Produção in vitro de embriões bovinos: utilização de diferentes fontes de gonadotrofinas na maturação dos oócitos

O presente trabalho foi conduzido com o objetivo de avaliar o efeito da utilização de diferentes fontes de gonadotrofinas para maturação in vitro dos oócitos bovinos fecundados e desenvolvidos in vitro sobre as taxas de clivagem (TC) e de blastocistos (TBL). Oócitos imaturos provenientes de ovários de vacas de abatedouro foram submetidos a maturação in vitro sob diferentes condições: meio TCM 199, acrescido de 10% de soro de vaca em estro (SVE), aditivos, hepes, NaHCO3, piruvato de sódio, antibióticos (meio B-199), 20 UI/mL de PMSG e 10 UI/mL de hCG (PMSG/hCG) ou meio B-199, acrescido de 5 mig/mL de FSH e 5 mig/mL de LH (FSH/LH). Seguidos 24 h de cultura a 38,5ºC em atmosfera com 5% de CO2, os oócitos maturos foram incubados com sêmen descongelado durante 18 a 21 horas. Após esse período, os oócitos foram transferidos para placas contendo microgotas de meio Ménezo suplementado com 10% de SVE e células epiteliais do oviduto bovino em suspensão, cobertas com óleo de silicone, os quais permaneceram em cultura por mais 9 dias. Os dados foram analisados pelo teste do Qui-quadrado. A TC e a TBL, para PMSG/hCG e FSH/LH, foram 60 e 13,9% e 61,2 e 10,6%, respectivamente. Não houve diferença entre os tratamentos com relação a TC ou a TBL. Esses resultados sugerem que ambas as fontes de gonadotrofinas podem ser utilizadas para maturação in vitro dos oócitos fecundados e desenvolvidos in vitro.

Plasma concentrations and placental immunostaining of interleukin-10 and tumor necrosis factor-alpha as predictors of alterations in the embryo-fetal organism and the placental development of diabetic rats

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

The first reported case of human tick paralysis in Brazil: a new induction pattern by immature stages

Tick paralysis (TP) is a rare disease with rapid progression and potential fatal evolution. Immediately after the diagnosis, removal of all ticks from the body of the patient is mandatory. The present study reports for the first time a human case of the disease in Brazil. The patient had loss of muscle strength, decreased reflexes and marked palpebral ptosis. Six hours after removal of the last tick, the ptosis improved and on the following day, the patient had near total regression of the symptoms. This report emphasizes the possible presence of similar cases that should be promptly diagnosed and quickly treated. A new induction pattern for TP in humans associated with immature stages of ticks is also presented.

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.

Ultrasound guidance is not necessary during easy embryo transfers

Purpose: To determine whether the use of ultrasound (US) to guide embryo transfer (ET) in a population previously defined as likely to have easy transfer would change the implantation and pregnancy rates in an ICSI program.Methods: A total of 100 patients identified as likely to have easy transfer after mock transfer were divided into two groups: Group I, US-guided ET (N = 50) and Group II, ET without the aid of US (N = 50).Results: Implantation and pregnancy rates were similar (p = 0.51, p = 0.29) for Group I (19.6%,42%) and Group II (16.3%,30%), as also was the abortion rate (p = 0.55) (Group I: 1/21; Group II: 2/15).Conclusion: As long as previous mock transfers are routinely performed during a cycle preceding assisted reproduction and the clinician considers transfer to be easy, ultrasound does not benefit the process of embryo transfer.

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.

Single-embryo transfer reduces clinical pregnancy rates and live births in fresh IVF and Intracytoplasmic Sperm Injection (ICSI) cycles: a meta-analysis

Background: It has become an accepted procedure to transfer more than one embryo to the patient to achieve acceptable ongoing pregnancy rates. However, transfers of more than a single embryo increase the probability of establishing a multiple gestation. Single-embryo transfer can minimize twin pregnancies but may also lower live birth rates. This meta-analysis aimed to compare current data on single-embryo versus double-embryo transfer in fresh IVF/ICSI cycles with respect to implantation, ongoing pregnancy and live birth rates.Methods: Search strategies included on-line surveys of databases from 1995 to 2008. Data management and analysis were conducted using the Stats Direct statistical software. The fixed-effect model was used for odds ratio (OR). Fixed-effect effectiveness was evaluated by the Mantel Haenszel method. Seven trials fulfilled the inclusion criteria.Results: When pooling results under the fixed-effect model, the implantation rate was not significantly different between double-embryo transfer (34.5%) and single-embryo transfer group (34.7%) (P = 0.96; OR = 0.99, 95% CI 0.78, 1.25). on the other hand, double-embryo transfer produced a statistically significantly higher ongoing clinical pregnancy rate (44.5%) than single-embryo transfer (28.3%) (P < 0.0001; OR: 2.06, 95% CI = 1.64,2.60). At the same time, pooling results presented a significantly higher live birth rate when double-embryo transfer (42.5%) (P < 0.001; OR: 1.87, 95% CI = 1.44,2.42) was compared with single-embryo transfer (28.4%).Conclusion: Meta-analysis with 95% confidence showed that, despite similar implantation rates, fresh double-embryo transfer had a 1.64 to 2.60 times greater ongoing pregnancy rate and 1.44 to 2.42 times greater live birth rate than single-embryo transfer in a population suitable for ART treatment.

Morbidity-mortality and performance evaluation of Brahman calves from in vitro embryo production

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

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.

Comparison of progesterone-based protocols with gonadotropin-releasing hormone or estradiol benzoate for timed artificial insemination or embryo transfer in lactating dairy cows

The objective was to compare two protocols for synchronizing ovulation in lactating Holstein cows submitted to timed AI (TAI) or timed ET (TET). Within each farm (n = 8), cows (n = 883; mean +/- SEM 166.24 +/- 3.27 d postpartum, yielding 36.8 +/- 0.34 kg of milk/d) were randomly assigned to receive either: 1) an intravaginal progesterone insert (CIDR (R)) with 1.9 g of progesterone + GnRH on Day -10, CIDR (R) withdrawal + PGF2 alpha on Day -3, and 1 mg estradiol cypionate on Day -2 (treatment GP-P-E; n(TAI) = 180; n(TET) = 260); or 2) a CIDR (R) insert + 2 mg estradiol benzoate on Day -10, PGF2 alpha on Day -3, CIDR (R) withdrawal + 1 mg estradiol cypionate on Day -2 (treatment EP-P-E; n(TAI) = 174; n(TET) = 269). Cows were subsequently randomly assigned to receive either TAT on Day 0 or TET on Day 7. Serum progesterone concentration on Day -3 was greater in GP-P-E than in EP-P-E (2.89 +/- 0.15 vs 2.29 +/- 0.15 ng/mL; P < 0.01), with no significant effect of group on serum progesterone on Day 7. Compared to cows submitted to TAI, those submitted to TET had greater pregnancy rates on Day 28 (44.0% [233/5291 vs 29.7% [105/354]; p < 0.001) and on Day 60 (37.6% [199/529] vs 26.5 [94/354]; P < 0.001). However, there were no effects of treatments (GP-P-E vs EP-P-E; P > 0.10) on synchronization (87.0% [383/440] vs 85.3% [378/443]), conception (TAI: 35.3% [55/156] vs 33.8% [50/148]; TET: 50.7% [115/227] vs 51.3% [118/230]) and pregnancy rates on Days 28 (TAT: 30.5% [55/180] vs 28.7% 150/174]; TET: 44.2% [115/260] vs 43.9% [118/2691) and 60 (TAI: 27.2% [49/80] vs 25.9% [45/174]; TET: 38.8% [101/260] vs 36.4% [98/269]). In conclusion, GP-P-E increased serum progesterone concentrations on Day -3, but rates of synchronization, conception, and pregnancy were not significantly different between cows submitted to GP-P-E and EP-P-E protocols, regardless of whether they were inseminated or received an embryo. (c) 2011 Elsevier B.V. All rights reserved.

Effects of postbreeding gonadotropin treatments on conception rates of lactating dairy cows subjected to timed artificial insemination or embryo transfer in a tropical environment

The objective of experiment 1 was to evaluate the effects of treatments with human chorionic gonadotropin (hCG) or GnRH 7 d after induced ovulation on reproductive performance of lactating dairy cows submitted to timed artificial insemination (TAI) or timed embryo transfer (TET). A total of 834 potential breedings were used from 661 lactating Holstein cows (37.3 +/- 0.3 kg of milk/d). Cows had ovulation synchronized and were assigned randomly to receive TAI on d 0 or TET on d 7. Within each group, cows were assigned randomly to receive on d 7 no additional treatment (control; n(TAI) = 156; n(TET) = 126), a 100 mu g i.m. injection of GnRH (n(TAI) = 155; n(TET) = 124), or a 2,500 TU i.m. injection of hCG (ITA = 151; n(TET) = 122). Postbreeding treatment affected the percentages of pregnant cows at TET on d 28 (control: 38.1%; GnRH: 52.4%; hCG: 45.1%) and on d 60 (control: 32.5%; GnRH: 41.1%; hCG: 38.5%), but postbreeding treatment did not affect percentages of pregnant cows at TAT on d 28 (control: 30.1%; GnRH: 32.2%; hCG: 32.4%) or on d 60 (control: 25.6%; GnRH: 27.1%; hCG: 29.8%). The objective of experiment 2 was to evaluate the effect of a treatment with GnRH 7 d after TET on reproductive performance of lactating dairy cows that received a previous GnRH treatment at TET. A total of 285 potential breedings were used from 257 lactating Holstein cows (35.1 +/- 0.8 kg of milk/d). Cows had ovulation synchronized and were assigned for TET on d 7. Immediately after TET, all cows were treated with a 100 mu g i.m. injection of GnRH. on d 14, cows were assigned randomly to receive (G7-14; n = 147) or not (G7; n = 138) an additional injection of GnRH. Pregnancy diagnosis were performed on d 28 and 60. The additional treatment with GnRH on d 14 did not affect the percentages of pregnant cows on d 28 (G7: 48.5%; G7-14: 42.9%) or on d 60 (G7: 39.8%; G7-14: 37.4%). In conclusion, treatment with GnRH or hCG 7 d after induced ovulation increased conception rates in lactating dairy cows submitted to TET, but not in cows submitted to TAI. Moreover, treatment with GnRH 7 d after TET did not enhance reproductive performance of lactating dairy cows that received a previous GnRH treatment at TET.

Use of oocyte transfer in a commercial breeding program for mares with reproductive abnormalities

In some mares with lesions of the reproductive tract, embryo collection and survival rates are low or collection of embryos is not feasible. For these mares, oocyte transfer has been proposed as a method to induce pregnancies. In this report, a method for oocyte transfer in mares and results of oocyte transfer performed over 2 breeding seasons, using mares with long histories of subfertility and various reproductive lesions, are described.Human chorionic gonadotropin or an implant containing a gonadotropin-releasing hormone analog was used to initiate follicular and oocyte maturation. Oocytes were collected by means of transvaginal ultrasound-guided follicular aspiration. Following follicular aspiration, cumulus oocyte complexes were evaluated for cumulus expansion and signs of atresia; immature oocytes were cultured in vitro to allow maturation. The recipient's ovary and uterine tube (oviduct) were exposed through a flank laparotomy with the horse standing, and the oocyte was slowly deposited within the oviduct. Oocyte transfer was attempted in 38 mares between 9 and 30 years old during 2 successive breeding seasons. All mares had a history of reproductive failure while in breeding and embryo transfer programs. Twenty pregnancies were induced. Fourteen of the pregnant mares delivered live foals. Results suggest that oocyte transfer can be a successful method for inducing pregnancy in subfertile mares in a commercial setting..