The Journal of Reproduction and Development Supplement

The aim of this study is to investigate the effect of progesterone (P4) on the multiple follicular growth during superovulation treatment in dairy cows. Animals were divided into three groups as follows; 1) Growing CL + CIDR (G1): Cows (n=4) were received a total dose of 28 A.U. FSH through the first 4 days (twice daily) after natural ovulation (day 0=d0). CIDR was inserted on d1 and removed 12 h after the last dose of the FSH. 2) Growing CL (G2): Cows (n=4, 7 trials) were treated similar to G1 but without CIDR. 3) CL absence (G3): Cows (n=4, 7 trials) received PGF_2α at 10 days after ovulation. After 36 h, all follicles (≥5 mm) were aspirated (d0). The FSH was given 24 h after aspiration as G1. The follicles were counted on d1, 3 and 5 in all groups. Blood was collected daily for 6 days. P4, E2, IGF-I and growth hormone (GH) were measured by EIA. While the P4 level was above 1 ng/ml from d2 to d5 in G1, it was under 0.5 ng/ml in the G3. P4 of G2 showed a gradual increase from 0.5 ng/ml at d1 to 2 ng/ml at d5. In G3, all cows showed an increase in E2 at d3 or d4 followed by an increase of IGF-1 within 24 h. GH increased concomitantly with the E2 increase in 4 trials out of 7. In contrast, the cows in G1 and G2 showed neither E2 nor IGF-1 increase. At d5, the number of follicles ≥8mm in G3 (22.8±2.0) was significantly (P<0.05) higher than G2 (9.2±2.9) and G3 (11.6±2.0). These data demonstrate that low P4 level during FSH treatment enhance multiple follicular growth and E2 secretion which followed by increase of IGF-1. Therefore, P4 may play a critical role in the superovulation response by controlling the multiple follicular growth.

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The rate of in vitro maturation (IVM) of canine oocytes has not improved in comparison to that of other mammalian species. This study aims to improve the efficiency of canine oocytes IVM using the antioxidant, extracellular superoxide dismutase (EC-SOD). Thus, the effect of conditioned medium of ECSOD transgenic mouse embryonic fibroblasts cultured with MEF culture medium (DMEM+ 5%FBS) for in vitro nuclear maturation in canine oocytes was investigated. In experiment I, oocytes were collected from the ovaries of domestic bitches, which were allotted to one of two groups: (1) TCM199 + 1% FBS (n = 108) or (2) DMEM+ 5% FBS (n = 112), cultured for 48 h and investigated for in vitro nuclear maturation of canine oocytes using Hoechst staining. Meiotic progression to metaphase II in group 1 was 1.8% compared to 1.8% in group 2. In experiment II, EC-SOD levels were examined in NTg-CMEF and Tg-CMEF at 0, 2 and 4 days obtained from EC-SOD transgenic mice generated in our laboratory. The concentration of EC-SOD in Tg-CMEF at day 2 was the highest for all groups (P < 0.05). EC-SOD levels in Tg-CMEF were higher than in NTg-CMEF; therefore, the efficiency of Tg-CMEF for IVM was investigated. In experiment III, oocytes were allotted to one of three groups: (1) Tg-CMEF at day 0 (n = 84), (2) Tg-CMEF at day 2 (n = 92) or (3) Tg-CMEF at day 4 (n = 98), cultured for 48 h and the IVM of canine oocytes investigated. The mean percentage of MII oocytes in IVM was 2.4, 4.4 and 2.0% for groups 1, 2 and 3, respectively. In experiment IV, the effects of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts

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We investigated the efficiency of invitro mauturation (IVM) as a basic experiment to study the development of canine oocytes after invitro fertilization (IVF). Therefore, we performed two-part experiments. First, experiment I compared the effects of TCM 199 without fetal bovine serum (FBS) to TCM 199 supplemented with 5 % FBS on the invitro nuclear maturation rate of canine oocytes. To investigate the efficiency of meiotic development to the metaphase II (MII) stages, 4.7 % (4/64) of all oocytes in TCM 199 without FBS developed to the MII stage compared with 1.7% (1/59) in TCM 199 with 5 % FBS for 48h. FBS did not increase invitro nuclear maturation. In experiment II, the cleavage rate of canine oocytes was investigated after heparin treatment on IVF. Canine oocytes were fertilized as four groups: Fert-TALP medium without heparin (FertI) or Fert-TALP medium supplemented with10,20,30ug/ml heparin (Fert II, Fert III, Fert IV, respectively). Oocytes for 24 h fertilization in Fert I were the highest of all of the groups as 6.5% (5/77) in Fert I, and developed to early morula. Especially, the oocytes cultured in Fert I for 24 h of insemination time had a higher rate of embryonic development than other groups. Therefore, we can assert that unlike in other mammals, heparin in canine IVF does not increase the efficiency of fertilization rate, and is not an important factor.

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There is no detailed report about the chromosome constitution in piglets derived from IVP system although high incidences of chromosomal abnormalities have been observed in preimplantation porcine embryos. In the present study, the chromosome constitution in porcine blastocysts and piglets derived from IVP system and piglets derived from in vivo was determined. Porcine oocytes were collected from 3-6-mm ovarian follicles and matured in modified NCSU-37 medium for 44 h. Following in-vitro fertilization with a final concentration of 1x10⁵ sperm/ml for 3 h, presumptive zygotes were cultured in vitro for 6 days. The resultant Day 6 blastocysts (Day 0 = day of IVF) were analyzed chromosomally. Leukocytes of 8 piglets derived from IVP (4 piglets) and in-vivo systems (4 piglets) were cultured in RPMI-1640 medium for 71 h, and then they were prepared as chromosome samples. The whole incidence of chromosomal abnormalities in IVP blastocysts was 45.3%. Incidences of polyploidy and mixoploidy, 23.8% and 16.2%, respectively, were higher than the incidence of haploidy, 4.3%. Between piglets derived from in-vivo and IVP systems, there was no difference in the average frequency of diploid cells, 96.7% and 95.9%, respectively. Some cells of hypodiploidy, hyperdiploidy and polyploidy were observed as chromosome imbalance in all samples. Tetraploidy, among several kinds of polyploidy, was the most frequently founded in both piglets derived from in-vivo and IVP systems. The results indicated that although high incidences of chromosomal abnormalities were observed in preimplantation porcine embryos, the borne IVP piglets were similar in normal chromosome constitution to in-vivo piglets.

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