Reliability of ultrasound for early sexing of goat fetuses derived from natural mating and from fresh, frozen and vitrified embryo transfer.

The aim of the present study was to identify the migration period of the genital tubercle and its later differentiation into external genital structures in fetuses derived from natural mating and fetuses from fresh, frozen and vitrified embryo transfer. A transrectal ultrasound with a double-frequency linear transducer (6.0 and 8.0 MHz) was used to monitor 123 goat fetuses, which were allocated to one of four groups: fetuses originating from controlled natural mating (G1, n = 32) and fetuses derived from fresh (G2, n = 34), frozen (G3, n = 30) and vitrified (G4, n = 27) embryo transfer. The transferable embryos were collected 7 days after mating by laparoscopy. Migration of the genital tubercle occurred significantly earlier (P < 0.05) in G1 than in G2, G3 and G4. The visualisation of the scrotum, prepuce and vulva occurred significantly earlier (P < 0.05) in G1 than in G2, G3 and G4. Our results show that fetal sexing is feasible after 55 days for fetuses from natural mating and after 60 days in fetuses from fresh and cryopreserved embryos. Thus, real-time ultrasonography is a reliable tool for fetal sex determination in goats after Day 50 of pregnancy, taking into account both the location of the genital tubercle and the identification of external genital structures.

Determination of the genital tubercle migration period in Morada Nova sheep foetuses by ultrasonography.

The aim of this study was to determine the period of genital tubercle (GT) migration using ultrasonography in Morada Nova sheep foetuses (n = 117) from natural mating (NM) and frozen embryo transfer (ET) to determine the window when foetal sexing can be determined. The examinations were performed using transrectal ultrasonography with a dual-frequency linear transducer (6.0 and 8.0 MHz) from day 30-54 of pregnancy at 48-h intervals. The period of GT migration of foetuses produced by NM varied from 36 to 46 days of pregnancy, resulting in an average of 39.5 +/- 2.9 days. For foetuses derived from ET, GT migration varied from 42 to 52 days of pregnancy with an average of 48.5 +/- 3.3 days, being possible the GT of foetuses from ET start to migrate 96 h later even if they are of the same gender. Migration of the GT occurred earlier (p < 0.05) in foetuses produced by NM and sexing accuracy for triplet pregnancies (77.8%) was significantly inferior (p < 0.05) to single (100%) and twin (92.9%) pregnancies for foetuses derived by NM. The results allow one to conclude that foetal sexing can be done from the 50th day onwards in foetuses produced by NM and from the 55th day onwards in foetuses derived from ET, and that multiple pregnancies compromise the sexing accuracy by ultrasonography.

Sexing of Dorper sheep fetuses derived from natural mating and embryo transfer by ultrasonography.

In order to improve fetal sexing in the Dorper sheep breed, the objective of the present study was to determine, by repeated ultrasonographic examinations, the migration period of the genital tubercle (GT) in sheep fetuses derived from natural mating or embryo transfer and to compare the accuracy of a single examination with repeated examinations at short intervals. For this purpose, transrectal ultrasound was performed, using a double-frequency linear transducer (6.0 and 8.0 MHz) for monitoring 51 sheep fetuses distributed in three experimental groups (EI, EII and EIII). The fetuses in EI (n = 23) and EII (n = 18) derived, respectively, from natural mating and embryo transfer were monitored at 48-h intervals from the 30th to 60th day of gestation and sexed based on the final location of the GT. The fetuses in EIII (n = 10), which originated from embryo transfer, were examined only once on the 65th day of gestation and sexed taking into consideration the final position of the GT and/or by identification of anatomical structures of external genitalia. The accuracy of fetal sexing was 91.3% (21 fetuses sexed/23 quantified) in EI, 88.9% (16 sexed/18 quantified) in EII and 100% (10 sexed/10 quantified) in EIII, without significant difference (P > 0.05) between experiments. Migration of the GT occurred earlier (P < 0.05) in fetuses produced by natural mating (43.0 +/- 2.8 days) than in those derived from embryo transfer (46.1 +/- 4.7 days). The results show that fetal sexing can be done from the 50th day onward in fetuses produced by natural mating and from the 60th day onward in fetuses derived from frozen embryos. It can also be concluded that repeated ultrasonographic exams in short time intervals do not maximise the accuracy of fetal sexing. In addition, real-time ultrasonography is a reliable tool for fetal sex determination in sheep after Day 50 of gestation, taking into account both the location of the GT and the identification of external genital structures.

Effect of retinoids and growth factor on in vitro bovine embryos produced under chemically defined conditions.

Experiments were conducted to investigate the beneficial effects of adding retinol (RT) and retinoic acid (RA) to bovine oocyte maturation media and insulin-like growth factor-I (IGF-I) to embryo culture under chemically-defined conditions. In Experiment 1.1, in vitro maturation (IVM) was performed in basic maturation media (bMM) and supplemented with 0.3microM RT or 0.5microM RA. For embryo development presumptive zygotes and embryos were placed in droplets of potassium simplex optimized medium (KSOM). Addition of RT and RA to bMM improved (p<0.05) blastocyst formation as compared with control treatments. In Experiment 1.2, using embryos originating from oocytes previously treated with RT and RA, the presumptive zygotes were placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The number of 2-4-cell stage embryos developing to the blastocyst and expanded blastocyst stages were greater (p<0.05) when embryo culture media was supplemented with IGF-I. In Experiment 2.1, IVM was conducted with bMM+FSH containing 0.3microM RT or 0.5microM RA. For embryo development, presumptive zygotes were placed in droplets of KSOM. Addition of RT or RA to IVM medium also enhanced (p<0.05) blastocyst formation. The supplementation of embryo culture media with IGF-I resulted in a greater number (p<0.05) of 2-4-cell stage embryos developing into blastocysts, expanded blastocysts and hatched blastocysts. In Experiment 2.2, using embryos originating from oocytes previously treated with RT and RA, presumptive zygotes were also placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The supplementation of embryo culture media with IGF-I resulted in a greater (p<0.05) number of 2-4-cell stage embryos developing to the blastocyst, expanded blastocyst and hatched blastocyst stages.

Effects of retinol on the in vitro development of Bos indicus embryos to blastocysts in two different culture systems.

The objective of this study was to evaluate the effect of retinol on the in vitro development of early embryos of cultured Bos indicus (Expt 1) to the blastocyst stage in medium simplex of optimization (KSOM) or sintetic fluid of oviduct (SOF) or co-cultured (Expt 2) with an oviduct cell monolayer (OCM) in KSOM or SOF. A total of 3149 cumulus-oocyte complexes obtained by aspirating follicles (2-5 mm diameter) from ovaries of slaughtered animals were selected for IVM and incubated in TCM 199 supplemented with 25 mM HEPES at 39 degrees C in air with 5% CO(2) and maximum humidity for 24 h. In vitro fertilization (IVF) was performed in modified defined medium (mDM) medium. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes were randomly allocated to the experimental groups. Zygotes cultured (Expt 1) in KSOM + retinol, KSOM, SOF + retinol and SOF were incubated in maximum humidity at 39 degrees C, 5% CO(2), 5% O(2) and 90% N(2). Zygotes co-cultured (Expt 2) in KSOM + retinol + OCM, KSOM + OCM, SOF + retinol + OCM and SOF + OCM were incubated at 39 degrees C, 5% CO(2). In both experiments media were partially changed 48 h after IVF and unfertilized ova were removed. Afterwards embryos were kept in culture or co-culture for further 9 days. In Expt 1, blastocyst rates (day 7) were 14.6% (KSOM + retinol), 15.8% (KSOM), 16.4% (SOF + retinol) and 15.9% (SOF). In Expt 2, the blastocyst rates (day 7) were 25.4% (KSOM + retinol + OCM) 14.2% (KSOM + OCM), 24.3% (SOF + retinol + OCM) and 15.9% (SOF + OCM). The same influence profile of retinol was observed in the formation of the expanded (day 9) and hatched (day 11) blastocysts. The results obtained in Expt 2 demonstrated that the addition of 0.28 microg/ml retinol to the embryo culture media used in this study had a significant (p < 0.05) positive effect on bovine early embryonic development, under the conditions tested, and can be used to enhance in vitro embryo production.

Ultrasonographic monitoring of 103 recipient mares of different reproductive status during the first 30 days after embryo transfers.

Ten pluriparous mares were used as donors to supply embryos which were transferred into 103 recipients, 31 of which were nulliparous, 34 were pluriparous and lactating, and 38 were pluriparous and non-lactating. The embryos were recovered eight days after ovulation and pregnancy was confirmed by ultrasound six days after the transfer; the length of the embryos was measured ultrasonographically on days 12, 14, 16, 18, 20, 25 and 30 after the embryo transfer. One hundred and fifteen of 200 flushes provided embryos, 12 being degenerate and 103 being viable embryos. From the 103 embryo transfers carried out, 51 pregnancies were confirmed by ultrasound within 30 days; 16 of the 31 nulliparous recipients became pregnant, 16 of the 34 pluriparous lactating recipients and 19 of the 38 pluriparous non-lactating recipients. There were no significant differences between the groups of mares in the mean (sd) rate of growth of their embryos between 12 and 30 days of gestation.