Ovaries and oocytes collection

Buffalo ovaries were collected from a local slaughterhouse (El-Batanoun–Menofeya governorate) during the breeding season (autumn and winter). Ovaries were removed immediately after slaughter in a thermos containing warm saline solution (0.9%; 30°C) supplemented with antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin). In the laboratory, ovaries were washed three times with warm (30°C) saline solution supplemented with antibiotics to remove adhering tissues and clotted blood on ovary surfaces. All visible follicles on surfaces (2–8 mm in diameter) were aspirated using a 20-gauge hypodermic needle attached to a sterile disposable 10 ml syringe. After aspiration, contents of the syringe were slowly dispelled into a sterile Petri dish (30 × 60 mm) to identify COCs under a stereomicroscope. Only compact cumulus–oocytes complexes (COCs) with at least four layers of complete cumulus cells and evenly granulated dark ooplasm were used for maturation.

Experimental design

Good quality COCs were randomly allocated during IVM (22 h) to one of four treatment groups: [1] control maturation medium (basic medium), [2] basic medium supplemented with ZnCl₂ (1.5 µg/ml), [3] Na₂SeO₃ (5 µg/l), or [4] ZnCl₂ + Na₂SeO₃ (1.5 µg/ml + 5 µg/l). These concentrations are based on Picco et al. (Reference Picco, Anchordoquy, De Matos, Anchordoquy, Seoane, Mattioli, Errecalde and Furnus2010) and Tareq et al. (Reference Tareq, Akter, Khandoker and Tsujii2012), respectively.

In the first four replicates, oocytes were matured for 22 h of IVM, fixed, and stained to evaluate the stage of nuclear maturation. Spent media were collected for biochemical assays of total antioxidant capacity (TAC), malondialdehyde (MDA) and hydrogen peroxide. Another four replicates were used for the control and treated COCs were used for RNA extraction. In total, 400 COCs were used in four independent replicates, allocating 25 COCs per treatment per replicate. RNA was reverse transcribed, and expression levels of antioxidant-related genes (SOD1, GPX4, CAT and PRDX1), antiapoptotic genes (BCL2 and BCL-XL) and proapoptotic genes (BAX and BID) were determined using a real-time polymerase chain reaction (PCR).

Preparation of maturation medium

Maturation medium was prepared as follows: 10 ml TCM-199 (Egyptian Organization for Biological Product and Vaccine, Agoza, Egypt), 6 mg/ml BSA(Sigma-Aldrich, A6003), 10 µg/ml epidermal growth factor (Sigma-Aldrich, E4127) and 50 µg/ml gentamycin (Sigma-Aldrich, G3632).

Medium was adjusted to pH 7.2–7.4 and 280–300 mOsm/l and sterile filtered using a 0.22-µm Millipore filter. Prepared maturation medium (500 µl/well) was placed into sterile 4-well dishes and equilibrated in 5% CO₂ in air at 38.5°C and high humidity for at least 2 h before the start of the experiment.

Oocytes maturation

Before in vitro maturation, recovered oocytes were washed twice in maturation medium in each treatment, then cultured in 4-well dishes (25 oocytes/well) and incubated for about 22 h in a CO₂ incubator (5% CO₂) at 38.5°C and maximum humidity.

Oocyte fixation and staining for nuclear maturation examination

After 22 h of maturation, COCs were removed from the maturation medium and denuded from cumulus cells by 5 min incubation and repeated pipetting in 2.9% sodium citrate. Oocytes (n = 15) in a drop of the appropriate medium were pipetted and mounted onto a glass slide. A coverslip with paraffin wax spots at every corner was put on the centre of a drop of the medium containing the oocytes. Thereafter, oocytes were examined under a stereomicroscope (Nikon SMZ645). The coverslip was pressed down on the oocyte until it was held in place. Oocyte fixation of the oocyte was carried out by placing the slides in a fresh mixture of acetic acid:ethanol (1:3, v/v) for 30 min. Slides were stained with a 1% aceto-orcein stain (1% orcein stain in 40% glacial acetic acid, w/v) for 3 min and washed with aceto-glycerol (3:1, v/v) then examined using phase-contrast microscopy (Leica DM500 with camera Leica ICC50HD) at high magnification (×400). The stage of nuclear maturation (Fig. 1) was defined using the morphology of the nuclear material (Hewitt et al., Reference Hewitt, Watson and England1998).

Figure 1.

Stages of nuclear maturation in buffalo oocytes determined by aceto-orcein staining after removal of cumulus cells. (A) Oocyte at germinal vesicle (GV) stage (vesicular nucleus with a distinctive nucleolus surrounded by fine filaments of chromatin). (B) Oocyte at germinal vesicle breakdown (GVBD) stage (chromatin condensation and disappearance of the nuclear vesicle). (C) Oocyte at metaphase of the first meiotic division (MI), formation of bivalents in which chromosomes are arranged equatorially on a metaphase plate. (D) Oocyte at anaphase of the first meiotic division (AI) stage, pulling apart of pairs of chromosomes to opposite ends of the meiotic spindle. (E) Oocyte at telophase of the first meiotic division (TI) stage, the two sets of chromosomes are separated. (F) Oocyte at metaphase of the second meiotic division (MII) stage, characterized by a tightly packed group of chromosomes in the form of the first polar body and another group that are well spread, allowing for the identification of individual chromosomes

Biochemical assay of maturation media

After 22 h of maturation, spent medium from each treatment was collected and preserved at −20°C until analysis. The concentrations of total antioxidants, TAC (Koracevic et al., Reference Koracevic, Koracevic, Djordjevic, Andrejevic and Cosic2001), MDA (Ohkawa et al., Reference Ohkawa, Ohishi and Yagi1979) and H₂O₂ (Aebi, Reference Aebi1984) were analyzed at wavelengths of 505, 534 and 510 nm, respectively in four runs using commercial kits (Biodiagnostic, Egypt) and spectrophotometry (Spectro UV-VIS Auto, UV-2602, Labomed, USA). All procedures were performed following the manufacturer’s instructions.

RNA isolation and cDNA synthesis

After maturation, control and treated COCs (pool of 25 COCs per replicate, four replicates) were washed twice in phosphate-buffered saline and snap frozen in cryo-tubes containing 20 µl of lysis buffer [0.8% IGEPAL, 40 U/µl RNasin (Promega, Madison, WI, USA), and 5 mM dithiothreitol (DTT) (Promega, Madison, WI, USA)]. Samples were stored in liquid nitrogen at −196°C until RNA isolation. Total RNA was isolated using a PicoPure^TM RNA isolation kit (MDS Analytical Technologies GmbH, Ismaning, Germany) following the manufacturer’s instructions. cDNA synthesis used oligo(dT)₂₃ primers, random primers, and SuperScript reverse transcriptase II (Invitrogen, Karlsruhe, Germany). In total, 1 μl of oligo(dT)₂₃ primer and 1 μl random primer were added to 10 μl of RNA sample, and the mixture incubated for 3 min at 70°C, then chilled on ice. Next, 8 μl of master mix containing 4 μl of 5× first strand buffer, 2 μl of 0.1 M DTT, 1 μl of dNTP (10 pmol/μl), 0.3 μl RNase inhibitor and 0.7 μl of SuperScript II™ reverse transcriptase (200 unit/μl) were added to the mixture and incubated for 90 min at 42°C, followed by heat inactivation for 15 min at 70°C. cDNA was stored at −20°C until further use.

Quantitative real-time PCR

Gene-specific primers were designed using PRIMER 3 Software (http://www-genome.wi.mit.edu/cgi-bin/prime/primer3-www.cgi). Primers design was based on cDNA sequences of eight transcripts deposited in GenBank (Table 1). Quantitative analyses of cDNA samples from groups of COCs were expressed relative to GAPDH gene expression (endogenous control) and run in separate wells using the Step One Plus real-time PCR system (Applied Biosystems). Independent qRT-PCR, four replicates per treatment, used a 20-μl reaction volume containing the iTaq SYBR Green master mix with ROX (Bio-Rad Laboratories, Munich, Germany), the diluted cDNA samples and specific forward and reverse primers. Thermal cycling parameters were: 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. After the end of the last cycle, a dissociation curve was produced by beginning fluorescence acquisition at 60°C and measuring at 7 s intervals until the temperature reached 95°C. Comparative cycle threshold (C_t) was used to quantify expression levels as previously described (Yadav et al., Reference Yadav, Singh, Singh, Saini, Palta, Manik, Singla, Upadhyay and Chauhan2013). Briefly, during data analysis, the C_t value of the housekeeping gene was extracted from the C_t value of the target gene to obtain change in C_t (∆C_t). The ∆C_t value of the target gene sample was subtracted from the calibrator (control) to get ∆∆C_t values. Difference in the transcript abundance for the target genes was calculated using 2^−∆∆Ct.

Table 1.

Details of primers used for quantitative real-time PCR

Statistical analysis

Mean percentages of parameters were statistically analyzed using analysis of variance (ANOVA) using the General Linear Model (GLM) procedures in SAS (SAS, 2007). Significant differences among treatments for maturation rate and oxidative biomarkers were tested using Duncan multiple range tests (Duncan, Reference Duncan1955) with significance levels of P < 0.05 and P < 0.0001, respectively. Values are expressed as mean ± standard error (mean ± SE).

Gene expression data were analyzed using the SAS GLM procedure. Differences in mean values were tested among treatment groups using ANOVA followed by Duncan multiple range tests with a significance level of P < 0.05.