Impact of glucose and pyruvate on adenosine triphosphate production and sperm motility in goats

Objective This study evaluates goat sperm motility in response to metabolic substrates and various inhibitors, aiming to assess the relative contribution of glycolysis and mitochondrial oxidation for sperm movement and adenosine triphosphate (ATP) production. Methods In the present study, two main metabolic substrates; 0 to 0.5 mM glucose and 0 to 30 mM pyruvate were used to evaluate their contribution to sperm movements of goats. Using a 3-chloro-1,2-propanediol (3-MCPD), a specific inhibitor for glycolysis, and carbonyl cyanide 3-chlorophenylhydrazone as an inhibitor for oxidative phosphorylation, cellular mechanisms into ATP-generating pathways in relation to sperm movements and ATP production were observed. Data were analysed using one-way analysis of variance for multiple comparisons. Results Sperm motility analysis showed that either glucose or pyruvate supported sperm movement during 0 to 30 min incubation. However, the supporting effects were abolished by the addition of a glycolysis inhibitor or mitochondrial uncoupler, concomitant with a significant decrease in ATP production. Although oxidative phosphorylation produces larger ATP concentrations than those from glycolysis, sperm progressivity in relation to these two metabolic pathways is comparable. Conclusion Based on the present study, we suggest that goat sperm use glucose and pyruvate to generate cellular energy through glycolysis and mitochondrial respiration pathways to maintain sperm movement.


INTRODUCTION
Cellular energy is an essential factor that supports the functionality of spermatozoa.The sustenance of sperm motility during their traversal within the female reproductive tract hinges on consistent and enduring energy.This delineates a probable rationale for the augmented requirement of adenosine triphosphate (ATP) by spermatozoa, surpassing other cellular entities.ATP, as the quintessential embodiment of cellular energy, assumes paramount importance.Sperm motility is highly dependent on the availability of ATP compared to other sperm functions such as capacitation, acrosome reaction, or sperm penetrability, accounting since it accounts for approximately 70% of ATP consumption [1].
Sperm produce ATP through glycolysis and oxidative phosphorylation localized to dif ferent regions of the cells.The localization within the principal piece underscores the significance of glycolysis in providing comprehensive support for tyrosine phosphorylation and hyperactivated motility.Several glycolytic enzymes have been identified to be situated within the fibrous sheath of the principal piece [2].Oxidative phosphorylation occurs in the mitochondria, which are tightly packed in the midpiece producing abundant ATP.Even though oxidative phosphoryla tion produces more ATP than glycolysis, there is a wide range of variation among species regarding the relative de pendency of ATP utilization.For example, the flagellar movement of murine and human sperm is supported by ATP generated from glycolysis, but this movement in boar and horses mainly uses oxidative phosphorylation [35].For goat sperm, the mechanism of ATP generation is still un known.
Glucose is the primary substrate for glycolysis.It is con verted into pyruvates and energy, including a total of 4 ATP, 2 nicotineamide adenine dinucleotide (NADH), and 2 pyru vates per glucose molecule [6].The pyruvates were used as a source for oxidative phosphorylation producing 32 ATP molecules [7].Some studies reported the effects of pyruvate and glucose utilization on sperm motility varying between species.Results in roosters indicated sperm motility requires ATP, resulting from glucose uptake and oxidative phosphor ylation [8].It is reported that glucose increases sperm velocity, increasing penetrability into eggs [9] but sperm motility and velocity depend on pyruvate in stallions and boar [10,11].Inadequate lactate and pyruvate as metabolic substrates for Oxphos lead to mitochondrial dysfunction resulting in in creased reactive oxygen species production and decreased motility [11].However, pyruvate does not affect sperm pen etration into eggs in rodents.Despite these findings, it has been unclear and debated in the field whether glycolysis or oxidative phosphorylation is the major contributor to ATP needed for sperm motility in goats.The present study evaluat ed the major contributor of ATP production in the relationship with sperm motility in goats.
Semen was collected from 3 fertile male Ettawa Crossbred goats using an artificial vagina.Furthermore, the semen from each buck was washed twice by centrifugation at 250 g for 5 min at RT with triscitrate buffer (pH 7.2) to exclude seminal plasma.All animal work was performed under the approval of the Research Ethics Committee of the Universitas Padjadjaran (approval no.39/UN6.KEP/EC/2023).

Sperm incubation and motility
A total of 10 7 sperm samples were incubated in triscitrate buffer (332 mM tris, 83 mM citrate, pH 7.2) at 37°C with 0 to 2 mM glucose, 0 to 30 mM pyruvate, 0 to 2 mM 3MCPD, and 0 to 80 μM CCCP for 0, 15, and 30 min.In addition, 3MCPD (a glycolysis inhibitor) and CCCP (a proton iono phore) were used to inhibit glycolysis and mitochondrial oxidative phosphorylation, respectively.Sperm motility was recorded using digital video microscopy for 5 s and was cat egorized as progressive, nonprogressive, and nonmotile [12].In brief, sperm that have forward progression (forward and slow) and sluggish motility were categorized as progressive motile sperm; nonprogressive motile sperm when sperm that vibrates in place; nonmotile for sperm with no move ment; and total motile sperm was the sum of progressive and nonprogressive motile sperm.Sperm motility profiles were then observed for spermatozoa incubated in combina tion with 0.5 mM glucose, 5 mM pyruvate, 0.5 mM 3MCPD, and 40 μM CCCP at 37°C for 30 min to determine the rela tive dependency of sperm motility on ATP providers.

ATP quantification
ATP concentration was quantified using the ATP biolumi nescence assay kit II (Roche, Germany).Sperm (1×10 7 ) were incubated with 0 or 0.5mM glucose, 5 mM pyruvate, 0.5 mM 3MCPD, and 40 μM CCCP in Triscitrate solution at 37°C for 30 min.After washing with Triscitrate solution, sperm were solubilized with lysis reagent and incubated at 25°C for 5 min.The supernatant was transferred to a fresh tube and mixed with luciferase reagent after centrifugation at 10,000 g for 1 min.The bioluminescence signal was mea sured using a multimode Tecan infinite M200PRO plate reader.

Statistical analysis
Multiple comparisons were performed using oneway analysis of variance, followed by Tukey's honestly significant differ ence.Results were expressed as mean±standard error of the mean and the differences were considered significant when there was a pvalue of <0.05.

RESULTS
The effect of glucose and incubation period was investigated by incubating sperm in 0, 0.5, 1, or 2 mM of glucose for 0, 15, and 30 min at 37°C.The results showed that there were no differences in total motile (77.2% to 84.5%), motile pro gressive (66.9% to 76.7%), and nonmotile (15.5% to 23.6%) when sperm were incubated in different levels of glucose for 0 to 15 min.However, nonprogressive motility in the group of sperm without glucose supplementation (4.5% compared with 17.6%) showed a difference.Total motile (79.0%com pared with 45.4%) and motile progressive (63.8% compared with 31.1%) were significantly greater in sperm incubated with 0.5 mM glucose than no glucose for 30 min (p<0.05;Table 1).There were no significant differences (p>0.05) in nonprogressive motile between glucose and no glucose supplementation groups in 30 min incubation (14.4% to 16.7%).Sperm without glucose supplementation had the highest nonmotile sperm (54.6% compared with 21% to 26%).The addition of 1 mM and 2 mM glucose did not result in a further increase in the value of sperm motility variables.Therefore, glucose has roles in sperm motility regulation as a glycolytic substrate, as shown in Table 1.
The importance of oxidative phosphorylation in goat sperm motility was examined by incubating sperm with 0, 20, 40, or 80 μM CCCP for 30 min in the presence of 5 mM pyruvate, followed by visual examination for motility analysis.The total and progressive motile values of sperm incubated with 5 mM pyruvate were larger (Figure 2; 70.3% and 55.0%, respectively) than those without pyruvate supplementation (45.8% and 32.9%, respectively).There were no significant differences in total motile when sperm incubated with 20 to 40 μM CCCP (62.0% to 58.5%) and progressive motile in 20 μM CCCP (45%) compared with pyruvateadded sperm.However, the addition of 40 μM CCCP decreased the pro gressive motile of sperm by 17.5%.The addition of 80 μM CCCP drastically decreased both total and progressive mo tile (25.4% and 0.4%, respectively).Furthermore, in response to 40 μM CCCP, nonprogressive motile (41.0%) was higher than that of sperm incubated without pyruvate and CCCP (12.9%), with pyruvate (15.2%), or pyruvate and 20 μM CCCP supplementation (17.0%) but did not differ from 80 μM CCCP group (25.0%).Likewise, the presence of CCCP increased nonmotile sperm in a dosedependent manner, indicating that mitochondrial respiration took part in the energy supply for the motility of goat sperm.
To examine the role of glycolysis and oxidative phos phorylation in goat sperm ATP production, cellular ATP concentration was quantified in sperm incubated with 3MCPD, CCCP, with or without glucose and pyruvate supplementation.In the presence of 5 mM pyruvate, the ATP concentration of sperm increased (mean increase of 185%), while 0.5 mM glucose increased ATP content (mean increase 72%) compared to those in sperm without sugars supplementation (Figure 3).The addition of inhibitors, either 3MCPD or CCCP decreased ATP content for all treat ments (91% to 93% vs pyruvate addition; 85% to 89% vs glucose addition), suggesting that pyruvate and glucose play a role in ATP production.There were no significant differences in ATP content among inhibitor groups.

DISCUSSION
Sperm motility is highly dependent on the availability of cel lular ATP.Despite the importance of metabolic substrates such as glucose and pyruvate for ATP production and sperm functions in goats, the regulatory mechanisms involved in energy metabolisms for sperm motility are poorly under stood.We found that ATP supports sperm motility in goats through glycolysis and oxidative phosphorylation.The re sults of the present study provide new insights into ATP generating pathways that support flagellar motility in goats.
Goat uterine fluid contains 0.5 mM of glucose [13], but the pyruvate concentration in the female reproductive tract is unknown.A previous study showed that 5 to 10 mM of pyruvatemaintained sperm progressivity for up to 3 days of storage in goats [14].Considering glucose concentration in uterine fluid and the effect of pyruvate concentration on sperm motility, the present study evaluated the effect of 0 to 2 mM glucose and 0 to 30 mM pyruvate on values of sperm motility variables.The concentration of 0.5 mM glucose was sufficient to maintain total motile, progressive motile, and nonmotile sperm for up to 30 min incubation.These results were consistent with previous studies where glucose was re ported to support the flagellar motility of sperm in different species [8,15,16].Previously, several glucose transporters were localized in the flagellar region in mammalian spermatozoa [17,18].The present study also found that the supplementa tion of 5 mM pyruvate was sufficient to support total motile, progressive motile, and maintain low nonmotile sperm.Pyru vate was also transported by monocarboxylate transporters across biological membranes and was metabolized through electron transfer in the respiratory chain to support motility [14,19].Even though glucose and pyruvate were important metabolic substrates for flagellar movements of sperm in goats, the cellular mechanisms should be analyzed.
In mammalian spermatozoa, energy metabolisms sup porting flagellar motility depend on glycolysis in the principal piece and oxidative phosphorylation in the mitochondria [4].Since 3MCPD is a potent inhibitor of glyceraldehyde 3phosphate dehydrogenase and CCCP is an uncoupler of mitochondrial oxidative phosphorylation, the effects were evaluated at 0 to 2 mM and 0 to 80 μM in the presence of glucose and pyruvate.The glycolysis inhibitor at ≥0.5 mM dramatically reduced total motile and progressive motile.A marked reduction in progressive motile was observed in the presence of ≥40 μM CCCP.In line with a previous study re garding the inhibitory effect of glycolysis using iodoacetate and mitochondrial respiration using rotenone or alphacya no4hydroxycinnamate (4CIN) on sperm motility in goats [14], our results indicate the functional importance of glycoly sis and oxidative phosphorylation in the flagellar movements of goat sperm.
In the present study, total motile was decreased by 3MCPD in the presence of glucose alone but slightly improved when pyruvate was also present.Similarly, the inhibition of total motile by CCCP with pyruvate alone was partially increased by the addition of glucose.The compensatory effect from either glucose or pyruvate addition was not observed in progressive motile.However, the suppression of mitochon drial respiration had greater efficacy in diminishing the population of progressively motile sperm, in comparison to the suppression of glycolysis.This observation implied the disparate roles of energy metabolic pathways in govern ing the flagellar motion of sperm.Several studies have documented functional differences in energy metabolic pathways on sperm motility in different species.For example, sperm motility in stallions depends heavily on oxidative phosphorylation [5], and glycolysis is the main ATPgener ating pathway for sperm motility in mice [3].
Results from a previous study of mouse sperm indicated higher cytoplasmic ATP content associated with higher swimming velocities [20].Even though pyruvateadded sperm produce more ATP concentration than glucose, sperm progressivity between the two metabolic substrates is com parable, which might suggest that ATP produced from glycolysis is efficient and effective in supporting the flagellar movement.Because flagellar movement is caused by the ac tivity of dynein ATPase that is localized along the entire length of the flagellum and depends on the ATP supply, it is reasonable that glycolysis occurred in the principal piece of the flagellum predominantly provides ATP for dynein ATPase of the flagella.Oxidative phosphorylation produces a large amount of ATP in the mitochondria but does not sufficiently propagate in the principal piece to support flagellar move ment [3].Furthermore, low ATP content was detected in sperm incubated either with 3MCPD or CCCP, resulting in low progressive motility, even though glucose or/and pyru vate were present, suggesting either glycolysis or oxidative phosphorylation can independently support sperm flagellar movement in goats.It was hypothesized that sperm could generate ATP from glycolysis when the substrates were present.Conversely, when there were oxidative phosphory lation substrates, sperm used those substrates through mitochondrial respiration in the midpiece to provide ATP for flagellar movement.The mechanisms of ATP generation by oxidative phosphorylation distributed to the entire flagellum should be investigated.In a preceding investigation concern ing sea urchin sperm, the phosphocreatine shuttle assumed the responsibility of conveying ATP from the mitochondrion to the remote flagellum [21].This finding propelled the sug gestion of a prospective inquiry into the functional attributes of phosphocreatine shuttle within goat sperm.

Figure 1 .
Figure 1.Motility characteristics of sperm incubated for 30 min in the presence of 3-chloro-1,2-propanediol (3-MCPD) with or without 0.5 mM glucose supplementation.Spermatozoa were incubated with 0.5, 1, or 2 mM 3-MCPD with 0-or 0.5-mM glucose supplementation for 30 min.Total and progressive motile were increased by glucose supplementation, but decreased in the presence of 0.5, 1, or 2 mM 3-MCPD.Contrarily, non-motile sperm was low in the presence of glucose but increased by 3-MCPD.Data are expressed as the mean±standard error of the mean (n = 6).a-d Different letters above columns indicate significant differences (p<0.05).

Figure 1 .
Figure 1.Motility characteristics of sperm incubated for 30 min in the presence of 3-Chloro-1,2propanediol (3-MCPD) with or without 0.5 mM glucose supplementation.Spermatozoa were incubated with 0.5, 1, or 2 mM 3-MCPD with 0-or 0.5-mM glucose supplementation for 30 min.Total and progressive motile were increased by glucose supplementation, but decreased in the presence of 0.5, 1, or 2 mM 3-MCPD.Contrarily, non-motile sperm was low in the presence of glucose but increased by 3-MCPD.Data are expressed as the mean±standard error of the mean (n = 6).a- d Different letters above columns indicate significant differences (p<0.05).

Figure 2 .
Figure 2. Motility characteristics of sperm incubated for 30 min in the presence of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) with or without 5 mM pyruvate supplementation.Spermatozoa were incubated with 20, 40, or 80 µM CCCP in Tris-citrate with or without 5 mM pyruvate for 30 min.Pyruvate supplementation increased the total and progressive motile of sperm, but the presence of CCCP abolished the increasing effect of pyruvate at 80 µM for total motile and 40 to 80 μM for progressive motile of sperm.No increasing effect of pyruvate was found on non-progressive motile, but the presence of 40 μM CCCP increased the percentage of non-progressive motile.The lowest percentage of non-motile sperm was found in the presence of pyruvate, but it is significantly increased by 80 μM CCCP.Data are expressed as the mean±standard error of the mean (n = 6).a-c Different letters above columns indicate significant differences (p<0.05).

Figure 2 .
Figure 2. Motility characteristics of sperm incubated for 30 min in the presence of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) with or without 5 mM pyruvate supplementation.Spermatozoa were incubated with 20, 40, or 80 µM CCCP in Tris-citrate with or without 5 mM pyruvate for 30 min.Pyruvate supplementation increased the total and progressive motile of sperm, but the presence of CCCP abolished the increasing effect of pyruvate at 80 µM for total motile and 40 to 80 μM for progressive motile of sperm.No increasing effect of pyruvate was found on non-progressive motile, but the presence of 40 μM CCCP increased the percentage of non-progressive motile.The lowest percentage of non-motile sperm was found in the presence of pyruvate, but it is significantly increased by 80 μM CCCP.Data are expressed as the mean±SEM (n = 6).Different letters above columns indicate significant differences (p<0.05).

Figure 4 .
Figure 4. Changes in sperm motility profile in response to metabolic substrates and inhibitors.The sperm motility analysis revealed increased total and progressive motility in response to glucose or pyruvate supplementation.3-Chloro-1,2-propanediol (3-MCPD) or carbonyl cyanide 3-chlorophenylhydrazone (CCCP) decreased total and progressive motile, even in the presence of both glucose and pyruvate.Non-progressive motile sperm tend to increase as a consequence of reduced progressive motile by inhibitors.Data are expressed as mean±standard error of the mean (n = 6).a-c Different letters above columns indicate significant differences (p<0.05).

Figure 4 .
Figure 4. Changes in sperm motility profile in response to metabolic substrates and inhibitors.The sperm motility analysis revealed increased total and progressive motility in response to glucose or pyruvate supplementation.3-Chloro-1,2-propanediol (3-MCPD) or carbonyl cyanide 3chlorophenylhydrazone (CCCP) decreased total and progressive motile, even in the presence of both

Table 1 .
Changes in sperm movement characteristics following incubation for 0 to 30 min with different levels of glucose (mM) supplementation Data are expressed as the mean ± standard error of the mean (n = 6).Within rows, different letters indicate a significant difference (p < 0.05).

Table 2 .
Changes in sperm movement characteristics following incubation for 0 to 30 min with different levels of pyruvate (mM) supplementation ab 22.2 ± 1.8 ab 21.5 ± 1.0 ab 21.6 ± 1.0 ab 50.8 ± 3.3 c 25.0 ± 1.4 a 26.4 ± 2.6 b 28.7 ± 1.2 b a-c Data are expressed as the mean ± standard error of the mean (n = 6).Within rows, different letters indicate a significant difference (p < 0.05).