
maintained under the same conditions which cows were housed.

Table 2
Composition of organic acid products (Perstorp Waspik BV, Waspik, The Netherlands) added
to total mixed ration (TMR).
Organic acid Ingredients (g/kg)

ProMyr TMR Flexible EN® (FLE) 350 – 450 formic acid

150 – < 250 propionic acid
100 – 200 sodium formate
ProMyr TMR Solid EN® (SOL) 750 – 850 sodium formate
100 – 150 potassium sorbate
50 – 100 silica, amorphous
30 – 70 propionic acid
10 – 40 glycerol propionate
ProMyr TMR Special EN® (SPE) 60 – 70 propionic acid
10 – 50 sodium formate
10 – 50 1,2,3-propanetriol, glycerol
200 – 300 glycerol propionate
ProMyr TMR Performance® (PER) 500 – 600 propionic acid
150 – 250 hexanoic acid
10 – 50 sodium formate
10 – 50 propanetriol, glycerol
150 – 250 glycerol propionates

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2.3. Feed intake and total-tract apparent digestibility

Cows were fed twice daily (0800 and 1300 h) targeting feeding rates between 50 and 100 g/kg. During the sampling period,
samples of corn silage and refusals were collected daily and composited into a single sample per cow per period. Samples of con-
centrate ingredients were collected once during each sampling period. Fecal samples were collected every 9 h on days 16, 17, and 18
of each experimental period. After samplings, fecal samples (∼100 g) were frozen and thawed by the end of each period to form
composite samples per cow per period.
Samples of feeds, orts, and feces were dried in a forced ventilation oven at 65 °C for 72 h and ground in a Wiley mill (MA340,
Marconi, Piracicaba, Brazil) through 1 mm and 2 mm sieves. Samples with 1 mm size were analyzed for DM (method 930.15, AOAC,
2000), crude protein (CP; N × 6.25, Kjeldahl method 984.13, AOAC, 2000), ether extract (EE; method 920.39, AOAC, 2000), acid
detergent fiber (ADF) and lignin (sa; method 973.18, AOAC, 2000), ash (method 942.05, AOAC, 2000), and neutral detergent fiber
using alpha-amylase (aNDF; Undersander et al., 1992) without the addition of sodium sulfite (TE-149 fiber analyzer, Tecnal, Pir-
acicaba, SP, Brazil). Feed samples were also analyzed for starch content through an enzymatic degradation (Amyloglucosidase®,
Novozymes, Curitiba, PR, Brazil) method and absorbances were measured on a semi-automatic spectrophotometer (SBA-200, CELM®,
São Caetano do Sul, SP, Brazil) according to Hendrix (1993).
Fecal, feeds, and refusals iNDF content were measured to estimate daily excretion of DM and nutrient digestibility. Feeds, orts,
and feces samples ground in 2 mm sieve were placed in non-woven bags (Casali et al., 2008; 20 mg DM/cm²) and incubated in the
rumen of two Holstein cows for 288 h according to Huhtanen et al. (1994). After the incubation period, samples were washed in
running tap water and analyzed for NDF content (iNDF), as described earlier. The daily fecal excretion was calculated as:
g
g ⎞ iNDF intake ( day )
Fecal excretion ⎛⎜ ⎟ =
g
⎝ day ⎠ iNDF excretion ( kg )

Dry matter and nutrient digestibility were calculated according to the equations:

% iNDF intake ⎞ ⎤
DM digestibility (%) = 100 - ⎡100 × ⎛
⎢
⎣ ⎥
⎝ % iNDF in feces ⎠ ⎦

% iNDF intake ⎞ ⎛ % nutrient in feces ⎞ ⎤

Nutrient digestibility (%) = 100 - ⎡100 × ⎛ ×
⎢
⎣ ⎝ % iNDF in feces ⎠ ⎝ % nutrient in the diet ⎠ ⎥
⎦

2.4. Sorting index

Samples of TMR and orts were collected on days 15 and 16 of each experimental period and analyzed for particle size distribution
using a Penn State Particle Separator (Nasco, Fort Atkinson, WI; Heinrichs and Kononoff, 2013). The sorting index was calculated as
described by Silveira et al. (2007), and the animal intake corresponding to each sieve was expressed by the percentage of total
predicted intake (on as-fed basis), using the formulas:

kg ⎞ ⎛ kg NM ⎞⎟ × PTMR ⎛⎜ kg ⎟⎞
Expected intake ⎛⎜ ⎟ = intake ⎜

⎝ day ⎠ ⎝ day ⎠ ⎝ kg ⎠

kg ⎞ ⎡ ⎛ kg ⎞ ⎛ kg ⎞ ⎤ ⎡ ⎛ kg ⎞ ⎛ kg ⎞ ⎤
Observed intake ⎛⎜ ⎟ =
⎢offer ⎜ day ⎟ × PTMR ⎜ kg ⎟ ⎥ − ⎢orts ⎜ day ⎟ × POrts ⎜ kg ⎟ ⎥
⎝ day ⎠ ⎣ ⎝ ⎠ ⎝ ⎠⎦ ⎣ ⎝ ⎠ ⎝ ⎠⎦

Sorting index=
observed intake ( )kg
day

expected intake ( )
kg
day

where, PTMR is the TMR particle size, and POrts is the particle size distribution in refusals. The sorting index of 1 means no sorting,
whereas < 1 indicates sorting against and > 1 means that cows sorted for feeds with a specific particle size.

2.5. Ruminal fermentation

On the 19th day of each experimental period, samples of ruminal digesta were collected from the cannulated cows before (0 h), 3,
6, 9, 12, and 15 h relative to the morning feeding. Immediately after each collection, ruminal liquid (250 mL) was obtained by
squeezing digesta through a four-layer cheesecloth. Ruminal fluid pH was measured using a digital pH-meter (MB-10, Marte
Cientifica, Santa Rita do Sapucaí, MG, Brazil). An aliquot (50 mL) of ruminal fluid sample from each time point was frozen for further
analysis of ammonia nitrogen (NH3-N) and short-chain fatty acids (SCFA) concentrations.
Ruminal fluid samples were centrifuged at 2000 × g for 15 min (at room temperature) and 1600 μL of the supernatant was
pipetted into a centrifuge tube and mixed with 400 μL of 1 M orthophosphoric acid for SCFA determination in the Laboratory of
Applied Microbiology of the Federal University of São Carlos (São Carlos, Brazil) by gas chromatography (AOC-20i, Stabilwax-DA™

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L.S. Gheller, et al. Animal Feed Science and Technology 261 (2020) 114406

30 m capillary column, 0.25 mm ID, 0.25 μm df, Restek®) using a flame ionization detector (Shimadzu© GC-2010 Plus Barueri, Brazil)
according to Del Valle et al. (2018).
Another 800 μL of the supernatant from each of sample was pipetted and mixed with 400 μL of sulfuric acid (1 N) for determi-
nation of NH3-N by the phenol-hypochlorite method (Broderick and Kang, 1980) and absorbances were measured on a microplate
reader (Biochrom Asys Microplate Reader, Biochrom, Cambridge, UK).

2.6. Milk production and composition

Cows were milked twice a day (0600 and 1600 h) and the production relative to each milking was electronically recorded (Alpro®,
DeLaval, Tumba, Sweden). Samples of milk (350 mL) were collected on days 15, 16, and 17 of each experimental period and analyzed
for fat, true protein, and lactose concentrations by infrared method (Lactoscan®, Entelbra, Londrina, Brazil). Milk production was
corrected to 3.5% fat (FCM) according to Sklan et al. (1992):

kg ⎞ ⎛ g ⎟⎞ × daily milk production ( kg )

FCM ⎛⎜ ⎟ = (0.432 + 0.1625 × fat in milk ⎜

⎝ day ⎠ ⎝ 100g ⎠ d

Samples of milk (10 mL) collected on day 16 of each experimental period were deproteinized with trichloroacetic acid (5 mL;
25%) according to Broderick and Clayton (1997), filtered on filter paper, and frozen. These samples were analyzed for urea con-
centration using colorimetric kits (urea: cat n. K-056; Bioclin, Belo Horizonte, MG, Brazil), and absorbances were measured on a
semiautomatic spectrophotometer (SBA 200, CELM®). Allantoin concentration in milk was determined according to Fujihara and
Yamaguchi (1978).

2.7. Nitrogen balance and microbial protein synthesis

Nitrogen balance was calculated as the difference between the nitrogen (N) intake (CP intake ÷ 6.25) and the sum of N excreted
in milk (milk CP ÷ 6.38), urine (method 984.13; AOAC, 2000), and feces (fecal excretion × N content). Microbial protein synthesis
was estimated after measuring the concentrations of purine derivatives (PD) in urine and milk according to Chen and Gomes (1992).
Urine samples (20 mL) were collected at the same time points of fecal samples. Immediately after each collection, urine samples were
diluted into sulfuric acid (0.036 N) in a v/v ratio of 1:4 to avoid bacterial destruction of PD.
Daily urinary excretion was estimated based on the urinary creatinine concentration, considering a daily creatinine excretion of
0.212 mmol/kg BW (Chizzoti et al., 2008). The total excretion of PD was calculated by the sum of allantoin (mol/d) in milk and uric
acid (mol/d) in urine. The concentrations of creatinine and uric acid were determined using commercial kits (Bioclin®) by an
enzymatic kinetic colorimetric reaction and absorbances were measured on a semi-automatic biochemical analyzer (SBA 200,
CELM®).
The absorbed PD was calculated according to the equation:
( PD− 0.385 × BW 0.75)
Absorbed PD (mmol/d) =
0.84
where PD is the sum of excreted PD, 0.385 × BW0.75 represents the endogenous excretion of PD, and 0.84 is the recovery of absorbed
PD (Verbič et al., 1990; Chen and Gomes, 1992).
The ruminal microbial N synthesis was calculated using the equation proposed by Chen and Gomes (1992):
(70 × Absorbed PD)
Microbial N (g/d) =
(0.83 × 0.134 × 1000)

considering 70 as the concentration of N in the PD (mg/mol), 0.134 as the purine to N ratio, and 0.83 the intestinal digestibility of
purines.

2.8. Blood parameters

On the 15th d of each experimental period, blood samples were collected by puncture of coccygeal vessels into vacutainer tubes
without clot activator (BD Vacutainer, Becton, Dickinson and Company, Franklin Lakes, NJ) 4 h after the morning feeding. Blood
samples were centrifuged at 2.000 × g for 15 min (at 25 °C) and serum obtained was frozen until analyses. Serum concentrations of
glucose and urea were analyzed using colorimetric biochemical kits (glucose: cat. n. K-082; urea: cat. n. K-056; Bioclin®).
Absorbances were measured on a semi-automatic spectrophotometer (SBA 200, CELM®).

2.9. Feeding activity

The feeding activity was recorded during 48 h, starting at 0600 h on day 13 d and ending at 0600 h on day 15 of each experi-
mental period. Cows were observed every 5 min for the following activities: eating, drinking, lying down, standing, and milking time
(Silva et al., 2016). Rumination time (min/d) was recorded by an electronic monitoring system (HealthyCow 24® Solution, SCR
Allflex, Netanya, Israel). According to these activities, the indices of rumination, idling [24 h - (consumption + feeding + milking)],

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and chewing (rumination + feeding) were calculated.

2.10. Statistical analysis

Data were analyzed using PROC MIXED of SAS 9.3 (SAS Institute, Cary, NC), according to the following statistical model:
Yijkl = μ + Qi + aj : i + Tk + Pl + eijkl

With: aj:i ≈ N (0, σa2 ) and eijkl ≈ N (0, σe2 ); where: Yijkl is the observed value of the response variable; μ is the overall mean; Qi is the
fixed block effect (i = 1–5); aj:i is the random animal effect within each Latin square (j = 1–25); Tk is the fixed effect of the treatment
(k = 1–5); Pl is the fixed effect of the experimental period (l = 1–5); eijkl is the experimental error; N stands for normal distribution
(Gaussian); σa2 is the variance associated with the random animal effect; and σe2 is the residual variance.
The ruminal fermentation data were analyzed according to the following model:
Yijklm = μ + Qi + aj : i + Tk + Pl + ωijkl + Hm + T × Hkm + eijklm

With: aj:i ≈ N (0, σa2 ), ωijkl ≈ N (0, σω2 ), and eijklm ≈ MVN (0, R); where: Yijklm is the observed value of the response variable; μ, Qi,
aj:i, Tk, and Pl were previously described; ωijkl is the residual error associated with cows within experimental period; Hm is the fixed
effect of sampling time (m = 1–6); T x Hkm is the interaction between treatment and time; eijklm is the experimental error; N stands for
normal distribution (Gaussian); σa2 is the variance associated with the random animal effect; σω2 is the residual variance associated
with the animals in each evaluation period; MVN stands for multivariate with normal distribution; and R is an array of variance and
covariance due to repeated measurements over time.
The TMR temperature data were analyzed according to the following model:
¯
Yijkl = μ + Bi + Tj + ωij + C1 (Xij − X ) + Hk + T × Hjk + eijkl

With ωij ≈ N (0, σω2 ), and eijkl ≈ MVN (0, R); where: Yijkl is the observed value of the response variable; μ is the overall mean; Bi is
the fixed effect of feed bunks (i = 1–30); Tj is the fixed effect of treatment (j = 1–5); ωij is the residual error associated with ex-
¯
perimental unit; C1 is a fixed effect of regression coefficient; Xij is the covariate measurement for feed bunk; X is the overall mean of

Table 3
Nutrient intake and apparent total-tract digestibility, and sorting index of dairy cows fed with a total mixed ration (TMR) treated with organic acids-
based products.
Item Treatmenta SEMb P-valuec

CON FLE SOL SPE PER C1 C2 C3 C4

Intake (kg/d)
Dry matter 23.6 24.5 24.6 24.5 24.0 0.207 0.021 0.31 0.82 0.27
Organic matter 21.8 22.6 22.6 22.4 22.3 0.194 0.037 0.45 0.97 0.92
Non-fiber carbohydrate 10.9 11.2 11.4 11.2 11.0 0.099 0.039 0.13 0.28 0.60
Neutral detergent fiber 7.62 7.97 7.93 7.88 7.92 0.072 0.014 0.61 0.80 0.80
Crude protein 3.73 3.84 3.83 3.81 3.80 0.033 0.094 0.55 0.95 0.96
Ether extract 0.813 0.841 0.839 0.827 0.816 0.008 0.18 0.11 0.91 0.53
DMI:BWd 36.7 37.9 38.0 37.8 37.8 0.410 0.028 0.70 0.87 0.98
NDF:BWe 11.9 12.5 12.4 12.3 12.2 0.140 0.024 0.43 0.72 0.71
Apparent total tract digestibility
Dry matter 0.725 0.731 0.728 0.730 0.728 0.0027 0.53 0.99 0.74 0.83
Organic matter 0.739 0.745 0.743 0.745 0.742 0.0029 0.50 0.89 0.74 0.73
Neutral detergent fiber 0.548 0.554 0.548 0.554 0.557 0.0038 0.55 0.64 0.60 0.85
Crude protein 0.724 0.722 0.720 0.729 0.722 0.0033 0.91 0.42 0.82 0.43
Ether extract 0.858 0.857 0.852 0.841 0.853 0.0043 0.50 0.40 0.69 0.37
Sorting indexf
> 19 mm 0.930 0.963 0.936 0.930 0.956 0.0084 0.33 0.67 0.24 0.26
19 - 8 mm 0.990 0.998 1.002 0.993 0.996 0.0014 0.028 0.069 0.33 0.38
8 - 4 mm 0.992 0.989 0.993 0.991 0.991 0.0012 0.71 0.99 0.31 0.87
< 4 mm 1.033 1.019 1.016 1.028 1.020 0.0022 0.0010 0.054 0.60 0.082

a
Treatments: control: (CON), basal diet without organic acids; formic acid 1 (FLE; ProMyr TMR Flexible®); formic acid 2 (SOL; ProMyr TMR
Solid®); propionic acid 1 (SPE; ProMyr TMR Special®); propionic acid 2 (PER; ProMyr TMR Performance®). All products were manufactured by
Perstorp Waspik BV (Waspik, Netherlands).
b
Standard error of the mean.
c
Probabilities for contrasts effects: C1 = control vs. organic acids; C2 = Formic acid (FLE + SOL) vs. Propionic acid (PER + SPE); C3 = Formic
acid 1 (FLE) vs. Formic acid 2 (SOL); C4 = Propionic 1 (SPE) vs. Propionic 2 (PER).
d
Dry matter intake : body weight ratio (g/kg BW).
e
Neutral detergent fiber intake : body weight ratio (g/kg BW).
f
When 1 means that there was no selection; values < 1 indicates sorting against; and values > 1 indicates sorting for particles on the particular
size range.

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the covariate measurements; Hk is the fixed effect of sampling time (k = 1–24); T × Hjk is the interaction between treatment and time;
σω2 is the residual variance associated with the feed bunk in each sampling time; eijkl is the experimental error. N and MVN were
previously described.
The following variance-covariance matrices were evaluated: autoregressive order 1, composite symmetric, Toeplitz and un-
structured. The matrix was selected based on the Bayesian criterion, using the lowest value.
The differences between treatments were analyzed according to the following orthogonal contrasts: C1: effect of the addition of
OA [control versus treatments (SPE + SOL + PER + FLE)]; C2: the OA type effect [formic acid (FLE + SOL) versus propionic acid
(PER + SPE)]; C3: the effect of different formic acid products (FLE versus SOL); and C4: the effect of different propionic acid products
(SPE versus PER). Significance level was set at P ≤ 0.05, and tendency was considered 0.05 < P ≤ 0.10. The highest P-value for
each significant variable was described in results section. The square root of sample size was considered as degrees of freedom for the
standard errors of the means.

3. Results

Total mixed ration treated with OA increased (P ≤ 0.039) DM, organic matter (OM), non-fiber carbohydrate (NFC), and NDF
intake, tended to increase CP intake (P = 0.094), and did not affect EE intake of cows (P = 0.18; Table 3). The ratios of DMI to BW
and NDF intake to BW were increased when treating TMR with OA. Despite the differences in feed intake, OA did not affect (P ≥
0.50) total tract digestibility of nutrients. Treating TMR with OA increased (P ≤ 0.028) sorting for feed particles between 8–19 mm
size and decreased sorting for feed particles shorter than 4 mm in relation to CON. Feeding TMR treated with OA composed mainly of
formic acid (FLE and SOL) tended to increase (P ≤ 0.069) sorting for feed particles between 8–19 mm size and tended to decrease
(P = 0.054) sorting for feed particles shorter than 4 mm in comparison with TMR treated with OA composed mainly of propionic
acid (SPE and PER).
Treating TMR with OA products decreased (P = 0.038) ruminal concentration of acetate and tended to decrease (P = 0.061)
ruminal concentration of total VFA (Table 4). Cows fed TMR treated with OA products had higher (P = 0.016) ruminal fluid pH in
comparison with CON group. Feeding TMR with OA products composed mainly of propionic acid resulted in greater ruminal fluid
propionate concentration and lower acetate to propionate ratio compared with OA products containing mostly formic acid. When
comparing the OA products composed mainly of propionic acid, TMR treated with PER tended to increase (P = 0.059) acetate to
propionate ratio in rumen of cows in comparison with (SPE).
Treating TMR with OA improved (P ≤ 0.046) FCM yield and protein content in milk (Table 5). In addition, TMR treated with OA
tended to increase (P ≤ 0.061) milk protein yield and lactose content. Organic acid-based products did not affect (P > 0.15) milk
yield and composition of cows. No treatment effect (P > 0.11) was detected for N balance and serum metabolites (Table 6). Organic
acids did not affect activity of cows, except for a trend to greater (P = 0.083) idling time (Table 7). Comparisons among OA products
revealed a greater time (P = 0.083) spent lying and a tendency (P = 0.093) to less time spent chewing for cows fed TMR with
propionic acid-based products (SPE and PER) compared with formic acid-based products (FLE and SOL). As expected, OA products
mitigated (P = 0.003) the increase in TMR temperature in relation to CON, especially after 10 h of exposure to air (Fig. 1).

Table 4
Ruminal volatile fatty acids (VFA) profile, pH, and NH3-N concentration of dairy cows fed a total mixed ration (TMR) treated with organic acids-
based products.
Item Treatmenta SEMb P-valuec

CON FLE SOL SPE PER C1 C2 C3 C4

VFA (mM)
Acetate 68.8 65.3 65.9 63.9 64.6 0.820 0.038 0.42 0.82 0.77
Propionate 26.7 24.5 23.9 27.1 25.2 0.615 0.15 0.041 0.66 0.15
Butyrate 17.6 16.8 16.5 16.7 16.4 0.341 0.14 0.81 0.77 0.74
BCFAd 4.83 4.63 4.80 4.73 4.73 0.095 0.47 0.93 0.35 0.99
Total VFA 118 111 111 113 111 1.61 0.061 0.77 0.98 0.59
pH 6.14 6.23 6.25 6.22 6.20 0.020 0.016 0.29 0.65 0.66
NH3-Ne (mg/dL) 15.3 15.2 14.4 14.5 14.5 0.339 0.42 0.71 0.48 0.95
A:Pf 2.68 2.72 2.80 2.47 2.63 0.041 0.69 < 0.001 0.32 0.059

No interaction effect between treatment and time (P ≥ 0.121) was observed for ruminal fermentation variables.
a
Treatments: control: (CON), basal diet without organic acids; formic acid 1 (FLE; ProMyr TMR Flexible®); formic acid 2 (SOL; ProMyr TMR
Solid®); propionic acid 1 (SPE; ProMyr TMR Special®); propionic acid 2 (PER; ProMyr TMR Performance®). All products were manufactured by
Perstorp Waspik BV (Waspik, Netherlands).
b
Standard error of the mean.
c
Probabilities for contrasts effects: C1 = control vs. organic acids; C2 = Formic acid (FLE + SOL) vs. Propionic acid (PER + SPE); C3 = Formic
acid 1 (FLE) vs. Formic acid 2 (SOL); C4 = Propionic 1 (SPE) vs. Propionic 2 (PER).
d
Branched-chain fatty acids.
e
Ammonia nitrogen.
f
Acetate to propionate ratio.

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L.S. Gheller, et al. Animal Feed Science and Technology 261 (2020) 114406

Table 5
Milk yield and composition of dairy cows fed a total mixed ration (TMR) treated with organic acids-based products.
Item Treatmenta SEMb P-valuec

CON FLE SOL SPE PER C1 C2 C3 C4

Yield (kg/day)
Milk yield 29.6 30.0 30.7 30.2 29.7 0.444 0.19 0.31 0.21 0.34
3.5% FCMd 30.3 31.3 32.2 31.3 30.8 0.466 0.046 0.15 0.20 0.47
Fat 1.08 1.12 1.18 1.12 1.13 0.021 0.051 0.34 0.10 0.73
Protein 0.97 0.99 1.01 1.00 0.98 0.014 0.061 0.41 0.26 0.29
Lactose 1.46 1.49 1.52 1.51 1.47 0.021 0.081 0.41 0.32 0.21
Milk composition (g/kg)
Fat 36.7 37.3 38.7 37.2 38.0 0.580 0.12 0.49 0.10 0.33
Protein 33.0 33.2 33.3 33.3 33.3 0.130 0.013 0.74 0.52 0.95
Lactose 49.4 49.7 49.8 49.9 49.7 0.180 0.051 0.77 0.76 0.38
e
MUN (mg/dL) 13.9 14.3 14.0 13.7 13.5 0.250 0.89 0.25 0.64 0.82
Efficiency
Milk : DMIf 1.25 1.22 1.25 1.23 1.22 0.019 0.20 0.51 0.20 0.64
FCM : DMIg 1.29 1.28 1.31 1.27 1.26 0.020 0.64 0.19 0.18 0.65

a
Treatments: control: (CON), basal diet without organic acids; formic acid 1 (FLE; ProMyr TMR Flexible®); formic acid 2 (SOL; ProMyr TMR
Solid®); propionic acid 1 (SPE; ProMyr TMR Special®); propionic acid 2 (PER; ProMyr TMR Performance®). All products were manufactured by
Perstorp Waspik BV (Waspik, Netherlands).
b
Standard error of the mean.
c
Probabilities for contrasts effects: C1 = control vs. organic acids; C2 = Formic acid (FLE + SOL) vs. Propionic acid (PER + SPE); C3 = Formic
acid 1 (FLE) vs. Formic acid 2 (SOL); C4 = Propionic 1 (SPE) vs. Propionic 2 (PER).
d
3.5% FCM = Fat corrected milk at 3.5%.
e
Milk urea nitrogen.
f
Milk yield to dry matter intake ratio.
g
3.5% Fat-corrected milk to dry matter intake ratio.

Table 6
Nitrogen balance, microbial protein synthesis, and serum metabolites of dairy cows fed a total mixed ration (TMR) treated with organic acids-based
products.
Item Treatmenta SEMb P-valuec

CON FLE SOL SPE PER C1 C2 C3 C4

N intake (g/d) 597 614 613 609 602 5.53 0.49 0.17 0.30 0.98
Fecal N (g/d) 205 205 215 215 212 3.22 0.67 0.36 0.58 0.30
Urinary N (g/d) 197 192 193 219 208 9.60 0.85 0.78 0.29 0.94
Milk N (g/d) 151 156 158 156 152 2.22 0.16 0.11 0.20 0.39
N balance (g/d N intake) 45.4 61.8 46.4 19.1 29.2 11.3 0.82 0.20 0.64 0.76
Microbial protein synthesis (kg/d) 2.04 1.89 2.22 1.88 2.19 0.099 0.95 0.92 0.28 0.31
Serum metabolites
Glucose (mg/dL) 66.1 63.8 64.0 63.9 62.8 0.719 0.13 0.72 0.96 0.60
Urea nitrogen (mg/dL) 37.0 37.8 38.0 35.6 37.8 0.593 0.79 0.26 0.88 0.14

a
Treatments: control: (CON), basal diet without organic acids; formic acid 1 (FLE; ProMyr TMR Flexible®); formic acid 2 (SOL; ProMyr TMR
Solid®); propionic acid 1 (SPE; ProMyr TMR Special®); propionic acid 2 (PER; ProMyr TMR Performance®). All products were manufactured by
Perstorp Waspik BV (Waspik, Netherlands).
b
Standard error of the mean.
c
Probabilities for contrasts effects: C1 = control vs. organic acids; C2 = Formic acid (FLE + SOL) vs. Propionic acid (PER + SPE); C3 = Formic
acid 1 (FLE) vs. Formic acid 2 (SOL); C4 = Propionic 1 (SPE) vs. Propionic 2 (PER).

4. Discussion

In agreement with our hypothesis, the addition of OA in the TMR was able to maintain the TMR temperature stable for a longer
period and increased DM intake of cows. Although no differences were detected in milk yield, OA treatments increased FCM pro-
duction. In view of the relatively small number of studies regarding the inclusion of OA in TMR, discussion was based the most on
results of OA inclusion during the ensiling process and their subsequent effects on performance of cows. Furthermore, the pre-
servative products used in this study included several components in relatively low content that were not discussed, such as po-
tassium sorbate, glycerol propionates, silica, and hexanoic acid.
Organic acids inclusion maintained the TMR temperature stable for longer periods. There was no difference, however, between
formic and propionic acid inclusion; thus, either the antimycotic effects from propionic acid (Santos et al., 2019a) or the inhibition of
fermentation by formic acid (Seppälä et al., 2016) were effective to preserve TMR. The barn temperature (24.4 ± 4.29 °C) where

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L.S. Gheller, et al. Animal Feed Science and Technology 261 (2020) 114406

Table 7
Feeding activity in of lactating cows fed a total mixed ration (TMR) treated with organic acids-based products.
Item Treatmenta SEMb P-valuec

COM FLE SOL SPE PER C1 C2 C3 C4

Activity (min/day)
Drinking 38.8 38.8 40.6 41.0 42.7 1.72 0.58 0.53 0.68 0.78
Eating 242 247 256 241 252 3.62 0.22 0.36 0.27 0.15
Lying down 710 697 676 715 709 11.0 0.42 0.040 0.24 0.76
Ruminating 466 477 482 474 474 6.92 0.31 0.50 0.65 0.97
Idlingd 991 982 975 984 977 3.96 0.083 0.73 0.37 0.44
Chewinge 709 728 738 707 725 8.30 0.15 0.093 0.47 0.20

a
Treatments: control: (CON), basal diet without organic acids; formic acid 1 (FLE; ProMyr TMR Flexible®); formic acid 2 (SOL; ProMyr TMR
Solid®); propionic acid 1 (SPE; ProMyr TMR Special®); propionic acid 2 (PER; ProMyr TMR Performance®). All products were manufactured by
Perstorp Waspik BV (Waspik, Netherlands).
b
Standard error of the mean.
c
Probabilities for contrasts effects: C1 = control vs. organic acids; C2 = Formic acid (FLE + SOL) vs. Propionic acid (PER + SPE); C3 = Formic
acid 1 (FLE) vs. Formic acid 2 (SOL); C4 = Propionic 1 (SPE) vs. Propionic 2 (PER).
d
Idling =24 h – (drinking + eating + milking).
e
Chewing = eating + ruminating.

Fig. 1. Total mixed ration (TMR) average temperature over 24 h-period.

Treatments: control: (CON), basal diet without organic acids; formic acid 1 (FLE; ProMyr TMR Flexible®); formic acid 2 (SOL; ProMyr TMR Solid®);
propionic acid 1 (SPE; ProMyr TMR Special®); propionic acid 2 (PER; ProMyr TMR Performance®). All products were manufactured by Perstorp
Waspik BV (Waspik, Netherlands).
Probabilities for treatment effect using orthogonal contrasts: C1: control vs organic acids; C2: Formic acid (FLE + SOL) vs Propionic acid
(PER + SPE); C3: Formic acid 1 (FLE) vs Formic acid 2 (SOL); C4: Propionic 1 (SPE) vs Propionic 2 (PER).
Probabilities (P): Time: P = < 0.001; Treatment vs. Time: P = 0.001; C1: P = 0.003; C2: P = 0.638; C3: P = 0.18, and C4: P = 0.25; Standard
error of the mean (SEM) = 0.144.
The addition of TMR and organic acid products occurred at time points 0 and 5 h or 08:00 and 13:00 h respectively.

TMR temperature was assessed remained within the range which silages are more susceptible to deterioration and yeast multi-
plication (Ashbell et al., 2002).
Addition of OA to TMR markedly increased the DMI of cows. The positive results of OA observed in DMI might be related to a
decrease in the concentration of hypophagic compounds in the TMR, such as butyrate, ammonia, and biogenic amines (Van Os et al.,
1995). For example, butyrate increases ruminal osmolality and reduces meal size (Allen, 2000). In addition, butyrate may stimulate
satiety signals through chemoreceptors in the rumen wall, liver, or portal system (Rook et al., 1965; Simkins et al., 1965; Urrutia
et al., 2019). Increases in silage ammonia concentration reduce feed acceptability by cows (Kertz et al., 1982), and biogenic amines
reduce ruminal motility and DMI (Scherer et al., 2015). The use of OA during the silage process reduces the accumulation of these
substances, maintaining silage nutritive value and avoiding losses during the ensiling period (Oliveira et al., 2017). Although the
concentrations of these hypophagic metabolites were not evaluated in the current study, OA have decreasead losses in the nutritionl
value of feed after exposure in similar conditions of the current study. The reduction in TMR pH due to OA inclusion preserves the
feed and increases its aerobic stability during the day, leading to positive impacts on feed intake (Daniel et al., 2013). Agreeing with
the current study, authors have reported lower temperature of refusals after 24 h in TMR treated with propionic acid (Kung et al.,
1998). Interestingly, OA products added to TMR have been associated with decreased DMI during the first few weeks after treatment

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L.S. Gheller, et al. Animal Feed Science and Technology 261 (2020) 114406

but returned to normal levels after four weeks or more (Krizsan et al., 2012; Daniel et al., 2013). The latter fact did not occur in our
study. Felton and DeVries (2010) negatively associated feed temperature with lower DMI. In this study, OA apparently avoided an
increase in TMR temperature and consequently, improved DMI of cows.
Organic acids inclusion altered the sorting index by increasing the intake of feed particles with 8–19 mm size and decreasing the
intake of feed particles shorter than 4 mm size. Sorting for longer particles increases the intake of physically effective fiber which
stimulates rumination, saliva production, and rumen buffering (Zebeli et al., 2006). In addition, formic acid increased the intake of
feed particles with 8–19 mm size in comparison with propionic acid. Because of decreased small particles intake, rumen VFA con-
centration was reduced. As expected, treatments with propionic acid increased ruminal concentration of propionate and decreased
acetate to propionate in the rumen fluid in relation to formic acid treatments. The decrease in ruminal acetate concentration by
treatments was not expected, since there was a greater intake of feed with longer particle size. Organic acid blends may have altered
the rumen microbial population or activity, and more trials should be carried to investigate the effects on rumen microorganisms. It is
worth mentioning that rumen VFA concentration does not correspond to the actual VFA production and differences in rumen volume
may hinder comparisons (Hall et al., 2015). Although there was a decrease in rumen acetate concentration with OA inclusion, most
VFA had a numerical decrease in concentration thus, affecting the total VFA concentration.
Although milk yield was not affected by treatments, FCM production increased with OA inclusion. Boosts in energy intake may
increase FCM (Khan et al., 2015). Milk solids yield tended to increase with OA inclusion likely because of the marginal increase in
milk yield. Greater DMI and less proteolysis, amino acid deamination and decarboxylation may have increased the availability of
metabolizable protein and energy for milk protein synthesis (Daniel et al., 2016; Oliveira el at., 2017). This response is observed with
silages treated with additives during the ensiling process (Nagel and Broderick, 1992; Oliveira et al., 2017; Muck et al., 2018). The
tendency for increased milk lactose content might be related with the greater ruminal propionate concentration observed in SPE
treatment. It is well known that propionate is a precursor for gluconeogenesis and glucose is a substrate for lactose synthesis (Wang
et al., 2016).
No differences in nutrient digestibility were detected in this study. The lack of effects in nutrient digestibility when incorporating
formic acid to the silage have been reported in literature (Halmemies-Beauchet-Filleau et al., 2013). Serum concentrations of glucose
and urea nitrogen were similar between treatments. In agreement with the current study, Nagel and Broderick (1992) and Daniel
et al. (2013) did not detect differences in blood concentrations of glucose and urea nitrogen of cows fed silage with formic acid or
TMR with. Treatments tended to decrease idling because of increased the time spent eating and drinking. Formic acid reduced the
time spent lying down and tended to increase chewing time in comparison with propionic acid, suggesting that formic acid may be
more beneficial to feed intake.

5. Conclusion

The addition of OA in TMR is an effective way to minimize increases in feed temperature while promoting DMI and FCM
production of dairy cows. Studies are needed to evaluate OA effects on rumen microorganism population, feed deterioration and
aerobic stability.

CRediT authorship contribution statement

Larissa S. Gheller: Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Methodology. Lucas G.
Ghizzi: Formal analysis, Investigation, Writing - review & editing. Júlia A. Marques: Investigation, Writing - review & editing. Caio
S. Takiya: Writing - review & editing. Nathália T.S. Grigoletto: Investigation, Writing - original draft. Mauro S.S. Dias:
Investigation. Tássia B.P. Silva: Investigation. Alanne T. Nunes: Investigation. Guilherme G. da Silva: Investigation. Luis G.X.
Fernandes: Investigation. Luciana N. Rennó: Supervision. Francisco P. Rennó: Conceptualization, Methodology, Supervision.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgments

Authors appreciate the Dairy Cattle Research Laboratory staff for animal handling and feeding. Authors are thankful to Perstorp
Waspik BV (Waspik, The Netherlands) for partial funding of this research. L. S. Gheller thanks the Coordination for the Improvement
of Higher Education Personnel (CAPES, Brasília, Brazil) and São Paulo Research Foundation (FAPESP, São Paulo, Brazil) for scho-
larships awarded [grant #2018/03652-2].

References

Allen, M.S., 2000. Effects of diet on short-term regulation of feed intake by lactating dairy cattle. J. Dairy Sci. 83, 1598–1624. https://doi.org/10.3168/jds.S0022-
0302(00)75030-2.
AOAC, 2000. Official Methods of Analysis, 17th Ed. Association of official analytical chemists, Arlington, VA, USA.
Ashbell, G., Weinberg, Z.G., Hen, Y., Filya, I., 2002. The effects of temperature on the aerobic stability of wheat and corn silages. J. Ind. Microbiol. Biotechnol. 28,

10
L.S. Gheller, et al. Animal Feed Science and Technology 261 (2020) 114406

261–263. https://doi.org/10.1038/sj/jim/7000237.
Broderick, G.A., Clayton, M.K., 1997. A statistical evaluation of animal and nutritional factors influencing concentrations of milk urea nitrogen. J. Dairy Sci. 80,
2964–2971. https://doi.org/10.3168/jds.S0022-0302(97)76262-3.
Broderick, G.A., Kang, J.H., 1980. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy Sci. 63,
64–75. https://doi.org/10.3168/jds.S0022-0302(80)82888-8.
Casali, A.O., Detmann, E., Valadares Filho, S.D.C., Pereira, J.C., Henriques, L.T., Freitas, S.Gd., Paulino, M.F., 2008. Influência do tempo de incubação e do tamanho de
partículas sobre os teores de compostos indigestíveis em alimentos e fezes bovinas obtidos por procedimentos in situ. R. Bras. Zootec. 37, 335–342.
Chen, X.B., Gomes, M.J., 1992. Estimation of Microbial Protein Supply to Sheep and Cattle Based on Urinary Excretion of Purine Derivatives – an Overview of
Technical Details. Rowett Research Institute: International Feed Research Unit, Bucksburnd, UK.
Chizzoti, M.L., Valadares, S.C., Valadares Filho, R.F.D., Chizzotti, F.H.M., Tedeschi, L.O., 2008. Determination of creatinine excretion and evaluation of spot urine
sampling in Holstein cattle. Livest. Prod. Sci. 113, 218–225. https://doi.org/10.1016/j.livsci.2007.03.013.
Daniel, J.B., Friggens, N.C., Chapoutot, P., Van Laar, H., Sauvant, D., 2016. Milk yield and milk composition responses to change in predicted net energy and
matabolizable protein: a meta-analysis. Animal 10, 1975–1985. https://doi.org/10.1017/S1751731116001245.
Daniel, J.L.P., Amaral, R.C., Sa Neto, A., Cabezas-Garcia, E.H., Bispo, A.W., Zopollatto, M., Cardoso, T.L., Spoto, M.H.F., Santos, F.A.P., Nussio, L.G., 2013.
Performance of dairy cows fed high levels of acetic acid or ethanol. J. Dairy Sci. 96, 398–406. https://doi.org/10.3168/jds.2012-5451.
Del Valle, T.A., Zenatti, T.F., Antonio, G., Campana, M., Gandra, J.R., Zilio, E.M.C., de Mattos, L.F.A., de Morais, J.G.P., 2018. Effect of chitosan on the preservation
quality of sugarcane silage. Grass Forage Sci. 73, 630–638. https://doi.org/10.1111/gfs.12356.
DeVries, T.J., von Keyserlingk, M.A.G., Beauchemin, K.A., 2005. Frequency of feed delivery affects the behavior of lactating dairy cows. J. Dairy Sci. 88, 3553–3562.
https://doi.org/10.3168/jds.S0022-0302(05)73040-X.
Felton, C.A., DeVries, T.J., 2010. Effect of water addition to a total mixed ration on feed temperature, feed intake, sorting behavior, and milk production of dairy cows.
J. Dairy Sci. 93, 2651–2660. https://doi.org/10.3168/jds.2009-3009.
Fujihara, S., Yamaguchi, M., 1978. Effects of allopurinol [4-Hydroxypyrazolo(3,4-d)Pyrimidine] on the metabolism of allantoin in soybean plants. Plant Physiol. 62,
134–138.
Goering, H.K., Gordon, C.H., 1973. Chemical aids to preservation of high moisture feeds. J. Dairy Sci. 56, 1347–1351. https://doi.org/10.3168/jds.S0022-0302(73)
85360-3.
Hall, M.B., Nennich, T.D., Doane, P.H., Brink, G.E., 2015. Total volatile fatty acid concentrations are unreliable estimators of treatment effects on ruminal fermentation
in vivo1. J. Dairy Sci. 98, 3988–3999. https://doi.org/10.3168/jds.2014-8854.
Halmemies-Beauchet-Filleau, A., Kairenius, P., Ahvenjarvi, S., Crosley, L.K., Muetzel, S., Huhtanen, P., Vanhatalo, A., Toivonen, V., Wallace, R.J., Shingfield, K.J.,
2013. Effect of forage conservation method on ruminal lipid metabolism and microbial ecology in lactating cows fed diets containing a 60:40 forage-to-concentrate
ratio. J. Dairy Sci. 96, 2428–2447. https://doi.org/10.3168/jds.2012-6043.
Haque, M.N., Chowdhury, R., Islam, K.M., Akbar, M., 2012. Propionic acid is an alternative to antibiotics in poultry diet. BJAS. 38, 115–122. https://doi.org/10.3329/
bjas.v38i1-2.9920.
Heinrichs, J., Kononoff, P., 2013. Evaluating Particle Size of Forages and TMRs Using the New Penn State Forage Particle Separator. Available at:. University Park, PA.
www.das.psu.edu/teamdairy/.
Hendrix, D.L., 1993. Rapid extraction and analysis of nonstructural carbohydrates in plant tissues. Crop Sci. 33, 1306–1311.
Huhtanen, P., Kaustell, K., Jaakkola, S., 1994. The use of internal markers to predict total digestibility and duodenal flow of nutrients in cattle given six different diets.
Anim. Feed Sci. Technol. 48, 211–227. https://doi.org/10.1016/0377-8401(94)90173-2.
Jongbloed, A.W., Mroz, Z., van der Weij-Jongbloed, R., Kemme, P.A., 2000. The effects of microbial phytase, organic acids and their interaction in diets for growing
pigs. Livest. Prod. Sci. 67, 113–122. https://doi.org/10.1016/S0301-6226(00)00179-2.
Kertz, A.F., Koepke, M.K., Davidson, L.E., Betz, N.L., Norris, J.R., Skoch, L.V., Cords, B.R., Hopkins, D.T., 1982. Factors influencing intake of high urea-containing
rations by lactating dairy cows. J. Dairy Sci. 65, 587–604. https://doi.org/10.3168/jds.S0022-0302(82)82236-4.
Khan, N.A., Yu, P., Ali, M., Cone, J.W., Hendriks, W.H., 2015. Nutritive value of maize silage in relation to dairy cow performance and milk quality. J. Sci. Food Agric.
95, 238–252. https://doi.org/10.1002/jsfa.6703.
Krizsan, S.J., Randby, A.T., Westad, F., 2012. Effect of acetic acid, caproic acid and tryptamine on voluntary intake of grass silage by growing cattle. Grass Forage Sci.
67, 361–368.
Kung Jr., L., 2010. In: Aerobic Stability of Silage, Proc. 2010 California Alfalfa and Forage Symposium and Crop/cereal Conference. Visalia, CA, USA.
Kung, L.J., Sheperd, A.C., Smagala, A.M., Endres, K.M., Bessett, C.A., Ranjit, N.K., Glancey, J.L., 1998. The effect of preservatives based on propionic acid on the
fermentation and aerobic stability of corn silage and a total mixed ration. J. Dairy Sci. 81, 1322–1330. https://doi.org/10.3168/jds.S0022-0302(98)75695-4.
Lambert, R.J., Stratford, M., 1999. Weak-acid preservatives: modelling microbial inhibition and response. J. Appl. Microbiol. 86, 157–164.
Mader, T.L., Davis, M.S., Brown-Brandl, T., 2006. Environmental factors influencing heat stress in feedlot cattle. J. Anim. Sci. 84, 712–719. https://doi.org/10.2527/
2006.843712x.
McCoy, G.C., Thurmon, H.S., Olson, H.H., Reed, A., 1966. Complete feed rations for lactating dairy cows 1, 2. J. Dairy Sci. 49, 1058–1063. https://doi.org/10.3168/
jds.S0022-0302(66)88017-7.
Moriel, P., Piccolo, M.B., Artioli, L.F.A., Santos, G.S., Poore, M.H., Ferraretto, L.F., 2016. Method of propionic acid-based preservative addition and its effects on
nutritive value and fermentation characteristics of wet brewers grains ensiled in the summertime. AAS. 32, 591–597. https://doi.org/10.15232/pas.2016-01513.
Muck, R.E., Nadeau, E.M.G., McAllister, T.A., Contreras-Govea, F.E., Santos, M.C., Kung, L., 2018. Silage review: recent advances and future uses of silage additives. J.
Dairy Sci. 101, 3980–4000. https://doi.org/10.3168/jds.2017-13839.
Nagel, S.A., Broderick, G.A., 1992. Effect of formic acid or formaldehyde treatment of alfalfa silage on nutrient utilization by dairy cows. J. Dairy Sci. 75, 140–154.
https://doi.org/10.3168/jds.S0022-0302(92)77748-0.
National Research Council (NRC), 2001. Nutrient Requirements of Dairy Cattle, 7th Revised Edition. National Academic Science, Washington, DC, USA.
Oliveira, A.S., Weinberg, Z.G., Ogunade, I.M., Cervantes, A.A.P., Arriola, K.G., Jiang, Y., Kim, D., Li, X., Gonçalves, M.C.M., Vyas, D., Adesogan, A.T., 2017. Meta-
analysis of effects of inoculation with homofermentative and facultative heterofermentative lactic acid bacteria on silage fermentation, aerobic stability, and the
performance of dairy cows. J. Dairy Sci. 100, 4587–4603. https://doi.org/10.3168/jds.2016-11815.
Rook, J., Balch, C., Johnson, V., 1965. Further observations on the effects of intraruminal infusions of volatile fatty acids and of lactic acid on the yield and
composition of the milk of the cow. Br. J. Nutr. 19, 93–99. https://doi.org/10.1079/BJN19650008.
Santos, J.P., Souza, V.C., Barbosa, E.F., Silva, R.B., Avila, C.L.S., Pereira, R.A.N., Lobato, D.N., Pereira, M.N., 2019a. Aerobic stability of total mixed ration with added
microbial growth inhibitors. J. Dairy Sci. 102 (Suppl. 1), 204 Abstract.
Santos, J.P., Souza, V.C., Barbosa, E.F., Silva, R.B., Pereira, R.A.N., Avila, C.L.S., Greco, L.F., Pereira, M.N., 2019b. Efficacy of chemical additives on microbial growth
and aerobic stability of total mixed ration. J. Dairy Sci. 102 (Suppl. 1), 204 Abstract.
Scherer, R., Gerlach, K., Südekum, K.H., 2015. Biogenic amines and gamma-amino butyric acid in silages: formation, occurrence and influence on dry matter intake
and ruminant production. Anim. Feed Sci. Technol. 210, 1–16. https://doi.org/10.1016/j.anifeedsci.2015.10.001.
Schingoethe, D.J., 2017. A 100-Year Review: total mixed ration feeding of dairy cows. J. Dairy Sci. 100, 10143–10150. https://doi.org/10.3168/jds.2017-12967.
Seppälä, A., Heikkilä, T., Mäki, M., Rinne, M., 2016. Effects of additives on the fermentation and aerobic stability of grass silages and total mixed rations. Grass Forage
Sci. 71, 458–471. https://doi.org/10.1111/gfs.12221.
Silva, T.H., Takiya, C.S., Vendramini, T.H.A., Jesus, E.Fde, Zanferari, F., Rennó, F.P., 2016. Effects of dietary fibrolytic enzymes on chewing time, ruminal fermen-
tation, and performance of mid-lactating dairy cows. Anim. Feed Sci. Technol. 221, 35–43. https://doi.org/10.1016/j.anifeedsci.2016.08.013.
Silveira, C., Oba, M., Yang, W.Z., Beauchemin, K.A., 2007. Selection of barley grain affects ruminal fermentation, starch digestibility, and productivity of lactating
dairy cows. J. Dairy Sci. 90, 2860–2869. https://doi.org/10.3168/jds.2006-771.
Simkins, K.L., Suttie, J.W., Baumgardt, B.R., 1965. Regulation of food intake in ruminants. 4. Effect of acetate, propionate, butyrate, and glucose on voluntary food

11
L.S. Gheller, et al. Animal Feed Science and Technology 261 (2020) 114406

intake in dairy cattle 1, 2, 3. J. Dairy Sci. 48, 1635–1642. https://doi.org/10.3168/jds.S0022-0302(65)88541-1.

Sklan, D., Ashkenazi, R., Braun, A., Devorin, A., Tabori, K., 1992. Fatty acids, calcium soaps of fatty acids, and cottonseeds fed to high yielding cows. J. Dairy Sci. 75,
2463–2472. https://doi.org/10.3168/jds.S0022-0302(92)78008-4.
Sousa, D.O., Hansen, H.H., Nussio, L.G., Nadeau, E., 2019. Effects of wilting and ensiling with or without additive on protein quality and fermentation of a lucerne-
white clover mixture. Anim. Feed Sci. Technol. 258, 114301. https://doi.org/10.1016/j.anifeedsci.2019.114301.
Undersander, D., Mertens, D.R., Theix, N., 1992. Forage analysis. National Forage Testing Association. USA. National Forage Testing Association, Omaha, NE p. 139.
Urrutia, N., Bomberger, R., Matamoros, C., Harvatine, K.J., 2019. Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in
lactating dairy cows. J. Dairy Sci. 102, 5172–5181. https://doi.org/10.3168/jds.2018-16024.
Van Os, M., Dulphy, J.P., Baumont, R., 1995. The effect of protein degradation products in grass silages on feed intake and intake behaviour in sheep. Br. J. Nutr. 73,
51–64.
Verbič, J., Chen, X.A., MacLeod, N., Orskov, E., 1990. Excretion of purine derivatives by ruminants. Effect of microbial nucleic acid infusion on purine derivative
excretion by steers. J. Agric. Sci. 114, 243–248. https://doi.org/10.1017/S0021859600072610.
Wang, B., Zhao, F.Q., Zhang, B.X., Liu, J.X., 2016. An insufficient glucose supply causes reduced lactose synthesis in lactating dairy cows fed rice straw instead of
alfalfa hay. J. Anim. Sci. 94, 4771–4780. https://doi.org/10.2527/jas.2016-0603.
Zebeli, Q., Tafaj, M., Steingass, H., Metzler, B., Drochner, W., 2006. Effects of physically effective fiber on digestive processes and milk fat content in early lactating
dairy cows fed Total Mixed Rations. J. Dairy Sci. 89, 651–668. https://doi.org/10.3168/jds.S0022-0302(06)72129-4.

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