Experimentelle Untersuchungen zur Effizienzsteigerung der In-vitro-Produktion von Rinderembryonen durch die Fertilisation aktivierter Oozyten sowie durch Vorbehandlung züchterisch wertvoller Schlachtkühe mit eCG

A. I. Centre, Neustadt a. d. Aisch, Dept. of Reprod. Med., Germany Research Institute for the Biology of Farm Animals Dummerstorf, Germany Institute of Reproductive Medicine, TiHo Hannover, Germany The present work aims to improve the developmental competence of in vitro matured (IVM), fertilized (IVF) and cultured (IVC) bovine oocytes on two different experimental ways.   For the first experiment the effect of a Ca-I-treatment (Ca-I A23187) before or immediately after the IVM to the activation mechanisms of the oocytes as well as to the developmental ability of the following fertilized and cultured oocytes was determined. The influence of the duration of IVM (18, 24 and 30 hours) as well as the existence of Ca2+ and Mg2+ in the activation medium and the cumulus oophorus was evaluated. The ovaries were transported from the slaughterhouse to the laboratory in 20-30 °C temperatured NaCl-solution. The ovaries were sliced to win the cumulus-oocyte-complexes (COKs). They were classified in 5 groups referring to the scheme of LEIBFRIED and FIRST (1979). Only oocytes of the groups 1 and 2 were used. The COKs were matured in TCM 199 containing FSH and 10 % ECS. The duration of IVM varied between 18, 24 and 30 h. After IVM oocytes were treated with Ca-I as COKs or as oocytes without a cumulus oophorus. The oocytes were denuded mechanically in 0,1 % hyaluronidase-solution. The cumulus-cell-suspension was used to perform a monolayer for later IVC. For activation the oocytes/COKs were treated in 20 mM Ca-I-solution (containing Ca2+ and Mg2+ or missing these ions) for 5 minutes and room temperature. IVF was conducted referring to the swim-up-method of PARRISH et al. (1986). In the fertilization medium 25 oocytes were incubated with 100.000 sperms for 22 h. Finally the oocytes were cultured in TCM 199 containing 10 % ECS for 9 days. Cleavage rate was determined on day 3, blastocyst rate on day 9. The following results were obtained: With the used IVP-technique a spontaneous activation rate of 12,5 % could be observed. A Ca-I-treatment led to an additional activation of bovine oocytes. The activation competence of Ca-I was dependent on the duration of IVM as well as the existence of the cumulus oophorus during the activation and Ca2+ and Mg2+ in the activation medium. A significantly increase of the activation rate could be reached by treating 24 h matured COKs (6,7 % vs. 19,6 and 22,1 %) as well as 30 hours matured oocytes without a cumulus oophorus in Ca2+- and Mg2+-missing (10,0 % vs. 47,1 %) Ca-I-solution. But no kind of Ca-I-treatment led to a development of bovine oocytes to morulae or blastocysts. The percentage of matured oocytes, reached in this experiment, was significantly higher after a maturation time of 24 h (93,5 %) than of 18 h (63,8 %). An additional Ca-I-treatment before the IVM could not improve the maturation rate. After IVF and IVC of oocytes the cleavage (60,6 % vs. 66,9 %; 70,9 % vs. 78,8 %) and blastocyst rate (with regard to all used oocytes: 24,6 % vs. 30,5 %; 18,6 % vs. 25,3 %) could be increased by extending the maturation time from 24 to 30 h. The existence of the cumulus oophorus during the IVF and IVC influenced the development of fertilized oocytes to blastocysts positively (blastocyst rate with regard to all used oocytes: 24,6 % vs. 18,6 %; 30,5 % vs. 25,3 %). The effect of a Ca-I-treatment after IVM on the developmental competence of oocytes after IVF and IVC was dependent on the duration of maturation as well as on the existence of Ca2+ and Mg2+ in the activation medium and the treatment of oocytes with or without a cumulus oophorus. The treatment of 30 h matured oocytes with Ca-I had no effect on their cleavage and blastocyst rate after IVF and IVC. A significantly decreased blastocyst rate could only be observed by treating cumulus free oocytes in a Ca-I-solution without Ca2+ and Mg2+ (blastocyst rate with regard to all used oocytes: 25,3 % vs. 8,3 %). Treating 24 h matured oocytes with Ca-I showed any negative effect on their developmental competence after IVF in no case. A significantly increase of the cleavage (60,5 % vs. 70,1 % and 84,2 %) and blastocyst rate (with regard to all used oocytes: 24,6 % vs. 40,3 % and 38,9 %) could be obtained by treating COKs with Ca-I. A Ca-I-treatment of COKs before IVM over 18 and 24 hours showed no influence on the maturation rate as well as on the cleavage and blastocyst rate after IVF and IVC. A significantly higher blastocyst rate could only be observed by treating COKs with Ca2+ and Mg2+-containing Ca-I-solution before a maturation time of   30 hours and a following IVF and IVC (blastocyst rate with regard to all used oocytes: 20,0 % vs. 36,8 %). Summary: In the used IVP-system activation of COKs was reached by treating them with Ca-I. Under special experimental conditions an improvement of the cleavage and the blastocyst rate was reached by treating bovine oocytes with Ca-I. In the second experiment the influence of a superovulation treatment with eCG Intergonanâ, Fa. Intervet) was tested on the oocyte quality and the results after IVP and embryo transfer from ovaries of slaughtered valuable breeding cows. The IVP-technique was used on the ovaries of 49 slaughtered valuable breeding cows. 20 cows were treated with 2.500 i.u. eCG 3 days before slaughter. During the treatment their oestral cycle was between the days 8 and 15 (V-group). 8 cows were treated under the same conditions but they got a placebo (K-group). In the O-group 21 animals were summarized, which could not be slaughtered under the defined conditions (unknown cycle, diseases, reproductive pathology,...). In this experiment the same IVP-technique was used as described in the first experiment, but oocytes of all qualitiy groups were used and treated separately to produce embryos. The oocytes were matured for 24 h and cultured for 9 days. The produced embryos were transferred directly to heifers or cryopreserved. Pregnancies were determined 6 weeks after the transfers. The results about births and physiology of the born calves were based on observations of the farmers.  The following results were obtained: On an average (mean ± SE) 43,0 ± 28,3 follicles could be counted on the ovaries of a cow (2-5 mm: 39,9; 5-8 mm: 3,0; >8 mm: 2,6). The ovaries of cows from the V-group possessed significantly more follicles with the sizes of 5-8 mm and >8 mm. The average number of oocytes per cow (mean ± SE) was 148,9 ± 86,8. 17,6 % could be graded as quality 1 and 2, 16,1 % as quality 3, 11,7 % as quality 4 and more than the half (54,6 %) as quality 5. The number as well as the quality of the oocytes could not be influenced by the pre-treatment of cows with eCG. Cows with ovarian cysts had a significant lower amount of oocytes with quality 1 and 2. After IVP of all gained oocytes (mean ± SE) 42,4 ± 26,7 2-8-cell stages and 17,0 ±12,5 blastocysts per cow could be produced on an average. This corresponds to a cleavage rate of 28,6 % and a blastocyst rate with regard to the cleaved embryos of 38,6 %. The blastocyst rate with regard to all used oocytes was 10,3 %. The individual influence of each cow was enormous (0-77 blastocysts/cow). The use of different bulls for IVF also led to significant differences in cleavage and blastocyst rates. The results of the V-group (cleavage rate: 28,8 %; blastocyst rate: 40,2 %) and the  K-group (cleavage rate: 34,3 %; blastocyst rate: 45,7 %) showed no significant differences. They were only significantly better than the cleavage (22,7 %) and blastocyst rate (30,1 %) of the O-group. From these results you can draw the conclusion that a pre-treatment of the donors with eCG cannot lead to higher cleavage and blastocyst rates after IVP. With 183 transferred embryos 70 pregnancies were established. This corresponds to an average pregnancy rate of 33,8 %. The pregnancy rate of the V- (39,9 %) and    K-group (39,6 %) was significantly higher than that of the O-group (21,8 %). 66 pregnancies could be analysed concerning their calving parameter. 67 calves     (1 twin) were born and 55 (82,1 %) were alive at the time of birth. 10,5 % was aborted during the 2nd and 5th month, 6 % died perinatal and 1,5 % were lost by euthanasia. The farmers reported from 26 calves that weighed more than 60 kg. In 21 cases (31,8 %) calves had to be born with additional help. 6 (9,1 %) calves had to be extracted by a caesarean. The sex ratio was shifted in favour of male descendants (1,4:1). Summary: The average blastocyst rate of 17 embryos per donor is very high. A pre-treatment of the donors with eCG cannot improve the blastocyst rate significantly. The phenomenon of the “large calf syndrome” concerning in vitro produced calves described in the literature can be observed in this experiment, too.