Cultura de embrião e indução de brotos in vitro para micropropagação do pinhão-manso

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Incubabilidade e Qualidade de Pintos de Ovos Matrizes de Frangos de Corte Submetidos a Estresse de Temperatura

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of Annona squamosa extract on early pregnancy in rats

Annona squamosa Linn., family Annonaceae, is said to show varied medicinal effects, including insecticide, antiovulatory and abortifacient. The purpose of present study was to investigate if A. squamosa seed aqueous extract, in doses higher than that popularly used to provoke abortion, interferes with reproductive performance, and to correlate the ingestion of this extract with possible alterations in rat embryonic implantation. Doses of 300 mg/kg (Treated Group I, n = 17) and 600 mg/kg (Treated Group II, n = 12) body wt. were administered by gavage, during days 1 to 5 of pregnancy (preimplantation period). The control group (n = 13) received water in the same manner, during the same period for comparison with experimental groups. The animals were euthanized on day 10 of pregnancy. Treatment of dams during the preimplantation period showed no signs of toxicity, and no alteration in the corpora lutea, implantations and embryo in terms of development numbers. The percentage of preimplantation and postimplantation losses in treated groups I and II did not differ from those of control. Treatment with aqueous extract of A. squamosa seeds caused no morphological change in the endometrium. The absence of morphological alterations in uterine epithelial cells in treated groups I and II permitted a viable embryonic implantation, as verified by the number of embryos in development at day 10 of pregnancy. Thus, A. squamosa seed aqueous extract did not interfere with the reproductive performance of pregnant rats.

Best site for embryo transfer: the upper or lower half of endometrial cavity?

BACKGROUND: the objective of the present study was to determine the importance of the site of embryo transfer (upper or lower half endometrial cavity) on implantation and clinical pregnancy rates. METHODS: A total of 400 transfers guided by ultrasound were randomly assigned to two groups according to the distance between the uterine fundus and the catheter tip at the time of embryo placement. Group I (n=200) consisted of transfers corresponding to a distance of <50% of the endometrial cavity length (ECL), i.e. transfer in upper half of the cavity; and group II (n=200) consisted of transfers corresponding to a distance of greater than or equal to50%, of the ECL, i.e. transfer in lower half of cavity. The Student's t-test, Mann-Whitney test and Fisher's exact test were used where appropriate. RESULTS: the general characteristics of the study population and the main transfer cycle characteristics had an equal distribution (P>0.05) between groups I and II. No significant difference in implantation or pregnancy rates was observed between groups I and II. CONCLUSION: the implantation or pregnancy rates were similar whether the embryos were deposited in the upper or lower half of the endometrial cavity.

Comparison of day 2 embryo quality after conventional ICSI versus intracytoplasmic morphologically selected sperm injection (IMSI) using sibling oocytes

Objective: To evaluate whether intracytoplasmic morphologically selected sperm injection (IMSI) could influence early paternal effects by observing embryo quality at day 2.Study design: The study included 30 couples with at least one of the following criteria: male factor infertility, at least 2 previous failures of implantation or previous miscarriages after IVF/ICSI. Sibling oocytes of each patient were randomly assigned to either the ICSI group or the IMSI group. For IMSI, spermatozoa were selected at 8400x magnification through an inverted microscope equipped with Nomarski differential interference contrast optics, Uplan Apo 100x oil/1.35 objective lens and variable zoom lens. For conventional ICSI, spermatozoa were selected at 400x magnification. An embryo was defined as top quality if there were four identical blastomeres on day 2 with no fragments or multinucleation of blastomeres. Data were analysed using the Wilcoxon and chi-squared tests. The significance level was set at P < 0.05. The variables were analysed in relation to the general population and the subpopulations with or without male factor.Results: A total of 331 MII oocytes (30 oocyte retrievals) were selected and injected by the ICSI (n: 172) or IMSI (n: 159) procedure. For IMSI, only spermatozoa classified as morphologically normal at high magnification were used. No differences (P > 0.05) in fertilisation rate (ICSI: 70.9%; IMSI: 70.4%), early embryo cleavage rate (ICSI: 66.9%; IMSI: 60.4%) or cleavage rate (ICSI: 99.2%; IMSI: 99.1%) were observed. on day 2, as compared to ICSI, IMSI provided a similar proportion of top quality embryos (ICSI: 57.8%; IMSI: 52.2%; P > 0.05). These results were not influenced by the presence or absence of male factor.Conclusion: In terms of embryo quality at day 2, IMSI had the same performance as conventional ICSI. However, we cannot exclude the possibility that IMSI effects occur only as a positive later paternal effect. (C) 2010 Elsevier B.V. All rights reserved.

Artificial oocyte activation using calcium ionophore in ICSI cycles with spermatozoa from different sources

The present study evaluated the effect of artificial oocyte activation (AOA) with calcium ionophore A23187 oil intracytoplasmic sperm injection (ICSI) cycles using spermatozoa from different sources. The 314 cycles evaluated were divided into three groups according to sperm origin, the ejaculated group (n = 92), the epididymal group (n = 82). and the testicular roup (n = 140). Each group was further split into experimental subgroups, depending oil whether or no AOA was performed. In additions the cycles of women younger than 36 years were evaluated separately. For each experimental group, ICSI outcomes were compared between subgroups. No significant difference was observed between subgroups for all sperm origin groups. When evaluating only the cycles of women younger than 36 years of age, AOA increased the percentage of high-quality embryos (74.5 versus 53.0%. P = 0.011) and the implantation rate (19.3 versus 10.5%, P = 0.0025) when it was used with ejaculated spermatozoa, and the percentage of high-quality embryos (64.4 versus 50.3%, P = 0.006) when epididymal spermatozoa were used. These results may suggest that both sperm maturity and oocyte quality play a role in oocyte activation. However. this study is to be continued to confirm these findings.

Artificial oocyte activation with calcium ionophore A23187 in intracytoplasmic sperm injection cycles using surgically retrieved spermatozoa

Objective: To evaluate the effect of artificial oocyte activation (AOA) on intracytoplasmic sperm injection (ICSI) cycles using surgically retrieved sperm.Design: Laboratory study.Setting: Fertility/assisted fertilization center.Patient(s): Couples undergoing surgical sperm retrieval for ICSI (n = 204).Intervention(s): Application of calcium ionophore A23187 for AOA.Main Outcome Measure(s): Cycles were divided into experimental groups according to the origin of the sperm used for injection and the type of azoospermia: [1] testicular sperm aspiration in nonobstructive-azoospermic patients (TESA-NOA group, n = 58), [2] TESA in obstructive-azoospermic patients (TESA-OA group, n = 48), [3] and percutaneous epididymal sperm aspiration in obstructive-azoospermic patients (PESA-OA, n = 98). For each experimental group, cycles where AOA was applied (subgroup: activation) were compared with cycles in which AOA was not applied (Subgroup: control). The fertilization, high-quality embryo, implantation, and pregnancy rates were compared among the subgroups.Result(s): For patients undergoing TESA, AOA did not improve ICSI outcomes for either type of azoospermia. However, for cases in which the injected sperm were retrieved from the epididymis, a statistically significantly increased rate of high-quality embryos was observed with AOA.Conclusion(s): Artificial oocyte activation may improve ICSI outcomes in azoospermic patients when epididymal, but not testicular spermatozoa, are injected. (Fertil Steril (R) 2009;92:131-6. (C)2009 by American Society for Reproductive Medicine.)

Decreased fertility in poor responder women is not related to oocyte morphological status

Introduction: In women showing impaired fertility, a decreased response to ovarian stimulation is a major problem, limiting the number of oocytes to be used for assisted reproduction techniques (ART). Despite the several definitions of poor response, it is still a matter of debate whether young poor responder patients also show a decrease in oocyte quality. The objective in this study was to investigate whether poor ovarian response to the superstimulation protocol is accompanied by impaired oocyte quality.Material and methods: This study included 313 patients younger than 35 years old, undergoing intracytoplasmic sperm injection. Patients with four or fewer MII oocytes (poor-responder group, PR, n = 57) were age-matched with normoresponder patients (NR, n = 256).Results: A higher rate of oocyte retrieval and a trend towards an increase in MII oocyte rate were observed in the NR group when compared to the PR group (71.6 +/- 1.1% and 74.1 +/- 1.0% vs. 56.3 +/- 2.9% and 66.5 +/- 3.7%; p < 0.0001 and p = 0.056, respectively). A trend toward increased implantation rates was observed in the NR group when compared to the PR group (44 and 24.5 +/- 2.0% vs. 28.8 and 16.4 +/- 3.9%; p = 0.0305 and p = 0.0651, respectively).Conclusions: Low response to ovarian stimulation is apparently not related to impaired oocyte quality. However, embryos produced from poor responder oocytes show impaired capacity to implant and to carry a pregnancy to term.

Relationship between visualization of meiotic spindle in human oocytes and ICSI outcomes: a meta-analysis

The objective of this meta-analysis was to investigate the influence of meiotic spindle visualization in human oocytes on intracytoplasmic sperm injection (ICSI) outcomes. Search strategies included on-line Surveys of databases (MEDLINE, em BASE, Science Citation Index, Cochrane Controlled Trials Register and Ovid). The fixed effect was used for odds ratio. Ten trials fulfilled the inclusion criteria comparing in-vitro and clinical ICSI outcomes with or without visualization of meiotic spindle in fresh and in-vivo matured oocytes. According to the meta-analysis, the results showed statistically significant higher fertilization rate (P < 0.0001) when the meiotic spindle was viewed than when it was not. Moreover, the percentage of pro-nuclear-stage embyros with good morphology (P = 0.003), cleavage rate (P < 0.0001), percentage of day-3 top-quality embryos (P = 0.003) and percentage of embryos that reached the blastocyst stage (P < 0.0001) were statistically significantly better among, embryos derived from oocytes in which meiotic spindle was viewed compared with those in which meiotic spindle was not observed. However, these differences were not observed in the clinical pregnancy or implantation rates. This observation has clinical relevance mainly in countries where there is a legal limit on the number of oocytes to be fertilized. However, additional controlled trials are needed to further confirm these results.

Effects of recombinant LH supplementation to recombinant FSH during induced ovarian stimulation in the GnRH-agonist protocol: a matched case-control study

Background: Some studies have suggested that the suppression of endogenous LH secretion does not seem to affect the majority of patients who are undergoing assisted reproduction and stimulation with recombinant FSH (r-FSH). Other studies have indicated that a group of normogonadotrophic women down-regulated and stimulated with pure FSH preparations may experience low LH concentrations that compromise the IVF parameters. The present study aimed to compare the efficacy of recombinant LH (r-LH) supplementation for controlled ovarian stimulation in r-FSH and GnRH-agonist (GnRH-a) protocol in ICSI cycles.Methods: A total of 244 patients without ovulatory dysfunction, aged < 40 years and at the first ICSI cycle were divided into two groups matched by age according to an ovarian stimulation scheme: Group I (n = 122): Down-regulation with GnRH-a + r-FSH and Group II (n = 122): Downregulation with GnRH-a + r-FSH and r-LH (beginning simultaneously).Result(s): The number of oocytes collected, the number of oocytes in metaphase II and fertilization rate were significantly lower in the Group I than in Group II (P = 0.036, P = 0.0014 and P = 0.017, respectively). In addition, the mean number of embryos produced per cycle and the mean number of frozen embryos per cycle were statistically lower (P = 0.0092 and P = 0.0008, respectively) in Group I than in Group II. Finally the cumulative implantation rate (fresh+thaw ed embryos) was significantly lower (P = 0.04) in Group I than in Group II. The other clinical and laboratory results analyzed did not show difference between groups.Conclusion: These data support r-LH supplementation in ovarian stimulation protocols with r-FSH and GnRH-a for assisted reproduction treatment.

Pregnancy outcomes in women with repeated implantation failures after intracytoplasmic morphologically selected sperm injection (IMSI)

Background: The purpose of this study was to compare laboratory and clinical outcomes of intracytoplasmic morphologically selected sperm injection (IMSI) and conventional intracytoplasmic sperm injection (ICSI) in couples with repeated implantation failures.Methods: A total of 200 couples with at least two prior unsuccessful ICSI cycles were enrolled: 100 couples were submitted to IMSI and 100 were submitted to routine ICSI. For IMSI, spermatozoa were selected at 8400x magnification using an inverted microscope equipped with Nomarski (differential interference contrast) optics. For conventional ICSI, spermatozoa were selected at 400x magnification. Clinical outcomes were evaluated between the two groups.Results: Study patients were comparable in age, number of treatment failures, aetiology of infertility, percentage of normal form assessed by MSOME (motile sperm organelle morphology examination), semen parameters, total number of oocytes collected, number of mature oocytes collected, total number of embryos transferred and number of high-quality embryos transferred. No statistically significant differences between the two groups were observed with regard to rates of fertilisation, implantation and pregnancy/cycle. Although not statistically significant, rates of miscarriage (IMSI:15.3% vs ICSI:31.7%), ongoing pregnancy (IMSI:22% vs ICSI:13%) and live births (IMSI:21% vs ICSI:12%) showed a trend towards better outcomes in the IMSI group. In addition, analysis of subpopulations with or without male factor showed similar results.Conclusions: Our results suggest that IMSI does not provide a significant improvement in clinical outcome compared to ICSI, at least in couples with repeated implantation failures after conventional ICSI. However, it should be noted that there were clear trends for lower miscarriage rates (approximate to 50% reduced) and higher rates of ongoing pregnancy and live births (both nearly doubled) within the IMSI group. Further confirmation as well as randomized large-scale trials are needed to confirm the beneficial effects of IMSI in couples with poor reproductive prognoses.

Efeito da microbiota cecal e do Lactobacillus salivarius inoculados in ovo em aves desafiadas com Salmonella enterica sorovar Enteritidis

Ovos embrionados provenientes de matrizes pesadas foram inoculados na câmara de ar com microbiota cecal total, microbiota cecal diluída e cultura de Lactobacillus salivarius, no 18º dia de incubação. Dois dias após o nascimento, as aves foram desafiadas com Salmonella enterica sorovar Enteritidis (SE) e, cinco dias após o desafio, avaliou-se a presença da bactéria no fígado e ceco. O efeito de exclusão competitiva, após o desafio com SE, somente foi observado pela ausência da bactéria no fígado das aves tratadas in ovo com L. salivarius. A inoculação in ovo de microbiota cecal indefinida ou diluída não reduziu a colonização de SE no fígado e no ceco das aves, incluindo, neste último, também o tratamento com L. salivarius. Nenhum dos tratamentos in ovo determinou índice de eclodibilidade superior a 65%.

Influence of sire and sire breed (Gyr versus Holstein) on establishment of pregnancy and embryonic loss in lactating Holstein cows during summer heat stress

Heat stress has negative effects on pregnancy rates of lactating dairy cattle. There are genetic differences in tolerance to heat stress; Bos taurus indicus (B. t. indicus) cattle and embryos are more thermotolerant than Bos taurus taurus (B. t. taurus). In the present study, the effects of sire and sire breed on conception and embryonic/fetal loss rates of lactating Holstein cows during the Brazilian summer were determined. In Experiment 1, cows (n = 302) were AI after estrus detection or at a fixed-time with semen from one Gyr (B. t. indicus) or one Holstein sire (B. t. taurus). Pregnancy was diagnosed 80 days after AI. In Experiment 2, cows (n = 811) were AI with semen from three Gyr and two Holstein sires. Pregnancy was diagnosed at 30-40 and at 60-80 days after AI. Cows diagnosed pregnant at the first examination but non-pregnant at the second were considered as having lost their embryo or fetus. Data were analyzed by logistic regression. The model considered the effect of sire within breed, sire breed, days postpartum, period of lactation, and AI type (AI after estrus versus fixed-time). There was no effect of the AI type, days postpartum or milk production on conception or embryonic loss rates. The use of Gyr bulls increased pregnancy rate when compared to Holstein bulls [9.1% (60/657) versus 5.0% (23/456), respectively, P = 0.008; data from Experiments 1 and 2 combined]. Additionally, in Experiment 2, cows inseminated using semen from sire #4 (Gyr) had lower embryonic loss (10%) when compared with other B. t. indicus (35.3% and 40%) or B. t. taurus sires (18.2% and 38.5%, P = 0.03). In conclusion, the use of B. t. indicus sires may result in higher conception rates in lactating Holstein cows during summer heat stress. Moreover, sire can affect embryonic loss and selection of bulls according to this criterion may result in higher parturition rates in lactating Holstein cows. (c) 2006 Elsevier B.V. All rights reserved.

Reproductive responses of rabbit does to a supplemental lighting program

The objective was to investigate whether the productivity of rabbit does can be improved, when natural photoperiod is decreasing, by adopting a supplemental lighting program. Three experiments were conducted involving two groups: control, submitted to the natural decreasing photoperiod, and supplemented with a lighting program which provided 14 h light/24 h beginning at 10 weeks of age. In the first experiment, 20 nulliparous does, 10 from each group, were euthanized 8 h after being presented to a buck; the overall number of follicles, whose diameter exceeded I mm, was determined macroscopically. The right ovaries were collected, histologically analyzed, and electronically measured. In the second experiment, 30 nulliparous does, 15 from each group, were presented to a buck (day 1). Receptive does were euthanized on day 8 to evaluate embryonic survival (number of normal embryos/ovulation rate). In the third experiment, 48 nulliparous does, 24 from each group, were followed from the first presentation to the buck until the weaning of the first litter. The effect of treatment on reproductive and body weight traits of does, and litter performance traits, at birth and weaning, was evaluated. The average number of follicles whose diameter exceeded 1 mm was higher in the treatment group (12.05 +/- 1.07 vs. 8.63 +/- 1.00, P=0.03 7). Receptive does of the treatment group had heavier ovaries relative to those of the control group (790 +/- 59 vs. 470 +/- 64 mg, P=0.004), whereas no treatment difference regarding this trait was found for non-receptive ones. Treatment had a favorable effect on pregnancy rate of total exposed and of receptive does (80.0% vs. 33.3%, P=0.01, and 92.3% vs. 50.0%, P=0.02, respectively). The number of underdeveloped embryos was lower (0.067 +/- 0.380 vs. 2.500 +/- 0.455, P=0.004), embryonic survival up to day 8, and uterus weight was higher in the treatment group (0.839 +/- 0.075 vs. 0.534 +/- 0.087, P=0.033 and 13.83 +/- 0.72 vs. 10.99 +/- 0.84, P=0.037, respectively). Number of presentations tended to be lower (1.32 +/- 0.17 vs. 1.75 +/- 0.16, P=0.077) and adjusted litter size in the first reproductive cycle tended to be higher (7.09 +/- 0.89 vs. 5.22 +/- 0.68, P=0.091) in the treatment group relative to the control.

Factors affecting conception rates following artificial insemination or embryo transfer in lactating holstein cows

The objective of this study was to evaluate the factors that may affect conception rates (CR) following artificial insemination (AI) or embryo transfer (ET) in lactating Holstein cows. Estrous cycling cows producing 33.1 +/- 7.2 kg of milk/d received PGF(2 alpha) injections and were assigned randomly to 1 of 2 groups (AI or ET). Cows detected in estrus (n = 387) between 48 and 96 h after the PGF2a injection received AI (n = 227) 12 h after detection of estrus or ET (n = 160) 6 to 8 d later (1 fresh embryo, grade 1 or 2, produced from nonlactating cows). Pregnancy was diagnosed at 28 and 42 d after estrus, and embryonic loss occurred when a cow was pregnant on d 28 but not pregnant on d 42. Ovulation, conception, and embryonic loss were analyzed by a logistic model to evaluate the effects of covariates [days in milk (DIM), milk yield, body temperature (BT) at d 7 and 14 post-AI, and serum concentration of progesterone (P4) at d 7 and 14 post-AI] on the probability of success. The first analysis included all cows that were detected in estrus. The CR of AI and ET were different on d 28 (AI, 32.6% vs. ET, 49.4%) and 42 (AI, 29.1% vs. ET, 38.8%) and were negatively influenced by high BT (d 7) and DIM. The second analysis included only cows with a corpus luteum on d 7. Ovulation rate was 84.8% and was only negatively affected by DIM. Conception rates of AI and ET were different on d 28 (AI, 37.9% vs. ET, 59.4%) and 42 (AI, 33.8% vs. ET, 46.6%) and were negatively influenced by high BT (d 7). The third analysis included only ovulating cows that were 7 d postestrus. Conception rates of AI and ET were different on d 28 (AI, 37.5% vs. ET, 63.2%) and 42 (AI, 31.7% vs. ET, 51.7%) and were negatively influenced by high BT (d 7). There was a positive effect of serum concentration of P4 and a negative effect of milk production on the probability of conception for the AI group but not for the ET group. The fourth analysis was embryonic loss (AI, 10.8% vs. ET, 21.5%). The transfer of fresh embryos is an important tool to increase the probability of conception of lactating Holstein cows because it can bypass the negative effects of milk production and low P4 on the early embryo. The superiority of ET vs. AI is more evident in high-producing cows. High BT measured on d 7 had a negative effect on CR and embryonic retention.