Examinando por Autor "Evangelista-Vargas, S."

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    ÍtemAcceso Abierto
    Assessment of spermatozoa in fertile alpaca (Vicugna pacos) males: study of sperm head morphometry using a nonautomated digital method and sperm morphology based on strict criteria
    (Blackwell Publishing Ltd, 2017) Evangelista-Vargas, D.; Evangelista-Vargas, S.; Valdivia, M.; Santiani, A.
    Although computer-assisted systems for sperm morphometry and morphological analysis are important tools in the study of male fertility, their use in extensive systems in alpacas is limited by factors such as the expense of equipment and the high altitudes of the Andean region. The objectives of this study were to evaluate alpaca sperm head morphometry using a nonautomated digital method and determine the frequency of sperm abnormalities based on strict criteria for sperm morphology in fertile male alpacas. Ejaculates (n = 15) from seven alpacas were collected, and sperm smears stained with modified Papanicolaou were processed. For morphometric analysis, 3,000 sperm (200 cells/sample) images were captured at 400× magnification and Quick Photo MICRO 3.0 software was used for manual measurement of basic (sperm head length, width, perimeter and area) and derived variables (ellipticity, shape factor, elongation and regularity). For morphology assessment, smears were observed at 1000× magnification according to WHO and strict criteria. Average morphometric parameters were length 5.48 μm, width 2.99 μm, perimeter 13.62 μm, area 12.43 μm2, ellipticity 1.86, shape factor 1.20, elongation 0.29 and regularity 1.05. Significant between-individual and within-individual differences were found in morphometric parameters. Based on morphometric study, sperm heads were classified as elliptical or normal (49%), long (18%), short (2%), pyriform (12%), round (9%), large (6%) and small (4%). Morphological analysis found no additional sperm head defects in 49% of normal sperm obtained by morphometry, although a 4% incidence of neck/mid-piece defects and a 16% incidence of principal-piece defects were found. We conclude that sperm head morphometry assessment in fertile alpacas using a nonautomated digital method is feasible, and that defects in sperm heads constitute the main morphological alteration (>50% of the sperm population), based on WHO and strict criteria. © 2016 Blackwell Verlag GmbH
  • Miniatura
    ÍtemAcceso Abierto
    Characterization of functional variables in epididymal alpaca (Vicugna pacos) sperm using imaging flow cytometry
    (Elsevier B.V., 2016) Santiani, A.; Ugarelli, A.; Evangelista-Vargas, S.
    Epididymal alpaca sperm represent an alternative model for the study of alpaca semen. The objective of this study was to characterize the normal values of some functional variables in epididymal alpaca sperm using imaging flow cytometry. Alpaca testicles (n = 150) were processed and sperm were recovered from the cauda epididymides. Only 76 samples with acceptable motility and sperm count were considered for assessment by imaging flow cytometry. Acrosome integrity and integrity/viability were assessed by FITC-PSA/PI and FITC-PNA/PI. Mitochondrial membrane potential (MMP) was assessed by MitoTracker CMXRos and MitoTracker Deep Red FM. Lipid peroxidation was evaluated using BODIPY 581/591 C11. Results show that the mean values for acrosome-intact sperm were 95.03 ± 6.39% and 93.34 ± 7.96%, using FITC-PSA and FITC-PNA, respectively. The mean values for acrosome-intact viable sperm were 60.58 ± 12.12% with FITC-PSA/PI and 58.81 ± 12.94% with FITC-PNA/PI. Greater MMP was detected in 65.03 ± 15.92% and 59.52 ± 19.19%, using MitoTracker CMXRos and MitoTracker Deep Red FM, respectively. Lipid peroxidation was 0.84 ± 0.95%. Evaluation of acrosome-intact and acrosome-intact viable sperm with FITC-PSA/PI compared with. FITC-PNA/PI or MMP with MitoTracker CMXRos compared with MitoTracker Deep Red FM were correlated (P < 0.05). The MMP using MitoTracker CMXRos was the only variable correlated (P < 0.05) with sperm motility (r = 0.3979). This report provides a basis for future research related to alpaca semen using the epididymal sperm model. © 2016 Elsevier B.V.
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    ÍtemAcceso Abierto
    Cryopreservation of Peruvian Paso horse spermatozoa: Dimethylacetamide preserved an optimal sperm function compared to dimethyl sulfoxide, ethylene glycol and glycerol
    (Blackwell Publishing Ltd, 2016) Santiani, A.; Evangelista-Vargas, S.; Vargas, S.; Gallo, S.; Ruiz, L.; Orozco, V.; Rosemberg, M.
    The objective was to evaluate the effect of different cryoprotectant agents in the cryopreservation of Peruvian Paso horse semen. Twenty semen samples were collected from five Peruvian Paso horse stallions. Each sample was divided into 12 parts to form the groups: dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol (GLY), at 3%, 4% and 5%. Samples were frozen using a rate-controlled freezer. Sperm parameters evaluated were motility and viability/acrosomal status. After thawing, progressive motility in DMA group was higher (p < .05) than in DMSO, EG and GLY groups. Similarly, viable acrosome-intact spermatozoa were higher (p < .05) using DMA in comparison with DMSO. No differences were found when comparing concentrations for any of the cryoprotectant agents. In conclusion, DMA seems to be a good cryoprotectant agent for the cryopreservation of Peruvian Paso horse stallion semen. © 2016 Blackwell Verlag GmbH.
  • Miniatura
    ÍtemAcceso Abierto
    Detection of intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) and lipid peroxidation during cryopreservation of alpaca spermatozoa
    (Blackwell Publishing Ltd, 2017) Evangelista-Vargas, S.; Santiani, A.
    Contents: The objective of this study was to detect changes in intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) production and lipid peroxidation during cryopreservation of alpaca spermatozoa. Twelve alpaca semen samples were conventionally cryopreserved. Intracellular superoxide anion and hydrogen peroxide were evaluated by fluorescence microscopy using dihydroethidium (DHE)/YO-PRO-1 and dichlorofluorescein diacetate (H2DCFDA)/propidium iodide (PI), respectively. Evaluations were performed during cooling curve at (1) 25°C, (2) 15°C, (3) 5°C/0 min, (4) 5°C/15 min, (5) 5°C/30 min and (6) after freezing/thawing. Evaluation of lipid peroxidation by measuring malondialdehyde (MDA) was performed at 25°C, 5°C/30 min and after thawing. Maximum percentages of total spermatozoa producing superoxide anion and hydrogen peroxide were found at 5°C/30 min (62.8 ± 6.3% and 30.5 ± 5.6%, respectively), and these results were higher (p < .05) than initial (25°C: 10.8 ± 3.8% and 6.8 ± 0.7%, respectively) and after thawing (29.8 ± 9.5% and 7.5 ± 1.8%, respectively) values. However, considering only viable spermatozoa, production of superoxide anion and hydrogen peroxide during overall stabilization at 5°C (>76% and >91%, respectively) and after thawing (74.9 ± 5.0% and 78.9 ± 2.2%, respectively) was higher (p < .05) than initial values at 25°C (38.7 ± 3.1% and 53.6 ± 2.0%, respectively). Lipid peroxidation at 25°C, 5°C/30 min, and post-thawing were 346.5 ± 99.8, 401.1 ± 64.8 and 527.7 ± 142.8 ng/ml MDA, respectively. These results showed that high percentage of viable alpaca spermatozoa produces intracellular reactive species oxygen (ROS) during the cryopreservation process of alpaca semen. © 2017 Blackwell Verlag GmbH.
  • Miniatura
    ÍtemAcceso Abierto
    Evaluación de la integridad acrosomal en espermatozoides epididimarios de alpaca mediante citometría de flujo [Evaluation of acrosome integrity in epidydimal alpaca sperm by flow cytometry]
    (Universidad Nacional Mayor de San Marcos, 2017) Ugarelli, A.; Evangelista-Vargas, S.; Santiani, A.
    The aim of this study was to determine the percentage of epidydimal sperm acrosome integrity using alpaca Arachis hypogaea (PNA) and Pisum sativum (PSA), combined with fluorescein isothiocyanate (FITC). Testicles (n=45) were obtained at Ninacaca municipal slaughterhouse (Pasco, Peru). Only 29 samples with motility higher than 30% and concentration higher than 50x106 sperm/ml were used. Sperm from cauda epididymis were recovered with 1 ml of Tris base solution, and then, washed by centrifugation at 600 g for 8 min and pellets were re-suspended in 300 μl of PBS. Each sample was divided into two aliquots and incubated for 8 min at 38 °C with FITC-PNA (0.5 μg/ml) or FITC-PSA (2.5 μg/ mL) together with propidium iodide (PI, 0.5 μg/ml) as a viability marker. Samples were evaluated by flow cytometry using a laser excitation 488 nm and emission detector channels 505-560 nm fluorescence (channel 02 for FITC) and 642-740 nm (Channel 05 for PI). Sperm emitting green fluorescence on Channel 02 were considered with acrosome damage while sperm emitting red fluorescence on Channel 05 were considered dead. The results showed 59.17 + 4.84 and 61.13 + 4.35% of live sperm with acrosome integrity when using FITC-PNA and FITC-PSA respectively. These results are an indicator of epididymal sperm acrosome integrity in alpaca and can provide the basis for studying sperm physiology in this specie.