FISHERY BULLETIN: VOL. 71, NO. 1 



MATERIALS AND METHODS 



ANIMALS 



Dasyatis sabina (Lesueur) were collected in 

 otter trawls. In the fall and winter stingrays 

 are most abundant in the shallow waters in the 

 Gulf of Mexico adjacent to Port Aransas, Tex. 

 In late spring the stingrays migrate into the bays 

 behind the line of barrier islands where they 

 were caught during the summer (Sage et al, 

 1972). 



INCUBATION TECHNIQUE 



Animals were killed by cutting across the hind 

 brain. The compact thyroid is located ventral 

 to the anterior end of the ventral aorta. The 

 thyroid was removed and divided into experi- 

 mental and control halves, and further divided 

 where necessary so that no piece of tissue was 

 larger than 5 mg. Preliminary experiments in- 

 dicated that the elasmobranch thyroid was slow 

 in responding to stimulation. Thyroid tissue 

 was therefore incubated for 3 days in 2 ml of 

 elasmobranch saline (Nicoll and Bern, 1964). 

 Antibiotics were added (Bakke et al., 1957) in 

 order to inhibit bacterial growth which might 

 result in the breakdown of the thyroxine re- 

 leased into the medium. The addition of anti- 

 biotics does not interfere with the ability of 

 thyroid glands to respond to TSH (Bakke et al., 

 1957; Sage, 1968a) , The incubation flasks were 

 gassed with 95% oxygen and 5% carbon dioxide 

 and shaken at 120 strokes/min at 30°C. This 

 temperature is within the normal environmental 

 range of D. sabina. Modification of the incu- 

 bation medium by the addition of 0.5 mg/ml 

 lactalbumin hydrolysate was found to increase 

 control rates but reduce the variability of the 

 response and was used in later experiments as 

 described in the text. 



Homogenates of whole pituitaries or various 

 regions of the stingray pituitary were made in 

 a glass homogenizer and added to the incuba- 

 tion media at a concentration of one pituitary 

 gland or region per thyroid gland. The homo- 



genates were added immediately prior to gassing 

 and adding of the thyroid tissue. 



THYROXINE ANALYSIS 



At the end of the 3-day incubation period 

 thyroid tissue was removed for histological ex- 

 amination, and the incubation media was centri- 

 fuged at 10,000 rcf for 10 min to remove cell 

 debris. Incubations were then stored below 0°C 

 until analyzed. Thyroxine was isolated by ion 

 exchange chromatography (Galton and Pitt- 

 Rivers, 1959) . The catalytic effect of iodine in 

 reducing eerie ions was used to quantify thy- 

 roxine iodine (Pileggi et al., 1961; Pileggi and 

 Kessler, 1968). Oxford Laboratories' (San Ma- 

 teo, Calif.) kit^ of reagents was used in the de- 

 terminations. The results were converted to 

 rates of thyroxine release per unit thyroid weight 

 per incubation, and the responses of treated 

 halves of the gland were then expressed as a 

 percentage of the matched control incubated 

 halves. Additives to the incubation media were 

 routinely analyzed but were invariably devoid 

 of thyroxine. 



HISTOLOGICAL METHODS 



At the end of the incubation period, thyroid 

 tissue was removed and fixed in mercuric formol 

 (90 parts saturated mercuric chloride to 10 parts 

 formaldehyde solution). Sections were cut in 

 polyester wax and stained with hematoxylin and 

 light green. The image of the thyroid follicles 

 was projected onto a sheet of paper, and a plan- 

 imeter was used to determine the percentage of 

 the area of follicle occupied by epithelium. 



Unstained thyroid sections were used for in- 

 terferometric determinations of mass per unit 

 area of the colloid (Bromage and Sage, 1968; 

 Sage, 1968b) . Such methods are very sensitive 

 in detecting changes in thyroid activity in both 

 teleosts (Bromage and Sage, 1968) and mam- 

 mals (Sage and Robins, 1970). 



^ Reference to trade names does not imply endorse- 

 ment by the National Marine Fisheries Service, NOAA. 



94 



