Paulo Fernandes de Lima

Comparison of different protocols used to induce and synchronize estrus cycle of Saanen goats

The objective of the present study was to determine the efficiency of different protocols in inducing and synchronizing the estrus cycle of Saanen goats by using new or reused synchro-mate-B (SMB) and controlled internal drug release (CIDR) in combination with either equine chorionic gonadotrophin (eCG) or cloprostenol. Female goats (n=120) were divided at random into six groups of 20 animals each. In the T1-SMB group, the females were injected intramuscularly (i.m.) with 2.5mg estradiol valerate+1.5mg norgestomet and received a subcutaneous ear implant containing 2.0mg of norgestomet for 9 days. On the day of implant removal, the animals received 100IU of eCG and 0.05mg of cloprostenol. In the T2-SMB group, the animals were identically treated, except that the ear implant they received had been previously used in cattle. In the T3-SMB group, the treatment was identical to that for T1-SMB, but eCG was not administered. In the T1-CIDR group, the animals were treated for 9 days with an intravaginal device inpregnated with 0.3g of progesterone and injected i.m. with 100IU of eCG and 0.05mg of cloprostenol on the day of implant removal. The animals of the T2-CIDR group were treated like those of the T1-CIDR group, except that the intravaginal implant they received had been previously used in goats. The animals of the T3-CIDR group were treated like those of the T1-CIDR group, but did not receive eCG. The percentages of estrus and fertility were 100/80% (T1-SMB), 90/80% (T2-SMB), 75/75% (T3-SMB), 100/95% (T1-CIDR), 100/100% (T2-CIDR) and 70/65% (T3-CIDR), respectively, with the results for both parameters being lower (P</=0.05) in the T3-SMB and T3-CIDR groups. It is concluded that auricular implants or intravaginal devices may be reused, at last one more time, because they are efficient for inducing and synchronizing estrus in cyclic dairy goats.

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 (RT) and retinoic acid (RA) on the in vitro development of pre-implantation goat embryos cultured in potassium simplex optimized medium or synthetic oviduct fluid or cocultured in oviductal cells monolayer either in potassium simplex optimized medium or synthetic oviduct fluid. A total of 2407 cumulus-oocyte complexes were aspirated from 2 to 6 mm ovarian follicles from slaughtered animals. Selected cumulus-oocyte complexes were subjected to in vitro maturation in TCM 199 for 24 h at 39 °C in an atmosphere of 5% (v/v) CO(2) in humidified air. In vitro fertilization was performed in modified defined medium. Eighteen hours after in vitro fertilization, cumulus cells were removed and presumptive zygotes were randomly distributed into experimental groups. In Experiment 1, presumptive zygotes were cultured in potassium simplex optimized medium, potassium simplex optimized medium + RT, potassium simplex optimized medium + retinoic acid, synthetic oviduct fluid, synthetic oviduct fluid + RT and synthetic oviduct fluid + RA at 39 °C in a humidified atmosphere of 5% (v/v) CO(2), 5% (v/v) O(2) and 90% (v/v) N(2). In Experiment 2, presumptive zygotes were cocultured in potassium simplex optimized medium + oviductal cells monolayer, potassium simplex optimized medium + RT + oviductal cells monolayer, potassium simplex optimized medium + RA + oviductal cells monolayer, synthetic oviduct fluid + oviductal cells monolayer, synthetic oviduct fluid + RT + oviductal cells monolayer and synthetic oviduct fluid + RA + oviductal cells monolayer in an atmosphere of 5% (v/v) CO(2) in humidified air. In both experiments, media were partially changed on day 2 after in vitro fertilization and unfertilized oocytes were excluded from the experiment. Embryos were cultured or cocultured for 8 days. In Experiment 1, there was no effect of RT or RA supplementation on the proportion of oocytes that reached the morula or blastocyst stages. By contrast, Experiment 2 demonstrated that the addition of 0.28 μg/ml RT and 0.5 μm RA to the embryo culture media stimulated (p < 0.05) development to the morula and blastocyst stages under the coculture conditions tested. In conclusion, retinoids play an important role in pre-implantation development of goat embryos and can be used to enhance in vitro embryo production.

Sexagem fetal em ovelhas Santa Inês por ultra-sonografia

The present study aimed to identify the sex and to determine the day of genital tubercle (GT) migration of ovine fetuses using real time ultrasonography. The sex was identified in Experiment (EI) taking into consideration the localization of GT and in Experiment II (EII) the presence of penis, prepuce and scrotal bag in male fetus and nipples, genital swelling and clitoris in female fetus. In EI, the females (n=17) were monitored with 12 hour intervals from the 35th to the 46th day of pregnancy, by transrectal via with linear transducer (6.0 and 8.0MHz). In EII, the females (n=30) with pregnancy period from 55 to 75 days were examined once only, using the same transducer and via used in EI. Among 17 females in EI, 11 (64.6%) fetuses were correctly sexed independent of single (7/11), twin (3/11) or triple (1/11) pregnancy In 6 (35.4%) pregnancies, 3 (17.7%) were twins, being impossible to sex one fetus of each pregnancy. In other 3 (17.7%) pregnancies the fetuses were correctly sexed, although the birth did not coincide with the quantification. In a male fetus of a single pregnancy, the migration of the GT began on day 37 of pregnancy and on the 46th day all the fetuses of the other pregnancies were correctly sexed. Among 30 females in EII, 16 (53.4%) pregnancies were single being sexed with accuracy of 100%. In other 14 (46.6%) remainder the pregnancies were twins, being impossible, in four cases, to be determined the sex of one of each twin. The incorrect diagnoses were fetuses sexed as females, however born as males. From the all born fetuses the total accuracy was 88.0% (EI) and 90.9% (EII), being not observed difference (P>0.05) between both experiments. The results allow to conclude that ultrasonography in real time is an efficient method to diagnose the fetal sex by visualization of GT, as well as by identification of penis, prepuce and scrotal bag in male fetus and nipples, genital swelling and clitoris in female fetus, since the scanning are performed from Day 50 of pregnancy

Use of steroid hormone treatments prior to superovulation in Nelore donors

The objective of this study was to evaluate the effectiveness of synchronization of follicular wave emergence using steroid hormone treatments in Nelore cows. Donors were placed into three groups. Those that were between days 9 and 12 of their cycle (estrus=day 0) formed the TI group (n=60), whilst those that were in any other stages of their estrus cycle constituted groups TII (n=60) and TIII (n=60). TI donors were submitted to a standard protocol of superovulation, however, TII and TIII donors were treated with the Syncro-Mate-B (SMB) or Controlled Internal Drug Releasing Device (CIDR-B) programs, respectively. Superovulation was induced with p-FSH, divided into eight decreasing doses at intervals of 12h. The donors received cloprostenol 48h after the beginning of the treatment and progestagens were removed 12h later. Artificial inseminations (AI) were done at 12 and 22h after the initiation of estrus and the embryo collections were done 7 days after AI. In the donors which displayed behavioral estrus, mean (+/-S.E.M.) total ova and viable (transferable) embryos were 15.8+/-1.4 and 8.3+/-1.0 (TI, n=56); 15.6+/-1.3 and 8.9+/-1.0 (TII, n=56); 17.3+/-1.0 and 9.9+/-0.9 (TIII, n=57), respectively, with no significant difference (P > or =0.05) among groups. In those animals that did not displayed behavioral estrus, the mean values of total ova and viable embryos were 3.5+/-1.6 and 0.7+/-0.5 (TI, n=4); 11.5+/-3.9 and 9.0+/-4.4 (TII, n=4); 8.7+/-5.0 and 5.0+/-2.9 (TIII, n=3), respectively, with no significant differences (P > or =0.05) among groups. Pregnancy rates of 62.2% (TI, n=235); 66.4% (TII, n=284) and 65.1% (TIII, n=244) were obtained with embryos transferred from these collections and did not differ significantly (P > or =0.05) among groups. It was concluded that the synchronization of the emergence of follicular waves in Nelore donors is usable and does not harm the efficiency of embryo transfer programs. In addition, in contrast to the standard superovulation protocol, this method permits the use of a large number of donors in a short time period, at any stage of the estrus cycle, minimizing the costs of embryo transfer.

Migration time of the genital tubercle in caprine and ovine fetuses: Comparison between breeds, sexes and species

The aim of this work was to determine the ideal moment to sex goat and sheep fetuses, to compare the average time of genital tubercle (GT) migration between sexes, breeds and species, and to evaluate the accuracy of fetal sexing between sexes. A total of 317 fetuses of 219 pregnant females were monitored at 24-hour interval, from days 30 to 60 of pregnancy in ewes, and from days 40 to 60 in goats. Examinations were performed using transrectal ultrasound equipped with a linear transducer of double frequency. Fetuses were identified as male when the GT was next to the umbilical cord and female when the GT was next to the tail. The average time of GT migration in ewes (41.3 +/- 3.1 days) was shorter (P < 0.05) than in goats (47.2 +/- 2.3 days)? In goats, the average time of GT migration of Saanen fetuses was later (P < 0.05) than in fetuses of other breeds, with no difference in the average time of GT migration between male (46.9 +/- 2.2) and female fetuses (47.4 +/- 2.4). In ewes, the average time of GT migration did not differ (P > 0.05) among breeds and sexes. In goat and sheep, no difference was noticed in the accuracy of fetal sexing between males and females (P > 0.05). The results show that fetal sexing in ewes must be done earlier than in goats, fetal sexing in Saanen goats must be performed later, and fetal sex does not influence the time of GT migration in either of the two species.

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.

The use of steroid hormones in superovulation of Nelore donors at different stages of estrous cycle.

The objective of the present study was to evaluate the superovulatory response and ova/embryo recovery from Nelore donors following treatment with a controlled internal drug releasing device and estradiol benzoate (CIDR-B program) at different stages of the estrous cycle. The control group (TI; n=40) received a standard superovulation protocol with females of this group being between days 9 and 12 of the estrous cycle (estrus = day 0). The donors that received a CIDR-B program containing 1.9 g progesterone and an intramuscular injection of estradiol benzoate (2 mg) were at day 0 (TII; n=30), between days 2 and 6 (TIII; n=30), days 7 and 12 (TIV; n=30), days 13 and 16 (TV; n=30) and days 17 and 20 (TVI; n=30) of the estrous cycle. Superovulation was induced with 400 IU of p-FSH, divided into eight decreasing doses (80/80; 60/60; 40/40; 20/20) at intervals of 12h. The donors received PGF2alpha (Cloprostenol) 48 h after beginning the treatment and CIDRs were removed 12h later. Artificial inseminations (AI) were performed 12 and 22 h after the initiation of estrus and embryos were collected 7 days after AI. The mean numbers (+/-S.E.M.) of total ova and embryos, viable (transferable) and degenerated embryos were 14.2+/-11.3, 7.4+/-6.9 and 3.2+/-3.5 (TI), 13.3+/-10.4, 7.1+/-6.2 and 3.3+/-4.3 (TII), 13.5+/-7.0, 8.1+/-6.7 and 2.3+/-3.0 (TIII), 17.4+/-9.9, 9.4+/-6.9 and 4.0+/-4.4 (TIV), 16.9+/-8.8, 9.8+/-8.1 and 2.7+/-2.5 (TV) and 13.0+/-7.8, 7.2+/-6.9 and 2.3+/-2.5 (TVI), with no significant differences (P>/=0.05) among groups. Pregnancy rates of 67.1% (TI; n=86/128), 60.8% (TII; n=59/97), 62.5% (TIII; n=73/115), 64.1% (TIV; n=84/131), 72.3% (TV; n=81/112) and 60.6% (TVI; n=63/104) were obtained with embryos transferred from these collections and did not differ significantly (P>/=0.05) among groups. The results of the present study allow us to conclude that a combination of steroid hormones may be used prior to superovulation in Nelore donors, at any stage of the estrous cycle without affecting the efficiency of embryo transfer programs.

Altas concentrações de FSH-p na maturação in vitro de oócitos Bos indicus

The aim of this work was to evaluate the efficiency of different concentrations of a commercial FSH-p on the nuclear maturation of Bos indicus oocytes, cleavage and in vitro development of embryos until blastocyst stages. The oocytes were selected and transferred to the maturation medium (TCM 199/25 mM HEPES) supplemented with different concentrations of FSH-p (T1 = 10mg/m ; T2 - 20mg/m ; T3 - 40mg/m) and after 24 hours of incubation, at 39oC in a moist 5% CO2 atmosphere. Some of the oocytes were removed and submitted to the analysis of nuclear maturation and the others were placed in the fertilization medium (mDM). After 18 hours of incubation, at the same atmosfhere condition mentioned above, the presumptive zygotes were transferred to the culture medium (KSOM) with a granulosa cells monolayer. The metaphase II, cleavage and blastocyst percentages were, respectively, 81.8/62.0/17.6% (T1), 55.6/64.0/19.5% (T2) and 50.0/65.0/16.3% (T3). The statistic analysis showed that a lower percentage of oocyte (P £ 0.05) treated with 20mg/m and 40mg/m of FSH-p reached the metaphase II stage and that the cleavage and blastocyst rate do not differ among treatments (P ³ 0.05). The results allow to conclude that the addition of 20mg/m and 40mg/m of FSH-p to the culture medium interfere in the nuclear maturation process, however all tested concentrations may be used without apparent damage to the cleavage and subsequent embryonic development.

Use of retinoids during oocyte maturation diminishes apoptosis in caprine embryos

Exposure of caprine oocytes and embryos to retinoids enhances embryonic development, but the mechanisms governing this phenomenon have not been characterised. The aim of the present study was to evaluate if the incidence of apoptosis is affected by the addition of retinyl acetate (RAc) and 9-cis-retinoic acid (RA) during in vitro maturation (IVM) of caprine oocytes. Embryonic development was recorded on days 3 and 8 post-fertilisation, and apoptosis was measured by caspase activity and DNA fragmentation (TUNEL assay). Control zygotes had lower capacity to cleave and reach the blastocyst stage (24.45 ± 2.32 and 5.32 ± 0.81, respectively) than those of RAc- (29.96 ± 1.62 and 7.94 ± 0.93, respectively) and RA-treated groups (30.12 ± 1.51 and 7.36 ± 1.02, respectively). Oocytes and blastocysts positive for TUNEL assay were more frequent, respectively, in the controls (8.20 ± 0.78, 8.70 ± 1.05) than in RAc (5.60 ± 0.52, 4.80 ± 0.51) and RA (6.40 ± 0.69, 5.40 ± 0.69). Caspase activity did not differ between control oocytes (7.20 ± 0.91), RAc (6.60 ± 0.68) and RA (7.30 ± 0.67), but it was reduced in RAc- (5.05 ± 0.62) and RA-treated blastocysts (5.75 ± 0.22) compared to controls (8.35 ± 0.71). These results indicate that the addition of retinoids during IVM increases the developmental potential of goat embryos with a concomitant reduction in apoptosis rates.

Use of retinyl acetate, retinoic acid and insulin-like growth factor-I (IGF-I) to enhance goat embryo production

Experiments were carried out to investigate the beneficial effects of retinyl acetate (RAc) and retinoic acid (RA) on goat oocyte maturation as well as the effects of insulin-like growth factor-I (IGF-I), RAc and RA during embryo culture under chemically defined conditions. In Experiment 1, in vitro maturation (IVM) was performed in a chemically defined basic maturation medium (bMM) supplemented with 0.3 μM RAc or 0.5 μM RA. Presumptive zygotes and embryos (2-4 cells) were cultured in droplets of potassium simplex optimised medium (KSOM); however, none of the embryos reached the blastocyst stage. In Experiment 2, oocytes were matured in bMM + RAc or bMM + RA. Presumptive zygotes and 2- to 4-cell embryos were placed in fresh KSOM droplets supplemented with RAc, RA, IGF-I, RAc+IGF-I or RA+IGF-I. In Experiment 1, addition of RAc and RA to bMM increased (P < 0.05) the proportion of 2- to 4-cell embryos reaching the morula stage as compared to the control. In Experiment 2, supplementation of embryo culture media with retinoids and IGF-I increased (P < 0.05) the proportion of 2- to 4-cell stage embryos developing to the morula and blastocyst stage. Our data demonstrate that goat embryo production in chemically defined media could be improved by exogenous RAc or RA and by the interaction between retinoids and IGF-I, and that goat embryos can be produced in vitro from oocytes following protocols similar to those currently used for cattle.