The preovulatory follicle of the domestic hen is almost certainly a steroid-producing structure (see Shahabi, Norton & Nalbandov, 1975). However (Gilbert, 1971 ). A cut, about 2 cm long, was made with a scalpel approximately along the line of the stigma, though the exact position is not critical. This step must be carried out quickly, with one sweeping stroke, and it should be completed before much escape of yolk occurs. Scissors may be used instead of a scalpel but they were found to be less satisfactory because there was a tendency for a point to penetrate too deeply into the yolk mass. Immediately after it had been cut the follicle was inverted over a suitable dish containing an aqueous medium and the follicular contents (yolk, perivitelline layer, granulosa and basal lamina) (Gilbert, 1971) were allowed to fall into the medium. The choice of medium depended on the purpose for which the granulosa cells were being harvested.If the operation has been done correctly, the entire theca (Text- fig. 1, PI. 1, Fig. lb) remains held in the forceps, without contamination by yolk or granulosa material, and it can be used for studies of thecal activity. The yolk, covered by the perivitelline layer, the granulosa layer and the basal lamina (Text- fig. 1, PL 1, Fig. 3), settles as an almost undisturbed sphere on the floor of the vessel. The split occurs at a position different from that occurring during ovulation when the granulosa cells and the basal lamina remain with the theca (PI. 1, Fig. la).Removal of the granulosa layer, together with its associated basal lamina and perivitelline layer, from the yolk was carried out under a low-power lens or dissecting microscope with a black back¬ ground. The cut ends of the composite membrane surrounding the yolk were located, grasped with fine dissecting forceps and gently pulled away from the yolk thereby everting the membrane over the surface of the yolk: this was made easier by using the yolk as a mass to pull against. Care was taken to protect the yolk from undue disturbance because yolk material dispersed throughout the medium prevented clear observation and caused unnecessary contamination of the preparation. It

The embryonic lifespan of the chick is 22 days. Development in the first day takes place in the oviduct, and in the remaining 21 days in the shelled egg. There have been few attempts to culture oviductal embryos, though methods covering the first few days of development in ovo are well established and a method for the final 18 days of development through hatching has recently been devised. I have now succeeded in culturing the fertilized ovum of the chick (Gallus domesticus) for the total embryonic period by growing it in a series of separate culture systems. This is the first report of a complete in vitro method for a homoiothermic animal. The technique opens the way to the investigation of developmental events in birds that require access to the embryo at the single-cell stage, and in particular to the genetic manipulation of the fertilized ovum.

ABSTRACT:The main histological abnormality in congenital fiber type disproportion (CFTD) is hypotrophy of type 1 (slow twitch) fibers compared to type 2 (fast twitch) fibers. To investigate whether mutations in RYR1 are a cause of CFTD we sequenced RYR1 in seven CFTD families in whom the other known causes of CFTD had been excluded. We identified compound heterozygous changes in the RYR1 gene in four families (five patients), consistent with autosomal recessive inheritance. Three out of five patients had ophthalmoplegia, which may be the most specific clinical indication of mutations in RYR1. Type 1 fibers were at least 50% smaller, on average, than type 2 fibers in all biopsies. Recessive mutations in RYR1 are a relatively common cause of CFTD and can be associated with extreme fiber size disproportion.

Summary. At peak laying periods the ovary of the domestic hen contained 30\p=n-\100small yolky follicles with diameters varying between 1 and 8 mm. In general, the number of these healthy follicles decreased with increasing size in that there were about 20 follicles with a diameter of 1\p=n-\2mm and 1 follicle (mean < 1) with a diameter of 7\p=n-\8 mm. The number of follicles with diameters > 8 mm (the hierarchy of large, yolky follicles) varied between 4 and 7. By using a dye-marker, growth from 3 to 5 mm was estimated to take 3 days, from 5 to 8 mm, 2 days and from 8 mm to ovulation, 6 days. No information was obtained for growth between 1 and 3 mm because the dye did not enter these smaller follicles. Between 5 and 25 small yolky follicles were atretic. The reduction in the number of follicles with time and the high incidence of atresia suggests that this is a normal fate of small yolky follicles in birds with a high rate of lay. In marked contrast, only one large yolky follicle was observed to be atretic throughout the whole experiment. These two very different levels of atresia serve to define two distinct groups of follicles in the size range studied. Ovulation rate appears to be the product of two complementary mechanisms, one for the initiation of growth and the other controlling the rate at which the small yolky follicles are lost through atresia.