Vitrification of buffalo (Bubalus bubalis) oocytes

The objective of the present study was to develop a method for the cryopres ervation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COC s) were obtained from slaughterhouse ovaries. Prior to vitrification of COC s 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), th e COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 degrees C). The COCs were then pla ced in 15-mu L of VS and immediately loaded into 0.25-mL French straws, eac h containing 150 mu L of 0.5 M sucrose in DPBS. The straws were placed in l iquid nitrogen (LN2) vapor for 2 min, plunged and stored in LN2 for at leas t 7 d. The straws were thawed in warm water at 28 degrees C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min an d then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffal o serum). The proportion of oocytes recovered in a morphologically normal f orm was significantly higher (98 and 88%, respectively; P<0.05), and the pr oportion 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 oocy tes, the oocytes were placed in 50-mu L droplets (10 to 15 oocytes per drop let) of maturation medium (TCM-199+15% FBS+5 mu g/mL FSH-P), covered with p araffin oil in a 35-mm Petri dish and cultured for 26 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees 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. (C) 2000 by Elsevier Sc ience Inc.