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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% and91%, 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.

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.