Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence.

Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.

Standardization of a Sex-Sorting Protocol for Stallion Spermatozoa by Means of Absolute RT-qPCR.

Sperm sexing is a technology that can generate great economic benefits in the animal production sector. Techniques such as sex-sorting promise over 90% accuracy in sperm sexing. However, for the correct standardization of the technique, some laboratory methodologies are required. The present manuscript describes in detail a standardized equine sperm sex-sorting protocol using an absolute qPCR-based methodology. Furthermore, the results of absolute qPCR were implemented and validated by generating equine/bovine heterologous embryos by intracytoplasmic sperm injection (ICSI) of presumably sexed equine spermatozoa into bovine oocytes using a piezoelectric system (Piezo-ICSI). Our results indicated that equine sex-sorting spermatozoa had a 97% and 94% certainty for X and Y sperm, respectively, while presumptive female and male equine/bovine hybrid embryos, generated by Piezo-ICSI, had an accuracy of 92% with respect to the desired sex. Therefore, it is concluded that the presented methodology is a reliable, cost-effective, and relatively simple option for standardizing sex-sorting of equine spermatozoa. This is supported by the results of the correct sexing of Piezo-ICSI heterologous embryos generated with the sexed spermatozoa, validating the correct sexing and viability of these gametes.

Effect of Atomized Black Maca (Lepidium meyenii) Supplementation in the Cryopreservation of Alpaca (Vicugna pacos) Epididymal Spermatozoa.

Artificial insemination is an important assisted reproductive technology that has been applied in several mammalian species. However, successful cryopreservation of semen of South American camelids has been limited, hindering the commercial application of artificial insemination in alpaca species. In this scenario, the addition of antioxidants to semen extenders provides a strategy to improve the freezability of mammalian sperm. Bioactive metabolites from natural extracts of black maca have shown valuable antioxidant properties. Thus, the objective of this study was to evaluate the effect of the addition of atomized black maca in the freezing medium of epididymal spermatozoa of alpacas. Fifteen pairs of epididymis were collected from a local slaughterhouse. Each sample was divided into six groups: (1) fresh, (2) yolk medium (YM), (3) 10 mg/mL maca, (4) 20 mg/mL maca, (5) 30 mg/mL maca, and (6) resveratrol (as an antioxidant control). Sperm cryopreservation was performed through the slow freezing method. Markers associated with functionality, such as motility, viability, and plasma membrane integrity, as well as markers associated with oxidative damage, such as DNA integrity, total ROS production, and mitochondrial function, were analyzed. The results show that the supplementation with black maca (20 mg/mL) improved the sperm motility, viability, plasma membrane integrity, and mitochondrial function evaluated according to an index of formazan deposits. Similarly, the ROS production decreased with maca at 20 mg/mL, although the DNA integrity did not show any differences among the groups. These results suggest that maca at 20 mg/mL has cytoprotective effects during freezing/thawing of epididymal sperm of alpaca species. Further research will be focused on assessing the effects of maca supplementation on semen extenders by using biomolecular markers (proAKAP4) associated with fertility.

Simple and Efficient Chemically Defined In Vitro Maturation and Embryo Culture System for Bovine Embryos.

Supplementation of the culture media for in vitro production (IVP) of bovine embryos with fetal bovine serum (FBS) is associated with inconsistent outcomes. The present study sought to replace FBS and BSA by insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). In Experiment 1, absence of FBS from maturation medium (MM) did not affect the rate of in vitro maturation, as assessed by the extrusion of the first polar body. However, when gonadotropins and FBS were removed from the MM, the maturation rate was significantly reduced even in the presence of growth factors. Therefore, gonadotropin-supplemented MM medium was established as the base medium for the defined maturation condition. In Experiment 2, the addition of growth factors to gonadotropin-supplemented MM medium supported similar maturation (~90%) compared to the undefined condition (FBS-carrying). In Experiment 3, the addition of growth factors to embryo culture medium showed similar in vitro competence compared to the undefined (FBS) control. In Experiment 4, completely defined conditions (absence of FBS and BSA during in vitro maturation and embryo culture) were tested. A higher cleavage was observed with FGF2 (86%) compared to EGF (77%) and the FBS control (77%), but similar blastocyst rates were observed for FGF2 (24%), EGF (19%) and the FBS control (25%). Embryo quality was similar among groups. Finally, post-thawing survival was higher for FGF2 (94%) compared to the FBS control (77%). Thus, we report a simple defined IVP system for bovine species that generates developmental outcomes and embryos of similar quality than those produced under conditions containing FBS.

High prevalence and clinical characteristics of respiratory infection by human rhinovirus in children from Lima-Peru during years 2009-2010.

INTRODUCTION: Human rhinovirus is a major cause of acute respiratory infections (ARIs) worldwide. Epidemiological data on human rhinovirus (RV) in Peru is still scarce, as well as its role in respiratory infections in children. Therefore, the aim of this study was to describe the prevalence of rhinovirus and to identify the circulating species in nasopharyngeal swabs from children with acute respiratory infections. MATERIALS AND METHODS: We analyzed nasopharyngeal swab samples that were collected from children younger than 17 years old, who had a clinical diagnosis of ARI from the "Hospital Nacional Cayetano Heredia" between May 2009 and December 2010. The original study recruited 767 inpatients with ARI, 559 samples of which were included and analyzed in the current study. Detection of rhinovirus and determination of rhinovirus species were characterized by PCR. RESULTS: Rhinovirus was detected in 42.22% samples (236/559), RV-A was detected in 10.17% (24/236) of the cases, RV-B in 16.53% (39/236), and RV-C in 73.31% (173/236). The age group with the highest number of cases was the 0-5 months group with 45.97%, followed by the 1-5 years group with 25.22%. Most of the positive RV cases, i.e., 86.44% (204/236), were hospitalized. The most common signs and symptoms found in patients who tested positive for RV were cough (72.88%), fever (68.64%), rhinorrhea (68.22%), and respiratory distress (61.44%). Infection with RV-A was associated with wheezing (p = 0.02). Furthermore, RV-C was related to cough (p = 0.01), wheezing (p = 0.002), and conjunctival injection (p = 0.03). A peak in RV-C cases was found in March (32 cases in 2010); June (18 cases in 2009 and 12 cases in 2010), which corresponds to the fall season in Peru; and also November (17 cases in 2009 and 4 cases in 2010), which corresponds to spring. RV-A and RV-B cases were constant throughout the year. CONCLUSION: In conclusion, we found a high prevalence of rhinovirus C infection among pediatric patients with acute respiratory infections in Lima, Peru. This viral infection was more common in children between 0 to 5 months old, and was associated with cough, wheezing, and conjunctival injection. Epidemiological surveillance of this virus should be strengthened/encouraged in Peru to determine its real impact on respiratory infections.

Effect of MnTBAP on in vitro capacitation of frozen-thawed stallion sperm.

In vitro manipulation of spermatozoa leads to deleterious changes of structure and function that occur mainly due to oxidative stress, therefore, prevention or treatment is a strategy to improve the functions of processed sperm. In the present study, the aim was to evaluate the effects of MnTBAP supplementation, a compound with antioxidant activity, on in vitro capacitation conditions of thawed equine sperm. For this purpose, stallion spermatozoa (2 × 106 cells/mL) were incubated in the sperm-TLP base medium for 4 h in which there were three different conditions: non-capacitating, capacitating, and capacitating plus 150 mM MnTBAP. There were incubations for 4 h at 37.5 °C in a humidified air atmosphere. Sample analysis was performed immediately after thawing (0 h), and at the end of the incubation period (4 h), unless otherwise indicated. The following variables were evaluated for spermatozoa: plasma membrane integrity and fluidity, acrosome integrity, intracellular calcium concentrations, intracellular pH, tyrosine phosphorylation, ATP concentrations, motility and heterologous zona-binding assay, using flow cytometry, fluorescent microscopy and/or chemiluminescence, depending on the most appropriate procedure for the variable being evaluated. Results indicated that capacitation-like changes were synergistically induced by the cAMP agonists, phosphodiesterase inhibitor and bicarbonate. The presence of bovine serum albumin was harmful to the plasma membrane. The MnTBAP supplementation had a positive effect on viability-related markers (plasma membrane integrity, membrane fluidity, associated with greater intracellular pH) when there were capacitating conditions. In conclusion, the activity of MnTBAP contributes to improving the in vitro incubation conditions of frozen-thawed stallion sperm.

Defective sperm head decondensation undermines the success of ICSI in the bovine.

The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whether in vitro maturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI, in vivo-matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, although in vitro-matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation by in vitro-matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu of in vitro-matured oocytes and to replicate the molecular changes associated with in vivo capacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species.

Sperm capacitation pretreatment positively impacts bovine intracytoplasmic sperm injection.

The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is low compared to other species due in part to inadequate egg activation and sperm nucleus decondensation after injection. We hypothesized that this low efficiency is due to the lack of complete sperm capacitation, so we evaluated the effects of isobutylmethylxanthine (IBMX) and methyl-ß-cyclodextrin (MßCD) on bovine sperm capacitation and on the preimplantation developmental potential of bovine embryos generated by ICSI. Treatment with IBMX and MßCD decreased sperm viability (between 13-30%); nevertheless, 0.4 mM IBMX and 1 mM MßCD increased (p < 0.05) capacitation metrics-that is, acrosome exocytosis, intracellular calcium level, plasma membrane fluidity, and tyrosine phosphorylation-compared to the control. After ICSI, embryos injected with IBMX- and MßCD-treated sperm showed similar cleavage to the untreated group (range 82-88%). Pronucleus formation rate was higher with MßCD-pretreatment (54%) compared to the control group (25%), and blastocyst rate was significantly improved with MßCD-pretreatment (24%) compared to the IBMX (18%) and control (17%) groups. Importantly, embryo quality-as assessed by the total number of cells, cell allocation, and apoptotic cell index-was not affected by the sperm treatments. In conclusion, MßCD pretreatment of sperm improved the efficiency of blastocyst production in bovine ICSI.

Improved preimplantation development of bovine ICSI embryos generated with spermatozoa pretreated with membrane-destabilizing agents lysolecithin and Triton X-100.

In cattle, intracytoplasmic sperm injection (ICSI) has a low efficiency. The acrosome content may be responsible for this effect because of the large amount of hydrolytic enzymes that are released within the oocyte. With the aim of removing the acrosome and destabilize the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX) at different concentrations. We evaluated the membrane integrity, the acrosome integrity, DNA integrity, and the variation of phospholipase C zeta. The rates of development (cleavage and blastocysts) were also evaluated along with pronuclear formation and the embryo quality. Spermatozoa incubated with LL and TX (0.01%, 0.02%, 0.03%, and 0.04%) decreased (P < 0.0001) sperm viability in a dose-dependent manner. The acrosome reaction was also increased (P < 0.0001) in all tested concentrations of LL and TX achieving 100% at 0.05% concentration in both treatments. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay reported an increase (P < 0.05) in DNA fragmentation only with the highest concentration of LL (0.06%), whereas all concentrations assessed of TX reported an increased respect to the control. Phospholipase C zeta expression decreased (P < 0.05) in spermatozoa treated with LL and TX at all concentrations tested. A higher cleavage rate was observed in ICSI-TX (66%) and ICSI-LL (65%) groups compared with the untreated control group (51%) and the blastocyst formation rate significantly increased in the ICSI-LL group (29%) compared with the control (21%). No differences were observed in the pronuclear formation and quality of the embryos. In conclusion, the destabilization of the plasma membrane and the release of the acrosomal content with LL and TX before ICSI improve the rate of embryonic development, without affecting the quality of the embryos produced by this technique.

Relaxant effects of a hydroalcoholic extract of Ruta graveolens on isolated rat tracheal rings.

BACKGROUND: Ruta graveolens L. (R. graveolens) is a medicinal plant employed in non-traditional medicines that has various therapeutic properties, including anthelmintic, and vasodilatory actions, among others. We evaluated the trachea-relaxant effects of hydroalcoholic extract of R. graveolens against potassium chloride (KCl)- and carbachol-induced contraction of rat tracheal rings in an isolated organ bath. RESULTS: The results showed that the airway smooth muscle contraction induced by the depolarizing agent (KCl) and cholinergic agonist (carbachol) was markedly reduced by R. graveolens in a concentration-dependent manner, with maximum values of 109 ± 7.9 % and 118 ± 2.6 %, respectively (changes in tension expressed as positive percentages of change in proportion to maximum contraction), at the concentration of 45 µg/mL (half-maximal inhibitory concentration IC50: 35.5 µg/mL and 27.8 µg/mL for KCl- and carbachol-induced contraction, respectively). Additionally, the presence of R. graveolens produced rightward parallel displacement of carbachol dose-response curves and reduced over 35 % of the maximum smooth muscle contraction. CONCLUSIONS: The hydroalcoholic extract of R. graveolens exhibited relaxant activity on rat tracheal rings. The results suggest that the trachea-relaxant effect is mediated by a non-competitive antagonistic mechanism. More detailed studies are needed to identify the target of the inhibition, and to determine more precisely the pharmacological mechanisms involved in the observed biological effects.

Relaxant effects of a hydroalcoholic extract of Ruta graveolens on isolated rat tracheal rings

BACKGROUND: Ruta graveolens L. (R. graveolens) is a medicinal plant employed in non-traditional medicines that has various therapeutic properties, including anthelmintic, and vasodilatory actions, among others. We evaluated the trachea-relaxant effects of hydroalcoholic extract of R. graveolens against potassium chloride (KCl)- and carbachol-induced contraction of rat tracheal rings in an isolated organ bath. RESULTS: The results showed that the airway smooth muscle contraction induced by the depolarizing agent (KCl) and cholinergic agonist (carbachol) was markedly reduced by R. graveolens in a concentration-dependent manner, with maximum values of 109 ± 7.9 % and 118 ± 2.6 %, respectively (changes in tension expressed as positive percentages of change in proportion to maximum contraction), at the concentration of 45 µg/mL (half-maximal inhibitory concentration IC50: 35.5 µg/mL and 27.8 µg/mL for KCl- and carbachol-induced contraction, respectively). Additionally, the presence of R. graveolens produced rightward parallel displacement of carbachol dose-response curves and reduced over 35 % of the maximum smooth muscle contraction. CONCLUSIONS: The hydroalcoholic extract of R. graveolens exhibited relaxant activity on rat tracheal rings. The results suggest that the trachea-relaxant effect is mediated by a non-competitive antagonistic mechanism. More detailed studies are needed to identify the target of the inhibition, and to determine more precisely the pharmacological mechanisms involved in the observed biological effects.

[Cataract blindness in people 50 years old or older in a semirural area of northern Peru]. / Ceguera por catarata en personas mayores de 50 años en una zona semirrural del norte del Perú

OBJECTIVE: To determine the prevalence of blindness and of cataract blindness in persons 50 years of age or older in Piura and Tumbes, which are two departments in northern Peru, and to describe the coverage and quality of cataract surgery services in that area, and the barriers that prevent access to those services. METHODS: Systematic sampling of persons 50 years old or older was done in Piura and Tumbes between August 2002 and March 2003, with 80 clusters of 60 people each being selected. Of the 4 800 persons chosen, 4,782 of them were examined, using a survey instrument that gathered general information on each person, the results of the visual acuity test and the lens examination, and information on cataract surgery or why that surgery had not been done. Visual acuity (VA) testing was done with a Snellen optotype with the letter "E," with sizes of 20/60 and 20/200 at distances of 6 m and 3 m, respectively, with the person using the visual correction (glasses) available. When the VA was less than 20/60 in one of the eyes, vision was tested with pinhole glasses. RESULTS: The prevalence of bilateral blindness (VA < 20/400) due to cataract, adjusted by age and sex, was 2.1% (95% confidence interval (CI): 1.7% to 2.6%). Among the 193 persons with bilateral blindness due to any cause, cataract was the cause in 104 of them (53.9%). The prevalence of blindness due to cataract or other causes increased with age and was higher in women than in men. Only 25% of the persons studied who needed cataract surgery had had that done. The prevalence of bilateral VA less than 20/200 due to unoperated cataract was 6.3% (95% CI: 5.3% to 7.3%); only 12% of the persons with that level of visual deficiency had had cataract surgery. The VA of 26% of the eyes operated on for cataract was lower than 20/200. The reasons given by persons who needed cataract surgery but who had not had it included not being able to pay for the operation (28%), lack of knowledge concerning cataracts (25%), fear of the operation (23%), and fear of completely losing their sight (17%). CONCLUSIONS: Most of the cases of blindness and of serious deficiency in visual acuity in persons 50 years old or older in Piura and Tumbes are due to uncorrected refractive defects, especially cataracts. The high prevalence of bilateral blindness due to cataracts (2.1%) could be reduced with measures that facilitate access to appropriate treatment.

Fundación oftalmologica del Norte en Piura / Foundation Ophthalmological of the North in Piura

En la ciudad de Piura, en la costa norte del país, trabaja esta institución. Se describe los logros alcanzados desde el año 1996, en localidades de la costa y sierra de esa área, con resultados muy promisorios.

Programas integrados de prevención de ceguera en el Perú: organización Peruana de lucha contra la ceguera (OPELUCE): resultados de 12 programas rurales y 19 urbano-marginales (1983-1986) / Integral programs of blindness prevention in Perú: organización Peruana de lucha contra la Ceguera (OPELUCE): results of 12 rural programs and 19 urban-marginal (1983-1986)

El Perú es un país con 18 millones de habitantes, de los cuales la tercera parte carece de atención ocular, siendo su tasa de ceguera el 0.6%, concentrándose el 82% de los Oftalmólogos en Lima Capital. No siendo eficiente la acción estatal en conducir la atención especializada a las poblaciones que la necesitan, la Organización Peruana de Lucha contra la Ceguera (OPELUCE) aborda esta problemática, y presenta su Modelo Integrado de Prevención de Ceguera que está aplicando en las zonas rurales del país desde hace 3 años, trascendiendo la agreste geografía que representa tener Costa, Sierra y Selva. Nuestro Modelo Integrado comprende: Asistencia clínica y quirúrgica, programa escolar, capacitación de maestros y personal de Salud, donación de material quirúrgico y anteojos, recopilación de datos, educación a la Comunidad y creación de un sistema de referencia de pacientes, programas de investigación, implementación de programas para los ciegos. Presentamos también los logros de nuestra Institución a través de doce (12) Programas Rurales y diecinueve (19) Programas en las zonas urbano-marginales. La problemática de la Prevención de la Ceguera en el Perú, al igual que en otros países en desarrollo, no es considerada entre las prioridades del Ministério de Salud. No existe, por consiguiente, un Programa de Salud Ocular que alcance precisamente a la población de las zonas urbano-marginales y peor aún, a los de las zonas rurales, permitiendo que los males que afectan a los ojos prosigan su libre evolución, conduciendo a la ceguera innecesaria a muchos enfermos. El Perú tiene aproximadamente 18 millones de habitantes, de los...

Hallazgos oftalmológicos en la comunidad Shipiba de Santa Teresita: tracoma, demostración citológica / Ophthalmological findings in the Shipiba community of Santa Teresita: tracoma, cytologic demonstration

En los meses de Junio y Agosto de l985, la Organización Peruana de Lucha contra la Ceguera, OPELUCE, realiza un estudio oftalmológico en la comunidad Shipiba de Santa Teresita, en Ucayali. Se examinó al 85% de la población, de los cuales el 74% padecía de conjuntivitis folicular. Se tomaron 38 muestras del tarso superior y en el 94% de ellas se hallaron Cuerpos de Inclusión de Halberstadter-Prowazek. También se encontró a la cuarta parte de la población con pterigion