ANESTHETIC EFFECTS OF MS-222 ON SCHIZOTHORAX O’CONNORI LLORD IN TWO SIZE RANGES

LIU Yan-Chao ， LIU Hai-Ping LIU Shu-Yun， LI Xi-Feng and SONG Xiao-Guang

(1. Institute of Fisheries Science， Tibet Academy of Agricultural and Animal Husbandry Sciences， Lhasa 850002， China;

2. Tibet Agriculture and Animal Husbandry University， Nyingchi 860000， China;3. Tanggu Township， Linzhou County， Lhasa 850000， China)

Abstract: This study examined anesthetic behaviors of Schizothorax o’connori Llord in two size ranges as induced by MS-222， to acquire useful information on anesthesia of said highland fish species in out-of-water activities (surgery， labeling， measurement， etc.). Test results indicated that for both large (25.0±1.5) cm and small-sized (14.8±2.3) cm specimens， respiration rates showed no significant change in anesthetic stages Ⅰto Ⅲ， and only begun to significantly decline after entering stage Ⅳ. The effective concentrations of MS-222 were 150—180 mg/L and 150 mg/L for two sizes， respectively. The 150—180 mg/L caused large specimens to enter anesthetic stage Ⅳ within 5min， and recovered within 5min; the 150 mg/L caused small specimens to enter stage Ⅳ within 5min， and recovered within 7min; both doses ensured a 100% survival rate after immersion for 20min. For large specimens anesthetized at 180 mg/L for 5min and exposed to air for 0—15min，there was no significant difference in recovery times (P＞0.05); for small specimens anesthetized at 150 mg/L for 5min and exposed to air for 0—15min， there were significant differences in recovery (P＜0.05).

Key words: MS-222; Anesthesia; Schizothorax o’connori Llord

Schizothorax o’connoriLlord is a highland coldwater fish species under the genusSchizothoraxHeckel， Schizothoracinae subfamily， Cyprinidae family. Its survival requires specific conditions in the aquatic environment， and will react strongly to any environmental disturbance. As rapid socioeconomic development takes place in Tibet，S. o’connoripopulations have become threatened by human activities like overfishing[1]， competition from invasive species[1]， and the species’ own slow growth[2]and late sexual maturity[3]. In recent years， researchers had explored areas related toS. o’connoriincluding its artificial fertilization[4]， large-scale domestication[5]， population dynamics[6]， fetal development[4]， and biological features[3]. However， no work has been done on its potential physiological changes and stress responses to stimuli during transportation， which have the potential to hinder its preservation[7]. From this perspective， the prevention of injury or death for local fish species under anesthesia can be a subject of remarkable value.

At present， anesthetics have been widely used in scientific procedures on fish species including surgery[8]， measurement， and labeling[9，10]. Anesthetics mitigate damage to fish bodies by suppressing activities of their central and peripheral nervous systems，and have also been used to increase the survival rate of fish populations during transportation[11]. Tricaine mesylate (MS-222) is an FDA-approved fish anesthetic that is quick to take effect or recover from， and safe to animal or human touch[12]. Its effectiveness has previously been shown in studies of three other highland Schizothoraxes:Schizothorax lissolabiatus[13]，Schizothorax wangchiachii[14]， andSchizothorax gra-hami[15]. In the former two cases， effective concentrations of MS-222 have been reported. No source has reported its effect on other species of the genus.

In this study， we utilized MS-222 to examine its anesthetic effects onS. o’connorispecimens in two size ranges， as well as specimens’ reactive behavior，to provide information useful for developing anesthesia techniques for highland Schizothoraxes.

1 Materials and Methods

1.1 Materials

SpecimensHealthy specimens ofS. o’connoriwere procured from a market for agricultural products in Bayi District， Nyingchi， Tibet Autonomous Region， China. The specimens were in two sizes: length(25.0±1.5) cm， weight (219.8±49.0) g， and length(14.8±2.3) cm， weight (46.9±20.3) g， respectivley.The specimens were kept in a concrete farm pond(5 m×2.5 m) at School of Tibet Agriculture and Animal Husbandry University， and fed until one day before the tests， using Salmofood produced in Chile by Vitapro Chile S.A.

AnestheticThe MS-222 anesthetic used in the tests was produced by Hangzhou Animal Medicine Factory (Hangzhou， China). It appeared as a white powder highly dissolvable in water， weighed with accuracy to 0.0001 g.

Other conditionsThe test used groundwater that was sufficiently aerated， at temperature (13±1)℃，with dissolved oxygen at 8.4 mg/L. Tests were performed in a plastic anesthetic container which is 25.1 cm high， 44.2 cm long， and 30.1 cm wide， using varying concentrations of MS-222. The water was slightly aerated with oxygen throughout each test.

1.2 Methods

Measuring effects of MS-222 on respiration rates on Schizothorax o’connori in two size ranges

Respiration rates were measured for specimens in each stage of anesthesia， using a gradient of concentration levels， with five specimens for each level:60， 90， 120， 150， 180， 210 and 240 mg/L. Specifically， this is done by first placing specimens in clean groundwater， then waiting for 3min to allow specimens to adapt to the environment， and finally adding anesthetics into water. Every 2 minutes， a record was made on the number of times a specimen breathed in a 20s interval. When the fish exhibited features of anesthetic stage Ⅳ， it would be immediately taken into clean groundwater for recovery， with recovery time and survival rate recorded. For each specimen， a blank control test was performed first， before starting the test with its designated concentration. Average respiration rate of five specimens was used as the rate for the given time segment.

Measuring effective concentrations of MS-222

Marking，et al.[16]posited that an ideal anesthetic concentration should achieve anesthesia in 3 minutes，and recovery in 5 minutes. After consulting extant literature (see Tab. 1) and accounting for characteristics ofS. o’connori， we defined effective concentration as follows: for larger specimens immersed in MS-222-infused anesthetic liquid， the effective concentration can induce stage IV of anesthesia in 5min， allow recovery in 5min， and allow a survival rate of 100%after immersing in the liquid for 20min; for smaller specimens， the effective concentration results in stage IV of anesthesia in 5min， recovery in 7min， in addition to a survival rate of 100% after immersing in the liquid for 20min.

Drawing from references [18]， [20]， and [21]， we designed a gradient of concentration levels: 90， 120，150， 180， 210 and 240 mg/L. Five specimens were tested with each concentration. When a specimen exhibited features of anesthetic stage IV， the duration until then would be recorded， and the fish would be immediately extracted from the liquid. We would then record its length and weight， immerse it in normal groundwater for recovery， and record its recovery time.

Measuring effects of exposure to open air on S. o’connori in deep anesthesiaTo evaluateS.o’connoribehavior in the context of surgery， labeling，measurement， and other out-of-water activities involving anesthesia， specimens were extracted from the liquid after being immersed in 180 mg/L MS-222 solutions for 5min. They were wrapped in wet towels，and exposed to open air for 0， 3min， 6min， 9min，12min， and 15min respectively before being placed into clear water. Recovery times were recorded. Five specimens were tested for each duration of exposure.

1.3 Data analysis

Tab. 1 Criteria for effective MS-222 concentration from extant literature

Excel 2003 and SPSS 21 were used for data processing and regression analysis. Significances were verified with one-way analysis of variance， and Tukey test (P=0.05).

2 Results and Analysis

2.1 Behavior criteria of S. o’connori in anesthesia and recovery

We based the criteria of anesthesia and recovery on those given by Liu，et al.[11，18]， with modifications made on account of actual behaviors ofS. o’connori(Tab. 2 and Tab. 3).

2.2 Effects of MS-222 on respiration rates of Schizothorax o’connori in two size ranges

Effects of MS-222 on respiration rates at various anesthetic and recovery stagesFor largesizedS. o’connori， their respiration rates show a tendency to increase in anesthetic stages Ⅰ—Ⅲ (Fig.1A); respiration rates sharply declined after stage Ⅳ;significant differences can be seen between respiration rates of stages Ⅰ—Ⅳ， Ⅴ and Ⅵ (P＜0.05). The rates display a gradual increase with progressing recovery stages (Fig. 1B)， with significant differences between stage I and stages Ⅱ—IV (P＜0.05).

For small-sizedS. o’connori， respiration rates appear stable in anesthetic stages Ⅰ—Ⅲ (Fig. 2A);rates rapidly slowed after stage Ⅳ， with significant differences between stages Ⅰ—Ⅲ， Ⅳ， and Ⅴ—Ⅵ(P＜0.05). Respiration rates gradually increased with progressing recovery stages (Fig. 2B) before stabilizing， with significant differences between stage Ⅰ and stages Ⅱ—Ⅳ (P＜0.05).

Tab. 2 Anesthetic stages and fish behavior characteristics

Tab. 3 Recovery process and fish behavior characteristics

Fig. 1 Respiration rates of large-sized S. o’connori under different concentrations in different anesthetic (A) and recovery (B) stages

Respiration rate changes of S. o’connori immersed in MS-222 solutions at seven concentrations for 20minDuring the 20min， large-sized specimens were immersed in the MS-222 solutions，their respiration rates each exhibited a trend of increase followed by decrease， indicating presence of stress responses at the start of anesthesia process. At lower concentrations (＜120 mg/L)， respiration rates would stabilize after following said trend， showing weaker stress responses. At concentrations 120—180 mg/L， respiration rates all displayed a trend from increase to decrease. Furthermore， at 120 mg/L， specimens could enter stage Ⅵ of anesthesia almost exactly at 20min， which would minimize the impact on their health. At higher concentrations (＞180 mg/L)，respiration rates would plummet after 2 to 4 minutes until reaching zero， indicating strong responses to this concentration range (Fig. 3A).

As large-sized specimens were placed into clear water for recovery， their respiration rates would display different trends depending on concentration， but all stabilized within a similar range at the end (Fig. 3B).

For small-sized specimens， there was also a trend of increase followed by decrease， indicating the presence of stress responses. The responses were relatively weak at 60 mg/L. At 90—180 mg/L， respiration rates showed a trend from increase to decrease.Particularly at 90 mg/L， specimens could enter stage VI of anesthesia almost exactly at 20min， which would minimize the impact. At higher concentrations(＞180 mg/L)， respiration rates would plummet after a short climb until reaching zero， indicating strong responses to this range (Fig. 4A).

Fig. 2 Respiration rates of small-sized S. o’connori under different concentrations in different anesthetic (A) and recovery (B) stages

Fig. 3 Respiration rate changes of large-sized S. o’connori immersed for 20min in 7 MS-222 concentrations (A， B)

Fig. 4 Respiration rate changes of small-sized S. o’connori immersed for 20min in 7 MS-222 concentrations (A， B)

As small-sized specimens were placed into clear water for recovery， their respiration rates would dis-play different trends depending on concentration， but all stabilized within a similar range at the end (Fig. 4B).

2.3 Effective concentrations of MS-222 for S. o’connori

Time until anesthetic stage Ⅳ and recovery for S. o’connori in MS-222As the concentration of anesthetic in water increased， the times gradually shortened for large-sized specimens to enter stageⅣ (P＜0.05). At MS-222 concentration 90 mg/L， most specimens took more than 700s to enter stage Ⅳ of anesthesia. Above 150 mg/L， all specimens entered stage Ⅳ within 300s (Fig. 5A).

When placed into recovery after being induced into anesthetic stage Ⅳ in 150—240 mg/L solutions，all large specimens fully recovered within 300s，without significant differences in recovery time(P＞0.05). Recovery was faster for concentrations above 150 mg/L. This may be due to that they can induce anesthesia faster， which means specimens stayed in anesthesia for shorter durations， and makes recovery easier (Fig. 5B).

Fig. 5 Induction time at anesthetic stage IV and recovery time (A， B) for large-sized S. o’connori under different concentrations

Fig. 6 Induction time at anesthetic stage IV and recovery time (A， B) for small-sized S. o’connori under different concentrations

Fig. 7 Effects of MS-222 concentrations on induction time (triangles) at anesthetic stage IV and recovery time (dots) of S. o’connori， large and small-sized (A， B)

For small-sized specimens， induction time into stage Ⅳ similarly shortened with concentration increase (P＜0.05)， except being slightly longer at 210 mg/L compared to 180 mg/L， which may be due to specimens having stronger stress responses to former concentration， thus prolonging their time to enter anesthesia. At 90 mg/L MS-222， most specimens took more than 500s to enter stage Ⅳ. Above 150 mg/L，all specimens entered stage Ⅳ within 300s (Fig. 6A).

When placed into recovery after being induced into anesthetic stage Ⅳ in 150—180 mg/L solutions，all small specimens fully recovered within 400s， with significant differences between concentrations(P＜0.05). Recovery was faster for 180 mg/L compared to other concentrations， which is likely due to its shorter anesthesia time making recovery easier.Recovery took longer for 210 mg/L compared to 240 mg/L， which may again be explained by specimens’stronger responses to 210 mg/L (Fig. 6B).

Through regression analysis (Fig. 7A)， the concentration-anesthesia time function for large-sized specimens can be obtained as:y=–3.6328x+935.07，R2=0.7226，P＜0.01， which is a negative correlation.They have the concentration-recovery time function:y=–0.6103x+371.23，R2=0.1767，P＜0.05， a negative correlation.

Regression analysis can similarly be applied to small-sized specimens (Fig. 7B)， to obtain concentration-anesthesia time function:y=–2.1594x+637.94，R2=0.5757，P＜0.01， a negative correlation. They have concentration-recovery time function:y=1.8196x+146.8，R2=0.2956，P＜0.01， a positive correlation.

Survival rates and recovery time of S o’connori immersed in MS-222 solutions in 20min

Tab. 4 Time until various stages of anesthesia， recovery time， and survival rate of large-sized S. o’connori immersed for 20min in 7 MS-222 concentrations

Tab. 5 Time until various stages of anesthesia， recovery time， and survival rate of small-sized S. o’connori immersed for 20min in 7 MS-222 concentrations

The specimens immersed in anesthetic solutions for 20min were moved into clear water for recovery(Tab. 4). At anesthetic concentrations 60—90 mg/L，large-sized specimens would not enter deep anesthesia， and their respiration rates at the end were slowed down but still regular. As the concentration increased， they took gradually shorter time to reach each stage of anesthesia. As they entered deep anesthesia， the specimens’ breathing became erratic and shallow， and operculum movements became smaller.

Specimens’ recovery time increased with anesthetic concentration， but shortened above 180 mg/L.This is because the time it took to reach stage Ⅵ was shorter at higher concentrations， thus shortening the time spent under anesthesia. One specimen perished at 210 mg/L， which lowered said concentration’s survival rate to 80%， while specimens at other concentrations all survived. In conclusion， the effective MS-222 mass concentration was determined to be 150—180 mg/L for large-sizedS. o’connori.

As MS-222 concentration increased， small-sized specimens took gradually shorter to enter various stages of anesthesia. But at 180 mg/L， specimens actually took longer to enter stages Ⅳ， Ⅴ and Ⅵ， which can be attributed to their stronger stress responses at said concentration. Their recovery time was less regular， but generally conformed to a trend of increasing with concentration. One specimen each perished at 180 and 120 mg/L， giving both a survival rate of 80%each， while specimens at other concentrations all survived. In overall， effective concentration was determined to be 150 mg/L for small-sizedS. o’connori(Tab. 5).

2.4 Effects of exposure to open air on S. o’connori in deep anesthesia

Using time until anesthetic stage Ⅳ and recovery (Fig. 6， Fig. 7)， and survival rates ofS. o’connoriimmersed for 20min (Tab. 4， Tab. 5)， we established effective anesthetic concentrations to be 180 and 150 mg/L respectively for our two size ranges， and then proceeded to air exposure tests under these concentrations.

Large-sized specimens were placed into a 180 mg/L MS-222 solution for 5min， then taken out for air exposure tests. When they were immersed in the solution， the specimens had been breathing shallowly and in anesthetic stages Ⅳ and Ⅴ. When first exposed to air， a few specimens stopped breathing， while the majority breathed with regularity. This was followed by increased amplitude in operculum movements， and faster respiration. Afterwards， some specimens started moving their tails， and slightly struggled. As air exposure went on， specimens struggled， regained their normal breathing rates， and gradually regained their balancing capability. The specimens were returned to water after exposure tests. Longer air exposure is shown to lead to shorter recovery in water. The longest recovery happened when specimens were immediately put into water recovery after anesthesia， at an average of 293s. The shortest occurred when specimens were exposed to air for 9min， at an average of 160s. Above 9min exposure time， recovery time would only slightly increase. In overall， recovery time does not differ much between different durations of air exposure (see Fig. 8A). Single-factor ANOVA suggests the length of air exposure does not significantly affect their recovery time (P＞0.05).

Small-sized specimens were placed into a 150 mg/L solution for 5min before being taken out. Specimens were in stages IV and V when in the solution. At the start of air exposure， they breathed weakly but with regularity. At exposure time 0min， all specimens could recover within 10min after being put into water recovery. At 3min， most specimens could regain balance around 6min in recovery. At 6min， most could regain balance around 4min. As the length of air exposure increased， recovery time display a trend of decrease， increase， and then stabilization. Recovery is longest without air exposure， at an average of 479s，and shortest with a 9min exposure， at an average of 212s. There are remarkable differences in recovery between air exposure durations (Fig. 8B). Singlefactor ANOVA suggests the length of air exposure has a significant effect (P＜0.05).

Fig. 8 Effects of air exposure duration on recovery time for large-sized (180 mg/L) and small-sized (150 mg/L) specimens (A， B)

3 Discussions

3.1 Effects of MS-222 on respiration rates

Respiration rate of fish in anesthesia is an indicator of the depth of its anesthesia， with slower rates indicating deeper anesthesia， and vice versa[18]. For MS-222 on both size ranges ofS. o’connori， respiration rates showed no significant change in anesthetic stages Ⅰ to Ⅲ， and only begun to significantly decline after entering stage Ⅳ， which suggests some importance in the transition from the minimal anesthesia of stage Ⅲ into stage Ⅳ. For large-sized specimens，both anesthesia time and recovery time are negatively correlated to concentration. Similar toSchizothorax wangchiachii[14]， for small-sized specimens， anesthesia time is negatively correlated to concentration，while recovery time is positively correlated. Largesized specimens took longer to enter anesthesia[22]，and anesthesia time is positively correlated to weight，which is similar to results on largemouth bronze gudgeon[23]. On the other hand， large specimens also had shorter recovery time[22]， which is negatively correlated to weight[24].

Higher MS-222 concentrations could reduce breathing rates faster， probably because they allowed specimens to take in more anesthetic in a shorter time via gill filaments. The anesthetic in the solution can be absorbed through gills and skin to arrive at the sensory center， and suppress the fish’s capabilities to react and move. This slows down its actions[25]， reducing its flapping of opercula， and drastically lowering its respiration rate. Since small-sized specimens had faster respiration than large-sized ones， they would absorb more anesthetic in a given duration， and enter anesthesia faster. The effects of anesthesia on respiration rates should be related both to characteristics of species and individual variations[22，26]， and the concentration of the drug[21].

3.2 Effective anesthetic concentration for S. o’connori

Effects of anesthesia are influenced not only by anesthetics， but also by factors related to the fish itself， typically species， size， and health， and environmental factors like water temperature and dissolved oxygen[11，27]. In general， MS-222 is noted for its quick induction， fast recovery， short time to achieve anesthesia， wide safety boundaries[28]， safety to human and fish health， and leaving low tissue residues[16]. It can slow down metabolism in fish species， reducing both their consumption of dissolved oxygen in water[26]and stress responses to being transported[29]， thereby increasing their survival rates[30].

This study has shown thatS. o’connoriindividuals at different sizes have different metabolic reactions to MS-222， likely with their surface area as an important factor on anesthetic effects[28]. Specimens at different sizes displayed different sensitivities to MS-222 concentrations. When large-sizedS. o’connoriwas placed into recovery after being induced into anesthetic stage Ⅳ at lower concentrations (＜150 mg/L)，its recovery time was likely prolonged by emergency responses. Recovery was faster for concentrations above 150 mg/L. When small-sizedS. o’connoriwas placed into recovery after being induced into anesthetic stage Ⅳ at particularly low (＜150 mg/L) and high concentrations (＞180 mg/L)， emergency responses also made recovery longer. Specifically， from this study， the effective mass concentration (EMC) is 150—180 mg/L for large-sizedS. o’connori， and 150 mg/L for small-sized ones. Specifics of these effects may warrant future studies.

Given comparable water temperature， these EMC results are slightly lower than those ofSiniperca chuatsi[21]andVerasper variegates[28]. Given comparable dissolved oxygen， they are close to those ofCynoglossus semilaevis[18]andSqualiobarbus curriculus[19]， slightly lower thanS. chuatsi[21]andV.variegates[28]， and higher thanScophthalmus maxinus[31](Tab. 6).

Tab. 6 Effective mass concentration of MS-222 for different fish species

Highland Schizothoraxes have strong tolerance for oxygen deprivation[32，33]in low-oxygen environments[34]. This is consistent with the finding that fish species with low oxygen requirements have longer anesthetic induction times than those with high requirements[35]. The low temperature of their habitat also has a suppressive effect on their sensory centers，giving them some resistance mechanism to negative environmental factors[28]. This factor can reduce their absorption rate of anesthetics， in turn increasing the concentration required to achieve anesthesia. A similar result was reported by Zhao，et al.[28]aboutV.variegates.

3.3 Air exposure of S. o’connori

S. o’connoritook the longest to recover from anesthetic stage IV when immediately put into recovery.Within 9min of exposure， recovery was faster when exposure time is longer; above 9min， recovery time slightly increased. Large-sized specimens do not differ much in recovery between different exposure durations. For small-sized specimens， respective recovery times after 6min and 9min of exposure are different from other groups. The variations or lack thereof may be explained by the time necessary for the anesthetic to be carried off by blood circulation around body[20]and broken down by metabolism， resulting in an equilibrium[21]that reduced suppressive effect on the brain.

Based on the results， in out-of-water activities forS. o’connori(e.g. surgery， labeling， measurement，etc.)， larger specimens (around 25.0 cm) are suited to an anesthetic concentration of 180 mg/L， and smaller specimens (around 14.8 cm) to 150 mg/L. In both cases， specimens should be kept exposed to open air for 9min after anesthesia is induced. This procedure should minimize damage to specimens， and quicken recovery.