Occurrence of oxidative stress in sheep during different pregnancy periods

www.afc.kg.ac.rs  Occurrence of oxidative stress in sheep during different pregnancy periods Tamer Tashla1, Milivoje Ćosić2, Vladimir Kurćubić3, Radivoj Prodanović1, Nikola Puvača1*  1 Department of Engineering Management in Biotechnology, Faculty of Economics and Engineering Management, Business Academy University, Cvećarska 2, 21000 Novi Sad, Serbia 2 Faculty of Agriculture, Bijeljina University, Pavlovića put bb, 76300 Bijeljina, Bosnia and Herzegovina 3 Department of Food Technology, Faculty of Agronomy Čačak, University of Kragujevac, Cara Dušana 34, 32102 Čačak, Serbia


Introduction
Free radicals are essentially unstable and reactive molecules formed as the by-products of oxidationreduction reactions and are categorized as reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Boukhenouna et al., 2018). Generally, the development of ROS is equivalent to their removal and animals are in the status of oxidative balance (Taverne et al., 2018). An inequity or imbalance can arise whenever the formation of ROS increases or there is a decrease in the rate of their removal through antioxidant systems Puvača et al., 2016;Tashla, 2021). This condition is called oxidative stress (Gessner et al., 2017). Oxidative stress means the status of oxidative overload, whether this term is used for the cell, organ or organism (Kryl'skii et al., 2019). Due to oxidative stress, the structure and functions of lipids, proteins, nucleic acids, and enzymes are damaged and consequently cause tissue damage (Barati et al., 2020). The oxidative damage of lipids through ROS is typically produced during various physiological statuses in a balanced, suitable amount of these radicals and is an adaptive defense against stress (Burgos-Morón et al., 2019). However, lipid peroxidation in excess is critical and initiates a selfstimulating chain reaction and consequently releases malondialdehyde (MDA) as a degradation by-product and is a crucial indicator of pro-oxidant evaluation of oxidative stress Beit-Hallahmi, 1972). Oxidative stress study is imperative in evaluating homeostasis disturbances and production in farm animals (Sinha et al., 2020). It is immensely hazardous because it does not reveal any symptoms and is identifiable with immense difficulty by various methods of analysis of antioxidant defense elements and products of oxidative stress in terms of ROS Tashla et al., 2019). Oxidative stress is a new field used to evaluate metabolic imbalances in farm animals for better maintenance and production of these animals. Very few conditions or statuses have been studied for determining the influence of oxidative stress in sheep when homeostasis is disturbed (Cao et al., 2020). There may be an imbalance in oxidative status during various physiological conditions like pregnancy (Cecchini et al., 2019), parturition (Alharthi et al., 2018), and lactation (Masters, 2018). Pregnancy is associated with dynamic fluctuations in metabolic activities, resulting in enhanced basal oxygen consumption and energy requirement (Lewis et al., 2020). Not only are appropriate hormones required for the establishment of the placenta, but extra nutrients are also needed for the development and growth of the fetus (Limesand et al., 2018). Thus, mother's reserves for nutrients are now mobilized to meet the demand, and consequently, the formation of ROS is enhanced, and both the fetus and the mother are facing oxidative stress. Negative energy balance is evident during late pregnancy, which consequently leads to oxidative stress, enhanced lipid peroxidation, and lowered activity of antioxidants (Kalyesubula et al., 2019). The National Research Council (NRC) has approved approximately 1.5 times more energy requirement of ewes for maintaining homeostasis in late pregnancy (Zhang et al., 2018). During parturition, the mother's physical attempt for fetus removal and hormones are the cause of metabolic imbalance and excess development of ROS. Increased demands for energy during early lactation initiate oxidative reactions, and electron flow increases, thus inducing ROS formation (Mavangira and Sordillo, 2018). For many centuries, sheep have been used for milk, meat, skin, fiber, manure, and physical work in different conditions (Tasić, 2018;Obućinski et al., 2019;Stojiljković et al., 2019). Some landless farmers in tropical arid areas keep sheep for personal use and sale. Sheep are very important due to their biological factors such as short generation interval, twinning, and short growth periods, and they do not require much space. Among various sheep breeds, the Lohi breed is famous for its high-quality meat and high growth rate and maximum income achieved through lamb production. It has a large and massive body with an average weight of 45-62 kg. Body color is white with a dark brown head, and drooping ears and tail are heavy and small. Lohi sheep account for 40% of national sheep production. Oxidative stress determination is the best tool for improved reproductive performance in sheep (El-Ratel et al., 2020) and cows (Majkić et al., 2017). As no significant information is available in the literature on Lohi sheep [48] on the physiological biomarkers concerning pregnancy and production at different stages, it is being hypothesized that these biomarkers during different stages of pregnancy and production will behave quite differently.
The paper aimed to evaluate oxidative stress during different pregnancy stages in Lohi sheep reared in Libya.

Materials and methods
A biological experiment with sheep was performed following the EU legislation and the Three Rs principle under the Directive 2010/63/EU.

Materials
All sheep selected for the investigations were healthy and cared for following all animal welfare standards. Early pregnant animals were fed alfalfa hay, corn silage, and concentrate; however, late pregnant animals were offered grass hay, wheat straw, corn, alfalfa, soybean, and wheat flour. The animals were given fresh clean water twice a day. All the animals were vaccinated as per the schedule and treated with antihelminthics after six months.
Clinically normal and healthy Lohi sheep aged 1.5-5 years were selected for the present study. Each group of animals was sub-grouped according to the stage of pregnancy into non-pregnant animals (control), early pregnant animals (40-50 days), mid pregnant animals (60-90 days), and late pregnant animals (100 days onward).

Blood sampling
Veterinarians were engaged in blood sampling during the studied period. Early morning blood samples were taken aseptically from the jugular vein from each animal at different stages of their pregnancy in the volume of 5 ml for EDTA-K2 and 5 ml for clotting.
Blood samples with anticoagulant and without anticoagulant were used for biochemical analysis.
The samples without anticoagulants were centrifuged at 167 × g for 15 minutes. The serum was harvested and preserved at -20 °C in small aliquots until further analysis in an accredited laboratory.

Biochemical analysis
To evaluate TAC, a semi auto-analyzer (Biosystems, BTS-330) was used for the spectrophotometric study. Monochromatic light (660nm wavelength) was selected from the spectrophotometer and used to heat the selected filter for about 5 min. Two hundred μl reagent was mixed with 5 μL serum/samples/standards. The first reading was taken before mixing reagent I with reagent II, which was used as a blank. Thereafter, 20 μL reagent II was added to the above mixture. This mixture was now incubated at 37°C for 5 min, and the second absorbance was read. Using the standard curve against the standards, delta absorbance was used to calculate TAC.
The oxidants present in the samples caused the oxidization of the O-dianisidine complex into ferric ion. Glycerol was used in this mixture to accelerate the reaction. Xylenol orange in the acidic medium formed a colored complex with ferric ions. The spectrophotometer was used to study color intensity, i.e. for the direct measurement of sample's oxidant molecules. Water was used for calibration purposes, and results were shown as μmol H2O2 equiv. L -1 . The assay was sensitive at 1.13 μmol H2O2 equiv. L -1, along with a precision rate less than 3% and linearity was shown to be 200 μmol H2O2 equiv. L -1 . Serum samples in aliquots of 35 μl were added into 225 μl reagent I, and the first absorbance was taken immediately. An 11 μl aliquot of reagent II was mixed in this serum sample and reagent I mixture, and after 4 minutes, final absorbance was read. Bichromatic wavelength containing main/primary 560 nm and 800 nm secondary/differential wavelengths was used for absorbance. The actual concentration of μmol H2O2 equiv. L -1 was calculated from the standard curve by using delta change in absorbance.
MDA was measured by the lipid peroxidation (LPO; μmol L -1 ) method. Kits (Abacum, UK) were equipped with components and instructions for their use and storage. LPO assay FTS reagent I, LPO assay FTS reagent II, lipid hydroperoxide standard, LPO Assay Extract R, and LPO assay Triphenylphosphine were prepared by using one vial of each component and stored as per instructions. Before initiating the LPO estimation method, lipid hydroperoxidase was extracted to form chloroform. One ml final volume of the assay was attained in each test tube. According to the instructions in the protocol, standards were run with each test simultaneously and in triplicate.
The method of Kostadinović et al. (2016) was used for the determination of serum SOD activity. Xanthine oxidase was employed to produce superoxide flux. Nitroblue tetrazolium (NBT) was used as an indicator of superoxide production. The degree of inhibition of the reaction unit of the enzyme was used to determine SOD activity, which would cause 50% inhibition for the reduction of NBT. Results were expressed in μ mL -1 . To prepare 0.052 sodium pyrophosphate buffer, 2.32 g sodium pyrophosphate was dissolved in 80 mL distilled water, and was adjusted to 8.3 by adding 1N HCL pH, then the volume was made up to 100 mL by distilled water and it was preserved at 4°C. For the preparation of 186 μM phenazinemethosulphate solution, 5.697924 mg phenazinemethosulphate was dissolved in 100 mL of distilled water. To prepare 300 μM nitrobluetetrazolium solution, 24.5292 mg nitrobluetetrazolium was dissolved in 100 mL of distilled water. To prepare 780 μM NADH solution, 51.74754 mg NADH was dissolved in 100 mL of distilled water. A total of 500 μL serum was added into 500 μL ethanol and 300 μL chloroform and centrifuged for half an hour at 18000 × g. A total of 900μL SOD reagent was prepared by mixing 0.1 mmol/L xanthine + 0.1 mmol/L ethylene diamine tetraacetic acid (EDTA) + 25 mmol/L nitro blue tetrazolium (NBT) + 50 mg serum (BSA) and 40 mmol/L Na2CO3 maintained at pH 10.2. The reagent was mixed with 50 μL of the supernatant removed after centrifugation, and incubation was done at 25°C for 20 min. By adding 1 mL CuCl2 (0.8 mmol/L), the reaction was stopped, and the absorbance of samples was read at 560 nm.
A spectrophotometer assay based on H2O2 was used to analyze catalase activity by Kostadinović et al. (2016) methodology. One ml substrate was prepared by mixing 65 μmol mL -1 H2O2 dissolved in 60 mmol L -1 sodium potassium buffer (pH 7.4). Incubation of 0.2 mL serum along with 1.0 mL substrate was done at 25°C for 1 min. The linearity of serum catalase was up to 100 KU L -1 . However, when catalase activity increased from 100 KU L -1 , phosphate buffer was diluted from 2 to 10 fold, and the assay was repeated. Under these circumstances, 1 unit catalase produced 1 μmol mL -1 H2O2 min -1 . The addition of 1.0 ml of 32.4 mmol L -1 ammonium molybdate [(NH4)6 Mo7O24.4 H2O] reaction was stopped by the production of yellow complex and this complex was read at 405 nm against blank 3.
Paraoxonase enzyme has the phenotypes PON-I, PON-II, and PON-III, with PON-I as the most active. PON I hydrolyzes several types of organophosphates such as paraoxon, aromatic esters such as phenylacetate, and lipid peroxidation products, and also reduces their accumulation. The rate of paraoxon enzymatic hydrolysis into p-nitrophenol was used to estimate PON I activity. A spectrophotometer was used to determine the color formed because of p-nitrophenol production. The primary hydrolysis rate or sensitivity of hydrolysis was constant until 5 min.

Statistical analysis
The data obtained were subjected to one-way analysis of variance (ANOVA) techniques. The data were analyzed by using the STATISTICA 13 statistical software. Duncan's Multiple Range test was applied to explain the significant difference between the stages of pregnancy.

Results and discussion
The results of oxidative stress evaluation are shown in Table 1. The total antioxidant status of Lohi sheep was observed by two-way ANOVA and the results showed significant (P < 0.05) differences. Lohi sheep exhibited a decrease in TAS concentration from non-pregnant to all subsequent stages, although only non-pregnant and advanced pregnant ewes were statistically different (P < 0.05). Lohi sheep showed higher values of TAS concentration through all stages. Findings have shown a significant decrease in TAS concentration from non-pregnant to advanced pregnant stages in sheep; however, significant results were given by non-pregnant and late pregnant ewes (Nawito et al., 2016). A new method to develop and evaluate TAS in animals was developed. Similar results were obtained by other studies, where the TAC value was highest in dry ewes and decreased with pregnancy progression (Nawito et al., 2016). As pregnancy is a stressful physiological condition, it causes an increase in cortisol concentration. The decrease in TAS might coincide with the absence of vitamins and mineral supplementation, i.e., exogenous antioxidants, during pregnancy. Similar results in reducing TAS during pregnancy were confirmed by different researchers (Nawito et al., 2016). Groups of sheep at various pregnancy stages showed significantly different results (P < 0.05) regarding the total oxidant status (TOS). The total oxidant status concentration increased from the first stage of non-pregnant animals to advanced pregnancy in the ewes. However, significantly (P < 0.05) different results were observed in mid and advanced pregnancy animals in ewes.
Similarly, TAS activity decreased just before parturition. TAC decreased during pregnancy as compared to non-pregnant groups. The oxidative stress index (OS 1), increa the steady gression of ls of total zymes, i.e., OD). er reports n-pregnant es in ewes e effect of resulted in 16). In our antioxidant superoxide oxide and CAT and nto water. n peroxide ter. (P < 0.05) t in noned in early, acts as an ainst lipid w density ding to our araoxonase in sheep groups. Non-pregnant animals had the maximum value of paraoxonase in our results. However, there is little information about serum PON-I activity in veterinary medicine, and scarce knowledge about the relationship between PON-I and arylesterase activity during reproductive performance and during lactation in sheep (Kanakkaparambil et al., 2009).

Conclusions
Prooxidant or antioxidant statuses and biochemical parameters are considered useful tools for evaluating oxidative stress during the physiological stress conditions of sheep pregnancy. Additionally, these tools might be helpful for improved management strategies under farm conditions in ewes. Animals face oxidative stress as indicated through an increase in the prooxidants MDA and TOS and a decrease in antioxidant capacity TAS, SOD, CAT, and PON-I with pregnancy progression. The use of oxidant and antioxidant markers and blood metabolic profiles is the recommended procedure for monitoring health status during this critical period and avoiding reduced productive performance and economic losses.