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Maturation and fertilization of bovine oocytes in vitro
THERIOGENOLOGY
MATURATION
AND FERTILIZATION
OF BOVINE OOCYTES -IN VITPO
R. Y. Lenzl R. L. Ax* and !. L. First1 G. D. Balll, M. L. Leibfriedl IDepartment oi Keat and An;mal Science *Department of Dairy Science University of Wisconsin Madison, WI 53706 The objective of this investigation was to determine some of the factors important for successful in vitro fertilization of -in vitro matured bovine The end-points xam were frequency of ovum penetration and oocytes. Ovaries and epididymides were obtained from a local formation of 2 pronuclei. Cumulus-oocyte complexes from l-5mn follicles were cultured slaughter plant. with FSH for 24h to facilitate cumulus and expansion oocyte nuclear maturation. oocytes were co-cultured with spermatozoa for an Subsequently, The sperm concentration during co-culture was 4 x 106 additional 24h. Experiment 1 examined preincubation of sperm/ml. sperm at a high concentration prior to insemination. The effects of the presence of hypotaurine and epinephrine during fertilization were also examined in this Experiment 2 dealt with the need for the presence of cumulus experiment. Experiment 3 was designed to examine the use of cells during fertilization. the stimulator of cumulus expansion prior to either FSH or CAR as FS!i (0.5 IU/ml) was present in the culture medium for the fertilization. 8-Br-CAR (10 mM) was present in the medium for entire maturational culture. only the initial 6h of the maturational period. Results of experiment 1 indicated that preincubation of sperm at a concentration of 1 x 108 sperm/ml for 3h prior to co-culture with ova was not beneficial to fertilization Fifty-eight : and 6l% of the ova were penetrated by preincubated and (P>.O5). Twenty Xand 36% of these, respectively, formed 2 control sperm, respectively. in addition, when 10 -H hypotaurine and 1 "M epinephrine were pronuclei. present during fertilization penetration rates (P=.O53) and the frequency of 2 Sixty-six % of the ova were pronuclei observed (Pc.05) were increased. penetrated by sperm and 41% of these formed 2 pronuclei when hypotaurine and When these agents were epinephrine were present in the culture medium. absent, 54% of the ova were penetrated and 19% of those penetrated formed 2 ihe results of experiment 2 indicated that the presence of cumuli pronuclei. were not necessary for penetration (P>.O5) but the frequency of ova that fowed 2 pronuclei was increased (Pc.05). When cumuli were left intact, 47% of the ova were penetrated and 72% of those penetrated formed 2 pronuclei. When cumuli were removed prior to co-culture with sperm, 57% were penetrated In addition, when cumuli removed-ova were and 35: formed 2 pronuclei. cultured in the presence of cumuli intact-ova, the frequency of 2 pronuclei The results of experiment 3 indicated that the observed was not increased. frequency of ova penetrated after incubation in either FSH or cAFP was not Similarly, the frequency of ova observed with 2 pronuclei different (P>.O5). Fifty Xof the ova incubated with FSB and 65% with was not different (P>.O5). Half of the ova penetrated in either group formed ? cAw were penetrated. in addition, ova matured in the presence of FSH or CAMP were pronuclei. In sunmary, penetrated at a higher frequency than untreated controls fP<.O5). these experiments demonstrated that in vitro matured bovine oocytes could be Additionally, the presence of expanded cumuli, fertilized in vitro. hypotaurine Find mphrine during co-culture with sperm improved the quality Preincubation of sperm at a high concentration prior to of fertilization. co-culture was not beneficial to fertilization.
112
JANUARY
1983 VOL. 19 NO. 1
MATURATION
AND FERTILIZATION
OF BOVINE OOCYTES -IN VITPO
R. Y. Lenzl R. L. Ax* and !. L. First1 G. D. Balll, M. L. Leibfriedl IDepartment oi Keat and An;mal Science *Department of Dairy Science University of Wisconsin Madison, WI 53706 The objective of this investigation was to determine some of the factors important for successful in vitro fertilization of -in vitro matured bovine The end-points xam were frequency of ovum penetration and oocytes. Ovaries and epididymides were obtained from a local formation of 2 pronuclei. Cumulus-oocyte complexes from l-5mn follicles were cultured slaughter plant. with FSH for 24h to facilitate cumulus and expansion oocyte nuclear maturation. oocytes were co-cultured with spermatozoa for an Subsequently, The sperm concentration during co-culture was 4 x 106 additional 24h. Experiment 1 examined preincubation of sperm/ml. sperm at a high concentration prior to insemination. The effects of the presence of hypotaurine and epinephrine during fertilization were also examined in this Experiment 2 dealt with the need for the presence of cumulus experiment. Experiment 3 was designed to examine the use of cells during fertilization. the stimulator of cumulus expansion prior to either FSH or CAR as FS!i (0.5 IU/ml) was present in the culture medium for the fertilization. 8-Br-CAR (10 mM) was present in the medium for entire maturational culture. only the initial 6h of the maturational period. Results of experiment 1 indicated that preincubation of sperm at a concentration of 1 x 108 sperm/ml for 3h prior to co-culture with ova was not beneficial to fertilization Fifty-eight : and 6l% of the ova were penetrated by preincubated and (P>.O5). Twenty Xand 36% of these, respectively, formed 2 control sperm, respectively. in addition, when 10 -H hypotaurine and 1 "M epinephrine were pronuclei. present during fertilization penetration rates (P=.O53) and the frequency of 2 Sixty-six % of the ova were pronuclei observed (Pc.05) were increased. penetrated by sperm and 41% of these formed 2 pronuclei when hypotaurine and When these agents were epinephrine were present in the culture medium. absent, 54% of the ova were penetrated and 19% of those penetrated formed 2 ihe results of experiment 2 indicated that the presence of cumuli pronuclei. were not necessary for penetration (P>.O5) but the frequency of ova that fowed 2 pronuclei was increased (Pc.05). When cumuli were left intact, 47% of the ova were penetrated and 72% of those penetrated formed 2 pronuclei. When cumuli were removed prior to co-culture with sperm, 57% were penetrated In addition, when cumuli removed-ova were and 35: formed 2 pronuclei. cultured in the presence of cumuli intact-ova, the frequency of 2 pronuclei The results of experiment 3 indicated that the observed was not increased. frequency of ova penetrated after incubation in either FSH or cAFP was not Similarly, the frequency of ova observed with 2 pronuclei different (P>.O5). Fifty Xof the ova incubated with FSB and 65% with was not different (P>.O5). Half of the ova penetrated in either group formed ? cAw were penetrated. in addition, ova matured in the presence of FSH or CAMP were pronuclei. In sunmary, penetrated at a higher frequency than untreated controls fP<.O5). these experiments demonstrated that in vitro matured bovine oocytes could be Additionally, the presence of expanded cumuli, fertilized in vitro. hypotaurine Find mphrine during co-culture with sperm improved the quality Preincubation of sperm at a high concentration prior to of fertilization. co-culture was not beneficial to fertilization.
112
JANUARY
1983 VOL. 19 NO. 1