The gender dependent influence of sodium bicarbonate supplementation on anaerobic power and specific performance in female and male wrestlers

The aim of this study was the assessment of progressive low-dose sodium bicarbonate (NaHCO3) supplementation on the anaerobic indices in two bouts of Wingate tests (WT) separated by wrestling-specific performance test and assessing the gender differences in response. Fifty-one (18 F) wrestlers completed a randomized trial of either a NaHCO3 (up to 100 mg·kg−1) or a placebo for 10 days. Before and after treatment, athletes completed an exercise protocol that comprised, in sequence, the first WT1, dummy throw test (DT), and second WT2. The number of completed throws increased significantly in males from 19.3 ± 2.6 NaHCO3pre to 21.7 ± 2.9 NaHCO3post. ΔWT2-WT1 improved particularly in the midsection of 30-s WT on NaHCO3. However, no significant differences were found in peak power (PP), power drop (PD) and average power (AP) (analyzed separately for each WT), and ΔWT2-WT1 in PP and PD. Interaction with gender was significant for AP, PP and PD, every second of WT1 and WT2, as well as DT test. In conclusion, our study suggests that the response to NaHCO3 may be gender-specific and progressive low-dose NaHCO3 supplementation allows the advantageous strengthening of wrestling-specific performance in males. It can also lead to maintenance of high anaerobic power mainly in the midsection of the 30-s Wingate test.


Results
Wingate anaerobic power indices. The interaction with gender was significant for average power (AP) (p < 0.0001), power drop (PD) (p < 0.0001), and peak power (PP) (p < 0.0001). However, no significant differences in AP, PD and PP were found after NaHCO 3 and PLA interventions neither in females nor in males (Table 1). There were no significant gender interactions for the differences between WT 2 and WT 1 (Δ WT 2 -WT 1 ) in AP, PD and PP. Δ WT 2 -WT 1 in AP, PP and PD were not significantly affected by NaHCO 3 and PLA treatments in both genders ( Table 2).
Blood sample analysis. Before and after supplementation no significant differences in glucose, lactate and pyruvate concentrations were found neither for female nor for male wrestlers (Table 3).

Discussion
In this study we showed that progressive supplementation of up to 100 mg·kg −1 sodium bicarbonate did not significantly influence AP, PD and PP characteristics in two Wingate tests. However, it improved power maintenance in the midsection of the 30-s Wingate test and performance in wrestling-specific dummy throw test. We observed that gender was a significant factor potentially influencing the effectiveness of such a treatment. Gender interaction was significant for AP, PD and PP, but possibly the dose was too small to elicit any significant improvement in those parameters in both males and females. Gender was also significant factor influencing the effect of NaHCO 3 on power in each second of the Wingate test and on performance in DT test. What is interesting, males significantly increased the number of throws in DT test, while females did not. That may suggest that the response to NaHCO 3 treatment is gender specific.
As previously observed, supplementation with NaHCO 3 may improve performance in combat sports [9][10][11] . NaHCO 3 resulted in improvement of boxing (punch efficacy: +5.4%) 10 , taekwondo (the total attack time in combat: +13%) 11 and judo (summed number of throws in three bouts of SJFT: +4 throws) 9 specific performance, respectively. In contrast, in our study performance in wrestling-specific DT improved significantly on NaHCO 3 with significant gender interaction. Then, when analysing genders separately we found that males increased the number of throws by ~11% (~2 throws), while no significant changes were observed in females. This slight, yet important change could contribute to winning in real wrestling competition. Previous studies on NaHCO 3 in combat sports did not include female athletes 9-12 . Gender differences in response to NaHCO 3 supplementation are especially worth discussing. Papers with female subjects are scarce. Only one of six studies on women showed the improvement after NaHCO 3 intake 21-26 . Kozak-Collins et al. 21 supplemented seven competitive female cyclists with either 300 mg•kg −1 NaHCO 3 or PLA (NaCl). 2 h after ingestion participants performed interval cycling protocol consisting of repeating intervals of 1 min 95%VO 2 max cycling and 1 min recovery at 60 W until exhaustion. They did not find any improvement in the  Table 2. The difference in power between WT 2 and WT 1 (Δ WT 2 -WT 1 ) before and after supplementation. Data are presented at mean ± SD. NaHCO 3pre , before sodium bicarbonate supplementation; NaHCO 3post , after sodium bicarbonate supplementation; PLA pre , before placebo; PLA post , after placebo. (2020) 10:1878 | https://doi.org/10.1038/s41598-020-57590-x www.nature.com/scientificreports www.nature.com/scientificreports/ number of completed intervals. In comparison, Price et al. 27 recruited only male subjects. Investigators also gave them NaHCO 3 or PLA (NaCl) before testing. Participants did two intermittent cycling trials comprised of repeated 3-min blocks (90 s at 40%VO 2 max, 60 s at 60%VO 2 max, 14-s maximal sprint, 16-s rest). Authors found that compared to PLA, power output was greater throughout exercise during the NaHCO 3 trial. Tiryaki and Atterbom 22 assessed the effect of NaHCO 3 on 600 m running time of trained females and found no differences (121.5 s on NaHCO 3 and 120.4 s on PLA). On the other hand, males improved running time in 400 m distance by 1.52 s on NaHCO 3 and in 800 m by 2.9s 28,29 . Even though there are no studies assessing the effect of NaHCO 3 on 600 m run in males, it can be expected that it would be also improved. Then, there are four studies on female team sports players [23][24][25][26] . Macutkiewicz and Sunderland 23 observed no influence of NaHCO 3 on Field Hockey Skill Tests and www.nature.com/scientificreports www.nature.com/scientificreports/ the Loughborough Intermittent Shuttle Test in elite female field hockey players. In comparison, Krustrup et al. 30 found 14% improvement in Yo-Yo intermittent recovery test level 2 performance (735 ± 61 m on NaHCO 3 vs 646 ± 46 m on PLA) in trained males. Moreover, Ducker et al. 31 and Miller et al. 32 observed improved repeated sprint capacity in males on NaHCO 3 . In a study by Ducker et al. 31 subjects did three sets of 6 × 20 m sprints with 4 min of recovery between sets. NaHCO 3 resulted in the best repeated-sprint performance. In a study by Miller et al. 32 male athletes were given NaHCO 3 or PLA and then performed repeated sprint cycling protocol comprising 10 × 6 s sprints with 60 s recovery. Total work completed during the repeated sprint protocol was higher in the NaHCO 3 condition (69.8 ± 11.7 kJ) compared with both -the control (59.6 ± 12.2 kJ) and PLA (63.0 ± 8.3 kJ) conditions. In a study on female team sports athletes NaHCO 3 failed to improve total work in prolonged intermittent www.nature.com/scientificreports www.nature.com/scientificreports/ sprint performance (IST) 24 . IST consisted of two 36-min "halves" of repeated ~2-min blocks: all-out 4-s sprint, 100 s of active recovery at 35%VO 2 max, and 20 s of rest. There was a trend toward improved total work in the second half, but it did not reach statistical significance (p = 0.08). Similarly, no improvement was observed in female water-polo players 25 . After the ingestion of NaHCO 3 or PLA the subjects performed a 59-min match-simulation test (MST) that included 56 ×10 m maximal-sprint swims. NaHCO 3 increased blood pH, but failed to improve mean sprint times. The only study to show improvement on NaHCO 3 in female athletes is a study by Delextrat et al. 26. Participants in that study were university basketball players. The supplementation protocol differed from all other studies. Athletes were supplemented with higher dose of NaHCO 3 (0.4 g•kg −1 compared to 0.3 g•kg −1 ) and it was a multiday (3 days) loading. NaHCO 3 improved mean values of sprint times, circuit times and jump height compared with PLA.
In summary, out of six trials on female athletes only in one NaHCO 3 was proven to be effective [21][22][23][24][25][26] . On the other hand, males seem to benefit more from the supplementation [27][28][29][30][31][32] . The reason for that might be in physiological differences. Females have smaller type II fibers than men 17,18 . Type II fibers rely predominantly on glycolytic energy system. It was shown that males have greater glycolytic capacity 19,20 . In addition, in females pH drops to a lesser extent that in males during the same type of exercise 20 . All of that would explain the gender differences in the response to NaHCO 3 supplementation observed in our and all previous studies.
Furthermore, it is important to observe that several bouts of intense exercise cause muscular fatigue, which may hamper performance during competition or training. In our study, power characteristics in WT 1 tended to be higher than in WT 2 ( Table 1). One of the factors contributing to fatigue is a decrease in intramuscular pH, which causes reduction in enzyme activation, competitive binding of H + to the active site of troponin, inhibition of oxidative phosphorylation and compromised resynthesis of phosphocreatine 6 . Sodium bicarbonate supplementation results in better buffering capacity of blood, which may increase the efflux of H + and La − out of muscle cells and decrease acidosis 4 .
It was previously established that the effect of NaHCO 3 supplementation may be pronounced predominantly in latter stages of exercise 9,13-15 . Artioli et al. 9 supplemented their athletes with 300 mg·kg −1 NaHCO 3 2 h before exercise. The performance test included four bouts of 30-s upper body WT tests. The significant changes in AP and PP were observed only in the two final bouts. This was attributed to improved resynthesis of phosphocreatine due to alkalosis caused by NaHCO 3 supplementation, since low intramuscular pH may hamper this process 9 .
Tobias et al. 14 assessed the effect of one week NaHCO 3 ingestion on four-bout upper-body WT performance. Single bout was 30 s long with the load of 5% body mass. Seven-day supplementation resulted in 8% increase in total work done (in all four bouts summed). However, when the bouts were analysed separately a significant increase in AP and PP was present only in the last bout (+9.4%, p = 0.038, and +13.7%, p = 0.018, respectively) 14 .
A subsequent study by Oliveira et al. 15 confirmed those results. They adopted a similar protocol of performance testing (four 30-s WT bouts for upper body interspersed by 3-min recovery) and also observed a significant increase in the total work done (+2.86%, p = 0.02) after 5-day NaHCO 3 supplementation compared to PLA. And again the difference was more pronounced in the last two bouts (sum of bout 3 and 4: +5.93%, p = 0.02).
Since aforementioned studies 9,14,15 showed that the effect of NaHCO 3 is apparent the most in latter stages of intense exercise, we aimed at assessing the gender-related effect of NaHCO 3 on the difference between the first and the second WT, which were additionally interspersed by DT. Dummy throw test is a highly strenuous test, specific to wrestling. It is comprised of two alternating modes -slow and fast 13 . The slow mode lasts 30 s, during which an athlete does four compulsory dummy throws. Whereas, in the quick mode an athlete performs as many throws as possible in 15 s. The test lasts 3 min and comprises four slow and four quick modes, so that it is highly exhausting. Thus, the participants of our study were already fatigued on the onset of the second WT. Even though the difference in PP between WT 2 and WT 1 tended to be improved by NaHCO 3 (by 35.1 W and 35.0 W in females and males, respectively), they were not statistically significant (Table 2).
Furthermore, innovative analysing (in the field of NaHCO 3 supplementation) of each second of WTs separately significant improvement (NaHCO 3post vs NaHCO 3pre ) in the difference in power between WT 2 and WT 1 were observed in seconds 12 th , 16 th and 21 st when all participants were taken together. In females the significant difference was apparent only in 21 st s (NaHCO 3post vs NaHCO 3pre ), whereas in males in 12 th s (NaHCO 3post vs NaHCO 3pre ). Compared to PLA, on NaHCO 3 the difference in power between WT 2 and WT 1 improved in seconds 12 th , 16 th , 17 th and 21 st in all participants. In females, significant improvement was observed in 21 st s and in males in 17 th s (NaHCO 3post vs PLA post ). It therefore seems reasonable to emphasize that most of the substantial effects were observed in the case of this supplementation protocol in the middle (12-21s) of the WTs.
In spite of the few significant differences observed in our study, we hypothesise that the dosage of NaHCO 3 might have been too small for female and male wrestlers to elicit more apparent improvements. We used up to 100 mg·kg −1 NaHCO 3 in days 8-10 of supplementation (Fig. 4). The dosage was well tolerated and did not cause any gastrointestinal (GI) problems, but the effectiveness was slight and moderate. Simultaneously, in previous  Table 3. Glucose, lactate and pyruvate concentrations before and after exercise tests in female and male wrestlers. Data are presented at mean ± standard deviation (SD). NaHCO 3pre , before sodium bicarbonate supplementation; NaHCO 3post , after sodium bicarbonate supplementation; PLA pre , before placebo; PLA post , after placebo.
Scientific RepoRtS | (2020) 10:1878 | https://doi.org/10.1038/s41598-020-57590-x www.nature.com/scientificreports www.nature.com/scientificreports/ studies higher doses were usually implemented 9,14,15,33 . IOC recommends the intake of 200-400 mg·kg −1 NaHCO 3 60-150 min prior to exercise 3 . However, in many athletes these doses result in GI distress 34 . This may prevent the practical use of supplementation with this compound in the conditions of natural high-intensity effort that is carried out, e.g. in combat sports. On the other hand, smaller doses might be ineffective. For instance, in nine healthy males the dose of 100 mg·kg −1 failed to induce alkalosis, increase base excess and had no influence on work output 35 . Furthermore, in six males McKenzie et al. 36 showed that even though induced alkalosis was greater with 300 than 150 mg·kg −1 NaHCO 3 , there were no differences in work produced (133.5 and 133.1 kJ, respectively) and time to fatigue in the last bout (106 and 110s) between those two doses. However, comparing all those results to ours is limited because all of them used acute supplementation protocol, while participants in our study www.nature.com/scientificreports www.nature.com/scientificreports/ ingested NaHCO 3 for ten days. In a previous study done by our lab, progressive-dose protocol of NaHCO 3 up to 150 mg·kg −1 was enough to improve CrossFit-like performance and ventilatory threshold 37 . However, NaHCO 3 supplementation protocol similar to the one used in the current study (10 days, up to 100 mg·kg −1 ) improved only time to PP in the second WT test with no further influence on anaerobic capacity and performance 13 . Nevertheless, we would like to highlight that in our research only highly-trained female and male wrestlers participated. Therefore, the observed changes related to males wrestling-specific performance and more effective maintenance of anaerobic power during high-intensity efforts, that can be considered beneficial at elite sport level, especially considering the short time duration of supplementation and a low dose of NaHCO 3 . It is worth bearing in mind, however, that a certain limitation to our study is the lack of verification of the bicarbonates concentration in the blood, which should be included in the subsequent studies, preferably in connection with the attempt to evaluate the effectiveness of supplementation of various doses of NaHCO 3 . Another limitation is the uneven distribution of participants in study groups. It is possible that if the number of participants was equal in each group the gender differences would be more pronounced.

Conclusions
Progressive low-dose NaHCO 3 supplementation allows in combat sports the advantageous suppression of fatigue-induced power decline in the midsection of the 30-s Wingate test and improvement in wrestling specific dummy throw test. The response to NaHCO 3 supplementation seems to be gender dependent. It appears that males can benefit more from the sodium bicarbonate supplementation, possibly because of physiological differences.

Methods
We would like to clarify that in this work we used the data previously collected in sodium bicarbonate studies involving wrestlers, which we conducted in our lab. We have already partially published the selected results obtained from most of the evaluated participants 13 . However, the data presented here was analyzed in a completely different fashion. The results for female and male wrestlers were analyzed separately to assess whether the response to the supplementation protocol may be determined by the gender of the athletes. We also focused on previously untouched aspects of the detailed change of power indices during each seconds of the Wingate test. Additional athletes were also included. Thus, we can unequivocally state that there is absolutely no risk of duplicate results, but we want to inform potential readers about the details of the data processing. Lastly, from a practical and scientific perspective, the research results which we have presented here are extremely valuable due to the innovative approach we have taken with NaHCO 3 supplementation, the detailed analysis of performance indices herein, and the accompanying assessment of gender-related responses to NaHCO 3 treatment.
Participants. Forty-six male and thirty-one female wrestlers were initially enrolled in this study. However, thirty-three male and eighteen female athletes participated in the study and were included in the analyses (Fig. 4). Anthropometric characteristics are presented in Table 4. The athletes were members of the Polish Wrestling National Team and/or competed in the highest level of Polish competitions. The inclusion criteria were good health, a valid medical clearance to participate in sports, a minimum of four years of combat sports experience, and doing at least four workout sessions (combat sport) a week. The exclusion criteria were current injury, any health condition preventing from participation, self-declared unwellness, and no interest in proper participation in study protocol.
All athletes reassured that they had not changed their life-styles, training regimen, diet or supplementation, and that they had not been using any medications and supplements with potential ergogenic effects, other than those supplied by the authors of this study. In accordance with the 1975 Declaration of Helsinki, before enrolment all participants had given their written consents to participate in the study protocol. Informed consents were also obtained from the parents of athletes under the age of 18 years, prior to participation in the study. The approval of the Bioethics Committee at Poznan University of Medical Sciences was obtained for this study. This trial was registered at Clinical Trials Gov (website: https://clinicaltrials.gov/ct2/show/NCT03406065; Clinical Trial Identification Number: NCT03406065). The study was registered retrospectively as registration was not   www.nature.com/scientificreports www.nature.com/scientificreports/ required when the study enrolment started. The authors confirm that all ongoing and related trials associated with this intervention are registered. The study complies with the CONSORT statement for randomized trials, as shown in Fig. 4.
Study design and protocol. The study was designed as randomized double-blind placebo-controlled parallel-group trail. The supplementation period lasted ten days. The participants were familiarized with the exercise testing protocol and the equipment on a preliminary meeting with the research team. Anthropometric measurements were also taken on the same day. When enrolled athletes were randomly divided into the treatment groups (the NaHCO 3 group or the PLA group). The random allocation sequence and matching were performed using stratified randomization via impartial biostatistics.
The experiment consisted of two separate visits (T 1 -T 2 ) for each participant. All testing was performed in natural conditions at the Central Olimpic Sports Centers (Spała, Zakopane) and Wrestling Training Centers (Poznań) in Poland. Throughout the study the participants were supplemented with either NaHCO 3 or PLA. Exercise tests were conducted before and after each trial at the same time of day. Testing sessions started between 7.30 and 10.00 a.m. each time. To maintain constant conditions the participants were asked to refrain from any strenuous exercise for 24h before the testing.
Supplementation. The participants were supplemented with NaHCO 3 for ten consecutive days. Initial dose was much smaller than the dose recommended previously 2,3 and was then increased gradually until 0.1 g•kg −1 was reached. This loading protocol was shown to eliminate any GI side effects 13,37 . Supplementation protocol is depicted in Fig. 4. Sodium bicarbonate was administered in the form of unmarked disc-shaped tablets (Alkala T, manufacturer-Sanum Kehlbeck GmbH & Co. KG, Germany). The tablets were ingested with at least 250 mL of water and could either be swallowed or dissolved in the mouth. Maltodextrin with NaCl served as PLA. It was administered in a similar tablet prepared by the same producer as of the NaHCO 3 tablets.
Daily doses of both NaHCO 3 and PLA were split into three even portions. On training days, the tablets were ingested in the morning, in the evening, and 1.5h before training session. On rest days, the supplements were administered in the morning, in the afternoon, and in the evening. To increase adherence the participants were also given personal supplementation plans.
Anthropometric measurements. Anthropometric measurements were taken in the fasted state in the preliminary visit in the morning. Body fat and free-fat mass were assessed based on air displacement plethysmography using the Bod Pod ® (Cosmed, Italy) 38 . Total body water and hydration level were assessed by means of bioelectric impedance, with Bodystat 1500 (Bodystat Inc., UK) 39 , and via urine specific gravity measurement, with URYXXON ® Relax (Macherey-Nagel, Germany).
Exercise tests. Every testing session consisted of two Wingate anaerobic tests interspersed with a dummy throw test. Wrestling-specific performance capacity was measured using a specific dummy throw test described previously 13 . Wingate tests were performed on a cycloergometer (Monark 894E, Sweden). All recommendations for such tests as proposed by Bar-Or were strictly followed 40 . External loading was set at 7.5% body weight. The first WT (WT 1 ) was performed 5 min before DT and the second (WT 2 ) 10 min after DT (Fig. 4). Prior to testing all athletes completed 5-min warm-up on cycloergometer at approximately 50 W power. During the test, the athletes were verbally encouraged to exert maximum effort. The recorded results were analysed using the Monark Anaerobic Test Software (ver. 3.0.1, 2009, Monark, Sweden). Blood samples analysis. Fingertip blood samples were taken twice, one sample before the WT 1 and the other 3 min after the WT 2 . During blood draws the participants seated in an upright position. Blood samples were immediately transferred to microtubes containing 500 µL of 0.6 M perchloric acid. Glucose concentration was measured using a colorimetric enzymatic method with glucose oxidase (Liquick Cor-GLUCOSE, Cormay, Łomianki, Poland). Lactate and pyruvate measurements were performed according to the method described previously 13 . All biochemical analyses were conducted using a Synergy 2 SIAFRT microplate multi-detection reader (BioTek, USA). Statistical analysis. The study was designed as a randomized parallel trial. Thus, in statistical analysis a mixed model of repeated measures with known error covariance matrix was used 41,42 . The random factor was participants nested in groups. Group stand for treatment (NaHCO 3 or PLA). Fixed factors were: period (NaHCO 3pre-WT1 , NaHCO 3pre-WT2 , NaHCO 3post-WT1 , NaHCO 3post-WT2 , PLA pre-WT1 , PLA pre-WT2 , PLA post-WT1 , PLA post-WT2 ), gender, times (period) (1-30 seconds of WT). Two-way interactions (gender × treatment, treatment × period, gender × period, and treatment × times (period)) and three-way interactions (gender × treatment × period, and gender × treatment × times (period)) were considered. Tested error covariance matrix structures included: Compound symmetry, Autocorrelation, Toeplitz and Unstructured. The choice of model with adequate covariance matrix structure was done according to Akaike information criterion 43 . Because gender and gender interactions with other factors were usually significant, those analyses were performed also for both genders separately. Statistical significance was set at p < 0.05. The assumptions of normality and homoscedasticity was tested using the Shapiro-Wilk test for normality. If data did not meet the assumptions then the Box-Cox transformation was used. Data were analyzed by own calculations and using the SAS 9.3 software program. Effect size was calculated as Cohen's ƒ2, as follows: f 2 = h 2 /(1 -h 2).