Total oxidizing and antioxidizing capacity, uric acid and albumin dynamics in bucking bulls under long-term acupuncture and training protocolsABSTRACT
This study aimed to evaluate oxidative stress parameters in bucking bulls submitted to acupuncture by measuring total anti oxidizing capacity (TAC-ABTS; TAC-ABTS+HRP), total oxidizing capacity (TOC; TBARS), albumin and uric acid levels. Thirty bulls were divided into two groups of 15 animals each: Group (GA) - acupuncture treated group, Group (GC) - control group. Measurements of parameters were performed 30 minutes before the jump (M0) and, 10 minutes (M10min), 12 hours (M12h), 24 hours (M24h), 48 hours (M48h) and 72 hours (M72h) after a jump. GA showed lower levels of TAC-ABTS in M0 (819,60μmol/L), and TAC-ABTS+HRP from M0 to M72h, while CG had a decrease in TAC-ABTS values in M10min (775,95μmol/L) and M12h (752,71μmol/L). GA presented higher levels of TOC in M12h (13,18μmol/L) and M24h (10,43μmol/L), and TBARS from M0 to M24h. GA had lower albumin levels in M0 (2,47g/dL), M10min (2,47g/dL) and M72h (2,41g/dL). GA showed higher levels of uric acid right after acute exercise in M10min (1,65mg/dL), M12h (1,67mg/dL) and M48h (1,65mg/dL). Acupuncture enhanced TAC, promoted a robust TOC, reduced albumin levels before and after acute exercise, and increased uric acid in bucking bulls.
Keywords:
oxidative stress; athlete; bovine; exercise; integrative medicine
RESUMO
Este estudo objetivou avaliar o estresse oxidativo em touros de rodeio submetidos à acupuntura, mediante mensuração da capacidade antioxidante total (TAC-ABTS; TAC-ABTS+HRP), da capacidade oxidante total (TOC; TBARS), dos valores de albumina e ácido úrico. Trinta touros foram divididos em dois grupos de 15 animais: grupo submetido à acupuntura (GA) e grupo controle (GC). As mensurações foram realizadas 30 minutos antes do salto (M0), 10 minutos (M10min), 12 horas (M12h), 24 horas (M24h), 48 horas (M48h) e 72 horas (M72h) após o salto. O GA apresentou valores inferiores de TAC-ABTS em M0 (819,60μmol/L) e TAC-ABTS+HRP de M0 a M72h, enquanto o GC apresentou diminuição de TAC-ABTS em M10min (775,95μmol/L) e M12h (752,71μmol/L). O GA demonstrou valores superiores de TOC em M12h (13,18μmol/L) e M48h (10,43μmol/L), TBARS de M0 a M24h. O GA apresentou valores inferiores de albumina em M0 (2,47g/dL), M10min (2,47g/dL) e M72h (2,41g/dL). O GA teve valores superiores de ácido úrico em M10min (1,65mg/dL), M12h (1,67mg/dL) e M48h (1,65mg/dL). A acupuntura otimizou a TAC, promoveu uma TOC mais robusta, reduziu as concentrações de albumina antes e após o exercício, bem como elevou o ácido úrico em touros de rodeio.
Palavras-chave:
estresse oxidativo; atletas; bovinos; exercício; medicina integrativa
INTRODUCTION
During biological reactions such as ATP production, oxidation of lipids, proteins, carbohydrates and deoxyribonucleic acid (DNA), a group of molecules called oxidizing substances is produced. (Colitt et al., 2019). These molecules are essential for organic functions, including cellular transcription and immune response (Davies, 2016). On the other hand, the increase in oxidizing substances in the circulation can induce the oxidation of other molecules without renewal potential and, consequently, lead to cellular damage, apoptosis or necrosis. (Calabrese et al., 2012).
To modulate and control the oxidizing substances production and their negative effects, the body produces antioxidant substances (AS) (Sun et al., 2019), which can be divided into enzymatic substances and non-enzymatic (e.g., vitamins E and A, uric acid and albumin) (Krishnaiah et al., 2011). The organic imbalance between the production of oxidizing substances and AS is called oxidative stress (OS) (He et al., 2017).
It is believed that during exercises, cellular oxidation may occur, therefore the evaluation of OS is a promising method for monitoring physical performance of athletic animals or for disease prognosis (Rosa and Vaisberg, 2002; Colitt et al., 2019). Data on OS in small ruminants and dairy cattle are found in the literature, but studies of OS in athletic bulls are absent. (Celi, 2010; Coleman et al., 2020).
The use of acupuncture in humans and animals to improve athletic performance is reported in the treatment of muscle, tendon, and ligament conditions. (Wang et al., 2020). Although there are scientific studies with evidence of acupuncture on hematological variables in rodeo bulls (Rosa et al., 2023), there are no experiments whose objective is to understand the effects of acupuncture on OS in athlete bulls.
There are different techniques to perform acupuncture, such as electroacupuncture, aquapuncture and dry need. (Angeli and Luna, 2008). It is known that acupuncture has the potential to cause analgesic effects, release endorphins promoting muscle relaxation and reduce the inflammatory response to exercise (Oh and Kim, 2022).
Acupuncture points can vary significantly across different animal species and purposes (Ding et al., 2023). Although the use of acupuncture in horses is well described to improve sports performance (Angeli and Luna, 2008), there are no studies involving the effect of acupuncture on OS in athletic bulls, and most of the knowledge for its application in sports medicine with bulls is based on horses.
Given the context, the present study aimed to evaluate the total antioxidant and oxidant capacity, albumin and uric acid levels in healthy bulls after six months of acupuncture associated with a training protocol. The hypothesis is that acupuncture may induce beneficial effects in terms of OS in rodeo bulls subjected to acute exercise. The rationale for this study is explained by the promising effect of acupuncture in athletes, which may contribute to the well-being of bulls used in sport.
MATERIALS AND METHODS
This study was approved by the Ethics Committee on the Use of Animals (CEUA) of the School of Veterinary Medicine and Animal Science at São Paulo State University (Unesp), Campus of Botucatu, São Paulo State, Brazil, under protocol 0083/2021.
Thirty healthy mixed breed bucking bulls, between 60 and 144 months of age (Mean ± SD; 98.9±24.7 months), with body mass between 890 and 1100kg (Mean ± SD; 967.5±48.9kg) and body condition score (BCS) ranging from 5 to 7 (Nutrient…, 2016), were eligible for study.
The inclusion criteria for the study were absence of alterations in clinical exam (Dirksen et al., 1993) and hemogram assessment (Kaneko et al., 2008), training for rodeo events for a minimum period of six months and having participated in at least one official competition. Bulls treated with any medication and who had undergone surgical procedures in the period of six months prior to the study were excluded from the evaluation.
During the study the bulls remained in a paddock of approximately 6 ha with Mombaça grass (Panicum maximum). Daily feeding was based on mean of 10kg corn silage per animal, mineral salt (Fosbovi pronto®, mineral supplement, Tortuga®, Brazil) and total feed mixture (340kg corn, 250kg soy, 330kg oat - 0,5% of the body mass) and water ad libitum. The study was conducted between the months of December and February at training centers for bucking bulls in the State of São Paulo, Brazil.
The animals were randomly allotted using a random number generator program (Randomizer®, Microsoft, USA), into two groups of 15 bulls each: Group 1 (GA) -bulls submitted to acupuncture; Group 2 (GC) - control group.
All the bulls included in the study underwent a six-month physical training protocol, always performed in the morning. The training consisted of walking in a sand circle (40 meters) for thirty minutes, alternating with trot gait for 5 minutes, two days a week (Monday and Friday), and jumping with a rider (average 70kg body mass) emulating a rodeo competition, with duration between 8-10 seconds, once a week (Wednesday).
Bulls included in the GA group underwent acupuncture sessions for six months (concurrently with the physical exercise protocol), performed every 14 days (on Tuesdays), by the same specialist, in the morning period. For acupuncture sessions the GA bulls remained at rest in a containment trunk, and 21G (30X0,80mm) dry needles (disposable hypodermic needle, Descarpak, Brazil) were applied perpendicular to the skin, without manipulation, maintained for 15 minutes and then were removed. The needels were applied in the following acupoints: B11 Da-zhu (cranial to the withers in the second thoracic intervertebral space), ID9 Qiang-feng (caudal border of the deltoid muscle), TA15 Bo-jian (dorsal border of the scapula), B23 Shen-shu (midline dorsal second lumbar intervertebral space), B25 Da-chang-shu (dorsal midline of fifth lumbar intervertebral space), B35 Hui-yang (muscular groove between the biceps femoris and semitendinosus muscles), VG2 Wei Gen (sacrococcygeal space), VB27 Wu Shu (Caudal to iliac spine, above wing of ilium) B28 Pangguangshu Bladder association point (lateral to dorsal midline, between second and third sacral vertebrae), E36 Hou-san-li (over cranial tibial muscle), B40 Wei zhong (center of the popliteal fossa), Bai Hui (midline of the lumbosacral space) (Kothbauer, 1999). The total session time lasted between 18 and 20 minutes per animal.
Although the GC were not subjected to acupuncture, all bulls in the control group remained for a similar amount of time to the GA in the containment trunk, but without receiving acupuncture or any other type of intervention.
Seven days after the training period in both groups (GC and GA) and the last acupuncture session in the AG group, all bulls were submitted to a single jump emulating the rodeo competition. Before the jump, the animals remained in the paddock at rest, without fasting food or water.
For the evaluation of OS parameters, blood collections were performed by venipuncture of the coccygeal vein, with the bulls under physical restraint in a cattle trunk. Eight mL of venous blood were collected using 21G (Microlance needles 25X0.8 mm, BD®, USA) needles and deposited in a vacuum dry tube containing clot activator (tube containing clot activator, Cralplast®, Brazil), and kept refrigerated and protected from light. One hour later, the tubes were centrifuged (5000rpm, 10 minutes) and the serum was separated and stored frozen (-20⁰C) until the moment of analysis.
The blood collections were performed 30 minutes before the jump (M0) and 10 minutes (M10min), 12 hours (M12h), 24 hours (M24h), 48 hours (M48h) and 72 hours (M72h) after the jump. OS parameters were evaluated in a semi-automated photocolorimeter method (BIO 2000®, BioPlus, Brazil). Total antioxidant capacity (TAC) was determined by inhibition of 2,2'-azino-bis 3-ethylbenzthiazoline -6-sulphonic acid (ABTS) cation reduction (TAC-ABTS) (Erel, 2004), inhibition of ABTS cation reduction associated with peroxidase (TAC-ABTS+HRP) (Rubio et al., 2016), cupric reducing antioxidant capacity (TAC-CUPRAC) (Rubio et al., 2016) and ferric reducing antioxidant power (TAC-FRAP) (Benzie and Strain, 1996). Additionally, non-enzymatic AS uric acid and albumin were determined using enzymatic-Trinder method and bromocresol green method, respectively, using commercial reagents following manufacturers’ recommendations (Labtest Diagnóstica SA, Brazil) (Almeida et al., 2021).
To measure the TOC, the xylenol orange colorimetric assay was used (Erel, 2005) and the lipid peroxidation was determined by thiobarbituric acid reactive substances (TBARS) (Hunter et al., 1985).
The design adopted for the study was a 2X6 factorial, with 2 treatments and 6 moments. The parameters were subjected to the Shapiro-Wilk normality test to verify normal distribution and the Bartlett test of homogeneity of variances to evaluate the equality of variances. For variables that met the assumptions of normality and homogeneity, ANOVA was used followed by Tukey's post-test to compare means. For variables that did not meet the assumptions, the median and interquartile deviation were used as descriptive measures, and data were analyzed using the Kruskal-Wallis test, followed by the Student-Newman-Keuls post-test. to compare the medians. Although the 2x6 factorial design suggests joint analysis of treatments and moments, the variables that did not present a normal distribution were analyzed using non-parametric methods, considered more robust to violations of statistical assumptions. The adopted approach allowed a reliable analysis, even without direct evaluation of the interactions between the factors. The analysis was carried out using the statistical software R (R Development Core Team, USA), considering a significance level of 5%.
RESULTS
Tables 1 and 2 show the TAC and TOC variations, while Fig. 1 illustrates the changes in values over time between groups. No significant variations were identified in relation to the TAC-CUPRAC and TAC - FRAP values. On the other hand, the TAC-ABTS values of the GC showed a decrease (p<0.05) between M0 and M10min or M12h, followed by an increase (p<0.05) between M10min and M48h, and M12h and M48h. In the intergroup evaluation, the animals from the GC showed higher values (p<0.05) compared to the GA at M0.
In the evaluation of TAC-ABTS+HRP, higher values (p<0.05) were always observed in the GC bulls. However, all values remained within the reference interval adopted for the species.
TOC values showed decrease (p<0.05) in GA bulls between M12h and M72h, and in the GC between the following moments: M0 and M12h or M24h. In the intergroup comparison, the GA bulls showed higher TOC values (p<0.05) compared to the GC at M12h and M24h. On the other hand, at M72h, GC bulls showed higher TOC values. Regarding the TBARS, it was observed that GA showed an increase in values (p<0.05) between M0 and M12h and M24h.
Values followed by different capital letters in the same column represent a significant intragroup difference (p<0.05). Values followed by different lowercase letters on the same line represent intergroup differences (p<0.05).
The values of non-enzymatic AS for both groups along the time points are shown in Table. 3. Bulls from the GC had higher albumin values (p<0.05) at M0, M10min and M72h, however, the values remained within of the reference limits for the species, which is the values of the GC at M0. Uric acid increased (p<0.05) between M12h and M72h in CG bulls; and in the intergroup evaluation, the GA bulls showed higher uric acid values (p<0.05) at the moments: M10min, M12h and M48h.
DISCUSSION
It is believed that acupuncture can increase the physical performance of athletes due to its anti-inflammatory and immunomodulatory properties (Angeli and Luna, 2008; Lu and Lu, 2013; Michelotto Júnior et al., 2014; Ondrejkovičová et al., 2016; Rizzo et al., 2017; Wang et al., 2020). Additionally, in the sporting context, oxidative imbalance was used to determine athletic performance in horses (Fazio et al., 2016; Williams, 2016).
The initial hypothesis was reached since, in general, the GA bulls showed effects of acupuncture on the OS after the jump. The lower values of TAC-ABTS (M0) and TAC-ABTS+HRP (M0 to M72h) show a possible long-term effect of acupuncture, which is the modulation of OS through the improvement of the antioxidant system (Bao et al., 2024). Although the GA TAC-ABTS levels were lower than GC only at M0, the values did not differ within the group at subsequent times, unlike the GC which showed a decrease in the same variable right after the jump until M12h. Furthermore, the lower levels of TAC-ABTS+HRP in GA, during all moments, and the absence of intragroup variations corroborate an optimization of the GA basal antioxidant system, reducing the need for increased antioxidant activity at rest and during acute exercise.
Acupuncture can modulate the OS by restoring the balance of Qi (vital energy) along the body's meridians, eliminating blockages that can promote dysfunction or imbalances (Chon and Lee, 2013). In Western medicine, Qi imbalance can be understood, along with other factors such as mitochondrial dysfunction, inflammation and increased local blood circulation, leading to OS (Chon and Lee, 2013). Acupuncture activates the sensory fibers of peripheral nerves, triggering modulatory changes that could stimulate the cholinergic system, reducing inflammatory responses and consequently improving the antioxidizing activity (Karavis, 1997). As a result, it could lead to a more effective anti oxidizing response to exercise, which in turn did not require an excessive production of AS before and after the exercise. According to Su et al., (2020), the acupuncture-induced antioxidant effect is caused by mitochondrial respiratory function improvement and mitophagy, which would promote an oxidative balance effect.
Mean and standard deviation, median and interquartile values of total antioxidant substances (TAC - ABTS, TAC-ABTS+HRP, TAC-CUPRAC and TAC-FRAP) of bucking bulls submitted to jumping and treated (GA) or not with acupuncture (GC), and evaluated 30 minutes before the jump (M0), 10 minutes (M10min), 12 hours (M12h), 24 hours (M24h), 48 hours (M48h) and 72 hours (M72h) after the jump
Median and interquartile values of total oxidant capacity (TOC) and lipid peroxidation products (TBARS) values of bucking bulls submitted to jumping and treated (GA) or not with acupuncture (GC), and evaluated 30 minutes before the jump (M0), 10 minutes (M10min), 12 hours (M12h), 24 hours (M24h), 48 hours (M48h) and 72 hours (M72h) after the jump
Change values in antioxidant substances (TAC-ABTS+HRP, TAC-ABTS; TAC-CUPRAC, TAC-FRAP), and total oxidant capacity (TOC), lipid peroxidation products (TBARS) of bucking bulls submitted to jumping and treated (GA) or not with acupuncture (GC), and evaluated 30 minutes before the jump (M0), 10 minutes (M10min), 12 hours (M12h), 24 hours (M24h), 48 hours (M48h) and 72 hours (M72h) after the jump.
Mean and standard deviation of the values of non-enzymatic antioxidant substances (albumin and uric acid) of bucking bulls submitted to jumping and treated (GA) or not with acupuncture (GC), and evaluated 30 minutes before jumping (M0), 10 minutes (M10min), 12 hours (M12h), 24 hours (M24h), 48 hours (M48h) and 72 hours (M72h) after the jump
The lack of difference in TAC-FRAP and TAC-CUPRAC values may be due to the fact that these laboratory methods measure a broader AS, including relatively stable enzymes such as superoxide dismutase and glutathione peroxidase, which maintain their respective functions after exercise (Bao et al., 2024).
At M12h, the GC showed an intragroup reduction in TOC levels in relation to M0, in addition to lower values when compared to the GA bulls. This may be related to several effects attributed to acupuncture in GA, which include adaptation to exercise through modulation of the hypothalamic-pituitary-adrenal (HPA) axis (Chon and Lee, 2013). It is known that acupuncture can increase the release of endorphins and other neuropeptides that regulate hormones such as cortisol and adrenaline, released during stressful situations like acute exercise (Chon and Lee 2013). After six months of treatment, a regulation in the HPA axis could lead to greater oxidative capacity in the GA bulls, allowing the organic system to cope with a higher oxidative demand during the jump and produce the necessary energy (Ding et al., 2023). Although GA bulls presented elevated TOC levels at M12h because of increased energy demand, at M72h the values were lower, both in intra- and intergroup comparisons, evidencing the greater antioxidant capacity of GA after acute exercise, discussed previously in this text.
According to Bao et al. (2024), the greater oxidative capacity promoted by acupuncture can also increase levels of OS markers, such as TBARS. From M0 up to M24h, TBARS values of GA bulls remained higher than those of GC. TBARS elevation levels may indicate intense lipid peroxidation, a situation that is commom after the mobilization of fatty acids for energy production during acute exercise (Han et al., 2020). The lipid peroxidation results in reactive molecules such as malondialdehyde, which in turn is measured by the TBARS (Hunter et al., 1985).
The lower albumin values in GA can be explained based on the HPA axis theory. According to Lim et al. (2010), in a systematic review they conclude that acupuncture has the potential to reduce the release of cortisol, which would reduce protein metabolism. Acupuncture can stimulate the parasympathetic nervous system and, consequently, inhibit the HPA axis, one of the most important pathways for the release of cortisol (Villas-boas et al., 2015). Apparently, in this study, the inhibition is not consistent, as in M12h there is no longer any difference in albumin levels between the groups.
Uric acid plays an important role as a free radical scavenger and protects tissues from oxidant damage when OS is established (Williams, 2016). Among the factors that can cause an increase in uric acid in the body, the intense metabolism of purines stands out, resulting from cell and tissue repair or renewal, a situation that occurs after acute exercise (Chen et al., 1990). During the cell renewal process, old cells undergo apoptosis, releasing their nucleotides, which are finally metabolized into uric acid. Acupuncture has the potential to enhance cellular and tissue repair by stimulating the release of growth factors such as endothelial, nervous and epidermal, in addition to increasing molecules of pro- and anti-inflammatory factors (interleukins and tumor necrosis factor), involved in repair and proliferation process (Li et al., 2021; Oh and Kim, 2022). This reasoning would explain the higher uric acid values in GA bulls, immediately after exercise in M10min and M12h.
The limitation of the study included the lack of measurement of specific antioxidant enzymes and cortisol. However, the most appropriate methods of determining OS for each situation, whether involving exercise physiology or disease conditions, as well as the methodologies for each species, still need additional research. Another factor in relation to this study was the lack of standardization of bulls regarding age group. In domestic animals, the older the individual, the lower the organism’s ability to deal with OS (Celi, 2010), a factor that may have interfered in the results. Throughout the study, bulls from both groups were kept together in paddocks, a situation that could lead to fights or stress, and possibly interfere with the variables measured.
CONCLUSION
Acupuncture modulates the OS by increasing total antioxidant capacity, promoting a more vigorous total oxidative capacity, reducing albumin levels before and after exercise, and optimizing purine metabolism, which increases body uric acid in bulls under long-term acupuncture and training protocol.
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