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OXYTETRACYCLINE MARKING STUDIES OF TILAPIA; Oreochromis niloticus

Conference Paper · January 2011

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 OXYTETRACYCLINE MARKING STUDIES OF TILAPIA;
Oreochromis niloticus
Y. M. Abdel-Hadi 1, 2, J. F. Craig 3, J. A. Babaluk 4
and R. Wastle 4

1
Aquaculture Dept., Faculty of Agriculture, University of Putra Malaysia (UPM), 43400,
Serdang, Selangor, Malaysia. Tel. 603-89464248, Fax: 89464102.
2
Central Laboratory for Aquaculture Research, Abbassa, 44662, Egypt.
E.mail address: ymhadi@yahoo.com
3
Whiteside, Dunscore, Dumfries, DG2 0UU, Scotland, U.K.
4
Freshwater Institute, 501 University Crescent, Winnipeg, Manitoba, Canada.

Abstract
Oxytetracycline (OTC) was intraperitoneally injected into tilapia, Oreochromis niloticus to
validate aging of this fish, to determine suitable marking dosages of OTC, to assess OTC-
induced mortality and to determine the rate of OTC incorporation into 4 bony structures
including scales, opercula, otoliths and pelvic fin rays' sections. The OTC was used in 3 doses;
12.5, 25 and 50 mg/kg of fish body weight. A total of 1200 fish with an average body weight
of 60 grams were used for the experiment (300 fish per OTC dose and another 300 fish were
injected with sterile saline solution as a control group). The fish of the 4 groups were
released in the water of 4 raceways at the World Fish Centre, Abbassa, Egypt and were kept
for an entire year. The results revealed that 1 annulus was laid down after the OTC mark on
all bony structures. OTC was incorporated into all examined bony structures. The clearest
OTC marks with minimal mortality were induced by the 25 mg OTC dosage. No significant
mortalities were noticed among the injected fish with OTC.
Key words: Oxytetracycline, tilapia, annulus, scale, operculum, otolith, fin rays.

Introduction
Until the 1970s, tetracycline had been used extensively in the field of aquaculture as an
antibacterial drug (Schnick, Meyer & Walsh 1986). OTC was found markedly effective in vitro
and in vivo against Aeromonas hydrophila micro-organism and the highest concentrations of
the drug were found in liver and bone tissues of the treated Oreochromis niloticus (Soliman
1994). Since it is incorporated into newly forming calcified tissue and is visible under
ultraviolet light as a fluorescent band (Bavelander & Goss 1962), it had been used periodically
by fisheries researchers as a marking tool (Emery & Wydoski 1987). Since that time,
tetracycline has been used widely as a means of marking fish (Babaluk & Craig 1990).
Applied at a known date, tetracycline has been used to validate methods of fish age
determination (Casselman 1974; Babaluk & Campbell 1987; McFarlane & Beamish 1987a).
Since tetracycline is incorporated relatively quickly into calcified structures (Campana &
Neilson 1982; Nagiec, Dabrowski, Nagiec & Murawska 1988), it can been used to verify the
existence of daily growth increments (Campana & Neilson 1982; Laurs, Nishimioto &
Wetherall 1985). High dosages of tetracycline will cause mortality so it is necessary to
determine a dosage that produces a suitable mark on calcified structures of a fish species
while causing minimal mortality (Kobayashi, Yuki, Furui & Kosugiyama 1964; McFarlane &
Beamish 1987b).
In this study, tetracycline was injected into tilapia, Oreochromis niloticus to validate 4 age
determination methods (scales, opercula, otoliths' and pelvic fin rays' sections) to assess
tetracycline-induced mortality, to determine how quickly it was incorporated into calcifying
tissues in fresh water and to determine the suitable marking dosage of tetracycline for this
species.

Materials and methods

Fish:
A number of 1200 fingerlings of monosex Oreochromis niloticus were used for the
study. The fish were acclimatized first and then were distributed into 4 raceways (300 per
each raceway). After that the fish were kept for 4 months before launching the experiment to
reach bigger sizes. The fish were fed on artificial ration of 25% protein in the rate of 3% of

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total fish biomass. Partial and periodical change of pond water was carried out at a monthly
basis. The average body weight at the time of arrival of the fish on May 1998 was 38 gram
and the average total length was 13 cm. While, the average body weight at the time of
injection of the OTC on September 1st, 1998 was 60 gram and the average total length was
15 cm.
Raceways: Four raceways were used for rearing the fish for an entire year, which was
the period of the experiment. The dimensions of each raceway were 50 meters long, 4
meters width and 1 meter of water depth. The experiment was carried out in the facilities of
the WorldFish Centre (The Regional Centre for Africa & west Asia) at Abbassa, Abou-
Hammad, Sharkia, Egypt) during the period from September 1998 until September 1999.
Anaesthesia: The fish were anaesthetized using MS222 in the rate of 75 mg/liter of water
and accompanied with sodium bicarbonate in the rate of 150 mg/l to avoid the expected
hyperacidity induced by MS222.
Selection of Oxytetracycline dosages: The fish in the 1st group were injected with OTC
in a dose of 12.5 mg/kg fish body weight and then released into the first raceway. The fish in
the 2nd group were injected with OTC in a dose of 25 mg/kg fish body weight and then
released into the second raceway. While, the fish in the 3rd group were injected with OTC in a
dose of 50 mg/kg fish body weight and then released into the third raceway. On the other
hand, the fish of the 4th group were injected with sterile distilled water and then released to
the 4th raceway to be kept as a control group. The fish were injected intraperitoneally using
an automatic syringe similar to that used in mass injection of chickens in poultry farms.
Preparation of the OTC injection solution: A commercial pharmaceutical preparation
known as Panterramycin (Pfizer) in a package of 50 cc was used as a stock solution of OTC.
Each 50 cc bottle contained Oxytetracycline in a concentration of 30 mg/cc. Two bottles (100
cc) were used for the experiment. This stock solution of OTC (100 cc) was diluted 10 folds in
a sterile distilled water (900 cc) to reach one liter of final injection solution of OTC. Thus,
each cc contained 3 mg of OTC. Each experimented dose was multiplied by the average fish
body weight (60 gram). The 50 mg/kg fish body weight x 60 gram = 3000 divided by 1000 to
convert kg to gram = 3 mg OTC (i.e. 1 cc of the injection solution) per fish. Thus, the 50
mg/kg b.wt. was achieved by injecting each fish 1 cc of the injection solution. Consequently,
the 25 mg/kg b.wt. was obtained by injecting half of the preceding dose, which was 0.5
cc/fish and finally 0.25 cc/fish for the 12.5 mg/kg b.wt. dosage. On the other hand, the fish in
the control group were divided into 3 groups (100 fish per each). The first group was injected
with 1 cc; the 2nd group was injected with 0.5 cc and the 3rd one with 0.25 cc of sterile
distilled water.
Injection of fish: All fish were injected in the early morning; at 7 a.m, to shun the hot
weather in the afternoon for minimal stress on the examined fish. The fish of the control
group were injected first with sterile distilled water using the automatic syringe before being
intermingled with OTC solution to avoid any undesirable entrance of OTC into the control fish.
Twenty fish were injected with distilled water and kept in a hapa for sampling. Then the fish
were injected with 50 mg OTC /kg b.wt. in the rate of 1 cc per fish and 50 injected fish were
kept in a hapa also for sampling directly after injection. After that, injection of the fish with
OTC in the rates of 25 and 12.5 mg/kg b.wt was carried out respectively. The injected fish
were then released into their respective raceways, where they were kept for 1 year.
Sampling regime: One fish from the fish kept in the control hapa and 5 from the fish
injected with 50 mg/kg.b.wt. were taken periodically after 6 hours, 12 hours, 24 hours, 48
hours, 72 hours, 96 hours, 1 week, 2 weeks and 4 weeks respectively. Periodical sampling of
5 fish from the 4 treatments in a monthly basis was carried out by partial fishing after half
emptying of water in the 4 raceways. Those 5 sampled fish from each treatment were
sacrificed. Each fish was numbered, weighed and the total body length was measured. The
fish were then dissected. Different bony structures were taken from each fish, which are
scales, opercula, otoliths and pelvic fins. Each bony structure was then put in a small
envelope and finally all structures were kept in larger envelopes containing a full data about
the fish.
Preparation of different bony structures for examination: The scales were soaked
for 5 minutes in enumerated ice cube trays containing water plus 2 drops of liquid soap to
facilitate the removal of mucus and tissue debris. The scale was then handled by a forceps,

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rinsed with water and rubbed by a paper tissue. After that, 3 dry scales were mounted
between 2 slides and compressed by means of an adhesive tape (El-Bouhy et al., 2002).
The opercula were handled by a forceps and put into boiling water several times then
cleaned using a paper tissue to remove the skin and tissue debris. The otoliths were
removed from the skull of the fish after beheading. Two longitudinal sections were made
along the skull passing the proximal rim of the 2 eye balls. Then the roof of the skull was
reflected and the brain was removed. The 2 otoliths were then found embedded in their
grooves, where they were removed by a forceps and put on a paper tissue. Finally, they were
dried gently between fingers & paper tissue and kept in a small envelope. The pelvic fin
rays were first trimmed and then placed in ice cube trays lined with Para film. After that they
were embedded in epoxy resin plus hardener in a ratio of 2:1 respectively. Finally the blocks
were left for 12 hours for hardening. The sections were carried out either manually using the
jeweler's hand saw or automatically using an isomet electric saw.
Examination of different bony structures: The opercula were examined whole on a
black background with a stereoscopic microscope with a hand-held reflective ultraviolet light
source. The viewing room was dark. Scales, otolith sections, and fin ray sections
were examined under reflected ultraviolet light using a fluorescence microscope.

Results
Age validation: There was quite a bit of growth on all structures after the OTC mark. It
also appears that the fish were at large long enough so that an “annulus” or “check” had
formed after the OTC mark. This was especially evident on the pelvic fin rays and opercula.
Thus, all examined bony structures were found to be reliable and valid for aging of
Oreochromis niloticus in Egypt. Time of annulus formation was on May depending on scales'
back calculations (Figure. 1). On the other hand, its time may be on April according to
opercula's back-calculations (Figure. 2). Different results were stated by Gladys et al., 2007
who found 2 annuli on the otoliths of Nile tilapia in 2 lakes in Uganda.

Figure. 1 Back-calculations using scales radii. R = Average total scale radius in mm (sampled from
5 fish). N.B. Notice the stunted growth during winter months (In January, February& March).

Fig. 1 The relationship between age, total

body length (T.L.) & scale radius (R)

350
Date
300
Average
250 T.L. mm
Length in mm

R mm
200
150
100
50
0
Date from September 1998 to
September 1999

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Figure. 2 Back-calculations using opercula radii. R = Average total operculum radius in mm (from 5
fish).

Fig.2 The relationship between age of fish, total

body length and operculum radius
300 Date in month

Total body length

250
in mm
total operculum
200 radius (R in mm)
Length in mm

150

100

50

0
Date from September 1998 to
September 1999

Oxytetracycline-induced mortality: The OTC-induced mortality was insignificant among

the experimented fish. No immediate mortality recorded in the day of OTC injection.
However, the fish injected with 25 mg/kg b.wt. of OTC showed the least mortality percentage
(0.3 %) followed by the 50 mg/kg b.wt. group (1.3 %). While, the fish in the 12.5 mg/kg
b.wt. group and the control one revealed relatively the highest mortality percentage (2.7 %).
Thus, the OTC dose of 25 mg/kg b.wt. was proved to be the best dose, which induced the
most powerful OTC mark with minimal mortality. Oxytetracycline uptake: All the three
dosages were sufficient to mark the various bony structures. OTC marks were clearly evident
on all structures from OTC-injected fish that were at large for one month. In all of these
cases, there was visible growth on the bony structure after the OTC mark.
- All control structures had no auto-fluorescence.
- Opercula: On the opercula from fish that had been injected and then sacrificed relatively
early, a yellow fluorescence was usually evident only on the edges or tips of spiny parts. As
time went on, the fluorescence tended to cover the whole surface of the opercula. Of the fish
that were at large for one month, a distinct “OTC line” was visible with subsequent growth on
the opercula also evident. As dense bone was being deposited in all planes, the fluorescence
that would have been deposited on the operculum was covered and became less evident.
Scales: All scales taken from relatively recent injected fish showed a yellow fluorescent glow
(quite dramatic when compared to the controls). Scales from fish that had been at large for
one month had a distinct yellow fluorescent line as well as the glow. The glow would
probably dissipate as the fish and scales grew.
Otoliths: It was more difficult to discern the fluorescence on otolith sections from the
recently injected fish. The OTC appears first near the sulcus of the otolith (Figure. 3). Since
the otolith is not vascularized (the other structures are), it theoretically, may take a longer
time to be deposited. These results agreed with those reported by Massou et al., 2004 who
got a clear OTC mark on the otoliths of Nile tilapia injected with 50 mg kg -1 live mass.
However, the results disagreed with those recorded by Wright et al., 2002 who stated that
difficulties in estimating age or backcalculating fish size from otoliths, however, have been
encountered in various fish species because of the appearance of checks resulting from
disruptions in otolith incremental deposition. These checks interrupt the regularity of the
primary increments.
Pelvic fin rays: OTC marks were relatively easy to discern although they were easier to see
on the machine-sectioned fins than hand-sawed sections.

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The fact that the fish had been at large for a lengthy period of time (nine months) made it
easier the discern of the OTC marks.
The optimum Oxytetracycline dosage: The 50 mg/kg dosage produced the
“strongest” mark on all structures although on the scales a relatively faint line was observed.
The scales had a florescent “glow” inside the OTC mark. The 25 mg/kg dosage produced
“good” marks on all structures (Figure. 3) although the marks were relatively faint on the
opercula and scales. The 12.5 mg/kg dosage produced faint marks on most of the
structures. All that was discernable on the opercula was a florescent glow (perhaps) on the
inner area of the bone.

Figure. 3 Transverse section of an otolith from a tilapia injected with 25 mg oxytetracycline/kg body weight on 1
September 1998 and recaptured on 2 September 1999 under (a) reflected ultraviolet light and b) transmitted white
light. Annuli are indicated by dots. Oxytetracycline mark (OTC) is also indicated. Bar = 0.5 mm.

Conclusion:
It could be concluded that tilapia fish aging is reliable in Egypt as a subtropical country
and can be used as a basis for stock assessment and other fisheries management
applications. The dose of 25 mg OTC/kg body weight for marking of tilapia is recommended
in subsequent work & future studies.
Acknowledgment
The authors are very grateful to the WorldFish Centre and its entire staff (Regional
Center for Africa and West Asia, Abbassa, Egypt), where the experiment was carried out.
Special thanks to Dr. Roger Row (the former deputy director general), Engineer Rezk Hara
and his team. The authors also appreciate the kind support of Dr. Ismail Radwan who
granted the fish of the experiment as a gift from his own hatchery in Kafr El-Sheikh, Egypt.

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