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Variation in ultrasound guided oocyte recovery in nellore cows per session and in vitro embryo production
Thefiogeno~gy
438
VARIATION IN ULTRASOUND-~/SIDED OOC r-rZ-'RECOVERY IN NELLORE COWS PER SESSION AND IN VITRO EMBRYO PRODUCTION. M.R. Watanabe~'2;Y.F. Watanabe~'3;P.H. Franceschini2; A. DayanS;R.B L6bo ~ !Department of Genetics - FMRP/USP, Av. Bandeirantes, 3900 - Cep: 14040-030 Ribeir~o Preto-S~o Paulo, Brazil 2Department of Animal Patology-FCAVJ/UNESP, Jaboticabal/SP 3Barba Agrieola e Comercial Ltda, Descalvado/SP The association of in vitro fertilization and repeated ovum pick up techniques appears to be an excelent alternative for superovulation to maximize production of embryos with of high genetic merit. The objective of this study was to evaluate this technique in indigenous Cows. Therefore, Nellore cows (n=6) 4-8 y of age were used for aspiration of ovarian follicles at 7 d intervals using an ultrasound (Aloka-500V) with a convex probe (5 MHz) connected to a vacuum pump (68 rnm Hg). Cumulus-oocyte complex were classified morphological criteria based of the cumulus cells and the citoplasm, and were transported to the laboratory located at 120 Km from the farm in TCM 199-HEPES+10%FCS maintaining 35°C, all ooeytes of one cow per droplet were in vitro matured (bicarbonatate-buffered TCM-199+10%FCS+0.5~tg FSH/ml+50~g LH/ml+l~tg estradiol/ml) in 50~tl overlaid with oil for 24h at 38.5°C in 5% CO2 in air. COCs were inseminated with frozen/thawed Percoll separated semen (10~ sperm cells/ml) in 50~tl of TALP medium+10~tg heparin/ml for 12 h. Presumptive zygotes with cumulus cells adhered were transferred to 50~tl CR2+10%FCS droplets. At 3 d post insemination cleavage rate was assessed, medium added and zygotes were stripped of cumulus cells. Blastocyst stage from days 6 to 8 post-insemination were transferred or vitrified. The results showed in table 1. Table 1. Recovery and in vitro developmental potential of oocytes from Nellore cows obtained by transvaginal ultrasound guided aspiration. Cows
Sessions
# Oocytes # Selected for # Cleaved a # Blastocysts b Recovered (n) IVF/IVC (n) Day 3 (n) Day 6-8 (n) A 7 18.0+4.9 (124) 12.0+3.1 (81 ) 6.0+_3.1 (42) 3.4+2,0 (24) B 7 9.0+1.5 (60) 5.0-+1.5 (36) 4.0+_1.4 (25) 1.1+0.6 (8) C 7 5.0-+1.1 (35) 4.0+_1.0 (25) 2.5+_0.5 (17) 1.4+0.7 (10) D 7 7.0+-1.6 (48) 5.0-+1.3 (36) 2.0+_0.9 (15) 1.1+1,0 (8) E 5 8.0-+1.5 (39) 7.0-+1.5 (33) 3.0-+1.2 (15) 1.0+0.8 (5) F 5 5.0+ 1.3 (26) 4.0+1.6 (17) 3.0+_1.3 (15) 0.6-+0.2 (3) Total 38 9.0+_3.7 (332) 6.0+_2.5 (228) 3.4+_1.4 (129) 1.53-+0.7 (58) Data are means per session +_SEMwith between (332) total numbers, obtained at 5-7 sessions (n=38) per cow of 6 cows. Significance was calculated by Chi-square at a: P<0,01 - b: P>0,05.
Although only a reduced number of transfers have been performed to date, (20 embryos in 15 recipients) we have verified the suitability of the technique with a pregnancy rate of 33% verified at 60 d post transfer. We can conclude that weekly ultrasound guided follicular aspiration in Nellore cows enables satisfactory results for in vitro embryo production, and that in overall it is possible to produce 1 gestation per weeks (Financial Support: CNPq-RHAE, FAPESP, FINEP, CAPES, Barba Agricola e Comercial Ltda).
438
VARIATION IN ULTRASOUND-~/SIDED OOC r-rZ-'RECOVERY IN NELLORE COWS PER SESSION AND IN VITRO EMBRYO PRODUCTION. M.R. Watanabe~'2;Y.F. Watanabe~'3;P.H. Franceschini2; A. DayanS;R.B L6bo ~ !Department of Genetics - FMRP/USP, Av. Bandeirantes, 3900 - Cep: 14040-030 Ribeir~o Preto-S~o Paulo, Brazil 2Department of Animal Patology-FCAVJ/UNESP, Jaboticabal/SP 3Barba Agrieola e Comercial Ltda, Descalvado/SP The association of in vitro fertilization and repeated ovum pick up techniques appears to be an excelent alternative for superovulation to maximize production of embryos with of high genetic merit. The objective of this study was to evaluate this technique in indigenous Cows. Therefore, Nellore cows (n=6) 4-8 y of age were used for aspiration of ovarian follicles at 7 d intervals using an ultrasound (Aloka-500V) with a convex probe (5 MHz) connected to a vacuum pump (68 rnm Hg). Cumulus-oocyte complex were classified morphological criteria based of the cumulus cells and the citoplasm, and were transported to the laboratory located at 120 Km from the farm in TCM 199-HEPES+10%FCS maintaining 35°C, all ooeytes of one cow per droplet were in vitro matured (bicarbonatate-buffered TCM-199+10%FCS+0.5~tg FSH/ml+50~g LH/ml+l~tg estradiol/ml) in 50~tl overlaid with oil for 24h at 38.5°C in 5% CO2 in air. COCs were inseminated with frozen/thawed Percoll separated semen (10~ sperm cells/ml) in 50~tl of TALP medium+10~tg heparin/ml for 12 h. Presumptive zygotes with cumulus cells adhered were transferred to 50~tl CR2+10%FCS droplets. At 3 d post insemination cleavage rate was assessed, medium added and zygotes were stripped of cumulus cells. Blastocyst stage from days 6 to 8 post-insemination were transferred or vitrified. The results showed in table 1. Table 1. Recovery and in vitro developmental potential of oocytes from Nellore cows obtained by transvaginal ultrasound guided aspiration. Cows
Sessions
# Oocytes # Selected for # Cleaved a # Blastocysts b Recovered (n) IVF/IVC (n) Day 3 (n) Day 6-8 (n) A 7 18.0+4.9 (124) 12.0+3.1 (81 ) 6.0+_3.1 (42) 3.4+2,0 (24) B 7 9.0+1.5 (60) 5.0-+1.5 (36) 4.0+_1.4 (25) 1.1+0.6 (8) C 7 5.0-+1.1 (35) 4.0+_1.0 (25) 2.5+_0.5 (17) 1.4+0.7 (10) D 7 7.0+-1.6 (48) 5.0-+1.3 (36) 2.0+_0.9 (15) 1.1+1,0 (8) E 5 8.0-+1.5 (39) 7.0-+1.5 (33) 3.0-+1.2 (15) 1.0+0.8 (5) F 5 5.0+ 1.3 (26) 4.0+1.6 (17) 3.0+_1.3 (15) 0.6-+0.2 (3) Total 38 9.0+_3.7 (332) 6.0+_2.5 (228) 3.4+_1.4 (129) 1.53-+0.7 (58) Data are means per session +_SEMwith between (332) total numbers, obtained at 5-7 sessions (n=38) per cow of 6 cows. Significance was calculated by Chi-square at a: P<0,01 - b: P>0,05.
Although only a reduced number of transfers have been performed to date, (20 embryos in 15 recipients) we have verified the suitability of the technique with a pregnancy rate of 33% verified at 60 d post transfer. We can conclude that weekly ultrasound guided follicular aspiration in Nellore cows enables satisfactory results for in vitro embryo production, and that in overall it is possible to produce 1 gestation per weeks (Financial Support: CNPq-RHAE, FAPESP, FINEP, CAPES, Barba Agricola e Comercial Ltda).