Effects of Hoechst 33342 staining and ultraviolet irradiation on mitochondrial distribution and DNA copy number in porcine oocytes and preimplantation embryos

Hoechst 33342 (H342), in combination with ultraviolet (UV) irradiation, is frequently used to aid or confirm the enucleation of porcine oocytes in somatic cell nuclear transfer programs. The exposure of oocytes to H342 and UV irradiation has a deleterious effect on the development of in vitro‐fertilized porcine oocytes, with increasing exposure to UV irradiation (up to 30 sec) having more drastic effects. It has been hypothesized that this decrease in embryonic development could be due to damage to the mitochondrial DNA (mtDNA). To investigate this hypothesis, we analyzed the mitochondrial distribution and DNA copy number of in vitro‐matured porcine oocytes exposed to H342/UV and the subsequent embryonic development compared with the mitochondrial distribution and DNA copy number of in vivo‐derived oocytes and embryos. Using quantitative, real‐time polymerase chain reaction (qPCR) protocols to analyze mtDNA and confocal laser scanning microscopy with MitoTracker Deep Red to determine mitochondrial distribution, we demonstrated that the simultaneous exposure of in vitro‐matured porcine oocytes to H342 staining and UV irradiation is associated with reduced oocyte developmental competence and abnormal mitochondrial distribution in the resulting cleaved embryos. In addition, 2‐ to 4‐cell embryos derived from oocytes exposed to H342/UV showed a significant decrease in mtDNA copy number. These results should be considered when H342/UV procedure is used during nuclear transfer in recipient porcine oocytes. Mol. Reprod. Dev. 79: 651–663, 2012. © 2012 Wiley Periodicals, Inc.     

Ecological risk assessment of the introduction of exotic carrageenophytes in the tropical Western Atlantic

The potential risks of cultivating carrageenophyte genera for commercial purposes in the circumtropical belt are debated. However, species introductions of two such genera, Kappaphycus Doty and Eucheuma J. Agardh, have been reported in 30 different locations in this region over the last 30 years. On several occasions, these introductions did not adequately evaluate potential environmental risks or were conducted without approval from local regulatory bodies. In the present paper, a working protocol is proposed for the quarantine and assessment of the possible effects of the introduction of Kappaphycus alvarezii and Kappaphycus striatus to shallow marine ecosystems of the tropical Western Atlantic. This protocol is based on field data following from the introduction of eucheumoids onto the Cuban shelf in the early 1990s. It was demonstrated that the propagation of either carrageenophyte in oligotrophic waters of the Cuban Archipelago did not pose a potential risk to the region’s biodiversity due to the synergic combination of high herbivory and low rates of growth. Physical features of the substrate and depth were the most important regulators of grazing. These environmental conditions restrict potential cultivation sites in the Cuban Archipelago to a few small regions where nutrient pulses are well established. In these areas, when the canopy of cultivated carrageenophytes is sufficiently high, a significant effect on benthic communities is observed. In consideration of the need to protect places with high intrinsic value, this fact should be considered during site selection.     

Effects of stretching on maximal anaerobic power: the roles of active and passive warm-ups

The purpose of the study was to provide practical suggestions on the effect of stretching on the maximal anaerobic power preceded by active or passive warm-up. To this aim, 15 relatively fit male subjects (age 23 +/- 0.2 years, height 177 +/- 2 cm, body mass 74 +/- 2 kg; [mean +/- SE]) randomly performed a series of squat jumps (SJ) and countermovement jumps (CMJ). Jumps were preceded alternatively by: i) passive stretching of lower limbs muscles; ii) active warm-up (AWU); iii) passive warm up (PWU); and iv) the joining of stretching with either active warm-up (AWU+S) or passive warm-up (PWU+S). In control conditions (C) only jumps were required. For the 2 jumps the flight time (Ft), the peak force (Pf), and the maximal power (Wpmax) were calculated. It resulted that Ft, Pf, and Wmax values were significantly higher: i) after AWU than after PWU and PWU+S in CMJ; and ii) in AWU as compared to those of other protocols of SJ. Stretching did not negatively affect the maximal anaerobic power, per se, but seems to inhibit the effect of AWU. The results suggested that AWU seemed to increase vertical jump performance when compared to PWU, presumably due to an increase in metabolic activity as a consequence of AWU, which did not occur in PWU, despite the same skin temperature. Passive stretching alone seemed not to negatively influence vertical jump performance, whereas, if added after AWU, could reduce the power output.     

Birth of piglets after deep intrauterine insemination with flow cytometrically sorted boar spermatozoa

The present study was carried out to determine the pregnancy rates, farrowing rates and litter size in sows with either induced or spontaneous ovulation inseminated with flow cytometric sorted spermatozoa using deep intrauterine insemination technology. Spermatozoa were stained with Hoechst 33342 and sorted by flow cytometry/cell sorting but not separated into separate X and Y populations. In Experiment 1, sows (n=200) were weaned and treated for estrus/ovulation induction with eCG/hCG. Inseminations with either sorted (70 or 140 million) or non-sorted (70 or 140 million) spermatozoa were done using a specially designed flexible catheter. Farrowing rates were 39.1 and 78.7% for 70 million of sorted and non-sorted, respectively, and 46.6 and 85.7% for 140 million of sorted and non-sorted, respectively (P<0.05). The litter size in sows inseminated with sorted spermatozoa showed a tendency to be lower than when non-sorted spermatozoa were inseminated. In Experiment 2, sows (n=140) were inseminated as in Experiment 1 except that natural estrus was used. The ovaries of these sows were evaluated by transrectal ultrasonography. Farrowing rates were 25 and 77.2% for 70 million of sorted and non-sorted, respectively, and 32 and 80.9% for 140 million of sorted and non-sorted, respectively (P<0.05). These results show that the Deep Intrauterine Insemination technology can be successfully used to produce piglets from sorted spermatozoa when sows are hormonally treated to induce synchronous post weaning oestrus and ovulation.     

Prevalence of Candida albicans and Trichomonas vaginalis in pregnant women in Havana City by an immunologic latex agglutination test

We aimed to estimate the prevalence of Candida albicans and Trichomonas vaginalis in immunocompetent pregnant women living in Havana City, Cuba, with or without symptoms of vaginitis, using a sample of 640 women from 6 Gyneco-obstetrics hospitals, which represents 2.5% of total yearly pregnant women. Diagnosis was made using a new latex agglutination kit (Newvagin C-Kure, La Habana, Cuba). Clinical sensitivity and specificity of this assay were validated against culture method, with 467 and 489 clinical specimens for Candida albicans and Trichomonas vaginalis, respectively. Results showed that the kit clinical sensitivity was 100% for Candida albicans and 86.7% for Trichomonas vaginalis compared with a clinical specificity of 93.3% for Candida albicans and 95.1% for Trichomonas vaginalis by culture. The prevalence of candidiasis was determined to be 42.3% (95% confidence interval [CI] 3.8%); the prevalence of trichomoniasis was 9.84% (95% CI 2.3%). In our sample, 48.7% of the women tested negative with respect to both candidiasis and trichomoniasis. Only 6.41% of the cases yielded inconclusive results. The test has high sensitivity, and our results indicate a relatively high prevalence of both infections. However, a significant difference (P < .001) was also observed in candidiasis and trichomoniasis prevalence among hospitals corresponding to the quantity of women with clinical vaginitis. No difference was observed between diabetics and nondiabetics, probably due to the special care of diabetic pregnant women. We conclude that the method is useful for this kind of vaginitis prevalence study and that candidiasis and trichomoniasis prevalences in pregnant women of Havana are 38.5% to 46.2 % (95% CI) and 7.5% to 12.1% (95% CI), respectively.     

Quantitative gene expression analysis in a brain slice model: influence of temperature and incubation media

We describe the RNA integrity (28S/18S ratio) and the messenger RNA (mRNA) expression of genes encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), microtubule-associated serine/threonine kinase 2 (Mast2), and β-actin in cortical brain slices incubated for up to 24 h in Ringer’s solution and Dulbecco’s modified Eagle’s medium (DMEM) at 25 and 37 °C. Our data reveal an optimal temporal working window between 1 and 6 h when slices are incubated in Ringer’s solution at 25 °C that allows experiments related to gene expression dynamics to be performed more suitably than those carried out at 37 °C. In addition, we show that reference gene expression may be modified in dynamic experiments and may compromise studies of gene expression.     

Intermittent Preventive Treatment of Malaria in Pregnancy with Mefloquine in HIV-Negative Women: A Multicentre Randomized Controlled Trial

Background

Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended by WHO to prevent malaria in African pregnant women. The spread of SP parasite resistance has raised concerns regarding long-term use for IPT. Mefloquine (MQ) is the most promising of available alternatives to SP based on safety profile, long half-life, and high efficacy in Africa. We evaluated the safety and efficacy of MQ for IPTp compared to those of SP in HIV-negative women.

Methods and Findings

A total of 4,749 pregnant women were enrolled in an open-label randomized clinical trial conducted in Benin, Gabon, Mozambique, and Tanzania comparing two-dose MQ or SP for IPTp and MQ tolerability of two different regimens. The study arms were: (1) SP, (2) single dose MQ (15 mg/kg), and (3) split-dose MQ in the context of long lasting insecticide treated nets. There was no difference on low birth weight prevalence (primary study outcome) between groups (360/2,778 [13.0%]) for MQ group and 177/1,398 (12.7%) for SP group; risk ratio [RR], 1.02 (95% CI 0.86–1.22; p = 0.80 in the ITT analysis). Women receiving MQ had reduced risks of parasitemia (63/1,372 [4.6%] in the SP group and 88/2,737 [3.2%] in the MQ group; RR, 0.70 [95% CI 0.51–0.96]; p = 0.03) and anemia at delivery (609/1,380 [44.1%] in the SP group and 1,110/2743 [40.5%] in the MQ group; RR, 0.92 [95% CI 0.85–0.99]; p = 0.03), and reduced incidence of clinical malaria (96/551.8 malaria episodes person/year [PYAR] in the SP group and 130/1,103.2 episodes PYAR in the MQ group; RR, 0.67 [95% CI 0.52–0.88]; p = 0.004) and all-cause outpatient attendances during pregnancy (850/557.8 outpatients visits PYAR in the SP group and 1,480/1,110.1 visits PYAR in the MQ group; RR, 0.86 [0.78–0.95]; p = 0.003). There were no differences in the prevalence of placental infection and adverse pregnancy outcomes between groups. Tolerability was poorer in the two MQ groups compared to SP. The most frequently reported related adverse events were dizziness (ranging from 33.9% to 35.5% after dose 1; and 16.0% to 20.8% after dose 2) and vomiting (30.2% to 31.7%, after dose 1 and 15.3% to 17.4% after dose 2) with similar proportions in the full and split MQ arms. The open-label design is a limitation of the study that affects mainly the safety assessment.

Conclusions

Women taking MQ IPTp (15 mg/kg) in the context of long lasting insecticide treated nets had similar prevalence rates of low birth weight as those taking SP IPTp. MQ recipients had less clinical malaria than SP recipients, and the pregnancy outcomes and safety profile were similar. MQ had poorer tolerability even when splitting the dose over two days. These results do not support a change in the current IPTp policy.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT 00811421","term_id":"NCT00811421"}}NCT 00811421; Pan African Clinical Trials Registry PACTR 2010020001429343 Please see later in the article for the Editors'' Summary