Shirley Evangelista-Vargas

Mitochondrial distribution and activity are closely associated with cytoplasmic maturation and developmental competence in mammalian oocytes; however, limited information is available regarding these processes in Vicugna pacos. This study aimed to characterize mitochondrial activity, aggregation, and distribution patterns in immature and in vitro mature Vicugna pacos oocytes. Mitochondrial fluorescence intensity ranged from 21.2 to 126 units, with mean values decreasing from grade I to IV. No significant differences were observed between immature (66.8 ± 22.7) and mature (59.2 ± 23.5) oocytes. Three distinct distribution patterns were identified: homogeneous non-peripheral (HoNP), heterogeneous peripheral (HeP), and heterogeneous non-peripheral (HeNP), with HeNP oocytes exhibiting the highest activity. During maturation, fluorescence intensity increased in HeNP and HeP but decreased in HoNP, suggesting distinct mitochondrial responses. Aggregation shifted from smooth in immature oocytes to granular in mature ones, and these two traits—HeNP distribution and granular aggregation—were consistently associated with higher mitochondrial activity. Overall, 78% of oocytes showed aggregation. These results demonstrate that mitochondrial remodeling accompanies oocyte maturation and that aggregation and distribution patterns may serve as biomarkers of oocyte quality. Further studies linking these features with fertilization and embryo development are needed to validate their predictive role in assisted reproduction of alpacas.

In remote alpaca breeding regions, access to advanced sperm analysis laboratories is limited. This study validates a practical cytometric method for evaluating sperm viability and acrosomal integrity in epididymal alpaca sperm using early fluorochrome staining, formaldehyde fixation, and intermediate storage. Thirty-two testes were transported at 5 °C, and spermatozoa were collected from the cauda epididymis. After morphometric screening, 26 samples were included. Aliquots were stained with Zombie Green (viability) and FITC–PSA (acrosomal integrity), at time zero. Each aliquot was divided for cytometric analysis at T0 (immediately), T24 (24 h after formaldehyde fixation) and T1w (1 week post-fixation). Fixed samples showed higher viability and acrosomal integrity values (T24: 70.75%, 97.24%; T1w: 71.80%, 97.21%) than T0 (67.63%, 95.89%). This may reflect fluorescence alterations associated with fixation. Strong correlations and Bland–Altman analysis confirmed consistency across time points. This method enables accurate sperm quality evaluation up to one week after collection, offering a useful tool for reproductive monitoring in field conditions without immediate analysis. Further research on ejaculated semen and field protocols is recommended.

Objective. To assess the oxidative stress on alpaca epididimal sperm frozen with different concentration of seminal plasma. Materials and methods. 29 post mortem alpaca epididymis were used. Epididymal sperm were obteined thought cuts and suspended in a diluent based on skim milk, egg yolk and fructose and dimethylacetamine. Samples were separated into four aliquots which were supplied with seminal plasma (v/v) in concentrations of 0, 10, 25 and 50% respectively; then, sperm motility was assessed before freezing; then, motility was assessed before freezing with an automatic freezer machine. After thawing, mitochondrial membrane activity, sperm viability, lipid peroxidation and sperm apoptosis were assessed using flow cytometry with images. Results. Sperm exposed to 50% seminal plasma showed the lowest post-thaw motility (3.5±3.7%) and the highest percentages of apoptotic cells (71.43±20.6%). No significant differences were found in the rest of parameters. Conclusions. This study constitutes non-conclusive evidence that presence of seminal plasma does not affect viability, mitochondrial activity, or lipid peroxidation of alpaca epididymal sperm during cryopreservation processes; however, when it is absent or added up to 25% in the cryopreservation process, it may be possible to obtain better results in terms of sperm motility and sperm apoptosis.

Objetivo. Desarrollar y evaluar primers para detectar el gen Protamina 3 (PRM3) en alpacas. Materiales y métodos. Los primers candidatos se diseñaron en base a las secuencias conservadas obtenidas del alineamiento del gen protamina 3 (PRM3) con la secuencia predicted del ARNm, ambos presentes en la base de datos del NCBI. Para la evaluación in silico, se enfrentaron los primers con el genoma de alpaca, y para la evaluación in vitro, se emplearon 51 muestras de tejido testicular. Resultados. Se obtuvieron 10 pares de primers. De estos, se seleccionaron los pares 1 y 6; denominados PRM3-P31 y PRM3-P32 y con amplicones de 240 pb y 328 pb, respectivamente. Además, la evaluación de subproductos se realizó mediante el programa Oligoanalyzer. Conclusiones. Se concluyó que el primer PRM3-P31 nos permite determinar la presencia del gen más seguridad que el otro primer seleccionado. Por lo tanto, fue posible desarrollar y evaluar primers para detectar la presencia del gen de la protamina 3 en alpacas.

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 evaluadas mediante citometría de flujo, utilizando un láser de excitación de 488 nm y canales detectores de emisión de fluorescencia de 505-560 nm (Canal 02 para FITC) y de 642-740 nm (Canal 05 para PI). Los espermatozoides que emitieron fluorescencia verde en el Canal 02 fueron considerados con daño acrosomal, mientras que los que emitieron fluorescencia roja en el Canal 05 fueron considerados muertos. Se obtuvo 59.17 ± 4.84 y 61.13 ± 4.35% de espermatozoides vivos con integridad acrosomal para FITC-PNA y FITC-PSA, respectivamente. Los resultados presentan un indicador de la integridad acrosomal en espermatozoides epididimarios de alpaca, que podría constituir una base para el estudio de la fisiología espermática en esta especie.

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.

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.