




































In ternationa l
Scholars
Journa ls

 

African Journal of Pig Farming ISSN 2375-0731 Vol. 5 (5), pp. 001-007, May, 2017. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Full Length Research Paper 

 

Erythrocyte osmotic fragility of pigs administered 
ascorbic acid and transported by road for short-

term duration during the harmattan season 

 
A. Y. Adenkola1*, J. O. Ayo2, A. K. B. Sackey3 and A. B. Adelaiye4

 
 

1
Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Agriculture, Makurdi, 

Nigeria. 
2
Department of Physiology and Pharmacology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria. 
3
Department of Surgery and Medicine, Faculty of Veterinary Medicine, Ahmadu Bello University Zaria, Nigeria. 

4
Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University Zaria, Nigeria. 

 
Accepted 12 October, 2016 

 
The experiment was carried out with the aim of investigating the effect of an antioxidant ascorbic acid on 
erythrocyte osmotic fragility of pigs transported by road for 4 h during the harmattan season. 16 pigs 
administered with ascorbic acid at the dose of 250 mg/kg per os and individually served as experimental 
animals and 13 others administered orally with sterile water were used as control animals. The animals were 
then transported for 4 h at a speed of 40 - 50 km/h covering a distance of 140 km. Blood samples for 
erythrocyte osmotic fragility determination which was done using standard procedure, were taken early in the 
morning a day before transportation, immediately after and a week after transportation. Erythrocyte osmotic 
fragility decreased significantly (P < 0.05) at NaCl concentration of 0.85, 0.80 and 0.70% in both experimental 
and control pigs following road transportation and the difference in the post-transportation values was higher 
(P < 0.05) in experimental compared to control pigs. The results indicated that ascorbic acid protected the 
integrity of the erythrocyte membrane in experimental pigs administered ascorbic acid following road 
transportation as demonstrated by lower percentage haemolysis immediately after road transportation and 
thus may alleviate the risk of increase in haemolysis due to road transportation stress in pigs during the 
harmattan season. 
 
Key words: Ascorbic acid, erythrocyte osmotic fragility, harmattan season, pigs, road transportation. 

 
INTRODUCTION 

 
In many parts of the world, including Nigeria, food ani-
mals are transported mainly by road. It has been esta-
blished that road transportation is stressful to livestock 
(Rajion et al., 2001; Giovagnoli et al., 2002; Adenkola and 
Ayo, 2009). During stress, there is an increase in 
generation of reactive oxygen species (ROS) in the body 
to a level that overwhelms tissue antioxidant defense sys-
tems (Akinwande and Adebule, 2003; Powers and 
Jackson, 2008). The mechanism of damage involves lipid 
peroxidation which destroys cell membranes with the  
 
 

 
*Corresponding author. E-mail:  aadenkola@yahoo.  com. Tel.: 
+234-805-4977696. 

 
 
 

 
release of intracellular compoents, such as lysosomal 
enzymes, leading to further tissue damage (Demir et al., 
2003). The ROS play a vital role in cellular and tissue 
damage (Tkaczyk and Vizek, 2007) and they have been 
demonstrated to have adverse effects on erythrocytes 
(Sumikawa et al., 1993; Avellini et al., 1995). The 
magnitude of stress in the body depends on the ability of 
the tissues to detoxify ROS (Williams et al., 2008) that is, 
their antioxidant defence. The ROS, including free radi-
cals, initiate many reactions which are deleterious to body 
cells (Sudha et al., 2007), if the ROS “quencher” is not 
available to terminate the reactions (Wulf, 2002; 
Akinwande and Adebule, 2003). Antioxidant supplemen-
tation, therefore, has been shown to be beneficial in 
stress-induced tissue damage (Senturk, 2001; Adenkola 



 
 
 

 

and Ayo, 2009). Vitamin C or ascorbic acid is a naturally 
occurring antioxidant (Sahin et al., 2001) and currently is 
the most widely used vitamin supplement throughout the 
world (Naidu, 2003). Ascorbic acid is an effective anti-
oxidant because it has an important metabolic role as a 
result of its reducing properties and function as an elec-
tron carrier. It can give up 2 electrons, and it is converted 
to dehydro-L-ascorbic acid (Rice, 2000; Sahin et al., 
2001). It has been established that ascorbic acid amelio-
rates heat stress and the adverse effects of stressful 
environmental conditions (Tauler et al., 2003; Adenkola 
and Ayo, 2006; Ayo et al., 2006; Minka and Ayo, 2007). 
The aim of the present study was to investigate the effect 
of ascorbic acid administration on the erythrocyte osmotic 
fragility of pigs transported by road for 4 h. 
 

 

MATERIALS AND METHODS 

 

Experimental site and meteorological conditions 

 

The experiment was performed at the faculty of veteri-
nary medicine, Ahmadu Bello, university, Samaru, Zaria 

(11
0
 10

/
 N, 07

0
 38

/
 E), located in the northern Guinea 

Savannah zone of Nigeria during the harmattan season. 
Harmattan season in Nigeria occurs between late-
November and early-March (Igono et al., 1982; Oladele et 
al., 2003). This zone is characterised by intensive live-
stock marketing and consequently, transportation of pigs, 
especially during the season. During the study period, 
wet and dry-bulb temperatures were determined before 
transportation at the experimental site using dry and wet-
bulb thermometers (Brannan, England) and rela-tive 
humidity (RH) was calculated using the manufacturer’s 
standard manual attached. The dry-bulb temperatures 
and RH were also recorded at 06:00, 13:00 and 18:00 h 
for three consecutive days post-transportation. 
 

 

Experimental animals and management 

 

29 local pigs served as subjects. They comprised males and 

non-pregnant, non-nursing females of different age groups, 

ranging from 9 to 12 months and bought in Zaria and its 

environs. The pigs were kept in a standard com-munal pen, 

made of concrete floor and iron walls with asbestos roofing. 

The pen measured 7.50 m x 2.55 m with half the length to 

the roof without block work, which provided adequate 

ventilation. The pigs were not res-trained inside the pen. 

They were kept under an intensive system of management. 

The pigs were pre-conditioned for 2 weeks before the 

commencement of the experi-ment. During the period, they 

were screened for haemo-parasites and endoparasites by 

taking their blood and faecal samples for analyses. Pigs 

found to be infected were treated using oxytetracycline 

(Kepro B. V
®

, Hollland) deep intramuscular at the dose of 20 

mg/kg and thiaben- 

  
  

 
 

 

dazole ((M.S.D AGVET
®

, U.S.A.) at the dose rate of 25 
mg/kg, respectively. 
 

 

Experimental design, transportation of animals and 
blood sample collection 

 

On the experimental day, 16 pigs were orally and 
individually administered ascorbic acid (Juhel® Nigeria 
Ltd., Enugu, Nigeria) at 250 mg/kg (Chervyakov et al., 
1977) dissolved in 20 ml of water, while 13 pigs which 
served as control were given 20 ml of sterile water. These 
administrations were made immediately before loading 
the pigs into the vehicle. Food and water were withdrawn 
12 h before the journey and throughout the journey 
period. The vehicle travelled from faculty of veterinary 
medicine, Ahmadu Bello university, Zaria on tarred road 
along Zaria-Jos road, covering a total distance of 140 km 
at a speed range of 40-50 km/h. The journey took 4 h 
(short-term duration), including stop-overs for police 
checkings. After completing the journey, the pigs were 
unloaded at the original loading point, fed and watered as 
they had been prior to the journey.  

Blood samples were taken early in the morning a day 
before transportation, immediately and a week after 
transportation. 5 mm of blood was drawn aseptically via 
the anterior vena cava using a 10 ml syringe and 18 

gauge x 1
1
/2 inch sterile needle from each animal. The 

blood was immediately poured inside a sample bottle, 
containing the anticoagulant, disodium salt of ethylene 
diaminetetra-acetic acid at the rate of 2 mg/ml of blood 
(Oyewale, 1992). After collection, the samples were 
transferred to Physiology Research Laboratory, Depart-
ment of Physiology and Pharmacology, Faculty of Veteri-
nary Medicine, Ahmadu Bello University, Zaria, where 
erythrocyte osmotic fragility test was carried out as des-
cribed by Faulkner and King (1970). 
 

 

Erythrocyte osmotic fragility determination 

 

Sodium chloride (NaCl) solution was prepared according 
to Faulkner and King (1970) in volume of 500 ml for each 
of the samples in concentrations ranging from 0.05 to 
0.85% at pH 7.4. A set of 10 test tubes, each containing 
10 ml of NaCl solution of concentrations, ranging from 
0.05 to 0.85%, were arranged serially in a test tube rack. 
One set was used to analyse each sample. The test 
tubes were labeled with corresponding NaCl concentra-
tion. 1 ml pipette was used to transfer exactly 0.02 ml of 
blood sample into each of the 10 test tubes. Mixing was 
performed by gently inverting the test tubes for about 5 
times. The test tubes were allowed to stand at room 

temperature (26-27
o
C) for 30 min. The contents of the 

test tubes were maintained at pH 7.4. Thereafter, the 
contents of the test tubes were re-mixed and centrifuged 
at 1,500 x g for 15 min. The supernatant of each test tube 



              

   Table 1.  Meteorological data from the study period pre-transportation.     
                

   Hour Ambient temperature (°C)    Relative Wind speed  

     Minimum Maximum   Dry-bulb  humidity (%)  (km/ day)  

   06: 00  13 24  14   24  226.43  

   13: 00  23 24  23   20  226.43  

   18: 00  21 22  21   19     

   Mean ± S. E. M 19.00 ± 3.1 23.33 ± 0.7  19.33 ± 2.7   21.00 ± 0.51     

 Table 2.  Meteorological data from the study period post-transportation.        
              

   Hour  Ambient temperature (C)   Relative   Wind speed 

     Minimum Maximum  Dry-bulb   humidity (%)  (km/ day) 

  06: 00  15 25   14    26   259.51 

  13: 00  24 26   24    20   259.51 

  18: 00  23 21   20    20     

  Mean ± S. E. M  20.67 ± 2.85 24.00 ± 1.53  19.33 ± 2.91  21.00 ± 0.67    
 

 

was transferred into a glass cuvette. The concentration of 
haemoglobin in the supernatant solution was measured 
using a spectrophotometer (Spectronic-20, Philip Harris 
Limited, Shenstone, UK) at 540 nm by reading the absor-
bance. The same procedure was repeated for every 
blood sample of each pig used for the study. The percent 
haemolysis was calculated using the formula (Faulkner 
and King, 1970). 
 

Haemolysis (%) = (OD of test/OD of distilled water) x 100 

 

Erythrocyte osmotic fragility curve was obtained by plot-
ting percent haemolysis against the saline concentra-
tions. 
 

 

Statistical analysis 

 

All data obtained were subjected to statistical analysis 
using student’s t-test. Data were expressed as mean ± 
standard error of mean. Values of P < 0.05 were 
considered significant. 
 
 
RESULTS 

 

Meteorological data 

 
The meteorological data from the study period are shown 
in Tables 1 and 2. The period was characterized by rela-
tively low values of minimum ambient temperature of 19.0  
± 3.1

o
C and low maximum ambient temperature of 23.3 ± 

0.7
o
C. The dry-bulb temperatures value obtained during 

the recordings was 19.3 ± 2.7
o
C. The harmattan season 

was characterized by relatively low humidity of 21.00 ± 
0.51%. The wind direction was North-east, and the speed 

 

 

was 226.43 km/ day.  
The meteorological data during the post-transportation 

period (Table 2) were similar to those obtained during the 
pre-transportation period (P > 0.05) (Tables 1 and 2). 
 

 

Effect of ascorbic acid administration on erythrocyte 
osmotic fragility of pigs transported by road for 4 h 

 

The minimum and maximum haemolysis of erythrocytes 
occurred at 0.85 and 0.20% in the experimental pigs, 
while the corresponding values in the control pigs were at 
0.85 and 0.50%, respectively, a day before the journey. 
All the values recorded at different concentrations before 
road transportation in experimental and control pigs 
(Figure 1) were not significantly (P < 0.05) different. Im-
mediately after the journey, the minimum and maximum 
values of haemolysis were obtained at 0.85 and 0.50%, 
respectively in the control pigs; while in the experimental 
pigs, the corresponding values were obtained at 0.85 and 
0.30%, respectively. At NaCl concentrations of 0.70, 0.80 
and 0.85%, significant (P < 0.05) differences existed 
between the values recorded in experimental and control 
pigs (Figure 2). However, on day 7 after transportation, 
significance difference (P < 0.05) existed at the concen-
tration of 0.40 and 0.50% between the experimental and 
control pigs (Figure 3), with the minimum value occurring 
at 0.85% in both experimental and control pigs and the 
maximum haemolysis occurred at 0.1 and 0.50% in the 
experimental and control pigs, respectively. 
 
 
DISCUSSION 

 
The results obtained in the present study demonstrated 
that the transported pigs were subjected to a cold and 



  
 
 

 
 120 

 

 100 
 

H
a

em
o

ly
si

s 80 
 

60  
  

%
 

 
 

 40 
 

 20 
 

 0 
   

0.85 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 
 

% NaCl Concentration 

 
Figure 1. Effect of ascorbic acid on erythrocytes osmotic fragility before 4 h of road transportation in pigs. 

 
 

 
 120 

 

 100 
 

 80 
 

o
ly

 s
is

 

 
 

H
ae

m
 

60  
 

 

%
 

 
 

 40 
 

 20 
 

 0 
   

0.85 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 
 

% NaCl Concentration 

 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 Experimental   
 Control  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 Experimental  
 Control 

 

 
Figure 2. Effect of ascorbic acid on erythrocytes osmotic fragility immediately (within 30 min) 4 h of road transportation in 
pigs. 



 
 
 
 
 
 
 
 
 
 
 
 

 

%
  

H
a

em
 o

ly
si

s 

 
 
 

 
120  
 
 

 
100 
 
 

 
80 

 
 

 
60 

 
 

 
40 

 
 

 
20 

 
 

 
0 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 Experimental   
 Control 

 

0.85 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 
 

% NaCl Concentration 

 
Figure 3. Effect of ascorbic acid on erythrocytes osmotic fragility on day 7 after 4 h of road transportation in pigs. 

 

 

dust-laden wind with low ambient temperature, classical 
of the harmattan season in the northern guinea savannah 
zone of Nigeria (Ayo et al., 1998a,b). Meteorological re-
sults obtained during the present study agree with the 
previous findings that the harmattan season is thermally 
stressful to pigs (Adenkola and Ayo, 2006; Adenkola et 
al., 2007).  

The erythrocyte osmotic fragility in hypotonic solution 
has been studied in various mammals (Olusanya and 
Adepoju, 1979; Oyewale, 1992) and it has been shown 
that it is related to geometric configuration of the erythro-
cytes, which in turn depends on the integrity of the cell 
membrane (Schalm et al., 1975). Before transportation, 
there was no significant difference between erythrocyte 
osmotic fragility of experimental and control pigs, but after 
the transportation the erythrocyte osmotic fragility was 
significantly different between the 2 groups. The re-sults 
showed, for the first time, that erythrocyte osmotic fragility 
test may serve as an indicator of road transport-tation 
stress in pigs and that it is of diagnostic value in stress 
due to road transportation of pigs during the har-mattan 
season.  

The maximum erythrocyte osmotic fragility obtained in 
this study disagreed with that of Oladele et al. (2003), 
who reported maximum erythrocyte osmotic fragility at 
0.15% NaCl concentration in chickens and guinea fowls 
during the harmattan season. The difference in the 

 
 

 

values may be attributed to species difference. The result 
of this study showed that road transportation stress, ap-
parently, mediates its adverse effects via oxidant mecha-
nisms. Thus immediately after transportation, haemolysis 
was higher in the control than experimental pigs. This 
finding was similar to those of Sumikawa et al. (1993) and 
Avellini et al. (1995) who observed that stress in-duced 
formation of free radicals which play a vital role in tissue 
damage and that they have deleterious effects on 
erythrocyte cytomembrane. Mossad et al. (2000) showed 
that haematological complications and autoimmne hae-
molytic anaemia observed in malignant lymphomas were 
attributed to oxidative stress. Thus, the results of the 
present study indicated that road transportation-induced 
stress enhances the generations of ROS, shown to in-
crease haemolysis (Langsford and Zydney, 1993). Al-
though free radicals were not measured directly in this 
study, it has been shown that they are generated in 
animals subjected to stress (Halliwell, 1996; Senturk et 
al., 2001; Chihuailaf et al., 2002). The observed increase 
in erythrocyte osmotic fragility in transported pigs fur-ther 
supports this fact. The higher haemolysis recor-ded in the 
control than experimental pigs post-transportation may be 
due to established impaired homeostatic mecha-nisms 
induced in animals not supplemented with ascorbic acid. 
The increase in ROS generation in the body, appa-rently, 
by road transportation stress on the body rendering cells 



 
 
 

 

more fragile and easily susceptible to hypotonic lysis. In 
contrast the lower percentage of haemolysis recorded in 
the experimental pigs was in agreement with 
observations of Senturk et al. (2001) and Candan et al. 
(2002) that ascorbic acid consolidates the integrity of 
erythrocyte membranes of and therefore reduces their 
oxidative damage. Oxidative stress occurs when the anti-
oxidant defence systems in the body are overwhelmed by 
free radicals (Williams et al., 2008). Ascorbic acid admini-
stration to experimental pigs apparently, reduced the 
intensity of oxidant stress by enhancing the antioxidant 
defense mechanisms and suppressing the transportation 
stress which greatly minimized the destruction of erythro-
cyte. Therefore, an increase in osmotic fragility of erythro-
cytes, indicating a higher percentage of haemolysis ob-
tained in control pigs may be due to exertional oxidant 
stress, prevented by ascorbic acid administration which 
increased the resistance of erythrocytes.  

It has been established that ascorbic acid ameliorates 
the adverse effects of environmental stress (Tauler et al., 
2003; Adenkola and Ayo, 2006; Minka and Ayo, 2007). 
Ascorbic acid has also been shown to prevent injurious 
effects of oxidants by the reduction of reactive oxygen 
and nitrogen species to stable molecules (Wilson, 2002). 
This fact may explain the finding in the present study that 
ascorbic acid administration in experimental pigs reduces 
erythrocyte osmotic fragility and the result is in agree-
ment with that of Candan et al. (2002) and Chihuailaf et 
al. (2002) that ascorbic acid is a stress induced “quen-
cher” capable of maintaining the integrity of the erythro-
cyte membrane.  

The results obtained in the present study, for the first 
time demonstrated that the administration of ascorbic 
acid prior to transportation of pigs is beneficial because it 
reduces the accompanied stress associated with road 
transportation notable erythrocytes’ fragility. It is, there-
fore, recommended that ascorbic acid be administered to 
pigs before transportation in order to reduce its adverse 
effects on pigs. 

 
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