REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 



1153 



very sensitive method has recently been 

 described by Grosvenor and Turner (1958). 

 Adult pigeons, Columba livia, between 240 

 and 360 gni. were kept in a room with a tem- 

 perature between 78 and 80°F., artificial 

 light-supplemented daylight during daylight 

 hours. Two solutions to be tested were in- 

 jected intradermally on opposite sides of 

 each crop sac. The volume injected was 0.1 

 ml. Four injections were made at 24-hovu- 

 intervals in the same area marked with a 

 nontoxic dye. The birds were killed 24 hours 

 after the last injection, and the crop sac dis- 

 sected and stretched over a light source. 

 The area stimulated was estimated with the 

 aid of plastic discs with diameters ranging 

 from 0.5 to 4.0 cm. With 15 pigeons per dose 

 level and a linear log dose-response rela- 

 tionshii^ was found for a range of 0.00072 

 to 0.02240 mg. per bird (1 mg. = 20 I.U.). 

 The index of precision was A = 0.11. 



ACTH. Bates, Lahr and Miller (1940) 

 used 2-day-old chicks for the assay of 

 ACTH. White Leghorn cockerels were in- 

 jected 3 times per day for 5 days. The 

 adrenals were weighed. Ten mg. of a par- 

 tially purified ACTH preparation resulted in 

 a 25 per cent increase in adrenal weight. 

 The assay, however, is relatively insensi- 

 tive. 



TSH. In order to compare different meth- 

 ods it was necessary to convert U.S. P. to 

 Junkmann-Schoeller (J.S.I units. In these 

 calculations 1 I.U. = 1 U.S.P. unit = 0.1 

 J.S. unit (Brown, 1959j. The use of chicks 

 for bioassay of TSH has been reviewed by 

 Turner (1950) and by Brown (1959). 



1. Gravimetric methods are too insensi- 

 tive to be of much value. 



2. Histometric methods. One-day-old 

 cockerels are injected five times at 12-hour 

 intervals and the chicks killed 12 hours 

 after the last injection. The thyroids are 

 removed, fixed in Bouin's solution, sectioned 

 at 4 to 6 /x, stained with hematoxylin and 

 eosin, or Mason's triple chrome stain. The 

 height of one cell in each of 100 acini is 

 measured, the cells to be measured being 

 selected at random or the height of the 

 highest and lowest cell in each of 50 acini is 

 measured. If the latter method is used the 

 acini are selected at random. In our labora- 

 tory the highest and lowest cell in each of 



20 acini is measured and with this method 

 0.003 U.S.P. units can be detected (van 

 Tienhoven, unpublished data). The assay 

 can be made more sensitive by adapting 

 Uotila and Kannas' (1952) technique of 

 l)rojecting the thyroid section on a screen 

 and estimating the percentage of epithelium. 

 Using this method, 0.0001 U.S.P. units can 

 be detected (Saatman and van Tienhoven, 

 unpublished data). 



3. Intracellular droplet method. In this 

 method 3-day-old chicks are injected with 

 the preparation to be assayed and the chicks 

 killed 2 hours later. The thyroids are re- 

 moved and fixed in Carnoy's fluid for 1 hour 

 at room temperature, embedded in paraffin, 

 sectioned at 4 /a, and stained with Heiden- 

 hain's azan. The droplets in 25 successive 

 cross sections of follicles are counted. The 

 assay is about 70 times as sensitive as the 

 gravimetric method and 5 times as sensitive 

 as the cell height measurement method. 



4. P'" depletion method. Bates and Corn- 

 field (1957) used 1-day-old chicks and in- 

 jected them with 0.2 ml. P=*i solution (2 to 

 3 fxc). After 24 hours counts were made in 

 vivo with the aid of a scintilation counter. 

 The assay solution was injected and simul- 

 taneously 0.2 cc. of a solution containing 8 

 /i.g. thyroxine and either 5.0 or 0.5 mg. pro- 

 pylthiouracil (PTU) was injected. The PTU 

 was injected to prevent re-utilization of 

 I''^\ and the thyroxine was used to inhibit 

 secretion of TSH from the chick's anterior 

 l)ituitary. This procedure was repeated daily 

 for 2 to 3 days and daily counts were made. 

 A linear relationship between log dose and 

 response was obtained. The index of ac- 

 curacy was A = 0.20. 



5. I^^^ accumulation. One-day-old cock- 

 erels are kept without food or water and 

 receive 5 injections at 12-hour intervals. 

 Twelve hours after the last injection 0.1 to 

 10 /AC I^^^ are given and the chicks are killed 

 5 hours later. Thyroids are removed and ra- 

 dioactivity counted. This test can detect 

 0.001 and 0.005 U.S.P. units, whereas the 

 histometric methods (cell height) could not 

 detect these levels (Shellabarger, 1954). 



Oxytocic principle. Adult chickens (1.8 

 to 2.2 kg.) are anesthetized with sodium 

 phenobarbital and blood pressure is taken 

 witli tlie aid of a mercurv manometer with 



