Verification of buffalo ( Bubalus bubalisi) oocytes

Abstract

The objective of the present study was to develop a method for the cryopreservation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Prior to vitrification of COCs in the vitrification solution (VS) consisting of 4.5 M ethylene glycol, 3.4 M dimethyl sulfoxide, 5.56 mM glucose, 0.33 mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), the COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 °C). The COCs were then placed in 15-μL of VS and immediately loaded into 0.25-mL French straws, each containing 150 μL of 0.5 M sucrose in DPBS. The straws were placed in liquid nitrogen (LN 2) vapor for 2 min, plunged and stored in LN 2 for at least 7 d. The straws were thawed in warm water at 28 °C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min and then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffalo serum). The proportion of oocytes recovered in a morphologically normal form was significantly higher (98 and 88%, respectively; P < 0.05), and the proportion of oocytes recovered in a damaged form was significantly lower (2 and 12%, respectively; P < 0.05) for the 3-min equilibration than for 1 min. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50-μL droplets (10 to 15 oocytes per droplet) of maturation medium (TCM-199+15% FBS+5 μg/mL FSH-P), covered with paraffin oil in a 35-mm Petri dish and cultured for 26 h in a CO 2 incubator (5% CO 2 in air) at 38.5 °C. Although the nuclear maturation rate did not differ between the 1- and 3-min equilibration periods (21.5 ± 10.7 and 31.5 ± 1.5%, respectively), the between-trial variation was very high for the 1-min period. This method of vitrification is simple and rapid, and can be useful for cryopreservation of buffalo oocytes.