Journal of Applied Animal Research

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/taar20

Investigation of the effectiveness of tomato pulp

on the in vitro fermentation of working horse diets

Kanber Kara

To cite this article: Kanber Kara (2022) Investigation of the effectiveness of tomato pulp on the
in vitro fermentation of working horse diets, Journal of Applied Animal Research, 50:1, 198-203,
DOI: 10.1080/09712119.2022.2054421

To link to this article: https://doi.org/10.1080/09712119.2022.2054421

© 2022 The Author(s). Published by Informa

UK Limited, trading as Taylor & Francis
Group

Published online: 31 Mar 2022.

Submit your article to this journal

Article views: 842

View related articles

View Crossmark data

Citing articles: 1 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=taar20
JOURNAL OF APPLIED ANIMAL RESEARCH
2022, VOL. 50, NO. 1, 198–203
https://doi.org/10.1080/09712119.2022.2054421

Investigation of the effectiveness of tomato pulp on the in vitro fermentation of

working horse diets
Kanber Kara
Faculty of Veterinary Medicine, Department of Animal Nutrition and Nutritional Diseases, Erciyes University, Kayseri, Turkey

ABSTRACT ARTICLE HISTORY

The aim of this study is to determine the effectiveness of dried tomato pulp, as a soluble dietary fibre Received 29 May 2020
source, using [0 (control group), 2.5, 5.0, 10.0, and 20.0% (experimental groups) as dry matter-DM] in Accepted 11 March 2022
horse total mixed ration (TMR) on the in vitro fermentation parameters. The in vitro total gas
KEYWORDS
production (TGP), methane emission, gas yield (GY24), metabolic energy (ME), in vitro true dry matter Gas production; in vitro
disappearance (T-DMd), short-chain fatty acids (SCFA) molarities, partial factor (PF24), microbial crude digestion technique;
protein production (MCP), and pH value at 24 h of fermentation in horse TMR’s were determined. In methane; dry matter
the study, in vitro TGP, GY24, PF24, ME, MCP, SCFA, and methane emission of horse TMR’s were not digestion
changed by 2.5 and 5.0% tomato pulp supplementation (P > 0.05). The in vitro TGP (73.6 vs. 85.6 and
105.9 mL/g DM), GY24 (189.4 vs. 210.8 and 286.3 mL for g T-DMd), PF24 (5.9 vs. 5.2 and 4.2 mg/mL for
GY24) ME (5.3 vs. 5.6 and 6.1 MJ/kg DM), MCP (270.1 vs. 258.2 and 215.1 mg/g DM), SCFA (0.32 vs. 0.37
and 0.46 mmol/0.2 g), and methane emission (0.28 vs. 0.33 and 0.48 ml/g DM) of horse TMR’s
positively affected by 10 and 20% tomato pulp supplementation (P < 0.05). The in vitro T-DMd values
of TMRs and pH value of in vitro digestion fluid were similar for both control and experimental groups
(P > 0.05). As a result, it was demonstrated that tomato pulp, which uses as dietary fibre and CP
source, did not affect negatively the digestion values of the working horse diet. However, it has been
considered that tomato pulp using at 10 and 20% ratios increased the in vitro digestion values of the
working horse diet. Determining the in vitro digestion levels of tomato pulp, which is a source of
soluble dietary fibre, for horses will guide future in vitro and in vivo studies.

Introduction
Horses are monogastric herbivores whose digestive system is
anatomically developed for them to ingest small amounts of
forage in a continuous manner. The digestive system of the
horse is quite unique. The system has a relatively small but
efficient stomach for grain utilization and a large caecum and
colon for utilization and fermentation of forage. The purpose
of the digestive system of the horse is to process feedstuffs
into their component nutrients for absorption and utilization
by the body (Frape 2004; Ralston 2021). Structural carbo-
hydrates, such as cellulose, along with other carbohydrates
that escaped digestion in the small intestine are fermented in
the large intestine. The microbial enzymes in the large intestine
can digest cellulose, starch, and sugars into volatile fatty acids
(VFA, in other words; short-chain fatty acids with C2–C3
carbons), which may supply up to 25% of the energy used by
the horse. Except in extreme stress conditions, bacterial
action in the large intestine of healthy adult horses produces
their daily B vitamin requirements. The caecum is the primary
site of water absorption. The rate of passage in the large intes-
tine is considerably slower than in other portions of the diges-
tive tract. The average rate of passage in the large intestine is
approximately 36–48 h (NRC 1989, 2007). The soluble undi-
gested fibres can be fermented in the colon, thereby producing
short-chain fatty acids (acetate, propionate, and butyrate) and
branched-chain fatty acids. The end products of the fermenta-
tion are used by different tissues in the body as a source of
energy (NRC 1989, 2007).
The digestibility of horse diets has been revealed by in vivo
and in vitro methods. While the in vivo method is the most
accurate, it is costly, time-consuming, labor-intensive, and
must be performed directly on a horse (Bush et al. 2001).
Apart from studies using direct horse feces inoculum plus
buffer mixture to detect in vitro digestion in horses, there are
studies using horse feces inoculum and fermentation
medium (vitamin, mineral, fatty acid, and yeast extract)
(Sweeney 2012; Kholif et al. 2015; Ersahince and Kara 2017;
Kara and Baytok 2017). Gas production profiles and parameters
are indicators of the rate and extent of degradability of feed-
stuffs in the horse’s caecum and ventral colon, while total
VFA and the type of VFA are fermentation end products from
microorganisms of materials escaping enzymatic digestion
and serve as energy sources for horses. An increase in propio-
nate can improve the horse’s overall energy balance, given
that propionate is glycogenic (Ersahince and Kara 2017).
Thus, feces as a microbial inoculum for in vitro digestibility
studies in horses has many practical and economic advantages
over caecal fluid as it does not require the use of surgically pre-
pared animals and can be collected from any individual or
several animals (Abdouli and Attia 2007; Earing et al. 2010).

CONTACT Kanber Kara kanberkara@erciyes.edu.tr

© 2022 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, dis-
tribution, and reproduction in any medium, provided the original work is properly cited.
 JOURNAL OF APPLIED ANIMAL RESEARCH 199

Cumulative gas production profiles of feeds have a wide In this study, it was aimed to reveal the fermentative digestions
variety, demonstrating the potential of the pressure transducer of a mixed ration (containing different ratios of tomato pulp)
technique to evaluate the digestive kinetics of feeds used in instead of a pure feedstuff. The aim of the present study was
horse diet (Murray et al. 2009). However, it is important to to determine the effect of 2.5, 5.0, 10.0, and 20.0% in horse
note that in vitro techniques are not designed to accurately total mixed ration tomato pulp on digestive parameters using
measure absolute digestibility but to estimate it and compare feces as inoculum.
the relative digestibility of different feeds (Tassone et al. 2019).
The fibrous feedstuffs in the horse diet can be forage (mainly
meadow hay and herbage) and concentrate feeds with high Materials and methods
fibre content (mainly grain bran, grain with husk, and agro- The samples of tomato pulp and feedstuffs
industrial by-products) (NRC 2007). Tomato-processing by-
product, also known as tomato pulp, consists of peel and Tomato pulp was collected as wet from a local factory in Kayseri
seeds and represents around 4% of the fruit weight (Del Valle province of Turkey. The wet tomato pulp was oven-dried
et al. 2006). The tomato industry generates large amounts of (Binder, Germany) at 65°C for 36 h. The feedstuffs of horse
by-products, and these by-products representing 10-30% of TMR were meadow hay, wheat straw, oat grain, wheat bran,
total processed tomatoes contain tomato seeds, peels, pulp, flaxseed meal, di-calcium phosphate (DCP), and salt. The
and cores (Rahmatnejad et al. 2009). Seeds and peels present dried tomato pulp and other feedstuffs samples which were
in tomato pulp (TP) consist of substances that are rich in nutri- grounded in size to pass through a 1 mm sieve (IKA Werke,
tional value (Isik and Topkaya 2016). The in vitro gas production Staufen im Breisgau, Germany) were mixed for laboratory
from insoluble fraction and potential gas production values of TMR’s (100 g, as DM). The control TMR included 20.9 Mcal
tomato pulp were higher than those of common fibrous feed- digestible energy (DE)/kg DM and 1045 g CP/day (Table 1).
stuffs (wheat bran and lucerne meal) and uncommon fibrous Oat grain and wheat bran in the control TMR of horses were
feedstuffs (maize bran, rice bran, lentil bran, and pomegranate reduced and replaced with tomato pulp between 2.5, 5, 10, and
peel) for herbivorous rabbit, which has a functional fibre fer- 20% as a DM basis. Each TMR sample was prepared at approxi-
mentation in the large intestine (Kara 2016). The tomato mately 500 g. The control and treatment TMR’s adjusted as iso-
pulp, which has 17% digestible dietary fibre, about 20% caloric and iso-nitrogenic (Table 1).
neutral detergent fibre (NDF), about 35–59% total dietary
fibre (TDF), 26% total sugars, 13.7–19.0% crude protein (CP),
Chemical analyses
7.55% pectins, and 55% non-structural carbohydrate, can be
a fibrous-energy-protein source for horse diet (Del Valle et al. The total mixed ration of a horse was milled through a 1 mm
2006; Kara 2016). sieve for use in chemical analysis and in vitro gas production.
Studies often evaluate pure ingredients without additives The ash, crude protein (CP) and ether extract (EE) contents
and are inoculated with faeces without prior enzymatic diges- were estimated according to the Association of Official Analyti-
tion (Ersahince and Kara 2017; Cagri and Kara 2018); however, cal Chemists (AOAC 1995; method 920.39; method 942.05;
there is limited information on the fermentation of horse method 942.01). The neutral detergent fibre (NDF), acid deter-
diets (Kara and Baytok 2017). However, positive and negative gent fibre (ADF), and acid detergent lignin (ADL) contents were
associative effects were observed when the mixtures were incu- analyzed using a glass crucible on FIWE3 fibre analyser (Velp,
bated, compared to the values expected from incubation of the Italy) (Van-Soest et al. 1991). The NDF was detected using
pure components (Niderkorn and Baumont 2009). Therefore, sodium sulfite and thermo-stable α-amylase (Megazyme,
there is a need to evaluate the fermentation of mixed diets Ireland) (aNDF). The NDF and ADF were given without ash
rather than pure ingredients. The tomato pulp can be an content (aNDFom and ADFom). Total starch contents of horse
ideal dietary fibre source for the large intestine and an total mixed ration with and without tomato pulp were analyzed
energy-protein source due to functional nutrient content its. using Megazyme assay (Megazyme, cat. no. K-TSTA-100A) pro-
cedures (Bray Business Park, Bray, Co. Wicklow, Ireland)
Total dietary fibre (TDF), soluble dietary fibre (SDF), and inso-
Table 1. Ingredients and chemical compositions of horse TMR used in the present luble dietary fibre (IDF) contents are determined according to
study. the AOAC enzymatic-gravimetric method (Prosky et al. 1988).
Feedstuffs Tomato pulp supplementation, % The basis of this method was the isolation of dietary fibre by
0 2.5 5 10 20 enzymatic digestion of the rest of the constituents of the feed-
Meadow hay 48.0 48.0 48.0 48.0 48.0 stuffs. The residue was determined gravimetrically. The starch
Wheat straw 27.0 27.0 27.0 27.0 27.0
Oat grain 16.0 16.0 16.0 12.0 2.0 of the feedstuff (approximately 1.0 g) was digested by combin-
Wheat bran 8.0 5.5 3.0 3.0 3.0 ing amylase (pH = 6.0; temperature, T = 100°C; time, t = 30 min)
Flax seed meal 7.0 7.0 7.0 7.0 7.0 and amyloglucosidase (pH = 7.5; T = 60°C; t = 30 min). The
Tomato pulp 0.0 2.5 5.0 10.0 20.0
DCP 0.3 0.3 0.3 0.3 0.3 protein of the feedstuff was digested with protease (pH = 4.5;
Salt 0.7 0.7 0.7 0.7 0.7 T = 60°C; t = 30 min). Ethanol (95%) was added to precipitate
DM (kg/day) 10.0 10.0 10.0 10.0 10.0 the soluble fibre and incubated for 60 min. Filtration was
DE (Mcal/kg KM) 20.9 20.9 20.9 21.0 20.8
CP (g/day) 1045 1036 1028 1039 1036 carried out in crucibles (Velp, Italy) by using a filtration
DM: Dry matter; DE: Digestible energy; DCP: Di-calcium phosphate; CP: Crude system (Velp Scientifica CSF 6, Italy). Protein and ash were
protein. measured in each residue in order to correct the values for
200 K. KARA

dietary fibre. Dietary fibre analyses were performed using (anaerobically) and used in the in vitro digestion technique.
Megazyme chemicals (Ireland) and were in duplicate. Analyses The diet for a horse, which had 550 kg body weight and mod-
were carried out as duplicates. Hemicellulose (HC), soluble fibre erate maintenance energy and nutrient requirement, was
(SF), water-insoluble pectin (WIP), and digestible fibre fractions studied as substrate. The in vitro digestion technique was
(DgF) levels of feedstuffs were calculated using the following carried out in glass syringes with 100 ml volume (Fortuna®,
formulas (Kara 2016): Poulten & Graf Ltd., Wertheim, Germany) using the medium
mixture, prepared according to Sunvold et al. (1995), and
HC(%, DM) = aNDFom (%)–ADFom (%)
Sweeney (2012). The samples (500 ± 10 mg as DM) were incu-
SF(%DM) = TDF (%)–aNDFom (%) bated with a medium mixture (30 ml) (Table 1) and feces inocu-
lum (5 ml) in glass syringes (n = 6). The syringes were closed
WIP(%) = SF(%)–SDF(%) using clips and then the initial volume was recorded and incu-
DgF(%) = HC(%) + SF(%) bated in a water bath with a thermostat (Special Waterbath,
Yapar Stainless Steel Ltd., Kahramanmaras, Turkey) at 39.0 ±
0.2°C up to 24 h. In addition, six blank syringes (no template;
medium mixture plus feces inoculum) were used to calculate
In vitro digestion technique for horses the total gas production.
The in vitro digestion technique in horses was carried out accord-
ing to Sunvold et al. (1995) and Sweeney (2012), which incu- Determination of in vitro total gas and methane
bated of feed sample in faeces inoculum and fermentation production and estimated fermentation parameters in
medium, which included solution A, solution B, trace mineral sol- horse TMR
ution, water-soluble vitamins, folate: biotin solution, riboflavin
solution, haemin solution, short-chain fatty acids, rezasurin, After measuring the volume (ml) of in vitro total gas production
yeast extract, trypticase, Na2CO3 and cystein-HCl*H2O (Table (TGP) at 24 h, the plastic syringe outlet was inserted into the
2). Feces samples used as an inoculum in the current study inlet of the methane analyser (Sensor, Europe GmbH, Erkrath,
obtained from two thoroughbred working horses (6–7 years of Germany) (Kara 2015). In the study, metabolizable energy
age, 480–500 kg of body weight) fed a diet containing about (ME) values short-chain fatty acid molarities (SFCA) of horse
70% forage and about 30% concentrate feed, on DM basis. TMR samples were calculated according to Kholif et al. (2015)
Feces samples were diluted at a 1:10 ratio with 0.9% sterile and developed by Menke et al. (1979). The gas yields (GY24),
serum physiologic solution (Polifleks, Polifarma, İstanbul, partial factor (PF24), and microbial crude protein production
Turkey) using a laboratory-type blender (Waring Products Div- levels (MCP) of the samples at 24 h were calculated using the
ision, Torrington, CT, USA). Diluted feces inoculum was filtered equations of Elghandour et al. (2015):
through four layers of cheesecloth under constant CO2 gas GY24 = [(GP24 × 103 ):T − DMd],
PF24 = T − DMd:GP24,
Table 2. Composition of in vitro fermentation medium.
Component Amount MCP(mg/gDM) = mgT − DMd–(mL gas × 2.2 mg/mL)
mL/L
Solution Aa 330.0 T-DMd: in vitro dry matter disappearance (mg) for g DM at
Solution Bb 330.0 24 h (mg/g DM).
Trace mineral solutionc 10.0
Water-soluble vitaminsd 20.0
GP24: volume (mL) of total gas produced by g DM at 24 h
Folate: biotin solutione 5.0 (mL/g DM).
Riboflavin solutionf 5.0
Haemin solutiong 2.5
Short chain fatty acidsh 0.4
Resazurinei 1.0
Determination of in vitro true dry matter
Distilled H2O 296.0 disappearance values
g/L
Yeast extract 0.5 Three of the fermentation syringes for horses were stopped
Trypticase 0.5 after 24 h, and then the in vitro true dry matter disappearance
Na2CO3 4.0
(T-DMd) values. The in vitro dry matter disappearance was
Cystein HCl*H2O 0,5
a determined by filtering the fermentation residues using a
Composition (g/L): NaCl, 5.4; KH2PO4, 2.7; CaCl2*H2O, 0.16; MgCl2*6H2O, 0.12;
MnCl2*4H2O, 0.06; CoCl2*6H2O, 0.06; (NH4)2SO4, 5.4. vacuum unit (Velp Dietary Fibre Analyzer, Italy) on pre-
b
Composition: K2HPO4, 2.7 g/L. weighed glass crucibles (Velp, porosity #2, Italy) the fermenta-
c
Composition (mg/L): ethylene diamine tetra acetic acid (disodium salt), 500;
tion residues, which was dried at 105°C and burning the
FeSO4*7H2O, 200; ZnSO4*7H2O, 10; MnCl2*4H2O, 3;H3PO4, 30; CoCl2*6H2O,
20; CuCl2*2H2O, 1; NiCl2*6H2O, 2; Na2MoO4*2H2O, 3. residual at 550°C. In vitro T-DMd was calculated as 1−[(DM
d
Composition (mg/L): thiamin-HCl, 100; d-pantothenic acid, 100; niacin, 100; pyr- residue−DM blank)/initial DM)] × 100.
idoxine, 100; p- amino benzoic acid, 5; vitamin B12, 0.25.
e
Composition (mg/L): folic acid, 10; d-biotin, 2; NH4HCO3, 100.
f
Composition: riboflavin, 10 mg/L in 5 mmol/L of Hepes.
g
Haemin: Haemin 500 mg/L of 10 mmol/L NaOH. Statistical analysis
h
Composition: n-valerate, iso-valerate, iso-butyrate and DL alpha- methyl buty-
rate, 250 mL/L. Statistical analysis of study data was performed using SPSS 17.0
I
Composition: 1 g resazurine/L distilled water. software (IBM Corp., Armonk, NY, USA). One-way variance
 JOURNAL OF APPLIED ANIMAL RESEARCH 201

analyses (ANOVA) were implemented for homogeneous var- colonic fermentation provided by high colon bacterial
iances. The linear, quadratic, cubic effects (polynomial contrast) enzymes in horses, digestion in the entire digestive tract
for using different levels of tomato pulp in TMRs were also should be provided. Dietary fibre mostly comprises carbo-
determined. Significance was defined at P values of <0.05. hydrate polymers, which resist hydrolysis by digestive
enzymes in the mammalian small intestine but can be fermen-
ted by large intestinal bacteria. One of the main benefits of
Results dietary fibre relates to its fermentability, which affects microbial
diversity and function within the gastro-intestinal tract, as well
The tomato pulp included 9.74% CP, 45.20% NSC, 43.78% TDF,
as the by-products of the fermentation process (Williams et al.
26.73% IDF, and 17% SDF in DM. Digestible fibre fractions
2017). Fibrous compounds in the diet of monogastric animals
(hemicellulose and soluble fibre) of tomato pulp was 29.63%
are important for intestinal fermentation and are divided into
of DM (Table 3).
soluble and insoluble dietary fibre. It has obvious that dried
The starch contents of control TMR and treatment TMRs with
tomato pulp, which is rich in SDF, will have a positive effect
tomato pulp were ranged from 8.53 to 6.90% in DM. The NDF
on large intestine fermentation in horses. In the in vitro diges-
contents of control TMR and treatment TMRs with tomato
tion experiment, adding tomato pulp up to 10% as DM to diet
pulp were ranged from 34.29 to 34.69% in DM. The CP contents
did not change the total gas production and estimated diges-
of all TMR’s were in among 10.62 and 10.28% in DM (Table 4).
tion values, but the addition of 20% significantly increased
The horse TMRs in the present study included 20.8–21 Mcal/
the in vitro gas production and estimated digestion values.
kg DM digestible energy (DE) and about 10% crude protein
Apart from the gases produced by colon fermentation, it is
(CP). In the present study, GY24, ME, SCFA, GY24, PF24, MCP,
understood that the rate of short-chain fatty acids, which is
and methane emission was not changed 2.5 and 5% tomato
the end product of carbohydrate fermentation, increases up
pulp (P > 0.05); but increased 10 and 20% tomato pulp (P <
to 20% with the use of tomato pulp, which is a soluble
0.05). The in vitro TGP for g DM of horse TMR’s at 24 h were
dietary fibre source, and colon health is positively affected.
ranged about 74 from 106 ml; and increased by 10 and 20%
Horses can provide long-term glucose from the short-chain
supplementation of tomato pulp (P = 0.035; cubic effect). The
fatty acids produced in their colons (NRC 2007). It has also
0.28 ml methane production of the control group reached
been understood that this carbohydrate source, which has a
0.48 ml for the treatment group with 20% tomato pulp (P =
low glycaemic index, is necessary for the health of the horses’
0.093). The ME values of horse TMR’s were ranged from 5.25
metabolism.
to 6.13 MJ/kg DM; and increased by 10 and 20% tomato pulp
Diet composition, particularly carbohydrate composition,
(P = 0.035). The T-DMd values of TMRs and pH of in vitro diges-
may affect the microbial population of the large intestine as
tion fluid were similar for treatment and experimental groups
well as the proportions of VFA that are produced. Feeding a
(Table 5).
high-starch diet (30% in DM) has been reported to decrease
the concentration of cellulolytic bacteria but increase the con-
centration of total anaerobic bacteria, lactic acid using bacteria,
Discussion and conclusion
lactobacilli, and streptococci in the caecum (Medina et al. 2002).
In vitro digestion techniques are widely used to understand Soluble DF can increase the viscosity of cyme depending on its
ruminal feed digestion in ruminants using inoculum with chemical structure. These dietary fibre compounds can lead to
rumen fluid content (Kara 2015, 2018, 2021). In recent years, a reduced glycaemic response, delaying gastric emptying and
digestion/fermentation values completed mixed feed or nutrient release as well as by inhibiting the action of α-
forage feedstuffs have been made as the in vitro for horses amylase, thus regulating blood glucose – a critical mechanism
using horse feces inoculum (Sweeney 2012; Kholif et al. 2015; related to the development of insulin resistance and then Type
Kara and Baytok 2017; Cagri and Kara 2018). However, in the 2 diabetes (Williams et al. 2017).
techniques made to understand the digestion of feeds in In the presented study, it is important to increase the levels
of soluble fibre source instead of starch as an energy source, to
provide long-term energy needed for horses, to increase the
Table 3. Fibre substances and some nutrient compositions of tomato pulp.
Nutrient matter %, DM Nutrient matter %, DM
Ash 15.29 HC 11.67
CP 9.74 SF 17.96 Table 4. Analysis values of horse TMR used in the present study.
EE 3.95 WIP 0.91 Tomato pulp supplementation, %
NSC 45.20 DgF 29.63
TDF 43.78 aNDFom 25.82 0.0 2.5 5.0 10.0 20.0
IDF 26.73 ADFom 14.15 Items (%, in DM) Control TMR Treatment TMR’s
SDF 17.05 ADL 6.12 CP 10.62 10.45 10.58 10.39 10.28
DM: Dry matter content as % in feed; Ash: Ash content as % in DM; CP: Crude EE 3.35 3.30 3.41 3.25 3.30
protein as % in DM; EE: Diethyl ether extract as % in DM; NSC: non-structural Ash 10.70 10.34 10.87 10.59 10.95
carbohydrates (100−Ash−EE−CP−aNDFom) (NRC 2001), aNDFom: Thermo- CF 15.21 15.25 15.74 16.29 16.97
stable α-amylase treated neutral detergent fibre content was corrected for NDF 34.29 34.78 35.45 35.96 34.69
ash as % in DM, TDF: Total dietary fibre as % in DM; SDF: Soluble dietary NFC 41.04 41.13 39.69 39.81 40.78
fibre as % in DM; IDF: Insoluble dietary fibre as % in DM; HC: Hemicellulose Starch 8.53 8.20 7.91 7.62 6.90
as % in DM (aNDFom −ADFom); SF: Soluble fibre as % DM (TDF- aNDFom); CP: Crude protein; NDF: assayed with a heat stable amylase and expressed exclu-
WP: Water-insoluble pectin as % DM (SF–SDF); DgF: Digestible fibre fractions sive of residual ash; EE: Diethyl ether extract; CF: Crude fibre; NFC: non-fibrous
(HC + SF) as % DM. carbohydrate; NFC is = 100%−(CP% + NDF% + EE% + Ash%).
202 K. KARA

Table 5. The in vitro fermentation parameters of horse diet include tomato pulp at different levels.
Tomato pulp supplementation, % P-values
0.0 2.5 5.0 10.0 20.0 SEM L Q C
Parameters Control TMR Treatment TMR’s
TGP 73.59b 72.83b 73.32b 85.60ab 105.88a 4.64 0.225 0.095 0.035
Methane 0.28b 0.20b 0.32b 0.33ab 0.48a 0.03 0.093 0.041 0.190
GY24 189.41b 191.89b 166.59b 210.77ab 286.31a 18.21 0.359 0.444 0.007
PF24 5.87a 5.97a 5.87a 5.21ab 4.23b 0.28 0.035 0.057 0.024
ME 5.25b 5.23b 5.24b 5.58ab 6.13a 0.12 0.234 0.099 0.031
MCP 270.10a 274.87a 269.20a 258.18a 215.07b 12.47 0.014 0.028 0.047
T-DMd 43.23 43.51 43.05 44.65 44.80 0.75 0.846 0.620 0.547
SCFA 0.32b 0.31b 0.32b 0.37ab 0.46a 0.02 0.218 0.098 0.033
pH 6.41 6.13 6.00 6.09 6.07 0.01 0.088 0.065 0.633
SEM: Standard error of means; L: linear; Q: quadratic, C: cubic; ME: Metabolizable energy, MJ/kg DM; TGP: Volume of in vitro gas produced at 24 hours of incubation, mL/
g DM, Methane: in vitro methane production as ml/g DM at 24 h (methane production, ml = (TGP, ml × methane as percent in TGP)/100), SCFA: molarities of short-
chain fatty acid for 0.2 g DM, GY24: gas yield is the total gas volume (mL) produced for g T-DMd, PF24: partial factor is ratio T-DMd to GP24 at 24 h (mg/mL GP24), MCP:
microbial crude protein produced at 24 h (mg/g DM).

number of probiotic bacteria in the intestine, and to maintain
blood glucose level. The starch level of forage materials used
in horse diets varies between about 1–3% in DM. The starch
level of Gramineae grain and by-products varies between 17
and 77% in DM (NRC 2007). Although the starch level was
reduced with tomato pulp supplementation instead of oat
grain and wheat bran in the treatment diets of the present
study, it was observed that there was no decrease in the in
vitro digestion level. There were even positive effects on
some digestive parameters. In the presented study, it is under-
stood that dried tomato pulp is a good source of soluble dietary
fibre. Determining the in vitro digestion levels of tomato pulp,
which is a source of soluble dietary fibre, for horses will guide
future in vitro and in vivo studies. It will be a guide for the
usability of sources containing soluble and insoluble dietary
fibre in different ratios in the diet of horses and for testing
this in vitro. Researchers will be able to base on these
findings in order to increase the diversity of dietary fibre com-
ponents, which are important for the health of horses.
Many studies about the reduction of methane emissions
from animal production (especially ruminant rearing systems)
have been carried out at the present time. Share in global
methane emissions of cattle has been demonstrated in pre-
vious studies (Kara et al. 2015). Despite the fact that the dairy
cattle produce annually 35–55 kg methane, the adult horse
produces annually 18 kg methane. Considering the horse
population in the world, the share of horses in animal-
derived methane is at a rate of 0.5–3% (Crutzen et al. 1986).
The rate of global methane emission of horse-derived
methane emission has significantly increased in last 10 years
by the increase of horse population in the world (Rafiu et al.
2012). In the present study, in vitro methane volumes produced
by horse TMR at different tomato pulp levels were 0.28–
0.48 ml/g DM at 24 h of in vitro incubation was similar to the
results of Kara and Baytok (2017) and Cagri and Kara (2018).
As it is understood from these values, methane is not produced
as much as ruminants (Ellis et al. 2007) in the digestive tract of
horses, and it is observed that the contribution of horses to
global warming is not as high as ruminants.
As a result, it was demonstrated that tomato pulp, which
was used as a dietary fibre and CP source, did not affect nega-
tively digestion values of ration in horses. However, it has been
considered that tomato pulp using, as a soluble dietary fibre
source, at 10 and 20% ratios in DM supplementation levels
increased the in vitro digestion values. But in the whole
gastro-intestinal tract, for example, gastric enzymatic, small
intestine; enzymatic and large intestine fermentative digestion
of horse diet with tomato pulp should be performed with an in
vitro model.
Disclosure statement
No potential conflict of interest was reported by the author(s).
ORCID
Kanber Kara http://orcid.org/0000-0001-9867-1344
References
Abdouli H, Attia SB. 2007. Evaluation of a two-stage in vitro technique for
estimating digestibility of equine feeds using horse faeces as the
source of microbial inoculum. Anim Feed Sci Technol. 132:155–162.
A.O.A.C. 1995. Official methods of analysis, 15th ed. Arlington, VA:
Association of Official Analytical Chemists.
Bush JA, Freeman DE, Kline KH, Merchen NR, Fahey GC. 2001. Dietary fat
supplementation effects on in vitro nutrient disappearance and in vivo
nutrient intake and total tract digestibility by horses. J Anim Sci.
79:232–239.
Cagri A, Kara K. 2018. The effect of safflower on the in vitro digestion par-
ameters and methane production in horse and ruminant. Acta Vet
Eurasia. 44:73–84.
Crutzen PJ, Aselmann I, Seiler W. 1986. Methane production by domestic
animals, wild ruminants, other herbivorous fauna, and humans. Tellus.
38:271–284.
Del Valle M, Camara M, Torija ME. 2006. Chemical characterization of
tomato pomace. J Sci Food Agr. 86(8):1232–1236.
Earing J, Cassill B, Hayes S, Vanzant E, Lawrence L. 2010. Comparison of in
vitro digestibility estimates using the Daisy II incubator to in vivo digest-
ibility estimates. J Anim Sci. 88:3954–3963.
Elghandour MMMY, Kholif AE, Bastida AZ, Martínez DLP, Salem AZM. 2015.
In vitro gas production of five rations of different maize silage and con-
centrate ratios influenced by increasing levels of chemically character-
ized extract of Salix babylonica. Turk J Vet Anim Sci. 39:186–194.
https://doi.org/10.3906/vet-1408-62.
Ellis JL, Kebreab E, Odongo NE, McBride BW, Okine EK, France J. 2007.
Prediction of methane production from dairy and beef cattle. J Dairy
Sci. 90:3456–3467.
Ersahince A, Kara K. 2017. Nutrient composition and in vitro digestion par-
ameters of Jerusalem artichoke (Helianthus tuberosus L.) herbage at
different maturity stages in horse and ruminant. J Anim Feed Sci. 26.

Frape D. 2004. Equine nutrition and feeding. 3rd ed. Hong Kong: Blackwell
Publishing Ltd.
Isik F, Topkaya C. 2016. Effects of tomato pomace supplementation on
chemical and nutritional properties of crackers. Ital J Food Sci. 28:525–
535.
Kara K. 2015. In vitro methane production and quality of corn silage treated
with maleic acid. Ital J Anim Sci. 14:718–722.
Kara K. 2016. Effect of dietary fibre and condensed tannins concentration
from various fibrous feedstuffs on in vitro gas production kinetics with
rabbit faecal inoculum. J Anim Feed Sci. 25:266–272.
Kara K. 2018. Estimated ruminal digestion values and digestion end-pro-
ducts of concentrated mix feed after in vitro treatment with propionic
acid. Vet Med. 63:537–545.
Kara K. 2021. Nutrient matter, fatty acids, in vitro gas production and diges-
tion of herbage and silage quality of yellow sweet clover (Melilotus offici-
nalis L.) at different phenological stages. J Anim Feed Sci. 30(2):128–140.
https://doi.org/10.22358/jafs/136401/2021.
Kara K, Baytok E. 2017. Effect of different levels of psyllium supplementation
to horse diet on in vitro fermentation parameters and methane emission.
J Fac Vet Med Istanbul Univ. 43:12–18.
Kara K, Guclu BK, Baytok E. 2015. Comparison of nutrient composition and
anti-methanogenic properties of different Rosaceae species. J Animal
Feed Sci. 24:308–314.
Kholif AE, Baza-Garcia LA, Elghandour MY, Salem AZM, Barbabosa A,
Dominguez-Vara IA. 2015. In vitro assement of fecal inocula from
horses fed on high-fibre diets with fibrolytic enzymes addition on gas,
methane and carbon dioxide productions as indicators of hindgut
activity. J Equine Vet Sci. https://doi.org/10.1016/j.jves.2015.11.006.
Medina B, Girard ID, Jacotot E, Julliand V. 2002. Effect of a preparation of
Saccharomyces cerevisiae on microbial profiles and fermentation pat-
terns in the large intestine of horses fed a high fiber or a high starch
diet. J Anim Sci. 80(10):2600–2609. https://doi.org/10.2527/2002.
80102600x.
Menke KH, Raab L, Salewski A, Steingass H, Fritz D, Schneider W. 1979. The
estimation of the digestibility and metabolizable energy content of
ruminant feedstuffs from the gas production when they are incubated
with rumen liquor. J Agr Sci. 93:217–222.
Murray J-A, Scott B, Hastie P. 2009. Fermentative capacity of equine faecal
inocula obtained from clinically normal horses and those predisposed to
laminitis. Anim Feed Sci Technol. 151(3–4):306–311.
JOURNAL OF APPLIED ANIMAL RESEARCH 203
National Research Council. 1989. Nutrient requirements of horses. 5th
Revised ed. Washington (DC): National Academy Press/NRC.
National Research Council. 2001. Nutrient requirements of dairy cattle. 7th
Revised ed. Washington (DC): National Academy Press/NRC.
National Research Council. 2007. Nutrient requirements of horses. 6th
Revised ed. Washington (DC): The National Academies Press. https://
doi.org/10.17226/11653.
Niderkorn V, Baumont R. 2009. Associative effects between forages on feed
intake and digestion in ruminants. Animal. 3:951–960.
Prosky L, Asp NG, Schweizer TF, DeVries JW, Furda I. 1988. Determination of
insoluble, soluble, and total dietary fiber in foods, food products: inter-
laboratory study. J Assoc Off Anal Chem. 71:1017–1023.
Rafiu OY, Noor ZZ, Abba AH, Hassan MAA, Din MFM. 2012. Greenhouse gas
emissions: quantifying methane emissions from livestock. Am J Eng Appl
Sci. 5:1–8.
Rahmatnejad E, Bojarpour M, Mirzadeh KH, Chaji M, Mohammadabadi T.
2009. The effects of different levels of dried tomato pomace on broilers
chicken hematological indices. J Anim Vet Adv. 8(10):1989.
Ralston SL. 2021. Nutritional requirements of horses and other equids. MSD
Manual, Veterinary Manual, Merck Sharp & Dohme Corp, Kenilworth (NJ)
USA. https://www.msdvetmanual.com/management-and-nutrition/
nutrition-horses/nutritional-requirements-of-horses-and-other-equids.
Sunvold GD, Fahey GC, Merchen NR, Reinhart GA. 1995. In vitro fermenta-
tion of selected fibrous substrates by dog and cat fecal inoculum:
influence of diet composition on substrate organic matter disappear-
ance and shortchain fatty acid production. J Anim Sci. 73:1110–1122.
Sweeney CR. 2012. In vivo and in vitro digestibility of a complete pelleted
feed in horses [Master of Science Thesis]. The Faculty of California
Polytechnic State University. San Luis Obispo, CA (USA). https://doi.
org/10.15368/theses.2012.167.
Tassone S, Renna M, Barbera S, Valle E, Fortina R. 2019. In vitro digestibility
measurement of feedstuffs in donkeys using the daisy(II) incubator. J
Equine Vet Sci. 75:122–126.
Van-Soest PJ, Robertson JB, Lewis BA. 1991. Methods for dietary fibre,
neutral detergent fibre and non starch polysaccharides in relation to
animal nutrition. J Dairy Sci. 74:3583–3597.
Williams BA, Grant LJ, Gidley MJ, Mikkelsen D. 2017. Gut fermentation of
dietary fibres: physico-chemistry of plant cell walls and implications
for health. Int J Mol Sci. 18(10):2203. https://doi.org/10.3390/
ijms18102203.