Effect of recombinant bovine somatotropin (bST) on follicular population and on in vitro buffalo embryo production

The objective of this study was to evaluate the effect of bovine somatotropin (bST) on ovarian follicular population in buffalo heifers and its influence on oocyte quality, recovery rates and in vitro embryo production. We tested the hypothesis that bST treatment in buffalo females submitted to an ovum pick-up (OPU) program Would improve the number of follicles recruited, oocyte quality and in vitro embryo production. A total of 10 heifers were assigned into two treatment groups: group bST (n = 5; receiving 500 mg of bST in regular intervals) and control group (n = 5; without additional treatment). Both groups were subjected to OPU sessions twice a week (every 3 or 4 days), for a total of 10 sessions per female, although due to procedural problems, only the first five OPU sessions produced embryos. The number of follicles and the diameters were recorded at all OPU sessions. The harvested oocytes were counted and classified according to their quality as either A, B, C, D or E, with A and B considered good quality. Cleavage and blastocyst production rates were evaluated 2 and 7 days after in vitro fertilization, respectively. The bST treatment increased the total number of antral follicles (> 3 mm in diameter; 12.2 compared with 8.7: p, < 0.05) and of small antral follicles (< 5 mm; 9.1 compared with 6.5; p < 0.05) per OPU session. The bST also tended to increase the number of oocytes recovered per session (5.2 compared with 4.1; p = 0.07), and enhanced the percentage of good quality oocytes (48.8% compared with 40.6%; p = 0.07), bST showed no effect on cleavage and blastocyst production rates (p > 0.05). The significant effects of performing repeated OPU sessions were decreasing the follicular population (p < 0.001) as well as the number of follicles aspirated (p < 0.001), and oocytes recovered (p < 0.02). In conclusion, bST treatment improves the follicular population, demonstrating its possible application in buffalo donors submitted to OPU programs. (c) 2008 Elsevier B.V. All rights reserved.

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)

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.

Effects of fetuin on zona pellucida hardening, fertilization and embryo development in cattle

Mammalian oocytes can undergo spontaneous meiotic maturation when they are liberated from their follicles and cultured in vitro; however, the zona pellucida (ZP) becomes resistant to chymotrypsin digestion, or hardens, when spontaneous maturation occurs in serum-free medium. Schroeder et al. [Biol. Reprod. 43 (1990) 891] described that fetuin, a component of fetal calf serum (FCS), inhibits ZP hardening during oocyte maturation. The aim of this experiment was to study the effect of the presence of cumulus cells and addition of hormones to maturation media on bovine zona hardening and embryo development in medium with and without fetuin. In Experiment 1, different concentrations of fetuin were added to the maturation medium. The time necessary for digestion of 50% of the ZP (d50) was not different when oocytes were matured in presence of 10% FCS, 1 mg/ml polyvinyl alcohol (PVA), or 4, 1 and 0.25 mg/ml of fetuin; cleavage rates were also similar. However, significantly more blastocysts (P < 0.05) were formed when FCS was used compared to PVA and 0.25 mg/ml of fetuin. In Experiment 11, we examined the influence of the presence of cumulus cells and hormones during the maturation of oocytes in media with PVA, BSA, FCS and fetuin. The d50 was significantly higher (P < 0.05) when oocytes were matured in presence of cumulus cells. The cleavage rate of cumulus-intact oocytes was similar for all groups. However, when oocytes were partially stripped before maturation, the cleavage rate was significantly higher (P < 0.05) when FCS or fetuin was used. In both stripped and non-stripped groups, significantly more blastocysts (P < 0.05) were formed when oocytes were matured with FCS compared to BSA and PVA. These results indicate that zona hardening, as described for mouse and human oocytes, does not have a large effect on bovine cumulus-intact oocytes. Apparently fetuin can be used as a substitute for FCS during bovine oocyte maturation, since it leads to similar developmental rates as FCS in intact and partially stripped oocytes. (C) 2002 Published by Elsevier B.V. B.V.