{"content":[{"text":"American J. Animal & Vet. Sci., 5 (2): 71-75, 2010","bbox":{"left":0.333333,"top":0.090314,"width":0.33321899999999993,"height":0.015355000000000008}},{"text":"embryos are different with relation to the species and for 3 and 5 min, respectively. Finally, the immature \n developmental stages. Previous studies have shown that oocytes were washed twice in HM for 5 min each and \n MII bovine oocytes have greater permeability to processed for in vitro maturation. \n cryoprotectants and water than Germinal Vesicle (GV) \n oocytes (Agca et al., 1998). However, there have been In Vitro Maturation (IVM): Immature oocytes were \n little fundamental studies which compare handling washed two times in medium 199 supplemented with \n temperatures specified for vitrification of immature 4 mg mL−1 BSA and washed again in maturation \n bovine species. The aim of the present study is to solution containing Hepes-buffered medium 199 \n determine the effects of three different equilibration supplemented with 10% fetal calf serum, 0.2 mM \n temperatures on in vitro viability and subsequent sodium pyruvate (P-5280), 50 µL mL−1 Gentamycin (G- \n embryo developments of immature bovine oocytes after 1264) and 1 µg mL−1 oestradiol-17β (E-8875). \n vitrification. Approximately 20-30 oocytes were incubated in 400 µL \n of maturation solution under mineral oil in 4-well plates \n MATERIALS AND METHODS for 22-24 h at 38.5°C under 5% CO2 atmosphere with \n high humidity. The vitrified separated oocytes also \n Reagents: Unless otherwise indicated, all chemicals underwent the same maturation process with control \n were purchased from Sigma-Aldrich (St. Louis, MO, group after warming. \n USA). \n Nuclear maturation stage: For the purpose of \n Oocyte collection: Ovaries were collected from local maturation determination, 24 h after maturation, \n abattoirs and transported to the laboratory within 3 h at oocytes were denuded using 0.1% (w/v) hyaluronidase \n 34-36°C in phosphate-buffered saline (PBS; P-4417) (Type 1-S) in Hepes-buffered medium 199 by \n containing penicillin-streptomycin (100,000 IU of vortexing. The cumulus-free oocytes were fixed in \n penicillin and 100 mg of streptomycin per liter). aceto-ethanol mixture (1:3, v/v) at 5°C for 24 h. Once \n Cumulus Oocyte Complexes (COCs) were obtained by fixed, oocytes in groups of 5-10 were mounted on \n slicing method in tissue culture medium 199 (Medium slides. Cover-slip with 4 spots of silicon glue at the \n 199, 12340; Gibco) containing 25 mM HEPES, Earl’s corners was gently pressed down until it touched and \n salts, L-glutamine and 2 mg mL−1 sodium bicarbonate secured the oocytes. Afterward, aceto-lacmoid stain \n modified by the addition of 4 mg mL−1 bovine serum was passed under the cover-slip and remained there for \n albumin (BSA, fraction V, A-3311) and gentamycin 5 min. Subsequently, a decolorizing solution (aceto- \n 50 µg mL−1 (G-1264). glycerol) was passed through to remove the stain \n residuals. Stained oocytes were examined under light \n Vitrification and warming: The vitrification protocol microscope (400x) for nuclear stages determination. \n was adapted from Kuwayama et al. (2005) with minor \n In Vitro Fertilization (IVF): The fertilization method \n modifications. Briefly, oocytes were washed twice in \n was previously described by Parrish al. (1988) with \n Holding Solution (HS, Hepes-buffered TCM medium et \n some modifications. Briefly, matured oocytes were \n supplemented with 20% Fetal Calf Serum, FCS) and \n washed three times in tyrode’s albumin lactate pyruvate \n kept there for about 15 min. Group of four COCs were \n buffered with HEPES Talp-Hepes (Parrish et al., 1988) \n incubated in the first Vitrification Solution (VS1; 7.5% \n and twice in fertilization medium. Viable spermatozoa \n DMSO and 7.5% EG in WS) for 10-12 min and then \n were obtained by centrifugation of frozen–thawed \n transferred to the second Vitrification Solution (VS2; \n spermatozoa on Bovipure (Nidacon International AB, \n 15% DMSO, 15% EG and 0.5 M sucrose in WS) for a \n Gothenburg, Sweden) discontinuous density gradient (2 \n further 60 sec. Instantly oocytes were loaded on a hand- \n ml of top-layer over 2 ml of bottom-layer) for 20 min at \n made cryostrip and submerged into Liquid Nitrogen \n (LN2) for storage. The time of exposure from VS2 to 300×g at room temperature. Viable spermatozoa \n LN2 was less than 90s. Vitrified samples were collected at the bottom of the tube, were washed with \n maintained in Liquid Nitrogen (LN2) for at least 10 days. 5 mL of sperm-TALP (Parrish et al., 1988) \n Immediately after removing Cryotop from LN2, supplemented by 6 mg mL−1 BSA (fraction V, A-3311) \n thin strip of Cryotop was submerged in 3-ml pre- and centrifuged for 10 min at 300×g. After \n warmed (39°C) HS plus 1 M sucrose (T1) and centrifugation 150 µL of the pellet was selected. \n smoothly tried to detach oocytes from Cryotop device. Spermatozoa were counted using a hemacytometer and \n Immature oocytes were left in T1 for 1 min and then diluted with IVF-Talp to give the final concentration of \n transferred to HS plus 0.5 and 0.15 M sucrose solution 1×106 spermatozoa mL−1. IVM-oocytes were transferred \n 72","bbox":{"left":0.117647,"top":0.118122,"width":0.764845,"height":0.7913209999999999}}],"images_bbox":[],"images_bbox_not_text_overlap":[]}