INTRODUCTION
Feed composition has a direct impact on the
qualitative and quantitative characteristics of the
gastrointestinal microbial community. Minerals and
vitamins are essential micronutrients that participate
in such vital processes as enzyme formation or the
synthesis and metabolism of hormones and vitamins.
They affect the nervous, cardiovascular, and endocrine
systems, as well as the activity of the endocrine glands
and the gastrointestinal tract.
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Micronutrient deficiency may trigger various
infectious and non-infectious diseases [1, 2]. A poorlybalanced
feed ration often leads to undesirable changes
in the microbiota of small ruminants. The resulting
digestive disorders cause various diseases and
eventually lead to poor livestock yield. Biofortification
fortifies animal diet with essential nutrients, thus
improving the chemical composition of meat. It renders
high-quality mutton that provides consumers with
essential microelements [3–9].
Practical microbiology gives scientific data on
the composition, role, or function of the microbial
community in the rumen content of small ruminants.
However, some of these methods have disadvantages
or limitations. For instance, researchers cannot choose
the optimal environment for microbial cultivation.
Fortunately, contemporary molecular genetic methods
make it possible to skip the stage of cultivation and
study microorganisms without the restrictions that
traditional diagnostic microbiology are prone to [10–14].
Small intestine (lat. intestinum tenue) of farm
animals absorbs nutrients from the chyme. It is in the
small intestine that the main digestion takes place,
and this is where most digestive enzymes come from.
Partially digested food leaves the stomach and enters
the duodenum, where it is processed by intestinal and
pancreatic juices and bile. The small intestine is where
digested food, toxins, poisons, medicinal substances,
etc. are absorbed into the bloodstream or lymphatic
channel [15–19].
The jejunum is somewhat structurally different from
other parts of the small intestine. Membrane digestion
is at its utmost in the upper parts of the jejunum. As a
result, its wall is thicker; it has more folds in the mucous
membrane, denser villi, and a more abundant blood
supply [20–22]. Therefore, the small intestine is a vital
system of animal body, and its flawless work is essential
for sheep farming, which proves the relevance of this
research.
Sheep farming needs new fundamental data on
the effect of biofortification on the bacterial rumen
community. Bacterial profile includes normal, opportunistic,
and pathogenic microflora, as well as
nonculturable and transit microflora that does not affect
the life of the animal. Light microscopy revealed the
morphology of the intestine and the main differences
between the samples obtained from animals fed with
Yoddar-Zn and DAFS-25.
The research objective was to assess the effect of
essential microelements on the ruminal microbiocenosis
and the microstructure of the jejunum in young rams.
STUDY OBJECTS AND METHODS
The next-generation sequencing (NGS) revealed
the digestive microflora of seven-month-old rams of
the Edilbaev breed. The experiment made it possible
to evaluate the effect of the feed additives Yoddar-Zn
(Material Specifications TU 10.91.10-252-10514645-
2019) and DAFS-25 (Material Specifications TU 10.91.
10-253-10514645-2019). The studies took place in the
laboratory of molecular genetic research of the Research
and Production Company BIOTROF (St. Petersburg,
Russia).
The feed additives were developed at the Volga
Region Research Institute for the Production and
Processing of Meat and Dairy Products. Both feed
additives contain Coretron, an enterosorbent used
in Russia in cattle diet, and cold-pressed pumpkin
cake, which served as a protein-carbohydrate
component (Tables 1 and 2) [6].
A scientific and economic experiment was necessary
to assess the effectiveness of various diets fortified with
organic microelements, i.e., mono- and di-iodotyrosines
and selenomethionine. After weaning from mothers
at the age of four months, 100 lambs of the Edilbaev
breed were divided into four groups, 25 animals in each.
The lambs were fed and fattened in the same way. On
day 105, when the animals were seven months old, they
were slaughtered by the traditional method according to
the Technical Regulations of the Customs Union on the
safety of meat and meat products TR TS 034/2013. Prior
to slaughter, all experimental animals had received no
food for 24 hours.
Yoddar-Zn is a source of bioavailable organic
iodine and zinc. It also contains iodized milk proteins
associated with amino acids and zinc compounds.
Yoddar-Zn owes its biological properties to bound iodine,
which is necessary for the biosynthesis of such thyroid
hormones as thyrotoxin and triiodothyropine. They are
important for metabolism and immune system [6].
The control group of young rams received 300 grams
of mixed fodder per head per day. The first experimental
group received daily the same mixed fodder together
Table 2 DAFS-25 feed additive
Ingredient Amount
Plant silicon
(diotomite Coretron), %
1.0
Pumpkin cake
protein-carbohydrate complex, %
99.0,
including 20.0
of pumpkin cake
Organic selenium
(selenomethionine), mg /100g
0.16
Table 1 Yoddar-Zn feed additive
Ingredient Amount
Plant silicon
(diotomite Coretron)
1.0
Iodine-containing
additive Yoddar-Zn, %
1.0
Pumpkin cake
protein-carbohydrate complex, %
98.0,
including 20.0
of pumpkin cake
Organic iodine
(mono- and di-iodotyrosines), mg/100g
3.0
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with 300 mg of Yoddar-Zn, the second experimental
group – 0.5 mg of DAFS-25, and the third experimental
group – a mix of these additives (300 and 0.5 mg).
The effect of the organic additives was studied
in vivo by comparing the microbiocenosis and microstructural
parameters of the small intestine in the
experimental and control groups of young rams.
The next generation sequencing (NGS) is currently
one of the most optimal research methods. NGS
technologies provide metagenomic studies of complex
microbial communities with a large volume of read
nucleotide sequences. This technology is much more
accurate than the Sanger sequencing in determining the
phylogenetic species of microorganisms [23].
The in vivo assessment of the impact on the intestinal
microbiocenosis took 105 days. Samples of the rumen
contents were put into sterile containers (Pan Eco,
Russia) immediately after the slaughter and tested for
microbial composition. Next step included histology of
jejunum samples. The preparations were stained with
hematoxylin and eosin to assess any possible changes in
the intestinal mucosa.
The bacterial content of the ram rumen was
analyzed by NGS method. Total DNA was isolated by
using the Genomic DNA Purification Kit (Fermentas,
Inc., Lithuania) according to the manual. The final
concentration of total DNA in the solution was
measured using a Qubit fluorimeter (Invitrogen, Inc.,
USA) with Qubit dsDNA BR Assay Kits (Invitrogen,
Inc., USA) according to the manual.
The NGS was performed on a second-generation
MiSeq sequencing platform (Illumina, Inc., USA) with
primers for the V3-V4 region of 16S rRNA; upstream
primer – 5´-TCGTCGGCAGCGTCAGATGTGTATAAG
AGACAGCCTACGGGNGGCWGCAG-3´; downstream
primer – 5´-GTCTCGTGGGCTCGGAGATGTGTATA
AGAGACAGGACTA-CHVGGGTATCTAATCC-3´ [24].
Libraries were prepared with Nextera® XT IndexKit
reagents (Illumina, Inc., USA); the PCR products were
purified with Agencourt AMPure XP (Illumina Inc.,
USA); the sequencing was performed with MiSeq®
ReagentKit v2 (500 cycle) (Illumina, Inc., USA) [25].
The obtained reads underwent overlapping, filtering
by Q30 quality, and primer trimming. The processing
involved the Illumina bioinformatics platform (Illumina,
Inc., USA). The quality control and assessment of the
taxonomic composition were carried out using the
QIIME2 v.2019.10 software (https://docs.qiime2.org)
and the Green-Genes database 13.5 (https://greengenes.
secondgenome.com).
Pieces of ram jejunum samples were removed by
preparation and fixed in 10% aqueous neutral formalin
solution at room temperature for 48 h. The selected
samples were removed from the fixing liquid and
washed under running water for 48 h. For dehydration,
the material was washed in alcohols of increasing
concentration from 50 to 96%. After that, the material
was embedded in paraffin shaped in paraffin blocks.
Sections of 5–8 μm were sliced with a sledge microtome,
deparaffinized, and stained by Ehrlich hematoxylin
and eosin dyes. Hematoxylin stains basophilic cellular
elements bright blue, while eosin alcohol acid dye stains
Y-eosinophilic cell elements pink. Basophilic structures
most often contain nucleic acids (DNA and RNA), i.e.,
nucleus, ribosomes, and RNA-containing cytoplasm
sections. Eosinophilic elements contain intra- and
extracellular proteins. Cytoplasm belongs to the main
eosinophilic environment, so its elements stain bright
red [1].
Microscopy involved a Levenhuk MED PRO 600
Fluo microscope, which is designed for transmitted light
brightfield microscopy or for a luminescent (fluorescent)
method (Magnification ×300).
The morphometric analysis of the obtained data
traced the thickness of the jejunum layers. The
experiment relied on a screw eyepiece micrometer
MOV-1-15× and an eyepiece ruler with 60 units of scale
division. The quantitative parameters of the histological
structures underwent further statistical processing.
Statistical processing of the obtained digital
data followed standard methods using the Microsoft
Excel 2010 (Microsoft Corp., USA) and the statistical
data analysis package StatPlus 2009 Professional 5.8.4
for Windows (StatSoft, Inc., USA). Student’s t-test was
applied to assess the reliability of data between the
experimental and control groups.
RESULTS AND DISCUSSION
This section describes the effect of feed additives
Yoddar-Zn and DAFS-25 in the diet of young Edilbaev
breed rams on their ruminal microbiocenosis and
jejunum microstructure.
The NGS analysis revealed the ruminal bacteria
community in the control and experimental groups.
The rumen samples contained 31 phyla of bacteria
and 1 phylum of archaea (Fig. 1). Firmicutes and
Bacteroides predominated with a total share of 86–94%.
The share of Actinobacteria, Spirochaetes, and
Candidatus Saccharibacteria was 1–6%. In the control
group, Firmicutes ranked first: their relative value
in the community was 65%, while the proportion of
Bacteroides was only 29.4%. This ratio was different
in the experimental groups. In the group that received
Yoddar-Zn, the proportion of Firmicutes and Bacteroides
was the same (42–43%). In the groups that received
DAFS-25 and DAFS-25 + Yoddar-Zn, the ratio of these
two phyla was reversed compared to the control group:
Bacteroides – 50–60%, Firmicutes – 30–35%.
At the level of orders, the community was dominated
by Bacteroidales, Erysipelotrichales, and Clostridiales.
Rams fed with DAFS-25 had a larger proportion of
Bifidobacteriales (5.8%). The control group had more
Erysipelotrichales – 28.8%.
Cellulolytic bacteria are important bacterial
community members. They break down the
fiber of plant foods and convert it to volatile fatty
acids. Cellulolytic bacteria in the rumen samples
were mainly represented by the bacterial families
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Clostridiaceae, Prevotellaceae, Flavobacteriaceae,
Eubacteriaceae, Lachnospiraceae, Ruminococcaceae,
and Thermoanaerobacteraceae, as well as by the
Bacteroidetes phylum.
The total proportion of cellulolytic bacteria was
different in all samples. The share of beneficial
cellulolytic bacteria ranged from 51.3 to 75.4%,
depending on the sample. The control group had the
smallest proportion of cellulolytic bacteria, while the
group that received DAFS-25 had the largest one. In the
groups treated with Yoddar-Zn and DAFS-25 + Yoddar-
Zn, the proportion of cellulolytic bacteria was 56.6 and
64.1%, respectively.
Lactate-utilizing bacteria are another important
group in the ruminal bacterial community. They ferment
lactic acid produced by bacteroids and lactic acid
bacteria and other organic acids into volatile fatty acids
used in metabolic processes.
The NGS analysis showed that the content of
Veillonellaceae lactate-utilizing bacteria was very large
in some samples. In the groups that received Yoddar-
Zn and DAFS-25 + Yoddar-Zn, their content was 20.6
and 12.9%, respectively, while the control group and the
experimental group fed with DAFS-25 alone, it was 9.1
and 5.1%, respectively. This indicator may demonstrate
that these bacteria are especially active in the sheep
rumen, depending on their physiological state of the
animal.
The share of bacterial pathogens was insignificant
in all samples and totaled about 0.5% in all groups.
Opportunistic Enterobacteriaceae were also represented
in a very small amount (≤ 0.1%) in all samples.
Prevotella appeared to be the dominant genus.
Its relative abundance in the experimental groups
exceeded the control (28.3, 38.9, and 33.4% vs. 22.8%).
Prevotella sp. often is the most numerous genera in
Figure 2 Rumen microbial community at the level of orders, %
0
10
20
30
40
50
60
70
80
90
100
Firmicutes Bacteroidetes Прочие
Actinobacteria Proteobacteria Spirochaetes
Euryarchaeota Acidobacteria Candidatus_Saccharibacteria
Chloroflexi Fibrobacteres Planctomycetes
Verrucomicrobia
0
10
20
30
40
50
60
70
80
90
100
Control Yoddar-Zn DAFS-25 Yoddar-Zn + DAFS -25
Firmicutes Bacteroidetes
Actinobacteria Proteobacteria
Euryarchaeota Acidobacteria
Chloroflexi Fibrobacteres
Others
Spirochaetes
Candidatus_Saccharibacteria
Planctomycetes
Verrucomicrobia
0
10
20
30
40
50
60
70
80
90
100
Control Yoddar-Zn DAFS-25 Yoddar-Zn + DAFS -25
Firmicutes Bacteroidetes
Actinobacteria Proteobacteria
Euryarchaeota Acidobacteria
Chloroflexi Fibrobacteres
Others
Spirochaetes
Candidatus_Saccharibacteria
Verrucomicrobia
0
10
20
30
40
50
60
70
80
90
100
Control Yoddar-Zn DAFS-25 Yoddar-Zn + DAFS -25
Firmicutes Bacteroidetes
Actinobacteria Proteobacteria
Euryarchaeota Acidobacteria
Chloroflexi Fibrobacteres
Others
Spirochaetes
Candidatus_Saccharibacteria
Planctomycetes
Verrucomicrobia
0
10
20
30
40
50
60
70
80
90
100
Control Yoddar-Zn DAFS-25 Yoddar-Zn + DAFS -25
Firmicutes Bacteroidetes
Actinobacteria Proteobacteria
Euryarchaeota Acidobacteria
Chloroflexi Fibrobacteres
Others
Spirochaetes
Candidatus_Saccharibacteria
Planctomycetes
Verrucomicrobia
0
10
20
30
40
50
60
70
80
90
100
Control Yoddar-Zn DAFS-25 Yoddar-Zn + DAFS -25
Firmicutes Bacteroidetes
Actinobacteria Proteobacteria
Euryarchaeota Acidobacteria
Chloroflexi Fibrobacteres
Others
Spirochaetes
Candidatus_Saccharibacteria
Planctomycetes
Verrucomicrobia
0
10
20
30
40
50
60
70
80
90
100
Control Yoddar-Zn DAFS-25 Yoddar-Zn + DAFS -25
Firmicutes Bacteroidetes
Actinobacteria Proteobacteria
Euryarchaeota Acidobacteria
Chloroflexi Fibrobacteres
Others
Spirochaetes
Candidatus_Saccharibacteria
Planctomycetes
Verrucomicrobia
0
10
20
30
40
50
60
70
80
90
100
Control Yoddar-Zn DAFS-25 Yoddar-Zn + DAFS -25
Firmicutes Bacteroidetes
Actinobacteria Proteobacteria
Euryarchaeota Acidobacteria
Chloroflexi Fibrobacteres
Others
Spirochaetes
Candidatus_Saccharibacteria
Planctomycetes
Verrucomicrobia
Figure 1 Rumen microbial community at phylum level, %
0
10
20
30
40
50
60
70
80
90
100
Bacteroidales Erysipelotrichales Selenomonadales
Coriobacteriales Bifidobacteriales Methanobacteriales
Lactobacillales Pseudomonadales Enterobacteriales
Bacillales Flavobacteriales
Clostridiales
Spirochaetales
Sphingobacteriales
Others
0
10
20
30
40
50
60
70
80
90
100
Control Yoddar-Zn DAFS-25 Yoddar-Zn + DAFS -25
Firmicutes Bacteroidetes
Actinobacteria Proteobacteria
Euryarchaeota Acidobacteria
Chloroflexi Fibrobacteres
Others
Spirochaetes
Candidatus_Saccharibacteria
Planctomycetes
Verrucomicrobia
0
10
20
30
40
50
60
70
Control Yoddar-Zn DAFS-25 Yoddar-Zn + DAFS -25
Firmicutes Bacteroidetes
Actinobacteria Proteobacteria
Euryarchaeota Acidobacteria
Chloroflexi Fibrobacteres
Others
Spirochaetes
Candidatus_Saccharibacteria
Planctomycetes
Verrucomicrobia
0
10
20
30
40
50
60
70
80
90
100
Control Yoddar-Zn DAFS-25 Yoddar-Zn + DAFS -25
Firmicutes Bacteroidetes
Actinobacteria Proteobacteria
Euryarchaeota Acidobacteria
Chloroflexi Fibrobacteres
Others
Spirochaetes
Candidatus_Saccharibacteria
Planctomycetes
Verrucomicrobia
0
10
20
30
40
50
60
70
80
90
100
Control Yoddar-Zn DAFS-25 Yoddar-Zn + DAFS -25
Firmicutes Bacteroidetes
Actinobacteria Proteobacteria
Euryarchaeota Acidobacteria
Chloroflexi Fibrobacteres
Others
Spirochaetes
Candidatus_Saccharibacteria
Planctomycetes
Verrucomicrobia
0
10
20
30
40
50
60
70
80
90
100
Bacteroidales Erysipelotrichales Selenomonadales
Coriobacteriales Bifidobacteriales Methanobacteriales
Lactobacillales Pseudomonadales Enterobacteriales
Bacillales Flavobacteriales
Clostridiales
Spirochaetales
Sphingobacteriales
Others
0
10
20
30
40
50
60
70
80
90
100
Bacteroidales Erysipelotrichales Selenomonadales
Coriobacteriales Bifidobacteriales Methanobacteriales
Lactobacillales Pseudomonadales Enterobacteriales
Bacillales Flavobacteriales
Clostridiales
Spirochaetales
Sphingobacteriales
Others
80
90
100
Bacteroidales Erysipelotrichales Selenomonadales
Coriobacteriales Bifidobacteriales Methanobacteriales
Lactobacillales Pseudomonadales Enterobacteriales
Bacillales Flavobacteriales
Clostridiales
Spirochaetales
Sphingobacteriales
Others
0
10
20
30
40
50
60
70
80
90
100
Bacteroidales Erysipelotrichales Selenomonadales
Coriobacteriales Bifidobacteriales Methanobacteriales
Lactobacillales Pseudomonadales Enterobacteriales
Bacillales Flavobacteriales
Clostridiales
Spirochaetales
Sphingobacteriales
Others
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sheep rumen. For instance, Prevotella also dominated in
a similar study by Cui et al. on the effect of selenium
feed additives on the microbial community in sheep [3].
Cui et al. also proved the significant effect of selenium
on ruminal bacterial populations and microbial
fermentation in the rumen in general.
Subdominant microorganisms in the rumen were
represented by the Dysgonomonas, Saccharofermentans,
Tangfeifania, and Treponema genera. Cui et al. showed
that the abundance of Saccharofermentans sp. was in
inverse relationship with selenium. Our research, on the
contrary, proved that the amount of Dysgonomonas sp.
and Prevotella sp. depended on the presence of selenium
in the diet.
To identify and evaluate the changes in the small
intestine wall, jejunum wall pieces were subjected to
microscopy [1].
This research of the effect of biofortification on
the microstructure of sheep jejunum yielded a more
accurate assessment of the safety of Yoddar-Zn and
DAFS-25 for small rumens [7, 8].
Light microscopy of the jejunum in all samples
revealed that the mucous membrane was well-structured,
with distinct layers. The mucous membrane of the
jejunum consisted of four layers: innermost mucosa
outermost, submucosa, muscularis (outer and inner
layers), and serosa. The columnar villi (Fig. 3) of the
mucosal epithelial layer were distinct and consisted of
a single-layer columnar epithelium lining the crypts.
The structure of the layer was dominated by goblet
cells and limbic epithelial cells, which produce mucus.
The lamina propria consisted mostly of cells and fibers
of loose fibrous connective tissue. The muscular layer
was represented by two distinct alternating layers of
myocytes: annular and longitudinal. The submucosa was
represented by loose fibrous tissue with clear contoured
blood and lymphatic vessels, as well as complex tubularalveolar
glands that produced intestinal juice.
The muscular membrane of the jejunum tissue had
two distinct layers of myocytes, which were separated
by a minimal layer of connective tissue. The structure
was clear; the cells were elongated and spindle-shaped.
On the outside, the jejunum was covered with a
serous membrane with layers of loose connective tissue
and mesothelium. The integrity of the latter was intact.
Figure 3 shows the mucous membrane of the jejunum
samples in the control group. The general histological
structure remained the same. We observed a slight
accumulation of mucus between the villi produced
by goblet cells. Epithelial cells were of an elongated
cylindrical shape. The glands of the lamina propria were
well expressed. The integrity of the layers was intact.
The jejunum samples in the experimental groups had
some histological features that differed from the control
group samples.
The jejunum of young rams that received Yoddar-Zn
had a single-layer cylindrical border epithelium on the
transverse sections of the villi (Fig. 4).
The lumen of the tubular glands looked deserted,
and the crypts were separated by a minimal layer of
Figure 3 Jejunum samples in control group. Epitheliocytes of
cylindrical villi and gland; stained with Ehrlich hematoxylin
and eosin. Magnification ×300
Figure 4 Jejunum samples in group fed with Yoddar-Zn.
Goblet cells of the villi are quite pronounced; stained
with Ehrlich hematoxylin and eosin. Magnification ×300
Figure 5 Jejunum sampled in animals fed with DAFS-25.
Epitheliocytes are cylindrical, the villi are distinct and
elongated; stained with Ehrlich hematoxylin and eosin.
Magnification ×300
Figure 6 Jejunum samples in animals fed with
DAFS-25 + Yoddar-Zn. The villi are distinct, with cylindrical
goblet cells; stained with Ehrlich hematoxylin and eosin.
Magnification ×300
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Giro T.M. et al. Foods and Raw Materials. 2022;10(2):310–317
connective tissue (Fig. 4). The muscular plate of the
mucosa was well expressed; the submucosa consisted of
connective tissue layers with elongated tubular glands.
The integrity of all membranes was intact.
Figure 5 shows the jejunum samples obtained
from animals that received DAFS-25. The cylindrical
epitheliocytes and the villi of the lamina propria were
distinct, with moderately pronounced glands with empty
lumens and numerous goblet cells. The integrity of the
membranes was intact: the muscle layers were separated
from each other by connective tissue. The serous tissue
was hardly developed.
Figure 6 shows the jejunum samples obtained from
animals that received DAFS-25+Yoddar-Zn. The organ
wall had a very obvious microstructure. The structure of
the mucous membrane of the small intestine was intact,
its constituent elements having clear contours. The
goblet cells and the single-layered columnar epithelium
were quite distinct. The villi were separated from each
other by a minimal layer of connective tissue. The
submucosa demonstrated contoured blood vessels, some
of which were filled with blood. This fact indicates a
more intensive metabolism in animals fed with DAFS-
25+Yoddar-Zn.
The myocytes of the muscular membrane are quite
clearly separated by loose fibrous connective tissue
with a minimal number of blood vessels. Muscle
cells corresponded to the state of contraction, i.e., the
cells were as if the muscle was contracted, and the
morphology of the early autolysis process.
The morphological analysis proved that the structure
of the jejunum wall in the control and experimental
groups was intact and typical. The layers had an integral
structure in all experimental groups. Samples obtained
from animals that received DAFS-25 + Yoddar-Zn
had the best developed structure.
Table 3 shows that the arithmetic mean value
of the thickness of the jejunum mucous layer was
19.40 ± 0.55 μm in the rams of the experimental groups,
which exceeded the control by 2.0 μm. The thickness
of the muscular membrane in experimental groups
also exceeded this indicator in the control group by an
average of 0.8–2.0 μm. The experimental rams also had
a slightly thicker serous layer.
The minimal thickening of the jejunum membranes
was minimal in the experimental groups,
the lowest observed in the animals that received
DAFS-25 + Yoddar-Zn. This fact may be an indirect
indicator of a more active digestion, a better digestibility,
and a greater absorption of feeds and nutrients into
the bloodstream.
CONCLUSION
Biofortification of young rams’ diet with essential
microelements had a positive effect on the quality and
quantity of the gastrointestinal microbial community,
which means a better digestion process and a greater
animal yield.
In the rumen samples, cellulosolytic bacteria,
which break down the fiber of plant foods into volatile
fatty acids, were mainly represented by Clostridiaceae,
Prevotellaceae, Flavobacteriaceae, Eubacteriaceae,
Lachnospiraceae, Ruminococcaceae, and Thermoanaerobacteraceae
families, as well as by the Bacteroidetes
phylum. The content of lactate-utilizing bacteria in the
rumen samples reached 40%, which may indicate a high
degree of activity of these bacteria, depending on their
physiological state of the animal.
The content of bacilli in the rumen samples
was ≤ 1%. The total proportion of pathogenic species
ranged from 0.2 to 6.3%. The experiment revealed ≥ 50
types of pathogenic microorganisms, which were most
abundant in the group fed with Yoddar-Zn + DAFS-25.
The pathogenic microorganisms belonged to erysipelothrix,
fusobacterial, and streptococci. The
content of porphyromonas reached 0.68% of total
microorganisms, while the proportion of Treponema in
the samples ranged from 0.6 to 1%. Lactobacilli were
represented mainly by Lactobacilliales (0.06–0.45%).
This fact may indicate a high degree of activity of
these bacteria in the sheep rumen, depending on their
physiological state of the animal.
The balance of the microflora in the sheep rumen
samples was good, and the amount of beneficial
microflora was enough to inhibit the pathogenic and
opportunistic bacteria.
The light microscopy revealed no adverse effect
of the feed additives DAFS-25 and Yoddar Zn on the
microstructural parameters of sheep jejunum. Therefore,
they can be recommended for fattening purposes in
industrial conditions.
The additives had no negative impact on the rumen
microbiocenosis and the jejunum microstructure.
The structure of the jejunum corresponded to the
morphological characteristics for this type and age of
Table 3 Wall thickness of the jejunum of seven-month-old rams fed with various feed additives
Research subject Wall thickness, μm
Mucus membrane Muscular membrane Serous membrane
Control 17.40 ± 1.07 8.30 ± 0.79 0.80 ± 0.51
Yoddar-Zn 19.10 ± 0.52 9.50 ± 0.81 0.90 ± 0.22
DAFS-25 19.40 ± 0.97 9.60 ± 0.79 0.90 ± 0.55
DAFS-25 + Yoddar-Zn 19.80 ± 0.97* 10.30 ± 0.71* 1.00 ± 0.44
*P ≤ 0.005
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farm animal in all the groups. A clearer micropicture of
the jejunum wall was revealed in the experimental group
of rams fed with DAFS-25 + Yoddar Zn.
The complex application of additives DAFS-25 and
Yoddar Zn helped optimize the processes of digestion,
absorption, and assimilation of feed nutrients, which
was partly confirmed by the minimal thickening of the
jejunum membranes.
Further research is needed to study the effect of
these additives on other important systems of animal
organism, e.g., digestive (liver), excretory (kidneys),
nervous (cortex and base of brain), and immune (spleen
and mesenteric lymph nodes) systems.
CONTRIBUTION
Authors are equally relevant to the writing of the
manuscript, and equally responsible for plagiarism.
CONFLICT OF INTEREST
The authors declare no conflict of interest.

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