Impact of vigorous effort on blood morphological indicators in triathletes participating in the XTERRA Poland 2017 competition Wpływ intensywnego wysiłku na wskaźniki morfologiczne krwi u triathlonistów startujących w zawodach XTERRA POLAND 2017

Purpose: The aim of the study was to determine the influence of participation in the XTERRA Poland 2017 triathlon on blood morphology indicators. Material and methods: The study was performed in a group among 10 triathletes aged 30-40 years. Blood was collected 24 hours before, immediately after , and 16 hours after the competition. Blood morphological indicators were evaluated using the ABXMicros60 analyser. Results: Comparison of the results among the first (24 hours before the competition), second (immediately after the competition), and third assessment (16 hours after the competition) revealed statistically significant changes for WBC [109/l], RBC [1012/l], HGB [g/l], HCT [l/l], MCV [fl] and PLT [109/l]. Conclusions: The intensity of effort in the XTERRA Poland 2017 triathlon competitors was confirmed in the results of blood morphology. Blood counts in those practicing triathlon well characterise the actual scope and direction of exercise changes and allow for the diagnosis of transient adaptive effects. The results of the research confirmed that vigorous physical effort during the triathlon increased leukocyte and platelet counts, but 16 hours after completing the competition, their value was close to baseline level. Most likely, this phenomenon was caused by the intense effort, stress or even eating a large meal before the competition. Analysing the red blood cell system showed a tendency towards decrease in the number of red blood cells, HGB and HCT both after the competition and 16 hours after its completion, which results from increased post-exercise haemolysis or the risk of anaemia. Słowa kluczowe triathlon, morfologia krwi Abstract Cel: Celem pracy jest uzyskanie odpowiedzi w jaki sposób udział w triathlonie XTERRA POLAND 2017 wpłynął na wskaźniki morfologii krwi. Materiał i metody: Badanie przeprowadzono w grupie 10 triathlonistóww wieku 30-40 lat. Krew pobrano 24 godziny przed zawodami, bezpośrednio po zawodach oraz 16 godzin po zakończeniu zawodów. Wykonano oznaczenia wskaźników morfologicznych krwi u triathlonistów w aparacie ABX MICROS 60. Wyniki: Analizując wyniki pomiędzy pierwszym pomiarem (24 godzin przed zawodami) a pomiarem drugim (bezpośrednio po zawodach) i trzecim pomiarem (16 godzin po) zaobserwowano zmiany istotne statystycznie dla: WBC [109/L], RBC [1012/L], HGB [g/L], HCT [L/L], MCV [fL], PLT [109/L].Cel: Celem pracy jest uzyskanie odpowiedzi w jaki sposób udział w triathlonie XTERRA POLAND 2017 wpłynął na wskaźniki morfologii krwi. Materiał i metody: Badanie przeprowadzono w grupie 10 triathlonistóww wieku 30-40 lat. Krew pobrano 24 godziny przed zawodami, bezpośrednio po zawodach oraz 16 godzin po zakończeniu zawodów. Wykonano oznaczenia wskaźników morfologicznych krwi u triathlonistów w aparacie ABX MICROS 60. Wyniki: Analizując wyniki pomiędzy pierwszym pomiarem (24 godzin przed zawodami) a pomiarem drugim (bezpośrednio po zawodach) i trzecim pomiarem (16 godzin po) zaobserwowano zmiany istotne statystycznie dla: WBC [109/L], RBC [1012/L], HGB [g/L], HCT [L/L], MCV [fL], PLT [109/L]. Medical Rehabilitation / Rehabilitacja Medyczna (Med Rehabil) 2020, 24 (2): 20-26 DOI: 10.5604/01.3001.0014.5097 eISSN 1896–3250 © AWF Krakow 21 Wnioski: Stopień zaawansowania wysiłku u zawodników startujących w triatlonie XTERRA POLAND 2017 został potwierdzony w wynikach morfologii krwi. Wskaźniki morfologii krwi u osób uprawiających triathlon dobrze charakteryzują faktyczny zakres i kierunek zmian wysiłkowych oraz pozwalają na diagnostykę przejściowych skutków adaptacyjnych. Wyniki badań potwierdziły, iż intensywny wysiłek fizyczny podczas triathlonu wpłynął na zwiększenie leukocytów i płytek krwi, jednak 16 godzin po zakończeniu zawodów ich wartość była zbliżona do stanu wyjściowego. Najprawdopodobniej zjawisko to zostało spowodowane intensywnym wysiłkiem, stresem czy nawet spożyciem dużego posiłku przed zawodami. Analizując układ czerwonokrwinkowy wykazano tendencję do zmniejszania się liczby krwinek czerwonych, HGB i HCT zarówno po zawodach jak i 16 godzin po zakończeniu zawodów co wynika z nasilonej hemolizy powysiłkowej czy zagrożeniem wystąpienia anemii.


INTRODUCTION
The triathlon is a very demanding discipline. This sport requires high endurance and physical condition of the body from athletes, which is associated with long-term training. Due to the specificity of this discipline, comprehensive preparation is required. Competitors must demonstrate high efficiency in each of the triathlon events (swimming, cycling, running). Preparation for the triathlon is individual in the case of each competitor. Diet, lifestyle and cooperation with a physician are important. A doctor examines parameters such as pulmonary ventilation per minute, blood pressure, cardiac volume per minute, heart rate and stroke volume 1,2 . Haematology also plays an important role in sports medicine. It is used for diagnosis, treatment and prevention. Constant monitoring of haematological indices allows assessment of physical performance, which is of great significance in endurance sports such as the triathlon. The results of all these tests help in choosing an individual training programme for each athlete. Physical effort is of great importance to the processes taking place in the body. It also influences changes in blood morphotic parameters. During activity, blood volume is reduced by up to 15% after just a few minutes of training. Physical training causes increased blood flow to the muscles and decreased blood flow to the internal organs. Blood pressure drops after exercise. The loss of water in the athlete's body is also significant. This affects the composition of the blood. Vigorous exercise causes an initial reduction in the amount of blood plasma, which only returns to the norm following exercise. Changes in blood counts also depend on the training and shape of the athletes. The less they are trained, the greater the changes can be observed [3][4][5] .

STUDY AIM
The aim of the study was to assess changes in blood counts in triathlon competitors. The following research questions were posed:

Characteristics of the studied competitors
The study group comprised men (n = 10) aged 32.8 ±3.1 years from the Active Side of Life Association (in PL: Stowarzyszenie Aktywna Strona Życia) in Kraków, regularly practicing the triathlon with an average training experience of 10 ±4.2 years. The sports level of the participants corresponded to sports class 1 and 2 (national level competitors). The training of triathletes was divided into 3 phases: base, specialisation and direct sports preparation. The triathlon season in Poland begins in May and ends in September. Getting the participants in shape for the competition is associated with the appropriate distribution of training over time. October is the period of de-training. From November to January inclusive, it is time to build the base. The period from February to April regards specialist preparation of athletes. After this specialist preparation, depending on the date of the competition, triathletes begin their direct competition preparation. The first stage of preparation, the base, is mainly work on strengthening the deep muscles and stabilising one's figure, as well as motor preparation. The athletes work to improve technique and motor coordination. Workouts are aerobic, at a moderate intensity level. The second stage of training is specialisation. The main goal of this stage is to improve the athlete's capabilities during exercise in the mixed zone and to prepare for subsequent subliminal work. The main training elements of this stage are perfecting runs and cycling. The specialisation stage introduces interval training with emphasis on the pace of running and cycling. The third stage is working on swimming technique. Interval workouts and running intervals are continued at a subliminal level. The training in the third stage is mostly focused on starting in the competition.

XTERRA POLAND Competition
The athletes participated in the XTERRA POLAND triathlon. The competition took place on August 13, 2017 at Zakrzówek Lagoon. The competitors covered the following distances: 1.5 kilometres (km) of swimming in the Zakrzówek reservoir, 36 km of cycling along the Bielańsko-Tyniecki Landscape Park route and 10 km of running in the Zakrzówek area. The mean (range) temperature during the sports competition was 31.2 ±3.1 C (25-33°C), relative humid-ity 69 ±8% (60-71%), and the dew point was 21 ±3°C (18-25°C). The swimming part was performed in a water tank with a water temperature of 20 ±1°C. At this stage of the race, all participants were equipped with wetsuits. During cycling, the competitors participating in the competition used bicycles with carbon or aluminium frames.
Before the start, the competitors were not instructed on the amount of fluids or energy products to be consumed during the competition in order to avoid the influence of these factors on the final result. However, after the competition, each participant was asked to estimate the amount of fluid consumed at each nutrition point. The average fluid intake of the competitors during the competition was 0.7 ±0.3 l of water and 1.5 ±0.5 l of isotonic drinks.

Measurement of morphological indices
Participants had their blood collected the day before the competition on August 12, 2017, on the day of the competition, immediately after the completion of all events and 16 hours after the competition. Approximately 10 millilitres (ml) of blood were collected into each Vacuette EDTAK2 tube. Blood was drawn in fasting state by a qualified nurse before the competition (Measurement 1) and 16 hours after the competition (Measurement 3) at the Laboratory of Blood Physiology of the University of Physical Edu-cation in Kraków and at the site of the competition (Zakrzówek -Krakow) immediately after the end of the competition (Measurement 2). The collected material was analysed at the Blood Physiology Laboratory of the University of Physical Education in Kraków. The study was approved by the Ethical Committee at the Regional Medical Chamber in Kraków (approv-

Statistical analysis
Data are presented as mean values and standard deviation. Normality of distributions was verified on the basis of the Shapiro-Wilk test. The assumption of sphericity was assessed using the Mauchsley test. Differences in the parameters examined before and after the triathlon were evaluat-ed using analysis of variance (ANO-VA) for repeated variables or, if the assumptions were not met, the ANO-VA test for Friedman's ranks was applied. Tukey's test or, respectively, the post hoc test for Friedman's ANOVA, being part of the Statistica Medical Kit software, were used for post-hoc evaluation. The significance level of α=0.05 was adopted in the analyses, which were performed using the Statistica 12 package (StatSoft®, USA).

RESULTS
Measurement 1 was performed 24 h before the triathlon, Measurement 2 -immediately after completing the triathlon, and Measurement 3 -16 hours following the end of the triathlon.
The mean values and standard deviation (SD) of the obtained results are presented in Table 1.

Analysis of results between Measurement 1 (24 h before competition) and 2 (immediately after the competition)
Analysis of the results between Measurement 1 (24 hours before the competition) and 2 (immediately after its completion) is presented in Table 1 and Figure 1.
Statistically significant changes were found. An increase in the number of WBC by as much as 56% was observed. The number of PLTs also Table 1 Mean values ± standard deviation (SD) for triathletes between Measurement 1 (24 hours before the competition),

(immediately after the competition) and 3 (16 hours after the competition) Index
Measurement 1

Analysis of results between Measurement 2 (immediately after the competition) and 3 (16 h after the competition)
Analysis of the results between the Measurement 2 (immediately after the competition) and 3 (16 hours after the competition) is presented in Table 1 and Figure 2. Statistically significant changes were noted. The number of WBCs decreased by 48.8%, while the number of PLTs also dropped by 20.5%. The number of RBCs fell again by 5.6%. Analysis of the results also showed a decrease in HGB by 10.9%, and HCT by 9.6%. In the analysis of the remaining indices, no statistically significant changes were observed.

Analysis of results between Measurement 1 (24 h before the competition) and 3 (16 h after the competition)
Analysis of the results between Measurement 1 (24 hours before the competition) and 3 (16 hours after the competition) is presented in Table 1 and Figure 3.
A decrease in the value of the majority of examined indices was observed. RBC decreased by 15.1%, HGB value by 16.1%, and HCT by 15.5%. The number of PLTs decreased by 13.7%. Analysis of the remaining indices did not show any statistically significant changes.

DISCUSSION
The aim of this study is to demonstrate the changes taking place in the morphotic elements of the blood among triathletes participating in the 2017 XTERRA POLAND competition before the start, immediately after and 16 hours after the competition. Based on the available liter-

Values of mean ± standard deviation (SD) for WBC among triathletes between Measurement 1 (24 h before the competition), 2 (immediately after the competition) an d 3 (16 h after)
ature and research published to date, there are not many studies in relation to the above-mentioned objective. The majority of morphological indices have been tested for runners and cyclists. According to Che-Hung Liu et al. 6 , the haematological indices: RBC, HGB, HCT and plas-ma were analysed in 19 runners taking part in the Ultramarathon in Taipei. No statistically significant changes were observed. In turn, according to Konstam et al. 7 , an increase in the number of RBCs was demonstrated for the whole group of cyclists participating in training. The number of RBCs decreased 20 minutes after the end of training, but did not drop to baseline level. However, in the group of 20 cyclists who participated in interval training, blood was collected before and immediately after training session, and the results indicated an increase in the number of WBC, RBC and PLT, as well as an increase in HGB and HCT 8 . Huskens et al. 9 tested 92 healthy volunteers taking part in an amateur cycling race who experienced a post-exercise increase in the number of PLT, where the effect was more intense in men than in women. Most likely, this phenomenon was due to the increased reactivity of pro-and anti-inflammatory processes. Natale et al. 10 conducted a study among a group of 14 men competing in a 100-km race. The results of this study before and immediately after the competition were compared, and an increase in the number of WBC was found. The authors suggest that such a change may be caused by damage to the muscle fibres. Galun et al. 11 conducted a trial in which 15 people participated in a 24-hour walk totalling 120 kilometers. After 16 hours of walking, the number of WBCs increased, and after another 16 hours, the number of WBCs fell to the value before the start. The authors of this study also suggest that these changes may be caused by muscle damage. Wells et al. 12 described a group of 6 men and 4 women running a marathon. The participants had their blood drawn 1 week before and immediately after the race, as well as 4, 8 and 24 hours after the competition. Several hours after the race, no changes in HCT, HGB concentration or RBC count were observed. The sample taken immediately after exercise showed an increase in the number of WBCs. In addition, immediately after the competition, the plasma volume was reduced by 8% in women and 13% in men. According to Davidson et al. 13 , in a group of 90 men and 25 women who ran a marathon, blood was drawn before and after the start, and the majority of the marathon runners experienced an increase in platelet counts as well as MCH and MCHC following the run. According to the authors, the probable cause of these changes was loss of water from the body. Other authors described research carried out among a group of 90 teenagers running a marathon 14 . Their blood was collected before, immediately after and 24 hours after the competition. There was a slight increase in the number of WBCs and a slight decrease in HGB and HCT immediately after the competition. The authors suggest that proper training of the athletes resulted in slight changes regarding morphotic elements of the blood 14 .
When analysing all the studied groups of triathletes (between Measurements 1, 2 and 3), a decrease in the RBC number was found, i.e. between Measurement 1 (24 hours before the competition) and 3 (immediately after the competition) by 5.6%, between Measurement 2 (immediately after the competition) and 3 (taken 16 hours after the competition), a decrease in the RBC by 5.5%; and between Measurement 1 (24 hours before the competition) and 3 (16 hours after the competition), a reduction in the RBC amount by 15.1% was noted. For HGB, between Measurement 1 (24 hours before the competition) and 2 (immediately after the competition) there was a decrease by 5.9%, between Measurement 2 (immediately after the competition) and 3 (16 hours after the competition) a decrease by 10.9% was found, and between Measurement 1 (24 hours before the competition) and 3 (16 hours after the competition), there was a reduction by 16.1%.
For HCT, between Measurement 1 (24 hours before the competition) and 2 (immediately after the competition), a reduction of 6.5% was shown, between Measurement 2 (immediately after the competition) and 3 (16 hours after the competition), there was a decrease by 15.5%, and between Measurement 1 (24 hours before the competition) and 3 (16 hours after the competition), a reduction by 15.5% was noted.
For HCT, between Measurement 1 (24 hours before the competition) and 2 (immediately after the competition), there was a decrease by 6.5%, between Measurement 2 (immediately after the competition) and 3 (16 hours after the competition), a decrease by 15.5% was observed, and between the Measurement 1 (24 hours before the competition) and 3 (16 hours after the competition), there was a reduction by 15.5%.
For MCV, between Measurement 1 (24 hours before the competition)

Values of mean ± standard deviation (SD) for PLT among triathletes between Measurement 1 (24 h before the competition), 2 (immediately after the competition) and 3 (16 h after)
and 2 (immediately after the competition) a decrease of 1.2% was shown.
Our results may find confirmation in the research by Rietjens et al. 15 , who also observed that the results of many elite (probably at Olympic distances) triathletes show a reduction in count of RBCs below the lower limit. The above results prove that this phenomenon was caused by post-exercise haemolysis. This is the phenomenon of a decrease in the number of red blood cells with the simultaneous transfer of haemoglobin to the blood plasma. Destruction of erythrocytes is caused by their squeezing through the capillaries and the influence of mechanical factors such as: increased acidity, pressure and body temperature, muscle contractions causing pressure on the vessels, oxidative stress and changes in red blood cell volume. Another cause of exercise-induced haemolysis is too low blood sugar (hypoglycaemia). In athletes, this is a temporary phenomenon caused by not consuming enough carbohydrates before strenuous exercise. Reduction in the number of erythrocytes among the studied triathletes may also be associated with excessive iron loss 16 . Currently, there are no established levels of iron requirements for athletes performing any specific disciplines. In people who train, iron resources are mainly excreted in urine and, to a lesser extent, in sweat. Dietary mistakes and even mechanical injuries should also be taken into account, which, through regular and prolonged foot-strike haemolysis on the ground, causes the destruction of red blood cells contained in the blood vessels of the lower limbs 3,4,17 . The etiology of this intravascular haemolysis has been linked to associated mechanical injuries running, hence the synonym "foot-strike haemolysis". Others have postulated that haemolysis may also be associated with other non-traumatic factors 18,19 . Researchers suggest that the decrease in haemoglobin may be due to haemolysis, which is a mechanism for destroying red blood cells during and after physical activity [20][21][22][23] . In addition, the decrease in haemoglobin may be due to haemodilution, which is associated with endurance training and occurs in athletes due to the increase in plasma volume 24 . In the research by scientists from Pavia Medical School, it has been proven that HCT reduction may occur in the case of high physical activity immediately after a long break from training 25 .
By analysing all the studied groups of triathletes (between Measurements 1, 2 and 3) for WBC, an increase by as much as 56% was shown between Measurement 1 (24 hours before the competition) and 2 (immediately after the competition), and between Measurement 2 (immediately after the competition) and 3 (16 hours after the competition), a decrease by 48.8% was indecated. The authors of studies conducted so far in athletes have noted that fluctuations in the number of white blood cells in the blood may be dependent on various factors. The increase in WBC counts is most often explained by a decrease in plasma volume during increased activity 26 . Scientists suggest that the number of white blood cells may also be affected by the consumption of a large meal before training, the vigorous effort itself, and even stress before the start or during the competition, which is manifested by an increase in adrenaline, noradrenaline or cortisol. This is confirmed in the research by Tota et al. 27 . According to Maron et al. 28 , increasing the number of leukocytes in the body confirms the principle that training improves the immunity of athletes. However, in a 4-year study on Spanish triathletes, it has been demonstrated that white blood cell counts are within the normal limits during both the pre-competitive and competitive periods 29 . However, 16% of triathletes in the Australian Institute of Sport study were neutropenic, while 5% showed signs of monocytopenia, respectively 30 . According to Philip et al 31 , neutropenic individuals are generally more susceptible to bacterial infections that can occur after inappropriate treatment or the occurrence of injuries to the skin. The cause of neutropenia is unclear. This may be due to the exercise-induced apoptosis of the neutrophil and the consequent shorter lifespan of the neutrophils.
When analysing all the studied groups of triathletes (between Measurements 1, 2 and 3), an increase in the number of PLTs was found, i.e. between Measurement 1 (24 hours before the competition) and 2 (immediately after the competition) by 7.8%, between Measurement 2 (directly after the competition) ) and 3 (16 hours after the competition), the number of PLTs decreased by 20.5%, and between Measurement 1 (24 hours before the competition) and 3 (16 hours after the competition) the number of PLTs decreased by 13.7%. One of the possible reasons for the increase in platelet count after vigorous training may be the loss of plasma in the blood after such exercise, and the decrease in PLT (within normal limits) in triathletes after competition as a return to baseline values. Vigorous exercise increases the activity of platelets and clotting factors, which ensures blood fluidity 31 . Górski 32 mentions an increase in the number of platelets after a single effort, but immediately states that the reason for this is difficult to explain. In other studies it has been shown that vigorous training increases blood clotting activity 33 . The triathlon has even been described as a high-risk sport, predisposing the athlete's body to blood clots 34 . In yet other studies on this subject, it has been demonstrated that vigorous exercise during a triathlon does not affect the risk mentioned here 35 .

CONCLUSIONS
The results obtained in the course of this research allow formulation of the following conclusions: 1. Blood counts in athletes practicing the triathlon well-characterise the actual scope and direction of exercise changes and allow for the diagnosis of temporary adaptive effects. 2. The results of the research confirmed that vigorous physical exercise during the triathlon increased leukocyte and platelet counts, however, 16 hours after the end of the competition, their values were similar to those obtained at baseline.