Introduction
A limiting factor for biotechnology in non-human primates (NHP) is handling of semen, usually ejaculated as liquid or coagulated fractions, the last fraction containing a substantial amount of viable sperm (Oliveira et al., Reference Oliveira, Santos, Leão, Brito, Lima, Sampaio and Domingues2016a, Reference Oliveira, Santos, Leão, Queiroz, Paim, Vianez-Junior and Domingues2016b; Lima et al., Reference Lima, Leão, Oliveira, Brito, Sampaio and Domingues2017). Semen coagulum from Saimiri genus can be partially liquefied by using extender ACP-118™ (ACP Biotecnologia™, Fortaleza, Ceará, Brazil) without causing sperm damage (Oliveira et al., Reference Oliveira, Santos, Leão, Brito, Lima, Sampaio and Domingues2016a, Reference Oliveira, Santos, Leão, Queiroz, Paim, Vianez-Junior and Domingues2016b). However the time taken remains a challenge, as at least 90 min are necessary for partial liquefaction (Oliveira et al., Reference Oliveira, Santos, Leão, Queiroz, Paim, Vianez-Junior and Domingues2016b), and this brings risks of oxidative stress if more time is needed to increase the amount of liquefied semen (Chatterjee and Gagnon, Reference Chatterjee and Gagnon2001). In NHP, the use of antioxidants resulted in improved post-thaw sperm motility even from ejaculates of low freezability (Dong et al., Reference Dong, Tollner, Rodenburg, Hill and VandeVoort2010). Among the available antioxidants, Trolox®, an analogue of vitamin E, has the ability to permeate cells and to protect the cell membrane due to its liposoluble and hydrosoluble properties. Extender supplementation with Trolox® improves human (40 µM Trolox®; Minaei et al., Reference Minaei, Barbarestani, Nekoonam, Abdolvahabi, Takzare, Asadi, Hedayatpour and Amidi2012) and feline (5 mM Trolox®; Thuwanut et al., Reference Thuwanut, Chatdarong, Techakumphu and Axner2008) sperm viability and motility after cryopreservation. To improve liquefaction outcome with long incubation times, we evaluated if Trolox® (100 or 150 µM) supported S. collinsi sperm quality after semen incubation in ACP-118™ for 90, 270 or 450 min.
Materials and methods
Five healthy, sexually mature (>5 years) S. collinsi males were selected based on body weight (1000±21 g), and testes consistency, symmetry and mobility. Males were housed in cages of 4.74×1.45×2.26 m (length, width, and height, respectively), under a natural photoperiod (12 h of light and 12 h of dark). The diet consisted of fresh fruits, vegetables, milk, cricket larvae (Zophobas morio), and a commercial pellet chow for primates. Water was available ad libitum. Semen was collected in the morning before feeding. For this, males were anaesthetised with ketamine hydrochloride [15 mg/kg, intramuscularly (IM); Vetanarcol; Köning S.A., Avellaneda, Argentina] and xylazine hydrochloride (1 mg/kg, IM; Köning S.A.). Under anaesthesia, the genital region was sanitized with a mild soap and distilled water (1:10) and gauze, and males were stimulated by electroejaculation (EEJ) (Autojac; Neovet, Uberaba, Brazil) (Oliveira et al., Reference Oliveira, Leão, Almeida, Santos and Domingues2015). If a male was unable to ejaculate after the session, no further attempts were made. Intervals between collections were of at least 30 days. In total, 19 ejaculates (three or four samples per animal) were collected. Immediately after ejaculation, conical tubes (1.5 ml) containing the semen were placed in a water bath at 37°C. Volumes of liquid and coagulated fractions were assessed in a graduated tube. After this step, each ejaculate was divided into three equal aliquots, which were diluted (1:1) in ACP-118™ alone or combined with 100 or 150 µM Trolox®. Sperm parameters were evaluated before (only liquid fraction) and after partial dilution (liquefaction) of the obtained coagula. Liquefaction was performed for three different incubation times (90, 270 or 450 min). These exposure times were selected based on experience under field conditions. Sperm concentration, motility, vigour and PMI were determined as previously described (Oliveira et al., Reference Oliveira, Leão, Almeida, Santos and Domingues2015). Dead sperm were labelled with propidium iodide (0.5 mg/ml; Sigma Chemical Co., St. Louis, MO, USA) and acrosome membrane damage was accessed with fluorescein-conjugated Pisum sativum agglutinin (FITC–PSA; 100 µg/ml; Sigma). Hoechst 33342 stain (40 µg/ml; Sigma) was added to detect the sperm under the microscope (Celeghini et al., Reference Celeghini, de Arruda, de Andrade, Nascimeto and Raphael2007). Data were analysed using the StatView 5.0 program (SAS Institute Inc., Cary, NC, USA). The animal was kept in the experimental unit. The effects of Trolox® concentration and incubation times on sperm motility, vigour, PMI and acrosome integrity were evaluated by two-way analysis of variance (ANOVA) with Tukey as the post-hoc test. Differences were considered significant when P-values were <0.05.
Results and Discussion
Means [± standard error of the mean (SEM)] of the collected volumes of liquid and coagulated seminal fractions were 85±10 µl (0–200 µl) and 280±51 µl (0–600 µl), respectively. Both fractions were transparent or opaque, and colourless, whitish or yellowish. Total sperm concentration was 24±19×106 sperm/ml. None of the tested treatments was effective to completely liquefy seminal coagulum and no differences in liquefied volumes were observed. This result indicated that extender should be supplemented with other compounds rather than only with antioxidant. Importantly, extender supplementation with antioxidant affected sperm parameters more significantly than did incubation time, and there was no interaction between time of liquefaction and treatment. It was possible to maintain sperm motility and vigour after 270 min of incubation at control levels only when extender was supplemented with Trolox® 100 µM. The same was observed for PMI after 450 min of incubation (Table 1). Acrosome membrane integrity was not affected by incubation time or treatment. The present results of Trolox®-supplemented extender were superior to those reported previously for Sapajus apella without Trolox® (Oliveira et al., Reference Oliveira, Miranda, Leão, Brito, Santos and Domingues2011). The effect of Trolox® on sperm motility is concentration dependent. Extender supplementation with Trolox® 60 μM was detrimental to human sperm motility, whereas no free radical activity was detected in the supplemented sperm samples (Donnelly et al., Reference Donnelly, McClure and Lewis1999). Possibly, there are differences in sperm susceptibility to lipid peroxidation, the set of intracellular and extracellular antioxidant systems that presents in semen differs between species, plus concentration of antioxidant added to the extender was too high for human sperm, leading to a pro-oxidant effect (Cao and Cutler, Reference Cao and Cutler1997) as observed with 150 μM Trolox®. Prevention of lipid peroxidation in the sperm membrane was reported in samples treated with Trolox® (Sarlós et al., Reference Sarlós, Molnár, Kókai, Gabor and Ratky2002). Lipid peroxidation caused failures in the metabolic rate mechanism resulting in cell death (Benzie, Reference Benzie1996). Once dead, the sperm released enzymes that had toxic effects on living sperm, causing changes in kinematic parameters such as decrease in sperm motility (Shannon and Curson, Reference Shannon and Curson1972). Therefore Trolox® could have helped motility indirectly by decreasing cell death. In conclusion, ACP-118TM with 100 μM Trolox® maintains sperm quality (motility, vigour and PMI) for at least 270 min of semen incubation at 37°C. Nevertheless, seminal coagulum was not completely liquefied even after 450 min.
Table 1 Percentages [mean±standard error of the mean (SEM)] of sperm motility and vigour, and plasma membrane integrity observed in the control, and after incubation in ACP-118 supplemented or not with Trolox (100 or 150 µM)
a,b Different lowercase letters in the same row indicate differences among treatments within the same incubation time and evaluated parameter (P<0.05).
A,B Different uppercase letters in the same column indicate differences among incubation times within the same treatments and evaluated parameters (P<0.05).
Financial support
This study was supported by the National Primate Center (CENP), CAPES (Brazil) and the Federal University of Pará (UFPA).
Conflicts of interests
The authors declare that there is no conflict of interest that can be perceived as prejudicing the impartiality of the research reported.
Ethical standards
All experimental protocols were approved by the System of Authorization and Information in Biodiversity (SISBIO/ICMBio/MMA no. 31542-2) and by the Ethical Committee in Animal Research of Evandro Chagas Institute (no. 0010/2011/CEPAN/IEC/SVS/MS).
