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Sylvaine Camous, Y. Heyman, Wided Méziou and Y. Ménézo

Summary. Early bovine embryos (1- to 8-cell stages) were recovered from superovulated heifers at slaughter on Days 2 or 3. Embryos were cultured for 3–4 days in Medium B2 supplemented with 15% (v/v) fetal calf serum in the absence (B2SS, 106 embryos) or presence of trophoblastic vesicles (B2SS + TV, 190 embryos).

At the end of culture, there were more (P < 0·001) morulae (≥16 cells) in B2SS × TV (46%) than in B2SS alone (18%) irrespective of the initial cell stage. More 8-cell embryos reached the 16-cell stage than did embryos with <8 cells (30% vs 15% in B2SS, P > 0·05; 70% vs 41% in B2SS + TV, P < 0·005). After culture, 102 morulae were transferred non-surgically to temporary recipient heifers (84 embryos cultured in B2SS + TV and 18 in B2SS). After 2 or 3 days, 14 out of 58 embryos from the B2SS + TV group and 3 out of 10 embryos from the B2SS group were recovered as blastocysts. Most blastocysts were deep-frozen and stored for several weeks. After thawing, 10 apparently normal embryos from the B2SS + TV group were transferred non-surgically into 10 recipient heifers. Four pregnancies were induced, but only one embryo survived to term (birth of a normal male calf).

It is concluded that trophoblastic vesicles release one or several unknown compound(s) normally present in vivo, promoting the cleavage of early bovine embryos.

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Y. Heyman, Sylvaine Camous, J. Fèvre, Wided Méziou and J. Martal

Summary. One or two trophoblastic vesicles (0·4–2 mm diam.) from cow (Day 14) or ewe (Day 11–13) embryos without their disc were transferred, after culture for 24 h, into recipients. Each vesicle was transferred into the uterine horn ipsilateral to the CL by the cervical route in heifers and surgically in ewes on Day 12 of the oestrous cycle.

In cows, daily measurements of plasma progesterone concentrations and checks for return to oestrus showed that the CL was maintained in 8 out of 12 recipients. These 8 cows had 25- to 37-day cycles while 4 recipient heifers returned to oestrus normally. Three recipients with an extended cycle were slaughtered. The dissected uterus showed that trophoblastic vesicles had developed in the uterine horns.

In ewes, the serum progesterone curve, determined in each recipient, showed that the CL was maintained in 7 out of 12 recipients. These 7 ewes had 20- to 54-day cycles and the other 5 ewes had a normal cycle of 15–19 days comparable to that of 17·0 ± 0·5 days for the 6 control ewes. Whenever the CL was maintained, high blood progesterone levels were followed by rapid luteolysis. In cattle and sheep, therefore, a trophoblastic vesicle transferred into the uterus can develop in vivo, secreting the embryonic signals when there is no embryonic disc control and transforming the cyclic CL into a CL of pregnancy in about 60% of the cases. These results indicate that the early signals inhibiting luteolysis may be of trophoblastic origin and confirm that after normal embryo transfer some of the late returns to oestrus may be due to the development of trophoblastic tissue only in utero.

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C. Vincent, V. Garnier, Y. Heyman and J. P. Renard

Summary. NBD–phallacidin revealed a polymerized actin distribution in the cortical region of the rabbit egg and along junctional feet. Staining with anti-alpha-tubulin antibody showed that the microtubule distribution was restricted to the barrel-shaped spindle. After cryoprotective treatment in the presence of propanediol, cortical polymerized actin was no longer visible within the egg and along junctional feet but filamentous actin was still present after treatment with dimethylsulphoxide. However, exposure to dimethylsulphoxide or propanediol led to the appearance of microtubules in the cytoplasm and to a disassembly of the spindle often associated with anomalies in chromosome position. Cytoplasmic microtubules formed by the action of propanediol were still present after freezing, thawing, and removal of the cryoprotectant, but after recovery of eggs in culture, they disappeared and barrel-shaped spindles were able to reform.

When the effect of propanediol addition on in-vivo fertilization and development of frozen oocytes was examined, 39% (79/200) of frozen oocytes were fertilized and 9% (9/105) developed to normal fetuses, compared to 81% (38/47) and 32% (12/38) respectively for unfrozen control oocytes.

Keywords: cryoprotectants; rabbit; oocytes; microtubules; microfilaments; freezing

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M Tamassia, Y Heyman, Y Lavergne, C Richard, V Gelin, JP Renard and S Chastant-Maillard

There have been few studies on a possible maternal influence on in vitro embryo production in cows. The objective of this study was to evaluate the maternal influence on oocyte production and in vitro blastocyst formation rate using repeated ovum pick-up and in vitro fertilization. Six contemporary cows raised on the same farm and with varied genetic origins were submitted to 42 weeks of ovum pick-up organized into four series. Collected oocytes were fertilized in vitro with spermatozoa from a different bull for each series. In total, 1933 oocytes were recovered from 3936 follicles with a recovery rate of 57.2% and a mean oocyte collection of 4.6+/-0.2 (mean+/-SEM) per animal per session. Animals were ranked according to their oocyte production. The best oocyte donor was the same female in all four series. No relationship was identified between oocyte production and blastocyst production rate (r=-0.08). The mean blastocyst rate was 28.8% with significant variation among animals. The best and the worst blastocyst producers were always the same animals independent of the semen used. The results of the present study support the hypothesis that in cattle, the oocyte donor influences the production of blastocysts. Furthermore, they demonstrate that oocyte and embryo production are independent factors. Further studies are necessary to identify the maternal or oocyte factors responsible for such differences.

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F. Revel, P. Mermillod, N. Peynot, J. P. Renard and Y. Heyman

The developmental competence of oocytes from 3-month-old calves was studied through in vitro maturation, fertilization and culture up to the blastocyst stage and by embryo transfer into a foster mother. Oocytes were recovered from antral follicles of calves after or without ovarian stimulation with exogenous FSH and their developmental potential was compared with that of oocytes recovered from cow ovaries. Fertilization and cleavage rates from calf oocytes did not differ significantly from those of cow oocytes. However, after 7 days of culture, the blastocyst formation rate was significantly lower for calves (9% and 11% for nontreated and treated animals, respectively) than for cows (over 20%). Transfer of blastocysts obtained from calf oocytes resulted in a lower pregnancy rate (1 of 23 recipients; 4%) than that achieved with cow oocytes (10 of 26; 38%). The recipient cow that was pregnant from calf embryos delivered a full-term live calf. These data show that some key regulative event that determines the ability to form blastocysts in cattle has not been fully achieved in oocytes from 3-month-old calves.

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B. Vigier, Dien Tran, F. du Mesnil du Buisson, Y. Heyman and Nathalie Josso

Summary. Monoclonal antibodies against bovine anti-Müllerian hormone (AMH) were used to study the hormone in cattle. Anti-Müllerian activity of testicular tissue, immunoreactive testicular AMH, serum AMH concentration and AMH production by incubated testicular tissue were detectable from 42 days, i.e. at the time of seminiferous tubule differentiation, and peaked between 50 and 80 days, when the Müllerian ducts regress in the male fetus. All the values stabilized at a lower level until 30 days after birth and then slowly decreased. At 18 months, only traces of AMH immunoreactivity were detectable in testicular tissue and serum concentration and AMH production by incubated testicular tissue were negligible; the main source of AMH in the adult animal was the rete testis fluid. Study of the disappearance rate of AMH from the serum of castrated calves gave a half-life of approximately 2 days for bovine AMH.

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D. Le Bourhis, P. Chesne, M. Nibart, J. Marchal, P. Humblot, J. P. Renard and Y. Heyman

The objectives of this study were to evaluate the efficiency and reliability of embryo sexing from isolated single blastomeres, and after nuclear transfer to examine the influence of the sex of donor embryos on development in vitro and in vivo up to calving. The sex of the donor embryo was determined by revealing a specific Y DNA sequence by PCR and electrophoresis after isolation of one, two, three, or more than five cells. The efficiency of sex determination was over 90% and reliability was 100% independent of the number of blastomeres used. In a second experiment, sex was determined from a single cell and the other cells were used for nuclear transfer. The effect of sex on in vitro development was studied in 386 male and 314 female reconstructed embryos derived from 19 male and 14 female parent embryos, respectively. Developmental competence in vitro of male and female constructs over 7 days was not statistically different (25.2 and 23.1% blastocysts on day 7, respectively; P > 0.05). After the transfer of predetermined male (n = 30) and female (n = 27) cloned embryos into recipient heifers, no effect of sex was observed on pregnancy rates at day 21, 35 and 90, or on calving rates (P > 0.05). These rates did not differ between single and twin transfer (P > 0.05). The sex of the calves born always corresponded to that determined from a single blastomere. These results show that sex can be determined accurately when using a single blastomere before nuclear transfer and that the sex of the parent embryo does not affect in vitro development or in vivo survival rates of cloned embryos.