UNIVERSITY OF AGRICULTURE, FAISALABAD

Sub-Campus Toba Tek Singh

SYNOPSIS FOR MSc (Hons) POULTRY SCIENCE

TITLE: EFFECT OF SCAVENGING-FEEDING ON PRODUCTION,

IMMUNITY AND GUT HEALTH OF RURAL CHICKEN AT DISTRICT
TOBA TEK SINGH

Name of the Student Muhammad Umer Farooq

Registration No. 2017-ag-8051

Abstract

The objective of this research is to investigate how scavenging feeding affects the production,
immunity, and gut health of rural scavenging chicken in spring season at District Toba Tek
Singh. For this purpose 60-Golden Misri adult females (age: 9-12 month) from four different
villages (Chak no 388JB, 290 GB, 323 JB, 287 GB) of district Toba Tek Singh will be
selected on random basis. The chicken will be weighed and then slaughtered to collect crop
and gizzard contents for proximate analysis. Blood samples from all birds will be collected to
determine complete blood count, antibody titers against Newcastle disease and Infectious
bronchitis disease and oxidative stress from serum. The giblets weight will also be recorded.
Samples for estimation of lactose and non-lactose fermenting bacteria will be collected from
duodenum, jejunum and ileum. The tissue samples from duodenum, jejunum and ileum will
be collected to carryout histology. Using the statistical analysis data will be examined by
technique of SPSS 18.0.0.

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 UNIVERSITY OF AGRICULTURE, FAISALABAD
Sub-Campus Toba Tek Singh
SYNOPSIS FOR MSc (Hons) POULTRY SCIENCE

I. TITLE: EFFECT OF SCAVENGING-FEEDING ON

PRODUCTION, IMMUNITY AND GUT HEALTH OF RURAL
CHICKEN AT DISTRICT TOBA TEK SINGH

II. a) Date of Admission 07-10-2021

b) Date of Initiation (Research) After approval
c) Probable Duration (Research) 01 semester
III. PERSONNEL

a) Name of the student Muhammad Umer Farooq

b) Registration Number 2017-ag-8051

IV. SUPERVISORY COMMITTEE

i. Supervisor __________________________________________
Dr. Umar Farooq ([email protected])

ii. Member ___________________________________________

Dr. Muhammad Farooq Khalid ([email protected])

iii. Member ___________________________________________

Mr. Umair Mahmood ([email protected])

V. Introduction

It is well established that type of feed play crucial role in development of gut
microflora which intern affects development of host gut and play a significant regulatory
function in immunity and general health (Wang et al., 2021). The intestinal health of chicken
has an impact on poultry productivity and is an integral subject of scientific research. In other
words, adverse effects on the gut health could partially affect the poultry health and disturb
nutrient uptake, digestion and absorption (Zhu and Jeorger, 2003).

The influence of gut micro flora on the host's physiological, metabolic,

immunological, digestive, and nutrient absorption has been demonstrated in multiple studies

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(Aziz et al., 2018). Research on poultry greatly emphasises the study of the intestinal growth
and bacterial community of several genetic lines of chickens. Improved health of rural
chicken will be attainable through the better knowledge of the microbiology and gut function
of chickens. (Goromela et al., 2007).

The small and large intestines make up the chicken's digestive system. The duodenum
and jejunum in particular are the main sites for digestion and nutrient absorption hence
ecology of the small intestine is crucial to the digestive tract's overall health. The food broken
down in stomach is moved down to small intestine. There is a lot of chyme-commensal
bacterial interaction in the small intestine. These microorganisms help break down proteins,
fats, and carbs into smaller molecules that can be easily absorbed by the small intestine
epithelium (Sarba et al., 2019). Several enzymes produced by Lactobacillus can improve
calcium absorption, lower pH, create acidic chemicals, and inhibit the colonization and
reproduction of other acid-resistant bacteria. By secreting protease and amylase the Bacillus
subtilis encourages protein digestion and absorption and enhance nutritional digestibility. In
addition to that the creation of short-chain fatty acids, the recycling of nitrogen, and the
production of amino acids, the cecal micro flora is believed to have a significant impact on
bird nutrition. Moreover it improves production of acetic and lactic acid, and also enhances
intestinal peristalsis that reduces excessive water absorption (Gadde et al., 2017).

The rural chicken feed on natural vegetation that consists of fibrous material. The
fibrous food effect digestion hence the diversity of the microflora. In order to understand the
effect of scavenging-feeding on nutrient intake, bird performance, gut health and immunity
this study has been planned. The objectives of the research are to find out the nutrients
composition of the crop and gizzard contents, estimate intestinal diversity of gram positive
and negative bacteria, immunity, egg characteristics and performance of the rural chicken
(Choi et al., 2015).

VI. Review of literature

Chen et al. (2022) studied the impact of Lactobacillus salivarius by giving oral
treatment to chicken and its effect on weight gain, immunity and intestinal health. For 5
weeks, chickens were given 109 colony-forming units (CFUs) of wild-type (WT). When L.
salivarius groups were administered to chickens, their body weight increased considerably

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when compared to the control group. Chicken faeces samples were used to identify the
microbial taxonomy in the small intestine and cecum on growth performance, fecal micro
flora, and immune responses. According to the findings it was shown that the chicken growth
performance with Lactobacillus salivarius resulted in an increase in weight gain and feed
conversion ratio.

Wang et al. (2021) reported that the intestinal micro flora is becoming more
acknowledged as an essential part of host immunity, metabolism and health. The
development of microbial community structures affecting the health and growth performance
of chickens depends mainly on early gut colonizers. They selected White Lohmann layer to
examine the diversity of micro biota . The intestinal contents of white Lohmann layer
chickens were collected and analyzed. The results showed that the gut microbiota may get
influenced by age and type of feed and it also has impact on immune system which has
important for gut health and immunological function.

Marzooqi et al. (2020) studied the diversity of major intestinal microfloral species of
local and commercial strains chickens by using 16s RNA sequencing technique. The diversity
of major microflora species of local omani chicken and commercial chicken were observed.
This study identified that the local breed had more intestinal diversity than the commercial
strain due to differences in feed and management of two groups. The results showed that
Lactobacillus bacteria was most abundant bacteria in omani chicken while Escherichia was
more abundant in broiler chickens. When compared to local chickens, the intestinal
microflora of commercial chickens showed a higher abundance of potentially pathogenic
bacteria like Clostridium and Campylobacter.

Sarba et al. (2019) investigated the presence of E. coli in internal organs of chickens.
Samples of intestine were taken from 13 birds of 5 selected farms of Ajmer suspected for
colibacillosis and subjected to bacteriology and biochemical examination. The results showed
that the samples of organs tested in the study were found to contain E. coli, with the liver and
spleen showing the greatest rates of isolation (70% and 68.3%, respectively). The frequency
of isolation was 55% and 51.7%, respectively, in the kidney and heart. The frequency of E.
coli isolation in the kidney and heart was lower than in the liver and spleen, but still
considerable.

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 Aziz et al. (2018) studied intestinal diversity in micro flora of chicken. Samples
(Duodenum, Jejunum and Ileum) were taken from 4 healthy 1 year-old rural chickens. The
dilution were made in the test tubes were manually poured on MacConkey agar plates. These
petri plates were then kept in incubator at 37°C for 48 h. A total of 20 replicates were made
by pouring the dilutions to obtain the bacterial colonies from birds in different farms of
Faislabad Pakistan. Findings of the study showed that rural chickens had more diverse
microbiota than that of commercial chickens. Especially (Lactobacillus) and
(Bifidobacterium) are two types of beneficial bacteria, were more prevalent in the indigenous
hens. These microorganisms are important for nutritional absorption, and gut health.

Ranjitkar et al. (2016) studied the composition of bacteria in crop, gizzard, ileum, and
cecum contents in comparison to the feeding and bird of different ages. Five birds were
randomly and cervical dislocation method was used to kill the birds. All birds were weighed
and the contents of the crop, gizzard and intestine were collected. Equal quantities of
intestinal contents from the four chickens of each group were collected. A commercial DNA
extraction kit was used for extracting the DNA from the samples. The extracted DNA was
then amplified using PCR method. The findings revealed that (Firmicutes) dominated the
bacterial communities in the gizzard and crop, whereas (Bacteroidetes) and (Proteobacteria)
dominated in the ileum and cecum. From the crop to the cecum, the relative abundance of
(Bacteroidetes) and (Proteobacteria) increased while the relative abundance of (Firmicutes)
decreased.

Salim et al. (2013) investigated the effect of direct feeding of microbes (DFM) as a
substitute to antibiotics and checked its effects on growth, performance, cecal microbial
species and carcase quality of rural chicken. One gram of the composite cecal sample from
each pen was diluted with 9 mL of 0.9% saline solution and mixed on a vortex mixer. Viable
counts of bacteria in the cecal samples were then conducted by plating serial 10-fold dilutions
(in 1% peptone solution) into Lactobacilli MRS agar plates. The results of this study showed
that adding DFMs to their feed enhanced the chicken growth and feed conversion ratio.
DFMs improved the hens' immunological responses resulted in increase in antibody synthesis
and lymphocyte proliferation. The microbial composition of the hens cecal microbial
population also showed changes, with increase in number of (lactic acid) bacteria and
decrease in number of potentially harmful bacterial species like (Escherichia coli).

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 Garrido et al. (2004) analyzed the changes in the normal intestinal microbiota balance
of hens exposed to acidified wood-derived litter. The non-acidified litter was used as a
control. The intestinal contents were collected in the test tubes from the lower ileum, vitelline
diverticulum and the ceca. Until inoculation and incubation, the samples were kept on cold
container. When the portion of the lower ileum was empty or contained little material, the
contents of the next 30 cm of the lower ileum were collected. Coliforms were seen on
MacConkey agar after incubation. The results showed that the chickens fed with control feed
as compared to those fed with organic acid-supplemented feed showed fewer
Enterobacteriaceae including Escherichia coli in their faecal and cecal samples. Acidified
litter helped to create a more ideal environment in the gut for beneficial bacteria.

VII. Materials and methods

A. Selection of Study area

Total 60 desi birds (Golden Misri) adult females (age: 9-12 months) will be selected
from four different villages (Chak no 388JB, 290 GB, 323 JB, 287 GB) of District Toba Tek
Singh on random basis. The study sites will be selected depending upon the diversity of flora
and fauna.
B. Collecting crop and gizzard contents

The hens will be slaughtered to collect crop and gizzard contents. At the time of
collection the crop and gizzard contents will be evaluated for the type of food material. The
food material will be separated to identify component of grass, insects etc. The crop and
gizzard contents will be stored separately in polythene zipper bags and placed in cold
container to transfer to laboratory facility. The crop and gizzard contents were heated at 60
°C in a hot air oven while 10 gm will be immediately placed in an oven at 105 °C for 24
hours to determine moisture. The proximate analysis of the crop and gizzard contents will be
carried out. The metabolizable energy, crude ash, crude fat, crude protein tests will be
performed to calculate the nutritional value of contents of crop and gizzard (AOAC, 2000)

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 C. Complete Blood Count(CBC)

Blood samples will be collected from the slaughtered birds. The samples will be
analyzed in laboratory for complete blood cell count (CBC) using method describe by
Olaniyi et al., (2012).
D. Estimation of Lactose and non-Lactose Fermenting Bacteria
The digesta samples will be collected from duodenum, jejunum and Ileum aseptically
for determination of concentration of Lactose and non-Lactose Fermenting Bacteria. The
MaCconkey Agar Media will be used for this purpose. After preparation of petri dishes using
standard procedure the intestinal digesta material will be poured on the petri dishes, followed
by incubation at 37°C for 48 hrs. After 48hrs the colonies on petri plates were counted and
presented in CFUS (Zhang and Kim, 2010).
E. Histology of Intestine

Duodenal, jejunal, and ilium samples of intestine will be collected for histopathology,
and they will be fixed in neutral buffered formalin followed by embedding, cutting, staining
using hematoxylin-eosin and processing for histology. Standard light microscopy will be used
to assess the histological sections. Measurements of the areas and depths of crypts, as well as
the lengths of the intestinal villus, will be made quantitatively using histomorphometry
(Nasrin et al., 2012).
F. Oxidative Stress
To measure the activity of SOD, CAT, and MDA levels in the blood by using the Eliza
kit method, blood samples will be taken at the time of Slaughter. The obtained blood will be
centrifuged for 10 minutes at 1000-3000 rpm to extract the serum after being stored at 4°C
for 24 hrs. The samples will be immediately analyzed to determine the levels of SOD, CAT,
and MDA (Kumar et al., 2010).

G. Immune Response
Blood samples will be taken from the brachial vein and serum will be separated after
centrifuge by putting the collected blood in slanted position for 15 to 30 mins. Serum will be
stored at -20 oC to check the serum antibody titers against NDV through HA/HI. The serum
antibodies titer against IBV through indirect ELISA (Vui et al., 2002)

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 H. Statistical Analysis

The SPSS General Linear Model procedure will be used to analyze the data
obtained from this experiment, and Turkey's test will be used to determine the statistical
difference of means (Steel et al., 1997).

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