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2az
<br />The Southwestern Natura[isl
<br />racycline hydrochloride (TC). Once deposited in
<br />bone, ALC fluoresces red and TC fluoresces yel-
<br />low when illuminated by ultraviolet (UV) light.
<br />Single or multiple treatments with either or each
<br />of these chemicals can be used to produce poten-
<br />tially long-term marks unique to different batches
<br />of fish with virtually 100% initial marking success
<br />(Tsukan.oto, 1985, 1988; Dabrowski and Tsu-
<br />kamoto,1986;Muth et a1.,1988; Brothers, 1990).
<br />Our objective was to determine optimum treat-
<br />ments for marking otoliths in late embryos and
<br />newly hatched larvae of razorback sucker. We
<br />evaluated fish survival and mark quality in dif-
<br />ferent ALC or TC concentrations and immersion
<br />durations for 1) single treatment of embryos with
<br />either chemical, producing single marks and 2)
<br />single treatment of larvae with each chemical,
<br />producing double marks.
<br />Fertilized razorback sucker eggs (one day old,
<br />postfertilization) were obtained from Dexter Na-
<br />tional Fish Hatchery and Technology Center, New
<br />Mexico, in March 1992 and 1993. Eggs were
<br />incubated in well water at 17°C and examined
<br />twice daily. Otoliths (sagittae and lapilli) were
<br />first observed in developing embryos about three
<br />days after fertilization, three days before hatch-
<br />ing.
<br />The single-mark experiment started one day
<br />after otoliths were first present in the embryos.
<br />A total of 1,440 viable eggs was divided into 72
<br />experimental groups (two maintained as con-
<br />trols), each with 20 specimens. Marking and
<br />rearing were conducted in 250-m1 plastic cups
<br />placed in a covered water bath at 17°C. Eggs were
<br />immersed in solutions of 5, 15, 25, 35, 50, 150,
<br />250, 350, or 450 mg ALC/1 distilled water or
<br />50,150, 250, 350, or 450 mg TC/1 distilled water.
<br />Alizarin complexone was first dissolved in 3 to 5
<br />ml of a 1-N KOH solution then diluted to volume
<br />(Tsukamoto, 1988); TC was soluble in water.
<br />Each test solution was adjusted to pH 6.8 to 7.2
<br />with tris buffer. Immersion durations for each
<br />ALC or TC concentration were 12, 18, 24, 30,
<br />and 36 h. Eggs were removed from test solutions
<br />immediately after treatment and incubated in well
<br />water. Larvae were reared for three days after
<br />hatching, and those surviving to the end of rearing
<br />were counted and preserved in 100% ethanol.
<br />Five preserved larvae were randomly selected from
<br />each experimental group for mark examination.
<br />Sagittae and lapilli were extracted, mounted whole
<br />in glycerin on glass slides, and examined with
<br />incident UV light under a compound fluorescent
<br />vol. 40, no. 2
<br />microscope for presence and intensity of the fluo-
<br />rescent mark. Intensity of the mark in otoliths
<br />from each larva examined was ranked as absent,
<br />faint, lucid, or bright with respective values of 0,
<br />1, 2, or 3. Values were averaged within each
<br />sample of five fish, and the mean was used as a
<br />subjective comparison of mark quality among ex-
<br />perimental groups.
<br />The double-mark experiment started one day
<br />after eggs not used in the single-mark experiment
<br />had hatched. A total of 2,200 larvae was divided
<br />into 110 experimental groups (two maintained as
<br />controls), each with 20 specimens. Marking and
<br />rearing were conducted in 1-1 glass beakers placed
<br />in a covered water bath at 17°C. Larvae were
<br />first immersed in buffered solutions of 12.5, 25,
<br />50, or 100 mg ALC/1 distilled water for 6, 12,
<br />and 18 h. Larvae were removed from test solu-
<br />tions immediately after ALC treatment, and
<br />numbers of dead larvae were recorded. Surviving
<br />larvae were reared in well water for 24 h. After
<br />the 24-h rearing period, larvae were next im-
<br />mersed in buffered solutions of 150, 250, or 350
<br />mg TC/1 distilled water for 6, 12, and 18 h. All
<br />possible combinations of ALC :TC concentra-
<br />tions and immersion durations were tested (total
<br />of ] 08 combinations). Larvae were removed from
<br />test solutions immediately after TC treatment and
<br />reared in well water for three days. Larvae sur-
<br />viving to the end of rearing were counted and
<br />preserved in 100% ethanol. Procedures for mark
<br />examination were the same as those described in
<br />the single-mark experiment; intensity of ALC
<br />and TC marks in otoliths was evaluated sepa-
<br />rately for each mark type.
<br />In the single-mark experiment, hatching suc-
<br />cess decreased and mark quality increased as im-
<br />mersion duration increased at ALC or TC con-
<br />centrations of >_ 150 mg/1. Lowest percent egg
<br />hatch ranged from 30 to 45% in treatments of
<br />250 and 350 mg ALC/1 for 36 h, 450 mg ALC/1
<br />for 24 to 36 h, 150 to 350 mg TC/1 for 36 h, and
<br />450 mg TC/1 for 30 and 36 h. Hatching success
<br />was 90 to 100% in remaining ALC or TC treat-
<br />ments. Percent egg hatch was 90 to 100% in con-
<br />trols. Survival of larvae during the first three days
<br />after hatching was 100% in all experimental
<br />groups.
<br />Otoliths from all larvae treated with ALC or
<br />TC as embryos that were examined by UV-light
<br />microscopy had fluorescent marks. Intensities of
<br />red ALC marks ranged from faint to lucid (mean
<br />quality values = 1.0 to 1.4) in treatments of 5 to
<br />1
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