V.B. Mutwedua, A.W. Nyongesac, J.A. Odumac, J.M. Kitaad, J.M.

Mbariab “Thermal Stress

Causes Oxidative Stress and Physiological Changes in Female Rabbits,” Journal of Animal
Physiology, January 2021

In this article, the authors investigated the effects of heat stress on oxidative stress status and
physiological changes using female New Zealand White rabbits. Temperature, humidity, and radiation variations
in the climate have been identified as potential hazards in the productivity and growth rates of all domestic
livestock species. Increases in ambient, high air humidity and temperature are said to make people
uncomfortable and increase stress levels, which slow down physiological and metabolic processes in animals.
As a homoeothermic animal, rabbits can regulate their body's heat input and output using physical,
morphological, biochemical, and behavioral processes to keep their body temperatures somewhat normal. The
connection between the oxidative there is little information in the literature about the physiological parameters,
stress status, and heat stress in female rabbits under various temperature conditions.

Objectives:
1. Assess the level of oxidative stress.
2. To determine the growth performances for female rabbits, as affected by different ranges of
temperature.
3. To determine behavioral alterations in female rabbits exposed to various temperature ranges.

Hypothesis:
1. If temperature and air humidity are both high, then it will be uncomfortable for animals and raise their
stress levels, which will slow down their physiological and metabolic processes.
2. If female rabbits raised under different temperature ranges, then it will affect female rabbit’s growth
performances
3. If female rabbits are exposed to higher temperature ranges, then it will exhibit significant behavioral
alterations compared to those exposed to lower temperature ranges
 Research Timeline

Animals and ● Female rabbits were purchased from a local recognized and licensed breeder and
housing transported to the animal house at the Department of Anatomy and Physiology, University of
Nairobi, Kenya
● Rabbits were housed in wire cages (0.8 × 0.6 × 0.6 m) at room temperature of 22 ± 4°C with
animal house relative humidity of 68 ± 5%
● Fecal matter and urine were removed from the cages and floor every morning.

Experimental ● All the 24 female rabbits were randomly assigned to 4 groups (T0: ambient temperature (19-
design 26°C), 127 T1: 27-28°C, T2: 31-32°C, T3: 35-36°C)
● heat was induced in each rabbit cage, using electrical heaters (brand: ARMCO) from 08:00
hours 129 to 16:00 hours followed by exposure to the normal air temperature as in the control
group
● The relative humidity and ambient temperature were recorded twice daily using an automatic
thermo-hygrometer.

Behavioral ● Anxiety was scored using behavioral indicators of pacing, scratch and self-directed behaviors
assessment ● Behaviors were evaluated during the last week of the experimentation.
● Following heat-induction, all behaviors were observed seven consecutive days before the
end of the trial from 10.00 hours to 12.00 hours of each observation day.
● With the use of a camera that was placed at a strategic position within the animal house, focal
subjects in individual cages were observed for their interest with empty toys and/or toys
enriched with food.

Blood and organ ● At the end of the experimental period (30 days), all animals were fasted for 24h and humanely
collections sacrificed by euthanizing
● 10 ml of the blood was collected
● The ovaries, uterus, lung, heart, liver and kidney were collected by dissection, washed using
saline solution and blot-dried for weight evaluation

Oxidative stress ● After harvesting and weighing, the kidney was quickly homogenized then centrifuged at 4800
biomarkers rpm for 60 min at 4°C
● Protein content in the supernatant was determined using bovine serum albumin as standard

Hematological ● Blood for hematological analysis was collected in a test tube with K3 EDTA anticoagulant and
and biochemical hematological parameters
analysis ● Meanwhile, 5 ml of blood for biochemical analysis collected in 195 tube free from
anticoagulant was centrifuged at 3000 rpm for 15 min and supernatant separated as serum
and preserved at -20°C

Statistical ● All data were submitted to analysis of variance using XL STAT for Windows 10 Software.
analysis ● Results are expressed as mean ± SD, and treatment effects among experimental groups
alongside controls assessed using one-way ANOVA.
Figure 1. Animals and housing

Figure 2. Experimental design

Figure 3. Behavioral assessment

Figure 4. Blood and organ collections

Figure 5. Oxidative stress biomarkers

Figure 6. Hematological and biochemical analysis

Figure 7. Statistical analysis

Table 1. Growth performances for female rabbits, as affected by different ranges of temperature

Animals exposed to temperatures of 31–32°C and 35–36°C experienced substantial reductions in final body
weight and body weight gain of 11% and 62%, respectively, when compared to animals from the control group
(19–26°C). The feed conversion ratio (64.81%) and water intake (24.19%), however, appeared to be trending
in the other direction, while there was no discernible difference between the treatment groups' average feed
intake and controls (P> 0.05).

Table 2. Behavioral alterations for female rabbits, as affected by different ranges of temperature

Table 2 displays the behavioral scores obtained from heat-stressed female rabbits. Animals in the control group
(19-26°C) and those exposed to 27-28°C showed no behavioral effects. Animals subjected to temperatures of
31–32°C and 35–36°C experienced dizziness. All animals subjected to temperatures of 31–32°C and 35–36°C
displayed a very weak response to the introduction of food (scoring 1) but became incredibly withdrawn to a
cage corner (score 4). Animals exposed to 19–26°C and 27–28°C, on the other hand, displayed a severe
response to the introduction of feed (score 4).

Table 3. Relative organs weight for female rabbits, as affected by different ranges of temperature

The temperature levels taken into consideration in this investigation had no discernible impact on the relative
weights of the heart and liver (P>0.05). However, when compared to animals exposed to the control group (19-
26°C) and the 27-28°C group, the relative weight of the lung and kidney was significantly increased (P 0.05) by
37.72% and 19.5%, respectively, in animals exposed to 31-32°C and 35-36°C, while the relative weight of the
ovaries and uterus decreased significantly (P 0.05) by 30.4% and 18.1%, respectively, at the same
temperatures.

Table 4. Hematological parameters for female rabbits, as affected by different ranges of temperature

Animals subjected to temperatures of 31–32°C and 35–36°C experienced significant decreases (P0.05) in Hb
of 23.64% and 12.73% in the RBC contents, although the same animals' total WBC, MCV, and LYM counts
increased, respectively, by 42.37%, 10.73%, and 15.53% when compared to controls.
When compared to the control and other treated groups, the PCV only reduced in the animals that were exposed
to 35–36°C. The heat stress had no significant impact on the other blood indicators, MCH, MCHC, and PLT.
Table 5. Serum biochemical parameters for female rabbits, as affected by different ranges of temperature

Total protein reduced by 12.02% in animals in the same groups compared to those of the control group (19-
26°C) and 27-28°C that were subjected to the heat stress, while creatinine, urea, and ASAT were each
substantially elevated (P 0.05) by 28.98%, 53.20%, and 23.31%, respectively. In treated groups compared to
controls, there was no statistically significant difference in cholesterol, ALAT, glucose, or total albumin.

Table 6. Oxidative stress biomarkers for female rabbits, as affected by different ranges of temperature

When compared to female rabbits exposed to 19–26°C and 27–28°C, kidney protein levels in those exposed to
31–32°C and 35–36°C significantly dropped by 3.55%. MDA concentration (74.29%) showed the opposite trend.
When compared to animals exposed to other temperature ranges, the activities of CAT, SOD, and GSH were
all significantly (P0.05) reduced in 35-36°C-exposed animals by 32.82%,
24.28%, and 34.34%, respectively.
Objectives Hypothesis Methods Results Discussion

Assess the level If female rabbits ● Female rabbits Their long exposure to Oxidative stress occurs
of oxidative are exposed to were purchased thermal stress leads to when the production of
stress long exposure of from a local the increase in free potentially destructive
heat stress, then recognized and radicals which may reactive oxygen species
it induce licensed breeder induce oxidative stress (ROS) exceeds the
oxidative stress and transported to body’s own natural
the animal house at antioxidant defense
the Department of (Tremellen, 2008).
Anatomy and
Physiology,
University of Nairobi,
Kenya
● Rabbits were
housed in wire
cages (0.8 × 0.6 ×
0.6 m) at room
temperature of 22 ±
4°C with animal
house relative
humidity of 68 ± 5%

To determine If female rabbits ● At the end of the The relative weights of The increased weight of
effect of heat raised under experimental period heart and liver were not lung in heat stressed
stress on relative high (30 days), all significantly affected rabbits reported in the
weight of some temperature,, animals were fasted (P>0.05) by the present study may be due
internal organs then it will affect for 24h and temperature levels to the larger air volumes
the weight of humanely sacrificed considered in this study required by these animals
female rabbit’s by euthanizing (Table 3). However, the to release heat to the
internal organs ● 10 ml of the blood relative weight of the environment through the
was collected lung and kidney were vapor released in the
● The ovaries, uterus, significantly increased breath (Marai et al., 2007).
lung, heart, liver and (P< 0.05), respectively The increase in weight of
kidney were by 37.72% and 19.5%, in the kidney could be due to
collected by animals exposed to 31- the intensive activity of
dissection, washed 32°C and 35-36°C while detoxification carried out by
using saline solution that of ovaries and this organ due probably to
and blot-dried for uterus decreased the increased insensible
weight evaluation significantly (P< 0.05) by loss of body water and salt.
30.4% and 18.1%
respectively at the same
temperatures when
compared to those
submitted to the control
group (19-26°C) and 27-
28°C.
To determine If female rabbits  Anxiety was Behavioral changes on Stress induces reactions
behavioral are exposed to scored using heat stressed female in rabbits including
alterations in higher behavioral rabbits were observed in behavioral modifications
female rabbits temperature indicators of the control group (19- in attempt to cope with the
exposed to ranges, then it pacing, scratch 26°C) as well as those stressor (Rafel et al.,
various will exhibit and self- exposed to 27-28°C. 2012). The appearance of
temperature significant directed Dizziness was observed dizziness in heat stressed
ranges. behavioral behaviors in animals exposed to animals reported in the
alterations  Behaviors were 31-32°C and 35-36°C. present study may be due
compared to evaluated All animals exposed to to the inhibition of
those exposed during the last 31-32°C and 35-36°C acetylcholinesterase
to lower week of the showed a very weak enzymes (AchE) by the
temperature experimentation response to feed heat thus resulting to
ranges. . introduction. accumulation of
 Following heat- acetylcholine in
induction, all cholinergic synapses
behaviors were (Sarkar et al., 2000).
observed seven
consecutive
days before the
end of the trial
from 10.00
hours to 12.00
hours of each
observation
day.
 With the use of
a camera that
was placed at a
strategic
position within
the animal
house, focal
subjects in
individual cages
were observed
for their interest
with empty toys
and/or toys
enriched with
food.

Results of this study indicated that exposure of female rabbits to 31-32°C and 35-36°C for 30
consecutive days impaired their growth performances, relative organ weights and caused some behavioral
abnormalities. Moreover, the thermal stress may be attributed to the oxidative stress and induce various
physiological changes in female rabbits.
Recommendations:
● Develop and test strategies for temperature management in rabbit husbandry and farming practices.
Implementing better climate control measures in rabbit housing could mitigate thermal stress and its
associated negative effects.
● Establish regular health monitoring protocols for female rabbits exposed to thermal stress. This could
include routine assessment of oxidative stress biomarkers and physiological parameters to detect early
signs of stress-related issues.
● Reduce stress levels by implementing environmental enrichment techniques in rabbit housing. This
might involve giving rabbits access to suitable shelter, air conditioning, and chances for mental and
physical activity.
● Ensure that rabbit owners and farmers have access to veterinary care and guidance on managing
thermal stress-related issues in their animals. Education and awareness campaigns may also be
beneficial.
● Advocate for the development and implementation of policies and regulations that promote the welfare
of rabbits in various settings, including farms, research facilities, and households, with a focus on
managing thermal stress effectively.

The concept of thermal stress causing oxidative stress and physiological changes in female rabbits is
closely related to cell signaling and cell communication as cells involve complex molecular and cellular
processes such as cell signaling and temperature sensing whereas cells have sophisticated mechanisms for
sensing changes in their environment, including temperature. Temperature-sensitive receptors on the cell
surface or within the cell can detect fluctuations in temperature. When cells sense thermal stress, it triggers
signaling pathways that lead to various cellular responses. These pathways often involve the activation of
specific proteins and molecules involved in cell signaling.
Oxidative stress is a result of cellular imbalance being upset by thermal stress, which increases the
generation of reactive oxygen species (ROS). Through cell-to-cell contact, this process causes physiological
changes in female rabbits. In summary, thermal stress induces oxidative stress and physiological changes in
female rabbits through complex cell signaling and communication, involving ROS as signaling molecules and
various pathways to adapt to the stressor.