Physiological Reports. 2023;11:e15801. 
 
  | 1 of 12
https://doi.org/10.14814/phy2.15801
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1 | INTRODUCTION
An important practical challenge in modern sports is to 
obtain objective information on an athlete's physical per-
formance and fitness during the training process. One of 
the most informative and popular methods for assessing 
the functional status of athletes is to study the anaerobic 
threshold (AT) (Ghosh, 2004; Solli et al., 2017). The AT 
represents the transition to an anaerobic mechanism of 
energy exchange when performing physical activity at 
submaximal and maximum power. The production and 
excretion of blood lactate are in equilibrium at the AT 
(MLSS, maximal lactate steady state), and a significant 
increase in the lactate level is observed (i.e., the lactate 
threshold) then the athlete transitions through the AT 
zone (Abreu et al.,  2016; Gobatto et al.,  2001). Despite 
numerous studies, the metabolic basis of AT has not 
been fully established. Studies of AT in humans are often 
Received: 20 March 2023 | Revised: 10 August 2023 | Accepted: 14 August 2023
DOI: 10.14814/phy2.15801  
O R I G I N A L  A R T I C L E
Lactate thresholds and role of nitric oxide in male rats 
performing a test with forced swimming to exhaustion
Natalya Potolitsyna
 |   Olga Parshukova |   Nadezhda Vakhnina |   
Nadezhda Alisultanova |   Lubov Kalikova |   Anastasia Tretyakova |   
Alexey Chernykh |   Vera Shadrina |   Arina Duryagina |   Evgeny Bojko
Institute of Physiology of Kоmi Science 
Centre of the Ural Branch of the 
Russian Academy of Sciences, FRC 
Komi SC UB RAS, Syktyvkar, Russia
Correspondence
Evgeny Bojko, Institute of Physiology 
of Kоmi Science Centre of the Ural 
Branch of the Russian Academy of 
Sciences, FRC Komi SC UB RAS, 
Syktyvkar, Russia.
Email: boiko60@inbox.ru
Funding information
Institute of Physiology of Kоmi Science 
Centre of the Ural Branch of the Russian 
Academy of Sciences, FRC Komi SC UB 
RAS, FUUU- 2022- 0063, Grant/Award 
Number: 1021051201877- 3
Abstract
The present study assessed a complex of biochemical parameters at the anaerobic 
threshold (AT) in untrained male Wistar rats with different times to exhaustion 
(Tex) from swimming. The first group of rats was randomly divided into six sub-
groups and subjected to a swimming test to exhaustion without a load or with a 
load of 2%– 10% of body weight (BW). In the first group, we established that for 
untrained rats, the load of 4% BW in the swimming to exhaustion test was optimal 
for endurance assessment in comparison with other loads. The second group of 
rats went through a preliminary test with swimming to exhaustion at 4% BW and 
was then divided into two subgroups: long swimming time (LST, Tex > 240 min) 
and short swimming time (SST, Tex < 90 min). All rats of the second group per-
formed, for 6 days, an experimental training protocol: swimming for 20 min each 
day with weight increasing each day. We established that the AT was 3% BW in 
SST rats and 5% BW in LST rats. The AT shifted to the right on the lactate curve 
in LST rats. Also, at the AT in the LST rats, we found significantly lower levels of 
blood lactate, cortisol, and NO.
K E Y W O R D S
anaerobic threshold, exhaustion, lactate biochemical indices, rats, swimming test
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided 
the original work is properly cited.
© 2023 Institute of Physiology of Komi Science Center of the UB of the RAS. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological 
Society and the American Physiological Society.
2 of 12 |   
POTOLITSYNA et al.
complicated by the infeasibility of complete control over 
the experiments, and in- depth deep, long- term and high- 
intensity studies of the AT phenomenon are not always 
possible in humans (Cholewa et al.,  2014). Therefore, 
to understand many fundamental aspects of exercise 
physiology, research using an adequate animal model is 
needed. Rodents are among the most popular and eas-
ily available laboratory animals for use in such models. 
Compared to human studies, working with animals al-
lows for greater control and regulation of environmental 
conditions and food intake. These studies make it possible 
to collect different types of tissues and perform a number 
of experimental manipulations that cannot be performed 
in humans (Cholewa et al., 2014). The results of studies 
on rats are traditionally projected onto humans (Voltarelli 
et al., 2002). There is evidence that rats adequately reflect 
the human response to physical activity based on the main 
biochemical parameters of blood (Goutianos et al., 2015). 
However, rodents and humans may not have similar reac-
tions to physical exercise (Greek et al., 2012; Rice, 2012).
The AT in animals is determined by various changes in 
blood biochemistry (Faude et al., 2009):
1. when animals achieve the reference lactate level in the 
blood (4 mmol/L) during exercise (Heck et al.,  1985);
2. when the first increase in blood lactate levels above the 
baseline level is detected (onset of blood lactate accu-
mulation) (Farrell et al., 2018; Faude et al., 2009);
3. when a notable bend (sharp change in curvature) in 
the lactate curve caused by massive lactate accumu-
lation during physical load is observed (Contarteze et 
al., 2008; Gobatto et al., 2001);
4. when undergoing a short period of submaximal load to 
induce hyperlactemia before starting the test with an 
increasing load (lactate minimum test, LMT) (Voltarelli 
et al., 2002).
In our opinion, fixed or precalculated lactate levels 
might not take into account considerable interindividual 
differences and differences between various lactate ana-
lyzers (Faude et al., 2009). Therefore, the most objective 
AT assessment method is by observing a sharp change 
(bend) in the lactate curve during exercise with an increas-
ing load. Then, the AT is determined as the physical load 
equivalent to MLSS; in other words, the highest exercise 
load at which the lactate levels in blood do not change sig-
nificantly (Contarteze et al., 2008; Faude et al., 2009; Heck 
et al., 1985). In the case of individual lactate curves, visual 
curve assessment is used. For groups, we believe a statisti-
cally significant increase in blood lactate levels in compar-
ison with the previous physical load is more appropriate.
It is also important to monitor other biochemical 
indicators that characterize the level of physiological 
adaptations of the body during exercise. Indicators in 
sports physiology, such as cortisol, catecholamines, glu-
cose, urea, and other metabolites, are most often used (De 
Araujo et al., 2016; Halson & Jeukendrup, 2004). Various 
studies have also proposed other markers of AT, includ-
ing blood catecholamines (Davies et al., 1974) and saliva 
amylase (Chicharro et al., 1999). We showed that elite ath-
letes (cross- country skiers) had a nitric oxide- dependent 
(NO- dependent) mechanism for regulating lactate levels 
during aerobic exercise, especially when working at the 
AT. In our previous work, we revealed a positive relation-
ship between NO metabolites and blood lactate at the AT, 
which was reversed at maximum load. This observation 
suggests the existence of an adaptive mechanism for reg-
ulating the level of lactate on the AT in highly qualified 
cross- country skiers (Parshukova et al., 2022).
The time of onset of the anaerobic (lactate) thresh-
old largely depends on the duration of the load (Pa-
padopoulos et al.,  2006; Roecker et al.,  1998; Weyand 
et al., 1994), physical fitness level (Støren et al., 2014; Tanji 
& Nabekura,  2019), intensity of movement (Wakayoshi 
et al., 1993), testing methods (Contarteze et al., 2008; De 
Araujo et al., 2016), and swimming patterns in swimmers 
(Dos Reis et al., 2018). Perhaps due to the influence of a 
large number of factors, research results are often contra-
dictory. Therefore, doubts are expressed that the AT (as an 
indirect method for determining aerobic endurance) re-
flects the optimal intensity of training, especially for elite 
athletes (Bosquet et al., 2002). Determination of the du-
ration of exercise to exhaustion is a direct method for de-
termining aerobic endurance and provides more complete 
and reliable information (Beck et al., 2014). There are few 
studies of the AT with measurements of the maximum du-
ration of physical activity to exhaustion in untrained rats.
Therefore, the hypothesis of the current study was that 
the characteristics of the AT will be different in rats dis-
playing different times to exhaustion (Tex) while perform-
ing the same swimming tests in the same conditions. The 
purpose of our study was to assess the biochemical param-
eters at the AT in untrained rats with different endurance 
levels performing a swimming test to exhaustion.
2 | MATERIALS AND METHODS
2.1 | Experimental animals
Our study used male Wistar rats (n = 60), aged 8 weeks at 
the beginning of the experiment, weighing 250– 300 g. Rats 
were housed in a room with a temperature of 21 ± 1°C 
and a controlled photoperiod (12 h of light/12 h of dark-
ness) on a standard vivarium diet, with access to water 
ad libitum. The protocol of the study was reviewed for 
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POTOLITSYNA et al.
compliance with the “Rules of the European Convention 
for the Protection of Vertebrates Used for Experimental 
and Other Scientific Purposes” and approved by the local 
Ethics Committee of the Institute of Physiology of the 
Komi Research Center of the Ural Branch of the Russian 
Academy of Sciences.
2.2 | Adaptation to water
Prior to experiments, all animals were adapted to water, 
with a subsequent recovery for 14 days (Brito et al., 2015). 
Adaptation consisted of keeping animals in shallow water 
at a temperature of 31 ± 1°C for 30 min for 14 days. The 
purpose of the adaptation period was to familiarize the 
animals with testing conditions while avoiding physical 
training adaptations.
2.3 | Experimental procedures
The swimming sessions were performed in cylindrical 
water tanks (height 60 cm × diameter 45 cm) with a water 
temperature of 30 ± 1°C and indoor air temperature of 
22 ± 1°C. The rats swam in individual tanks with desatu-
rated water. After weighing the animals, a metal weight 
of the necessary mass was affixed to the base of the tail 
using elastic nontraumatic tape. A stopwatch was started 
when the animals were placed in the water. Exhaustion 
was defined as the animals being incapable of staying on 
the water surface, the loss of symmetrical movements dur-
ing swimming, or the animals remaining underwater for 
more than 10 s (Chimin et al., 2013). No deaths occurred 
during or after exercise in any of the animal subgroups.
2.4 | Method for assessing physical 
activity to exhaustion
Rats (n = 50) were randomly divided into six groups. Each 
group performed a swimming test to exhaustion with one 
of the following swim loads (SL): without load (SL0, n = 9) 
or with a load of 2% (SL2, n = 8), 4% (SL4, n = 8), 6% (SL6, 
n = 10), 8% (SL8, n = 8), and 10% (SL10, n = 7) of body 
weight (BW). After the animals achieved exhaustion, they 
were immediately removed from the water tank, dried, 
anesthetized, and sacrificed via decapitation.
2.5 | Method of measuring the AT
To determine the AT, we used the method of Gobatto 
et al. (2001) with modifications (Figure 1). A total of 10 
rats were used in the study. All rats had previously gone 
through the test to exhaustion with a load of 4% BW. 
Based on this test, all rats were divided into two groups: 
rats with a long swimming time (time to exhaustion, 
Tex > 240 min, LST, n = 5) and rats with a short swimming 
time (Tex < 90 min, SST, n = 5). After this test, all rats were 
allowed to recover for 1 week. One rat from each group 
was decapitated to assess the recovery of the blood bio-
chemistry parameters after exertion to exhaustion (recov-
ery control after swimming to exhaustion). The remaining 
rats were subjected to physical activity to assess the AT. 
FIGURE 1 
The protocol for 
anaerobic threshold evaluation in rats.
1st day 
0% BM 
swim 20 min 
2nd day 
2% BM 
swim 20 min 
3rd day 
3% BM 
swim 20 min 
4th day 
4% BM 
swim 20 min 
5th day 
5% BM 
swim 20 min 
6th day 
6% BM 
swim 20 min 
Swimming test to exhaustion with 4% of body weight 
Recovery control after 
swimming 
to exhaustion 
n=2 
LST 
Swimming, long time 
Tex>240 min, n=4 
Separation into groups by duration of the swimming 
SST 
Swimming, short time 
Tex<90 min, n=4 
Anaerobic threshold test 
One week recovery 
Sacrifce, blood sampling 
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POTOLITSYNA et al.
Each animal participated in six experimental tests over 
6 days with a 24- h interval between tests. Each test con-
sisted of continuous swimming for 20 min with a load of 
0%, 2%, 3%, 4%, 5%, or 6% BW in a tank filled with de-
saturated water at a temperature of 31 ± 1°C. Immediately 
after performing the exercise, the rats were removed from 
the water, and their tails were heated with warm water 
and dried with a towel. Blood samples were taken from 
the tail vein using a syringe and placed in heparinized Ep-
pendorf tubes (1.5 mL capacity). After performing the test 
with last load (6% BW), the rats were sacrificed.
We determined the AT by observing a sharp increase 
in blood lactate using the lactate curve obtained by per-
forming physical load tests (Faria et al.,  2021; Faude 
et al.,  2009). The increase between the two consecutive 
loads was required to be statistically significant. The AT 
in this case was the lower of the two consecutive physi-
cal load intensities, the one after which the increase was 
observed.
2.6 | Blood samples and analyses
The lactate levels in the blood samples collected from 
the caudal veins were determined using a lactate ana-
lyzer (Accutrendplus, Roche Diagnostics GmbH). Blood 
(mixed, arteriovenous) after decapitation was collected 
in tubes containing heparin and centrifuged at 2400 rpm 
for 10 min at 4°C. The samples were frozen and stored at 
−40°C. Plasma levels of lactate (Sentinel Diagnostics), 
urea, glucose, and cortisol (all from Human GmbH) were 
measured using immunoenzyme assays (ChemWell 2900 
biochemical analyzer). Levels of NO in the plasma were 
measured using the Griess reaction by evaluating stable 
metabolites of NO, including nitrites (NO2) and nitrates 
(NO3), which were merged together as an index (NOx). 
These methods were previously described (Parshukova 
et al., 2020, 2022).
2.7 | Statistical analysis
All values are expressed as the means ± SD. Statistical 
analyses were performed using Statistica 8.0 (Statsoft). 
The statistical significance of differences between the SST 
and LST groups was estimated using the Mann– Whitney 
(U) test. For comparisons of multiple independent groups, 
we used the Kruskal– Wallis test. For comparisons be-
tween workload groups within a corresponding group, we 
used Friedman ANOVA and Kendall's coefficient of con-
cordance. When necessary, the Newman– Keuls post hoc 
comparison test was used. The statistical significance level 
was set at p < 0.05.
3 | RESULTS
The times of swimming to exhaustion and the parameters 
of blood biochemistry in rats performing the Tex test with 
various loads are presented in Table 1.
The total swimming time of rats performing the test to ex-
haustion expectedly decreased with increasing tail weights 
and showed significant variation between individual rats. 
The greatest variation between minimal and maximal 
times of swimming was observed in the group swimming 
with weights of 0%– 6% BW. All rats within each group were 
clearly divided by the duration of swimming into LST and 
SST groups (Table 2). The duration of swimming did not ex-
ceed 4 min in the SL8 and SL10 groups, and these values did 
not differ significantly between the SL8 and SL10 groups.
The concentrations of glucose and lactate in the blood 
of rats after exercise to exhaustion generally tended to 
be higher as the weight of the attached load increased. 
However, the picture became clearer when the rats were 
separated within each group according to the duration of 
swimming. Lactate and glucose levels in LST rats were sig-
nificantly lower than those in SST rats.
The average values of the other parameters did not show 
regularities or trends that corresponded with differences in 
load weights or the duration of swimming in groups with-
out separation. However, the differences became apparent 
when the groups were divided into SST and LST groups. 
The concentrations of cortisol were higher in SST rats than 
in LST rats, and the concentrations of urea were lower. 
Levels of nitric oxide metabolites were also notable. The 
NOx index was significantly higher in LST rats than in SST 
rats. The significant differences in NOx values primarily de-
pended on the levels of NO3, and the levels of NO2 showed 
no significant differences between groups.
3.1 | Anaerobic threshold
Based on the time of swimming to exhaustion, all rats 
were divided into rats that swam for more than 240 min 
(LST, n = 4) and rats that swam for less than 90 min (SST, 
n = 4). The dynamics of lactate and the level of the AT in 
these groups are shown in Figure 2.
The dynamics of lactate in venous blood differed in 
these two groups of rats. In the SST group, we observed a 
sharper increase in this indicator, and starting at the load 
of 3% BW, we registered a statistically significant differ-
ence from the first data point. However, the most signifi-
cant increase in lactate levels was detected at a load of 4% 
BW. Further testing of rats at loads of 5%– 6% BW did not 
show significant changes in lactate levels compared with a 
load of 4% BW. Therefore, the AT in SST rats was assessed 
at the level of 3% BW. The lactate curve in LST rats had a 
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POTOLITSYNA et al.
TABLE 1 Swimming time and biochemical parameters of blood in untrained male rats (all group) after performing swimming tests to exhaustion with different loads.
Work load groups
Swimming time, 
min
Glucose, 
mmol/L
Lactate, 
mmol/L
Cortisol, ng/
mL
Urea, mmol/L
NOх, μmol/L
NO2, μmol/L
NO3, μmol/L
SL0
419.7 ± 229.1
4.9 ± 1.5
4.1 ± 2.5
21.0 ± 3.3
8.0 ± 3.5
37.0 ± 21.8
5.2 ± 2.3
31.8 ± 21.7
*SL6, SL8, SL10
*SL10
*SL8
*SL10
SL2
148.6 ± 130.2
5.2 ± 2.3
6.8 ± 4.4
19.8 ± 8.3
5.3 ± 1.8
27.7 ± 13.5
9.1 ± 2.1
18.5 ± 13.9
*SL4
*SL4
*SL4
*SL4
*SL4
SL4
89.1 ± 86.9
4.7 ± 2.4
5.7 ± 4.8
23.7 ± 5.5
5.1 ± 2.0
40.9 ± 8.2
3.9 ± 0.8
37.0 ± 7.8
*SL2, SL10
*SL2, SL8
*SL10
*SL2, SL8
*SL2
*SL2, SL6, SL8, SL10
SL6
59.9 ± 83.1
6.0 ± 2.8
11.0 ± 6.7
23.0 ± 5.0
6.3 ± 1.4
22.1 ± 13.0
6.4 ± 3.0
15.7 ± 15.1
*SL0
*SL4
SL8
3.0 ± 1.1
8.6 ± 2.9
14.2 ± 1.4
16.1 ± 2.4
5.0 ± 2.0
12.3 ± 2.5
5.6 ± 1.4
6.8 ± 2.7
*SL0
*SL0, SL4
*SL4
*SL4
SL10
2.2 ± 0.4
9.8 ± 1.8
13.3 ± 1.3
16.5 ± 3.8
2.8 ± 1.1
14.1 ± 2.2
6.6 ± 1.9
7.5 ± 1.7
*SL0
*SL0, SL4
*SL0, SL4
*SL4
Note: The values were expressed as means ± SD.
*The differences between workload groups are significant at <0.05 for the Kruskal– Wallis ANOVA test.
TABLE 2 Swimming time and biochemical parameters of blood in short swimming time (SST) and long swimming time (LST) male rats after performing swimming tests to exhaustion 
with different loads.
Work load 
groups
Swimming time, min
Glucose, mmol/L
Lactate, mmol/L
Cortisol, ng/mL
Urea, mmol/L
NOх, μmol/L
NO2, μmol/L
NO3, μmol/L
SST
LST
SST
LST
SST
LST
SST
LST
SST
LST
SST
LST
SST
LST
SST
LST
SL0
168.0 ± 24.0
545.5 ± 163.5
4.9 ± 2.3
5.0 ± 1.3
7.2 ± 1.6
2.5 ± 0.7
24.5 ± 2.2
19.2 ± 2.0
7.2 ± 5.5
8.5 ± 2.6
23.0 ± 18.7
42.0 ± 8.2
5.8 ± 2.1
5.2 ± 2.4
17.3 ± 19.9
36.8 ± 19.5
*SL6
# 
*SL6
*SL6# 
# 
SL2
48.0 ± 14.2
282.7 ± 58.0
6.4 ± 2.4
3.5 ± 0.2
10.3 ± 0.5
2.1 ± 0.6
25.6 ± 5.5
15.4 ± 3.7
4.3 ± 0.7
6.6 ± 2.1
20.0 ± 3.5
37.9 ± 11.1
10.0 ± 1.1
8.0 ± 2.9
10.1 ± 2.4
29.9 ± 15.4
*SL6
# 
# 
# 
# 
# 
SL4
29.2 ± 7.4
189.0 ± 48.8
5.8 ± 2.5
2.9 ± 0.5
8.3 ± 4.1
1.3 ± 0.2
26.0 ± 5.9
19.9 ± 1.0
4.7 ± 2.1
5.7 ± 2.0
37.0 ± 8.1
47.3 ± 2.2
3.6 ± 0.7
4.2 ± 1.0
33.4 ± 7.6
43.0 ± 3.1
*SL6# 
*SL6
*SL6
*SL6 # 
*SL6
*SL6
SL6
8.4 ± 6.8
180.0 ± 2.0
7.0 ± 7.4
3.0 ± 2.8
15.0 ± 1.7
1.5 ± 0.3
26.2 ± 2.9
19.1 ± 3.7
5.7 ± 1.3
7.7 ± 0.5
15.4 ± 8.7
37.8 ± 1.6
6.8 ± 3.6
5.5 ± 0.4
8.6 ± 12.0
32.3 ± 1.8
*SL0, SL2
*SL4# 
# 
*SL0, SL4
*SL4 # 
# 
*SL4
# 
*SL4
# 
Note: The values were expressed as means ± SD.
*The differences between workload groups (SST, LST accordingly) are significant at <0.05 for the Kruskal– Wallis ANOVA test.
#The differences between SST and LST groups are significant at <0.05 for the Mann– Whitney (U) test.
6 of 12 |   
POTOLITSYNA et al.
flatter slope than the SST curve and was characterized by 
a more gradual increase in the concentration of lactate in 
the blood with the increasing weight of the attached loads. 
The most significant increase in this indicator occurred 
when the rats performed the test at a load of 6% BW. There-
fore, the AT for the LST group was set at a load of 5% BW.
The concentrations of lactate, glucose, and cortisol in 
arteriovenous blood collected after decapitation of rats 
were higher in SST rats than in LST rats (Table 3).
Urea levels in the SST and LST groups showed no signif-
icant differences. The NOx levels were significantly higher 
in SST rats than in LST rats. The levels of NO2 and NO3 
did not reveal significant differences in the arteriovenous 
blood of either group of rats. However, we observed higher 
values of both metabolites in SST rats than in LST rats.
4 | DISCUSSION
Only a few studies have described swimming Tex in rats 
(Beck & Gobatto, 2013; Travassos et al., 2018; Venditti & 
Di Meo, 1996). Some groups used arbitrary loads without 
describing the reasoning for weight choice (Travassos 
et al.,  2018; Venditti & Di Meo,  1996). Other research-
ers used a method in which the load to exhaustion was 
based on a preliminary calculation of AT. For example, 
Beck et al. (2014) used the minimum lactate level on the 
“U- shaped” lactate curve obtained after the blood LMT as 
the AT. The loads that the rats were subjected to in this 
test were then used as benchmarks for the swimming to 
exhaustion experiment. However, the method of calculat-
ing AT significantly affects the interpretation of the re-
sults and makes it impossible to compare the results with 
those of other studies. Therefore, we evaluated Tex in rats 
swimming to exhaustion using various loads in the first 
stage of our study (Table 1). Predictably, the increase in 
the load weight affected the duration of the swim and re-
duced it from several hours to several minutes. However, 
the swimming time also strongly depended on the endur-
ance of the rats. For most load weights, Tex was divisible 
into two groups (Table  2): SST and LST. Loads heavier 
than 8% BW were too heavy for untrained rats (Gobatto 
FIGURE 2 Blood lactate levels in tail vein blood of rats from SST and LST groups. SST— swimming for short time, LST— swimming for 
long time. The values are expressed as means ± SD. The statistical significance of differences between SST and LST groups was estimated 
using Mann– Whitney (U) test; p- values were considered significant at *p < 0.05. The statistical significance of differences between workload 
groups within corresponding group (SST or LST accordingly) was estimated using the Friedman ANOVA and Kendall coefficient of 
concordance, and is shown in italics. When necessary, the Newman– Keuls post hoc comparison test was used. Statistical significance is 
indicated in comparison with the specified load weight at #p < 0.05.
p=0.0021
p=0.0019
#0
#0,2,3
#0,2,3
#0,2,3
*
*
*
*
#0,2
#0,2,3,4
2
3
4
5
6
7
8
0
1
2
3
4
5
6
7
Lactate, mmol/l
Load weight (% of body mass)
SST
LST
TABLE 3 Biochemical parameters of arteriovenous blood of sacrificed rats after performing the last load (6% BW).
Groups
Lactate, 
mmol/L
Glucose, 
mmol/L
Cortisol, ng/
mL
Urea, 
mmol/L
NOx, 
μmol/L
NO2, 
μmol/L
NO3, 
μmol/L
SST
10.3 ± 4.4
9.5 ± 0.8
34.2 ± 9.3
4.0 ± 1.0
28.1 ± 1.8
9.5 ± 1.6
18.6 ± 3.1
LST
6.3 ± 0.7
8.5 ± 0.7
19.1 ± 3.2
4.5 ± 1.1
24.3 ± 1.8
8.0 ± 1.1
16.3 ± 2.9
p- valueSST- LST
0.049
0.126
0.049
0.512
0.049
0.126
0.512
Note: Data are presented as the means ± SD. p- valueSST- LST— The statistical significance of differences between short swimming time (SST) and long swimming 
time (LST) groups was estimated using the Mann– Whitney (U) test. p < 0.05 are shown in bold.
   | 7 of 12
POTOLITSYNA et al.
et al., 2001), and no differences were observed in this case. 
Despite the lack of similar studies in the literature, there 
are fragmentary results on rats swimming to exhaustion. 
For example, Venditti and Di Meo (1996) showed a Tex 
close to our results (294 ± 32 min) in untrained rats (swim-
ming to exhaustion) with a load of 2% of BW. Beck and 
Gobatto (2013) reported that rats swam 108 ± 46 min with 
a load weight of 5% BW, which is less than the load of 
4%– 6% BW in our study. A greater variation in Tex was 
described in Travassos et al. (2018).
Rats in the Travassos et al. (2018) study swam at a load 
of 6% BW from 3 to 22 min and were divided into low per-
formance (Tex = 3– 12 min) and high performance (Tex = 12– 
22 min) groups depending on the time of exhaustion. 
Notably, the authors excluded all rats that swam longer than 
22 min. The variability in the duration of swimming with 
the load of 6% BW was larger in our study, and therefore, the 
division into endurance groups was different. All rats that 
swam less than 21 min with a load of 6% BW were included 
in the SST group, and rats that swam approximately 180 min 
were included in the LST group. Therefore, the study of un-
trained rats using various load weight protocols allows com-
parison of metabolic changes in LST and SST animals and 
identifies the best load weight for the test.
We showed that the blood biochemistry changes in dif-
ferent load weights and the time of swimming to exhaus-
tion also significantly differed between the LST and SST 
rats. The SST rats had higher levels of lactate, glucose and 
cortisol, and the LST rats had higher levels of urea and 
nitric oxide. Notably, these differences became more ob-
vious with increases in the load weight while swimming.
Despite all of the rats being of the same age, being 
housed in the same conditions, being fed the same diet, 
and having no previous training, some rats showed higher 
inborn physical endurance. These results are fully con-
sistent with the hypothesis that genetics is an important 
determinant of the response to physical activity (Koch 
et al.,  2005) and may affect the features of anatomy 
(Britton & Koch,  2001), pulmonary function (Kirkton 
et al., 2009), insulin response (Schwarzer et al., 2021), and 
the predominant type of skeletal muscle fibers (Abernethy 
et al., 1990). It was expected that the metabolism in rats 
with different physical endurance would be different. A 
significant increase in blood lactate at the lower loads in 
SST rats reflects lower aerobic capacities, and hypoxia oc-
curs faster in these rats under high- intensity physical exer-
cise. Howlett et al. (2009) showed that SST rats had VO2max 
and oxygen transfer in skeletal muscles that was 50% lower 
than those in higher endurance rats, despite having higher 
absolute muscle mass. The maintenance of glucose levels 
in hypoxia is provided primarily by glycolysis and glycog-
enolysis (Brooks & Mercier,  1994; Emhoff et al.,  2013). 
With sufficient oxygen supply during prolonged physical 
exercise, there is a higher fat utilization. The increased 
contribution of lipids to energy metabolism makes it pos-
sible to significantly increase endurance during physical 
exercise (Brooks & Mercier,  1994; Nosaka et al.,  2009). 
There are more data on a more complex system of regu-
lation of lipid metabolism depending on the intensity of 
exercise (Lyudinina et al., 2018; Romijn et al., 1993).
Physical exercises also stimulate increases in cortisol 
levels. This hormone plays a significant role in acceler-
ating lipolysis, ketogenesis, and proteolysis (Del Corral 
et al., 1998). The level of cortisol increases in proportion to 
the intensity of exercise, but the final level depends on the 
total duration of exercise. Moderate-  and high- intensity 
exercises increase the levels of circulating cortisol. In con-
trast, low- intensity exercise does not lead to an increase 
in cortisol levels (Del Corral et al., 1998; Hill et al., 2008).
The levels of cortisol did not show significant differ-
ences with respect to Tex in our study. However, cortisol 
levels were higher in SST rats than in LST rats, especially 
at loads of 2%– 4% BW. Perhaps, this result occurred be-
cause of the different behaviors of rats when performing 
the test and the levels of individual stress. Glucocorticoids 
in rodents are often used as biomarkers of stress, with 
cortisol reacting faster during severe acute stress, unlike 
corticosterone, which is associated more with adaptation 
during chronic stress (Gong et al., 2015).
NO is another metabolite that allows adaptation to 
significant physical exercise (Oral, 2021). It is a signaling 
molecule with a wide variety of effects in mammals, the 
most well- known of which is the regulation of local vaso-
motor tone and resistance to microvascular flow (Baskurt 
et al., 2011). Skeletal muscles of rodents contain unusu-
ally high concentrations of nitrates compared to blood and 
other tissues, which indicates the high importance of ni-
tric oxide for their body (Piknova et al., 2015). Nitric oxide 
has an extremely short half- life of only a few milliseconds 
in biological tissues, and it is important that it is con-
stantly produced at its sites of effect (Jones et al., 2021). 
Experimental data indicate that physical exercises lead to 
an increase in the enzymatic synthesis of nitric oxide and 
activation of the associated vascular control mechanisms 
(Baskurt et al., 2011). We previously found a positive cor-
relation between nitrogen oxide and lactate at the AT and 
a negative correlation at maximum load in elite cross- 
country skiers possessing high endurance (Parshukova 
et al., 2020). The higher level of NOx we obtained in LST 
rats, but not SST rats, is consistent with these findings. It 
characterizes a more adequate response of the vascular 
bed in response to physical exercise and allows better con-
trol of vascular tone for a longer time. The increase in NOx 
levels in LST rats was observed primarily due to the NO3 
fraction. Under conditions of normal and increased oxy-
gen consumption by tissues, NO is formed enzymatically 
8 of 12 |   
POTOLITSYNA et al.
via the oxidation of L- arginine, and the final metabolite of 
this process is primarily NO3 (Cubrilo et al., 2011).
Our study showed that the use of a load weight of 4% 
BW was the most informative for studying the level of 
physical endurance in untrained rats. At this load weight 
and a background of a wide Tex range, the rats showed 
significant changes in most of the biochemical indices as-
sessed in our study, which included the most informative 
indices in relation to the problem under discussion.
4.1 | Anaerobic threshold
There are a large number of methods for studying the 
lactate threshold in rats (Faude et al., 2009). The choice 
of method depends on the research goals. However, it 
is also important to consider the natural abilities of rats 
and our capacity to project the results on humans in the 
future. The optimal method for determining the AT in 
rats is swimming with increasing load. Several studies 
of the lactate threshold in swimming rats found that 
loads of 4%– 6% BW were more often used (Contarteze 
et al., 2008; Gobatto et al., 2001; Voltarelli et al., 2002). 
For example, Gobatto et al. (2001) showed that the AT 
corresponded to 6% BW at a blood lactate concentra-
tion of 5.5 mmol/L. Another study established the AT 
at a weight of 4.0% BW and a lactate level of 5.2 mmol/L 
(Abreu et al., 2016). Similar lactate values were shown 
at an AT with a load of 4.5% BW (Zhouab et al., 2018). 
However, these studies do not mention individual en-
durance variation in rats. The results of our study 
showed that this characteristic of laboratory animals 
may significantly shift the AT to the left or the right on 
the lactate curve. The ATs in SST and LST rats were 3% 
BW and 5% BW, respectively. The lactate curve of SST 
was less flat than that of LST. For increasing endur-
ance, it is generally recognized that a shift of the lactate 
curve to the right is interpreted as an increase in physi-
cal performance, and a shift to the left is considered a 
deterioration in endurance (Abreu et al.,  2016; Faude 
et al.,  2009). The lactate concentration at the AT was 
also different and higher in SST rats than in LST rats 
(5.8 mmol/L vs. 5.2 mmol/L). A lower lactate level at the 
end of physical exercise in LST rats may be associated 
with a lower rate of lactate accumulation and/or a lower 
metabolic clearance of lactate (Donovan & Brooks, 1983; 
Yang et al., 2020). Higher endurance augments capaci-
ties for lactate production, disposal, and clearance (Mes-
sonnier et al., 2013). Our data are generally consistent 
with the results of other studies, although no data on the 
AT when swimming in rats with a load below 4% were 
found. However, rats with higher endurance were likely 
included for various reasons in the described studies 
(Abreu et al.,  2016; Contarteze et al.,  2008; Gobatto 
et al., 2001; Voltarelli et al., 2002; Zhouab et al., 2018).
Biochemical data from the arteriovenous rat blood as-
says (Table  3) also demonstrated significant differences 
between the SST and LST rats. Because the collection of 
arteriovenous blood occurred within 3– 5 min after the last 
collection of blood from the caudal vein, the data on lactate 
from arteriovenous blood showed higher values relative to 
lactate from the caudal vein. The most significant increase 
was observed in SST rats, which reflected their lower re-
covery abilities compared to LST animals. Glucose, corti-
sol, and NOx levels were also significantly higher in the 
SST rats. This pattern of blood biochemistry generally 
characterizes more significant rearrangements and higher 
stress levels in SST rats than in LST rats at a similar level 
of physical exercise. Notably, the increase in NOx levels 
in this test occurred due to an increase in nitrites (NO2), 
unlike in swimming to exhaustion. Under hypoxic con-
ditions, NO2 is an alternative source of nitric oxide syn-
thesis (Gladwin et al., 2000; Schulman & Hare, 2012) and 
participates in adaptation to hypoxia caused, for example, 
by physical exertion (Gladwin et al., 2000). The current 
understanding of nitrite- dependent mechanisms of adap-
tation to hypoxia is based on data on the reduction of NO2 
by oxygen- dependent and hypoxic nitrite reductase (Glad-
win & Kim- Shapiro, 2008).
NO is a mediator of skeletal muscle function and af-
fects cellular respiration and contractility. In working skel-
etal muscle, inhibition of NOS improves the economy of 
muscle contraction, decreases the outflow of lactate from 
the muscles, and reduces the oxygen cost (Krause & Van 
Etten, 2005). Thiol groups, reactive metal ions in the pro-
teins' active centers, can interact with NO, which leads to 
various responses and further biological events in skeletal 
muscles. NO- mediated reactions inhibit heme- containing 
proteins, such as cytochrome C oxidase, thus interfering 
with the function of cytochrome C oxidase in cell respi-
ration (Borutaite & Brown, 1996). Inhibition of this en-
zyme and of the sarcoplasmic reticulum Ca2+- ATPase in 
fast- twitch and slow- twitch skeletal muscle fibers by NOS- 
generated NO may also lead to inhibition of mitochondrial 
respiration in skeletal muscle (Klebl et al., 1998). More-
over, aconitase and respiratory chain complex I can also 
be targeted by NO (Clementi et al., 1998). NO is crucial 
for the activation and inhibition of ryanodine receptors 
(RyRs) (Stamler & Meissner, 2001), which play a decisive 
role in the release of Ca2+ into the cytosol and therefore 
in muscle excitation and contraction (Mazzone & Carme-
liet, 2008). In our experimental work, we have shown that 
elite athletes (cross- country skiers) have an NO- dependent 
mechanism for regulating lactate levels during aerobic ex-
ercise, especially when working at the AT. In particular, at 
the AT, we have revealed a positive relationship between 
   | 9 of 12
POTOLITSYNA et al.
NOx (nitric oxide metabolites) and blood lactate, with that 
relationship being reversed at maximum load. This obser-
vation suggests the existence of an adaptive mechanism 
for regulating lactate levels at the AT in highly qualified 
cross- country skiers (Parshukova et al., 2022).
Therefore, our data provide a new understanding of 
the role of NO- dependent mechanisms in the phenome-
non of AT.
5 | CONCLUSION
We found that the level of individual endurance signifi-
cantly affected the AT in untrained rats. The AT in SST 
rats and 5% BW in LST rats. These groups also had dif-
ferent blood biochemistry profiles at the AT and after 
swimming to exhaustion. There was a shift in the AT to 
the right side on the lactate curve in the zone of the AT 
in LST rats compared to SST rats, and the levels of lactate, 
glucose, cortisol, and NOx were lower. At the end of the 
exercise to exhaustion, SST rats had higher blood levels 
of lactate, glucose, and cortisol, and LST rats had higher 
levels of urea and NOx.
AUTHOR CONTRIBUTIONS
Evgeny Bojko, Nadezhda Vakhnina, Natalya Potolit-
syna conceives and designed the experiments; Natalya 
Potolitsyna, Nadezhda Vakhnina, Nadezhda Alisyl-
tanova, Lubov Kalikova, Anastasia Tretyakova, Alexey 
Chernykh, Vera Shadrina, Arina Duryagina performed 
experiments; Natalya Potolitsyna, Olga Parshukova, 
Lubov Kalikova analysed data; Natalya Potolitsyna, 
Olga Parshukova, Lubov Kalikova, Nadezhda Vakhnina 
interpreted results of experiments; Natalya Potolitsyna 
prepared figures; Natalya Potolitsyna, Alexey Chernykh 
drafted 
manuscript; 
Natalya 
Potolitsyba, 
Evgeny 
Bojko, Olga Parshukova, Nadezhda Vakhnina, Alexey 
Chernykh edited and revised manuscript; Natalya Po-
tolitsyna, Evgeny Bojko, Olga Parshukova, Nadezhda 
Vakhnina, Alexey Chernykh approved final version of 
manuscript.
ACKNOWLEDGMENTS
The study was conducted within the framework of the 
research work of the Institute of Physiology of Kоmi Sci-
ence Centre of the Ural Branch of the Russian Academy of 
Sciences, FRC Komi SC UB RAS, FUUU- 2022- 0063 (No. 
1021051201877- 3).
FUNDING INFORMATION
This research did not receive any specific grant from fund-
ing agencies in the public, commercial, or not- for- profit 
sectors.
CONFLICT OF INTEREST STATEMENT
No conflicts of interest, financial or otherwise, are de-
clared by the authors.
ETHICS STATEMENT
The Ethics Committee of the Institute of Physiology of the 
Russian Academy of Sciences, Syktyvkar approved the 
experimental design and protocol of the study. The study 
was performed in accordance with the ethical standards 
as laid down in the 1964 Declaration of Helsinki and its 
later amendments.
ORCID
Natalya Potolitsyna 
 https://orcid.
org/0000-0003-4804-6908 
Evgeny Bojko 
 https://orcid.org/0000-0002-8027-898X 
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How to cite this article: Potolitsyna, N., 
Parshukova, O., Vakhnina, N., Alisultanova, N., 
Kalikova, L., Tretyakova, A., Chernykh, A., 
Shadrina, V., Duryagina, A., & Bojko, E. (2023). 
Lactate thresholds and role of nitric oxide in male 
rats performing a test with forced swimming to 
exhaustion. Physiological Reports, 11, e15801. 
https://doi.org/10.14814/phy2.15801

Lactate thresholds and role of nitric oxide in male rats performing a test with forced swimming to exhaustion.
Potolitsyna, Natalya,Parshukova, Olga,Vakhnina, Nadezhda,Alisultanova, Nadezhda,Kalikova, Lubov,Tretyakova, Anastasia,Chernykh, Alexey,Shadrina, Vera,Duryagina, Arina,Bojko, Evgeny