Effect of adenosine concentration on quality of cooled ram semen

This study analyzed the adenosine concentration on quality of ram semen in different refrigerated storage times at 5 °C. The design was in blocks with repeatedmeasures factorial design. Five levels of adenosine and four refrigeration times were considered as fixed effects and the animal as random effect (block). Ram semen was diluted with Andromed® diluent (control) and increasing adenosine levels (0.5, 0.75, 1, and 1.5%). The samples were taken from refrigeration after 4, 8, 12, and 16 h and evaluated for progressive rectilinear sperm motility, sperm vigor, and membrane integrity (supravital test). Data were subjected to analysis of variance with Tukey’s test. The regression analysis was conducted to evaluate behavior of adenosine levels. Factor interaction for rectilinear progressive motile variable was verified. There was a quadratic effect of progressive rectilinear motility in function of adenosine levels for each cooled storage time studied, with recommended maximum adenosine concentration of 0.81 and 1.04% at 4 and 8 h, respectively. Adenosine also promoted a protective effect on the membrane integrity. Adenosine added to the diluent increases sperm motility and vigor and protects the sperm membrane integrity.


Introduction
Artificial insemination with cooled semen has advantages over its use in reproductive programs, since the refrigeration process allows the spermatozoa to remain viable longer than fresh semen due to metabolic reduction (Allai et al., 2015;Falchi et al., 2018). However, normal metabolism of sperm cells promotes the release of metabolites called reactive oxygen species (ROS), and the excessive production of ROS causes a process known as oxidative stress (Maxwell and Watson, 1996), which is aggravated by the refrigeration process (Zalata et al., 1998).
To avoid the oxidative stress caused by refrigerated semen, some studies have been carried out in different animal species testing substances, some with antioxidant potential (Mata-Campuzano et al., 2014;Affonso et al., 2017), aiming at maintaining spermatozoa quality in a diluent for longer duration.
Adenosine is a purinergic nucleoside formed by a glycosidic bond between a purine adenine base and a D-ribose (Polosa, 2002;Polosa and Holgate, 2006). The literature describes nucleotides and nucleosides as participants in male genital system functions and proposes the presence of purine receptors on Effect of adenosine concentration on quality of cooled ram semen Bezerra et al.
2 the cell surface of this system. These functions would be related to fertility and reproduction control (Fredholm et al., 1996;Rodrigues et al., 2000;Bellezza and Minelli, 2017).
The presence of A 2 adenosine receptors in spermatozoa was evidenced in previous studies (Fraser, 1990;Fraser and Duncan, 1993;Fénichel et al., 1996). These receptor agonists could stimulate the production of cyclic AMP (Liguori et al., 2005). In addition, cyclic spermatic AMP would be deeply involved in spermatic function control (Tash and Means, 1988), contributing to ATP formation and increasing sperm motility and vigor.
Adenosine could also have a protective effect on sperm plasma membrane as a potential antioxidant, since this nucleoside is released into different tissues under oxidative stress (Masino et al., 1999). Moreover, when these tissues are treated with adenosine and/or its agonists, significant improvement in lipid peroxidation (Yavuz et al., 1997) and recovery from cellular damage (Almeida et al., 2003;Masino et al., 1999) occur.
Adenosine action as an additive in diluent for animal semen has not been identified; therefore, the objective of this study was to evaluate nucleoside concentrations on quality of refrigerated ram semen at different cooled storage times.

Material and Methods
All procedures performed during the execution of this work were in accordance with the ethical principles of animal experimentation of the National Animal Experimentation Council and were approved by local Comissão de Ética no Uso de Animais (CEUA), protocol no. 037/2017 (CEUA) and 23084.011913/2017-21 (UFRA).
Six adult rams (Santa Inês breed) presenting a good body condition score (3, in a scale of 1 to 5) were used. Semen collections were performed every three days, yielding a total of six viable ejaculates from each animal, totaling 36 ejaculates.
The samples were taken from males with the aid of estrogen-treated ewes with 1 mg estradiol cypionate (E.C.P -Zoetis ® ) and appropriately contained. After semen collection, samples were conditioned in a water bath at 37 °C for analysis.
The ejaculates considered for this study had minimum physical patterns of 0.5 mL total volume, sperm vigor of 3, and minimum sperm motility of 80% (CBRA, 2013).
One drop of semen from each ejaculate was placed on a previously heated slide at 37 °C. In a microscope with 10X magnification, the wave motion (spermatic mass movement) was evaluated. Subsequently, a coverslip heated at 37 °C was placed on the semen drop to evaluate rectilinear spermatic motility and sperm vigor in 400X magnification (CBRA, 2013). All analyses were performed by a trained laboratory technician.
Sperm concentration (spz/mL) was calculated by spectrophotometry (Spectrum Lab 22 PC ® ), adding 20 μL of fresh semen in 8 mL of saline solution (distilled water and sodium chloride -3%) in a test tube.
In a 1.5-mL plastic tube containing 1 mL of buffered formal-saline (Hancock, 1956), semen aliquots were conditioned to turbid the solution for spermatozoa morphological analysis by wet preparation. Two hundred cells per slide were assessed using a phase-contrast microscope at 1000X, and sperm defects were determined in percentage (CBRA, 2013).
Sperm viability (membrane damage) were evaluated using eosin-nigrosin stain by supravital test. One hundred cells per slide were assessed and classified as live (% of cells uncolored) and dead (% of cells colored in pink or purple), using a phase-contrast microscope at 1000X (Smith and Murray, 1997). The analysis was conducted according to Mayer et al. (1951) and Swanson and Bearden (1951) methodology.
Following determination of sperm concentration, dilutions of each treatment were performed to maintain a concentration of 50 million spermatozoa per 0.25 mL straw. The semen was diluted with the commercial extender Andromed (Minitube ® ) (control), and increasing adenosine (Sigma ® ) levels (0.5, 0.75, 1, and 1.5 %).
Before cooled storage, samples were identified and placed on the semen freezing machine (3000 -TK ® ) until the refrigeration stage. After 4, 8, 12, and 16 h of cooled storage, samples were conditioned in a 37 °C water bath and evaluated for rectilinear sperm motility, sperm vigor, and supravital test.
The experimental design was in blocks with repeated-measures factorial design. Five levels of adenosine and four refrigeration times were considered as fixed effects and the animal as random effect (block). Data were subjected to the D' Agostino test for analysis of normality. All dependent variables had a normal distribution and were subjected to analysis of variance with comparison of means using Tukey's test. Regression analysis was developed for the different levels of adenosine and cooling times. All analyzes were performed at 5 % level of probability.
The effect of adenosine levels and refrigeration times were evaluated according to the following model: in which Y ijk is the quantitative response variable, μ is the overall mean, B k is the block effect (animal), α i is the fixed effect of adenosine level, β j is the fixed effect of cooled storage time; (α×β) ij is the interaction effect, and e ijk is the random error.
No significant interaction between adenosine levels and refrigeration time effect for sperm vigor and supravital test was observed. In view of this, the effect of each factor was studied separately.
Higher values (P≤0.05) were recorded for sperm vigor and supravital test, in the treatments containing 0.5 and 0.75% adenosine compared with the others, and equal between these two treatments (P>0.05) ( Table 2). The supravital test and sperm vigor displayed a quadratic effect, showing an optimal adenosine level of 0.82 and 0.67%, respectively (Figures 2 and 3).

Discussion
Fresh semen values were similar to those observed in experiments that used Santa Inês breed in the same location (Maia et al., 2011;Frazão Sobrinho et al., 2014).
Adenosine monophosphate-activated protein kinase (AMPK) is located in the head and the midpiece of the sperm (Zhu et al., 2018), and its presence promotes adenosine rephosphorylation to form adenosine monophosphate (AMP) (Rodrigues et al., 2000). Therefore, adenosine added to the dilution at 0.5 to 1% concentrations may have favored AMP formation, with consequent energy production, observed by the increase in motility and sperm vigor in the different refrigeration times studied. The increase in the values of these variables should provide an increase in fertilization rate, since fertilizing capacity and sperm hyperactivation closely depend on the production of adenosine triphosphate (ATP) in midpiece of the spermatozon.  Effect of adenosine concentration on quality of cooled ram semen Bezerra et al. 5 An increase in ATP production and sperm motility as well as an improvement in membrane integrity and in the acrosome reaction was observed in goat sperm when researchers added acadesin (AMP analog) in goat semen dilution (Zhu et al., 2018). Other authors observed that AMP produced by adenosine kinase has effects on sperm motility of mice (Vadnais et al., 2013).
The beneficial adenosine effect on maintaining the quality of cooled ram semen may also be related to the presence of A 2 adenosine receptors in spermatozoa, as evidenced in previous studies (Fraser, 1990;Fraser and Duncan, 1993;Fénichel et al., 1996).
A 2 receptor agonists of this nucleoside could stimulate the cyclic AMP production in human (Liguori et al., 2005) and mouse (Fraser and Duncan, 1993) spermatozoa. Cyclic spermatic AMP would be deeply involved in spermatic function control (Tash and Means, 1988), as well as in motility control and fertilization capacity (Harrison, 2003). Shen et al. (1993) observed sperm motility increase in humans following the administration of an A 2 -specific receptor agonist. Thus, purinergic nucleoside addition may work as an A 2 agonist, which contributes to ATP formation, increasing sperm motility and vigor up to 16 h on refrigeration compared with the control treatment.
Likewise, adenosine presence, mainly at 0.5 and 0.75% concentrations, prolonged the spermatozoa quality subjected to refrigeration up to 16 h. On the other hand, when adenosine was added in a higher