Effects of exposure to heavy metals on viability, maturation, fertilization, and embryonic development of buffalo (Bubalus bubalis) oocytes in vitro

Abstract

The aim of the present study was to examine the effect of heavy metals, cadmium and lead, on buffalo oocyte viability and in vitro development. Oocytes were aspirated from ovaries of slaughtered buffaloes. Only viable and metabolically active oocytes with more than three layers of cumulus cell layers and homogeneous ooplasm were selected. Effects of nine concentrations (0, 0.005, 0.05, 0.5, 1.0, 1.5, 2.5, 5, and 10 μg/mL) of cadmium or lead on buffalo oocyte viability, morphological abnormities, maturation, and embryonic development in vitro were studied. Oocytes were cultured for 24 h and then checked for viability (0.05% trypan blue staining for 2 min), morphological abnormalities, and reduction assay by MTT test in experiment 1. The doses of cadmium and lead causing 100% oocyte death (1-day culture) were determined (experiment 2). In experiment 3, viable oocytes were matured in vitro in media containing different levels of cadmium or lead and then inseminated in vitro with frozen-thawed spermatozoa, and the resultant cleaved embryos were cultured in a control embryo culture medium for 8 days. In experiment 4, oocytes were cultured in control oocyte maturation medium, then fertilized, and the resultant embryos were cultured in media containing different levels of cadmium or lead for 8 days. The number of cells in the trophectoderm and inner cell mass (ICM) and the total cell counts (TCN) of blastocysts derived by in vitro culture of two- to four-cell-stage embryos (produced in control medium) in media containing 0, 0.005, 0.05, 0.5, and 1.0 μg/mL of cadmium or lead were analyzed by differential staining technique (experiment 5). Cadmium and lead were found to have a dose-dependent effect on viability, morphological abnormities, maturation, cleavage and morula/blastocyst yield, and blastocyst hatching. A significant decline in viability of oocytes was observed at 1.0 mg/mL cadmium or lead compared to the control group. The doses of cadmium and lead causing 100% oocyte death (1-day culture) were 18 and 32 μg/mL, respectively. Cadmium and lead at 1.0 and 2.5 μg/mL, respectively, caused a significant reduction of maturation of oocytes compared to the lower concentrations. No cleavage or morulae/blastocysts were produced when the oocytes/embryos were cultured in media containing 2.5 and 5.0 mg/mL of either cadmium or lead, respectively. Similarly, no morulae/blastocysts were produced from cleaved embryos cultured in media containing 2.5 and 5.0 μg/mL cadmium and lead, respectively. The developmental block, degeneration, and asynchronous divisions were higher in embryos exposed to cadmium than in those exposed to lead. TCN and number of cells in ICM were significantly lower in blastocysts derived from two- to four-cell-stage embryos cultured in media containing heavy metals. In conclusion, cadmium and lead lowered the viability and development of buffalo oocytes but at a concentration higher than that estimated in the body fluids and environment. Cadmium was found to be more ovotoxic than lead.