- Email: [email protected]
Evolution of day 8 and 9 in vitro derived bovine blastocysts, fertilized with two different bulls
Theriogenology
EVOLUTION
213
OF DAY 8 AND 9 IN VITRO DERIVED BOVINE BLASTOCYSTS, FERTILIZED WITH TWO DIFFERENT BULLS
B. Avery and D. Quetglas Department of Reproduction, Royal Veterinary and Agricultural University, 13 Btilowsvej, DK-1870 Frederiksberg C, Denmark The aims of this study were to compare blastocyst rates, speed of development, and number of blastomeres from bovine in vitro derived Day 8 (BL-XB-H), and Day 9 blastocysts (XB-H), sired by semen from two bulls of different breeds (Jersey and Holstein Friesian Dairy Breed). The embryos were produced by a standard method of in vitro maturation for 24 h in TCM-199 with serum and gonadotropines, in vitro insemination for 22 h with frozenlthawed washed spermatozoa in TALP-medium with heparin, and in vitro coculture in MenezoB2 medium with a suspension of bovine oviduct epithelial cells for 9 days post insemination Oocytes were randomly assigned to at 39°C in 5% CO, in air and high humidity. insemination with either of the two bulls. A total of 69 blastocysts were collected at Days 8 and 9; from the Jersey bull the numbers of Day 8 and 9 blastocysts were 8 and 27, respectively, from the Holstein bull the numbers were 8 and 26, respectively. The embryos were stained with the fluorescent dye Hoechst 33342, for counting of blastomeres. This technique might underestimate the number of blastomeres by 10 to 20%. The major findings are presented in the table. The Jersey bull consistently gave higher blastocyst rates (35+5%, 1283 oocytes, 8 replicates) than the Holstein bull (24f7%, 1258 oocytes, 6 replicates), in spite of the use of optimal spermatozoa and heparin concentrations in the fertilization medium. The embryonic speed of development was identical between the two bulls. It seemed as though the morphology of the blastocysts was slightly better for the Jersey than the Holstein bull, although this difference was subtle, and at the hatched stage of development the blastocysts did not differ visibly. Within each stage of development (BL-BL; XB-XB; H-H), no difference in numbers of blastomeres were noted between the two bulls, nor did the numbers of blastomeres differ for the same embryonic stage, whether collected at Day 8 or 9. There was a significant increase in the number of blastomeres from increasing stages of development (BL-XB-H). In conclusion, this study shows that the two tested bulls had different capabilities to produce blastocysts, but that number of blastomeres and speed of development were identical. This difference could be associated with the breed. Table. Numbers of blastomeres (Mean&SD) from blastocysts, blastocysts, collected at Day 8 and 9.
expanded
Stage
Holstein Bull
Jersey Bull
BL
68+10* (n=6)
88+16* (n=3)
133+29’ (n=7)
147*27’ (n=15)
191*40c (n=21)
202+34’ (n=I7)
H
and hatched
n: No. blastocysts. Results analyzed by t-test and by Tukey Kramers Multiple Comparisons test. Numbers with different superscripts differs (PcO.05). BL:blastocyst; XB:expanded blastocyst; H:hatched blastocyst. Supportedby the Animal Biotechnology Research Center.
EVOLUTION
213
OF DAY 8 AND 9 IN VITRO DERIVED BOVINE BLASTOCYSTS, FERTILIZED WITH TWO DIFFERENT BULLS
B. Avery and D. Quetglas Department of Reproduction, Royal Veterinary and Agricultural University, 13 Btilowsvej, DK-1870 Frederiksberg C, Denmark The aims of this study were to compare blastocyst rates, speed of development, and number of blastomeres from bovine in vitro derived Day 8 (BL-XB-H), and Day 9 blastocysts (XB-H), sired by semen from two bulls of different breeds (Jersey and Holstein Friesian Dairy Breed). The embryos were produced by a standard method of in vitro maturation for 24 h in TCM-199 with serum and gonadotropines, in vitro insemination for 22 h with frozenlthawed washed spermatozoa in TALP-medium with heparin, and in vitro coculture in MenezoB2 medium with a suspension of bovine oviduct epithelial cells for 9 days post insemination Oocytes were randomly assigned to at 39°C in 5% CO, in air and high humidity. insemination with either of the two bulls. A total of 69 blastocysts were collected at Days 8 and 9; from the Jersey bull the numbers of Day 8 and 9 blastocysts were 8 and 27, respectively, from the Holstein bull the numbers were 8 and 26, respectively. The embryos were stained with the fluorescent dye Hoechst 33342, for counting of blastomeres. This technique might underestimate the number of blastomeres by 10 to 20%. The major findings are presented in the table. The Jersey bull consistently gave higher blastocyst rates (35+5%, 1283 oocytes, 8 replicates) than the Holstein bull (24f7%, 1258 oocytes, 6 replicates), in spite of the use of optimal spermatozoa and heparin concentrations in the fertilization medium. The embryonic speed of development was identical between the two bulls. It seemed as though the morphology of the blastocysts was slightly better for the Jersey than the Holstein bull, although this difference was subtle, and at the hatched stage of development the blastocysts did not differ visibly. Within each stage of development (BL-BL; XB-XB; H-H), no difference in numbers of blastomeres were noted between the two bulls, nor did the numbers of blastomeres differ for the same embryonic stage, whether collected at Day 8 or 9. There was a significant increase in the number of blastomeres from increasing stages of development (BL-XB-H). In conclusion, this study shows that the two tested bulls had different capabilities to produce blastocysts, but that number of blastomeres and speed of development were identical. This difference could be associated with the breed. Table. Numbers of blastomeres (Mean&SD) from blastocysts, blastocysts, collected at Day 8 and 9.
expanded
Stage
Holstein Bull
Jersey Bull
BL
68+10* (n=6)
88+16* (n=3)
133+29’ (n=7)
147*27’ (n=15)
191*40c (n=21)
202+34’ (n=I7)
H
and hatched
n: No. blastocysts. Results analyzed by t-test and by Tukey Kramers Multiple Comparisons test. Numbers with different superscripts differs (PcO.05). BL:blastocyst; XB:expanded blastocyst; H:hatched blastocyst. Supportedby the Animal Biotechnology Research Center.