Effect of ascorbic acid administration on erythrocyte osmotic fragility in rabbits (Oryctolagus cuniculus) subjected to road transportation

Abstract

The physiological responses of erythrocytes of rabbits transported by road for 2 h under hot-humid climate and the ameliorating effect of ascorbic acid (AA) were investigated. Nine rabbits served as experimental (treated) and were administered AA orally at a dose of 100 mg/kg body weight, while seven other rabbits served as control and were given equivalent of sterile water only. Thirty minutes after AA administration, the rabbits were transported. The erythrocyte osmotic fragility (EOF) values recorded in the control rabbits immediately after transportation and three days post-transportation were significantly (P < 0.05) higher than the pre-transportation values and the corresponding values obtained in treated rabbits. They were indicated by an increase in haemolysis on each point of the fragility curve. The result suggested that road transportation resulted in haemolysis of erythrocytes, which lasted for three days after transportation. Thus, the rabbits may require more than three days after transportation for their erythrocytes to return to base-line values. In treated rabbits, the transportation had no significant (P > 0.05) effect on the EOF. The result demonstrated that 2 h of road transportation of rabbits under adverse climatic conditions resulted in oxidative damage of erythrocyte membrane, which was alleviated by AA administration.

Keywords:

• ascorbic acid
• erythrocyte fragility
• haemolysis
• rabbit
• transportation stress

Introduction

There has been a rapid increase in rabbit (Oryctolagus cuniculus) production over the past years due to its use as a laboratory animal, source of good meat, fancy fur quality and as the third most available pet animal, apart from the cat and the dog. Thus, there is an increasing need to transport these animals from one place to the other. It has been established that road transportation is a major stress factor in farm and laboratory animals, exerting deleterious effects on health, performance and, ultimately, product quality (SCAHAW 2002, 2004; Lambertini et al. 2006; Minka & Ayo 2007; Averos et al. 2008). The major stress factors responsible for the loss of welfare during transportation are rounding-up, handling, loading, sudden and progressive changes in weather conditions, novelty of environment, noise and vibration from the vehicle, social disorder and deprivation of food and water. This subject has been adequately reviewed in many livestock species (Minka & Ayo 2009; Giannetto et al. 2011; Piccione et al. 2013). Obviously, the movement of rabbits from one laboratory to the other for the purpose of research may compromise welfare and expected experimental results, especially if the rabbits are transported under adverse environmental conditions and without adequate time to acclimatise. There exists strong legislation by the European Union and many other developed countries on the issue of animal welfare during transportation and the application of the three replacement, reduction and refinement (Rs; EFSA 2004, 2011; LASA 2005). Results of some studies have suggested management practices, such as administration of electrolytes (Schaefer et al. 1997) and antioxidant vitamins (Minka & Ayo 2007, 2010a) towards the alleviation of road transportation stress in livestock, including poultry. However, detailed legislations and studies on the effects of transportation in rabbits are inadequate (Lambertini et al. 2006; Liste et al. 2008, 2009), and management strategies towards ameliorating transportation stress in rabbits are currently lacking in the available literature. Haematological examination is often used in the assessment of the health status and adaptability of animals to various stress factors, including stress due to transportation by road (Tuli et al. 1995; Knowles et al. 1999; Minka & Ayo 2007, 2008). Of particular importance in this aspect are the erythrocytes, which are prone to oxidative damage by free radicals, often generated excessively during stressful conditions (Bansal et al. 1996). Erythrocyte osmotic fragility (EOF) test has been used experimentally as a measure of erythrocyte viability and clinically as a diagnostic tool (Orcutt et al. 1995; Minka & Ayo 2010b). Recently, EOF was demonstrated to be of value in the assessment of oxidative stress and as a biomarker of stress in humans during exercise (Tauler et al. 2003), in transported pigs (Adenkola & Ayo 2009; Asala et al. 2011) and goats (Minka & Ayo 2010b).

Ascorbic acid (AA) belongs to the first line of antioxidant defence system against a host of stress factors, especially oxidative stress (Balz 2003; Tauler et al. 2003). It is a potent chain-breaking antioxidant, involved in the prevention and termination of free-radical chain reactions and propagation, occurring in stressful situations (Balz 2003; Powers & Jackson 2008). AA has been suggested to be the most powerful and effective scavenger of reactive oxygen species than any other endogenous antioxidant in an aqueous environment (Balz 2003). The administration of the vitamin in humans has been shown to alleviate adverse effects of free radicals generated during exercise (Tauler et al. 2003). The free radicals generated as a result of the action of many stress factors, including road transportation that overtaxes the homeostatic mechanisms of the animals, overwhelm the natural antioxidants in the body (Minka & Ayo 2008, 2010a; Urban-Chmiel et al. 2009; Piccione et al. 2013). To date, there are no studies in the available literature on the effect of stress due to road transportation on EOF of rabbits and measures to mitigate the stress. Such information, if available, may improve the welfare of rabbits and allow the transporters to give sufficient time for acclimatisation, which will improve the quality of research findings in rabbits after transportation.

The aims of the present study were to establish the recovery period of erythrocytes of rabbits subjected to usual transportation under hot-humid conditions and to suggest the use of an antioxidant vitamin, AA as an ameliorating agent against transportation stress in rabbits.

Materials and methods

Animal management

Sixteen healthy rabbits belonging to both sexes, aged between 8 and 12 months and weighing between 1200 and 1800 g were used for the study. The rabbits were of different crossbreeds (New Zealand and local breed), characteristic of the rabbits reared in the zone of study. The rabbits were reared in standard individual metallic cages of 60 × 25 cm in the Livestock Farm, College of Agriculture and Animal Science, Kaduna (10° 31′ N, 7° 26′ E).

In tropical Africa, rabbits are commonly housed in individual cages, except for mating purposes when they are housed in pairs (Schiere & Corstiaensen 2004). The rabbits were given access to pelleted feed (proximate analysis: 14.5% crude protein, 7.2% crude fibre, 7% fat, 0.8% calcium and 0.4% phosphorus) and water ad libitum.

Experimental sites and procedures

The study was carried out during the peak of the hot-humid (rainy season) from 30 August to 9 September 2011 in Kaduna and Zaria (11° 04′ N, 7° 42′ E), located in the Northern Guinea Savannah zone of Nigeria. The transportation procedures which included handling, crating, loading and transportation proper were done according to guidelines on the transportation of laboratory animals (LASA 2005).

Dry- and wet-bulb temperatures were recorded during the study period seven days before and after transportation at 07:00, 14:00 and 18:00 h using a dry- and wet-bulb thermometer (COCET, Shenzhen = Guangdong, China). The relative humidity (RH) was calculated from the values obtained using the instruction attached by the manufacturer. Similarly, the dry-bulb temperature (DBT) and RH were recorded just before the transportation, 30 and 90 min into the journey, and on arrival at Zaria (at 2 h of the transportation) when the journey was completed. Three days to the transportation, the rabbits were randomly divided into treated and control groups, comprising nine (six males and three females) and seven rabbits (five males and two females), respectively, and base-line blood values were obtained.

On transportation day, 30 min before loading for transportation, the treated rabbits were administered orally using a gavage (Schiere & Corstiaensen 2004) with AA at the dose of 100 mg/kg, dissolved in 5 ml of sterile water. The control rabbits were administered similarly, but with only 5 ml of sterile water. During the administration of AA, the rabbits were placed in a restraining device to avoid unnecessary struggling and injury. Thereafter, the rabbits were transported on asphalt dual-carriage road, using a Honda vehicle (1989 Honda, Wagon-MPG model, stocked in rabbit cages at 350 cm² per rabbit) from Kaduna to Samaru-Zaria, covering a distance of 80 km at an average speed of 50 km/h. The stocking density used in the present study was near the normal commercial practice in the zone and was similar to 360 cm² per rabbit used by Liste et al. (2008, 2009), but less than the 600 cm² per rabbit recommended by EFSA (2004). The journey lasted 2 h. Blood samples were collected from all the rabbits on arrival, and subsequently on the third and seventh day of post-transportation period. Food and water were withdrawn from the rabbits 8 h before transportation and during the transportation period (Dal et al. 1997).

Measurement of EOF

At each period of blood sampling, about 2 ml of blood was collected from the rabbits via puncture of antecubical vein into a sterile test tube, containing an anticoagulant, sodium ethylenediaminetetraacetic acid at the ratio of 1 ml of blood to 1.5 mg of the anticoagulant. The needles were removed so as to prevent mechanical haemolysis, when the blood was being transferred to the collecting tubes. The test tubes, containing the collected blood, were quickly kept in ice pack and taken to the laboratory, where the blood samples were analysed for EOF. The EOF was determined as described by Beutler (1983). Briefly, 10 µl of blood sample collected from the rabbits was added to tubes with increasing concentrations of buffered-saline solution (pH: 7.4; 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9% of NaCl). The tubes were incubated at room temperature at 26°C for 30 min after mixing the blood gently. Thereafter, the samples were centrifuged at 150 × g for 10 min and the supernatant was collected. A spectrophotometer (Shimadzu UV160U UV-VIS, Shimadzu Corp, Kyoto, Japan) was used to determine the optical density of the supernatant at 540 nm. Haemolysis in each tube was expressed as a percentage, taking 100% as the maximum value of the absorbance of the distilled water (0% NaCl concentration).

Statistical analysis

The Number Cruncher Statistical System (NCSS) package was employed (NCSS, Kaysville, UT, 2001). The data obtained were expressed as mean ± standard error of the mean (mean ± SEM) and were subjected to Student's t-test to determine the difference between experimental and control. Repeated measures of ANOVA and Tukey's post-hoc test were used to determine the effects of sampling periods. Values of P < 0.05 were considered significant.

Results

Dry-bulb and relative humidity

The average DBT at the farm level before transportation was 33.0 ± 0.58°C with a maximum value of 34.0°C and minimum of 20.5°C, while the RH was 45–59%. The values were not significantly different from those recorded during the post-transportation period. During transportation the average DBT inside the vehicle was 31.3 ± 0.5°C, and the value fluctuated between the maximum and minimum values of 34°C and 30.0°C, respectively. The average value of RH recorded inside the vehicle was 59.3 ± 4.01%, and the maximum and minimum values were 61 and 48%, respectively (Table 1).

Table 1. Meteorological conditions inside the vehicle during the 2-h transportation period.

	Ambient temperature (AT, °C)			Relative humidity (RH, %)
Period	Mean	Maximum	Minimum	Mean	Maximum	Minimum
Pre-transport^1	27.6 ± 0.5	28.4	26.6	53.5 ± 5.4	59	48
30 min	30.5 ± 0.3	31.0	30.0	58.5 ± 5.2	59	58
1 h 30 min	31.4 ± 0.2	31.7	31.0	59.5 ± 4.3	61	58
2 h	32.4 ± 0.1	34.0	31.5	60.0 ± 3.2	61	59
Mean ± SEM	31.3 ± 0.5^a	41.3 ± 0.6^b	42.5 ± 0.4^c	59.3 ± 4.01	60.3 ± 5.2	58.3 ± 4.2

Note: Mean values with different superscripts alphabets within a row differ significantly (P < 0.05).

¹ Not included in calculation of the mean values.

Erythrocyte osmotic fragility

The mean EOF values (% NaCl concentration at 20, 50 and 80% haemolysis) for treated and control rabbits at pre- and post- transportation periods are shown in Table 2. The pre-transportation values of EOF recorded in both treated and control rabbits were not significantly different (P > 0.05). The median corpuscular fragility (50% haemolysis) of the erythrocytes occurred at NaCl concentration of 0.4–0.5%, while the highest (maximum, 80%) percentage haemolysis occurred at 0.1 and 0.2% NaCl concentrations in treated and control rabbits, respectively (Table 2).

Table 2. Mean ± SEM erythrocyte osmotic fragility values (% NaCl concentration at 20, 50 and 80% haemolysis) for treated (n = 9) and control (n = 7) rabbits at pre- and post-transportation periods.

Post-transportation period
	Pre-transportation		Immediately after		3 days		7 days
Percent	Treated	Control	Treated	Control	Treated	Control	Treated	Control
Minimum (20%)	0.589 ± 0.001^a	0.590 ± 0.001^a	0.712 ± 0.002^b	0.900 ± 0.005^c	0.701 ± 0.004^b	0.800 ± 0.001^c	0.600 ± 0.005^a	0.590 ± 0.003^a
Median (50%)	0.478 ± 0.002^a	0.500 ± 0.001^a	0.488 ± 0.003^a	0.700 ± 0.001^b	0.580 ± 0.001^a	0.667 ± 0.002^b	0.450 ± 0.001^a	0.451 ± 0.001^a
Maximum (80%)	0.100 ± 0.002^a	0.200 ± 0.002^a	0.140 ± 0.001^a	0.401 ± 0.001^b	0.180 ± 0.001^a	0.365 ± 0.001^b	0.110 ± 0.001^a	0.101 ± 0.003^a

Note: Mean values with different superscripts within a row differ significantly (P < 0.05).

Immediately (15 min) after transportation and on the third day following the transportation, the EOF rose from the pre-transportation values in both treated and control rabbits. However, the increase in fragility was significantly greater (P < 0.05) in control than in treated rabbits, as evidenced by a significant rise in the minimum (20%), median (50%) and maximum (80%) haemolysis values. Immediately and three days after transportation, 50% of the erythrocytes were haemolysed at NaCl concentrations of 0.6–0.7% in the control rabbits, and the values were higher (P < 0.05) than the corresponding values of 0.4–0.5% obtained in the treated rabbits. Similarly, the differences in the values between the treated and control rabbits were significant (P < 0.05) at 20 and 80%. Seven days after transportation, the EOF of the rabbits returned to base-line values (Table 2).

Discussion

The present result showed that rabbits subjected to the study had pre-transportation EOF values similar to those found in healthy rabbits (Thomason 1970; Kogawa et al. 1978), which suggested that the rabbits used in the present study were healthy. The DBT and RH values recorded were outside the recommended thermoneutral values of 10–30°C and 45–60%, respectively, established for rabbits (Animal Welfare Institute 2004). Thus, the thermal environment data showed that the rabbits were subjected to high AT and high RH, shown to induce heat stress in rabbits (Liste et al. 2009).

The significant increase (P < 0.05) in EOF of the rabbits immediately and three days after the transportation, especially in control rabbits, demonstrated that rabbits transported by road for 2 h had an increase in EOF. Furthermore, the transportation resulted in an increase in susceptibility of the erythrocytes to haemolysis. The rise in EOF obtained in the present study, due to handling, loading and transportation, was similar to the results obtained in pigs (Adenkola & Ayo 2009; Asala et al. 2011) and goats (Minka & Ayo 2010b), transported by road under hash environmental conditions. Although free radicals are constantly produced under normal conditions, the body is equipped with natural antioxidants that scavenge free radicals before they cause any damage to cells. However, as the stress factors continue to act on the body, an imbalance is created between pro-oxidants and antioxidants (Tauler et al. 2003).

The increase in EOF in the present study may be attributed to the effects of transportation procedures on the rabbits, which included rounding-up, handling, loading and vehicle motion and vibration, noise from the engine or moving vehicle, travel sickness and introduction to a new environment. In addition, the exposure of the rabbits to incremental thermal environment with temperature values higher than the recommended upper critical value of 30° C (Liste et al. 2009) may result due to heat stress. Although the rabbits were transported during the wet (rainy) season, the period was characterised by high ambient temperature and high RH, especially in the afternoon hours. High ambient temperature and high RH have been shown to induce heat stress in livestock and may increase free-radical generation in rabbits (Thomason 1970; Orcutt et al. 1995; Anwar et al. 2010) and in humans (Tauler et al. 2003; Powers & Jackson 2008). Besides, Liste et al. (2008) reported that transportation of rabbits even under optimal conditions is stressful to the animals.

The results of EOF obtained in this study demonstrated an increase in the membrane fragility of the erythrocytes of the control rabbits, apparently due to changes in the molecular properties of the erythrocyte membrane induced by free radicals. This factor has been reported to cause metabolic decay of the erythrocyte membrane, hence, its haemolysis (Piccione et al. 2007; Asala et al. 2011). Free radicals have been reported to initiate lipid peroxidation, which is the oxidative deterioration of the membrane lipids (Powers & Jackson 2008). The EOF result suggests the presence of anaemia in the rabbits and that the transportation stress factors decreased their natural antioxidant defence systems. Although the quantity of antioxidant was not measured in this study, EOF has been used and accepted as a biomarker of stress, a diagnostic tool for assessing transportation stress in animals and an appropriate model for the study of oxidative stress (Anwar et al. 2010; Minka & Ayo 2010b). The present result demonstrated, for the first time, the effect of road transportation stress under adverse climatic conditions on the erythrocyte fragility of rabbits, and that road transportation of rabbits resulted in oxidative damage of the erythrocyte membrane.

It is apparent from the result of this study that transportation by road may exert profound effects on rabbits in ways that may not be immediately obvious and that the animals require careful monitoring and adequate adaptation period following the transportation. The post-transportation EOF values obtained in rabbits administered with AA were not significantly different (P > 0.05) from the pre-loading values. This finding suggests for the first time that AA protected the erythrocytes from haemolysis in rabbits subjected to 2-h road transportation under unfavourable climatic conditions. The result clearly showed that AA administration 30 min prior to transportation ameliorated the adverse effects of handling, loading and road transportation on rabbits. The procedures are known to be stressful and associated with an increase in cortisol; the chief hormone of stress, responsible for several oxidative damages (Thomason 1970; Odore et al. 2004; Anwar et al. 2010). Similar ameliorating effects of AA against EOF were reported in transported pigs (Adenkola & Ayo 2009; Asala et al. 2011) and goats (Minka & Ayo 2010b) under extreme thermal environment. The mechanism by which AA protected the erythrocytes of the rabbits from haemolysis may be through the inhibitory role of AA on corticosterone (Karanth et al. 2000; Balz 2003), and its detoxifying and scavenging effects on free radicals generated during transportation (Seibert et al. 2001; Urban-Chmiel et al. 2009; Minka & Ayo 2010a). Besides, AA has been demonstrated to reduce the capacity of O₂ consumption through tissue oxidative metabolism and decrease heat load by enhancing mechanism of thermoregulation via increasing heat loss (Kuth & Forbes 1993), which may also decrease the generation of free radicals and further reduce lipopeoxidation.

The ameliorating effect of AA against haemolysis observed in the present study suggested that the administered AA replenished and booted the concentration of serum AA. Hemila (1997) earlier reported that AA at a dose of 1 g/day increases whole blood AA by 100–150% in humans. Since AA is cheap, easy to administer, non-toxic and tolerated at a high dose with no withdrawal period, it may be used to mitigate transportation stress and reduce the recovery period of rabbits following transportation.

In general, the results showed that rabbits that were not given AA and transported for 2 h under high ambient temperature, and high RH may require more than three days for the erythrocytes to fully recover from stress due to road transportation during hot-humid conditions. The results disagree with the findings of Liste et al. (2009), who did not measure EOF, but showed that 8-h lairage was enough for rabbits to recover from stress due to 3-h transportation by road. Similarly, studies in rats and in rabbits have recommended adaptation periods of 3 days and 48 h, respectively, yet periods of 3–5 days after transportation have been recommended for rats used in toxicology studies (Damon et al. 1986; Toth & January 1990; van Ruiven et al. 1998). The variations in the results may be due to differences in transportation conditions, breed of rabbit, season and variables measured. De la Fuente et al. (2004) showed that different seasons and stocking density exert different effects on transported rabbits, with rabbits transported in summer having higher level of stress than those transported in winter.

Conclusion

In conclusion, 2-h road transportation of rabbits during the wet season resulted in a significant increase in EOF. The administration of AA to rabbits 30 min before transportation decreased EOF induced by the transportation. AA mobilised some defence and compensatory mechanisms of the rabbits and protected the erythrocytes from the deleterious effects of road transportation.

Acknowledgements

The authors are grateful to the laboratory staff of the College of Agriculture and Animal Science, Kaduna, and Mr E. N. Nwosu of the Department of Veterinary Physiology and Pharmacology, Ahmadu Bello University, Zaria, Nigeria for their technical assistance.