The Effect of Supplementation Probiotic on Morphometric Rat Intestine (Rattus

Novergicus)

Lovita Adriani1, Ronny Lesmana2,3

*Corresponding Author : Lovita Adriani

Tel : +62-818-0200-0111, Email : lovita_yoghurt@yahoo.co.id

Ronny Lesmana
Tel : +62-877-3667-1707,Email : Ronny@unpad.ac.id

1
Faculty of Animal Husbandry, Universitas Padjadjaran. Jalan Raya Bandung-Sumedang km 21,
West Java, Indonesia, 45363.
2
Division of Physiology Molecular, Departement of Anatomy, Physiology and Biology Cell,
Faculty of Medicine, Universitas Padjadjaran. Jalan Raya Bandung-Sumedang km 21, West Java,
Indonesia, 45363.
3
Central Laboratory, Universitas Padjadjaran. Jalan Raya Bandung-Sumedang km 21,
Jatinangor, West Java, Indonesia, 45363.
Title of The Manusricpt : The Effect of Supplementation Probiotic on Intestine Morphometrics at Rats
(Rattus Novergicus).

ABSTRACT

Introduction : Probiotics are a microorganism that can modify the microflora in the body, it can replace the
pathogenic microbe with non-pathogenic microbe for the body in the human’s intestine and improve the ecosystem
of intestinal flora. however there is limited information about role of probiotics in altering morphometrics of the
intestine.
Objective : The aim from this study is to know the effect of supplementation probiotic at fermented cow’s milk
and fermented soy milk on intestine morphometric in female rats (Rattus norvergicus) like height, width-apical
and width-basal.
Method : This research used female whistar rat with total 45 and they were housed in cage with the size 50 cm
x 47 cm x 45 cm. The rats were acclimatized for two weeks and given the normal peller and water ad libitum,
prior to dietary manipulation. The research method used a Completely Randomized Design (CRD) with 5
treatmens and 3 replications and every unit was occupied by 3 rats. The treatment consisted of T0 only basal
feed (control), T1 basa feed with 100% fermented cow milk, T2 basal feed with fermented cow milk 50% +
fermented soy milk 50%, T3 basal feed with fermented cow milk 75% + fermented soy milk 25%, T4 basal feed
with fermented cow milk 25% + fermented soy milk 75%. The sample taken and tested is jejenum and ileum.
Then data of height, width-apical, and width- basal of jejenum and ileum was analyzed using variance analysis
and if there were significant differences, further testing would use Tuckey’s test .
Result and Discussion: Based on the research that have been conducte, it knowed if height, width-apical and
width-basal of ileum at all treatment happen decrease than control, Results reflect that treatments doesn’t affect
significant improvement to the morphometric ileum. Interestingly, in the jejunum, we observed that
probiotic supplementation alters the jejenum morphometric . It is confirmend by an increase of height,
width-apical and width-basal of jejunum. Best formulation of the administration is fermented cow milk 25%
and fermented soy milk 75% gives the best impact on morphometric jejenum.
Conclusion : Taken together, supplementation of probiotic at fermented cows’s milk and fermented soy milk
gives the positive impact on morphometric jejenum and The treatment is given the best result on morphometric
jejunum is T4 with combination fermented cow milk 25% and fermented soy milk 75%.
Keyword :ileum, jejenum, probiotics, morphometric, rat
INTRODUCTION

Probiotics are a microorganism that can modify the microflora in the body, it can replace the pathogenic
microbe with non-pathogenic microbe for the body in the human’s intestine [1]. Probiotics are microorganism
consumed either in the form cell or fermented product gives the advantage for improving indigenous microflora
character [2]. Besides on that probiotics are a microorganism have the ability to improve the ecosystem of
intestinal flora and to maintaint the host. Probiotics have to reach colon in order to decrease the number of
pathogenic bacterias in the colon also shows its physiology role in maintaining the balance microflora at the
gastrointestinal tract [3].
Probiotics have many functions for the body, especially for improving the ecology bacteria at intestine
,preventing intestinal cancer and increasing the digestibility of nutrients [3-4]. Besides that probiotics have the
ability to increase the activity of digestive enzymes in the intestine so that the absorption of nutrients will become
maximum. It can increase the area for absorption nutrient at Intestine with influence the intestinal anatomy like
density and size of small intestine villi [5]. In this research, milk either cow’s milk or soy is fermented by
probiotic bacteria. Probiotics are fermented with several materials like soy milk, cow’s milk then it will be
searched its the best combination. Soy is a group of oligosaccharides contains sucrose and raffinose it is difficult
to digest and to absorb by the intestine. However, it helps a growth substrate for non-pathogenic bacteria in the
intestine [6]. Lactic acid bacteria in fermented soy milk have the ability in increasing isoflavone digestibility [7].
Isoflavones are secondary metabolite compounds that are usually synthesized by plants. In the soybean, there
are 2 to 4 mg/g isoflavone contents. Kind of isoflavones are genistein, daidzein and glisten [8]. Antioxidants
have been known to have the ability in improving intestinal villi because it gives an impact on increasing
absorption area. Fermentation of lactic acid bacteria also result in hydrolysis of soy protein hydrolysis of soy
protein into short peptides like Phe-Asp-His-Val-Glu and PheAsn- His-Leu-Asp-His, which are able to decrease
DPPH free radicals, thus acting as antioxidants [8].
Some study of intestine morphometric measurement have been done by Lovita [20] regarding the effect
of supplementation probiotic on morphometric chicken intestine. The benefit of supplementation probiotics on
gut performance and morphology that by dietary means. It is possible to give a positive impact for developing
of the gut and for providing a competitive benefit in favor of beneficial bacteria. It can alter not only gut dynamics
but also many physiologic processes [7-9]. In the present study, evaluation of fermented soy and cow
utilizing probiotic on small intestine morphometry of rats was done to evaluate the effect of fermented
soy and cow as probiotic on small intestine morphometry of rats.

MATERIALS AND METHODS

Animal : Forthy- Five Female Rat Wistar with age 10-12 weeks old, 200-250 gram body weigh with a total of
45. Female Wistar rats from Biofarma Laboratory, Bandung, Indonesia, age of 10-12 weeks with body weight
200-250 gram were used in this study. Rats were housed in a cage with the size 50cm x 47cm x 45cm (3-4
rats/cage). The rats were acclimatized for two weeks given the normal pellet and water ad libitum, prior to dietary
manipulation.
All experiment procedures were approved by Health Research Ethic Committee Faculty of Medicine Universitas
Padjadjaran No. 875/UN6.KEP/EC/2018. There were 5 treatments and 3 replications then every unit was
occupied by 3 rats. T0 only basal feed (control), T1 basal feed with 100% fermented cow milk, T2 basal feed
with fermented cow milk 50% + fermented soy milk 50%, T3 basal feed with fermented cow milk 75% +
fermented soy milk 25%, T4 basal feed with fermented cow milk 25% + fermented soy milk 75%.
Processing Probiotic : Fermentation of cow milk and soy milk made in the laboratorium and The assessment
was conducted at the Faculty of Animal Husbandry, University of Padjadjaran. Probiotics used in Fermentation
of cow’s milk and soybean milk Streptococcus thermophillus, Lactobacillus bulgaricus, Lactobacillus
acidophilus, and Bifidobacterium bifidum. The result of fermentation used as probiotic and given to the rat. The
dose of fermented milk was 1.25% with ratio (1:1) based on the research had been conducted by Lovita [19].
Preparation Process : Preparation process consists of several phase, and can be seen in the following these
image

Morphometry Measurement : Jejunum samples and Ileum samples collected from the small intestine of rats
in each treatment group. Then these samples washed with physiological NaCl, then saved in Bouin for a day. The
samples dehydrated using alcohol with different concentrations 70%,80%, 90%, and 100%. After that, the
samples cleaned using xylol and alcohol 100% 3 times for 5 min each in 1:3. 1:1, 3:1 ratios. The samples
infiltrated in an oven at 56oC using xylol and paraffin solutions in the ratio of 3:1. 1:1, 0:1 in sequences for 15
min. Preparation sample used paraffin embedding procedures with sectioning at 6-8 μm thikness and kept for 1
day. Then the samples added Haematoxylin-Eosin and soaked in haematoxylin ehrlich for 2-10 min. HNO3 0.5%
in 70% ethanol used to reduce the excess color. Alcohol and xylol were used to wash the samples. n the end, the
sample was covered with a mounting agent and looked under a microscope.
Data Analyze : The data analysis was done to know the effect of each treatment. Data analyze used one-
way analyzed of variance (ANOVA). Then the results had been obtained presenting as mean ± standard
error. The significant result will be investigated using Tuckey’s test by considering differences significant
at p < 0.05.
RESULT AND DISCUSSION
Based on the research that has already been carried out, it is obtained data regarding height, basal
wide, and apical wide of jejunum villi and ileum villi. The data showed in Table 1 and Table 2.

Table 1. The Effect of Supplementation Probiotic on Morphometrics Ileum

T0 T1 T2 T3 T4
H-ILEUM 429.77±26.99 391±45.09 355.82±24.04 450.99±24.04 364.6±40.12
WA-ILEUM 145±23.00 125.10 ±15.52 118.4±18.76 109.37±17.23 104.68±11.63
WB-ILEUM 159.28±24.87 154.13±27.17 137.47±22.230 142.02±62.41 121.11±15.72
Note : T0 basal diet (control), T1 basa feed with 100% fermented cow milk, T2 basal feed with
fermented cow milk 50% + fermented soy milk 50%,T3 basal feed with fermented cow milk 75%
+ fermented soy milk 25%, T4 basal feed with fermented cow milk 25% + fermented soy milk
75%.H-ILEUM (Height Ileum), WA-ILEUM (Width Apical Ileum). WB-ILEUM (Width Basal
Ileum)

Based on (Table 1) can be explained if supplementation probiotic on morphometric ileum doesn’t give
the positive impact. Based on Table 1 can be explained if supplementation probiotic on morphometric ileum
doesn’t give a positive impact. It is proved at each treatment happening to decline like the height of ileum the
width-apical of ileum, and the width-basal of the ileum. The height of ileum at T1 occurs 8.8 % decreasing
compared with T0 (control), likewise with T2 and T4 each occurs decreasing by 17% and 15%. The width-apical
of ileum at T1 occurs 13% decreasing likewise T2 18.34 %, T3 24.5 %, and T4 27,9%. The width-basal of ileum
at T1occurs decreasing 3.1% compared with T0 (control) likewise with T2 13.69 %, T3 10,8%, and T4 23.98%.
Those all show if ileum doesn’t give an improvement. Figure 1 shows the appearance of the jejunum structure in
each treatment.
 T0 T1 T2 T3 T4

The figures 1 showe visualization of ileum villi in each treatment and replicates T0 = Treatment with
basal feed, T1= basa feed with 100% fermented cow milk, T2 = basal feed with fermented cow milk
50% + fermented soy milk 50%, T3 = basal feed with fermented cow milk 75% + fermented soy
milk 25%, T4 = basal feed with fermented cow milk 25% + fermented soy milk 75%.

Tabel 2. The Effect of Supplementation Probiotic on Morphometrics Jejenum

T0 T1 T2 T3 T4
H-
JEJUNUM 362.82±22.51 412.6±25.94 463±18.83 364.29 ± 16.45 443.54±45.04
WA-
JEJUNUM 96.31 ±8.52 103.49±12.07 108.16±11.55 114.61±11.55 126.20±37.45
WB-
JEJUNUM 109.15±13.34 116.12±11.63 123.10±14.74 121.09±12.33 154.43±28.62
Note : T0 only basal diet (control), T1 basa feed with 100% fermented cow milk, T2 basal feed with
fermented cow milk 50% + fermented soy milk 50%,T3 basal feed with fermented cow milk 75%
+ fermented soy milk 25%, T4 basal feed with fermented cow milk 25% + fermented soy milk
75%. H-ILEUM (Height Jejunum), WA-ILEUM (Width Apical Jejunum ). WB-ILEUM (Width
Basal Jejunum )

Based on (Table 2) can be explained if supplementation probiotic gives a positive in improving

morphometrics jejunum. It is proved all treatment happening escalation compared with T0 (control). The
height of jejunum at T1 happens 13 % increase compared with T0 (control) likewise T2 27%, T3 0.45%
and T4 22.25 % its all happen to increase. The Widht-apical of jejunum at T1 happens a 7.2% increase
compared with T0 (control) likewise T2 12.30 %, T3 19 % and T4 31 %. The Widht-Basal of jejunum at
T1 happens a 6.4% increase compared with T0 (control) likewise T2 12.7%, T3 10.9 % and T4 41.5%.
Furthermore, based on statistic analyze supplementation probiotic give significant increasing (P<0.05) on
the height of jejunum, the width-apical of jejunum and the width-basal of jejunum. Figure 2 shows the
appearance of the jejunum structure in each treatment.
 T0 T1 T2 T3 T4

The figures 2 shows visualization of jejunum villi in each treatment and replicates T0 = Treatment
with basal feed, T1= basa feed with 100% fermented cow milk, T2 = basal feed with fermented cow
milk 50% + fermented soy milk 50%, T3 = basal feed with fermented cow milk 75% + fermented
soy milk 25%, T4 = basal feed with fermented cow milk 25% + fermented soy milk 75%.

Probiotics have a function for changing gene expression in intestinal cells [10], that influence of
intestine morphometric. Based on this research is known if improving intestine morphology by
supplementation probiotic occurs at jejunum part where the height of villus, width-basal of villi and width-
apical of villi at all treatments occurs escalation compared with T0 (control). Jejunum has a function to
absorb nutrient in the form of minerals, electrolytes, carbohydrates, protein, and fats that are consumed in
the form of food [11].
The supplementation of probiotics can increase the height of villi according to [12] reported that the
height of villi at jejunum increase with administration of the multi-microbe probiotic product. The rat is
supplemented orally high, low, and moderate dose of Bifidobacterium sp after 2 and 4 weeks of application. It
is known to occur escalation of the height of villi [13]. Shirkey at al [14] declare if villi are the longest part in
the jejunum of pigs which supplemented by Lactobacillus fermentum. The increasing of the height of villi leads
to increase surface area, at the same time also the digestion and the absorption of disaccharides and dipeptides
will promote. It is indicated that longer villi have a correlation with activation of cell mitosis [15].
The supplementation probiotics are able to increase the width-apical of jejunum and the width-basal of
jejunum, because of the increasing of the short-chain fatty acids and the decreasing the production of ammonium
[16]. Short-chain fatty acids have a function to stimulate the propagation of intestinal epithelial cells. The
decreasing of ammonium production maintains the pH condition of the intestine in the acid. The increasing of
the width-apical and the width-basal of jejunum is associated with a larger area of the villi surface for the
absorption into the bloodstream [17]. Furthermore, besides it can increase the width-basal of jejunum and the
width-apical of jejunum. The supplementation of Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium
bifidum, and Enterococcus faecium can increase a surface area of villi [18]. The increasing of surface area will
promote nutrient absorption. The increase absorptive area is useful because digested nutrients pass into the villi
through diffusion, so the effectiveness of diffusion increases.
CONCLUSION
The administration of probiotics in fermented cow milk and soy milk gives the significant effect to
height, width-apical and width-basal on villi of jejunum and ileum. Even though,in the ileum doesn’t give a
positive impact, however, the administration of probiotics give a positive impact on morphometric jejunum. The
best treatment give the maximum effect is T4 with combination 25% fermented cow and 75% fermented soy
milk.
ACKNOWELDGEMENTS
The authors would like to thank a group of students who helped in the study. The authors also wish to
thank Academic Leadership Grant (ALG) Project, University Padjadjaran, Bandung for funding the
experiment.
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