Research in Veterinary Science 139 (2021) 112–120

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Research in Veterinary Science

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Effect of dietary supplementation with grape residue flour on weight gain,

metabolic profile, leukogram, proteinogram and antioxidant response in
suckling lambs
Vitor L. Molosse a, Guilherme L. Deolindo a, Bruno G. Cécere a, Hiam Marcon b, Gilneia da Rosa a,
Marcelo Vedovatto c, Claiton A. Zotti d, Anielen D. Silva e, Mateus Fracasso e, Vera M. Morsch e,
Raissa A. Carvalho f, Wanderson A.B. Pereira f, Aleksandro S. Da Silva a, b, *
a
Postgraduate Program in Animal Science, State University of Santa Catarina (UDESC), Chapecó, SC 89815-000, Brazil
b
Department of Animal Science, UDESC, Chapecó, SC 89815-000, Brazil
c
State University of Mato Grosso do Sul, Aquidauana, MS 79200-000, Brazil
d
Department of Animal Science, University of West Santa Catarina, 89820-000 Xanxerê, SC, Brazil
e
Graduate Program of Toxicological Biochemistry, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil
f
Veterinary Medicine, Instituto Federal Catarinense (IFC), Concordia, SC, Brazil

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

Keywords: The objective of this study was to determine whether the addition of grape residue flour (GRF) in the diet of
Lamb suckling lambs would improve their health and consequently enhance their growth. We used 48 lambs, 30 days
Performance of age divided into four treatments with four repetitions each, with each repetition consisting of three animals.
Antioxidant
The groups were identified as follows: G-0%, used as a control (without GRF) and G-0.5%, G-1% and G-2%
Immune system
referring to treatment with grape residue flour in doses of 0.5%, 1%, and 2% of inclusion in the concentrate,
respectively. Lambs in groups G-1% and G-2% had higher final body weights, weight gain and average daily gain
(ADG) compared to the other groups. GRF dietary supplementation had good antioxidant potential, being able to
stimulate glutathione S-transferase (GST) activity and consequently reduce levels of reactive oxygen species
(ROS) in lambs that consumed the highest dose of GRF (G-2%). GRF supplementation improved humoral re­
sponses, with increased serum levels of heavy-chain and light-chain immunoglobulins; however, there was a
reduction in serum ceruloplasmin levels in these lambs. We observed higher concentrations of glucose and tri­
glycerides in lambs in the 2% group. There were lower lymphocyte counts in lambs that received GRF. Lambs
supplemented with the highest doses of GRF (G-1% and G-2%) had lower total bacterial counts in the feces. We
conclude that the use of GRF in the supplementation of suckling lambs improved animal health, as it stimulated
the antioxidant and immune systems and consequently favored their growth.

1. Introduction non-toxic compounds with antioxidant potential (Negrette-Guzmán

et al., 2013). In general, these additives donate electrons and neutralize
Meeting the demand for food for the population, while guaranteeing free radicals, resulting in the prevention of cell injuries (Saeidnia and
the integrity of animal health, as well as sustainable production, are Abdollahi, 2013).
among the greatest challenges for the global agriculture and livestock The use of by-products from agro-industries in the formulation of
industries. It is necessary to use alternatives to minimize negative im­ ruminant diets can be a strategy to lower the cost of production, as well
pacts on the sheep production chain, and, consequently, to enable ani­ as the need to recycle waste that would otherwise be destined for
mals to express their full genetic potential. Thus, the search for disposal (Guerra-Rivas et al., 2016). According to data of IBGE - Instituto
alternatives has been widely researched and in particular, there are feed Brasileiro de Geografia e Estatística (2020), the southern Brazilian re­
additives that in recent years have increasingly consisted of natural, gion was responsible for approximately 61% of the total grape

* Corresponding author at: Postgraduate Program in Animal Science, State University of Santa Catarina (UDESC), Chapecó, SC 89815-000, Brazil.
E-mail address: aleksandro_ss@yahoo.com.br (A.S. Da Silva).

https://doi.org/10.1016/j.rvsc.2021.07.004
Received 18 August 2020; Received in revised form 16 June 2021; Accepted 1 July 2021
Available online 6 July 2021
0034-5288/© 2021 Elsevier Ltd. All rights reserved.
V.L. Molosse et al. Research in Veterinary Science 139 (2021) 112–120

production in 2020; about 72% of the total Brazilian area destined for marketed for human consumption, with nutraceutical purposes. The
this agribusiness is concentrated in the southern region of the country. feed samples were analyzed according to Association of Official
Consequently, the southern Brazilian region also has a large amount of Analytical Chemists (1997): dry matter (DM), method 930.15; crude
waste from grape processing (19 to 25% is waste-peel and seed) (Dwyer protein (CP), method 976.05; ethereal extract (EE), method 920.39 and
et al., 2014; Mendes et al., 2013). Commonly, this dry residue is ashes, method 942.05. The concentration of neutral detergent fiber
composed of peel and stalk (8–20%), residual pulp (5–10%) and seeds (NDF) and acid detergent fiber (ADF) were done according to the
(38–52%) (Christ and Burrit, 2013; Brenes et al., 2016). methodology of Van Soest et al. (1991). The values (g/kg) were as fol­
Grape seed and peels (Vitis vinifera) contain flavonoids (catechin, lows: dry matter (DM), 974; crude protein (CP), 98.9; ethereal extract
epicatechin, procyanidins and anthocyanins), phenolic acids, and (EE), 79; neutral detergent fiber (NDF), 679.9.
resveratrol, all of which have been shown to have functional activities
(Sato et al., 2001). Natural antioxidants can be extracted from the grape 2.2. Animals and experimental design
seed. Among these are flavonoids and proanthocyanidins that act as
scavengers of free radicals and promoting vasodilation, inhibiting en­ 2.2.1. Pre-experiment: care for lambs
zymes such as phospholipase, cyclooxygenase, and lipoxygenase, in Prior to the experiment, these lambs remained with their mothers for
addition to acting as reducers in lipid peroxidation processes (Fascina the first 3 days of age, for adequate colostrum intake, and later were
et al., 2012). Today, resveratrol is one of the most studied phenolic housed in collective pens with a maximum capacity of four animals,
compounds in the world, mainly because of its anti-inflammatory, where they received liquid diet (fresh sheep’s milk) through a collective
antioxidant, and immunomodulatory properties. Resveratrol is known feeder (Milk Bar®) in the proportion of 1500 mL/lamb/day; they also
to minimize the effects of apolipoprotein-B peroxidation, associated received ad libitum concentrate with 22% crude protein, up to 30 days
with low-density lipoprotein (LDL), resulting in the restoration of of age.
glutathione in plasma and tissue (Sahin et al., 2010). There are also
beneficial cardiovascular effects secondary to the antioxidant function 2.2.2. Experiment
and antiplatelet activities of resveratrol (Matos et al., 2012). However, it The experiment was carried out on a commercial farm located in the
is important to know that there are legislative constrains that rule the city of Chapecó (South, Brazil). We used 48 Lacaune male lambs [10.52
management of grape processing (in particular winemaking) residues in ± 2.81 kg of body weight and age of 30 ± 3 days]. We used only males in
different countries, but there are possible strategies for a sustainable, this study for the purpose of standardizing the experimental groups, and
lawful, integral, and remunerative recovery of grape processing residues the greatest number were males in the batch of lamb available on the
(Spigno et al., 2017). farm.
The inclusion of grape residue flour (GRF) as a feed supplement is a The pens were made up of sawdust bed, and, had automatic drinkers
promising alternative for improving animal performance and meat and two feeders. We use curtain management associated with fans to
quality; as was recently verified in dairy sheep farming (Alba et al., maintain the thermal comfort of the animals in the installation during
2019) and laying poultry farming (Reis et al., 2019). In pigs, supple­ the experiment; as well as daily cleaning of the stalls (also highlight to
mentation with resveratrol improves meat quality (Zhang et al., 2015), move the bed).
as well as reducing levels of palmitic acid, stearic acid and arachidic acid To carry out the experiment, the animals were divided into four
and increasing linoleic acid in the subcutaneous fat of pigs (Yan and treatments with four repetitions and three lambs per repetition. Each
Kim, 2011). Sheep intake concentrates supplemented with either 10% repetition corresponds to pens that contained three lambs. The groups
grape pomace or 5% grape seed during pregnancy and lactation; how­ were identified as follows: G-0%, used as a control (without GRF) and G-
ever, the by-products added to the diets was a good way to reduce costs 0.5%, G-1% and G-2% referring to the treatment with grape residue
on feeding and waste, but they were not able to provide a healthier fatty flour in doses of 0.5%, 1% and 2% of inclusion in the concentrate,
acid profile, neither in milk nor in the meat of the suckling lambs respectively. The diets were formulated to meet or exceed the lamb’s
(Resconi et al., 2018). According to Goñi et al. (2007), dietary supple­ requirements, according to nutritional requirements (National Research
mentation with grape marc improves the antioxidant capacity in breast Council – N.R.C, 2007; Table 1). The corn was ground in a knife-type
and thigh meat. In addition, according to the literature, the addition of grinder, using a number 2 sieve; and so the corn ground into small
antioxidants extracted from grapes improves the shelf life of meat and fragments in order to improve digestibility.
meat products, protecting against deterioration (Brannan, 2008). The daily feeding of the animals was carried out twice a day (at 07:00
Resveratrol supplementation in poultry had a growth-promoting effect, and 17:00). At both time points, the liquid feed (sheep’s milk) was first
being able to improve immunity and oxidative status, reflected in better supplied using a collective feeder (Milk Bar®). During the experiment,
productive performance (Alagawany et al., 2015). the milk amount was reduced, i.e., 1000 ml, 750 ml, 500 ml, 250 ml/
Considering the studies already carried out and the characteristics of lamb/day between days 1–7, 8–14, 15–21 and 22–30 of the experiment,
the substances present in the grape residues, we have hypothesized that respectively. This reduction was necessary because the animals were
the inclusion of grape skin flour and grape seed in lamb feed promoted weaned at 60 days of age. After the milk consumption, the concentrate
the activation of the antioxidant and immunological responses. There­ was provided according to the animals’ body weight (2% of BW between
fore, the objective of this study was to determine whether the addition of day 1 to 15 days old and 2.5% of BW between 16 and 30 days old).
GRF in the diet of suckling lambs would improve their health and Concentrate intake was 100% of that offered for all treatments and
consequently enhance their growth. repetitions, throughout the experimental period.
After the total ingestion of the concentrate, each pen received corn
2. Materials and methods silage ad libitum. Free access to silage by lambs is a common practice on
the experimental farm where the experiment was carried out. We tried
The project was approved by the Ethics Committee on Animal to measure silage intake, but it was not possible to do it safely and
Research at the University of the State of Santa Catarina (protocol reliably of data. This is because the lambs played with the silage, letting
number: 1932030419), following guidelines from CONCEA/Brazil. it fall to the ground, leading to waste due to the animals having been
housed in a floor shed, with food passing through the cracks, thereby
2.1. Grape residue flour preventing reliable measurement of the amount of silage. The feeders
with silage of all treatments and repetitions, during all moments of the
The GRF used was purchased from a local company (Natural Prod­ day had this food available; this allows us to state that silage con­
ucts Company; Essential®) in 2019, at a cost of US$ 5.46 per kg, and it is sumption was not limited in this study. The silage leftovers in the feeders

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Table 1 2.4. Growth performance

Ingredients used to formulate the basal concentrate of lambs, and chemical
composition in diet (silage and concentrate) and chemical composition of feed The lambs were weighed to check average body weight at five time
used in the diet of lambs supplemented with grape residue flour. points (days 1, 7, 15, 21, and 30) using a digital scale. From this infor­
Nutrients Concentrate (%) mation, we calculated weight gain (final BW – initial BW) and average
G-0% G- G-1% G-2% daily gain [(final BW – initial BW) / number of days] in each experi­
0.5% mental period (1–15 d; 15–30 d; 1–30 d).
Ingredient
Ground corn 56.89 56.45 56.00 55.10 2.5. Sample collection
Soybean meal 38.11 38.05 38.00 37.90
Grape residue flour – 0.5 1.00 2.00
Premix1 4.00 4.00 4.00 4.00 Blood was collected on days 1, 15 and 30 of the experiment from the
Calcitic limestone 1.00 1.00 1.00 1.00 jugular vein. Fecal samples were collected directly from the rectal am­
pule, to avoid environmental contamination. Whole blood was collected
Chemical composition (g/ Corn Concentrates
kg)2 Silage G-0% G- G-1% G-2% in two tubes: without anticoagulant to obtain serum and with EDTA as
0.5% anticoagulant for complete blood counts. The blood collected without
Dry matter 343.8 888.0 893.2 886.2 888.0 anticoagulant was centrifuged at 3500 rpm for 10 min and the serum
Crude protein 81.4 231.9 222.5 226.3 225.3
was stored at − 20 ◦ C until analysis. All biochemical analyses took place
Ether extract 14.5 23.8 27.4 28.1 27.0
NDF 481.4 133.0 158.7 164.4 150.9
simultaneously within 15 days after the end of the experiment.
ADF 279.3 41.7 44.5 53.6 57.6
1
Premix composition: phosphorus (min 30 g/kg), calcium (min 200 g/kg, 2.6. Blood analysis
max 280 g/kg), sulfur (min 28 g/kg), sodium (min 45 g/kg), potassium (min. 25
g/kg), cobalt (min 20 mg/kg), chromium (min 5 mg/kg), iron (min 450 mg/kg); 2.6.1. Hematological analysis
iodine (min 25 mg/kg), magnesium (min 20 g/kg), manganese (min 650 mg/ The total erythrocyte and leukocyte count, as well as the hemoglobin
kg), selenium (min 6.7 mg/kg), zinc (min. 805 g/kg) vitamin A (min 100,000 (Hb) content were measured using a CELM® 530 semi-automatic
IU/kg), vitamin D (min 22,000 IU/kg), vitamin E (min 300 IU/kg), Saccharo­ analyzer. The hematocrit was obtained after capillary centrifugation
myces cerevisiae 25 × 109and sodium monensin (1400 mg/kg).
2
(1000 rpm for 5 min). The differential leukocyte count was performed
Note: DM (dry matter), NDF (neutral detergent fiber), ADF (acid detergent
on blood smears stained with commercial stain (Romanowsky method)
fiber), EE (ether extract).
using a 1000× magnification light microscope (Feldman et al., 2000).
The blood count analysis was performed within 2 h after sample
were discarded prior to providing the new feed with concentrate and
collection.
silage, in order to have fresh food throughout our research.
The experiment lasted 30 days (lambs aged between 30 and 60 days),
2.6.2. Serum biochemistry
i.e. final period of the lambs’ suckling period. Between days 1 and 30 of
Serum levels of total proteins (PT), glucose, albumin, and urea were
the lambs age, they were used as a period of adaptation to the concen­
measured using the Bio-2000 BioPlus® semi-automatic analyzer and
trate, when the consumption of concentrate differed greatly between
commercial kits (Analisa®, Gold Analisa Diagnóstica, Belo Horizonte,
animals (according to the practical experience of our research group).
Brazil) following the recommendations of the manufacturer by enzy­
This was the main justification for the experiment not having started
matic or kinetic methods, depends on the evaluated variable. Globulin
right after the lambs were born, as adaptation is extremely necessary (i.
levels will be obtained with mathematical calculation (total proteins –
e. animals need to learn to consume), and the animals still prefer milk
albumin).
during the first 30 days of life.
Data collection and samples were taken three times during the trial:
2.6.3. Oxidant/antioxidant status
day/sample 1 (30 days of life), day/sample 15 (45 days of life) and day/
sample 30 (weaning – 60 days of life); as described in detail below.
2.6.3.1. Glutathione S-transferase. The activity of serum glutathione S-
transferase (GST) was measured based on the method described by
2.3. Analysis of the chemical composition of feed
Habig et al. (1974) and was expressed as U GST/mg of protein.
In the laboratory, the samples were pre-dried in a forced-air oven at
2.6.3.2. Superoxide dismutase. The activity of the superoxide dismutase
55 ◦ C for 72 h, then removed from the oven and weighed again to
enzyme (SOD) was measured using the methodology described by
determine the partial dry matter content, followed by grinding in a
McCord and Fridovich (1969), where the speed of the formation of the
Wiley-type mill (Marconi, model: MA340), using a 1-mm mesh sieve.
adrenochrome, measured at 480 nm in a medium containing glycine, to
The pre-dried and ground samples were subjected to the heating at
determine the value of SOD. The result was expressed as IU/mg of
105 ◦ C to obtain DM, and later to obtain the mineral material in a muffle
protein.
at 600 ◦ C (Silva and Queiroz, 2002). CP was determined by the micro-
Kjeldahl method (Method 984.13, Association of Official Analytical
Chemists, 1997). To determine the levels of neutral detergent fiber 2.6.3.3. Lipid peroxidation. Serum lipid peroxidation was measured as
(NDF), the samples were packed in polyester bags (Komarek, 1993) and amount of thiobarbituric acid reactive substances (TBARS) according to
were treated with neutral detergent solution in an autoclave at 110 ◦ C Jentzsch et al. (1996). The reaction was read in spectrophotometer at
for 40 min (Senger et al., 2008); for concentrate samples, we included 535 nm. The result was expressed as nmoles malondialdehyde/ml of
α-amylase (Mertens, 2002). The concentrations of acid detergent fiber serum.
(ADF) were determined according to AOAC (Association of Official
Analytical Chemists, 1997, method 973.18). Results are presented in 2.6.3.4. Reactive oxygen species. The determination of reactive oxygen
Table 1. species (ROS) was based on the technique described by Ali et al. (1992).
The samples were diluted 1:10 with 10 nM Tris (pH 7.4) and 5 μL of
dichlorofluorescin diacetate (DCFH-DA) in methanol was added over 15
min, at a temperature of 37 ◦ C, to form a non-fluorescent dichloro­
fluorescin (DCFH). The formation of dichlorofluorescein (DCF;

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fluorescent DCFH) was monitored using spectrophotometry at 525 nm. observation.

The formation of ROS was determined using a standard curve DCF in All other variables of the study were analyzed as repeated measures
methanol (0.05–1.0 μM). The results were expressed as U DCF/mg and tested for fixed effects of treatment, day, and treatment × day, using
protein (Ali et al., 1992). pen (treatment) and animal (pen) as random variables and the statistical
model was:
2.6.4. Proteinogram
yijkl = μ + Ti + Dj + P(T)k + A(P)l + (TD)ij + εijkl
Polyacrylamide gel electrophoresis containing sodium dodecyl sul­
fate (SDS-PAGE) was performed according to the technique described by Where: Yijkl = observation of the effect of treatment i per days of
Fagliari et al. (1998) using mini-gels (10 × 10 cm). The gels were stained collection j in pen(treatment) k, and animal(pen) l, where μ = overall
with Coomassie blue and were photographed to identify and quantify mean; Ti = effect of treatment i, where i = 1 (G-0%), 2 (G-0.5%), 3 (G-
protein fractions using Labimage 1D software (Loccus Biotechnology). A 1%) and 4 (G-2%); Dj = effect of days of collection (j = 0, 15 and 30); P
standard containing fractions with molecular weight between 10 and (T)j = effect of pen (treatment) j (j = 12 pens); A(P)k effect of animal
250 KD (Kaleidoscope - BIORAD) was used as a reference. (pen) k (k = 48 animals); TDij = interaction between treatment i and day
j; Ɛijkl = random error associated with each observation.
2.7. Feces microbiology All results obtained on d 0 for each variable were included as cova­
riates in each respective analysis, but were removed from the model
Stool samples collected on days 1 and 30 were placed in isothermal when P > 0.10. The compound symmetric covariance structure was
boxes and analyzed within 2 h. The total bacterial count (TBC) was selected for concentration of AST and GGT and the first order autore­
performed using 1 g of each stool sample, diluted in 9 mL of Buffered gressive covariance structure was selected for all others variables. The
Peptone Water in sterile test tubes, homogenized in a vortex shaker covariance structures were selected according to the lowest Akaike in­
resulting in the 10− 1 dilution, and the remaining dilutions were per­ formation criterion. Means were separated using PDIFF and all results
formed until achieving 10− 6, always inoculating 1 mL of the previous were reported as LSMEANS followed by SEM. Significance was defined
dilution in 9 mL of Buffered Peptone Water. Then, 200 μL of the 10− 6 when P ≤ 0.05, and tendency when P > 0.05 and ≤ 0.10.
dilution of each sample was inoculated in a Petri dish previously pre­
pared with standard agar for counting and incubated in a bacteriological 3. Results
oven at 37 ◦ C for 48 h. We used a colony counter and the results were
expressed as colony forming units per mL (CFU/mL). 3.1. Growth performance

The results of growth performance are shown in Table 2. Effects of

2.8. Economic viability: gross margin
treatment × day and treatment were detected (P ≤ 0.05) for body weight
(BW) and the animals G-1% and G-2% had higher weight at d 30 than G-
In order to determine the economic viability of including GRF in the
0% animals. The lambs G-1% and G-2% showed greater weight gain
diet of lactating lambs, an analysis of gross profit over the period was
(GP) and average daily gain (ADG) (P ≤ 0.04) from 15 to 30 d in relation
carried out. For that, the values of total variable costs and total revenue
to the lambs G-0%. The lambs G-1% and G-2% presented greater weight
were considered only related to the breastfeeding period (experiment
gain (WG) and ADG (P ≤ 0.04) from 0 to 30 d in relation to the lambs G-
period). The calculation of gross profit is based on the subtraction be­
0%. The consumption of concentrate did not differ statistically for
tween total revenue and its variable costs. Variable costs are all costs
consumption of concentrate (Supplementary material).
that fluctuate according to production, and can be divided into opera­
tional costs (electricity, labor, fuel, among others) and food (bulky,
concentrated, minerals, among others). In the present study, we 3.2. Serum clinical biochemistry
considered as variable costs only the costs of total concentrate
consumed, considering that all other expenses were similar in the The results of serum biochemistry are shown in Table 3. No effects of
different groups, therefore, disregarded in the analysis of gross profit. treatment × day and treatment (P ≥ 0.11), for AST, GGT, total protein,
Taking into account that, at the end of the experiment, the animals albumin, globulin, or urea were detected. However, treatment effects (P
would not be commercialized, the total revenue was calculated from an = 0.01) were detected for the serum glucose concentration, i.e., the G-
estimate, considering the following formula (similar to Chikwanha et al., 2% animals had higher concentrations of glucose than the G-0% and G
2019): Income = (weight gain in period × Carcass yield) × Meat price. − 1% animals. Treatment effects (P = 0.05) were observed for the serum
concentrations of triglycerides; the G-2% lambs showed higher values
than the others.
2.9. Statistical analysis

We use a completely randomized design here. All dependent vari­ 3.3. Hematological variables
ables were tested for normality using the Univariate procedure of SAS
(SAS Inst. Inc., Cary, NC, USA; version 9.4) and all variables were nor­ The hematological variables are shown in Table 4. There were no
mally distributed. Then, all data were analyzed using the MIXED pro­ effects of treatment versus day and treatments (P ≥ 0.11) for erythro­
cedure of SAS, with the Satterthwaite approximation to determine the cytes, hematocrit, hemoglobin, leukocytes, neutrophils, neutrophils/
denominator degrees of freedom for the test of fixed effects. Weight gain lymphocytes ration, monocytes and eosinophils. However, treatment
and average daily gain were tested for fixed effect of treatment using pen effects (tendency; P = 0.08) were detected for lymphocyte number. The
(treatment) and animal(pen) as random effects as follow the statistical G-2% animals showed lower values compared to the G-0% and G-1%
model: animals.

yijk = μ + Ti + P(T)j + A(P)k + εijk

3.4. Serum oxidant and antioxidant status
Where: Yijk = observation of the effect of treatment i on pen(treat­
ment) j, and animal(pen) k where μ is the overall mean; Ti = effect of Hematological variables are shown in Table 5. No effects of treat­
treatment i, where i = 1 (G-0%), 2 (G-0.5%), 3 (G-1%) and 4 (G-2%); P ment versus day and treatment (P ≥ 0.32) were found for TBARS or SOD.
(T)j = effect of pen (treatment) j (j = 12 pens); A(P)k effect of animal However, effects of treatment versus day (P = 0.05) were detected for
(pen) k (k = 48 animals); Ɛijk = random error associated with each the serum concentration of ROS, i.e., G-2% lambs showed lower values

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V.L. Molosse et al. Research in Veterinary Science 139 (2021) 112–120

Table 2
Growth performance of lambs supplemented with grape residue flour (G).
Variables Treatments1 SEM P-value

G-0% G-0.5% G-1% G-2% Treat Treat×day

Body weight (BW), kg 0.05 0.01

d0 10.49 10.44 10.53 10.57 0.44
d 15 13.96 14.31 14.24 14.43 0.44
b ab
d 30 17.69 18.39 18.63 a 19.37 a
0.44
Weight gain (WG), kg
d 0 to 15 3.46 3.87 3.71 3.85 0.62 0.47
d 15 to 30 3.74c 4.03bc 4.71a 4.94a 0.81 0.04
d 0 to 30 7.20c 7.90bc 8.10ab 8.80a 0.34 0.01
Average daily gain (ADG), kg
d 0 to 15 0.230 0.258 0.247 0.256 0.04 0.47
d 15 to 30 0.249c 0.268b 0.314a 0.329a 0.05 0.04
d 0 to 30 0.240c 0.263bc 0.270ab 0.293a 0.01 0.01
The treatments G-0%, G-0.5%, G-1% and G-2% represent 0, 0.5, 1 and 2% of grape residue flour in the concentrate, respectively. a–b
1
Tends to differs (P ≤ 0.10) between treatments.

Table 3
Biochemical profile of lambs supplemented with grape residue flour (G).
Variables1 Treatments2 SEM P-value

G-0% G-0.5% G-1% G-2% Treat Treat×day

AST (U/L) 87.33 92.71 107.25 99.54 6.00 0.11 0.47

GGT (U/L) 100.71 103.00 104.29 108.79 4.28 0.58 0.94
Glucose (mg/dL) 70.67b 76.46ab 73.71b 82.20a 2.52 0.01 0.59
Total protein (g/dL) 5.16 4.77 4.95 4.85 0.11 0.44 0.64
Albumin (g/dL) 2.55 2.33 2.29 2.44 0.09 0.21 0.95
Globulin (g/dL) 2.66 2.44 2.66 2.41 0.10 0.28 0.33
Urea (mg/dL) 24.51 28.25 26.58 27.96 1.52 0.33 0.43
Triglycerides (mg/dL) 31.67b 31.12b 30.37b 36.7a 1.80 0.05 0.62
1
AST, aspartate aminotransferase and GGT, gamma-glutamyl transferase.
2 a–b
The treatments G-0%, G-0.5%, G-1% and G-2% represent 0, 0.5, 1 and 2% of grape residue flour in the concentrate, respectively. Differs (P ≤ 0.01) between
treatments.

Table 4
Hematological variables of lambs supplemented with grape residue flour (G).
Variables Treatments1 SEM P-value

G-0% G-0.5% G-1% G-2% Treat Treat×day

Erythrocytes (x106 μL) 7.07 6.68 6.92 7.00 0.22 0.61 0.94
Hematocrit (%) 35.50 35.12 34.83 36.46 0.61 0.27 0.87
Hemoglobin (g/dL) 11.26 11.09 11.01 11.56 0.17 0.11 0.59
Leucocytes (x103/μL) 9.77 9.23 9.44 9.06 0.60 0.85 0.15
Neutrophils (x103/μL) 4.17 3.81 3.90 4.00 0.33 0.78 0.52
Lymphocytes (x103/μL) 5.28a 5.20ab 5.26a 4.77 b 0.40 0.08 0.15
Neutrophil/Lymphocyte ratio 0.98 0.81 0.83 0.93 0.08 0.48 0.79
Monocytes (x103/μL) 0.26 0.22 0.27 0.23 0.04 0.80 0.74
Eosinophils (x103/μL) 0.06 0.07 0.05 0.06 0.02 0.85 0.44

The treatments G-0%, G-0.5%, G-1% and G-2% represent 0, 0.5, 1 and 2% of grape residue flour in the concentrate, respectively.

Table 5
Serum levels of oxidants and antioxidants of lambs supplemented with grape residue flour (G).
Variables1 Treatments2 SEM P-value

G-0% G-0.5% G-1% G-2% Treat Treat×day

TBARS 1.83 1.92 1.74 1.65 0.10 0.32 0.57

ROS 0.30 0.05
d0 26.88 19.45 23.61 23.31 25.08
d 15 47.01 37.68 18.51 39.81 25.08
d 30 79.57a 49.74ab 58.16ab 18.42b 25.08
SOD 9.74 8.55 8.72 9.43 0.81 0.69 0.64
GST 0.17 0.04
d0 1.88 2.00 2.10 1.94 0.23
d 15 1.91 2.03 2.36 2.14 0.23
d 30 1.78b 1.56b 2.68a 2.78a 0.23
1
Lipid peroxidation (TBARS - nmol MDA/mL), reactive oxygen species (ROS - U DCFH/mg of protein) superoxide dismutase (SOD - U SOD/mg of protein) and
glutathione S-transferase (GST - U GST/mg of protein).
2
The treatments G-0%, G-0.5%, G-1% and G-2% represent 0, 0.5, 1, and 2% of grape residue flour in the concentrate, respectively.

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V.L. Molosse et al. Research in Veterinary Science 139 (2021) 112–120

than did G-0% animals on day 30. Effects of treatment versus day (P =
0.04) were detected for GST, i.e., G-1% and G-2% lambs showed higher
values than did G-0% and G-1% lambs on day 30.

3.5. Proteinogram

The results obtained for the constituents of the serum proteinogram

are shown in Fig. 1. Effects of treatment versus day (P = 0.01) and
treatment (P = 0.01) were found for ceruloplasmin, transferrin, light
chain Ig, and IgA. The G-0.5%, G-1% and G-2% animals had lower
values of ceruloplasmin on days 15 and 30 than did G-0% animals. The
G-1% and G-2% animals had lower transferrin values on day 15 than did
Fig. 2. Bacterial account in feces of lambs supplemented with grape residue
G-0% and G-0.5% animals; on day 30, G-0% and G-2% animals had
flour (G). The treatments G-0%, G-0.5%, G-1% and G-2% represent 0, 0.5, 1,
higher values than did G-0.5% or G-1% animals. G-1% and G-2% lambs and 2% of grape residue flour in the concentrate, respectively. a–b Differs (P ≤
had higher values of heavy chain Ig on day 30 than did G-0% or G-0.5% 0.05) between treatments each respective day. Vertical bars represent the SEM.
animals. G-2% animals showed higher values of light chain Ig on day 15
and 30 than did other groups. G-0.5% and G-1% animals had higher IgA
(Supplementary material). As the animals that consumed diets con­
levels than did G-0% and G-2% animals on day 15; on day 30, G-0.5%, G-
taining GRF had greater weight gain; as a result, economic viability
1%, and G-2% animals had higher values than G-0% animals.
calculations show that it is profitable for the farmer to include GRF in
the lamb diet; for example, the inclusion of 2% in the concentrate allows
3.6. Total bacterial counts a profitability of 1 dollar per lamb in 30 days of experiment (Supple­
mentary Material).
The results obtained for total bacterial counts are shown in Fig. 2.
Effects of treatment versus day (P = 0.05) and treatment (P = 0.01) were 4. Discussion
detected for TBC on day 30, where the G-1% and G-2% animals had
lower values than did the G-0% and G-0.5% animals. Dietary supplementation with GRF was associated with higher final
body weight, weight gain and ADG of lactating lambs, as observed by
3.7. Economic viability Zhao et al. (2018) who supplemented male lambs from cross 1/2 Dorper
(♂) × 1/2 Small thin-tailed (♀) weaned for 74 days with 10% grape
The addition of GRF left the diet at a higher cost, although it had marc. These authors found that dietary supplementation with grape
higher income and balance when compared to the control diet marc stimulated development of the rumen, abomasum, and large

Fig. 1. Serum concentration of ceruloplasmin, transferrin, immunoglobins heavy (IgH) and light (IgL) chain and immunoglolins A of lambs supplemented with grape
residue flour (G). The treatments G-0%, G-0.5%, G-1% and G-2% represent 0, 0.5, 1 and 2% of grape residue flour in the concentrate, respectively. a–b Differs (P ≤
0.05) or tends to differ (P ≤ 0.10) between treatments each respective day. Vertical bars represent the SEM.

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V.L. Molosse et al. Research in Veterinary Science 139 (2021) 112–120

intestine. Although these authors did not analyze the mechanism dietary supplementation of grape seed proanthocyanidin extract (250
involved in the further development of these organs and the relationship and 500 mg/kg) improved the antioxidant capacity of broiler chickens
with the highest performance, it is known that tannin, a phenolic com­ infected with aflatoxin B1.
pound, contained in grape marc, can interfere with the gram-negative Because the components of the grape residue have anti-inflammatory
microbiome of the rumen, leading to attenuation of ammonia produc­ characteristics (Li et al., 2001; Colombo et al., 2019; Rodríguez-Mor­
tion and subsequent reduction of cellulite and proteolytic bacteria gado et al., 2015), we believe that the consumption of GRF by lambs
(Abarghuei et al., 2010). As a result, there is a lower demand for energy reduced counts lymphocytes, inflammatory cells that can exacerbate an
by these bacteria, which may explain the higher concentration of blood inflammatory response when present in high numbers. It is already
glucose detected in the present study. However, there is also the hy­ known that inflammatory processes consume large amounts of ATP to be
pothesis that supplementation with grape flour alters a ruminal micro­ efficient; consequently, this reflects negatively on the zootechnical
biota in order to favor the production of propionate, increasing the performance of animals, as it reduces ATP in muscle tissue and as a
availability of glucose precursors in hepatocytes. In addition, previous result, animals gain less weight. The anti-inflammatory effect of GRF
studies concluded that the inclusion GRF in the diet of animals had a occurs via inhibition of the NF-kB pathway through the activation of
growth-promoting effect associated with increased activity of intestinal PPAR-gamma (Martinez-Micaelo et al., 2015). We also observed
digestive enzymes (Huang et al., 2012; Xie et al., 2012), immunological decreased serum levels of ceruloplasmin, an acute-phase protein whose
stimulation (Xie et al., 2012; Zhou et al., 2013) and increased antioxi­ levels elevate to help the organism eliminate the cause of the imbalance
dant capacity (Wang et al., 2008; Zhao et al., 2013; Lu and Zhai, 2014). and restore homeostasis (Cerón et al., 2005). This effort by the organism
In lamb production, numerous factors induce oxidative stress and can trigger systemic effects such as hyperthermia, and elevated blood
consequently stimulate or deplete antioxidant defenses. In the present cortisol concentrations (Eckersall, 2000), which are not desirable for
study, we found that the consumption of GRF stimulated the antioxidant animals. In summary, grape flour modulated immune function in suck­
system and thereby reduced injurious oxidative reactions that consume ling lambs and had an indirect effect on the weight gain. The anti-
energy and lead less weight gain. According to the literature, oxidative inflammatory effect was not as marked as the effect on laying hens
stress is a consequence of an abnormal increases in the production of (Reis et al., 2019) or dairy sheep (Alba et al., 2019). This may be because
ROS, resulting in adverse health conditions and reduction in production young lambs have developing immune systems.
efficiency (Yuan et al., 2007). In the present study, GRF dietary sup­ The consumption of GRF (1% and 2%) by lambs reduced the bacte­
plementation acted as a strong antioxidant, an effect also observed in rial counts in the feces, an expected result, due to fact that the GRF has
broiler chickens (Abu Hafsa and Ibrahim, 2018), quails (Sahin et al., functional compounds with antimicrobial and prebiotic characteristics
2012), pigs (Kafantaris et al., 2018), laying hens (Reis et al., 2019), as (Katalinić et al., 2010; Agte et al., 2010) capable of modulating animal
well as in meat and meat products (Amin and Edris, 2017; El-Zainy et al., microbiota (Etxeberria et al., 2013). The effect on bacteria did not
2016). A study with lactating lambs and post-lactation showed that interfere with performance; however, it is important to perform studies
supplementation with grape marc increased the expression and activity to identify which bacteria (pathogenic or non-pathogenic) cause growth
of GST, an enzyme involved in the metabolism of glutathione (GSH) compromise. It is important to remember that non-pathogenic bacteria
(Hayes et al., 2005), resulting in improvements in redox status (Kafan­ also trigger important functions both for the health of the animal in­
taris et al., 2016). Similar to the current study, there was greater GST testine and for digestibility and growth rate (Montagne et al., 2003). In
activity in lambs that consumed GRF. The compounds present in the GRF agreement with these results, Kafantaris et al. (2016) demonstrated that
have functional capacity to act negatively on the concomitant genera­ grape marc supplementation in lambs had beneficial effects on fecal
tion of ROS (Zhao et al., 2018; Sahin et al., 2012). Rubiolo et al. (2008) microflora. These authors found decreased counts of harmful bacteria
observed that resveratrol stimulated Nrf2, leading to positive regulation such as Salmonella, E. coli, Shigella, Yersinia, and Proteus as well as
of antioxidant enzymes in rats, consequently stimulating their antioxi­ increased counts of the beneficial bacteria of the genus Bifidobacterium.
dant capacity (Alía et al., 2003; Kafantaris et al., 2016). This information Overall, the data suggest that similar effects on the intestinal microbiota
is important, because resveratrol is one of the main components present may have occurred in our study because the animals gained weight.
in the grape; today with several known biological and medicinal prop­ In our study, there was economic viability with the use of GRF in the
erties (Berman et al., 2017). Therefore, both the properties of GRF and lambs’ diet, similar effect described by Chikwanha et al. (2019) for
resveratrol alone are desirable in animal diets, because they promote castrated Dohne Merino lambs, who found a gross margin returns were
health. greatest for 12.2% grape pomace diet. In beef cattle, Tayengwa et al.
The consumption of GRF by lambs stimulated a humoral immune (2020) could observe higher income on feed costs in Angus steers sup­
response in which serum levels immunoglobulins were elevated, in an­ plemented with 150 g/kg of dry grape pomace compared to traditional
imals that consumed the highest percentage of GRF (2%) in the diet and diets. The profitability between 0.86 and 1 dollar per lamb that
had the lowest lymphocyte counts. When the immune system is acti­ consumed the GRF, shows that it is advantageous to use this additive,
vated, it generates large amounts of free radicals that turn compromise even when it is purchased in markets specialized in natural products,
its functions (Catoni et al., 2008). Thus, the use of GRF in the formu­ when it has added value for being in the processed form of flour. It is
lation of diets has antioxidant potential that minimizes side-effects and important to note that the farmer may have a higher income if his farm is
immunodeficiencies. Flavonoids have direct effects on a variety of anti- close to the grape processing industries, where the processing residue
inflammatory and pro-inflammatory cellular functions (Catoni et al., often has low acquisition costs or only transportation costs, as it is
2008); for example, procyanidins present in the GRF are capable of donated by many companies in southern Brazil, for example. Therefore,
stimulating immune responses (Percival, 2009). In the present study, we the residue of wine production is shown as an alternative for feeding
observed improvements in humoral responses of suckling lambs, with lambs; as well as it could have other applications like oils, alcohols,
increased levels of heavy and light chain immunoglobulins, e.g. IgA. The cosmetic extracts, among others.
increase in the humoral response in this critical phase is of great value,
because, during this period, the animals have very low concentrations of 5. Conclusion
gamma globulins and their immune systems are not yet fully responsive,
leaving them vulnerable. Hao et al. (2015), in a study with weaned The use of grape flour in the supplementation of suckling lambs’
piglets supplemented with 100 mg/kg and 150 mg/kg grape seeds, stimulation of the antioxidant and immunological systems, known to be
showed improvement in humoral and cellular immunity, with an in­ effective responses to improve animal health, consequently, was re­
crease in serum levels of IgG, IgM, C4 and IL-2, similar to our results with flected in the greater weight gain of lambs. In practice, we found that the
increased immunoglobulins. Ali Rajput et al. (2017) observed that inclusion of GRF in the concentrate raised its cost; but lambs that

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consumed this additive had greater weight gain. Thus, the inclusion of Fagliari, J.J., Santana, A.E., Lucas, F.A., Campos, E., Curi, P.R., 1998. Constituintes
sanguíneos de bovinos recém-nascidos das raças Nelore (Bos indicus) e Holandesa
GRF was economically viable when considering gross income.
(Bom taurus) e de bubalinos (Bubalis bubalus) raça Murrah. Arq. Bras. Med. Vet.
Zootec. 50, 253–262.
Acknowledgements Fascina, V.B., Sartori, J.R., Gonzales, E., Carvalho, F.B.D., Souza, I.M.G.P.D.,
Polycarpo, G.D.V., Stradiotti, A.C., Pelícia, V.C., 2012. Phytogenic additives and
organic acids in broiler chicken diets. R. Bras. Zootec. 41, 2189–2197.
We thank the research funders (CAPES/Brazil, CNPq/Brazil, Feldman, B.F., Zinkl, J.G., Jain, N.C., 2000. Veterinary Hematology. Williams & Wilkins,
FAPESC/Santa Catarina/Brazil, UDESC/Santa Catarina/Brazil). We are p. 1344.
grateful to the Federal Institute of Santa Catarina/Brazil, Concórdia Goñi, I., Brenes, A., Centeno, C., Viveros, A., Saura-Calixto, F., Rebolé, A., Arija, I.,
Estevez, R., 2007. Effect of dietary grape pomace and vitamin E on growth
campus for the proteinogram analysis. We are grateful for all the help of performance nutrient digestibility, and susceptibility to meat lipid oxidation in
the coworkers involved and Cabanha Chapecó for providing the animals chickens. Poult. Sci. 86, 508–516.
and the structure for the experiment. Guerra-Rivas, C., Vieira, C., Rubio, B., Martínez, B., Gallardo, B., Mantecón, A.R.,
Lavin, P., Tanso, T., 2016. Effects of grape pomace in growing lamb diets compared
with vitamin E and grape seed extract on meat shelf life. Meat Sci. 116, 221–229.
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org/10.1016/j.rvsc.2021.07.004. weaned piglets. Livest. Sci. 178, 237–242.
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