Reprod. Dom. Anim., 27,44-45 (1992) @ 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0936-6768

Department of Embryo Transfer, All Union Insti tute of Animal Husbandry, iVloscow Region, Podolsky District, Dubrovitsy 142012, USSR.

In vitro Maturation and In vitro Fertilization of Bovine Oocytes*

N. Sergejew and F. Baibekow

A total of 121 ovaries was collected from slaughtered Holstein Frisian heifers of different age and were transported in warm physiological saline within 2-2 1 /2 hours to the laboratory. All follicles between 1 to 6 mm in diameter were punctured and a total of 507 oocytes was collected in tubes. After 1 0 minutes of sedimentation com- pact cumulus oocytes complexes with a dense cytoplasm were isolated under a stereo microscope. TCM 199 supplemented with 10% FCS was used as basic, medium. Prior to maturation oocytes were washed three times in TCM 199 supplemented with sodiurnbicarbonate, IIEPES lactate and pyruvate. The culture medium in method 1 was supplemented additionally with 10 pg/ml FSII and 20% estrous cow serum (pH 7.4, 290 mOsm). In method I1 only TCM 199 supplemented with 2% FCS and anti- biotics were used. The oocytes were cultured in droplets covered with mineral oil for 24 hours (37’C, 5 % COz ) and were classified for maturation based on the cumulus expansion.

In vitro fertilization

Frozed-thawed semen from 2 bulls was capacitated following the procedure described by Pdrrish (1986). In vitro fertilization in method I was performed in microdrops (fertilization medium “Fert-TALP”, 37,5 ’ C, 5 % C 0 2 , max. humidity) and after one hour of equilibration epinephrine and heparin were added to the fertilization medium. Subsequent to a 20 hour incubation period in the present of spermatozoa the fer- tilized oocytes were transfered into TCM 199 supplement with 20% FCS. In method I1 BO medium was used for fertilization which was only supplement with caffein and heparin.

Co-culture

In order to collect oviductal epithelia cells oviducts were flushed with modified TCM 199 supplemented with 10% FCS. The epithelia cells were treated with EDTA-trypsin and subsequently washed twice with ‘I’CM 199. After a culture period of 48 hours the monolayer had grown sufficiently and the supernatant was removed and used as conditioned medium.

Results

In this study in vitro maturation and fertilization procedures were used as described by Berg and Brem (1989) and Waldmann (1991) (method I ) and were compared to those described by Shimohira (1990) (method 11). After a 24 hour period of incubation 8 1 % of the oocytes (method I ) or 35 % (method 11) were matured. Out of 474 fertilized oocytes 242 (5 1 X) started cleavage within 24 hours when they were treated as described in method I whereas 3 1 % of the oocytes

* Vortrag gehalten anlaBlich der 18. Jahrestagung der deutschen Embryotransfergruppe in Trap- penkamp 1991.

In vitro Maturation and In vitro Fertilization of Bovine Oocytes 45

Table 1: Culture of in vitro fertilized oocytes with different media

Culture cleavage rate 16-cell stage morula early blastocyst n/n (W n/n (W n/n (W nln (%)

method I 2421474 1231474 331474 141474

method I1 10133 3/33 1/33 0133

(52) (26) ( 7 ) (3)

(31) (9) (3)

Total 252/507 126/507 341507 141507 (41,5) (17,5) (5) ( 1 9 5 )

cleaved when they were fertilized according to method 11. Development beyond the 8-cell-stage was observed in 123 (26%) cases (method I ) respectively in 3 cases (9%) (method 11). Only 7 % ( 3 %) developed t o later stages and only 3 % (method I) reached the blastocyst stage because of the 8-cell-stage-block. The co-culture of early embryos either with epithelia cells o r after supplementation with conditioned medium improved the developmental capacity t o later stages. Although it is not clear how the mechanism of the monolayer system work, it seems likely that the oviductal epithelia cells secrete substances like growth factors. Some embryos were stained, others were transfered t o recipient heifers unsurgically. Out of 8 (early blastocysts) transfers 3 pregnancies were obtained. This supports the positive effect of the co-culture-system to improve the development after in vitro fertilization.

References

Berg, U. & G. Brem, 1990: In-vitro production of bovine blastocysts by in vitro maturation and fertilization of oocytes and subsequent in vitro culture. Zuchthygiene 24, 134-139. - Parrish, J.J., J.L. Susko-Parrish, M.L. Leibfried-Rutledge, E.S. Critser, W.H. Eyestone & N.L. First, 1986: Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 25, 59 1-599. - Wald- mann, U.R., 199 1 : Ergebnisse von In-vitro-Befruchtungsversuchen mit Rinderoozyten unter Be- riicksichtigung ihrer Entwicklungsfahigkeit nach Transfer auf Empfangertiere. Hannover, Tier- arztl. Hochsch.. Diss.