Oxidative costs of reproduction in mouse strains selected for different levels of food intake and which differ in reproductive performance

Oxidative damage caused by reactive oxygen species has been hypothesised to underpin the trade-off between reproduction and somatic maintenance, i.e., the life-history-oxidative stress theory. Previous tests of this hypothesis have proved equivocal, and it has been suggested that the variation in responses may be related to the tissues measured. Here, we measured oxidative damage (protein carbonyls, 8-OHdG) and antioxidant protection (enzymatic antioxidant activity and serum antioxidant capacity) in multiple tissues of reproductive (R) and non-reproductive (N) mice from two mouse strains selectively bred for high (H) or low (L) food intake, which differ in their reproductive performance, i.e., H mice have increased milk energy output (MEO) and wean larger pups. Levels of oxidative damage were unchanged (liver) or reduced (brain and serum) in R versus N mice, and no differences in multiple measures of oxidative protection were found between H and L mice in liver (except for Glutathione Peroxidase), brain or mammary glands. Also, there were no associations between an individual’s energetic investment (e.g., MEO) and most of the oxidative stress measures detected in various tissues. These data are inconsistent with the oxidative stress theory, but were more supportive of, but not completely consistent, with the ‘oxidative shielding’ hypothesis.


A-Reactive oxygen metabolites (ROMs) and Non-enzymatic antioxidant capacity (OXY)
The principle of ROMs assay is that the iron released from serum protein under an acid medium attacks hydroperoxide molecules present in serum and generate free radicals. These radicals react with chromogen to produce a stable coloured complex that can be measured by spectrophotometer. The serum and standard (10 μl each) were separately added to 1 ml of a reaction mixture containing 0.01 M acetetic acid/sodium acetate buffer (PH=4. 8) and N, N -diethyl-p-phenylenediamine as chromogen, and then incubated for 90 min at 37°C. When the reactive oxygen metabolites react with chromogen, they produce a complex colour in which its intensity is directly proportional to their concentration.
After incubation, the intensity of the colour was spectrophotometrically read at 505 nm at 37°C. The concentration of ROMs was calculated by dividing the absorbance of the sample and absorbance of the standard and multiplied by the concentration of standard. All samples, standards, and blanks were made in duplicate. The dROMS assay was expressed as mg H 2 O 2 /dL equivalents.
The OXY assay is based on the detecting the ability of non-enzymatic antioxidants to quench the oxidation effects of hypochlorous acid (HOCl). This assay has been previously used to measure total antioxidant capacity in different species [1][2][3][4][5][6] .The serum and standard were first diluted 1:100 with distilled water and then 10 μl from each of them was added to the 2ml eppendorf tube in duplicate. One millilitre of oxidant solution (HCIO based) was added to the blank, standard, and sample tubes and incubated for 10 min at 37°C. After incubation, the solution was poured into a cuvette already containing 10 μl of chromogen. The intensity of the colour was inversely proportional to the concentration of non-enzymatic antioxidants capacity in which the change in the colour was spectrophotometrically read at 505 nm at 37°C.
The concentration of OXY was calculated by using this formula OXY = (absorbance of blank-absorbance of sample)/(absorbance of blank-absorbance of standard)* standard. All samples, standards, and blanks were made in duplicate. The OXY was expressed as mM of HCIO.

B-Enzymatic antioxidant activities (CAT, SOD, and GPx)
Catalase activity is based on detecting the amount of H 2 O 2 that was decomposed by catalase activity using KMnO 4 . One hundred -eight microliters of supernatant was incubated with 18 ߤl of 10% Triton x100 on the ice for 30 minutes. For liver samples, the samples were diluted 1:20 with ice-cold 50Mm phosphate buffer before adding Triton x100. Sixty microliter of background sample (ice-cold 50Mm phosphate) and each sample was added to a 2 ml eppendorf tube in triplicate. Diluted H 2 O 2 (6mM) was added to the tube and incubated on ice for 3 min. The reaction was stopped by adding H 2 SO 4 (3M). Finally, KmnO 4 (2 mM) was added to the tube and absorbance was measured at 480nm. One unit of the enzyme activity was equivalent to k (U/min), where k=log (S o /S 3 ) × (2.3*V t * DF)/ (V S *T*X). S o = difference between absorbance of standard and background, S 3 =difference between absorbance of standard and sample, V t =total volume of sample, H 2 O 2 , and H 2 SO 4 , DF=dilution factor, V S = volumes of sample, T= incubation time, and X= amount of total protein (mg/ml).
The total SOD activity is based on the inhibition of the auto-oxidation of pyrogallol by the enzyme in the sample (with and without SOD) at 25°C and then followed kinetically at 420nm for 120 seconds with 2 seconds interval..The total mixture of reaction was constituted from 780 μl (50 mM Tris buffer), 10 μl supernatant of liver sample, and 10 μl of pyrogallol. For brain and mammary gland samples, 40 μl of their supernatants and 10 μl of pyrogallol were added to the reaction mixture in a total volume of 800 μl. The measurement was firstly preceded by a blank, containing only 10 μl pyrogallol in 790 μl Tris buffer, and followed by measuring a mixture containing the sample in triplicate. One unit of SOD activity was expressed as the amount of the enzyme causing 50% inhibition of pyrogallol oxidation. SOD activity was expressed as U/mg protein, where SOD activity = (% inhibition*DF*V t )/ (50*V S *X). DF=dilution factor, V t =total volume of sample, VS= volume of sample, and X= amount of total protein (mg/ml).
The GPx activity is based on the oxidation of NADPH by GPx in the presence of reduced glutathione (GSH) and hyroperoxide at 25°C. The total mixture of reaction was constituted from 4.28 mM sodium azide (to inhibit catalase activity), 1.07 mM EDTA, 4.286 mM GSH, 0.214 mM NADPH, and 1 U/mL of glutathione reductase in ice-cold 50-mM phosphate buffer. Twenty-five microliters of H 2 O 2 and a measured amount of sample, depending on the tissue (10, 25, and 50 μl in liver, mammary gland, and brain, respectively), were added to the reaction mixture. Reactions were followed kinetically at 340 nm for 60 seconds with 2 seconds interval in a total volume of 700 μl. The spontaneous NADPH oxidation reaction in the absence of enzyme was measured using a background (25 μl H 2 O 2, 10 μl of 50 mM Tris buffer, and 665 of reaction mixture) and then subtracted from the assay values. One unit of GPx was defined as the amount of enzyme that oxidized 1 mmol of NADPH per minute in the presence of reduced glutathione. Absorbance was read on a SPECTRA maxPlus microplate spectrophotometer (Molecular Devices Corp. Sunnyvale, CA, USA) and analysed using SOFT max Pro software (Molecular Devices Corp.).
Background and samples were made in triplicate. GPx activity was expressed as nmole NADPH/ min/ mg protein, where GPx activity = (absorbance of sample/ min*Vt*DF)/ (0.00622*Vs*X). The activity of all enzymes was expressed per mg protein. Total protein content for different tissue samples was measured using the method of Bradford 7 . Protein content of the supernatant was measured using a Bradford assay (Quick Start Bradford protein essay kit 2; Biorad Laboratories, Hemel Hempstead, UK).

C-DNA damage (8OHdG) measured by ELISA and HPLC-ECD methods
For ELISA method, 100 mg of frozen liver tissue was homogenised with 1 ml of cold Electrochemical detection was carried out using Dionex UltiMate 3000 HPLC electrochemical cell 6011 RS, with 8OHdG being detected at potential +300 mV while dG was detected at +800 mV. Prior to injection, samples were passed through 0.2µm RC filters (Phenomenex). Determination of 8OHdG was performed by injecting 10 μL while 1μL of diluted dG (10x times) was used for determination of dG. Standards and samples were made in duplicate and the damage expressed as a ratio between 8OHdG and 10* 6 dG.