Rumen fluid from donor cows fed different additives can affect the in vitro fermentation parameters

Abstract

This study assessed the impact of rumen ‘fluid not adapted’ (FNA) and of rumen ‘fluid adapted’ (FA) to three additives on in vitro kinetics of gas production and end products of fermentation.
The experiment was performed according to a Latin Square Design of 4 cows and 4 experimental periods. The dry cows received a total mixed ration designed for lactating cows without (control) or with 1 g/d of one of three additives (allyl-sulphide, cinnamaldehyde, limonene) that were selected for their known effects on rumen fermentation. The collected rumen fluids (FNA and FA, respectively) were used as inoculum of 4 consecutive in vitro incubations (1 for each experimental period) adding or not adding 30 mg of one of the three pure compounds. The incubations were performed using a commercial equipment to evaluate the kinetics of gas production
and collect the end products of fermentation. The results indicated that FA did not influence any fermentation parameters compared to FNA. However, when allyl-sulphide was added in vitro, the effects of this compound tended to be more pronounced with FA than with
FNA. This experiment highlights that the three tested pure additives, which show activity on in vitro fermentations, can alter the in vitro activity of rumen fluid collected from cows fed with these compounds. Therefore, the administration of pure additives directly to the cows can influence the rumen microbial activity and the response of in vitro experiments.

Full text

Italian Journal of Animal Science
ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/tjas20
Rumen fluid from donor cows fed different
additives can affect the invitro fermentation
parameters
Giulia Rossi, Selene Massaro, Sheyla Arango, Sarah Currò, Mauro Spanghero,
Diana Giannuzzi, Stefano Schiavon, Lucia Bailoni & Franco Tagliapietra
To cite this article: Giulia Rossi, Selene Massaro, Sheyla Arango, Sarah Currò, Mauro
Spanghero, Diana Giannuzzi, Stefano Schiavon, Lucia Bailoni & Franco Tagliapietra
(2025) Rumen fluid from donor cows fed different additives can affect the invitro
fermentation parameters, Italian Journal of Animal Science, 24:1, 53-60, DOI:
10.1080/1828051X.2024.2442044
To link to this article: https://doi.org/10.1080/1828051X.2024.2442044
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RESEARCH ARTICLE
Rumen fluid from donor cows fed different additives can affect the in vitro
fermentation parameters
Giulia Rossi
a
, Selene Massaro
a
, Sheyla Arango
b
, Sarah Curr�
o
b
, Mauro Spanghero
c
, Diana
Giannuzzi
a
, Stefano Schiavon
a
, Lucia Bailoni
b
and Franco Tagliapietra
a
a
Dipartimento di Agronomia, Alimenti, Risorse naturali, Animali e Ambiente (DAFNAE), University of Padova, Legnaro, Padova, Italy;
b
Dipartimento di Biomedicina Comparata e Alimentazione (BCA), University of Padova, Legnaro, Padova, Italy;
c
Dipartimento di
Scienze Agroalimentari, Ambientali e Animali, University of Udine, Udine, Italy
ABSTRACT
This study assessed the impact of rumen ‘fluid not adapted’ (FNA) and of rumen ‘fluid adapted’
(FA) to three additives on in vitro kinetics of gas production and end products of fermentation.
The experiment was performed according to a Latin Square Design of 4 cows and 4 experimen-
tal periods. The dry cows received a total mixed ration designed for lactating cows without
(control) or with 1g/d of one of three additives (allyl-sulphide, cinnamaldehyde, limonene) that
were selected for their known effects on rumen fermentation. The collected rumen fluids (FNA
and FA, respectively) were used as inoculum of 4 consecutive in vitro incubations (1 for each
experimental period) adding or not adding 30 mg of one of the three pure compounds. The
incubations were performed using a commercial equipment to evaluate the kinetics of gas pro-
duction and collect the end products of fermentation. The results indicated that FA did not
influence any fermentation parameters compared to FNA. However, when allyl-sulphide was
added in vitro, the effects of this compound tended to be more pronounced with FA than with
FNA. This experiment highlights that the three tested pure additives, which show activity on
in vitro fermentations, can alter the in vitro activity of rumen fluid collected from cows fed with
these compounds. Therefore, the administration of pure additives directly to the cows can influ-
ence the rumen microbial activity and the response of in vitro experiments.
HIGHLIGHTS
The pure compounds influence in vitro rumen fermentations.
The administration of additives to cows can affect the activity of rumen fluid and the
response to in vitro experiments.
ARTICLE HISTORY
Received 21 August 2024
Revised 20 November 2024
Accepted 9 December 2024
KEYWORDS
Pure extract; gas production
kinetic; methane
production; rumen fluid
Introduction
In vitro methods are a quick and cost-effective tool for
evaluating feed quality and screening the effects of
additives and bioactive compounds (e.g. plant com-
pounds with antimicrobial properties) on ruminal fer-
mentation (Klevenhusen et al. 2012; Palangi and
Lackner 2022). Typically, rumen fluid is collected from
donor cows fed diets with no additive supplemented
(fluid not adapted, FNA) and is used as inoculum for
incubation of short duration (usually 24–48 h). The
incubation of an FNA over a short period might gener-
ate misleading interactions between the rumen micro-
bial population and the tested additives leading to a
scarce efficacy of additives requiring long fermentation
time to be effective, or to promote additives that are
active in short-term incubations but not in the long-
term (Calsamiglia et al. 2007). Another interesting
issue, little explored in the past, is to see whether the
fermentative properties of the rumen fluid is altered if
the donor cows are fed specific additives.
Typically, in vitro systems are used to test the fermen-
tative properties of feeds or additives incubated in
rumen fluid with a standard diet and a buffered
medium. A different approach could involve feeding ani-
mals with additives and evaluating the fermentative
properties of rumen fluid collected from these animals
(rumen fluid adapted, FA). This approach could combine
the advantage of in vivo trials (long-time adaptation and
CONTACT Selene Massaro selene.massaro@phd.unipd.it
� 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the
posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
ITALIAN JOURNAL OF ANIMAL SCIENCE
2025, VOL. 24, NO. 1, 53–60
https://doi.org/10.1080/1828051X.2024.2442044

direct evaluation of in vivo effects) with those of in vitro
techniques (standardization of experimental conditions,
reduced cost, evaluation of parameters difficult to meas-
ure in vivo, such as fermentation process dynamics and
methane production kinetics).
Therefore, the research objectives were: (1) to
investigate whether rumen fluid from cows adapted or
non-adapted in vivo to a specific additive, with recog-
nised effects on ruminal fermentation, can influence
the ruminal fermentation tested in vitro. (2) to investi-
gate whether specific additives behave similarly when
fermented in vitro using rumen fluid from cows
adapted or non-adapted to the same additive.
Materials and methods
Ethical approval
The administration of additives to the cows and the
collection of rumen fluid were authorised by the Ethics
Committee for the Care and Use of Experimental Ani-
mals at the University of Padova (Prot. n. 120869).
In vivo experimental design, animals, diets
This paper is part of Dr. Giulia Rossi’s PhD thesis, which
focused on testing different additives to reduce enteric
methane emissions from dairy cattle (Rossi 2017).
The in vivo trial was conducted at the experimental
farm of the University of Padova (Legnaro, Padova,
Italy). A total of four dry cows, homogeneous in age
and parity, were housed in individual pens one week
before the start of the experiment. The cows were ran-
domly assigned to four experimental groups according
to a 4 4 Latin square design and fed with a Total
Mixed Ration (TMR) composed by: Corn silage 291 g/
kg DM; Corn-barley based mixture 233 g/kg DM;
Sunflower-soybean based mixture 173 g/kg DM; Alfalfa
hay 123 g/kg DM; Ryegrass hay 120 g/kg DM; Sugar
beet pulp 37 g/kg DM; Mineral and vitamin mixture
13 g/kg DM; Linseed 10 g/kg DM. The TMR chemical
composition was: dry matter, 462 g/kg as fed; Crude
Protein (CP), 130 g/kg; Ether Extract, 34 g/kg; NDF,
375 g/kg; ADF, 219 g/kg; ADL, 37 g/kg; Starch, 253 g/
kg; Not Starch Carbohydrates (NSC), 401 g/kg; Ash,
60 g/kg. For feeding adaptation, the cows received a
TMR either without supplementation (control, CTR) or
with the addition of one of three additives (1 g/d)
known for their effects on rumen fermentation
(Palangi and Lackner 2022): limonene (Merck, Milan,
Italy; 183164; Purity 97%; LIM), cinnamaldehyde
(Merck, Milan, Italy; W228613; Purity 95%; CIN), and
allyl sulphide (Merck, Milan, Italy; A35801; Purity 97%;
ALL). The TMR was primarily composed of wheat and
corn silages (355 and 257 g/kg DM respectively), with
contents of crude protein, lipids, aNDF, and starch of
154, 33, 378, and 238 g/kg DM, respectively.
The dosage of each additive (1 g/d) was determined
on the basis of the amounts commonly used for feeding
dairy cows (Benchaar et al. 2006), aiming to inhibit
methane emissions without negatively affecting rumen
fermentation activity and animal performance. The daily
amount of TMR fed to each cow was calculated accord-
ing to the nutrient requirements of dairy cows estab-
lished by the NRC (2001) and ranged between 14.1 to
16.9 kg of DM for the four dry-cows. Similarly, to ensure
complete ingestion of the additives, the TMR was distrib-
uted in two daily feedings: an initial portion of approxi-
mately 10 kg DM, with or without supplementation, was
distributed in the morning, while the remaining TMR,
without supplementation for all cows, was distributed in
the afternoon after the complete consumption of the
first portion. To ensure even distribution of the additives
in the TMR the whole individual doses of additives were
diluted in 150 mL of water and sprayed over the TMR of
the cows during the morning meal. Each of the four
consecutive experimental periods lasted a total of
21 days, of which 14 days involved the administration of
the additives, followed by a 7–day transition period dur-
ing which all cows received the TMR without supple-
mentation. According to Warner (1962), a 21–day
experimental period is considered sufficiently cautious to
prevent any carryover effects of dietary treatments on
subsequent experimental phases. The TMR was collected
during each experimental period, bulked and analysed
for proximate composition in three replicates (AOAC
20th edition, 2016).
Rumen fluid collection and in vitro experimental
design
All the in vitro procedures were carried out considering
the guidelines of the Italian Journal of Animal Science.
In each experimental period (21 d), the day after
the last administration of the additives (24 h), the
rumen fluids for in vitro tests were collected from
each cow before morning feeding, using an oesopha-
geal probe managed by a well-trained technician to
minimise salivary dilution (Tagliapietra et al. 2012). The
four rumen fluids were stored separately in four ther-
mal flasks pre-heated at 39 ± 0.5 C, taken to the
laboratory, and filtered through 3 layers of cheesecloth
to eliminate residual feed particles. The pH of the
rumen fluid was immediately tested to verify potential
saliva contaminations.
54 G. ROSSI ET AL.

To dose the additives in the fermentative bottles,
solutions were prepared for the three tested additives,
each consisting of 25 mL of 96% ethanol (v/v) and
750 mg of cinnamaldehyde, limonene, or allyl-sulphide.
The four in vitro tests (one for each experimental
period) were conducted immediately after collecting
the rumen fluid. The in vitro tests utilised a fully auto-
mated commercial wireless apparatus that allows con-
tinuous measurement of gas emissions (AnkomRF GP
System, Ankom Technology
V
R
, NY, USA), as described
by Tagliapietra et al. (2010). This system consists of 44
bottles equipped with pressure sensors and an
antenna for wireless connection to a computer.
In each in vitro incubation, ten experimental treat-
ments were compared using ruminal fluid collected
from cows either fed or not fed with additives (FNA or
FA), with or without the addition of the same addi-
tives also in vitro. In Table 1are listed and described
the 10 experimental treatments and are provided the
corresponding abbreviation codes. The ten experimen-
tal treatments were incubated in four replicates, along
with four bottles without substrate (blanks) containing
only buffer solution and ruminal fluid collected from
each cow (one blank per ruminal fluid). In total, 44
bottles were incubated in each incubation and
repeated in 4 consecutive experimental periods using
the rumen fluid collected from the cow fed with (FA)
and without (FNA) the additives according to the Latin
Square Design (in total 176 observations).
In each fermentation bottle (volume 317mL), the fol-
lowing components were added: 1.000 ± 0.0010g of
substrate (the same TMR fed to the cows, ground
through a 1 mm sieve), 100 mL of buffered medium
(Menke and Steingass 1988), 50 mL of ruminal fluid,
and 1 mL of ethanol-additive solution to achieve an
additive concentration of 30 mg per gram of dry matter
of the substrate. To standardise the in vitro conditions,
the control and blank bottles were also filled with 1 mL
of ethanol. The amount of ethanol added in each bottle
resulted in a concentration (0.7% v/v) which is consid-
ered safety for microbial growth and activity (Benchaar
et al. 2007). The bottles were placed in a ventilated
incubator for 24 h at 39.0 ± 0.4 C, and the gas was
automatically released at a fixed pressure of 6.8 kPa.
The gas released from each bottle was collected in
sealed plastic bags for subsequent analysis. During the
incubation, the kinetics of gas production were continu-
ously measured (Figure 1), the gas production rate was
calculated from 0 to 3 h (GPR 0–3h, mL/h/g DM) and
from 3 to 6h (GPR 3–6h, mL/h/g DM), and the cumula-
tive gas production at 24 h (GP24, mL/g DM).
Samples collection and analysis at the end of
in vitro fermentation
At the end of each incubation run a sample of 9 mL of
fermentation gas was collected using vacutainer tubes
both from the headspace of each bottle and from the
respective sealed plastic bags. The same day, these
gas samples were analysed for methane (CH4) concen-
tration as described by Cattani et al. (2014).
As soon as the bottles were opened, the fermentation
liquid was analysed for pH with a potentiometer (Bench
pH/ion metre, Oakton Instruments, Vernon Hills, USA),
and a 5 ml sample was stored in 10ml tubes containing
1 ml of metaphosphoric acid (25%, w/v) to stop the
microbial activity. The VFA profile of buffered rumen
fluid was determined by HPLC (Thermo-Finnigan, CA,
USA) as described by Tagliapietra et al. (2010).
The fermentation medium was filtered into pre-
weighed crucibles (30 mL, Robu Glasfilter-Ger€
ate
GMBH
V
R
Pore 2, Hattert, Germany) and analysed for the
NDF fibre fraction using a fibre analyser (FIWE 6;
VELP
V
R
Scientifica, Milan, Italy). The analysis was con-
ducted with the addition of alpha-amylase, without
sodium sulphite, and included ash (aNDF). The degrad-
ability of the fibre (NDFd) and true dry matter digest-
ibility (TDMd) were calculated as described by Goering
and Van Soest (1970).
Statistical analysis
All data (mean of 4 replicates) were analysed using the
PROC MIXED procedure of SAS 9.4 (SAS Institute Inc.,
Cary, NC, USA) with a linear mixed model that included
treatment (n¼10) as a fixed factor, and experimental
period (n¼4) and period cow interaction (n¼16) as
random factors. For all the parameters, the Bonferroni
adjustment was used for multiple-comparison
Table 1. Description of the 10 experimental treatments
obtained using ruminal fluid collected from cows either fed
(FA) or not fed (FNA) with additives, with or without the add-
ition of the same compounds also in vitro (cinnamaldehyde,
CIN; limonene, LIM; allyl-sulphide, ALL). The last column
reports the treatment code.
Treatment code Type of diet fed to the cows
Type of additive
In vivo
1
In vitro
2
FNA Without additive (FNA) – –
FNAþCIN Without additive (FNA) – CIN
FNAþLIM Without additive (FNA) – LIM
FNAþALL Without additive (FNA) – ALL
FA
CIN
With additive (FA) CIN –
FA
CIN
þCIN With additive (FA) CIN CIN
FA
LIM
With additive (FA) LIM –
FA
LIM
þLIM With additive (FA) LIM LIM
FA
ALL
With additive (FA) ALL –
FA
ALL
þALL With additive (FA) ALL ALL
1
1 g/d of in vivo additive dose;
2
30 mg/bottle of in vitro additive dose.
ITALIAN JOURNAL OF ANIMAL SCIENCE 55

correction. Additionally, for each additive, orthogonal
contrasts were performed to evaluate the effect on fer-
mentative parameters of: (i) the addition of the additive
to the rumen fluid non-adapted (‘FNA’ vs
‘FNA þadditive’); (ii) the comparison between the two
types of rumen fluid (‘non-adapted’ vs ‘adapted’, ‘FNA’
vs ‘FA’); (iii) the addition of the additive to the rumen
fluid adapted (‘FA’ vs ‘FA þadditive’).
Results
The in vitro addition of cinnamaldehyde in combination
with rumen fluid adapted (FA) or non-adapted (FNA)
did not have a significant effect on fermentation proc-
esses (Table 2). Although the cumulated gas production
after 24 h of fermentation (GP24) was not affected by
the additives, a reduction in the rate of GP was
observed during the first 3 h of incubation when CIN
was added in vitro both to FNA (FNA vs. FNAþCIN;
p¼0.003) and to FA (FA
CIN
vs. FA
CIN
þCIN; p¼0.002)
and the peak of GP was delayed by about 6 h (from 3
to 9h after the start of the incubation, Figure 1A).
When limonene was added in vitro to FNA, the
NDFd, GP24, and CH
4
production values were reduced,
respectively, by 31% (p<0.001), 20% (p<0.001), and
31% (p<0.001). Similar results were obtained when
limonene was added to FA
LIM
. Furthermore, the propor-
tions of the main VFA and the proportion of methane
in total GP24 (CH
4
mL/100 mL GP24) were modified
(p<0.01) and, as shown in Figure 1B, the kinetics of
GP were similar to those of CIN, although more pro-
nounced with a clear differentiation of FNAþLIM and
FA
LIM
þLIM from FNA and FA, respectively (p<0.001).
The addition of allyl-sulphide to the FNA resulted in a
shift from acetic acid (-28%) to propionic (þ12%) and
butyric acid (þ16%), along with a decrease of both CH
4
production (-34%; p<0.001) and proportion (-28%;
p<0.001). Whereas, it had a minor effect on NDF degrad-
ability (-6%; p¼0.09) and on GP24 (-8%; p¼0.04). When
allyl-sulphide was added to the FA, there were notable
Figure 1. Effect of the pure extracts (A) cinnamaldehyde, CIN; (B) Limonene, LIM; (C) Allyl-sulphide, ALL) on the rate of gas pro-
duction kinetics (mL/h/g DM): each graph reports the kinetics obtained by fermenting the dairy cow diet using as inoculum the
rumen fluid collected from cows not fed (fluid non-adapted, FNA) or fed (fluid adapted, FA) with the pure additives, both without
or with the addition of the same additive (CIN, LIM or ALL) in vitro. Standard error of mean of gas production rate at 3, 6, 12 and
24 h of incubation were 2.57, 0.80, 0.61 and 0.50 mL/h per g of DM, respectively. (n¼16).
56 G. ROSSI ET AL.

effects on the aforementioned parameters (VFA, CH
4
and
GP production) and all the effects were more pronounced,
for example, GP24 was reduced by −15% (p<0.001). In
addition, allyl-sulphide incubated with FA also changed
the rate of gas production in the first 3 h of fermentation
(-20%; p<0.001) (Figure 1C).
For all the fermentation parameters considered, the
use of rumen fluid adapted to the three additives (FA)
did not significantly alter the fermentation processes
compared to the use of rumen fluid non-adapted
(FNA). A trend towards different effects of the two
rumen fluids was observed only in cows fed with lim-
onene, where a 9% reduction in butyric acid percent-
age was noted with FA
Lim
compared to FNA (p<0.09).
Discussion
In contrast to previous studies, the experimental design
employed in this trial aims to test the effects of pure
compounds by feeding them to cows and using
adapted or non-adapted rumen fluid as inoculum for
in vitro incubations. The underlying hypothesis assumes
that the administration of additives to cows can have
an impact on the activity of rumen fluid when used as
an inoculum for in vitro incubations. Therefore, the
effect of compounds was tested in three ways: (i) using
the ‘conventional’ in vitro procedure: FNA vs FNA þpure
compound; (ii) testing the rumen fluid adapted or non-
adapted to the pure compound as inoculum of fermen-
tation (FNA vs FA); (iii) combining the previous two
procedures testing the effects of the pure compound
in vitro using the adapted rumen fluid as inoculum (FA
vs FAþpure compound). Dual-flow continuous ferment-
ers are used to simulate the rumen environment by
incubating rumen fluid with tested compounds for a
specified period (e.g. 10 days). This approach allows for
monitoring the temporal evolution of the compound’s
impact on rumen fermentations and determining
whether rumen microorganisms exhibit adaptive capa-
bilities in response to these compounds (Cardozo et al.
2004; Busquet et al. 2005; Castillejos et al. 2007).
However, under these in vitro conditions, the ruminal
microbial consortium undergoes an intense selective
process, and in vivo validation of these results remains
necessary. Only the studies of Mlambo et al. (2007) and
Klop et al. (2017) have used the in vitro batch system to
compare the activity of rumen fluids collected from ani-
mals adapted or non-adapted to specific additives on
Table 2. In vitro degradability of fibre aNDF (NDFd, g/kg aNDF) and of true dry matter (TDMd, g/kg DM), rate of gas production
from 0–3 h (GPR 0–3h, mL/h/g DM) and from 3–6 h (GPR 3–6h, mL/h/g DM), cumulated total gas production after 24 h (GP 24h,
mL/g DM), volatile fatty acid profile (mol/mol % VFA), methane production (CH4, mL/g DM) and proportion (% GP) as affected
by the two types of rumen fluid (fluid non-adapted, FNA; fluid adapted, FA) and by the in vitro addition of cinnamaldehyde
(CIN), limonene (LIM), and allyl-sulphide (ALL). (n¼16).
Treatments
NDFd TDMd GPR 0–3h GPR 0–6h GP 24h Acetate Propionate Butyrate CH4
g/kg NDF g/kg DM mL/h/g DM mL/h/g DM mL/g DM %VFA %VFA %VFA mL/g DM % GP
Fluid Non-Adapted (FNA) 476 787 18.4 17.3 287 57.2 19.1 16.3 50.4 15.4
Cinnamaldehyde (CIN)
FNAþCIN
1
452 776 14.5 16.4 274 56.9 18.8 16.7 47.3 15.0
FA
CIN2
461 778 16.0 15.9 259 57.9 19.8 15.2 43.4 14.5
FA
CIN
þCIN
3
435 768 12.4 14.7 260 57.1 20.4 15.3 41.0 13.7
Limonene (LIM)
FNAþLIM
4
327 725 11.7 14.6 230 54.7 21.2 17.3 34.8 12.7
FA
LIM5
465 781 16.7 16.7 266 58.3 20.1 14.7 43.2 14.1
FA
LIM
þLIM
6
313 718 9.5 12.6 196 55.6 21.1 16.7 30.1 11.8
Allyl-sulphide (ALL)
FNAþALL
7
446 775 17.7 15.9 265 51.1 21.5 19.0 33.4 11.1
FA
ALL8
466 781 18.3 14.5 280 57.5 20.1 15.0 52.0 16.4
FA
ALL
þALL
9
464 780 14.6 16.5 243 51.9 22.4 17.8 28.3 10.2
SEM 2.1 0.8 2.57 0.80 14.3 0.72 0.98 0.67 4.37 1.03
Treatment P-value          
Contrasts
Cinnamaldehyde
FNA vs. FNAþCIN 0.15 0.15 0.003 0.08 0.09 0.62 0.74 0.33 0.40 0.70
FNA vs. FA
CIN
0.59 0.59 0.51 0.20 0.17 0.51 0.60 0.27 0.25 0.52
FA
CIN
vs FA
CIN
þCIN 0.15 0.15 0.002 0.05 0.94 0.23 0.48 0.85 0.55 0.46
Limonene
FNA vs. FNAþLIM <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.004 0.05 <0.001 0.01
FNA vs. FA
LIM
0.70 0.70 0.64 0.60 0.30 0.28 0.48 0.09 0.24 0.39
FA
LIM
vs FA
LIM
þLIM <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.19 <0.001 <0.001 0.02
Allyl-sulphide
FNA vs. FNAþALL 0.09 0.09 0.55 0.01 0.04 <0.001 0.001 <0.001 <0.001 <0.001
FNA vs. FA
ALL
0.73 0.73 0.99 0.84 0.74 0.72 0.46 0.19 0.79 0.48
FA
ALL
vs FA
ALL
þALL 0.89 0.89 0.001 0.09 <0.001 <0.001 0.004 <0.001 <0.001 <0.001
1
FNA with the addition in vitro of CIN;
2
FA to CIN;
3
FA to CIN with the addition in vitro of CIN;
4
FNA with the addition in vitro of LIM;
5
FA to LIM;
6
FA
to LIM with the addition in vitro of LIM;
7
FNA with the addition in vitro of ALL;
8
FA to ALL;
9
FA to ALL with the addition in vitro of ALL.
ITALIAN JOURNAL OF ANIMAL SCIENCE 57

diets fermentation, but they did not adopt a Latin
square design model to evaluate the effect of the
rumen fluid donor on in vitro parameters.
Additive effect (FNA vs FNA þcompound)
The results of this study indicated that the three tested
additives had distinct effects on rumen fermentations,
consistent with previous research (Crane et al. 1957;
Kamel et al. 2009; Cattani et al. 2016). These trials found
that comparable doses of garlic (allyl-sulphide) and limon-
ene additives reduced in vitro total gas and methane pro-
duction. Allyl-sulphide emerged as an effective additive
for manipulating rumen fermentation, as it altered the
volatile fatty acid profile, decreased the acetate/propion-
ate ratio (-20,6%) and decreased the methane production
(-34%) without relevant impact on fibre degradation
(NDFd −6%). These results are consistent with those
reported in previous research (Cattani et al. 2016). Such
effect on rumen fermentation could be attributed to the
relatively short incubation time (24 h), which was probably
insufficient for the rumen population to adapt, making
the fermentation sensitive to the in vitro addition of allyl-
sulphide and limonene. This hypothesis is supported by
several authors (Cardozo et al. 2004; Molero et al. 2004;
Castillejos et al. 2007), who noted that rumen bacteria can
develop a tolerance mechanism also in vitro, but the pro-
cess requires much more than 24 h of fermentation. In the
present research, the in vitro supplementation of cinna-
maldehyde to the fluid non-adapted produced only weak
effects on fermentations, decreasing the rate of gas pro-
duction but without affecting overall gas and methane
production or feed degradation. Recently, we observed
similar effects on feed degradability and gas production
kinetics but also noted a reduction of methane and a
change in volatile fatty acid production (Cattani et al.
2016). Current literature highlighted that the role of cinna-
maldehyde on modulating the fermentation is still contro-
versial, with the main effects appearing to be related to
the use of greater doses compared to those used in the
present study (200 mg/L of fermentation fluid)
(Calsamiglia et al. 2007).
Rumen fluid type effect (FNA vs FA)
The in vitro rumen fermentations were not affected by
the use of rumen microbial inoculum either adapted
(FA) or non-adapted (FNA) to the additive and this
could be attributed to the low doses of additive
included in the diet (1 g/kg DM) This dosage was close
to those commonly administered to the dairy cows
(Benchaar et al. 2006) and was chosen in an attempt
to reduce methane emission while avoiding inhibition
of microbial activity and diet degradation, therefore, it
is not known if a higher dosages might promote a
larger response. Additionally, it cannot be excluded
that the microbial population might have developed a
tolerance mechanism to the additives during the
in vivo administration period, allowing the microorgan-
isms to mitigate the biological activity of these sub-
stances (Poulopoulou and Hadjigeorgiou 2021). The
development of such a tolerance mechanism is plaus-
ible, as the 14–day feeding period for the additives
was probably sufficient for the adaptation of rumen
microflora, which typically require about 10 days to
acclimatise to new dietary components (Warner 1962).
The activity of ruminal fluids collected from cows
fed with additives may be attributed to changes or
adaptations of the microbial population but also to
the direct presence of the additive within the fluid. In
this study, ruminal fluid samples were collected 24 h
after the final additive doses were administered to the
cows. Notably, the tested additives are highly volatile;
for instance, studies in humans have detected allyl-sul-
phide in milk within minutes post-ingestion, with
nearly complete disappearance within 4 h (Scheffler
et al. 2016). Thus, while a residual presence of addi-
tives in the sampled ruminal fluids cannot be com-
pletely excluded, its concentration is likely minimal.
Additives effect on adapted rumen fluid (FA vs
FA þpure compound)
When cinnamaldehyde and limonene were added in vitro
to rumen fluid collected from cows adapted with the same
additives (FA), the fermentation profiles were comparable
to those previously described when the additives were
added to the rumen fluid non-adapted (FNA). In this case,
therefore, both adapted and non-adapted rumen fluids
exhibited not only comparable fermentative patterns but
also similar microbial activity when the additives are added
in vitro. The in vivo administration of these additives
appears to have no effect on in vitro fermentations, at least
under the present experimental conditions. Conversely,
the impact of allyl-sulphide on rumen fermentations was
much greater when this additive was fermented in vitro
with the rumen fluid adapted compared to that non-
adapted. For instance, when allyl-sulphide was incubated
with FNA, it reduced the gas production rate at the begin-
ning of the fermentation by 3% and the total gas produc-
tion by 7%, whereas when it was incubated with FA, gas
production was reduced by 20% and 13%, respectively.
Similarly, the use of allyl-sulphide in FNA did not change
the peak of GP, while the use of this additive in FA
delayed the peak from 3 to 6 h of fermentation.
58 G. ROSSI ET AL.

Therefore, the impact of allyl-sulphide on fermentations
was increased when incubated with the rumen fluid of a
cow fed the same additive (Figure 2). Under the present
experimental conditions, these results support the
hypothesis that the use of rumen fluids adapted or non-
adapted to a pure compound can modify the response of
short-term in vitro batch systems. However, this phenom-
enon is additive dependent. In previous experiments,
Mlambo et al. (2007) and Klop et al. (2017) did not
observe an adaptation of the rumen fluid to the in vitro
presence of the same compound. Actually, the Authors
evaluated the effect of pure additives on rumen fermen-
tations using rumen fluids collected from cows that were
either fed or not fed the same additives and observed
that these effects showed an apparently additive pattern.
In other words, these Authors observed that the effects of
the additives on in vitro fermentative were cumulative
with those of rumen fluid adapted or not adapted to the
same additive.
Conclusions
Our study highlights that the additives, exhibiting inhibi-
tory activity during in vitro fermentations (short term
effect), did not alter the rumen fluid activity and
response of in vitro fermentations when fed to cows
whose rumen fluid was used as inoculum (long term
effect). However, in our experimental conditions (e.g. 14
d adaptation, 1 g/d), the rumen fluid of cows fed
(adapted) with a specific compound enhanced the
in vitro system responsiveness to the same compound.
On this basis, in vitro techniques can be used to test the
microbial activity of rumen fluids collected from cows
adapted for a certain period to the presence of specified
compounds, to highlight any defence mechanisms
developed by the microflora against the same additives.
Further investigation is needed to explore the
potential of using short-term batch culture systems to
evaluate the effects of specific compounds on rumen
fermentation by comparing the activity of rumen fluid
collected from both adapted and non-adapted cows.
Acknowledgments
This work is part of the PhD thesis of Doctor Giulia Rossi car-
ried out within the PhD course in Animal and Food Science
(University of Padova, 2017, Cycle XXX). The authors would like
to express their gratitude to Roberto Mantovani for his valu-
able support in the statistical analysis of the experimental data.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Ethical approval
The animal study protocol was approved by the Ethics
Committee for the Care and Use of Experimental Animals of
the University of Padova (Prot. n. 120869).
Funding
Furthermore, this research was funded by the University of
Padova with the project BIRD213117/2021 ‘Development of a
new methodology for the assessment of ruminal methane
emissions using in vitro techniques’, BIRD222027/2021 ‘In vitro
rumen fermentation lab (FerLab) to support studies on the
environmental sustainability of livestock farms’, and
DOR2158177/21 ‘Studio delle procedure di conservazione del
liquido ruminale da utilizzare come inoculo microbico in vitro.’
ORCID
Selene Massaro http://orcid.org/0009-0006-9111-7977
Sheyla Arango http://orcid.org/0000-0002-4175-2650
Sarah Curr�
o http://orcid.org/0000-0003-1879-1309
Mauro Spanghero http://orcid.org/0000-0001-9782-8194
Figure 2. Effect of rumen fluid, used as in vitro inoculum, collected from cows not fed with allyl-sulphide (fluid non-adapted,
FNA) or from those fed with allyl-sulphide (fluid adapted, FA
ALL
) and incubated adding the allyl-sulphide in vitro (þALL) or not on
(A) cumulated gas production at 24 h (mL/g DM) and on (B) rate of gas production at 3 h (mL/h/g DM). (n¼16).
ITALIAN JOURNAL OF ANIMAL SCIENCE 59

Diana Giannuzzi http://orcid.org/0000-0003-2975-0385
Stefano Schiavon http://orcid.org/0000-0002-5539-8947
Lucia Bailoni http://orcid.org/0000-0003-3027-2288
Franco Tagliapietra http://orcid.org/0000-0002-0593-1600
Data availability statement
The results and analyses presented in this paper are freely
available upon request.
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