During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 μg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome-reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 μM/μg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.

Cooled semen has been used routinely to prolong sperm viability until artificial insemination time. However, spermatozoa are subjected to oxidative stress. The aim of the present work was to investigate the protective and antioxidant effect of the milk proteins lactoferrin (Lf) and caseinate added to equine semen cooling extenders. Semen from six stallions was cooled at 5 °C after resuspension with C1) milk- and glucose-based, C2) 0.6% caseinate, C3) C2 + Lf 200 μg ml , C4) C2 + Lf 500 μg ml and C5) C2 + Lf 1000 μg ml extenders, and kept at 5 °C for 24 h. Sperm motility characteristics and intact membrane rates were not different among the treatments (P > 0.05). As a result of the cooling process, the nitrite concentration increased significantly in the cooled semen (69.6 ± 78.9 μm per ×10 spermatozoa) compared with the fresh semen (8.6 ± 1.9 μm per ×10 spermatozoa). In contrast, the H O concentrations were lower in the 0.6% caseinate extender (265.9 ± 221.3 μm per ×10 spermatozoa) than in the milk extender (430.9 ± 199.8 μm per ×10 spermatozoa, P < 0.05), showing an antioxidative effect of the caseinate compared with the milk. However, in all groups, hydrogen peroxide concentrations were similar to the undiluted fresh semen (332.8 ± 151.3 μm per ×10 spermatozoa). Caseinate showed to be as efficient as milk to protect equine-cooled spermatozoon.

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During cryopreservation, the sperm were submitted to an increased generation of reactive oxygen species. Furthermore, because of the large portion of seminal plasma removal, there is a decrease of sperm antioxidant protection. Addition of antioxidants proteins found in seminal plasma, such as lactoferrin (Lf) and catalase (Cat), to the freezing semen extenders could protect the sperm during cryopreservation. Lactoferrin is a transferrin, which prevents the hydroxyl radicals generation, and Cat plays an antioxidant role. The aim of this study was to determine the effects of Lf and Cat supplementation to the INRA 82 freezing extender (Battelier et al. 1997) on sperm motility parameters and membrane functionality of stallion frozen semen. Semen from 6 stallions was collected with an artificial vagina, diluted with Kenney extender (1 : 1), and centrifuged (500 × g, 10 min). The supernatant was discarded, and sperm number per milliliter was calculated. Semen was resuspended with 3 extenders to 100 × 106 sperm mL–1. The treatments were distributed in (F1) control, INRA 82 freezing extender (Battelier et al. 1997), (F2) F1+ 500 μg mL–1 lactoferrin, and F3) F1 + 200 IU mL–1 catalase. Semen samples were packaged in 0.5-mL straws and cooled to 5°C (0.27°C min–1). For semen freezing, the straws were laid over the LN vapor for 20 min and plunged into the LN. The straws were thawed at 37°C for 30 s. Motility parameters of frozen semen were determined using a computer sperm cell analysis, and sperm membrane functionality was assessed by the hyposmotic swelling test (Lagares et al. 1998). The data were analysed using Friedman test using stallion as a block. A probability of P < 0.05 was considered significant. There was no significant difference between the percentage of total sperm motility (median, minimum-maximum value; F1: 29.9, 11.0–82.7; F2: 49.8, 7.7–55.2; F3: 39.8, 5.7–92) and progressive sperm motility (F1: 7.1, 3.2–23.3; F2: 13.4, 2.6-22.4; F3: 15.6, 1.1–29.6), and functional sperm membrane (F1: 26.7, 14.7–56.2; F2: 50.5, 15.7–61.7; F3: 46.6, 13.8–50.9) with regard to the treatment. However, the velocity parameters: velocity average path (F1: 29.3, 22.1–33.80; F2: 34.6, 24.8–44; F3: 35.7, 18.2–42.6), velocity curvilinear (F1: 36.9, 30.5–45.1; F2: 42.5, 34.7–51; F3: 44.6, 25.5–50.9), and velocity straight line (F1: 23.4, 17–3.60; F2: 28.9, 18.8–38.2; F3: 26.6, 13.6–37.2) in the treatment with Lf (F2) were higher compared with the control (F1; P < 0.05). These results corroborate with studies reporting the lack of positive effect on equine sperm motility when antioxidants were added to skim milk-based extenders. Although the addition of Lf or Cat to skim milk-based extenders did not improve the motility sperm characteristics and sperm membrane functionality, more studies about the positive effect of Lf on the velocity parameters are necessary. Lactoferrin could then play an important role on the oxidative metabolism, which provides energy to the sperm movement.Acknowledgments to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, for the financial support.

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