Individual differences in temperature perception: evidence of common processing of sensation intensity of warmth and cold

The longstanding question of whether temperature is sensed via separate sensory systems for warmth and cold was investigated by measuring individual differences in perception of nonpainful heating and cooling. Sixty-two subjects gave separate ratings of the intensity of thermal sensations (warmth, cold) and nociceptive sensations (burning/stinging/pricking) produced by cooling (29 degrees C) or heating (37 degrees C) local regions of the forearm. Stimuli were delivered via a 4 x 4 array of 8 mm x 8 mm Peltier thermoelectric modules that enabled test temperatures to be presented sequentially to individual modules or simultaneously to the full array. Stimulation of the full array showed that perception of warmth and cold were highly correlated (Pearson r = 0.83, p < 0.05). Ratings of nonpainful nociceptive sensations produced by the two temperatures were also correlated, but to a lesser degree (r = 0.44), and the associations between nociceptive and thermal sensations (r = 0.35 and 0.22 for 37 and 29 degrees C, respectively) were not significant after correction for multiple statistical tests. Intensity ratings for individual modules indicated that the number of responsive sites out of 16 was a poor predictor of temperature sensations but a significant predictor of nociceptive sensations. The very high correlation between ratings of thermal sensations conflicts with the classical view that warmth and cold are mediated by separate thermal modalities and implies that warm-sensitive and cold-sensitive spinothalamic pathways converge and undergo joint modulation in the central nervous system. Integration of thermal stimulation from the skin and body core within the thermoregulatory system is suggested as the possible source of this convergence.     

Interactions between Chemical and Thermal Cutaneous Stimuli: Inhibition (Counterirritation) and Integration

Methyl salicylate, a commonly used chemical counterirritant, was applied topically to the forearm to determine whether a nonpainful chemical irritation could inhibit the perception of another (weaker) chemical irritation. In the first experiment, sensations of irritation (burning and stinging) produced by a 10% solution of methyl salicylate were significantly attenuated when a 15% solution of the same chemical was applied to the opposite forearm. In the second experiment, neither the perception of warmth nor the heat pain threshold was affected by application of 10% or 15% methyl salicylate to a site 10 cm from the thermal stimulus. Inhibition did, however, occur in the opposite direction: Chemical irritation was reduced after the thermal stimulus reached a painful level. In the third experiment, a 15% solution of methyl salicylate was applied immediately adjacent to the thermal stimulus, with the result that ratings of warmth intensity increased rather than decreased, and perceived irritation was again attenuated following a painful heat stimulus. Overall, the results indicate that (1) chemical counterirritation can occur at nonpainful levels; (2) the resulting inhibition is confined to the nociceptive system; and (3) when the nociceptive and warmth systems are activated together, the tendency is toward integration rather than inhibition.     

Interactions between chemical and thermal cutaneous stimuli: inhibition (counterirritation) and integration.

Methyl salicylate, a commonly used chemical counterirritant, was applied topically to the forearm to determine whether a nonpainful chemical irritation could inhibit the perception of another (weaker) chemical irritation. In the first experiment, sensations of irritation (burning and stinging) produced by a 10% solution of methyl salicylate were significantly attenuated when a 15% solution of the same chemical was applied to the opposite forearm. In the second experiment, neither the perception of warmth nor the heat pain threshold was affected by application of 10% or 15% methyl salicylate to a site 10 cm from the thermal stimulus. Inhibition did, however, occur in the opposite direction: Chemical irritation was reduced after the thermal stimulus reached a painful level. In the third experiment, a 15% solution of methyl salicylate was applied immediately adjacent to the thermal stimulus, with the result that ratings of warmth intensity increased rather than decreased, and perceived irritation was again attenuated following a painful heat stimulus. Overall, the results indicate that (1) chemical counterirritation can occur at nonpainful levels; (2) the resulting inhibition is confined to the nociceptive system; and (3) when the nociceptive and warmth system are activated together, the tendency is toward integration rather than inhibition.     

Site-dependent and subject-related variations in perioral thermal sensitivity

The Marstock method of limits was used to obtain thresholds for detection of cooling, warming, cold pain and heat pain for 34 young adults, upon eight spatially matched sites on the left and right sides of the face, the right ventral forearm and the scalp. Male and female subjects were tested by both a male and a female experimenter. Neither the experimenter nor the gender of the subject individually influenced the thresholds. The thermal thresholds varied greatly across facial sites: sixfold and tenfold for cool and warmth, respectively, from the most sensitive sites on the vermilion to the least sensitive facial site, the preauricular skin. Warm thresholds were 68% higher than cool thresholds, on average, and 12% higher on the left compared to the right side of the face. The mean cold pain threshold increased from 21.0°C on the hairy upper lip to 17.8°C on the preauricular skin. Sites on the upper lip were also most sensitive to noxious heat with pain thresholds of 42–43°C. The scalp was notably insensitive to innocuous and noxious changes in temperature. For the sensations of nonpainful cool and warmth, the more sensitive a site, the less the estimates of the thresholds differed between subjects. In contrast, for heat pain, the more sensitive a site, the more the estimates differed between subjects. Subjects who were relatively more sensitive to cool tended to be relatively more sensitive to warmth. Subjects’ sensitivities to nonpainful cool and warmth were less predictive of their sensitivities to painful cold and heat, respectively. Short-term within-subject variability increased with the magnitude of the thresholds. The lower the threshold, the more similar were repeated measurements of it, within a 5–25?s period.     

Is there modal opponency in the thermal senses?

1. 1.The sensations evoked by pairs of distinct thermal stimuli applied to the back of the hand were studied in 17 volunteer subjects. Four stimulus combinations were used; neutral-cold (NC), neutral-neutral (NN), neutral-warm (NW), and cold-warm (CW).

2. 2.The subjects were first asked to estimate the magnitude of the thermal sensations evoked by the thermal stimuli. On average, the four pairs were reported as increasing magnitude in the following order: NC, CW, NN, and NW, seeming to suggest that the subjects experienced the cold-warm combination as a composite sensation of cold and warmth intermediate between pure cold and pure warmth.

3. 3.When asked only to detect the presence of a cold stimulus, the subjects performed as well for the CW combination as for the CN combination. This second result indicates that the reported composite magnitude of CW does not result from a true opponency of cold and warmth but from a cognitive combination of distinct sensations of cold and warmth.

Respirometry and swimming dynamics of the giant australian cuttlefish,sepia apama (mollusca,cephalopoda)

Swimming dynamics of the giant Australian cuttlefish, Sepia apama, were investigated using swimtunnel respirometry. Relationships between jet pressure, fin frequency, swimming speed and oxygen consumption were defined. Laboratory calibration of swimming parameters is necessary to allow estimates of swimming costs in the field.

Jet pressure was the best predictor of oxygen consumption with an averaged equation of MO₂?=?722 (jet pressure)?+?107?r ²?=?0.51. Individually, fin frequency and jet pressure correlated highly to swimming speed, but due to the complicated usage of finning and jetting, the correlation between swimming speed and oxygen consumption was weaker. Cuttlefish were not optimal swimtunnel subjects and could not swim at high speeds for extended periods. At 15°C and a swimming speed of 0.06?m?s^?1, the gross cost of transport was calculated to be 10.1?kg^?1?m?^?1, with a net cost of 4.1?kg^?1?m^?1.     

Synthetic heat at mild temperatures

"Synthetic heat", also known as the heat grill illusion, occurs when contact with spatially adjacent warm and cold stimuli produce a sensation of "heat". This phenomenon has been explained as a painful perception that occurs when warm stimulation inhibits cold-sensitive neurons in the spinothalamic tract (STT), which in turn unmasks activity in the pain pathway caused by stimulation of C-polymodal nociceptors (CPNs). The "unmasking model" was tested in experiment 1 by combining warm (35-40°C) and cool ( &#83 27°C) stimuli that were too mild to stimulate CPNs. After discovering that these temperatures produced nonpainful heat, experiment 2 was designed to determine whether heat could be induced when near-threshold cooling was paired with mild warmth, and whether lowering the base temperature for cooling would increase the noxious (burning, stinging) components of heat for fixed cooling steps of 1-3°C. Cooling by just 1°C from a base temperature of 33°C led to reports of heat on more than 1/3 of trials, and cooling by just 3°C evoked heat on 75% of trials. Lowering the base temperature to 31 or 29°C increased reports of heat and burning but did not produce significant reports of pain. Perception of nonpainful heat at such mild temperatures indicates either that cold-sensitive nociceptors with thresholds very similar to cold fibers innervate hairy skin in humans, or that heat can result from integration of warm fiber and cold fiber activity, perhaps via convergence on nonspecific (e.g., WDR) neurons in the STT.     

Double thermal transitions of type I collagen in acidic solution

Contributed equally to this work. To further understand the origin of the double thermal transitions of collagen in acidic solution induced by heating, the denaturation of acidic soluble collagen was investigated by micro-differential scanning calorimeter (micro-DSC), circular dichroism (CD), dynamic laser light scattering (DLLS), transmission electron microscopy (TEM), and two-dimensional (2D) synchronous fluorescence spectrum. Micro-DSC experiments revealed that the collagen exhibited double thermal transitions, which were located within 31–37?°C (minor thermal transition, T _s?～?33?°C) and 37–55?°C (major thermal transition, T _m?～?40?°C), respectively. The CD spectra suggested that the thermal denaturation of collagen resulted in transition from polyproline II type structure to unordered structure. The DLLS results showed that there were mainly two kinds of collagen fibrillar aggregates with different sizes in acidic solution and the larger fibrillar aggregates (T _p2?=?40?°C) had better heat resistance than the smaller one (T _p1?=?33?°C). TEM revealed that the depolymerization of collagen fibrils occurred and the periodic cross-striations of collagen gradually disappeared with increasing temperature. The 2D fluorescence correlation spectra were also applied to investigate the thermal responses of tyrosine and phenylalanine residues at the molecular level. Finally, we could draw the conclusion that (1) the minor thermal transition was mainly due to the defibrillation of the smaller collagen fibrillar aggregates and the unfolding of a little part of triple helices; (2) the major thermal transition primarily arose from the defibrillation of the larger collagen fibrillar aggregates and the complete denaturation of the majority part of triple helices.     

Sleep pattern is associated with adipokine levels and nutritional markers in resident physicians

Shift work and long hours of work are common in medical training and have been associated with a higher propensity for developing nutritional problems and obesity. Changes in leptin and ghrelin concentrations – two hormones that contribute importantly to the central regulation of food intake – are poorly described in this population. The aim of this study was to identify possible negative associations between sleep patterns, nutritional status and serum levels of adipokines. The study included 72 resident physicians (52 women and 20 men) who underwent the following assessments: nutritional assessment (3-day dietary recall evaluated by the Adapted Healthy Eating Index), anthropometric variables, fasting metabolism, physical activity level, sleep quality and sleepiness. Resident physicians with poor sleep quality reported greater weight gain after the beginning of residency (5.1 and 3.0?kg, respectively; p?=?0.01) and higher frequency of abnormal waist circumference (44.2 and 17.6%, respectively; p?=?0.04) than those with better sleep quality. Mean ghrelin concentration was greater in volunteers with poor sleep quality (64.6?±?67.8 and 26.2?±?25.0?pg/mL, respectively; p?=?0.04). Women identified as having excessive daytime sleepiness had lower levels of leptin (9.57?±?10.4?ng/mL versus 16.49?±?11.4?ng/mL, respectively; p?=?0.03) than those without excessive sleepiness. Furthermore, correlations were found between hours of additional work per week and: intake of cereals, bread and pasta (r?=?0.22, p?=?0.01); intake of servings of fruits (r?=??0.20; p?=?0.02) and beans (r?=??0.21; p?=?0.01); and global score for Adapted Healthy Eating Index (r?=??0.23; p?=?0.008; Table 3). The sleep quality total score correlated with servings of beans (r?=??0.22; p?=?0.01) and servings of oils (r?=?0.23; p?=?0.008). Significant correlations were found between mean of time of sleep and servings of cereals, bread and pasta (r?=?0.20; p?=?0.02), servings of meat (r?=??0.29; p?=?0.02) and cholesterol levels (r?=?0.27; p?=?0.03). These observations indicate that sleep patterns and long working hours of resident physicians are negatively associated with biological markers related to central food control, the lipid profile, cholesterol levels and eating healthy foods. These factors may predispose these shift workers to become overweight and develop metabolic disorders.     

The thermal physiology of Stenopelmus rufinasus and Neohydronomus affinis (Coleoptera: Curculionidae), two biological control agents for the invasive alien aquatic weeds,Azolla filiculoides and Pistia stratiotes in South Africa

Water lettuce, Pistia stratiotes, and red water fern, Azolla filiculoides, are floating aquatic macrophytes that have become problematic in South Africa. Two weevils, Neohydronomus affinis and Stenopelmus rufinasus, are successful biological control agents of these two species in South Africa. The aim of this study was to investigate the thermal requirements of these two species to explain their establishment patterns in the field. Laboratory results showed that both weevils are widely tolerant to cold and warm temperatures. The critical thermal minima (CT_min) of N. affinis was determined to be 5.58?±?0.31°C and the critical thermal maxima (CT_max) was 44.52?±?0.27°C, while the CT_min of S. rufinasus was 5.38?±?0.33°C and the CT_max?44.0?±?0.17°C. In addition, the lower lethal temperatures were ?9.85?±?0.06°C for N. affinis and ?6.85?±?0.13°C for S. rufinasus, and the upper lethal temperatures were 42.7?±?0.85°C for N. affinis and 41.9?±?2.52°C S. rufinasus. Using the reduced major axis regression method, the development for N. affinis was described using the formula y?=?12.976x?+?435.24, while the development of S. rufinasus was described by y?=?13.6x?+?222.45. These results showed that S. rufinasus develops twice as fast as N. affinis. Using these formulae and temperature data obtained from the South African Weather Service, N. affinis was predicted to complete between 4 and 9 generations per year in South Africa, while S. rufinasus was predicted to complete between 5 and 14 generations per year around the country. These results suggest that both species should not be limited by cold winter, nor warm summer temperatures, and should establish throughout the ranges of the weeds in South Africa.     

Characteristics of attention-related body sensations. Temporal stability and associations with measures of body focus,affect, sustained attention,and heart rate variability

Aim: This study investigated the temporal stability and correlates of attention-related body sensations that emerge without external stimulation during rest and due to focused attention on a body part.

Materials: To assess attention-related body sensations, participants were asked to focus on a freely chosen body area with closed eyes, and had to report whether the sensation of that area had changed. Self-report questionnaires were used to assess various aspects of body focus (body awareness, body responsiveness, somatosensory amplification, subjective somatic symptoms), and positive and negative affectivity. Previous experiences in body–mind therapies were also measured. PEBL Continuous Performance Test was used to assess sustained attention. Heart rate variability scores were based on a 3-minute long resting heart rate measurement.

Methods: Fifty-eight university students (22.3?±?3.95 years; 34 females) participated in the study. The stability of attention-related body sensations was measured 8?weeks later on a randomly chosen sub-group (n?=?28).

Results: Attention-related body sensations showed a mediocre temporal stability (r_ρ?=?0.47, p?=?0.012). People reporting attention-related body sensations showed significantly higher body awareness, somatosensory amplification, and resting heart rate; and marginally higher somatic symptoms. No relation was found with body–mind practice, body responsiveness, positive and negative affect, the vagal component of heart rate variability, and performance in the sustained attention task.

Conclusion: Attention-related sensations are relatively stable over time. They are connected to some, but not to all of the aspects of body focus. Further studies are needed to elaborate the influencing stable and situational factors.     

Site-dependent and subject-related variations in perioral thermal sensitivity

The Marstock method of limits was used to obtain thresholds for detection of cooling, warming, cold pain and heat pain for 34 young adults, upon eight spatially matched sites on the left and right sides of the face, the right ventral forearm and the scalp. Male and female subjects were tested by both a male and a female experimenter. Neither the experimenter nor the gender of the subject individually influenced the thresholds. The thermal thresholds varied greatly across facial sites: sixfold and tenfold for cool and warmth, respectively, from the most sensitive sites on the vermilion to the least sensitive facial site, the preauricular skin. Warm thresholds were 68% higher than cool thresholds, on average, and 12% higher on the left compared to the right side of the face. The mean cold pain threshold increased from 21.0 degrees C on the hairy upper lip to 17.8 degrees C on the preauricular skin. Sites on the upper lip were also most sensitive to noxious heat with pain thresholds of 42-43 degrees C. The scalp was notably insensitive to innocuous and noxious changes in temperature. For the sensations of nonpainful cool and warmth, the more sensitive a site, the less the estimates of the thresholds differed between subjects. In contrast, for heat pain, the more sensitive a site, the more the estimates differed between subjects. Subjects who were relatively more sensitive to cool tended to be relatively more sensitive to warmth. Subjects' sensitivities to nonpainful cool and warmth were less predictive of their sensitivities to painful cold and heat, respectively. Short-term within-subject variability increased with the magnitude of the thresholds. The lower the threshold, the more similar were repeated measurements of it, within a 5-25 s period.     

Comparison of unpleasant and pain thresholds of thermal stimuli in the orofacial regions: a psychophysical study using quantitative sensory testing in healthy young men

Abstract

Purpose/Aim: To gain a better understanding of the psychophysics of thermal pain perception in a clinical setting, this study investigated whether thermal thresholds of unpleasantness are different from pain thresholds of cold and heat stimuli. Of particular interest was the relationship between unpleasantness and pain thresholds for cold vs heat stimuli.

Material and methods: Thirty healthy male volunteers (mean age 26.1?years, range 23 to 32?years) participated. Thermal detection, cold pain (CPT) and heat pain (HPT) thresholds were measured at 5 trigeminal sites by the method of limits using quantitative sensory testing (QST), followed by cold unpleasant (CUT) and heat unpleasant (HUT) thresholds.

Results: The temperatures at which individuals first reported thermal sensations as unpleasant or painful substantially differed among subjects. CUT exhibited a higher mean value with less variability than CPT, and HUT presented a lower mean than HPT (p?<?.001). As with CPT, CUT did not show any significant difference between the test sites. On the other hand, HUT, like HPT, exhibited site differences (p?<?.001). There was moderate correlation between CUT and CPT, whereas HUT and HPT were strongly correlated. The relationship between unpleasant and pain thresholds of cold vs heat stimuli was significantly different even when controlling for test site variability (p?<?.001).

Conclusion: These findings indicate that unpleasant and pain thresholds to thermal stimuli differ in healthy young men. Of particular note is the distinct relationship of unpleasant and pain thresholds of cold vs heat stimuli, revealing the thermal difference in temperature transition from unpleasantness to pain.     

Effect of alcohol consumption on peripheral blood Alu methylation in Korean men

Alcohol use disorders (AUD) are defined as alcohol abuse and alcohol dependence, which create a substantial public health problem worldwide. To date, no therapeutic can effectively solve these problems. They are complex diseases characterized by both genetic and environmental factors. DNA methylation can act as a downstream effector of environmental signals and account for multi-factorial nature of the disease. Global DNA methylation of peripheral blood cells has recently been proposed as a potential biomarker for disease risk. Alu elements host one-quarter of CpG dinucelotides in the genome to function as proxies for global DNA methylation. In this study, we evaluated the Alu methylation in the peripheral blood DNA of healthy volunteers and AUD patients using the pyrosequencing technology. The Alu methylation level is significantly higher in AUD compared to healthy controls (23.4?±?1.6 versus 22.1?±?1.0, t?=?7.83, p?<?0.0001). Moreover, significant correlation was found between Alu methylation and alcohol use disorders identification test score (r?=?0.250, p?<?0.0001), alcohol problem (r?=?0.294, p?<?0.0001), and life position (r?=?–0.205, p?=?0.0005). Overall, these novel findings indicate that alcohol-related increase in Alu methylation might play a complex role in the etiology and pathogenesis of AUD. Further studies are required to elucidate the mechanisms underlying this relationship.     

Effects of apelin-13 in mice model of experimental pain and peripheral nociceptive signaling in rat sensory neurons

Objective: Apelin-13 is an endogenous peptide with potential analgesic action, although the sites of its analgesic effects remain uncertain and the results are even controversial with regard to its pain modulating action. This study evaluated the possible pain-modulating action of peripherally administered apelin-13 using heat-induced, withdrawal latency to the thermal stimuli, acute pain model in mice. Involvement of peripheral mechanisms was tested, by using the intracellular calcium concentrations as a key signal for nociceptive transmission, in cultured rat dorsal root ganglion (DRG) neurons. Methods: DRG neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium-sensitive dye Fura-2 acetoxymethyl ester (1?µM). Intracellular calcium responses in individual DRG neurons were quantified by ratiometric calcium imaging technique. Results: Peripheral injection of a single dose of apelin-13 (100?mg/kg and 300?mg/kg) significantly decreases the latency to painful stimuli in a dose and time-dependent manner (p?<?0.01, p?<?0.05, respectively, n?=?8 each). Apelin-13 (0.1?µM and 1?µM) did not produce a significant effect on cytoplasmic Ca²⁺ ([Ca²⁺]_i) responses, evoked by membrane depolarization, in cultured rat DRG neurons. Conclusion: Together these results indicate that apelin-13 can cause increased pain sensitivity after peripheral administration, but this effect does not involve calcium mediated signaling in primary sensory neurons.     

Cutaneous thermal thresholds of tropical indigenes residing in Japan

The purpose of this study was to examine the deacclimatization of the cutaneous thermal sensations of tropical indigenes residing in temperate climates. Tropical indigenes (n=13) who were born and raised in tropics but had resided in Japan for 5–61 months participated in this study, along with temperate indigenes (n=11). Their cutaneous thermal thresholds for warm, cool, hot, and cold sensations were measured in 12 body regions using a thermal stimulator controlled by a Peltier element and a push button switch. Subjects pressed the button-switch as soon as they perceived a feeling of being ‘slightly warm’, ‘slightly cool’, ‘hot’, or ‘cold’ from a neutral thermal state. Our results showed that: (1) among the tropical indigenes, no significant relationship was found between the duration of their stay in Japan and their cutaneous thermal thresholds; (2) the tropical indigenes were, on average, 3.3, 3.5, 4.2, and 7.3 °C less sensitive to warm, hot, cool, and cold sensations, respectively, than the temperate indigenes (P<0.05); and (3) the inter-threshold sensory zones between cutaneous warmth and coolness, and hot and cold sensations were wider among the tropical indigenes than among the temperate indigenes. It was concluded that the nature of the heat acclimatization of the cutaneous thermal thresholds for the tropical indigenes was retained despite their residence in a temperate climate for up to 61 months, indicating that they had more blunted perceptions of both warming and cooling than the temperate indigenes.     

Differences in circadian patterns between rural and urban populations: An epidemiological study in countryside

The physiological pattern of the sleep–wake cycle is influenced by external synchronizing agents such as light and social patterns, creating variations in each individual’s preferred active and sleep periods. Because of the demands of a 24-h working society, it may be imperative for many people to adapt their sleep patterns (physiologically) to their daily activities. Therefore, we analyzed the difference in sleep patterns and chronobiological parameters between an essentially rural farming and urban small-town populations. We studied 5942 subjects (women, 67.1%, N?=?3985; mean age, 44.3?±?13.1 years), from which the chronotype, circadian sleep pattern, and period of light exposure were collected using the Munich Chronotype Questionnaire (MCTQ). A structured questionnaire was also made for collection of social and demographic information. Compared with the urban population (N?=?3427, 57.7%), the rural population (N?=?2515, 42.3%) presented a more predominantly early sleep pattern, as determined by the mid-sleep phase (rural: 2.26?±?1.16; urban: 3.15?±?1.55; t-test, p?<?0.001). We also found less social jetlag (rural: 0.32; urban: 0.55; Mann–Whitney U test, p?<?0.001) and higher light-exposure (rural: 9.55?±?2.31; urban: 8.46?±?2.85; t test, p?<?0.001) in the rural population. Additionally, the rural population presented a higher prevalence of psychiatric disorders (rural: 156, 6.20%; urban: 165, 4.80%; Chi-square, p?<?0.05), and a lower prevalence of metabolic diseases (rural: 143, 5.70%; urban: 225, 6.60%; Chi-square, p?<?0.05). The significant difference in sleep parameters, chronotype, and light exposure between groups remained after multivariate regression analysis (r^2?=?0.41, F?=?297.19, p?<?0.001, β?=?1.208). In this study, there was a significant difference between the rural and urban populations in natural light exposure and sleeping patterns. Because of agricultural work schedules, rural populations spend considerable time outside that is an obligation related to work schedules. Our results emphasize the idea that latitude may not be the main factor influencing individual circadian habits. Rather, circadian physiology adapts to differences in exposure to light (natural and artificial) as well as social and work schedules.     

Generation of reactive oxygen species during cryopreservation may improve Lilium × siberia pollen viability

During biotic and abiotic stress in plants, reactive oxygen species (ROS) may play two very different roles: high ROS concentrations can exacerbate damage, whereas low concentrations can activate defense responses. The aim of this study was to investigate the relationship between ROS generation and pollen viability after cryopreservation. ROS generation was detected from ‘Siberia’ (Lilium?×?siberia) pollen using flow cytometry with 2′,7′-dichlorodihydrofluorescein diacetate as a fluorescent probe. Pollen viability was determined by 2,3,5-triphenyltetrazolium chloride staining. ROS generation was slightly increased by rapid cooling (26.13?±?4.74 vs. 15.80?±?2.30 for fresh pollen) and significantly increased by vitrification (49.74?±?1.43; P?<?0.01). Pollen viabilities after rapid cooling and vitrification were significantly increased (58.88?±?3.76% and 70.35?±?2.90%, respectively) over that of fresh pollen (46.65?±?1.61%; P?<?0.01). No significant differences in ROS generation were associated with cold acclimation at different temperatures before rapid cooling. However, sharp decreases in viability were observed with cold acclimation at 4°C and ?20°C relative to rapid cooling without acclimation (P?<?0.01). We observed nonsignificant decreases in ROS generation among vitrification treatments that omitted different steps and a significant decrease when the unloading step was omitted (P?<?0.05). Pollen viabilities were significantly reduced when the loading or dehydration steps were omitted (P?<?0.01). No significant differences were observed in ROS generation or pollen viability among the treatments when 200 U/ml catalase was added to different solutions used in the vitrification process. Comprehensive analysis of all data indicated a positive correlation between ROS generation and pollen viability (r?=?0.651, P?<?0.001). Therefore, increasing ROS generation during cryopreservation may improve the viability of ‘Siberia’ pollen.