Influence of Probiotic Supplementation on Oxidative Stress and Lipid Disorders in Quail

| Oxidative stress and ROS generation which induced via free radicals which up-raised by consumption of oxidized oils rich in polyunsaturated fatty acids, triggers inflammatory changes, DNA damage and oxidative induced tissue damage mainly in liver and kidney. Several feed additives were used to counteract such type of cellular stressors especially those which have antioxidant properties. In our current study, we checked the benefits of using lactobacillus acidophilus which are considered as feed additives and a novel inhibitor of food-induced oxidative stress. Our results confirmed the nobility of the lactobacillus acidophilus in counteracting and prevention of all cellular damage especially in liver and kidney which induced by oxidized oils consumed by quails. Furthermore its ability to reduce cholesterol levels in the blood and creating a balance between the pro-oxidant and the antioxidant levels. Our results strongly suggested the lactobacillus acidophilus can act as a potent prophylactic inhibitor of the cellular oxidative stress induced by the oxidized oils in food.


Advances in Animal and Veterinary Sciences
May 2020 | Volume 8 | Issue 5 | Page 479 To control OS several additives were used in food and feed, especially additives which have antioxidant properties such as Vit A, Vit C, tocopherols, selenium, polyphenols, palmitic acid and sesamol (Parkash & Kumar 2010).
Probiotics nowadays announced to hamper OS. As probiotics are pure cultures of normally inhabitant microorganisms isolated from milk of different ruminant species and colonized aseptically to be served as food or feed additives for its beneficial health values (Khalifa and Noseer, 2019). Huss et al 2008 have been used the quails instead of mice as an experimental model in research plans due to ease housing and management in collecting the blood samples besides it is economic.
This experiment was designed to evaluate the influence of probiotic against OS generated by oxidized oils in quail ration.

ExpErIMEntal DEsIgn
All experimental procedures were reviewed and approved by the Animal Care and Use Committee of Aswan University, Aswan, Egypt.
A total number of 45 quails aged 15 days old were introduced and housed together for 5 days and feeding on the same basal diet. Then the 45 birds were divided into three equal groups (n= 15), the first group fed on the basal diet and act as control, the 2 nd group (OXO) received basal diet mixed with oxidized oil (2%) and the 3 rd group (OXP) fed on oxidized oil 2% of basal diet plus probiotic in water in a dose of 2 g/L.
All groups were housed in clean cages at temperature arranged between 25:27 ºC.
The oxidized oil was added to diet as a mean of developing and generating OS.

oxIDIzED oIl
The oxidized oil was prepared thermally by heating at 180 Cº for 48 hours in a 1000 ml beaker in the oven and preserved at -20 °C. The chemical and physical analytical tests were performed to compare between fresh oil and thermally oxidized oil as Iodine Value. Through titration of fresh and oxidized oil against iodine. (Shafaeizadeh et al., 2011).

probIotIcs sourcE
The probiotics used in this experiment (consists of mixture of lactobacillus acidophilus 6 billion/cfu, Saccharomyces cervices 2 billion/cfu in a water soluble dextrose carrier) was purchased commercially and added to the drinking water as recommended dose.

ratIon forMatIon
The basal ration used in this experiment is in referring to that used by (Khalifa and Noseer, 2019).

saMplE collEctIon
The blood samples were collected from heart puncture of a live quail at age of 25, 35 & 45days with 10 days intervals. The blood samples were leaved to coagulate and centrifuged at 3000 rpm, and then the serum obtained and admitted for biochemical determination by micro lab 300 devices (ID 6788483548 France).

bIochEMIcal paraMEtErs
The next tests were applied on serum for evaluation of the probiotic effect against OS. Cholesterol, alanine transaminase (ALT) and aspartic acid transaminase (AST) as indicators to liver function, Kidney function expressed as creatinine & uric acid ,Oxidative stress biomarkers malonialdehyde (MDA) and total antioxidant capacity (TOC).

cholEstErol
Cholesterol measured by using 10ul of serum + 1ml of reagent at 546 nm wave length. Spectrum kits (Young et al., 1975).

KIDnEy functIon
Kidney test uric acid investigated by 1ml of serum + 1ml of reagent at wave length 546 and creatinine measured by 100 ul of serum + 1ml of working reagent at wave length 492 (Tietz, 1986) spectrum kits.

oxIDatIvE strEss
MDA was measured in serum by mixing 0.2 ml +1ml of chromagen boil in water bath for 30 min and read against 534 wave length (Ohkawa et al., 1979). According to explained in Biodignostic commercial kits. TOC was measured in serum by incubating 0.02 ml+0.5 R1 for 5 min at 37ºC and add 0.5 of working reagent at 505 wave length (Koracevic et al., 2001).

statIstIcal analysIs
Data, bars and markers in the figures were represent the mean ± s.d. We used the two-tailed Student's t-test, we compared each of a number of treatments with a single control. Differences were considered statistically significant when the P value and the significance were measured

rESultS And dIScuSSIon
Addition of the probiotics is a novel prophylactic way for avoiding the excess generation of ROS, hydrogen peroxides, other toxins and cellular stressors via promotion of beneficial microorganism in gut so it demonstrated as natural antioxidants (Mishra et al., 2015). Probiotics are member of live micro-organisms safe species exerts general beneficial health effects according to (FAO/WHO 2002). Lactobacillus species are considered as a beneficial example which exceed more than 50 species, and can be taken from fermented food like yogurts and dietry supplements .moreover lactobacillus species are mostly efficient for poultry (Bernardeau & Vernoux 2013). Probiotics bacteria have multiple therapeutic effects especially for regulating gut function. Probiotics are able to lower the cholesterol level due to inhibition of hydroxymethyl-glutaryl-coenzyme A (HMG COA), which is a key regulatory enzyme for cholesterol biosynthesis and converts cholesterol in the intestine to coprostanol which easily excreted (Khalifa and Noseer, 2019).
In this study the total cholesterol was increased in the quails feed on oxidized oil (OXO), while the group fed diet with probiotics (OXP) showed lower levels almost nearby to control group Figure 1.   In this study the oxidized oil found to be exerted harmful effects causing several alterations of lipid metabolism and also decreases activities of antioxidant enzymes and the major organ involved in lipid metabolism is the liver. Figure 4 and 5 measuring indicator enzymes of liver function tests such as alanine transaminase (ALT) and aspartic acid transaminase (AST) levels in serum of treated group with oxidized oil without antioxidants (OXO) was increased indicating negative liver function and consequently liver damage, while Oral administration of Lactobacillus acidophilus (OXP) significantly reduced the ALT and AST levels in serum of mice (Lin et al., 2018). Thermally treated oil can convert fatty acid configuration from cis to trans isomers. Trans isomers increases the incidence of hypertension and through on the broad line lead to cardiovascular disease (Bryk et al., 2011). We confirmed the decrease of cardiac biomarker AST,which is decreased in OXO group in compare to OXP & control group. As well as Haque et al. (2017) confirmed that decreased levels of transaminase enzymes ALT and AST indicated less or no liver damage while treated group with the oxidized oil (OXO) caused nephrotoxicity and hepato-toxicity seen by increased level of liver and kidney functions. nephrotoxicity and hepatotoxicity seen by increased level of liver and kidney func-

Advances in Animal and Veterinary Sciences
May 2020 | Volume 8 | Issue 5 | Page 481 tions. Therefore increased AST/ALT ratio predictive of the progression of fibrosis especially with AST elevated levels in non -alcoholic fatty liver diseases (NAFLD). (Botros and Sikria, 2013). Senanayake et al. (2015) found that mathematical transaminase ratio of AST/ALT increased with age especially in ALT in control and OXP treatment groups due to high metabolic demand by hepatocyte than OXO treated due to liver damage. Only ALT levels predict the progression to metabolic syndrome. While in the OXO treated group ( Figure 6) the AST/ALT ratio had lower due to liver damage and it worth noting that there weren't any general accepted reference of De Ritis ratio in birds.  Most antioxidants are heat labile and thermal heated oil more prone to oxidation leading to disturbance of endogenous antioxidants of living organisms & couldn't fight the oxidative stress. The mechanism of antioxidant system is to combine with free radicals, and so inactivated them thus increment of the free radicals intracellular concentration and increase the TOC (Mielnik et al., 2003). So increasing MDA levels in our study related to increase oxidative stress of OXO group in compared to OXP & control groups of quail may be triggerd by free radicals (Aytekin et al., 2015, Esgalhado et al., 2015. Also feeding oxidized oil lower the efficiency of antioxidant enzymes system and so decrease (TOC) & lower the capacity of scavenging free radicals, causing metabolic oxidative stress in quail consumed ration supplemented with oxidized oil (OXO) group in countrary to OXP & control groups. (Liang et al., 2015) (Figure 8).   Tan et al. (2018) had been referred to effect of lipid per-oxidation caused by oxidized oil in ration which known via the elevated levels of thiobarbituric acid reactive substances (MDA) in the internal organs which causing vigorous damage to these organs and the inflammatory changes which occurs and lower the ability to counteract the free radicals. Our study agreed with such hypothesis as we proven that; the quail fed only on the oxidized oils (OXO)

Advances in Animal and Veterinary Sciences
May 2020 | Volume 8 | Issue 5 | Page 482 showed remarkable and significant decrease in the antioxidant capacity in compare with the control group and the group which fed on probiotics plus oxidized oils (OXP) Figure 7. Thus MDA level measured in which considered as maker of oxidative stress-induced liver injury and oral administration of lactobacillus species inhibited increase of MDA level in serum or liver of oxidized oil induced oxidative stress mice (Lin et al., 2018).

concluSIonS
The probiotics especially lactobacillius spp have a significant effect in reducing oxidative stress. Probiotics must be provided in daily diets in order to preserve liver vitality. In this study we proven the efficacy of using the probiotic lactobacillus spp as a prophylactic feed additives to counteract the ROS generation and the cellular damage which induced by the free radicals produced by the oxidized oils in the ration.
A further work still needed to declare the actual mechanism by which the probiotic lactobacillus blocking the ROS generation via molecular analysis ROS/ antioxidant pathways and its gene regulation especially its role in regulation of the NRF2 gene which regulates the expression of antioxidant proteins that protect against oxidative damage triggered by ROS generation.