Scientific Papers

Protective effect of soursop (Annona muricata linn.) juice on oxidative stress in heat stressed rabbits | Journal of Animal Science and Technology


The study was conducted at the rabbit unit of Teaching and Research Farm, Department of Agricultural Technology, Federal Polytechnic, Ado-Ekiti, Nigeria. The study area is located between latitude 7o3′7″N 7′12″N and latitude 5o11″E and 5o 31″E. The temperature-humidity index (THI), an indicator of thermal comfort level for animals in an enclosure was calculated as modified by Marai et al. [5] and given as:

$$ \mathrm{THI}=\mathrm{t}-\Big[\left(0.31-0.31\ \mathrm{x}\ \mathrm{RH}\ \mathrm{t}-14.4\right) $$

Where RH = relative humidity /100. t = ambient temperature.

The values of THI obtained for rabbit are classified as:

< 27.8 °C = absence of heat stress,

27.8–28.9 °C = moderate heat stress,

28.9–30 °C = severe heat stress and.

above 30 °C = very severe heat stress (Marai et al.,) [5].

The study was conducted between December 2017 and January 2018 with average daily THI of 29.94 ± 1.56 and 28.59 ± 3.10 at the rabbit unit, which is the peak of the dry season in the study area at the time.

Collection and extraction of soursop juice

Ripe soursop (Annona muricata) fruit was harvested fresh and it was extracted in the Teaching and Research Farm of the Agricultural Technology, Federal Polytechnic Ado –Ekiti, Nigeria. The fruits were peeled, weighed and the fruit pulp blended (without the seeds) with the ratio of 1:2 (pulp: distilled water). The soursop juice was clarified with juice extractor (Mikachi model No 1706). The juice obtained was designated as soursop juice (SSJ) and kept frozen 4 °C in disposable 5 ml sterile sample bottles until its required for use.

Experimental animal and management

Sixty (60) mixed breed (New Zealand white x Chinchilla) male rabbit bucks kept in a battery cage system kept in an open-sided house were used for the experiment. The rabbit house has natural lighting program (12D:12 L), with the dynamic ventilation system. The experimental units are made of wire mesh double sided boxes of 1.1sq.m. The animals were fed ad libitum with diets containing crude protein 17.05%, digestible energy 2592.06 Kcal/kg, crude fibre 10.02%, calcium 0.45% and phosphorus 0.21% as shown in Table 1. Freshwater was made available to the animals always. Other routines and periodic management practices necessary for rabbit production were carried out.

Table 1 Gross composition of experimental diet

The bucks were adult of 12-18 months old with the average weight of 1826 ± 8.35 g. 60 bucks were randomly allotted to five experimental treatments of four replicates (3 bucks per replicate) per treatment in a completely randomised design. SSJ was administered per kg body weight (BW), designated as treatment 1 to 5, administered 0.55mlkg−1BW distilled water (control), 0.55 mlkg−1BW SSJ, 1.11 mlkg−1BW SSJ, 1.67 mlkg−1BW SSJ and 2.22 mlkg−1BW SSJ respectively. Each treatment consists of 12 bucks. The soursop juice was administered orally to the rabbit in the morning between 8:00 am and 8:30 am daily to all bucks in a study that lasted for eight weeks.

Sample collection and analysis

A baseline blood sample was collected from all rabbits at least temperature humidity index, to serve as positive control as non-heat stressed treatment. The fastened blood sample was collected at 28 days and 56 days of the study. Blood was collected through the marginal ear vein into a sample bottle. Serum was separated by centrifugation (Medi Scan centrifuge model No 800D) and stored at -2 °C before analysis. Serum obtained was assayed for total protein, albumin, cholesterol, and triglyceride using commercial assay kits and its procedure, lipid peroxidation, reduced glutathione, catalase, and superoxide dismutase activity using standard procedures.

Superoxide dismutase (SOD); to 2.1 ml of 50 m M butter, 0.02 ml of enzyme source and 0.86 ml of distilled water. The reaction is initiated with 0.02 ml of 10 mM pyrogallol and change in absorbance monitored at 420 mm. One unit of SOD is defined as that amount of enzyme required to inhibit to auto-oxidation of pyrogallol by 50℅ in standard assay system of 3 ml. The specific activity is expressed as unit /min/mg protein.

Catalase activity; the assay system contain 1.9 ml,0.05 M buffer PH 7.0 and 1.0 ml 0.059 M water. The reaction is initiated by addition of 0.1 ml enzyme source. The decrease in absorbance is monitored at 1 min interval for 5 min at 240 min and activity is expressed as moles of water decomposed /min/mg protein.

The assay for lipid peroxidation; The reaction mixture in a total volume of 3.0 ml contained 1.0 ml serum, 1.0 ml of TCA (0.67%). All the test tubes were placed in a boiling water bath for a period of 45 min. The tubes were shifted to the ice bath and then centrifuged at 2500 rpm for 10 min. The amount of malondialdehyde (MDA) formed in each of the samples was assessed by measuring the optical density of the supernatant at 532 nm.

The glutathione (GSH) content; in which 1.0 ml of PMS fraction (10%) was mixed with 1.0 ml of sulphosalicylic acid (4%).The sample was incubated at 4 °C for at least 1 h and then subjected to centrifugation at 1200 rpm for 15 min at 4 °C. The assay mixture contained 0.4 ml filtered aliquot, 2.2 ml phosphate, buffer (0.1 M, PH 7.4) and 0.4 ml DTNB (10 mM) in a total volume of 3.0 ml. The yellow colour developed was read immediately at 412 mm.

Statistical analysis

Data obtained were subjected to analysis of variance at α = 0.05, the general linear model procedure of SAS, while means were separated using Duncan’s multiple range test of same software.

The statistical model is as the following:

$$ {\mathrm{Y}}_{ijl}=\upmu +{\mathrm{B}}_i+{\mathrm{e}}_{ijl} $$

Where Yijl represents the value of serum biochemical parameters and oxidative status measured in the lth animal; μ is the overall mean for each character; Bi is the fixed effect of ith Soursop (SSJ) was administered 5 levels (i = 0.55 mlkg−1BW water (control), 0.55 mlkg−1BW SSJ, 1.11 mlkg−1BW SSJ, 1.67 mlkg−1BW SSJ and 2.22 mlkg−1BW SSJ); and eijl is the random residual effect.



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