Introduction
Sapajus apella presents a high degree of seminal coagulation, which does not liquefy spontaneously after ejaculation (Dixson & Anderson, Reference Dixson and Anderson2002). Hence, sperm from S. apella suffer stress during coagulum liquefaction, followed by the stress caused by the freezing process (Oliveira et al., Reference Oliveira, Miranda, Leão, Brito, Santos and Domingues2011; Leão et al., Reference Leão, Miranda, Brito, Lima, Santos and Domingues2015). Although seminal freezing has been described in S. apella using TES–TRIS and coconut-derivate extenders, sperm parameters such as motility and vigour decrease significantly after seminal coagulum liquefaction, cooling, freezing, and thawing, sometimes being almost null (Oliveira et al., Reference Oliveira, Miranda, Leão, Brito, Santos and Domingues2011; Leão et al., Reference Leão, Miranda, Brito, Lima, Santos and Domingues2015). Similar effects have been observed in other species of neotropical primates such as Saimiri collinsi (Oliveira et al., Reference Oliveira, Leão, Almeida, Santos and Domingues2015, Oliveira et al., Reference Oliveira, Santos, Leão, Queiroz, Paim, Vianez-Júnior and Domingues2016a,Reference Oliveira, Santos, Leão, Brito, Lima, Sampaio and Dominguesb), S. vanzolinii, S. cassiquiarensis and S. macrodon (Oliveira et al., Reference Oliveira, Santos, Leão, Queiroz, Paim, Vianez-Júnior and Domingues2016ab). It is known that the use of extenders will dilute the concentration of natural antioxidants present in the seminal plasma (Agarwal et al., Reference Agarwal, Sharma, Nallella, Thomas, Alvarez and Sikka2006), and cryopreservation leads to an increased production of reactive oxygen species (ROS) (Aitken & Baker, Reference Aitken and Baker2006). Consequently, a redox system imbalance causes the impairment of sperm's capacity to fertilize due the injury of the plasma membrane (Taylor et al., Reference Taylor, Roberts, Sanders and Burton2009), and decrease in sperm motility (Kefer et al., Reference Kefer, Agarwal and Sabanegh2009). Catalase is an antioxidant present in seminal plasma and plays a role in counteracting oxidative stress (Mora-Esteves & Shin, Reference Mora-Esteves and Shin2013). The beneficial effect of catalase on sperm viability and motility has been reported for human (Moubasher et al., Reference Moubasher, El Din, Ali, El-Sherif and Gaber2013). In non-human primates the use of catalase has been investigated in the rhesus macaque (Macaca mulatta) (Dong et al., Reference Dong, Correa and Yandevoort2009; McCarthy & Meyers, Reference McCarthy and Meyers2011), and was found to decrease lipid peroxidation (McCarthy & Meyers, Reference McCarthy and Meyers2011), and to improve post-thaw sperm motility (Dong et al., Reference Dong, Correa and Yandevoort2009). Our aim was to evaluate the effect of semen extender supplementation with catalase at two different concentrations (10 and 50 µg/ml) on sperm morphology, motility, vigour, and plasma membrane integrity during the processes of seminal liquefaction, cooling, and freezing–thawing.
Materials and Methods
This study was approved by the Ethical Committee in Animal Research (no. 013/2009/CEPAN/IEC/SVS/MS) and by the System of Authorization and Information in Biodiversity (SISBIO/ICMBio/MMA no. 34009-3). Six S. apella males were provided by the National Primate Center (Ananindeua, Brazil). Length, width, height, circumference and volume of both testes were measured. Animals were stimulated using a rectal electro-ejaculation (EEJ) procedure (Oliveira et al., Reference Oliveira, Miranda, Leão, Brito, Santos and Domingues2011). Two extenders were prepared, representing the A and B fractions. The A fraction was used for liquefaction of the seminal coagulum and consisted of 5.84 g ACP 118® (ACP Biotecnologia®, Fortaleza, Ceará, Brazil) diluted in 50 ml of ultrapure water. The B fraction was used for freezing and was composed of 60% A fraction, with a final concentration of 20% egg yolk, and 3% glycerol (Sigma Chemical Corporation, St. Louis, MO, USA). Both A and B fractions were either or not supplemented with the antioxidant enzyme catalase (Sigma), as follows: (i) fractions A and B without catalase (Control); (ii) fractions A and B with 10 µg/ml catalase; or (iii) fractions A and B with 50 µg/ml catalase. Both concentrations were reached after semen dilution in the extenders. After collection, semen coagulum was divided into three equal parts, each one was diluted (1:1) in fraction A of the tested treatments (Control, or 10 µg/ml or 50 µg/ml catalase) and maintained at 37°C in a water bath. Seminal coagulum in each extender was mechanically dissociated (Oliveira et al., Reference Oliveira, Miranda, Leão, Brito, Santos and Domingues2011) until a volume of >100 μl of liquid fraction was obtained. Samples were cooled down from 37°C to 4°C within 90 min (–0.4°C/min). An equal volume of precooled material (4°C) corresponding to the B fraction from each treatment was added in three steps interspersed with 10 min breaks. The spermatozoa were drawn into 0.12 ml plastic straw (IMV, L'Aigle, France) and sealed with metal beads. Straws were placed horizontally on a rack 10 cm above the surface of liquid nitrogen. Twenty minutes later, they were submerged directly into liquid nitrogen for 1-week storage. For thawing, straws were kept in a water bath (37°C) for 30 s, and thawed semen was evaluated microscopically. Samples were evaluated for sperm motility, vigour, plasma membrane integrity, and morphology. All evaluations were performed under a light microscope (Nikon, Tokyo, Japan) at ×100 magnification. Sperm vigour was evaluated on a scale of 0 to 5 (Oliveira et al., Reference Oliveira, Miranda, Leão, Brito, Santos and Domingues2011). Sperm motility was evaluated with the help of an optical microscope, and expressed as the percentage of cells actively moving (Dong et al., Reference Dong, Rodenburg, Huang and VandeVoort2008). For this, no motility was considered 0, slight movement with greater than 75% of sperm showing vibration only was represented by 1, moderate forward movement in about greater than 50% of sperm was represented by 2, forward movement in about 70% of sperm was represented by 3, and when 90% or greater than 95% of sperm presented very active forward movement, scales 4 and 5 were used. Sperm motility was expressed as the percentage of cells actively moving in a forward direction. Sperm vibrating in place were not considered to be motile (Dong et al., Reference Dong, Rodenburg, Huang and VandeVoort2008).
Sperm plasma membrane integrity and morphology, including morphologic defects, were evaluated as described previously (Oliveira et al., Reference Oliveira, Miranda, Leão, Brito, Santos and Domingues2011). Sperm motility, plasma membrane integrity and morphology were compared using analysis of variance (ANOVA) and vigour was compared using the Kruskal–Wallis test. A P-value < 0.05 was considered to be statistically significant. When an animal provided more than one ejaculate, the mean value was used for statistical analysis as a unit, and not all the values individually from a single animal.
Results
All the males (n = 6) used in this study were healthy during the experiments and presented symmetrical testes with normal consistency and mobility. Mean (±SD) body weight was 3.915 ± 3 kg. Testicular biometry is presented in Table S1. In total, 26 semen collection trials (at least two attempts in each of the six males) were performed resulting in seven ejaculates from four males (Table 1). Before liquefaction, the seminal coagulum presented a yellowish colour and was opaque. Mean (±SD) collected volume of coagulated semen was 410 ± 129 μl (250–600 μl; min–max). After liquefaction some seminal micro-clots were still present, but without interference with semen manipulation and cooling or freezing procedures. No significant effect of liquefaction or cooling on the evaluated semen parameters was observed (Table 2). However, freezing–thawing led to a significant decrease in sperm motility and vigour, as well as impaired plasma membrane integrity. Exposure to catalase during dilution and cooling did not improve sperm motility, or plasma membrane integrity. Similar results were observed when sperm motility and vigour were evaluated after freezing–thawing. However, cryopreservation solution supplementation with 50 µg/ml catalase maintained plasma membrane integrity similar to that of fresh sperm (Table 2). Liquefaction, cooling, and freezing–thawing did not affect sperm morphology, independently of extender supplementation with catalase (Table 3).
Table 1 Mean (±SD) data of ejaculates (number), sperm motility (%), vigour (grade), and sperm plasma membrane integrity (PMI; %)
Table 2 Motility, vigour, and plasma membrane integrity (PMI) of Sapajus apella sperm
A,B Different uppercase letters indicate differences between means comparing diluted, cooled and freezing–thawing within the same group; P < 0.05.
Table 3 Mean (± SD) percentages of morphologically normal sperm and sperm with pathologic defects
a After liquefaction.
Discussion
The limited capacity of storing antioxidant enzymes due to small cytoplasm, combined with a rich membrane with unsaturated fatty acids makes sperm susceptible to oxidative stress and to lipid peroxidation (Aitken & Baker, Reference Aitken and Baker2006; Agarwal et al., Reference Agarwal, Virk, Ong and du Plessis2014). Thus, supplementation of the extender with antioxidant has been reported as an alternative to protect the sperm against ROS during semen cryopreservation. Usually the targets are the plasma membrane integrity and sperm motility (Aitken & Baker, Reference Aitken and Baker2006; Moubasher et al., Reference Moubasher, El Din, Ali, El-Sherif and Gaber2013). No effect was observed when extenders were supplemented with catalase during liquefaction and cooling. This can be simply because no expressive deleterious effects were observed during these procedures, without a need to include catalase. Catalase is already naturally found in cells, and the stress levels occurred during the present liquefaction and cooling processes did not require exogenous catalase. Catalase was efficient to maintain sperm motility after the cryopreservation process. Differently from our findings, the use of catalase (200 IU/ml) was associated with an improved human sperm viability, increase in percentage of progressive motility, as well as decrease in DNA damage, when compared with ejaculates cryopreserved without catalase (Moubasher et al., Reference Moubasher, El Din, Ali, El-Sherif and Gaber2013). In this study, all treatments had partially recovered post-thaw sperm motility in S. apella, differently from the data reported by Oliveira et al. (Reference Oliveira, Miranda, Leão, Brito, Santos and Domingues2011) who described absence of motility after cryopreservation in TEST and in natura coconut water extenders. This discrepancy may be due the extenders used by Oliveira et al. (Reference Oliveira, Miranda, Leão, Brito, Santos and Domingues2011), as well as the cryopreservation protocol performed within 4.8 hours, while in the present study all handling took 3.4 h, a factor that influence ROS production (Kothari et al., Reference Kothari, Thompson, Agarwal and Plessis2010). Considering that supplementation with catalase was effective in maintaining the plasma membrane integrity in frozen–thawed sperm, this antioxidant may have minimized the effects of lipid peroxidation on S. apella sperm membrane (Mccarthy & Meyers, Reference McCarthy and Meyers2011). However, it is not advised to simply supplement extender with catalase, and other strategies or antioxidants should improve not only sperm membrane integrity, but motility post-thaw.
Acknowledgements
The authors thank CENP for the logistical support. D.L. Leão was supported by CNPq, Brazil.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S0967199416000447
