Endorsed by: Dean/Director/ Deputy Dean/

Deputy Director IACUC / 101

IACUC office use only

Date of receive:
Date of review:
Date of meet:
Date:

INSTITUTIONAL ANIMAL CARE & USE COMMITTEE

UNIVERSITI PUTRA MALAYSIA

Animal Utilisation Protocol (AUP) - Research

This completed Animal Utilisation Protocol (AUP) needs to be submitted to
The Secretariat, Institutional Animal Care and Use Committee, c/o Unit of Ethics Research,
Level 5, Office of the Deputy Vice Chancellor (Research & Innovation), Universiti Putra Malaysia,
and approved by UPM IACUC prior to commencement of the animal study.
Direct all enquiries to iacuc@upm.edu.my or 03-9769 1431

PROJECT TITLE:
(Related to the animal work only and must include the animal model to be used in the study)

dd/1mm/yr
1dd/mm/yr
Starting Date: 1st /10/2023 Completion Date: 10th /12/2023
Application form should be submitted at least 2 months prior to commencement of animal study

1. PERSONNEL
Name Institution/Department Phone Number/e-mail Signature

Principal Investigator: 1. Department of Food +60 19-2659710 /

Sciences, Faculty of norhasnida@upm.e
Food Science and du. my
DR. NORHASNIDA Technology, UPM
ZAWAWI
2. Laboratory of Halal
Science Research, Halal
Products Research
Institute, UPM
ALL other personnel involved in
the project:
Please indicate role (co-researcher,
technical staff, RA, GRA, student)

1. Functional +601127893562
STELLAMARIS KEMBABAZI kembabazi.stella@g
(GRA) Carbohydrate and
mail.com
Protein Research
Laboratory, Faculty of
Food Science and
Technology, UPM

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2.Department of Food
Science and
Technology
Faculty of Science
Kyambogo University

If you need more space for animals involved,

please insert new rows

Attending veterinarian:
(Please also read and sign on
Appendix 1)

2. RESEARCH PROJECT INFORMATION:

For RESEARCH, is this a pilot / preliminary study? [ ✓ ] YES [ ] NO

Has funding been approved for this study?

[ ] No, applying for funds

[✓ ] Yes – Provide Grant 3240324247

No:
*Please attach a copy of approval letter(s).

Peer Review for Scientific Merit of Research Studies has been performed by:
[ ] Granting Agency:
[ ] Other (Specify):

*Please provide a copy of scientific reviewer’s comments.

Purpose of Animal Use (check one):

[ ] Studies of a fundamental nature in sciences relating to essential structure or function (i.e. biology,
psychology, biochemistry, pharmacology, physiology, behaviour, etc.)
[✓] Studies for scientific purposes that relate to human or animal disease or disorders.
[ ] Studies for regulatory testing of products for the protection of humans, animals, or the environment.
[ ] Studies for the development of products or appliances for human or veterinary medicine
[ ] Education and training of individuals in institutions or facilities
[ ] General operating protocols (for routine management of herds/colonies)
[ ] Diagnostic testing

Classification (check one):

[ ] Acute - utilising an animal for a brief period (less than 24 hrs.), followed by euthanasia or return of the
animal to source, or humanely killing an animal upon receipt or after a brief housing period during which
time no manipulations other than standard management procedures are performed, i.e. anaesthetized
without recovery, euthanised for tissue collection, etc.

[✓] Chronic - maintaining the animal and performing experimental procedures during this time, i.e. feeding
trials, antibody production, breeding colony, recovery surgery.

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Category of Invasiveness (check one):

A Involve either no living materials or use of no living materials, or use of plants, bacteria, protozoa,
- studies on tissues obtained from autopsy or slaughterhouse.
THIS CATEGORY DOES NOT NEED AN AUP

[ ]B Experiments on vertebrates species, expected to produce little or no discomfort

- mere restraint for blood sampling, injection of harmless substance, physical examination,
- experiment on completely anaesthetised animals which do not regain consciousness, food/water deprivation for
few hours, standard methods of euthanasia (anaesthetic overdose or sedation/light anaesthesia follow by
decapitation)

Experiments that involve some minor pain/discomfort for short duration to vertebrate species
[✓ ] -exposure of blood vessels, implant chronic catheters, behavioural study involving short-term stressful restraint,
C immunization employing Freund’s adjuvant, surgery under anaesthesia resulting in minor post-surgical discomfort

[ ]D Experiments that involve significant but unavoidable stress or pain to vertebrate species
-deliberate induction of behavioural stress, major surgical procedure resulting in significant post-operative
discomfort, induction of anatomical/physiological deficit resulting in pain/distress, application of noxious stimuli
from which escape is impossible, prolonged (> several hours) physical restraint, procedures that produce pain in
which anaesthetics are not used (toxicity testing with death as end-point, production of radiation sickness, certain
injections, stress and shock research resulting in pain approaching pain tolerance threshold/point of intense
reaction)

[ ]E Procedures that involve inflicting severe pain near, at, or above the pain tolerance threshold of unanaesthetised,
conscious animals
-use of paralytic agent alone for surgical restraint without use of anaesthetics, severe burn or trauma infliction on
unanaesthetised animals, inescapable severe stress or terminal stress

Please refer Appendix on Categories of Invasiveness in Animal Experiments.

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3. LAY SUMMARY (250 words maximum)

In LAY TERMINOLOGY, please provide concise summaries of the proposed animal study.
Avoid use of technical jargon.

a) Brief research background and objectives for the proposed animal study.

Background:
Starch is classified as resistant, slowly digestible, and rapidly digestible starch depending
on the hydrolysis time in the small intestines. The activity of α-amylase activity is in such
a way that when the dextrin hydrolysis in the small intestine epithelial lining takes between
20 to 120 minutes, the starch is regarded as rapidly digestible (RDS) and slowly digestible
starch (SDS) respectively (Englyst & Cummings, 1985).
Resistant starch (RS) on the other hand isn’t hydrolyzed within 120 minutes, it continues
to large intestines for fermentation by the microbiota into short-chain fatty acids
(SCFAs; Dupuis et al., 2014).

RS is classified as type I, II, III, IV, and V (Englyst and Cummings, 1985).
RSI is enzymatically inaccessible starch common to grains; type II is typically
granular starch found in raw potato and bananas; type III is retrograded starch,
chemically modified starch (type IV) while type V is amylose-lipid complex,
for example, stearic acid-complexed high amylose starch (Englyst and Cummings, 1985).
RSIII is of particular interest because it is thermally stable (Haralampu, 2000),
and easily fermented by the intestinal microflora (Lehmann et al., 2002) into butyrate;
a substrate and signal metabolite for activation of the proliferation of probiotic microbes
in the gut (Sajilata et al., 2006; Marques et al., 2019). In addition, RSIII has a unique
structure unlike other types of RS that is able to bind bile salts, thus preventing their
reabsorption in the ileum, consequently stimulating their production in the liver, and
overall increasing the utilization of cholesterol
(Dongowski et al., 2005; Bojarczuk et al., 2022).

In addition to the prebiotic role, RS confers other physiological functions like

reduction in risk of colon cancer, hemorrhoids, diverticulosis, constipation, increased fecal
bulking, modulation of blood glucose level, and blood cholesterol level
(Sharma & Yadav, 2008). Studies have also shown that RS improves the
textural properties of baked products (Khan et al., 2020), it is thus, important for
food scientists to formulate functional foods that exploit both the physiological
and textural properties of RS.

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a) Brief research background and objectives for the proposed animal study.

However, for the intake of RS to cause significant physiological importance,

the daily carbohydrate intake should be 10% to 20% RS (Roman and Martinez, 2019).
Yet, on the contrary, most commercial food products have less than 5% RS per serving.
White bread is the most consumed bakery product that contributes to the
highest proportion of carbohydrates in the diet, yet, it is a high glycemic index food with severe
outcomes on health especially for overweight, obese, and diabetic patients
(Harvey, 2010; Roman and Martinez, 2019). This study intends to determine
the anti-hypercholesterolaemic effects of Resistant Starch Type III rich bread in Diet-Induced
Obese Rats.

Objectives:
1. 1. To determine the effect of RS-III rich-bread on the hypercholesterolaemic and
glucose tolerance in high-fat diet-induced obese rats
2. To determine the effect of RS III-rich bread on the fermentability of short-chain
fatty acids and microbiome in the gut of high-fat diet-induced obese rats.

References

Englyst, N., & Cummings, H. (1985). Englyst1985.Pdf. January, 778–787.

Dupuis, J. H., Liu, Q., & Yada, R. Y. (2014). Methodologies for Increasing the Resistant
Starch Content of Food Starches: A Review. Comprehensive Reviews in Food Science
and Food Safety, 13(6), 1219–1234. https://doi.org/10.1111/1541-4337.12104
Haralampu, S. G. (2000). Resistant starch - a review of the physical properties and
biological impact of RS3. Carbohydrate Polymers, 41(3), 285–292.
https://doi.org/10.1016/S0144-8617(99)00147-2
Lehmann, U., Jacobasch, G., & Schmiedl, D. (2002). Characterization of resistant starch
type III from banana (Musa acuminata). Journal of Agricultural and Food Chemistry,
50(18), 5236–5240. https://doi.org/10.1021/jf0203390
Sajilata, M. G., Singhal, R. S., & Kulkarni, P. R. (2006). Resistant starch - A review.
Comprehensive Reviews in Food Science and Food Safety, 5(1), 1–17.
https://doi.org/10.1111/j.1541-4337.2006.tb00076.x
Marques, L. A., Cazarin, C. B. B., Bicas, J., Maróstica, M. R., Carrilho, E., & Bogusz, S.
(2019). Determination of short chain fatty acids in mice feces by capillary
electrophoresis. Journal of the Brazilian Chemical Society, 30(6), 1326–1333.
https://doi.org/10.21577/0103-5053.20190031
Dongowski, G., Jacobasch, G., & Schmiedl, D. (2005). Structural stability and prebiotic
properties of resistant starch type 3 increase bile acid turnover and lower secondary
bile acid formation. Journal of Agricultural and Food Chemistry, 53(23), 9257–9267.
https://doi.org/10.1021/jf0507792
Bojarczuk, A., Skąpska, S., Mousavi Khaneghah, A., & Marszałek, K. (2022). Health
benefits of resistant starch: A review of the literature. Journal of Functional Foods,
93(May). https://doi.org/10.1016/j.jff.2022.105094
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a) Brief research background and objectives for the proposed animal study.
Sharma, A., & Yadav, B. S. (2008). Resistant starch: Physiological roles and food
applications. Food Reviews International, 24(2), 193–234.
https://doi.org/10.1080/87559120801926237

Khan, A., Siddiqui, S., Ur Rahman, U., Ali, H., Saba, M., Andleeb Azhar, F., Maqsood Ur
Rehman, M., Ali Shah, A., Badshah, M., Hasan, F., & Khan, S. (2020).
Physicochemical properties of enzymatically prepared resistant starch from maize
flour and its use in cookies formulation. International Journal of Food Properties,
23(1), 549–569. https://doi.org/10.1080/10942912.2020.1742736
Roman, L., & Martinez, M. M. (2019). Natural and Commercial Alternatives. Foods.
Harvey, P. (2010). FOOD FORTIFICATION: THE 2008 UGANDA FOOD
CONSUMPTION SURVEY. May.

b) Anticipated impact and potential benefits to human and/or animal welfare.

With the growing trends of diet-related non-communicable diseases (NCDS, overweight,

and obesity), consumers are becoming increasingly more conscious of their diets since nutritional
intervention is the most recommended remedy. This study will open doors to the possibility of consuming
white bread without worries of NCDS but rather obtaining the physiological functions of RS.

4. ANIMAL MODEL
Justify the species and/or strain used for this research purpose. Please provide references for the proposed
animal model or disease/condition (e.g. diabetes mellitus, osteoarthritis).

A number of animal models have been used, for example non-human primates, large mammals
such as pigs, dogs, and cats, and smaller animals such as rabbits, rats, and mice. Larger animals
are naturally difficult to maintain, require higher maintenance costs, and hence are not as
commonly used as compared to the smaller animals. Among the smaller animal models, rodents
are the first choice of users due to their smaller body size, omnivorous nature, and non-wild
tranquil behavior, which allows for ease of handling for researchers as well as significantly
reduced dietary and housing costs.

Diet-induced Obesity (DIO) is commonly induced in animal models by feeding rats with a
high-fat diet, with lipids contributing about 45 to 60% of calories (de Moura e Dias et al., 2021).
This feeding is able to induce human-like obesity since it increases adipose deposition and
stimulates the onset of hypertension and insulin resistance. DIO is easily induced in rats within
four weeks and its effects can last for eight weeks.

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Justify the species and/or strain used for this research purpose. Please provide references for the proposed
animal model or disease/condition (e.g. diabetes mellitus, osteoarthritis).

References
de Moura e Dias, M., dos Reis, S. A., da Conceição, L. L., Sediyama, C. M. N. de O., Pereira, S.
S., de Oliveira, L. L., Gouveia Peluzio, M. do C., Martinez, J. A., & Milagro, F. I. (2021).
Diet-induced obesity in animal models: points to consider and influence on metabolic
markers. Diabetology and Metabolic Syndrome, 13(1). https://doi.org/10.1186/s13098-021-
00647-2
Cereal Chemistry, 78(6), 680–689. https://doi.org/10.1094/CCHEM.2001.78.6.680
Cunha, L. F., Ongaratto, M. A., Endres, M., & Barschak, A. G. (2021). Modelling
hypercholesterolaemia in rats using high cholesterol diet. International Journal of
Experimental Pathology, 102(2), 74–79. https://doi.org/10.1111/iep.12387
Křížova, E., Šimek, V., Abelenda, M., & Puerta, M. (1996). Food intake and body weight in rats
with daily food-availability restrictions. Physiology and Behavior, 60(3), 791–794.
https://doi.org/10.1016/S0031-9384(96)00161-8

5. ALTERNATIVES
a) Explain the necessity of using animals in this project, and why alternatives (in-vitro and ex-vivo systems) to
replace the use of animals would be inappropriate to meet your project or teaching objectives.
Please provide references.

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The in vitro experiments of DIO do not completely mimic the in vivo obesity environment, with
significant differences including hormones and cytokines, and bio metabolites like glucose and
fatty acids existing between the vivo and vitro (Ghanemi et al., 2022).
On the other hand, in vivo, studies have been used as substitutes for humans to study DIO and its
related biomarkers. Different strains of rats tend to show significant lipid accumulation in the
adipose and the liver when used for DIO studies, in addition to the similar pattern of response to
the oral glucose tolerance tests and insulin load (Li et al., 2020).
Thus, to be able to study the hypercholesterolaemic effect of type resistant starch rich bread, in
vivo studies using a rat model is very suitable.
References
Ghanemi, A., Yoshioka, M., & St-Amand, J. (2022). In Vitro Mimicking of Obesity-Induced
Biochemical Environment to Study Obesity Impacts on Cells and Tissues. Diseases, 10(4),
76. https://doi.org/10.3390/diseases10040076
Li, J., Wu, H., Liu, Y., & Yang, L. (2020). High fat diet induced obesity model using four strains
of mice: kunming, c57bl/6, balb/c and icr. Experimental Animals, 69(3), 326–335.
https://doi.org/10.1538/expanim.19-0148

b) Indicate any alternatives to animal use that are already incorporated into the project design (in-vitro & ex-
vivo systems).

Sensory evaluation will be carried out on the bread and the bread formulation with the best
attributes for customer acceptability will be used for this study. Sensory evaluation and the
optimisation of the bread formulation will be done in Uganda.

6. ANIMAL USE
a) List ALL ANIMALS involved in the study.
Quantity Species/Strain Weight/ Gender Accommodation Experimental Room*
Age Building [Eg: procedure room/area, or
hatchery/pond/tank (for aquatic animals)]

48 Sprague 190 ± male Animal House, Animal House, Faculty of

Dawley Rats 20 g Faculty of Medicine & Health Sciences,
(5-7 Medicine & UPM
weeks Health
old) Sciences,
UPM
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Quantity Species/Strain Weight/ Gender Accommodation Experimental Room*

Age Building [Eg: procedure room/area, or
hatchery/pond/tank (for aquatic animals)]

If you need more space for animals involved,

please insert new rows
*Please specify if experiments on infectious diseases can be carried out in the proposed area/room. Please note that experiments
involving infectious diseases that can be a threat to healthy animals cannot be permitted in the same facility.

b) Explain how the total number of animals to be used was determined.

e.g. 6 animals x 3 treatments = 18 animals. Include a flow chart or table if necessary.

8animals (2controlsx 4 treatments)=48

Week Treatment
Rats Normal (16) Obese (16) Control Control
(number) Group1( Group2(8)
8)
0 Acclimatization
1 25-35g of 25-35g of 40g of cafeteria HFD 25-35g 25-35g of
2 RSIII RS bread White altromin
3 bread (Std RS- bread (standard)
4 (EBF- wheat feeds
wheat composite)
composite)

5 25-35g of 25-35g of
6 RS bread RS bread
7 (EBF-wheat (EBF-wheat
8 composite) composite)
9 Plus 40g
HFD

KEY
DIO rats-diet induced obese rats
RSIII bread (EBF) - bread made out of a composite of Extruded banana flour (EBF) and wheat flour
HFD- High-fat diet
RS bread (Std-RS-wheat composite) - bread made out of a composite of Nutriose and wheat flour.
The percentage substitution for Nutriose will be equivalent to that of EBF

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b) Explain how the total number of animals to be used was determined.

e.g. 6 animals x 3 treatments = 18 animals. Include a flow chart or table if necessary.

c) Indicate consideration given to reduce the use of animals in the project design.

Justifications for:
a. Selection of HFD
High fat (45% energy from fat, i.e., food with 22% fat) Cafeteria foods will be selected
and fed to the rats to induce DIO. This amount of fat has been proven sufficient to induce
DIO in rats in 4 weeks.

b. Use of standard RS (NUTRIOSE®06FM; Roquette)

This powder has been used in studies that compare the effect of resistant starch
(dextrin/maltodextrin) on DIO and its markers in human clinical trials

c. Feeding during evening

Rats are generally nocturnal and feed more in the night than during the day. The feeds
shall be given in the night to ensure maximum feeding.

d. Use of weaned male rats: Male Sprague–Dawley rats were highly susceptible to DIO by an
HFD.
e. Cafeteria feeding: studies have shown that during the early stages of diet exposure, rats
exposed to the cafeteria diet snacked more than their chow-fed counterparts; and the early
levels of snacking were directly related to terminal body weights

References

Aliasgharzadeh, A., Dehghan, P., Gargari, B. P., & Asghari-Jafarabadi, M. (2015). Resistant
dextrin, as a prebiotic, improves insulin resistance and inflammation in women with type 2
diabetes: A randomised controlled clinical trial. British Journal of Nutrition, 113(2), 321–
330. https://doi.org/10.1017/S0007114514003675

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c) Indicate consideration given to reduce the use of animals in the project design.

South, T., Holmes, N. M., Martire, S. I., Westbrook, R. F., & Morris, M. J. (2014). Rats eat a
cafeteria-style diet to excess but eat smaller amounts and less frequently when tested with
chow. PLoS ONE, 9(4), 1–12. https://doi.org/10.1371/journal.pone.0093506

Taraschenko, O. D., Maisonneuve, I. M., & Glick, S. D. (2011). Sex differences in high fat-
induced obesity in rats: Effects of 18-methoxycoronaridine. Physiology and Behavior,
103(3–4), 308–314. https://doi.org/10.1016/j.physbeh.2011.02.011

7. SOURCE
Indicate the source or supplier:
[ ✓ ] UPM Animal Resource Unit [ ] Client Owned [ ] Client Donated [
] Resident Animal
[ ] Wildlife [ ] Field studies [ ] UPM Herd / Flock [ ] Local suppliers/farms
[ ] Other institution(s) [ ] Import (attach health certificate & import permit)
[ ] Transfer from other researcher/ research (AUP No:____________________________)
For the above, please provide details:
SPECIES SOURCE/SUPPLIER ADDRESS/ PHONE MODE AND
LOCATION NUMBER CONDITION OF
TRANSPORTATION

Sprague Comparative Medicine Comparative +60 17-404 Air-conditioned

Dawley and Technology Unit Medicine and 9122 (En. transport,
rats (COMeT), Institute of Technology Unit Saiful transported to
(male) Bioscience, UPM (COMeT), Qushairi) Animal House in
Institute of Faculty of
Bioscience, Medicine and
Universiti Putra Health Sciences,
Malaysia, 43400 UPM
UPM Serdang,
Selangor

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If you need more space for animals

involved, please insert new rows

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8. ANIMAL CARE & HUSBANDRY

a) Specify provisions of basic requirements for each species/strain of animals used
(For guide on species care and husbandry requirements, please refer to the UPM Code of Practice for the Care and Use of
Animals for Scientific Purposes at http://www.rmc.upm.edu.my/dokumen/PTPPY1_92272_upm_code_of_practice.pdf).

Species/strain 1: Sprague-Dawley rats

i. Caging: [✓ ] Plastic [ ] Metal [ ] Aquarium [ ] Tank [ ] Others-
specify_____________
For aquaculture research, please specify size & volume of space : ________________________
ii. Stocking density: 2 animal per cage (cage/pen/paddock/tank dimension or floor space)
iii. Flooring/Bedding:[ ] Wood slatted [ ] Wire mesh [ ] Wood shaving [ ] Newspaper

[✓ ] Others Corncob bedding

iv. Temperature of room: [ ] Not regulated [ ✓] Regulated at 25⁰C-30⁰C
v. Ventilation: [ ] Not regulated [ ✓ ] Regulated

vi. Feed: [ ✓] Custom-formulated [✓ ] Commercial – name of manufacturer Altromin

International
vii. Water: Source Normal Saline Delivery: [ ] Bottle [ ✓]] Water bowl
[ ] Others________________

Species/strain 2:_____________
(please copy items above )

b) Specify the frequency of the following activities (if applicable) and who will be performing
Activity Frequency Performed by (name)
Feeding 2/day Stellamaris Kembabazi
Changing water bottle/bowl/tank 2/day Stellamaris Kembabazi
Changing bedding/litter tray/filter 2/day Stellamaris Kembabazi
Changing/cleaning cage/pen/tank/filter 2/week Stellamaris Kembabazi

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c) Specify any enrichment provisions, i.e. space, specific materials or objects provide,
if any. (e.g. tissue paper, cardboard shelter, cardboard tube etc.)

Tissue and shredded papers will be provided as the enrichment.

9. PROCEDURES

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a) Using a FLOW DIAGRAM WITH TIMELINE, describe sequence of research procedures that involve animals in
this project. THE COMPLETE FLOW DIAGRAM WITH TIMELINE SHOULD ONLY DESCRIBE THE PROCEDURES
FROM THE POINT OF PURCHASE OR PROCUREMENT TO WHEN AND HOW THE ANIMALS ARE EUTHANISED .
Please provide reference(s) where appropriate.
In cases of surgical procedures, description of the following should be included; patient preparation before
surgery, pain and distress management, frequency of monitoring during and post-surgery as well as technical
description of surgical procedures and post-operative care.

FLOW DIAGRAM MUST BE CONCISE

Week Treatment
Sample test No. Normal DIO Control
collected Rats group1 group2 group1 group2 group1 group2

1,5,9 Saphenous Lipid profile 8 8 8 8 6 6

vein blood
Blood from Glucose 8 8 8 8 6 6
tail tolerance
test
Feces SCFAS 6 6 6 6 6 6
Food Food 8 8 8 8 8 8
remains consumption
Body weight 8 8 8 8 8 8
Liver liver weight 6 6 6 6 6 6
colon microbiome 6 6 6 6 6 6
2,3,4,6, Blood from Glucose 8 8 8 8 6 6
7,8 tail tolerance
test
Feces SCFAS 6 6 6 6 6 6
Food Food 8 8 8 6 6
remains consumption
Body weight 6 6 8 8 6 6

SCFAS- short chain fatty acids

Treatment group Group 1 Group 2
Normal RSIII bread with extruded banana flour RSIII bread with Nutriose
flour
DIO RSIII bread with extruded banana flour RSIII bread with extruded
banana flour and HFD
Control White bread Normal standard feeds

Reference(s):

Yu, W., Zhang, Z., Pan, W., Guan, W., Lin, Q., Xia, W., Li, T., & Hsieh, E. (2021). Comparison of
Differences in Bone Mineral Density Measurement With 3 Hologic Dual-Energy X-Ray
Absorptiometry Scan Modes. Journal of Clinical Densitometry, 24(4), 645–650.
https://doi.org/10.1016/j.jocd.2021.01.003
Rashighi, M., & Harris, J. E. (2017). 乳鼠心肌提取 HHS Public Access. Physiology & Behavior,
176(3), 139–148. https://doi.org/10.1053/j.gastro.2016.08.014.CagY

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b) List ALL procedures, manipulations, and/or measurements that will be performed on the
animals.

PROCEDURE(S) Compound No. of Frequency State

E.g. transportation, physical restraint, blood sampling, name, dosage, animals and category of
administration of compounds or chemicals, injection of route & volume involved duration invasiveness
anaesthetics, analgesics, antibiotics, behavioural test, *B-E (Please
euthanasia etc. (if applicable) refer to page 2)
(Please list procedures in sequence)

1 Standard Normal diet 20-35g/day 48 1/daily A

2 RSIII bread (Extruded banana 20-35g/day 8 1/daily A
flour
Altromin standard feeds 20-35g/day 6 1/daily A
RSIII bread (Standard RS) 20-35g/day 8 1/daily A
White bread 20-35g/day 6 1/daily A
2 Body weight measurements - 48 1/week B
(9times)
3 Oral glucose tolerance test i) Oral i) 1/week B
(OGTT) administration
of 0.2 g/100 g
b.w. glucose in
an overnight-
fasted state
ii) Blood will 48
be collected ii)9
via tail vein times /
(0.01-0.1 ml) OGTT
at intervals of procedure
0, 30, 60, 90,
and 120 min
for blood
glucose
measurement
4 High fat diet 40g 16 1/daily A
(4weeks)
5 Body composition analysis using - 18 1,5&9 B
dual-energy X-ray Week
absorptiometry (DEXA) body (3times)
scan
6 Fecal collection using metabolic - 48 Once per B
cages Week
(9times)

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PROCEDURE(S) Compound No. of Frequency State

E.g. transportation, physical restraint, blood sampling, name, dosage, animals and category of
administration of compounds or chemicals, injection of route & volume involved duration invasiveness
anaesthetics, analgesics, antibiotics, behavioural test, *B-E (Please
euthanasia etc. (if applicable) refer to page 2)
(Please list procedures in sequence)

7 Blood collection - 48 Once per B

Week
(i) Tail vein
(9times)
(ii) (ii) Saphenous vein

8 Colon microbiome - 18 1,5&9 B

Week
(3times)
9 Blood triglyceride, cholesterol, - 48 Once per B
HDL-C and LDL-C Week
(9times)
10 Anesthesia - 30 1,5&9 B
Week
(3times)
*Indicate the Category of Invasiveness as stated in page 2 for each procedure listed.
Please consult your AV for procedures involved the usage of drug (dosage, route, volume etc.).

c) List ALL individuals who will carry out the procedures listed in 9 b).
Provide their technical qualifications and relevant experience in performing these procedures.
Name* Procedure(s) to be performed Qualifications/experience with these
(list the corresponding procedure number as procedures
listed in table 9b above e.g. 1, 2, 3)

Stellamaris Kembabazi 1,2,3,4,5,6,7,8,9 BSc. Biochemistry, MSc.

Biochemistry

If you need more space, please insert new rows

*All individual names should also be listed in page 1. Procedure involved the usage of controlled drug should be done by AV.

d) Specify the criteria used to assess the level of anaesthesia required for invasive procedures
(if relevant)

[ ] Not applicable [ ] Respiratory rate [ ] Heart rate [ ] Corneal reflex [ ✓ ] Toe pinch [ ✓] Tail
pinch
[ ] Response to procedures [ ] Others – specify:

e) Specify the methods/criteria for monitoring the condition/level of pain and distress of the
animals following the above listed procedures.
(Please attach Template of Animal Assessment/Monitoring Sheet)

[ ] Not applicable [✓ ] Loss of appetite [✓ ] Loss of weight [ ] Restlessness

[✓ ] Laboured breathing [✓ ] Loss/reduce mobility [✓ ] Abnormal resting posture [✓ ]

Unresponsiveness

[✓ ] Failure to show natural inquisitiveness [ ] Failure to groom/unkempt appearance

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d) Specify the criteria used to assess the level of anaesthesia required for invasive procedures
(if relevant)
[ ] Red stains around eyes of rats [ ] Guarding/protecting painful area
[ ] Licking, biting, scratching, shaking of affected area
[ ] Others – specify:

f) Specify frequency of animal observations:

1. Daily husbandry routine: ______times per hour / day / week *(delete where not relevant)
2. Following experimental procedures: ______times per hour / day / week *(deletel where not relevant)

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10. EXPERIMENTAL AND/OR ANIMAL USE ENDPOINT

When experimental procedures produce animals that may become ill, it is necessary to define an endpoint to
ensure that an experimental animal’s discomfort, pain and/or distress is terminated, minimised or reduced.
a) List any clinical conditions or abnormalities expected as a result of the proposed study (e.g. behavioural
changes such as increased grooming, vocalization or postural changes, or physical abnormalities such as
anorexia, dehydration, diarrhoea, etc.). All expected clinical abnormalities that could arise need to be
included in Animal Assessment/Monitoring sheet as stated in 9e).

b) List the criteria to trigger the decision to remove an animal from the experiment, or to terminate the
experiment (e.g. moribound, 20% body weight loss for a week, etc.). If death is required as the endpoint,
please justify.

Extreme loss in body weight (20% reduction in 1 week) and is moribund.

11. DISPOSAL OF ANIMALS

SPECIES, TO BE RETAINED/ SOLD TO/ DONATED
TO/ TRANSFERRED TO/ ADOPTED BY
quantity
(specify location or to/by whom and purpose
if animals are retained)
TO BE EUTHANISED (specify method/drug/dose. If
a physical method of euthanasia is to be used i.e.
cervical dislocation, justify its use)
CARCASS DISPOSAL (specify method)

Sprague Euthanasia (terminal blood collection)

Dawley while under anaesthesia (intraperitoneal
rats, 30 injection with ketamine (100 mg/ kg b.w.)
+ xylazine (10 mg/ kg b.w.))
- Dispose via clinical waste

12. EMERGENCY VETERINARY CARE

Is routine veterinary care appropriate for animals in this project? [ ✓] YES
[ ] NO
If NO, attach specific instructions in case an emergency should arise.
IN THE EVENT OF AN ANIMAL HEALTH EMERGENCY, IF THE PERSONNEL LISTED IN SECTION 1 COULD NOT BE
CONTACTED, THE DECISION OF A CLINICAL VETERINARIAN APPOINTED BY THE IACUC WILL BE FINAL.

13. HAZARDS:

Does this project involve hazardous agent or animal? [ ] YES [✓ ]

NO
If YES, please complete the details as below.
TYPE SPECIFY AGENT, DOSAGE, ROUTE, FREQUENCY
Radio-Isotope N/A
Carcinogen N/A
Dangerous chemical N/A
Contagious pathogen to N/A
humans [ ] animals [ ]
*Recombinant DNA/RNA N/A
Other (e.g. *GMO, electroshock) N/A

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Precaution step(s) and/or special animal care or containment procedure required for the hazard(s):

*Project involving Recombinant DNA/RNA/GMO needs to apply for acknowledgement (contain

used) or approval (field trial) from National Biosafety Board or UPM Institutional Biosafety
Committee (IBC).
14. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE APPROVAL
Upon approval, a protocol number will be assigned. This number must be used when ordering
animals. This Animal Utilisation Protocol is valid for the duration of the project pending on submission
of annual progress report by the investigator and recommendation by the animal facility manager.
PRINCIPAL INVESTIGATOR’S DECLARATION TICK
By signing this form, I certify that:

1. this project will be conducted in accordance with the Animal Welfare Act 2015
(https://www.aaalac.org/resources/Malaysia.pdf), UPM policy and Code of Practice for
the Care and Use of Animals for Scientific Purposes
(http://www.tncpi.upm.edu.my/upload/dokumen/20180522091735PTPPY1_92272_ethi
cspolicy.pdf) and Institutional Care and Use Committee (IACUC) guidelines, and any
other applicable federal/state laws and regulations.
2. the information provided in this AUP is complete and accurate.
3. the proposed experimental activities described above have not been carried out by
myself or other researchers in this institution or elsewhere.
4. all activities are designed to assure that pain/distress/discomfort of animals is
minimized.
5. all personnel listed in section 9c are aware of, and will follow the approved procedures
outlined in this form. They will be appropriately trained and qualified, and that I am
responsible for the supervision, training, and work of said personnel.
6. I will maintain appropriate animal records (e.g. animal monitoring sheet, veterinary
care, euthanasia, surgery, anesthesia etc.).
7. veterinary care will be available when necessary,and provided by the qualified
attending veterinarian (AV). I will immediately notify his/her regarding any unexpected
study results that negatively impact the animals, and any unanticipated pain or
distress, morbidity or mortality will be documented and reported to the IACUC.
8. the information provided in this AUP will be kept current and any changes must be
notified by submitting Form IACUC/105. I aware that IACUC approval must be
obtained prior to performing the revised animal procedures described therein.
9. I understand that approval of proposed project is valid for a maximum of one (1) year
from the date of approval. I aware that extension of the approval need to be requested
at least one (1) month prior to project completion by submitting Form IACUC/106.
10. I will notify and submit Form IACUC/106 following the completion of project. I
understand that my new AUP application will not be processed before report
submission.
11. I understand that the IACUC may approve the application as submitted, required
modifications in order to receive approval, or rejected, and the approval may be
subject to further review.
By submitting this form, I have read and understood the above declaration.

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Project Title:

Signature and stamp of Principal Investigator:

Date:

Appendix 1

ATTENDING VETERINARIAN’S DECLARATION TICK

By signing this form, I certify that:

1. I should provide input in protocol review, the development of study removal criteria, and
responsible conduct of research activities and can be invited to attend the IACUC
meeting together with the research team if required.
2. I oversee the well-being and clinical care of animals used in research, testing and
teaching. The responsibility extends to monitoring and promoting animal well-being at
all times during animal use and during all phases of the animal’s life. Well-being is
determined by considering physical, physiological and behavioural indicators.
3. I shall provide guidance to investigators and all personnel involved in the care and use
of animals to ensure appropriate husbandry, handling, medical treatment,
immobilization, sedation, analgesia, anaesthesia and euthanasia.
4. I shall provide guidance and oversight to surgery and perioperative care involving
animals in accordance with current established veterinary medical and nursing
procedures, if applicable.
5. I am expected to carry out daily observation of all animals in the study project to assess
their health and well-being. However, the daily observation of animals may be
accomplished by someone else other than myself provided that there is a mechanism
of direct and frequent communication between the researchers and I so that timely and
accurate information on problems of animal health, behaviour, and well-being is
conveyed to me.
6. if I am on leave or will be otherwise unavailable to provide any general or emergency
veterinary care, interim arrangements are made to ensure that there is always ready
access to veterinary care. Timely provision of veterinary medical care and emergency
veterinary care is always available after working hours, on weekends, and on holidays.
7. any unethical events or animals are not kept to optimum welfare care or found during
an audit will be reported to the IACUC. I aware that the IACUC will initiate
investigations where the researchers and I can be summoned for explanation and
deliberation on the matter.
8. following the completion of project, I will notify the PI to submit a final report to the
IACUC on the care and ethical use of animals in the project, including animal
monitoring log if necessary.

By submitting this form, I have read and understood the above declaration.

Project Title:

Name of Principal Investigator:

Signature and stamp of Attending Veterinarian: Annual Practicing Certificate number:

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Date:

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