Collection, aspiration, and classification of buffalo oocytes

Buffalo ovaries were collected from the Delhi abattoir. These ovaries were washed three or four times with antibiotic fortified (400 IU/ml penicillin and 500 μg/ml streptomycin) isotonic normal saline (32–37°C). The washed ovaries were transported to the laboratory within 6 h of slaughter. The oocytes were aspirated from surface follicles (2–8 mm diameter) (Madan et al., Reference Madan, Das and Palta1996; Shahid et al., Reference Shahid, Jalalib, Khanc and Shamid2014) with the help of an 18–19-gauge needle attached to a 10 ml syringe containing the aspiration medium (TCM-199 + 0.3% BSA + 0.0001 g/ml l-glutamine + 50 μg/ml gentamicin sulfate). The purpose of the follicles, 2–8 mm in diameter, was to retrieve numerous, and good quality oocytes for IVF experiments. The content of the syringe was collected in a 15-ml centrifuge tube to settle out the collected cells. The sediment at the bottom contained the oocytes, granulosa cells, and other cellular debris, which was poured into a 100 mm × 100 mm Petri dish. The oocytes were screened and washed with washing medium [TCM-199 + 10% fetal bovine serum (FBS) + 0.81 mM sodium pyruvate + 0.0001 g/ml l-glutamine + 50 μg/ml gentamicin sulfate]. The aspirated oocytes were graded according to the following criteria:

• Grade A: Compact cumulus–oocyte complexes (COCs) with an unexpanded cumulus mass having ≥5 layers of cumulus cells with homogenous evenly granular ooplasm.

• Grade B: COCs with 2 to 4 layers of cumulus cells and homogeneous evenly granular ooplasm.

• Grade C: Oocytes partially or wholly denuded or with expanded or scattered cumulus cells or with an irregular and dark ooplasm.

• Grade D: Oocytes with deformed or irregular ooplasm without cumulus cells.

These different grades of immature oocytes were used to analyze the expression of the pluripotency-associated genes in the study.

Preparation of buffalo follicular fluid

Buffalo ovaries were collected from Delhi abattoir and transported to the laboratory under cool conditions (∼4°C) within 6 h post collection. Follicular fluid was aspirated from all visible surface follicles (4–8 mm in diameter) using a 23-gauge needle. The follicles of about 4–8 mm diameter were chosen to aspirate the follicular fluid. Small-sized follicles of 2–3 mm diameter would have very less amounts of follicular fluid, therefore these small follicles were ignored, whereas large-sized follicles only (4–8 mm diameter) were targeted to aspirate the follicular fluid for IVF experiments. The cellular debris was removed by centrifugation at 12,000 rpm for 30 min at 4°C at least two times. The supernatant was collected and sterilized by passing through a 0.22-μm Millipore filter. The follicular fluid was distributed in 1 ml aliquots in microcentrifuge tubes and stored at −20°C until further use. This follicular fluid was used for the whole study to minimize the variation in gene expression data.

In vitro maturation of oocytes

Different grades of immature oocytes, i.e. A, B, C and D types, were used for expression of the pluripotency-associated gene transcripts. Buffalo immature oocytes were washed five or six times with washing medium and then twice with IVM medium (TCM-199 + 10% FBS + 5 μg/ml porcine FSH + 1 µg/ml estradiol-17β + 0.81 mM sodium pyruvate + 0.0001 g/ml l-glutamine + 5% buffalo follicular fluid + 50 μg/ml gentamicin sulfate). Approximately 20 A and B grades of immature oocytes were cultured in 100-μl droplets of IVM medium overlaid with sterile mineral oil in 35-mm Petri dishes and cultured for 24 h in a humidified CO₂ incubator (5% CO₂ in air) at 38.5°C. These matured oocytes were in vitro fertilized and then cultured until day 7 of post IVF. Petri dishes carrying IVM droplets (containing few cumulus/granulosa cells to the surface of the droplet) were kept at 38.5°C in a humidified CO₂ incubator. These IVM droplets were used for in vitro culture of the presumptive zygotes/embryos. Several trials were conducted to obtain the different stages of embryos, i.e. 2-cell, 4-cell, 8–16 cell, morula, and blastocyst to analyze the expression of the pluripotency-associated gene transcripts under the study.

Sperm preparation and in vitro fertilization (IVF)

Spermatozoa preparation for fertilization has been described by earlier workers (Chauhan et al., Reference Chauhan, Singla, Palta, Manik and Madan1998). Briefly, two French mini straws of frozen–thawed buffalo semen (Bull No. MU-5596) were washed twice with Brackett and Oliphant (BO) medium (medium containing 10 µg/ml heparin, 137.0 µg/ml sodium pyruvate, and 1.94 mg/ml caffeine sodium benzoate). The pellet was resuspended in ~0.5 ml capacitation and fertilization BO medium (washing BO medium containing 10 mg/ml fatty acid-free BSA). In vitro matured oocytes were washed twice with fertilization BO medium and transferred to 50-μl droplets of BO medium (~20 oocytes/droplet). For IVF, the spermatozoa suspended in 50 μl of BO medium (~3 million spermatozoa/ml) were added to the droplets containing the mature oocytes. IVF was conducted in 50-µl droplets of BO medium for which 50 µl of processed semen was added in each droplet (each droplet containing approximately 150,000 sperm). These droplets were covered with sterile mineral oil and placed in a CO₂ incubator (5% CO₂ in air) at 38.5°C for 18 h.

In vitro culture (IVC) of embryos

At the end of sperm–oocyte incubation period, cumulus cells were washed off from the oocytes by gentle pipetting/vortexing. Oocytes were then washed in a modified Charles Rosenkrans medium with amino acids (mCR2aa) containing 0.6% BSA fraction V and cultured for 48 h post-fertilization. After that, the embryos were moved to IVC medium (mCR2aa + 0.6% BSA fraction V + 10% FBS) and cultured in 100 µl IVC medium on the original beds (IVM droplets in which maturation of the cumulus–oocyte complexes (COCS) was performed) of granulosa cells for 9 days in a humidified CO₂ incubator (5% CO₂ in air) at 38.5°C.

During maturation, the COCs were removed from the droplets, however some cumulus/granulosa cells were shed and sloughed off from the matured COCs and became attached to the surface of the droplets. These granulosa cells may grow further and create bedding for the growing embryos. The presumptive zygotes/embryos were cultured in these droplets after IVF. The medium was replaced with 50% fresh IVC medium at 48 h intervals.

RNA isolation and cDNA preparation

Total RNA was isolated from different grades of immature oocytes separately and different stages of in vitro fertilized (IVF)-produced buffalo embryos using the TRIzol method (Invitrogen). Ten immature oocytes of each grade (A, B, C and D grades) were taken for RNA isolation. IVF-produced embryos from different trials were pooled and the RNA was isolated from each stage using TRIzol reagent. Similarly, different stages of embryos, namely 2-cell (n = 90), 4-cell (n = 340), 8–16 cell (n = 205), morula (n = 135), and blastocyst (n = 129) collected from different trails, were used for RNA isolation. About 200 µl TRIzol reagent was added for each grade of immature oocytes and mixed thoroughly by vortexing for 4–5 min, then kept at room temperature for 10 min. Total RNA was isolated from immature oocytes as per the manufacturer’s protocol (Invitrogen). Here, 500 µl TRIzol was added for each grade of embryo, and other reagents were added according to the manufacturer’s recommendations. The dried RNA pellet was dissolved in 10 µl (for immature oocytes) and 20 µl (for embryos) diethylpyrocarbonate (DEPC)-treated water. RNA was treated with 1 µl DNase I (Fermentas) at 37°C for 30 min to remove any DNA contamination. DNase I was inactivated using DNase inactivation reagent (Ambion) as per the manufacturer’s recommendations. RNA was quantified using a NanoQuant reader (Tecan).

cDNA was prepared from total RNA of different grades of immature oocytes and various stages of embryos using a single-stranded cDNA synthesis kit (RevertAID, Fermentas). About 5 µl (10–50 ng/µl) of total RNA of oocytes and embryos were used for cDNA preparation; 1 µl oligo(dT) (supplied with the kit) and 6 µl of DEPC water were added and mixed. The samples were incubated at 65°C for 5 min followed by 4 µl of 5× RT buffer, 1 µl of Ribolock (RNase inhibitor, 20 U/µl), 2 µl of 10 mM dNTPs mix, and 1 µl of Moloney murine leukemia virus reverse transcriptase (M-MLV RT, 200 U/µl) was added in each tube. A negative RT control (all components except reverse transcriptase) was include to negate DNA contamination in cDNA synthesis. Samples were incubated at 42°C for 1 h followed by 70°C for 5 min. The quality of cDNA was checked by measuring the A₂₆₀/A₂₈₀ OD ratio and the quantity was calculated by NanoQuant reader (Tecan).

Amplification of cDNA by GAPDH and pluripotency-associated genes

For expression of the individual genes under the study, primers were designed using Primer3 software based on published nucleotide sequences and on the sequence homology (ClustalW software) between mouse, human, sheep, goat, pig, and cattle. After synthesizing the cDNA from oocytes and embryos, cDNA were confirmed by presence of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene. For this purpose, initially, a reverse transcription-PCR (RT-PCR) assay was performed on cDNA of different grades of immature oocytes and various stages of embryos to detect the presence of GAPDH as a housekeeping marker gene. The GAPDH gene was amplified using forward primer (5´-CTCCCAACGTGTCTGTTGTG-3´) and reverse primer (5´-TGAGCTTGACAAAGTGGTCG-3´) (Coussens and Nobis, Reference Coussens and Nobis2002). Expression levels of the pluripotency-associated genes (NANOG, OCT4, SOX2, c-MYC and FOXD3) were analyzed in immature oocytes and various stages of buffalo embryos using RT-PCR. The PCR reaction components were: 1× PCR buffer with NH₄(SO4)₂, 1.5 U Taq DNA polymerase, recombinant (Fermentas), 200 μM dNTPs mixture, 1.5 mM MgCl₂ and 10 pM of each primer, and 3 µl (~600 ng) of template cDNA. RT-PCR amplification reaction conditions for pluripotency genes were as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for the 30 s, annealing at 56°C/57°C for 30 s, and extension at 72°C for 30 s. The final extension was carried out at 72°C for 10 min for all genes. Negative RT and negative PCR controls were included to check DNA contamination and ascertain the authenticity of PCR amplification. PCR primer sequences, annealing temperature, and their product sizes are given in Table 1.

Table 1. Primer sequences for the pluripotency-associated genes

Relative abundance of pluripotency-associated genes in buffalo oocytes and embryos using RT-qPCR

Reverse transcription qPCR (RT-qPCR) was performed on a CFX 96 system (Bio-Rad) using SYBR green (double-stranded DNA specific fluorescent dye), i.e. Light Cycler 480 SYBR Green I Master (2×) (Roche Diagnostics, GmbH, Germany) and Maxima SYBR green/fluorescein qPCR Master Mix (2×) (Fermentas). Here, 2.0 µl of cDNA (200 ng/µl) from each grade of oocytes and each stage of embryos were added to the 5.0 µl of 10× SYBR green master mixture (containing dNTPs, Taq DNA polymerase, MgCl_2, and buffer) in triplicate along with two no template controls (NTC). About 0.2 µl of each gene-specific forward and reverse primer was mixed with the above sample and then the reaction was adjusted to 10 µl with nuclease-free water. The reaction was prepared in 96-well plates in triplicate with two NTC for each gene. Amplification conditions for all pluripotency genes were as follows: initial denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 10 s, annealing X°C (respective temperature for each gene listed in Table 1) for 25 s, and extension at 72°C for 25 s. A melting curve was given from 65 to 95°C with 0.5°C increments for 0.05 min.

The specificity of RT-qPCR was confirmed by analysis of the melting curve through CFX manager software. Amplification results were analyzed by considering the C_t value for the target gene in the oocytes and embryos as a calibrator, and the housekeeping GAPDH gene was used as the control (Normalizer). After each reaction, a dissociation curve (melting curve) was run to check the specificity of the PCR product. During analysis, the C_t value for the housekeeping gene was subtracted from the C_t value of the target gene to obtain the change in C_t (ΔC_t). The ΔC_t value of the calibrator was subtracted from the ΔC_t of the target sample to get the ΔΔC_t values (Livak and Schmittgen, Reference Livak and Schmittgen2001). The ΔΔC_t method was used to calculate the expression fold difference in mRNA transcript for the target genes using the equation 2^-∆∆Ct, by assuming that the PCR amplification efficiency was two (Livak and Schmittgen, Reference Livak and Schmittgen2001).

Statistical analysis

Data were analyzed using SYSTAT v.7.0 software (SPSS Inc. Chicago, IL, USA). Data were grouped and presented as means ± SE and subjected to analysis of variance. Pairwise comparison of means was carried out using the least significance difference test and SYSTAT v.7.0. Differences were considered significant at P < 0.05.