Evaluation of marking and tagging methods for genetic studiesin carp

Y BASAVARAJUt, B S RENUKA DEVI, G MUKTHAYAKKA, L PURUSHOTHAM REDDY,G C MAIR*, E E RODERICK* and D .1 PENMAN**

Fisheries Research Station, University of Agricultural Sciences, Hesaraghatta, Bangalore 560089, India*Univeristy of Wales Swansea, Singleton Park, Swansea, SA2 8PP, Wales, UK**Institute of Aquaculture, Universit), of Stirling, Stirling FK9 4LA, Scotland

tCorresponding author (Fax, 91-80-8466451; Email, frs.oda@axcess.net.in).

A variety of marking and tagging methods were tested on common carp (Cyprinus carpio L.) with the aimof identifying suitable methods for genetic studies in this and other species of carp. Elastomer and Alcianblue dye marking; Cold and Silver nitrate branding; Floy, Fingerling, Carlin disc and visible implant tags;and fin clipping were all tested on a range of sizes of common carp (from mean weights of 10-25 g upto 600-800 g). The branding and tagging methods tested did not give satisfactory retention rates. A combinationof elastomer marking and fin clipping was then tested as a method for strain identification in a growthcomparison trial on catla (Catla catla Hamilton) and found to be satisfactory for this purpose. Passiveintegrated transponders (PIT) tags were used to individually identify catla of wild or hatchery origin beinggrown for use as broodstock. These had almost 100% (98.8%) retention rates, but are expensive comparedto most other tagging methods.

.Introduction

Tagging and marking is used in fisheries research andmanagement for estimating fish population sizes, migra-tion studies, growth rates in natural environments andgenetic research with a great deal of research on manyspecies but largely excluding some species groups suchas the carp (Parker et al 1990). In genetic improvementprogrammes it is often essential to mark individual fish,families or strains. This helps in comparison of perform-ance of different groups, identification of broodstock forspawning, etc. Limitations in facilities and experimentaldesign considerations often make it desirable to conductperformance trials in communally stocked tanks or ponds,where reliable marking techniques are essential in orderto discriminate between the mixed stocks. Since consid-erable money and time is spent on developing a geneticimprovement programme, it is most desirable that reliable,cost-effective and adaptable marking, tagging methodsare developed.

Nielsen (1992) lists seven main categories of taggingtechniques, namely (i) external tags, (ii) external marks,(iii) internal tags, (iv) natural marks, (v) biotelemetrintags, (vi) genetic identifiers, and (vii) chemical marks.The choice of the tagging/marking technique dependsmainly on the value of the fish and the number ofindividuals, batches or strains being handled and theobjectives for which the technique is used. The designof a genetic improvement programme may be influencedby the availability of appropriate marking/tagging tech-niques. For example the number of different strains orfamilies in a selective breeding programme which couldbe communally evaluated u~ing external marks as iden-tifiers would be a function of the number of availablemarks (for example, different colours of dyes) and thenumber of potential marking sites on the fish using aparticular technique. The intensity of selection wouldalso be influenced by the number of fish that couldrealistically be tagged or marked.

A number of these marks and tags have been tried

Keywords. Marking; tagging; genetics; carp

J. Biosci., 23, No.5, December 1998, pp 585-593. @ Indian Academy of Sciences 585

586 Y Basavaraju et at

2.ld Silver nitrate marking: Large scales generallycover the flanks and the belly of carp making theselocations unsuitable for external marks. Hence differentshapes of marks were made on the opercular regionusing silver nitrate pencils.

on different species for different purposes with varyingdegree of success (Saxena et al 1979; Jhingran et al1981; Khan et al 1988; Roy et al 1991 on carp andHerbinger et al 1990 on Atlantic salmon).

As part of a project on the genetic improvement ofcultivable carp, several marking and tagging methodswere evaluated, using the common carp as the main testspecies. The objective was to establish reliable methodsof tagging and marking for carp for use in such research,principally for identification of individual brood fish andfor marking strains and families for communally stockedgrowth trials.

2.le T-bar anchor tags (Flay tag): These are numberedplastic tags with a 'T' shaped head (Floy Tag andManufacturing Inc., Seattle, USA). The needle on thepurpose-built tagging gun (supplied with the tags) wasinserted into the dorsal musculature of each fish bylifting a scale and pushing the needle at a 450 angle.The trigger was then pressed to release the T -shapedhead into the muscle, leaving the numbered portion ofthe tags sticking out from the fish.

2.

Materials and methods

The trials on the common carp (Cyprinus carpio L.)and catla (Catla calla Hamilton) were conducted in twolocations, at the Fisheries Research Station, Hesaraghatta,Bangalore and at the fish farm of the State Department,Department of Fisheries at BR Project (BRP), Shimoga(300 km from Bangalore). All fish were anaesthetizedusing 250 ppm benzocaine (unpublished results) beforeapplying tags or marks.

2.1

f Fingerling tags: These are small numbered plastictags (F1oy Tags and Manufacturing Inc., USA) of differentcolours attached to a thread. Using a needle, the mono-filament thread was passed thrQugh the body above thelateral line on the upper portion of the caudal peduncleor through the muscle at the base of the dorsal fin raysand tied over the top of the fish. The fish were treatedwith tetracycline (by dipping in a 10 ppm solution for1-2 min) after the tagging operation to reduce the chanceof wound infection.

2. Details of tags/marks used and applicationprocedures

2.1

g Carlin disc: These are larger numbered plastictags of different colours with two threads. The tags wereattached to the fish by passing both threads separatelythrough the muscle at the base of adjacent dorsal finrays using a needle. A knot was tied on the other sideof the body to secure the tags.

2.1a Elastomer dye marking: The visible implant fluo-rescent elastomer developed by and purchased ffom NorthWest Marine Technology Inc., Seattle, USA, providesexternally visible, subcutaneous marks. The systemutilizes a biocompatible, two part elastomer materialwhich contains fluorescent colouring. Within 1-2 h, themixture cures to a solid elastomer having fluorescentpigments in a cohesive, well defined biocompatible mark.

The two components were mixed using the doublebarrelled syringe provided and dispensed into I mlsyringes fitted with 24, 22 or 19 g needles. The needleswere inserted below the skin on the ventral surface ofthe fish and the elastomer injected subcutaneously. Anyexcess elastomer remaining on the surface was wipedoff immediately to prevent the formation of an elastomer"tail" which could have caused loss of the injectedelastomer if this caught on nets or rough surfaces.

2.1

h Visible implants: Alphanumerically coded tags(from Northwest Marine Technology Inc., Seattle, USA)were inserted just below the dorsal fin ray and beneaththe thin membrane covering a part of the operculumusing a special flattened syringe (supplied with tags).

2.1

i Fin clipping: The pectoral or pelvic fin wasclipped at the base, while the upper or lower caudal finlobe was removed using a pair of scissors.

2.1j Passive integrated transponder tags: These glass-encased electronic transponder tags (AVID NorthwestMarine Technology Inc., Seattle, USA) were insertedinto the peritoneal body cavity of catla. This was doneeither by using a scalpel to cut an aperture in the bodywall and forceps to insert the tag through the apertureor using the injecting applicator needle supplied withthe tags. All tags and dissecting equipment were sterilizedwith ethanol before use. The entry wound for the tagwas dusted with OrahesiveTM powder containing anantibiotic (Mair 1989). This formed a gel on contact

2.1 b Alcian blue dye marking: Saturated Alcian bluesolution was injected just beneath the epidermal layerat the base of one of the paired ventral fins. Any excessdye was wiped off.

2.1c Cold branding: A round copper rod having adiameter of 5 mm with a smooth rounded end was heldin liquid nitrogen until the temperature equilibrated andthen used to mark the fish on the hard portion of itsoperculum.

587Tagging and marking for carp

mately 150 m2 were used with equal densities of eachstock in each pond. The fingerlings were marked atmean weights of 9.3 to 9.8 g and grown in the pondsfor 24 weeks, when the experiment was terminated dueto problems associated with heavy rainfall. Fish weresampled every three weeks for weighing and measuring,at which times they were identified to hatchery stockorigin by the elastomer/fin clipping markings. Fin clip-pings were repeated as necessary at each sampling, whilethe elastomer marking was reinforced, where required,nine weeks after the start of the trial.

with water helping to seal the aperture in the body walland reducing the risk of infection. Each tag had anindividual code which is read by a battery-poweredreader held close to the fish.

2.2 Common carp tagging and marking trials

2.4 Tagging of carla broodstock

Passive integrated transponder (PIT) tags were used tomark fish from hatchery and wild sources for future useas broodstock. In June and July 1996, 209 fish from1994, 1995 and 1996 year classes (mean group weightsranging from 73.13:t 8.27 to 1555.75:t 79.30 g) weretagged and placed into earthen ponds. In mid-November1996, these fish were sampled and the tags checked(167 out of 209)

At Hesaraghatta, the medium size group B fish (35-70 g) were stocked in 200 m2 concrete ponds in duplicatewhile the larger group C fish (> 100 g) were stocked ina 200 m2 manured earthen pond. In addition to regularfertilization with manure, stocked fish were fed with aI : I mixture of groundnut oil cake and rice bran at 5%of their body weight/day, with a single morning feed.Fish were sampled once every three weeks to assesstheir growth rate, health and retention of tags/marks.The quantity of feed to be given was also regulated atthe time of sampling.

At BRP Farm, the smallest group A fish (10-25 g) werestocked in a 400 m2 concrete pond while the largest groupD fish (600--800 g) were stocked in an 800 m2 earthenpond. Ponds were manured and fed as described above.

These experiments were conducted between Februaryand July 1995 with the period between tagging and finalassessment of the retention rates ranging from 129-152days (tables 2-5). The retention rate for each tag ormark was calculated as:

3. Results

(Number of fish recovered with tag or mark) x 100(Initial number of fish marked with this technique)

The loss rate for each technique using this methodfor calculating retention includes both fish which havesurvived but lost the tag or mark, and fish which havedied during the trial. An adjusted retention rate estimatewhich took account of this mortality was calculated forgroup A as follows (assuming mortality rate to be equalfor all tagging and marking methods used in this group,as there was no evidence for differential mortality inother groups)

(Retention rate) x (Initial total number offish in the group)(Final total number of fish in the group)

2.3 Catla growth trial

Subsequent to the initial trials on common carp, elastomermarking and fin clipping were used to mark catlafingerlings from three different hatcheries for stockingin a communal growth trial. The combinations were: TBDam (TBD)--elastomer at the base of the pelvic finand anal fin clipping; BRP--elastomer at the base ofleft pectoral fin and lower caudal fin clipping: KRP-elastomer at the base of right pectoral fin and uppercaudal fin clipping. Three replicate ponds of approxi-

A summary of tag/mark retention according to initialsize of fish and tagging/marking method is presented intable 1. The .details of the individual trials are presentedin tables 2-5.

Some of the techniques tried were found to beunsuitable or ineffective during or shortly after applicationof the tag or mark. For example, trials with cold brandingindicated that marks were not clearly visible. This wasalso .the case with silver nitrate marking, the marks werevery clear immediately after marking but they tended tofade after 24 h. Carlin discs were found to be too largefor small fish. It was also difficult to locate suitablepositions for inserting visible implant tags. They weretried at different places such as below the ventral sideof lip, below the jaws and in between dorsal fin rays(in salmonids and some other fish, these tags are insertedinto the clear tissue immediately behind the eye; Anon.1995). However all tags were lost within 24 h. Hence,these aforementioned methods were not tried on manyfish. Only dye marking, fin clipping, fingerling and floytags were tried on larger number of fish.

Tagging and marking did not result in significantmortality in three of the four group (see tables 2-5).The low survival seen in group A at BRP could probablybe attributed to management (for reasons beyond ourcontrol the fish were moved from one pond to anotheronce during the experiment) rather than to the effect oftagging or marking itself.

From table 2 it can be seen that only Alcian bluemarking and caudal fin (upper and lower lobes) clipping

Y Basavaraju et at588

marking and Alcian blue gave significant rates of retentionfor the larger fingerlings used in group B at Hesaraghatta.

Using a subjective scale for rating the intensity ofthese marks at the end of the trials (no mark + to ++++score for highly visible marks), the Alcian blue appearedto give more intense marks at the end of the trial. Thismay be influenced by the greater tendency of Alcianblue to spread out under the skin on initial injection,while the elastomer remains in a more compact markdue to its greater initial viscosity and curing properties.The visibility of the elastomer marks can be greatlyenhanced by exposure to UV light, which causes fluore-

had adequate retention with no major differences in thesethree methods. All other tags/marks were lost. Retentionrates for caudal fin clipping (i.e., those in which it couldstill be detected despite regeneration) were 40% and28.9% respectively for the upper and lower lobes in thesmallest group of fish (67.5% and 48.7% respectivelyafter adjustment for mortality). Fins regenerated fullyand rapidly but in those in which the mark was 'retained',it was possible to detect that they had been clipped dueto "kinks" in the caudal lobes and the slight change inthe colour of the regenerated lobes.

As can be seen from table 3, only elastomer dye

Table Retention (%) of various tagging/marking methods in common carp ofvarying sizes.

Elastomer 72.0

(50.0)

Alcian blue 24.0(50.0)[40.5]

80.0(30.0)

Cold branding 0.0(19.0)

0.0(1.0)

Silver nitrate 0.0(70.0)

[0.0]

0.0(10.0)

Floy tag 10.0

(20.0)

6.7

(30.0)

0.0

(20.0)

3.3(30.0)

Fingerling tag 0.0(10.0)[0.0]

Carlin disc 47.4(19.0)

0.0(8.0)

20.(10.

O.(10

100.(20

Visible implant 0.0{10.0)

40.0(50.0)[67.5]28.9(45.0)[48.7]

0.0(95.0)[0.0]0.0

(90.0)[0.0]

Fin clip(upper caudal)

96.7(30.0)

Fin clip(lower caudal)

Fin clip(pelvic)

96.7(30.0)

Fin clip

(pectoral)96.7(30.0)

Number of fish recovered at the end of the trial with sample size (number of fishtagged/marked per group) indicated in parentheses and the adjusted retention rate (groupA only) in square brackets. Empty cells indicate where certain tagging methods werenot tried in some size groups.

59.0(100.0)

37.9(58.0)

0

0)00)00)

Tagging and marking for carp 589

Table 2. Results from comparative trials (over 129 days) of various tagging and markingmethods on common carp size group A at BRP Fish Farm.

Control 90 ]0.7 1920 60.3 463.5

Totalmar

Totalfish

410" 45

400 237(59.3%)

UC, upper caudal fin lobe clipped; LC, lower caudal fin lobe clipped; Pelv., one pelvic fin clipped;Pect., one pectoral fin clippedOJncludes fish which had lost marks or tags .I7fhe figure [in brackets] is retention rate adjusted for survival ratecSome of the fish were marked or tagged using more than one technique, accounting for the factthat the total number of marks or tags exceeds the total number of marked/tagg~d fish.

Table 3. Results from comparative trials (over 151 days) of various tagging and markingmethods on common carp size group B.

No. offish

Initial meanwt. (g)

Final meanwt. (g)

Retention

(%)Weight gain

(%)Mark/tagNo.

recovered

Elastomer

Alcain blue

Cold branding

Floy tags

Fingerling tagsVisible implants

Control

100

58

19

2030

10

0

44.0

37.0

67.973.9

52.)

30-50"

59

220

22

0

152b

99.5

95.7

59.037.9

0.0

10.0

6.70.0

125.0158.6

130.0

150.0

75.9

187.9

102.6

Total no. oftags/marks

Total no. of fish

237 85

237 240C

(101.3%)

OWeight range, means not availablebIncludes fish which had lost marks/tags"Exceeds initial total. No obvious source for the excess fish was identified.

no. ofks/tags

no. of

590 Y Basavaraju et al

scence of the orange dye (UV light was not used for of retention. In addition Carlin discs gave a retentionthe subjective scoring just described). rate of 47.4% but as mentioned above were considered

In the second trial at Hesaraghatta on the larger group to be unsuitable for smaller fish, the major target ofC fish (100-200 g), Alcian blue again had a high rate these experiments.

Table 4. Results from comparative trials (over ISI days) of various tagging and markingmethods on common carp size group C.

No. offish

Initial meanwt. (g)

No.recovered

Final meanwt. (g)

Retention(%)

Weight gain(%)Mark/tag

50 167.38 36

0

0

9

0

530

157.35 72.00.00.0

47.40.0

-5.9Elastomer

Cold branding

Floy tagsCarlin disc

Visible implants

Control

-123.32

198.80

175.00

2019

8

0

-174.22

-

-12.3

98 47Total no.of tags/marks

Total no. of fish 98 98(100.0%)

a As there were no untagged fish at the beginning of the experiment, this represents the fish which

had lost marks or tags.

Table 5. Results from comparative trials (over 129 days) of various tagging and markingmethods on common carp size group D.

No. offish

Initial meanwt. (g)

No.recovered

Final .meanwt. (g)

Retention(%)

Weight gain(%)Mark/tag

Alcian blue(pectoral fin)Alcian blue(pelvic fin)Silver nitrate

Floy tagCarlin disc

Visible implant

Fin (UC)

Fin (LC)

Fin (Pelv.)

Fin (Pect.)

Control

20 650.33 589.5 55.0 -9.1

30 733.33 29 673.90 96.7 -8.1

10

30

10

10

20

30

30

30

50

700.00

766.66

700.00

750.00

800.00

633.33

683.33

750.00

300-500

0 0.03.3

20.00.0

100.0

96.7

96.7

96.7

-

530.()()

575.()()

-

-30.78

-23.33

-13.4

-3.1

-13.7

-10.1

20

20

29

29

29

960

-

692.50

613.80

589.50

673.90

Total No. oftags/marks

Total No. of fish

220 150

237b 240

(100-0%)

UC, Upper caudal fin lobe removed; LC, lower caudal fin lobe removed; Pelv., one pelvic finremoved; Pect., one pectoml fin removedQlncludes some fish which had lost marks or tagsbSome of the fish were marked or tagged using more than one technique, accounting for the factthat the total number of marks or tags exceeds the total number of marked/tagged fish.

Tagging and marking for carp 591

In the largest fish (group D at BRP), most marksapplied were effective. Regenerated fins were easilydetected giving high rates of retention (96-100%). Alcianblue was again effective, especially when injected at thebase of the pelvic fin (96.7%).

Growth was not affected in small sized groups butmarginal reduction in weight was noticed in all largerfish in size groups C and D ranging from 120-750 g(tables 4 and 5). No specific reasons could be attributedfor this although the most likely reason is that most ofthe fish in this range were mature and in spawningcondition. Fish were taken out from these ponds forroutine seed production work and released back afterspawning. Hence, low weights recorded at the time oftermination of the experiment might be due to the lossof weight of gonads which can account for 15-20% ofthe total body weight. Loss of condition due to poorfood supply and/or poor water quality may also be afactor. In these groups all fish lost weight, regardlessof tagging methods used indicating that the weight lossis not a factor related to the principal objective of theexperiment. Very few fish lost both sets of marks inthe catla growth trial (see figure I). In. fact as shownin figure I most fish could be identified to hatcherystock origin at each sampling period on the basis ofelastomer mark alone, particularly after this was reinforcednine weeks after the start of the trial. However, the finclipping was useful for identifying the fish which hadlost the elastomer marks and it was then much easierto reapply elastomer marks when necessary. Very goodtag retention was seen in the evaluation of PIT tags ina range of sizes of catla high fish. From a total of 167PIT tagged catla sampled, the tag could be detected withth~ reader in ]65 (98.8%) fish 4-5 months after theywere tagged.

was satisfactory for producing good quality marks insalmon (Herbinger et al 1990), it proved not to be usefulfor the carp in the present trials.

Elastomer dye marking was tried on carp for the firsttime and the results appear to be satisfactory, withslightly higher retention rates than Alcian blue across arange of sizes of fish especially when marks are viewedwith the aid of UV light. The cost of marking usingelastomer is, however, considerably higher and the elasto-mer is more time consuming to prepare and apply tothe fish.

Fin clipping, which is a simple technique to employ,was found to be very useful in larger fish where regenera-ted fins could easily be distinguished from unclippedfins. The regenerated fins developed a faint red colouron the regenerated portion, which made clipped finsdistinct from unclipped. However, this method was notuseful for fish weighing less than 109 as a singlemarking technique as the regeneration was very fast andthe regenerated fin was difficult to distinguish from theunclipped fin. This was observed in all the fins clipped.Alagaraja and Gupta (1976) and Jhingran et al (1981)also concluded that regeneration of clipped fins occurredrapidly but that fin clipping did not appear to affect thegrowth and survival of the fish. The reduction in weight

100

~---

--~90

~ 80~

-;.~ 70~=

rI)

~

~

~

~60

4.

Discussion

f

~It is quite clear that of the techniques tested here onsmaller fish « 1 00 g), elastomer or Alcian blue dyemarking and possibly caudal fin clipping appear to bethe onJy suitable methods. Khan et al (1988) in theirstudy on dye marking of carp reported that M-Procianblue was a promising marker for the Indian major carpand common carp. While evaluating jet injection ofAlcian blue and cold branding as markers for the Atlanticsalmon.. Herbinger et al (1990) reported that Alcian bluemarks were quite cJearly visible even after 6 months ofrearing. The fading of marks after relatively longerperiods is influenced by the growth rate. Thus underconditions of rapid growth, Herbinger et al (1990) con-cluded that the jet injection of Alcian blue would besatisfactory up to 1.5 years. With less stringent require-ment (10-20% loss acceptable), Alcian blue could remainsatisfactory for longer periods. Although cold branding

--!:F,.

:IAII

10 20Weeks after stocking

Figure 1. Data from the catla growth trial. Three ponds wereeach stocked with fingerlings from each of three hatcheries(TB Dam, BRP and KRP). The different hatchery stocks weremarked at the start of the trial using combinations of elastomerand fin clipping (see text). The elastomer marks were reinforcednine weeks after the start of the trial (indicated by the arrowhead), while fin clips were redone every sampling period wherenecessary. Samples were taken every four weeks from 8-24weeks after stocking. (0), Sample size expressed as meanpercentage of number of fish originally stocked in pond. (~,Mean percentage of fish whIch could be identified to hatcheryon the basis of elastomer marking. (. ), Mean percentage offish which could not be identified to hatchery stock by eithermarking method.

592 Y Basavaraju et al

few years in the possibilities for the use of geneticmarkers such as microsatellite DNA loci, as an alternativeto physic~l tagging (Castelli et al 1990). Due to thehigh levels of variation found at such loci, they offerthe possibility of identifying individuals from mixedgroups to strains and even to family (e.g., full-sibs orhalf-sibs) by comparison of their DNA with that ofparents. This would permit fish to be mixed at the frystage without any type of physical marking, grown toan appropriate stage and identified to family by typingDNA from several microsatellite loci (Wright and Bentzen1995). This should offer considerable advantages inimproving the validity and resolving power of suchgrowth comparisions and avoid the necessity of expensivetagging techniques such as PIT tags. We are currentlydeveloping microsatellite DNA loci for application inselective breeding programme in catla.

Acknowledgements

This work was conducted as a part of the project onGenetic Improvement of Carp funded by the Departmentfor International Development, Fish Genetics Programme,University of Wales, Swansea, UK (R 6059). We thankShri S N Shanmukha, Director of Fisheries for allow-ing us to use departmental facilities and Shri TVenkateshappa, Deputy Director of Fisheries, Shimogaand Shri Mahadevappa, BR Project Fish Fam1 and hisstaff for providing facilities.

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seen in the present study in one group was common toall the fish in that group and there was no evidence toindicate that anyone tagging method was more detri-mental to growth than any other for any size group offish. Roy et al (1991) also reported that clipping ofpectoral fins did not affect growth and survival of catlaor rohu, while it caused marginal reduction of growthin rnrigal and silver carp.

The external tags tested appear not to be useful forcornmon carp under these conditions. All the tags evalu-ated exhibited high loss rates although they are success-fully used in research on other species. Earlier attemptsto use external tags on Indian major carp were also notsuccessful, with a very low recovery rate, ranging from0.8-6% (Saxena et at 1979). A wide range of types ofexternal tags exists and there are various possibilitiesfor location and methods of attachment, but significantimprovements on the retention rates observed by Saxenaet at (1979) and in this study would be necessary formost applications.

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MS received 22 September 1997; accepted 14 September 1998

ColTesponding editor: SAMIR BHATTACHARYA