A decrease in lactate concentration in the incubation

A decrease in lactate concentration in the incubation medium was observed in samples treated with heparin and 2′,5′-dideoxyadenosine compared with the control (<0.05; Fig. 1B). While, the combination of 2′,5′-dideoxyadenosine and hyaluronic acid, did not affect lactate concentrations in the incubation medium compared with the control (>0.05; Fig. 2B).
Discussion In the present study we compared the oxidative metabolism and intracellular signals of heparin, the glycosaminoglycan used routinely to induce sperm capacitation prior to IVF in bull, with the effects of hyaluronic acid, another physiological glycosaminoglycan also present in the reproductive tract of the female, which began to be studied in the last decades due to its effects enhancing sperm events such as capacitation and acrosome reaction, motility, sperm selection and in vitro fertilization in numerous species. Previously, we have demonstrated that the hyaluronic pepstatin effect indicated that capacitation induction is not only dose dependent but also dependent on incubation time (1000μg/ml with 60min incubation were the optimal conditions to capacitate), so hyaluronic acid treatment had a dynamic response because the percentages of capacitated sperm differed at different incubation times and concentrations (Fernández and Córdoba, 2014). Heparin also has a dynamic response respect to optimal conditions to induce capacitation, as demonstrated by different authors (Fukui et al., 1990, Córdoba et al., 1997). Therefore, in this study enzymatic activities were determined at 15 (for heparin) or 60 (for hyaluronic acid) minutes incubation, i.e. the minimal concentration and minimum incubation time of capacitation inductors to ensure sperm viability and plasma and mitochondrial membrane integrity, as optimal conditions to induce capacitation of cryopreserved bull sperm, in order to register metabolism and signaling parameters in capacitation status. The conversion of lactate into pyruvate, catalyzed by LDH, is accompanied by NADH production and consequently by a change in the redox balance, which may be associated with alterations in reactive oxygen species concentrations (Galardo et al., 2014). There are several studies which report this enzyme has an important role in sperm capacitation in different species such as mice (Duan et al., 2003), dogs (Kawakami et al., 2006) and cattle (Córdoba et al., 2007), and it was also shown that boar sperm can efficiently metabolize both citrate and lactate through a pathway regulated by LDH (Medrano et al., 2006). In the present study, differential variations of LDH activity and lactate concentration in the incubation medium were detected in capacitation with heparin or hyaluronic acid. With hyaluronic acid-induced capacitation, an increase in LDH activity and lactate concentration were detected in incubation medium, while with heparin treatment these parameters were less compared with its control. Therefore, the metabolic pattern observed in heparin capacitation induction would be primarily linked to mitochondrial oxidative phosphorylation, also indicated by the significant increase in oxygen consumption caused by this inducer, in contrast to the lesser respiration rate of hyaluronic acid-capacitated sperm described previously (Fernández and Córdoba, 2014a). The metabolic changes required for sperm capacitation produced by the latter glycosaminoglycan are characterized by lactate production and basal mitochondrial respiratory activity. Lactate is present in seminal fluid and female genital tract secretions in high concentrations and can be metabolized by human (Hereng et al., 2011), rat (Galardo et al., 2014) and bull sperm (Fernández and Córdoba, 2014b). Both lactate and pyruvate can enter and exit the sperm cell through two different types of monocarboxylate transporters (MCTs) in the cytoplasmic and mitochondrial sperm membranes. These transporters are located in the sperm head (MCT1) and tail (MCT2) and are mainly involved in LDH shuttle (Gladden, 2004). Evidence was found that the uptake and output of lactate and pyruvate in the mitochondria would be mediated by a translocase (Milkowski and Lardy, 1977, Córdoba et al., 2007).