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Use of in vitro fertilization for production of calves from involuntary cull cows
Theriogenology
39:320,
1993
USE OF IN VITRO FERTILIZATION FOR PRODUCTION OF CALVES FROM INVOLUNTARY CULL COWS D. Stringfellow, M. Riddell, K. Riddell R. Carson, R. Smith, and B. Gray Department of Pathobiology, College of Veterinary Medicine Auburn University, Alabama 36849-5519 USA Many cows of high genetic merit must be culled prematurely, having produced few or no calves. The main purpose of this study was to assess the feasibility of using a system for in vitro maturation of oocytes (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) of IVF embryos to produce calves from these involuntary cull cows. Specific objectives were to determine the number of cumulus-oocyte-complexes (COCs) that could be collected per cow, and the numbers of cleaved zygotes and morulae/blastocysts that could be produced per cow when commercially frozen semen was used for fertilization of matured oocytes. For these experiments, 15 Holstein and 2 Jersey cows (age 2.3 to 7.0 years) from experimental dairy production units within the Auburn University system were used. Cows were culled for a variety of reasons, including mastitis, injury, and reproductive failure. Both ovaries and oviducts were removed surgically from each animal. COCs were recovered from both ovaries using a slicing technique and cultured for 24 h in 50 1.11drops of TCM-199 supplemented with 20% estrous cow serum (ECS). The epithelial lining was stripped from the oviduct ipsilateral to a corpus hemorrhagicum or early corpus luteum. The resulting sheets of bovine oviductal epithelial cells (BOEC) were broken into small pieces by aspiration through a 25 gauge needle. The small pieces of BOEC were incubated in TCM-199 with 10% ECS for 24 or 48 h before use in IVF or IVC drops, respectively. Matured oocytes were cocultured for 16 h with sperm (about 5~10~) and BOEC in 100 ~1 drops of TALP medium supplemented with 6 mg/ml BSA and 20 &ml heparin. Sperm were separated by a swim-up procedure from semen that had been cryopreserved for routine artificial insemination. Presumptive zygotes were cultured in 100 1.11drops of TCM-199 supplemented with 10% ECS and BOEC. All microdrops in these procedures were overlaid with silicone oil, and incubations were at 39°C in a highly humidified atmosphere of 5% CO, and air. Approximately 48 h after beginning IVC, cultures were examined for cleavage, and on Days 6 and 7, the numbers of morulae and blastocysts were determined. Embryos from 5 cows were transferred to recipients that were examined for pregnancy at about 40 days after fertilization. An average of 43.9 (range 7 to 129) COCs per cow were collected. After IVM and IVF of these oocytes, an average of 38.9 (range 5 to 119) presumptive zygotes were placed into IVC. Further, an average of 15.6 (range 1 to 60) cleaved zygotes and 5.3 (range 0 to 20) morulae or blastocysts were observed at Days 2 and 7, respectively. Transferable embryos were produced from 16 of the 17 cows. Nine embryos from five donor cows were transfered to recipients. Four of the nine recipients were confirmed to be pregnant after 40 days.
320
Copyright 0 1993 Butterworth-Heinemann
39:320,
1993
USE OF IN VITRO FERTILIZATION FOR PRODUCTION OF CALVES FROM INVOLUNTARY CULL COWS D. Stringfellow, M. Riddell, K. Riddell R. Carson, R. Smith, and B. Gray Department of Pathobiology, College of Veterinary Medicine Auburn University, Alabama 36849-5519 USA Many cows of high genetic merit must be culled prematurely, having produced few or no calves. The main purpose of this study was to assess the feasibility of using a system for in vitro maturation of oocytes (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) of IVF embryos to produce calves from these involuntary cull cows. Specific objectives were to determine the number of cumulus-oocyte-complexes (COCs) that could be collected per cow, and the numbers of cleaved zygotes and morulae/blastocysts that could be produced per cow when commercially frozen semen was used for fertilization of matured oocytes. For these experiments, 15 Holstein and 2 Jersey cows (age 2.3 to 7.0 years) from experimental dairy production units within the Auburn University system were used. Cows were culled for a variety of reasons, including mastitis, injury, and reproductive failure. Both ovaries and oviducts were removed surgically from each animal. COCs were recovered from both ovaries using a slicing technique and cultured for 24 h in 50 1.11drops of TCM-199 supplemented with 20% estrous cow serum (ECS). The epithelial lining was stripped from the oviduct ipsilateral to a corpus hemorrhagicum or early corpus luteum. The resulting sheets of bovine oviductal epithelial cells (BOEC) were broken into small pieces by aspiration through a 25 gauge needle. The small pieces of BOEC were incubated in TCM-199 with 10% ECS for 24 or 48 h before use in IVF or IVC drops, respectively. Matured oocytes were cocultured for 16 h with sperm (about 5~10~) and BOEC in 100 ~1 drops of TALP medium supplemented with 6 mg/ml BSA and 20 &ml heparin. Sperm were separated by a swim-up procedure from semen that had been cryopreserved for routine artificial insemination. Presumptive zygotes were cultured in 100 1.11drops of TCM-199 supplemented with 10% ECS and BOEC. All microdrops in these procedures were overlaid with silicone oil, and incubations were at 39°C in a highly humidified atmosphere of 5% CO, and air. Approximately 48 h after beginning IVC, cultures were examined for cleavage, and on Days 6 and 7, the numbers of morulae and blastocysts were determined. Embryos from 5 cows were transferred to recipients that were examined for pregnancy at about 40 days after fertilization. An average of 43.9 (range 7 to 129) COCs per cow were collected. After IVM and IVF of these oocytes, an average of 38.9 (range 5 to 119) presumptive zygotes were placed into IVC. Further, an average of 15.6 (range 1 to 60) cleaved zygotes and 5.3 (range 0 to 20) morulae or blastocysts were observed at Days 2 and 7, respectively. Transferable embryos were produced from 16 of the 17 cows. Nine embryos from five donor cows were transfered to recipients. Four of the nine recipients were confirmed to be pregnant after 40 days.
320
Copyright 0 1993 Butterworth-Heinemann