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Effects of Neem Tree Leaf Extract

(Azadirachta I Ndica A. Juss) as Defaunating
Agent of Sheep (Ovis Aries L.) Fed Napier Grass
(Pennisetum Purpureum Schumach)
Arnold Capin Valleser, MSc, Dolly Ann Dela Cruz Caraca, MAEd,
Bachelor of Science is Agriculture Department Bachelor of Science is Agriculture Department
Philippine Society of Animal Science Visayas Chapter, Carcar City College
Carcar City College Carcar City Cebu, Philippines
Carcar City Cebu, Philippines

Abstract:- The effectiveness of neem tree leaf extract The presence of billions of bacteria, and thousands of
(NTLE) as defaunating agent was compared with sodium protozoa, and fungi per ml rumen fluid as agents of
lauryl sulphate (SLS). Ten (10) sheep was used in the digestion makes it possible f or ruminants to consume and
comparison of defaunating agents, the neem tree leaf utilize highly fibrous feeds (Muchira, 2020; Eugene et al.,
extract was found to cause fluctuations in ruminal pH, 2004). However, it is very important that the balance of
similar to that of sodium lauryl sulphate, effectively microbial population in the rumen is maintained at a proper
reducing protozoal counts (from 296,000 cells/ml to 1,000 ratio to ensure efficient breakdown of consumed roughages.
cells/ml) and increasing bacterial population (from In instances when supply of amino acids (from protein) in
10,780 colony forming units/ml to 950,000 colony the rumen is limited, the protozoan, which require amino
forming units /ml) in the rumen of sheep. It is therefore acid-N f or growth and normal function (Schwab, and
strongly advised to use neem tree extract as defaunating Broderick 2017; Jouany, 1996), will engulf bacteria f or
agents for sheep fed Napier grass to defaunated sheep in amino acid supply. As a consequence, bacterial population
order to maximize food utilization and animal declines, affecting the overall microbial Fermentation in the
performance. rumen by reducing the rate at which bacteria colonize
ingested food particles (Owens and Basalan, 2016; Hungate,
Keywords:- Defaunation, Neem Tree, Rumen Protozoal, 1966; Coleman, 1975).
Rumen Bacterial.
Manipulation of the microbial ecosystem by
I. INTRODUCTION defaunation (process of making the rumen free of protozoa)
increases bacterial population and the consequent capture of
The inventory data on goat and sheep in the country dietary nitrogen in the rumen, thereby improving the
from the year 1999 to 2002 showed that sheep population in efficiency of feed utilization. The removal of protozoa in the
the country was less than 1% of the total sheep population in rumen results not just in the increase of the amount of
Asia which became a concern of the Philippine Council for protein but also energy due to the elimination of nutritional
Agriculture, Forestry and Natural Resources Research and competition between bacteria and protozoa in utilizing
Development (PCARRD). Hence, innovation that can assist carbohydrate. Defaunation has been carried out using
the sheep industry became one of the Council’s research detergents, and the commonly used is sodium lauryl
priority areas, to solve nutritional problems leading to sulphate (Santra et al., 2007; Santra and Karim, 2000). The
significant growth performance and production of the economic benefits of defaunation using these chemicals had
animal. been proven scientifically. Due to the high cost, pollution
through accumulation of sodium ions and the dangers to
Like other ruminants, nutritional diet is the primary people and animals due to its corrosive nature of using this
factor to be considered in sheep raising. As pointed out by chemical, there is then a need f or alternative defaunating
Lee (2008), nutrition of the animal is related to its growth agent.
performance, and innovation such us rumen manipulation or
bypass protein supplementation are promising strategies to
provide efficient nutrition of the animal (Leng, 1991).

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There is wide array of plants that can be used as C. Preparation and Feeding of Experimental Animals
defaunating agents (Monforte-Briceno et al., 2005; Serohi et Prior to the conduct of the study, the animals were
al., 2009). Plant extracts containing high levels of saponins, dewormed and given vitamin ADE through injection. The
flavonoids, and tannins had been found to reduce population sheep in this experiment were fed ad libitum using napier
of protozoa, decrease methane production and induce grass (Pennisetum purpureum Schumach). The feeding of
extensive stimulation of microbial metabolism resulting in experimental animals was done for 14 days. Experimental
enhanced flow of microbial protein from the rumen, animals were fed at 8:00 A.M. and at 4:00 P.M daily.
increased efficiency of feed utilization, and improved Drinking water was made available at all times, and the
nutrition of animal (Teferedegne, 2000; Patra et al., 2006; animal shed was cleaned daily.
Tekeli et al., 2007). However, effectiveness of plant extracts
as defaunating agent would depend upon the source, and the D. Dietary Treatments and Experimental Set-up
type and level of secondary metabolites present such as Ten (10) sheep were weighed and randomly distributed
saponins, flavonoids and tannins (Patra et al., 2006). Neem to the two treatments and confined individually in open-top
(Azadirachta indica A. Juss) plant extract has high metabolism cages (Betsil, 1985). The treatments consisted
concentration of secondary metabolites and, therefore, can of t wo (2) types of defaunating agents as follows:
be a good alternative (Chauhan et al., 2004). Neem is a fast-  Neem tree leaf extract
growing tree, belonging to family Meliacea, and has a  Sodium lauryl sulpahte
strong root system. It is tagged as “a tree f or solving the
global problems” because of its potential in solving human E. Administration of Defaunating Agents
health, agronomical and environmental problems. It is used In this study, the method of Ankrah et al., (1990b) was
as defaunating agent in buffalo as reported by Chaunhan et employed for defaunation, with some modifications.
al., (2004).
Day 1-3 Feed given was reduced to half of the ad
The study's goals are to assess the effectiveness of libitum intake, and at the same time the defaunating agent
sodium lauryl sulphate and neem tree extract as defaunating was administered every day in 3 consecutive days by
agents for sheep fed Napier grass. drenching.

II. MATERIALS AND METHODS Two (2) hours post-dosing in day 3, the animal was
drenched with 200 ml of a substrate solution of flour and
A. Preparation and Dosing of Neem Tree Leaf Extract sugar to sustain the bacterial population in the rumen when
Fresh l eaves of neem tree were collected excluding the the animals were fed half full.
t wigs. The collected l eaves were weighed and washed with
tap water to remove foreign particles. Juice of the neem tree Day 4 Start of feeding the experimental animals ad
l eaves was extracted with tap water as the extracting agent ibitum ration until the end of the experiment.
by the use of a blender. About 1 kilo of neem tree leaf and 1
liter of tap water or 1:1 ratio was used to extract 1 liter of Day 6 The second dose of defaunating agent was
concentrated neem tree juice (Fig. 2). The extracted juice administered to the animal, while being fed ad libitum.
was strained with cheesecloth and administered fresh to the
animal, or stored for not more than 24 hours before Day 14 The defaunating agent was administered at
administration. Only 200 ml of the juice extract was 50% of the dose without feed restriction.
drenched to each animal per administration. The first dose
was done for three (3) consecutive days at 7:00 A.M., the F. Rumen Fluid Collection
next dose was on the 6th day, and the third dose was on the The 10 ml sample of rumen fluid was collected through
14t h day after the first dose. stomach tubing using a human stomach tube (fringe 18 x
20 125 cm), as shown in Figure 4. The collection was done one
hour after every dose of the defaunating agents (Jouany et
B. Preparation and Dosing of Sodium Lauryl Sulphate al., 1981). The initial collection was done night before the
Solution administration of the first dose of the defaunating agents,
Sodium laury lsulphate powder (Fig. 3) was dissolved and the second was done on third day which was done one
in distilled water before being administered to the animal. hour after administering the last dose. The third collection
The rate of administration was at 0.08 gram of powder was done at day seven, while the last collection was done at
dissolved in 10 ml of water per kg body weight (Santra and day fourteen (Rowe et al., 1985). This is the one
Karim, 2002) by drenching. The first dose was done for recommended since defaunating agents can affect the rumen
three (3) consecutive days at 7:00 A.M., followed by a ecosystem, thus, there was sampling on the 3r d day during
second dose on the 6t h day, and a third dose on the 14t h “adaptation period” and on the 9th day by which the rumen
day. ecosystem was believed to have stabilized. All rumen fluid
samples were added with 40 ml formal saline (9g sodium
chloride/l) solution upon collection (Rowe et al., 1985).

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G. Data Gathered III. RESULTS AND DISCUSSION

 Ruminal pH Determination The study used a botanical leaf extract of neem tree as
The pH of the rumen fluid was determined using pen defaunating agent in comparison to sodium lauryl sulphate
type pH meter. Determination of pH was done before and (SLS). The use of neem tree leaf extract (NTLE) is assumed
one hour after administration of defaunating agents to the to be ecologically friendly compared t o t he commercially
animal during the first dose at day 1-3, during the second available SLS.
dose at day 6, t o t he last dosage at day 14.
A. Ruminal pH
 Protozoal Count As presented in Table 1 and Figure 1, the pH rapidly
The collected rumen fluid sample was immediately increased as defaunating agents were administered, then
placed into the test tube and was serially diluted to 1:10 continuously declined in the second until the third dose. The
dilutions (Rowe et. al., 1985) within one hour after result is expected f or SLS being an alkali, but NTLE
collection. Protozoa was counted through the bright line exhibited similar effects as that of the commercial
standard 1/10 mm deep counting chamber method using a defaunating agent. It was also observed that rumen pH
microscope and hand tally counter. The protozoal counts rapidly declined after the first dose of the defaunating agent
were then expressed as: Cell counts (cc/ml) = Number of in three consecutive days.
protozoal cells x 2500 x dilution rate
Detergents had been used effectively as defaunating
 Bacterial Count agents (Santra et al., 1994). SLS had been primarily used as
The rumen fluid was immediately placed into the test one of the ingredients in making commercial soap and tooth
tube with rubberized cover and was brought to the paste, therefore, it has the capacity to kill protozoa in the
laboratory. The rumen fluid samples were serially diluted rumen. In the experiment of Benge (1986), NTLE and oil
from 1:10 to 1:1,000,000 dilutions (Rowe et. al., 1985) and was utilized in making soap as well. NTLE appeared to be
poured into the prepared medium containing using MRS comparable to SLS in effecting sudden increases and
agar. The medium was incubated for 18 to 24 hours using consequent drop in rumen pH, therefore it has a great
anaerobic chamber (GasPak) system. The bacterial colonies potential in killing protozoa in the rumen since protozoa are
were counted by the use of a Suntex Colony counter, and very susceptible to fluctuations in rumen pH. Within the
expressed as “colony forming unit’s /ml fluids as follows: normal limits of rumen pH from 6.2 to 6.8, population of
protozoa in the rumen is high (Shriver et. al., 1986).
Colony Forming Unit (cfu/ml) = Number of bacterial However, the sudden rise in rumen pH after administration
colonies x dilution rate of defaunating agents eliminate protozoa, allowing bacteria
to flourish rapidly with a consequent reduction in rumen pH
 Analysis of Data (Santra et al., 1995).
All data were analyzed by unpaired t -Test using
Statistical Package for the Social Science (SPSS) version 12
computer package.

Table 1: The Fluctuations in Rumen Fluid pH of Sheep Fed Napier Grass and Defaunated using NTLE and SLS.
1st dose day 2nd dose day 3rd dose day
1-3 6 14
Treatment
Before After Before After Before After
Neem Tree Leaf Extract (A) 6.8 7.1 6.3 7.0 6.0 6.9
Sodium Lauryl Sulphate (B) 6.8 7.1 6.4 7.0 6.9 7.0
p-value 0.092 0.08 0.407 0.303

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Fig 1: The Rumen pH Values Sheep Fed Napier Grass as Offered by the Type of Defaunating Agent

B. Rumen Protozoal Count

As presented in Table 2 and Figure 2, the influence of defaunating agents on the rumen pH apparently affected the protozoal
population in the rumen. The protozoal count rapidly declined after the first dose of NTLE and SLS in three consecutive days, and
decline continuously until the 3rd dose in day 14. The reduction in protozoal count by NTLE was comparable to that of SLS,
showing no significant difference (P<.01) between the two defaunating agents.

However, the defaunation treatment, did not completely eliminate all protozoa in the rumen, and the possibility of a shift in
the composition towards the more acid-resistant protozoa such as the Isotrichia and Entodinium species (Nagaraja and Towne,
1990) is the likely explanation. The mode of action on the NTLE defaunating agent on the protozoal population is not known, but
the secondary metabolites present in NTLE (Biu et al., 2009) may have effectively affected the attachment of protozoa (Sarwar et.
al., 1999), reducing its function to utilize fiber particles (Varga and Kolver, 1997; Santra and Karim, 2002), which eventually
facilitated the killing action of the extract.

Table 2: The Ruminal Content Protozoal Count (cell Count /ml) in Sheep Fed Napier Grass Defaunated using NTLE and SLS
Initial 1st dose day 2nd dose day 3rd dose day
Treatment 1-3 6 14
Neem Tree Leaf Extract 2960,00 23,000 11,000 1,000

Sodium Lauryl Sulphate 330,500 21,000 11,250 625

p-value 0.171 0.495 1.000 0.422
Cc/ml – Cell count x 2500 x 10 (dilution)

Fig 2: The Ruminal Protozoal Count Sheep Fed Napier Grass as Offered by the Type of Defaunating Agent.

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Fig 3: Photomicrograph of Species of Ciliated Entodiniomorphid protozoa Observed in the Rumen Fluid of Sheep in this
Experiment.

C. Rumen Bacterial Count

The key benefit of defaunation is the consequent increase in bacterial population in the rumen which are considered as key
microbes of the ruminant animal for the digestion of feedstuffs in the ruminant. They are much affected when protein deficient
diet is fed to the ruminant animal and the protozoa in the rumen starts to feed on bacteria, reducing its population and, thus, its
digestive action.

As shown in Table 3 and Figure 3, the bacterial population in the rumen fluid increased, with more rapid increase after the
2nd and 3rd dose of NTLE and SLS. Comparing this with protozoal count in Table 2, this is also the stage when protozoal count
was at its lowest, indicating the inverse relationship of these organisms in the rumen. There was no significant difference between
NTLE and SLS in effecting increases in bacterial population, indicating the effectivity of NTLE as a defaunating agent and its
comparability to SLS. The moment protozoa lost their attachment to the fiber particles, bacteria must have penetrated aggressively
into starch particles and utilized dietary nitrogen (Miresan et al., 2006), leading to the multiplication of the number of bacteria,
especially amylolytic bacteria (Kurihara et al., 1978).

Table 3: The Rumen Bacterial Colony Forming Unit (cfu / ml) in Sheep Defaunated NTLE and SLS and Fed Napier Grass.
Initial 1st dose day 2nd dose day 3rd dose day
Treatment 1-3 6 14
Neem Tree Leaf Extract 10,780 12,480 702,000 950,000

Sodium Lauryl Sulphate 10,840 13,140 680,00 947,500

p-value 0.853 0.609 0.010 0.132

The figures appeared to be underestimation because these are expressed as colony forming units / ml rumen fluid (Figure 4)
and not individual bacterial count. Beside, a large number of bacteria are non-culturable (Kamra, 2005) and may also depend on
the type of diet. For example, the increased supply of rumen degradable protein, facilitate the growth of bacteria in the rumen
allowing increases in its number, and lead to increased microbial protein synthesis.

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Volume 9, Issue 8, August – 2024 International Journal of Innovative Science and Research Technology
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Fig 3: The Rumen Bacterial Count (cfu / ml) in Sheep Fed Napier Grass and Defaunted using NTLS or SLS.

Fig 4: Bacterial Colony Forming Units (cfu) in Cultured Rumen Fluid of Sheep Fed Napier Grass and Defaunated using NTLE
and SLS.

IV. ACKNOWLEDGMENT [3]. Simon Muchira, and Gathira Peter (2020). Application
of Molecular Techniques to Overcome Challenges of
I would like to express my sincere gratitude to the Ruminant Nutrition in the Tropics. Institute of
Department of Science and Technology - Science Education Biological, Environmental and Rural Sciences,
Institute (DOST-SEI) Advanced Science and Technology Aberystwyth University.
Human Resource Development Program (ASTHRDP) Chromeextension://Efaidnbmnnnibpcajpcglclefindmka
Philippine Council for Agriculture, Aquatic Resources j/Https://Pure.Aber.Ac.Uk/Ws/Portalfiles/Portal/42133
Research and Development (PCARRD), and Carcar City 694/Muchira_Simon_Peter_Gathira.Pdf
College for providing me with the invaluable opportunity to [4]. M. Eugene, H. Archimede, B. Michalet-Doreau and G.
be a graduate scholar. Your support has been instrumental in Fonty (2004). Effect of Defaunation on Microbial
my academic growth and professional development. Activities in The Rumen of Rams Consuming A Mixed
Diet (Fresh Digitaria Decumbens Grass And
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