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Effect of Polyvinylpyrrolidone on Vitrification of Buffalo (bubalus Bubalis) Oocytes

Jannatul Bari
Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M.N. Islam
Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md Hasanur Alam
Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

A. Khatun
Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M.A. Hashem
Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. Moniruzzaman
Corresponding author:
Department of Animal Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Vitrification, a method of rapid cooling, is an alternate cryopreservation method of oocytes and embryos. The present study was aimed to examine the effect of polyvinylpyrrolidone (PVP) on the vitrification of buffalo oocytes. Cumulus oocyte complexes (COCs) with fully grown oocytes (120-130 µm in diameter) were aspirated from slaughtered buffalo ovaries for vitrification. COCs were treated with equilibration solution at room temperature for 5 min and then transferred to a vitrification solution for 1 min. Then the COCs were submerged into liquid nitrogen (-196?C) for a while using cryotops. The COCs were thawed, diluted, and washed in a washing solution for 5 min, respectively. Vitrified oocytes were incubated for in vitro maturation (IVM) at 38.5⁰C under an atmosphere of 5% CO₂ in the air for 24 hrs. Cumulus cells surrounding the oocytes were removed mechanically, oocytes were fixed in acetic acid and ethanol, and stained with aceto-orcein to examine the meiotic stages of oocytes. The numbers of morphologically normal oocytes after vitrification were higher in 5% PVP than 0 and 10% PVP groups. A proportion of oocytes treated with 5% PVP reached the metaphase II (MII) stage while none of the oocytes from 0% and 10% PVP groups developed beyond anaphase I and metaphase I (MI) stages, respectively. These results show that PVP can be used as a cryoprotectant for the vitrification of buffalo oocytes.

Keywords: Buffalo, Cryopreservation, In vitro maturation, Oocytes, PVP

Location: In Bangladesh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Buffalo

Objectives:

To examine the effect of replacing DMSO with PVP on vitrification solution for cryopreservation of buffalo oocytes.

Methodology:

Chemicals: All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise mentioned. Collection of Cumulus Oocyte Complexes (COCs): Buffalo ovaries were collected from a local slaughterhouse and transported to the laboratory in 0.9% normal saline. The ovaries were washed in Dulbecco’s phosphate buffer saline (DPBS) solution supplemented with gentamycin sulfate (50 mg/mL) once and rinsed three times in DPBS. Visible antral follicles (4–6 mm in diameter) were aspirated using a 20-gauge needle attached to a 10 ml syringe to collect COCs. The COCs were screened under a stereomicroscope and washed three times in TCM-199 (pH 7.4, Nissui Pharmaceutical, Tokyo, Japan) containing 0.85 mg/mL NaHCO₃, 0.08 mg/mL gentamycin sulfate and 25 mM HEPES in a plastic dish (No. 1008, Falcon, Becton Dickinson and Company, Franklin lakes, NJ, USA) for vitrification. COCs containing healthy oocytes (120–130 µm in diameters) were selected based on their morphological appearances (uniformly granulated cytoplasm surrounded by multilayered compact cumulus cells) for vitrification [36]. Vitrification and Warming of Oocytes: Vitrification of COCs was performed following the procedure of our previous reports with some modifications. Briefly, the basic solution was TCM-199 containing 2.5 mg/mL HEPES, 2.47 mg/mL NaHEPES, 0.35 mg/mL NaHCO₃, and 0.05 mg/mL gentamycin sulfate. The equilibration solution was TCM-199 containing 7.5% (v/v) ethylene glycol (EG), 7.5% (v/v) dimethyl sulfoxide (DMSO) and 20% fetal bovine serum (FBS). The vitrification solution consisted of 15% (v/v) EG,15% (v/v) DMSO, 0.5M sucrose and 20% FBS in M-199 in control (0% Polyvinylpyrrolidone; PVP). In other two groups DMSO was replaced with 5 or 10% PVP (molecular weight 360,000). The warming solution was 20% FBS, 0.5 M sucrose in M-199 that contained 0, 5 or 10% PVP depending on the PVP concentrations of the vitrification solution. The dilution solution was 20% FCS in M-199 containing 0, 5 or 10% PVP. The washing solution contained 20% FCS in M199. In Vitro Maturation (IVM): The basic medium for oocyte maturation was TCM199 supplemented with 0.1 mg/ml sodium pyruvate, 0.08 mg/ml gentamycin sulfate, 5% (v/v) FBS and 100 ng/ml follicle-stimulating hormone (FSH; NIDDK, Washington, DC, USA). The vitrified and thawed COCs were washed three times in the IVM medium. COCs were placed in 100 µL droplet of IVM medium in 35 mm Petri dish under mineral oil, and incubated at 38.5^oC, 5% CO₂ in humidified air for 24 hrs. After 24 hrs, oocytes were observed under the microscope for cumulus expansion. Statistical Analysis: All data were subjected to one-way ANOVA, and the significance of difference among means was determined by Duncan's Multiple Range Test (DMRT). All statistical analyses were conducted using SPSS (IBM SPSS Statistics 22) software for Windows. Values of P < 0.05 were considered significant.

Source of Information: Journal of Buffalo Science, 2020, 9, 152-158

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The numbers of oocytes recovered after vitrification did not differ among the treatment groups. However, the number of morphologically normal oocytes was significantly (P<0.05) higher in 5% PVP than that of other groups. In a higher concentration (10%) and without PVP oocytes underwent to various abnormal morphological changes, e.g., shrinkage of oocytes cytoplasm, denudation of oocytes and dissociation of cumulus cells, etc. A proportion of vitrified oocytes treated with 5% PVP reached the MII stage while none of oocytes in 0or10% PVP group progressed beyond Anaphase I and MI stage, respectively. The percentage of oocytes at the MI stage was higher in 5% PVP (40%) than in other groups. The cumulus expansion in 5% PVP treated oocytes were also comparable with that in control group, although there were no significant differences among the PVP treated groups. The numbers of morphologically normal oocytes after vitrification were higher in 5% PVP than 0 and 10% PVP groups. A proportion of oocytes treated with 5% PVP reached the metaphase II (MII) stage while none of the oocytes from 0% and 10% PVP groups developed beyond anaphase I and metaphase I (MI) stages, respectively. These results show that PVP can be used as a cryoprotectant for the vitrification of buffalo oocytes.

Knowledge Management: Journal