The aim of this study was to characterize the semen of the Peruvian Hairless Dog. Fifteen males, aged 1 to 5 years, registered by the Kennel Club Peruano were used to collect semen (one ejaculated per male) by digital manipulation. A basic spermatogram was performed and macroscopic (volume and colour) and microscopic (sperm motility and concentration, using light field microscopy) characteristics were recorded. Additionally, sperm function tests were performed: sperm viability (using SYBR-14 and propidium iodide), mitochondrial membrane potential (MMP, using MitoTraker Deep Red FM), and acrosomal integrity (using FITC-PSA and propidium iodide) and analysed by flow cytometry with image analysis. The following values ​​were found (mean ± 95% CI): 6.1 ± 2.7 ml volume, opalescent colour; 66.7 ± 10.5% motility; 262.7 ± 20.4 x 106 sperm/ml sperm concentration; 77.7 ± 9.7% of sperm via...

Aim: To evaluate two extenders and two cryoprotectant agents (CPA) for alpaca semen cryopreservation. Methods: Semen samples were obtained from four adult alpacas (Lama pacos) and frozen using extender I (TRIS, citrate, egg yolk and glucose) or extender II (skim milk, egg yolk and fructose), each containing either glycerol (G) or ethylene glycol (EG) as CPA. Consequently, four groups were formed: 1) extender I-G; 2) extender I-EG; 3) extender II-G; and 4) extender II-EG. Semen was diluted in a two-step process: for cooling to 5 °C (extenders without CPA), and for freezing (extenders with CPA). Viability and acrosome integrity were assessed using trypan blue and Giemsa stains. Results: When compared, the motility after thawing was higher (P < 0.05) in groups II-EG (20.0 % ± 6.7 %) and II-G (15.3 % ± 4.1 %) than that in groups I-G (4.0 % ± 1.1 %) and I-EG (1.0 % ± 1.4 %). Viable sperm...

The aim of this study was to characterize the semen of the Peruvian Hairless Dog. Fifteen males, aged 1 to 5 years, registered by the Kennel Club Peruano were used to collect semen (one ejaculated per male) by digital manipulation. A basic spermatogram was performed and macroscopic (volume and colour) and microscopic (sperm motility and concentration, using light field microscopy) characteristics were recorded. Additionally, sperm function tests were performed: sperm viability (using SYBR-14 and propidium iodide), mitochondrial membrane potential (MMP, using MitoTraker Deep Red FM), and acrosomal integrity (using FITC-PSA and propidium iodide) and analysed by flow cytometry with image analysis. The following values ​​were found (mean ± 95% CI): 6.1 ± 2.7 ml volume, opalescent colour; 66.7 ± 10.5% motility; 262.7 ± 20.4 x 106 sperm/ml sperm concentration; 77.7 ± 9.7% of sperm via...

The aim of this study was to determine the percentage of viability in alpaca epididymal spermatozoa before and after the cryopreservation process by flow cytometry. A total of 46 alpaca testes were collected from a slaughterhouse in Pasco, Peru and 41 of them were used with sperm motility ≥30% and sperm concentration ≥50x106 sperm/ml. Spermatozoa were recovered from the tail of the epididymis with 1 ml of skimmed milk-based extender, separating into two aliquots to evaluate viability before and after the cryopreservation process. The extender was removed by washing by centrifugation with PBS, the pellets were resuspended in 100 μl of PBS, adding 0.5 μl of SYBR-14 (100 nM) and 0.5 μl of propidium iodide - PI (12 μM), and incubated for 10 min at 38 °C. The evaluation of sperm viability was performed by flow cytometry with image analyzer, using an excitation laser of 488 nm an...

Membrane potential is the electrical difference between the inside and outside of a cell, and depolarization is the reduction of the negative value of this potential. This study aimed to determine the state of the plasma membrane potential of alpaca spermatozoa after cryopreservation, using fluorescence intensity as a measure. In total, 32 epididymal sperm samples subjected to a standard cryopreservation protocol were analysed. Each sample was evaluated before and after the process. The fluorochrome DISBAC2(3) was used at 15 µm for 30 min and 12 µM propidium iodide (PI) at 37 °C for 10 min to analyse membrane depolarization and viability, respectively. Measurements were performed with a flow cytometer containing an image analyser, using a 488 nm excitation laser and the detection channels Ch03 and Ch05 for DisBAC2(3) and PI, respectively. The mean fluorescence intensity of DisBAC2(3)...

The aim of this study was to determine the percentage of viability in alpaca epididymal spermatozoa before and after the cryopreservation process by flow cytometry. A total of 46 alpaca testes were collected from a slaughterhouse in Pasco, Peru and 41 of them were used with sperm motility ≥30% and sperm concentration ≥50x106 sperm/ml. Spermatozoa were recovered from the tail of the epididymis with 1 ml of skimmed milk-based extender, separating into two aliquots to evaluate viability before and after the cryopreservation process. The extender was removed by washing by centrifugation with PBS, the pellets were resuspended in 100 μl of PBS, adding 0.5 μl of SYBR-14 (100 nM) and 0.5 μl of propidium iodide - PI (12 μM), and incubated for 10 min at 38 °C. The evaluation of sperm viability was performed by flow cytometry with image analyzer, using an excitation laser of 488 nm an...

The aim of this study was to demonstrate, by flow cytometry, that the percentage of alpaca spermatozoa with high mitochondrial membrane potential (MMP) is significantly reduced after the cryopreservation process. Forty-one alpaca testicles were obtained from a local slaughterhouse in the Ninacaca district, Pasco, Peru. The spermatozoa were recovered from the tail of the epididymis with an extender based on skim milk and only those samples with minimum 30% motility and 50x106 spermatozoa/ml were cryopreserved. The MMP evaluation was carried out before and after the cryopreservation process. Each sample was incubated for 10 minutes at 38 °C with MitoTracker Deep Red (100 nM) to determine the percentage of high MMP by imaging flow cytometry. The effect of cryopreservation on the percentage of spermatozoa with high MMP was evaluated by a paired t-student test and the percentage of motility ...

The aim of this study was to determine the effect of cryopreservation on acrosomal integrity in viable alpaca sperm. Samples from 46 alpaca testicles that had motility >30% and sperm concentration >50x106 sperm/ml were processed. Sperm recovered from the tail of the epididymis were separated into two 500 µl aliquots. The first aliquot was for the initial fresh evaluation and the second aliquot was frozen in straws and stored in liquid nitrogen until the evaluation of acrosomal integrity. For the evaluation of viability and acrosomal integrity, 100 µl of each sample were incubated with 2.5 µl of FITC-PSA (100 µg/ml) and 0.5 µl of propidium iodide (PI, 2.4 mM) for 10 min at 38 °C. Immediately afterwards, the samples were evaluated by flow cytometry, acquiring 10 000 events compatible with sperm per sample. FITC-PSA and PI were excited with a 488 nm laser, and fluorescence ...

The aim of this study was to demonstrate, by flow cytometry, that the percentage of alpaca spermatozoa with high mitochondrial membrane potential (MMP) is significantly reduced after the cryopreservation process. Forty-one alpaca testicles were obtained from a local slaughterhouse in the Ninacaca district, Pasco, Peru. The spermatozoa were recovered from the tail of the epididymis with an extender based on skim milk and only those samples with minimum 30% motility and 50x106 spermatozoa/ml were cryopreserved. The MMP evaluation was carried out before and after the cryopreservation process. Each sample was incubated for 10 minutes at 38 °C with MitoTracker Deep Red (100 nM) to determine the percentage of high MMP by imaging flow cytometry. The effect of cryopreservation on the percentage of spermatozoa with high MMP was evaluated by a paired t-student test and the percentage of motility ...

The aim of this study was to determine the effect of cryopreservation on acrosomal integrity in viable alpaca sperm. Samples from 46 alpaca testicles that had motility >30% and sperm concentration >50x106 sperm/ml were processed. Sperm recovered from the tail of the epididymis were separated into two 500 µl aliquots. The first aliquot was for the initial fresh evaluation and the second aliquot was frozen in straws and stored in liquid nitrogen until the evaluation of acrosomal integrity. For the evaluation of viability and acrosomal integrity, 100 µl of each sample were incubated with 2.5 µl of FITC-PSA (100 µg/ml) and 0.5 µl of propidium iodide (PI, 2.4 mM) for 10 min at 38 °C. Immediately afterwards, the samples were evaluated by flow cytometry, acquiring 10 000 events compatible with sperm per sample. FITC-PSA and PI were excited with a 488 nm laser, and fluorescence ...

El objetivo del presente trabajo fue determinar el porcentaje de integridad acrosomal en espermatozoides epididimarios de alpaca utilizando Arachis hypogaea (PNA) y Pisum sativum (PSA), conjugadas con isoticionato de fluoresceína (FITC). Se recolectaron 45 testículos de alpaca obtenidos del Camal Municipal de Ninacaca, provincia de Pasco (Perú) y se utilizaron 29 muestras recuperadas de la cola del epidídimo con motilidad mayor de 30% y concentración mayor de 50x106 espermatozoides/ml. Los espermatozoides fueron suspendidos con 1 ml de solución Tris base, se lavaron por centrifugación a 600 g por 8 min y los pellets fueron re-suspendidos en 300 μl de PBS. Cada muestra fue dividida en dos alícuotas e incubadas por 8 min a 38 °C con FITC-PNA (0.5 μg/ml) o FITC-PSA (2.5 μg/ml), junto con un marcador de vitalidad (ioduro de propidio [PI], 0.5 μg/ml). Las muestras fueron evaluada...

The aim of this study was to determine the effect of the type (dimethylacetamide [DMA] and dimethylformamide [DMF]) and concentrations (1, 3.5, 7%) of cryoprotective agents on three sperm parameters during the cryopreservation process of epididymal alpaca sperm. A 2x3 factorial design consisting of the two cryoprotectants and the three concentrations was used. The parameters of sperm viability (SYBR-14/PI) and mitochondrial membrane potential (MitoTracker Deep Red FM) of sperm of 40 epididymis were evaluated by flow cytometry and motility by microscopy after thawing. 111/5000. A higher percentage of motility after thawing was found with AMD (14%) than with DMF (10%) (p=0.001). In addition, there was a significant effect of the cryoprotectant concentration in all the parameters evaluated (p<0.001), being better at concentrations of 1 and 3.5% than with 7%. No significant effect of the ...

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 (cha...

The aim of this study was to determine the effect of the type (dimethylacetamide [DMA] and dimethylformamide [DMF]) and concentrations (1, 3.5, 7%) of cryoprotective agents on three sperm parameters during the cryopreservation process of epididymal alpaca sperm. A 2x3 factorial design consisting of the two cryoprotectants and the three concentrations was used. The parameters of sperm viability (SYBR-14/PI) and mitochondrial membrane potential (MitoTracker Deep Red FM) of sperm of 40 epididymis were evaluated by flow cytometry and motility by microscopy after thawing. 111/5000. A higher percentage of motility after thawing was found with AMD (14%) than with DMF (10%) (p=0.001). In addition, there was a significant effect of the cryoprotectant concentration in all the parameters evaluated (p<0.001), being better at concentrations of 1 and 3.5% than with 7%. No significant effect of the ...

The aim of the study was to determine the acrosome integrity (AI) in alpaca epididymal spermatozoa, both fresh unfixed and fixed with 2% formaldehyde, using the flow cytometry technique with FITC-PSA as fluorochrome. Epididymal sperm were obtained from 26 testes and grouped into 0-hour, 24-hour and 1-week samples. The samples were incubated with FITC-PSA (concentration 2.5 µg/mL). The 24-hour and 1-week samples were kept refrigerated for the corresponding time. For evaluation, formaldehyde was removed by washing with PBS. Sperm without green fluorescence were considered to have an intact acrosome. The results showed that the AI percentages were 95.89 ± 4.35% (0 hours), 97.24 ± 3.36% (24 hours) and 97.21 ± 3.49% (1 week). Pearson correlation between unfixed samples with samples fixed for 24 hours and 1 week showed strong and positive correlations (r=0.86 and r=0.84, respectively). Gra...

The aim of this work was to determine the morphology and growth kinetics of an isolate of primary cells from passages 1-3 of the alpaca placental chorion (aPChC) as stem cell precursors. Five alpaca placentas were processed by mechanical enzymatic disintegration for primary isolation of nucleated cells, evaluating the shape, length, width, and kinetics of these cells, as well as the population of cells obtained per gram of placental chorion tissue. The results in cell isolation yielded 6x105 nucleated cells/g of chorion tissue. The morphometry evaluated from passage 0 to 3 showed longitudinal elongation ranging from 110.99 ± 27.16 μm to 298.24 ± 52.06 μm, respectively, while the measure of the cell width ranges from 30.97 ± 8.53 μm to 48.17 ± 11.27 μm. The growth kinetics expressed as cumulative population doubling level per passage number were 2.07, 1.65, 1.06 and 0.30. The morp...

The study aimed to determine the proportion of alpaca (Vicugna pacos) sperm cells with caspases-3/7 in an activated form. The epididymis of 23 pairs of testicles obtained from the Municipal Slaughterhouse of Huancavelica, Peru, were used. In the laboratory, an extender (fat-free milk, egg yolk and fructose) was used to obtain sperm cells from the caudal part of the epididymis. The cells were washed twice with 1 mL of PBS and centrifuged. It was added 100 µL of CellEvent™ Caspase 3/7 Green Detection Reagent to the product and incubated at 37.5 °C for 30 min, while 0.5 µL of Propidium Iodide was added 10 minutes before the end of the incubation, as a marker of cell death. The reading was done by flow cytometry. The mean ± standard deviation of sperm cells with caspase 3/7 activation was 36.21 ± 11.25%, confidence interval from 31.34 to 41.08% and a coefficient of variation of 3...