Reproduction in the Female

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22 Reproduction in the Female A. L. JOHNSON Department of Biological Sciences University of Notre Dame Notre Dame, Indiana 46556

I. ANATOMY OF THE FEMALE REPRODUCTIVE SYSTEM

I. Anatomy of the Female Reproductive System 569 A. Ovary 569 B. Oviduct and Sperm Storage Glands 574

The right ovary and oviduct are present in embryonic stages of all birds, but the distribution of primordial germ cells to the ovaries of the chicken becomes asymmetrical by day 4 of incubation; by day 10 regression of the right oviduct is initiated under the influence of Mullerian inhibiting substance (Hutson et al., 1985). The reproductive system of galliformes consists of a single left ovary and its oviduct, although on occasion a functional right ovary and oviduct may be present. Among the falconiformes and in the brown kiwi, both left and right gonads and associated oviducts are commonly functional, although the ovaries may be asymmetrical in size; in sparrows and pigeons, about 5% of specimens have two developed ovaries (see Romanoff and Romanoff, 1949; Kinsky, 1971).

II. Breeding and Ovulation–Oviposition Cycles 575 A. Ovulation–Oviposition Cycle and Rate of Lay 575 B. Parthenogenesis 577

III. Ovarian Hormones 577 A. Steroid Production and Secretion 578 B. Nonsteroidal Hormones and Growth Factors 580

IV. Hormonal and Physiologic Factors Affecting Ovulation 580 A. Hormones and Ovulation 580 B. Hormones during Sexual Maturation and Molt 583 C. Effect of Light on the Ovary and Ovulation 583 D. Photorefractoriness 584 E. Molt 584

V. Oviposition 584 A. B. C. D. E. F.

Neurohypophyseal Hormones 584 Prostaglandins 584 Postovulatory Follicle 585 Preovulatory Follicle 585 Other Factors 585 Broodiness and Nesting Behavior 585

A. Ovary The left ovary is attached by the mesovarian ligament at the cephalic end of the left kidney. The number of oocytes of the chick embryo increases from approximately 28,000 on the 9th day of development to 680,000 on the 17th day and subsequently decreases to 480,000 by the time of hatching, when oogenesis is terminated. The ovary of the immature bird consists of a mass of small ova, of which at least 2,000 are visible to the naked eye. Only a relatively few of these (250–500) reach maturity and are ovulated within the life span of most domesticated species, and considerably fewer mature in

VI. Composition and Formation of Yolk, Albumen, Organic Matrix, and Shell 586 A. B. C. D. E.

Yolk 586 Albumen 586 Organic Matrix 587 Layers of Crystallization 588 Calcium Metabolism 589

References 591

Sturkie’s Avian Physiology, Fifth Edition

569

Copyright 䉷 2000 by Academic Press. All rights of reproduction in any form reserved.

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wild species. The functionally mature ovary of the hen is arranged with an obvious hierarchy of follicles and in its entirety weighs 20–30 g. Commonly, there are four to six large yolk-filled follicles 2–4 cm in diameter accompanied by a greater number of 6- to 12-mm follicles in which yellow yolk deposition has been initiated and numerous small white follicles ⬍6 mm (Figure 1). The ovary receives its blood supply from the ovarian artery, which usually arises from the left renolumbar artery but may branch directly from the dorsal aorta (Hodges, 1965). Within the ovary, bloodflow is greatest to the five largest preovulatory follicles (Scanes et al., 1982; see Chapter 7 for further details). The ovarian artery divides into many branches and leads to a single follicular stalk, usually from two to four separate arterial branches. All veins from the ovary unite into the two main anterior and posterior veins, which subsequently drain into the posterior vena cava. Several workers have shown that the ovary is well innervated by both adrenergic and cholinergic fibers (Gilbert, 1969; Dahl, 1970b; Unsicker et al., 1983) and that a greater number of neurons are present within the theca layer as a follicle progressively matures. It is now clear that such innervation provides a variety of neurochemical (e.g., catecholamines) and neurohumeral factors (e.g., neurotropins, vasoactive intestinal peptide, substance P, calcitonin gene-related peptide) to the ovary and its follicles; such factors have been proposed to function in diverse roles such as in the organization of primordial follicles in the embryonic and early posthatch

ovary and the differentiation of preovulatory follicles in the adult ovary. 1. Ovarian Follicle The follicle consists of concentric layers of tissue that surround the oocyte and yolk, including: (1) the oocyte plasma membrane, (2) the perivitelline layer, (3) granulosa cells, (4) basal lamina (basement membrane), and (5) the theca (interna and externa) (Figure 2). The follicle is highly vascularized except for the stigma (the point of rupture during ovulation), which contains a lesser number of underlying small veins and arteries (Nalbandov and James, 1949). The main arteries from the stalk are directed toward the fastest growing follicles, branch into arterioles, and pass through the theca to the basal lamina to form arterial capillaries (Dahl, 1970a). The germinal vesicle (blastoderm) is localized within the germinal disc region of the granulosa layer. The avian oocyte remains arrested at the first meiotic prophase stage during follicle development and resumes meiosis (undergoes germinal vesicle breakdown) 4– 6 hr prior to ovulation. Granulosa cells of prehierarchal follicles are closely packed and cuboidal, and the granulosa cell layer may be several cells deep. As the follicle grows exponentially in diameter during the rapid growth phase, the granulosa cells become squamous in shape and form a single cell layer. Spaces and cell junctions form between granulosa cells to facilitate transport of yolk. Despite the dramatic increase in the circumference of the granulosa layer during this time relatively little cell proliferation occurs, except within the germinal disk region. Granulosa cell DNA synthesis and proliferation are 5- to 10-fold higher in prehierarchal follicles when compared to granulosa cells from hierarchal follicles; moreover, DNA synthesis is 2-fold higher in granulosa cells within the germinal disk region when compared to cells from the outer layer region (Tilly et al., 1992). The basal lamina of the hen follicle is approximately 1 mm thick and consists of collagen and the glycoprotein, fibronectin, among other components; these are deposited by the adjacent granulosa and theca layers. Fibronectin production and secretion is regulated by a variety of gonadal hormones and gonadotropins (Conkright and Asem, 1995). 2. Postovulatory and Atretic Follicles

FIGURE 1 Prehierarchal and hierarchal follicles of the laying hen ovary. F1, F2, F3, F4, F5, five largest hierarchal follicles; POF, postovulatory follicle; SWF, small white follicles, SYF, small yellow follicles. (Reprinted with permission from A. L. Johnson (1990), CRC Crit. Rev. Poult. Biol. 2, 319–346. Copyright CRC Press, Boca Raton, Florida.)

The postovulatory follicle (POF) contains the granulosa and thecal layers which remain subsequent to ovulation (Figure 1). The POF of the hen is metabolically active at a progressively lower rate for several days after ovulation, as indicated by the decreasing presence of enzymatic activity (Chalana and Guraya, 1978). Struc-

Chapter 22. Reproduction in the Female

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FIGURE 2A Diagrammatic relationship of follicle tissue layers immediately surrounding the oocyte with special emphasis on the coated vesicles of the perivitelline layer. (Redrawn from Wyburn et al., 1966, with permission of Cambridge University Press.)

tural resorption of the POF occurs, in part, via the process of apoptosis (Tilly et al., 1991b) (Figure 3) over a period of 6–10 days in the chicken and several months in the mallard. The POF may influence nesting behavior and has been demonstrated to be functional in timing oviposition (Gilbert et al., 1978). There is no structure in birds functionally analogous to the corpus luteum of mammals. Follicles that initially begin to grow but fail to reach the fully differentiated stage at which they are ovulated become atretic. The death and resorption of ovarian follicles also occurs via apoptosis (Tilly et al., 1991b); atresia is characterized by pronounced oligonucleosome formation within the granulosa cell layer, with considerably less apoptotic cell death within the theca layer (Figure 3). The subsequent reabsorption of the oocyte and yolk occurs either via the vascular system (involu-

tion atresia) or by the rupture of the thecal layers and slow loss of the yolk into the peritoneal cavity (bursting atresia). Atresia occurs with high incidence among prehierarchal follicles (⬍9 mm in diameter) and under normal physiological conditions is absent among preovulatory hierarchal follicles (Gilbert et al., 1983). Natural instances of atresia among hierarchal follicles occur, for example, subsequent to a change from the egg-laying state to incubation and broody behavior and with the onset of molt. Atresia of hierarchal follicles can also be induced by prolonged daily administration of equine chorionic gonadotropin (eCG) ( Johnson and Leone, 1985), by hypophysectomy (Yoshimura et al., 1993a), or by destruction of the germinal disk region (Yoshimura et al., 1994). The high rate of atresia in prehierarchal follicles correlates with the susceptibility of the granulosa cell layer

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FIGURE 2B Electron micrograph of tissue layers depicted in Fig. 2A. Abbreviations are as labeled in Fig. 2A. (From Perry and Gilbert, 1979, and from Agricultural Research Council Poultry Research Centre Report, Roslin, Midlothian, Scotland, 1980.)

to undergo apoptosis (Figure 3); by contrast, granulosa cells from hierarchal follicles are inherently resistant to undergoing cell death. A current area of intense research centers on investigations of cellular mechanisms mediating follicle atresia via apoptosis in vertebrate species. Apoptosis (sometimes inappropriately referred to as programmed cell death; see Wyllie, 1992) is an evolutionarily conserved process of cell death which serves to selectively eliminate cells/tissues during tissue remodeling or in the course of normal tissue turnover. Among the factors proposed to mediate cell viability, or alternatively, cell death are the Bcl-2-related family of proteins, the interleukin converting enzyme (ICE)-related family of enzymes, as well as a select group of protooncogenes (e.g., c-myc) and tumor suppressor genes (e.g., p53) (Williams and Smith, 1993; Wyllie, 1994; Tilly et al.,

1995a,b). It is significant to note that: (1) genes from each of these groups are expressed in developing chicken ovarian follicles, (2) the level of constitutive expression for each gene (death inducing/death suppressing) is correlated to inherent granulosa cell susceptibility or resistance to cell death, and (3) the level of mRNA expression for such genes is generally regulated by the same physiological factors (e.g., growth factors and gonadotropins) that promote hen follicle viability ( Johnson et al., 1993, 1997a,b). 3. Growth of the Follicle and Deposition of Lipid Growth of the follicle can be divided into three phases: (1) slow growth of follicles 60–100 애m in diameter, lasting months to years; (2) a several-month period


A PREOV. 17 h

6 h

POSTOV. 1 d

0.5 h

2-3 d

4-5 d

B N G

MA T

G

GA T

G

T

FIGURE 3 (A) Oligonucleosome formation, the hallmark of apoptosis, in preovulatory (Preov) and postovulatory (Postov) hen follicles. Abbreviations, h, hours before ovulation; d, days following ovulation. (B) Presence of oligonucleosomes in granulosa (G) and theca (T) tissues from morphologically normal (N), mildly atretic (MA), and grossly atretic (GA) follicles. (Adapted from Tilly et al., 1991b.)

of increasingly rapid growth that consists mainly of deposition of yolk protein; and (3) a rapid growth phase during the final 6–11 days prior to ovulation in domestic fowl, ducks, and pigeons (up to 16 days in some penguins; Grau, 1982) when the majority of yolk protein and lipids are deposited. In this final phase of development, the follicle of the domestic hen will, on average, transport up to 2 g of yolk protein per day, grow in diameter from 8 to 37 mm, and increase in volume by a factor of 3500 to 8000. Yolk protein formation occurs in the liver and is regulated primarily by gonadotropin and steroid hormones. Transport from the liver to the ovary occurs by the blood. Following release from capillaries within the theca layer, the plasma-borne precursors vitellogenin (VTG, a phosphoglycolipoprotein) and very low density lipoprotein (VLDL, functions mainly to transport triglycerides, phospholipids, and cholesterol) pass across the basement membrane and through gaps between granulosa cells to the plasma membrane of the oocyte (Figure 2). Translocation of VTG and VLDL across the oocyte plasma membrane is a receptor-mediated event

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(Shen et al., 1993). The receptor for VTG/VLDL is localized within coated vessicles of the oocyte plasma membrane but is not expressed in extraovarian tissues. Moreover, this receptor is absent in a genetically altered, nonlaying strain of chickens (the restricted ovulator, R/ O; Barber et al., 1991). The lack of VTG/VLDL receptor expression in this strain results in large accumulations of VTG and VLDL in the blood and apparently prevents ovarian follicles from entering the rapid growth phase of development. Following transport across the plasma membrane, VTG and VLDL become localized to yolk spheres where proteolytic processing to phosvitin, lipovitellin, triglycerides, cholesterol, and phospholipids by cathepsin D occurs (Retzek et al., 1992). Lipids and protein are deposited into the growing follicle at about the same ratio for most of the growth phase, but during the final rapid growth phase, relatively more lipid is incorporated. It has been suggested that deposition of yolk into the maturing follicle is terminated by 24 hr before ovulation. The ultrastructure of developing follicles has been well described (Wyburn et al. (1965), Rothwell and Solomon (1977), Perry et al. (1978a,b), and Gilbert et al. (1980)). It has been proposed that in chickens the decrease in egg production with age is in part caused by both an increase in the incidence of atresia and a reduction in the number of follicles reaching the final phase of rapid growth; that is, fewer follicles receive a proportionately greater quantity of yolk, resulting in larger sized eggs (Williams and Sharp, 1978; Palmer and Bahr, 1992). Mechanisms controlling development and maintenance of the follicular hierarchy are poorly understood. Pituitary gonadotropin involvement is indicated by the finding that hypophysectomy rapidly leads to follicular atresia, whereas ablation and treatment with a chicken pituitary extract results in the maintenance of the normal follicular hierarchy. Moreover, daily injections of follicle-stimulating hormone (FSH; 200 애g porcine FSH) or eCG (25 IU) were found to decrease the rate of atresia and increase the number of growing follicles in aging hens (Palmer and Bahr, 1992). On the other hand, daily injections of eCG at higher doses (75 IU) to intact laying hens stimulates numerous small follicles to enter the rapid growth phase and results in the elimination of the follicular hierarchy and the cessation of ovulation ( Johnson and Leone, 1985). Thus, levels of circulating FSH would appear to play a critical role in establishing and maintaining the preovulatory hierarchy as well as in regulating the rate of follicle atresia. Inhibin is one hormone that has recently been shown to directly influence circulating levels of FSH. This biologically active glycoprotein is a member of the transforming growth factor beta (TGF웁) superfamily of peptides and is composed of an 움- and 웁-

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subunit. In the hen ovary, inhibin is produced at highest levels by the four largest preovulatory follicles; surgical removal of preovulatory follicles results in a 80% decline in circulating inhibin levels and in a significant elevation in plasma FSH, but not luteinizing hormone (LH) ( Johnson, P.A. et al., 1993).

B. Oviduct and Sperm Storage Glands The oviduct of the hen is derived from the left Mullerian duct. In the chicken, the left oviduct develops rapidly after 16 weeks of age and becomes fully functional just prior to the onset of egg production. The oviduct consists of five distinguishable regions: infundibulum, magnum, isthmus, shell gland, and vagina. (For details on general oviduct histology, see Aitken (1971) and King (1975); on the infundibulum and magnum, see Wyburn et al. (1970); for the isthmus, see Hoffer (1971) and Solomon (1975); and for the shell gland, see Breen and DeBruyn, 1969, Nevalainen (1969), and Wyburn et al. (1973).) A dorsal and a ventral ligament suspend the oviduct within the peritoneal cavity. Over recent years, the chicken oviduct has provided the molecular biologist with a superb model for the study of steroid hormone regulation of gene expression in eukaryotes, and in particular, the regulation of ovalbumin synthesis by steroids (see, for example, Schrader et al., 1981). 1. Infundibulum Subsequent to ovulation, the ovum is engulfed by the infundibulum (a structure not directly connected to the ovary) where it resides for approximately 18 min (range, 15–30 min). Occasionally, the ovum fails to be picked up by the infundibulum (an ‘‘internal ovulation’’) and is reabsorbed in 24 hr or less. Infundibular activity appears not to be controlled by ovulation per se, as foreign objects placed into the abdominal cavity prior to ovulation will also be taken up, and there are reports that entire unovulated follicles may be engulfed and later laid as fully developed eggs. Fertilization of the ovum occurs in the infundibulum, and it is here that the first layer of albumen is produced in the chicken. 2. Magnum The ovum next passes to the largest portion of the oviduct, the magnum (a length of 33 cm in the chicken), where in the mature female the majority of albumen is formed. Estrogen stimulates epithelial stem cells to develop into three morphologicaly different cell types: tubular gland cells, ciliated cells, and goblet cells. Tubular glands are responsible for the production of ovalbumin, lysozyme, and conalbumin under the stimulatory

control of estrogen, while goblet cells synthesize avidin following exposure to progesterone and estrogen (Tuohimaa et al., 1989). A measurable amount of calcium secretion occurs within the magnum, but at no time during the laying cycle is calcium secretion greater than the basal rate of secretion found in the shell gland (Eastin and Spaziani, 1978a). The ovum remains in the magnum approximately 2–3 hr. 3. Isthmus The isthmus is clearly distinguishable from the magnum. It has a thick circular muscle layer muscle, and glandular tissue is less developed compared to the magnum. This tissue is characterized by a layer of epithelial cells with underlying tubular gland cells. Both inner and outer shell membranes are formed during the 1- to 2-hr (mean time, 1 hr and 14 min) passage through the isthmus. There is some evidence to suggest that shell formation, particularly the mammillary cores, is initiated in the distal portion of the isthmus. 4. Shell Gland The shell gland (uterus) is characterized by a prominent longitudinal muscle layer lined medially with both tubular gland and unicellular goblet cells. Prior to calcification, the egg takes up salts and approximately 15 g fluid into the albumin from the tubular glands, a process termed ‘‘plumping’’; this fluid contains carbonic anhydrase, acid phosphatase, and esterase activty plus bicarbonate and a variety of additional ions (Salevsky and Leach, 1980). The ovum remains in the shell gland for 18–26 hr depending on cycle length. Calcification within the shell gland is associated with stimuli initiated by ovulation or by neuroendocrine factors that control and coordinate both ovulation and calcium secretion. Additional evidence suggests that distension of the shell gland by the egg is not a stimulus for initiating a high rate of calcium secretion, which is characteristic of calcification, nor is autonomic innervation involved (Eastin and Spaziani, 1978a). Calcification of the egg at first occurs slowly, increases to a rate of up to 300 mg/hr over a duration of 15 hr, then again slows during the last 2 hr before oviposition; shell pigments (primarily protoporphyrin and biliverdin) are deposited via ciliated cells of the shell gland epithelium during the period of 3 hr through 0.5 hr prior to oviposition. 5. Vagina The vagina is separated from the shell gland by the uterovaginal sphincter muscle and terminates at the cloaca. There are numerous folds of mucosa, which are


lined by ciliated and nonciliated cells, and a few tubular mucosal glands which may have a secretory function. The vagina has no role in the formation of the egg, but, in coordination with the shell gland, participates in the expulsion of the egg. In domestic fowl, spermatozoa are stored in specialized sperm-storage tubules located in the uterovaginal region and remain viable for a period of 7–14 days in the chicken and for greater than 21 days in the turkey hen. Uterovaginal junction glands are apparently devoid of innervation and contractile tissue, but possess a welldeveloped vascular system (Gilbert et al., 1968; Burke et al., 1972; Tingari and Lake, 1973). Evidence suggests that spermatozoa fill the uterovaginal glands in a sequential fashion without mixing so that with successive inseminations, sperm from the latest insemination is most likely to fertilize an ovum. Following oviposition of each egg, spermatozoa are released from these tubules by an unknown mechanism and migrate to the infundibulum for fertilization (Zavaleta and Ogasawara, 1987). 6. Blood Flow and Innervation to the Oviduct The blood supply to the oviduct and shell gland of the domestic hen has been described by Freedman and Sturkie (1963) and Hodges (1965). For a review of the vasculature to the oviduct of additional avian species, see Gilbert (1979). Blood flow to the shell gland of the hen is increased during the presence of a calcifying egg compared to times when no egg is present (Scanes et al., 1982). For additional details on blood flow, see Chapter 7. The oviduct is innervated by both sympathetic and parasympathetic nerves. Innervation of the infundibulum is via the aortic plexus and the magnum by the aortic and renal plexuses (Hodges, 1974). Sympathetic innervation of the shell gland is via the hypogastric nerve, which is the direct continuation of the aortic plexus. Parasympathetic pelvic nerves, which constitute the left pelvic plexus, arise from the pelvic visceral rami of spinal nerves 30–33. 7. Oviduct Motility Cilia are found along the entire length of the oviduct, and a likely function of these cilia is that of sperm transport. Egg transport is primarily accomplished by contractions of the oviduct; oviduct musculature functions as a stretch receptor and the mechanical stimulus is produced by the ovum itself (Ariamaa and Talo, 1983). Changes in electrical activity and oviduct motility have been recorded in the magnum, isthmus, and shell gland during the ovulatory cycle, with the greatest frequency of electrical activity and contractions occurring in the

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shell gland at the time of oviposition (Shimada, 1978; Shimada and Asai, 1978). Both 움- and 웁-adrenergic receptors are present throughout the length of the oviduct and have been shown to affect oviduct motility (Crossley et al., 1980).

II. BREEDING AND OVULATION– OVIPOSITION CYCLES Breeding cycles among species of birds can be classified according to the length of the cycle and the time of the year at which each species becomes reproductively active. Continuous breeders, such as the domestic hen or Khaki Campbell duck, are, under optimal conditions, reproductively active throughout the year. Most wild species that breed in temperate, subarctic, and arctic zones display yearly cycles, while birds adapted to tropical or desert climates may breed with cycles less than a year, at 6-month intervals, or when favorable conditions exist (opportunistic breeders) (for further details, see Lofts and Murton, 1973; Wingfield and Farner, 1993). Wild birds usually lay one or more eggs in a clutch and then terminate laying to incubate the eggs. The number of eggs per clutch and total number of clutches vary with the species and season. For example, some birds, such as the auk or sooty tern (Sterna fuscata), lay a single egg to incubate. The king penguin (Aptenodytes patogonica) will lay but one egg and breeding will not necessarily occur on a yearly basis. In contrast, the European partridge will lay a single clutch per year consisting of as many as 12–20 eggs. The pigeon usually lays two eggs per clutch and averages eight clutches per year. The interval between clutches is approximately 45 days in the fall and winter and from 30 to 32 days in the spring and early summer. Finally, clutch size of the bobwhite quail declines with advancing season, from a mean high of 19.2 eggs in early May to 11.3 eggs in late July. In some species, when eggs are destroyed or removed from the nest early enough in the breeding season birds may produce an additional clutch (indeterminate layers; an example is the duck Anas platyrhynchos) (Donham et al., 1976). Determinate layers fail to lay additional eggs on the removal or destruction of eggs in the nest. In seasonal (or noncontinuous) breeders, the ovary undergoes periods of growth and regression. The weight of the European starling ovary may vary from 8 mg during the regression phase to 1400 mg at the height of the breeding season.

A. Ovulation–Oviposition Cycle and Rate of Lay The ovulation–oviposition cycle (the time from ovulation of an ovum to the oviposition of the egg) of the domestic hen generally ranges from somewhat longer

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than 24 hr (24⫹) to 28 hr in length, and ovulations proceed uninterrupted for several days or as long as 1 year or more in extreme instances. The number of eggs laid on successive days is called a sequence, and each sequence is separated by 1 or more pause days on which no egg is laid. The term clutch is sometimes used synonomously with sequence, although the former term is generally considered more appropriate to describe the group of eggs laid by nondomesticated species prior to incubation behavior. The 24⫹-hr ovulation–oviposition cycle is characteristic of the chicken, turkey, bobwhite quail, and several other species including the Japanese quail. By contrast, the interval between successively laid eggs in the pigeon has been reported to be 40–44 hr and in the Khaki Campbell duck is 23.5–24.5 hr (see Johnson, 1986). In the domestic hen, the longer the sequence, the shorter the duration of the ovulation–oviposition cycle. The delay, or ‘‘lag,’’ in hours between the oviposition of successive eggs in a sequence is not constant (Table 1). The differences in lag time within a sequence represent mainly variations in the amount of time between oviposition and the subsequent ovulation; this interval ranges from 15 to 75 min. It is clear that even when the lag between the oviposition of successive eggs approaches 24 hr there remains a progressive shift toward laying the egg later and later in the day. As the sequence becomes shorter, the lag becomes greater and the length of the ovulation–oviposition cycle deviates more from 24 hr. The total lag time between the first ovulation of a sequence (C1 ovulation) and last ovulation of a se-

quence (Ct ovulation) is greater in chickens (4–8 hr depending on sequence length) than in Japanese quail (1.5–5 hr). The normal release of LH in the hen is restricted to a 4- to 11-hr period (the ‘‘open period’’) beginning at the onset of the scotophase (dark phase). The timing and regulation of the ‘‘open period’’ is as yet incompletely understood. For further details concerning the ovulation–oviposition cycle and the sequence, see Fraps (1955), van Tienhoven (1981), and Etches (1990). A chicken typically ovulates the first egg of a sequence early in the photophase, and the timing of ovulation is synchronized by the onset of the scotophase. In comparison, Japanese quail ovulate the Cl egg 8–9 hr after the onset of the photophase, and this appears to be synchronized by the timing of the light phase. The rate of lay refers to the number of eggs laid in a given period of time, irrespective of the regularity or pattern of laying. For example, a chicken laying at the rate of 50% for 60 days produces 30 eggs, the same number of eggs as another that lays at a rate of 75% for 40 days. On reaching sexual maturity (at 앑20–22 weeks of age), the domestic hen lays with an erratic pattern (sequences with 2 or more pause days or occasionally more than 1 egg per day) and with a high incidence of abnormal (soft-shelled and double-yolked) eggs for the first 2 weeks. Six to 10 weeks after the onset of egg laying, production peaks (frequently at a rate of 90% or more) and gradually decreases over a period of 40–50 weeks, depending on whether the chicken is an egg- or meat-type breed. Subsequently, egg production progressively declines and the hen begins a period of

TABLE 1 Egg Production in the Domestic Hen Housed under a Photoperiod of 14 hr L:10 hr Da Rate of egg production (%) 66

75

80

83

86

88

89

90

Number of eggs in continuous sequence 2 Position in sequence 1 09:05 2 13:30 3 4 5 6 7 8 9 Average interval between consecutive eggs: 28:25 a

3

4

5

6

7

8

9

08:41 11:26 15:15

08:01 10:09 12:03 15:23

07:24 09:22 10:26 11:46 15:05

07:33 09:20 10:34 11:30 12:43 15:40

07:45 09:12 10:10 10:51 11:39 12:38 15:28

07:36 08:59 09:57 10:26 10:55 11:43 12:49 15:44

07:18 08:39 09:25 09:44 09:53 10:26 10:59 12:02 15:26

27:17

26:27

25:55

25:37

25:17

25:09

25:0

Lights on 06:00 hr. From Etches and Schoch (1984).


molt. A typical laying strain of chicken will produce 280 or more eggs in a 50-week production period.

B. Parthenogenesis Parthenogenesis (development from an unfertilized oocyte) has been documented in turkeys; 32–49 % of infertile eggs may initiate development, but the embryos usually die at an early stage (Olsen, 1960). Genetic selection can increase the incidence of parthenogenesis in turkeys, and viable poults are homozygous diploid males that are often sexually competent. Cytologic studies indicate that parthenogenesis in turkeys is initiated from a haploid oocyte and proceeds to the diploid state after a meiosis that is unaccompanied by cytokinesis (mitosis)

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(see van Tienhoven, 1983). Parthenogenesis occurs much less frequently in chickens, while its occurrence in nondomesticated species has apparently not been documented.

III. OVARIAN HORMONES While much work has been published concerning the expression of steroidogenic enzymes and the production and metabolism of ovarian steroids in preovulatory, hierarchal follicles of the domestic fowl, only recently has the steroidogenic profile of prehierarchal (⬍9 mm) follicles been described (Figures 4 and 5; see, for instance, Tilly et al., 1991a; Kato et al., 1995; Tabibzadeh et

FIGURE 4 Steroid synthesis by granulosa and theca tissues from prehierarchal (⬍9-mm diameter) and hierarchal follicles of the laying hen. Note that while a three-cell model of steroidogenesis is proposed for hierarchal follicles, the granulosa layer of prehierarchal follicles produces virtually no steroids. 17움OH-ase, cytochrome P450 17움-hydroxylase; 3웁-HSD, 3웁-hydroxysteroid dehydrogenase; FSH-R, folliclestimulating hormone receptor; LDL, low density lipoprotein; LH-R, luteinizing hormone receptor; SCC, cytochrome P450 cholesterol side-chain cleavage; VIP-R, vasoactive intestinal peptide receptor.

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FIGURE 5 Luteinizing hormone- and follicle-stimulating hormone-induced progesterone production in granulosa cells from follicles at different stages of development. Abbreviations: rhFSH, recombinant human FSH; oLH, ovine LH. (From Johnson, 1993.)

al., 1995). Moreover, while numerous additional factors (i.e., prostaglandins, neurohormones, growth factors) and/or their receptors are known to be expressed in ovarian tissues, the function(s) for many of these has yet to be fully established. The present discussion emphasizes recent research (derived almost entirely from domesticated fowl) related to steroid synthesis and the process of ovarian follicle differentiation. For more detailed information, refer to Johnson (1986,1990,1994). There is comparatively less information about the endo-

crinology of reproduction in wild species; however, for a comprehensive discussion of annual reproductive cycles see Wingfield and Farner (1993).

A. Steroid Production and Secretion 1. Hierarchal Follicles Fully potentiated steroid production by preovulatory follicles is best described by a three-cell model (Porter et al., 1989; Nitta et al., 1991) and occurs predominantly


via the ⌬4 steroidogenic pathway. The granulosa layer produces predominantly progesterone which serves as a precursor for androstenedione and testosterone synthesis by the theca layer and to a lesser extent by granulosa cells (Figure 4). Progesterone is also the primary steroid involved in potentiating the preovulatory surge of LH that precedes ovulation by 4–6 hr. The theca layer also expresses cytochrome P450 cholesterol sidechain cleavage (P450scc) activity; however, the predominant steroid product of the theca interna layer is androstenedione while cells in the theca externa synthesize estrogen. Steroid production by granulosa and theca cells is predominantly under the regulatory control of LH acting via the adenylyl cyclase/cAMP second messenger signaling pathway (Bahr and Calvo, 1984; Tilly and Johnson, 1989). LH receptor mRNA is expressed in granulosa and theca tissue of all hierarchal follicles ( Johnson et al., 1996), and LH-induced adenylyl cyclase responsiveness increases within the granulosa layer as a follicle approaches the time of ovulation. By contrast, FSH binding to membranes and FSH-induced cAMP formation and steroidogenesis is low to nonexistent in hierarchal follicles (Ritzhaupt and Bahr, 1987; Johnson, 1993). The theca cell adenylyl cyclase system is highly sensitive to LH, but is essentially unresponsive to FSH. Finally, there is some evidence for LH-stimulated steroid production occurring within both follicle layers via inositol 1,4,5-trisphosphate (IP3) formation and calcium mobilization (Hertelendy, 1989; Levorse et al., 1991). In contrast, activation of the diacylglycerol/protein kinase C second messenger system attenuates LHinduced steroid production by granulosa and theca cells (Tilly and Johnson, 1991). Among the potential physiological factors that may act via protein kinase C are included growth factors [e.g., transforming growth factor 움 (TGF움) and epidermal growth factor (EGF)] and prostaglandins. Nevertheless, it is significant to note that while TGF움 attenuates LH-induced steroid production in the F1 granulosa cells, the extent of this inhibition is limited to approximately 50% of maximal stimulation. It is proposed that this inhibitory action is related to the termination of the preovulatory progesterone surge. Of additional interest is the observation that LHinduced cAMP accumulation and androstenedione production from the theca layer dramatically decline within the F1 follicle (Marrone and Hertelendy, 1985); this ‘‘desensitization’’ may be mediated via protein kinase C activation. Presumably, the decrease in the rate of progesterone metabolism to androstenedione ensures maximal progesterone secretion at the time of the preovulatory LH surge. Arachidonic acid may function as an additional hormone-induced second messenger and has been de-

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termined to inhibit LH-induced progesterone production from F1 granulosa cells. While this inhibitory action occurs independently from protein kinase C activation, the specific endocrine/paracrine factor(s) that activate this system has yet to be identified ( Johnson and Tilly, 1990a,b). Finally, the requirement and involvement of a number of ions (e.g., Ca2⫹, Na⫹, K⫹,Cl⫺) and ion channels in granulosa cell steroid production has been investigated (e.g., Morley et al., 1991; Asem and Tsang, 1989). 2. Prehierarchal Follicles and Stromal Tissue The theca layer of prehierarchal (⬍8-mm diameter) follicles, as well as stromal tissue from prepubertal pullets and laying hens, is steroidogenically active, both occurring predominantly via the ⌬5 steroidogenic pathway (Figure 4) (Kowalski et al., 1991; Levorse and Johnson, 1994). By contrast, granulosa cells from prehierarchal follicles are incapable of synthesizing progesterone due to the lack of P450scc activity, although they do possess the ability to produce cAMP in response to FSH treatment (Tilly et al., 1991a). While granulosa cells from prehierarchal follicles express 3웁-hydroxysteroid dehydrogenase (3웁-HSD) activity, it is unclear whether any significant amount of progesterone is produced by this cell layer in vivo. As selection of a follicle into the hierarchy is proposed to occur (presumably at a rate of one per day) from a pool of approximately 8 to 14 6- to 8-mm diameter follicles, it has been proposed that the process of follicle recruitment is coupled to the initial expression of LH receptor mRNA and the acquisition of functional P450scc enzyme activity within the granulosa layer. Expression of P450scc mRNA and the initiation of enzyme activity in vitro is induced by culture with FSH, and this induction is prevented by coculture with TGF움 or EGF (Li and Johnson, 1993). This latter finding is of significance as such results may help to explain how prehierarchal follicles are maintained in a relatively undifferentiated state in vivo despite their continued, daily exposure to circulating concentrations of FSH. Once the selection of a follicle occurs, granulosa cells begin the transition from being predominantly FSH and VIP dependent (granulosa cells from follicles up to approximately 12-mm in diameter) to becoming primarily LH dependent (hierarchal follicles) (Figure 5). 3. Postovulatory and Atretic Follicles Progesterone content of the most recent postovulatory follicle declines during the first 15 hr after ovulation (Dick et al., 1978), although LH-stimulable adenylyl cyclase activity is present for at least 24 hr after ovulation

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(Calvo et al., 1981). Both the enzymes 3웁-HSD and 17웁hydroxysteroid dehydrogenase have been identified in follicles during the early stages of atresia; however, this activity is rapidly lost as atresia progresses.

B. Nonsteroidal Hormones and Growth Factors Prostaglandins E and F have been identified in preovulatory and postovulatory follicles (Hammond et al., 1980; Shimada et al., 1984); however, prostaglandin production is not stimulated by gonadotropins in vitro (Hertelendy and Hammond, 1980). Norepinephrine (NE) and to a lesser extent epinephrine (EPI) and dopamine are found predominantly within the theca layer of preovulatory follicles, and tissue levels of EPI and NE peak approximately 6–9 hr prior to ovulation (Bahr et al., 1986). Vasoactive intestinal peptide (VIP) is localized within nerve terminals of the theca layer and is proposed to act on the adjacent granulosa layer via the adenylyl cyclase/cAMP second messenger system. For instance, VIP has been found to stimulate steroidogenesis and plasminogen activator activity in preovulatory (F1) follicle granulosa and theca cells ( Johnson and Tilly, 1988; Tilly and Johnson, 1989) and to promote differentiation and suppress apoptosis in 6- to 8-mm follicle granulosa cells ( Johnson et al., 1994; Flaws et al., 1995). The neurohypophyseal hormones arginine vasotocin and mesotocin are produced in granulosa and theca tissue of hierarchal follicles, and levels of each vary with a differential pattern during the ovulatory cycle (Saito et al., 1990). Several locally produced growth factors and their receptors have recently been implicated in regulating ovarian function via paracrine effects, including TGF움 and its receptor, the EGF receptor ( Johnson, 1994; Onagbesan et al., 1994), TGF웁 (Law et al., 1995), nerve growth factor (NGF) and the p75 (low affinity) NGF receptor ( Johnson, unpublished data), stem cell factor and its receptor, c-kit ( Johnson, 1993; Jensen and Johnson, 1995), and insulinlike growth factor I (IGF-I) (Roberts et al., 1994). It is inevitable that many additional physiologically relevant growth factors and receptors have yet to be identified within avian ovarian tissue.

IV. HORMONAL AND PHYSIOLOGIC FACTORS AFFECTING OVULATION Ovulation in the domestic hen generally follows an oviposition within 15–75 min except for the first ovulation of a sequence, which is not associated with oviposition. Neither the premature expulsion of the egg (which

can be induced with prostaglandins or other agents) nor a delayed oviposition (affected by epinephrine or progesterone) influences the time of ovulation. Ovulation occurs via a rupture along the follicle stigma, and this process likely involves multiple, often redundant, factors which include proteolytic enzymes (e.g., collagenase, plasminogen activator), vasoactive substances and the process of apoptosis. For additional information, see Johnson (1986).

A. Hormones and Ovulation 1. Luteinizing Hormone Releasing Hormone Avian species express two different forms of luteinizing hormone-releasing hormone (LHRH-I and LHRHII; see Chapter 14); however, only LHRH-I appears to be directly involved in regulating LH secretion (Sharp et al., 1990). Injection of LHRH-I induces ovarian steroid production and premature ovulation, whereas in vivo administration of LHRH antiserum has been shown to block ovulation. These effects, however, are mediated via the pituitary and LH secretion, and there is no evidence of a direct ovulation-inducing effect of either LHRH moiety within the ovary. 2. Luteinizing Hormone Plasma concentrations of LH in the domestic hen (and most other birds studied to date) peak 4–6 hr prior to ovulation (Figure 6) ( Johnson and van Tienhoven, 1980). It is likely that this preovulatory surge provides a direct stimulus for germinal vesicle breakdown and for subsequent ovulation. Some workers have reported an additional peak of LH at 14–11 hr prior to ovulation

FIGURE 6 Diagramatic representation of circulating concentrations of ovarian steroids and pituitary gonadotropins during the ovulatory cycle of the hen. A4, androstenedione; DHT, 5움-dihydrotestosterone; E1, estrone; E2, estradiol-17웁; FSH, follicle-stimulating hormone; LH, luteinizing hormone; P, progesterone; T, testosterone. (Reprinted with permission from A. L. Johnson (1990), CRC Crit. Rev. Poult. Biol. 2, 319–346.)


(Etches and Cheng, 1981); however, the significance of this second peak has yet to be determined. In addition to these ovulatory–oviposition cycle-related peaks there occurs a crepuscular (occurring at the onset of darkness) peak of LH, which has a periodicity of 24 hr and has been proposed to act as a timing cue for the subsequent preovulatory LH surge ( Johnson and van Tienhoven, 1980; Wilson et al., 1983). Injection of LH into laying hens almost always increases plasma concentrations of progesterone, estrogens and androgens; however, the ovulatory response is dependent on the stage within the sequence. For instance, LH treatment 14–11 hr prior to the first ovulation of a sequence results in premature ovulation, whereas the same treatment before a midsequence ovulation commonly results in follicle atresia and blocked ovulation (Gilbert et al., 1981). Passive immunization of laying hens with antiserum generated against partially purfied chicken LH results in the cessation of ovulation for approximately 5 days and extensive atresia of existing follicles (Sharp et al., 1978). 3. Follicle-Stimulating Hormone A rise in plasma follicle-stimulating hormone (FSH) has been observed 15 hr prior to ovulation in the domestic hen (Scanes et al., 1977a) (Figure 6), and this occurs coincident with an increase in FSH binding to ovarian tissue (Etches and Cheng, 1981). Other researchers find less pronounced cycle-related fluctuations in circulating FSH (Krishnan et al., 1993). Endocrine mechanisms regulating pituitary release of avian FSH are not clear. For instance, Hattori et al. (1986) have reported that LHRHI induces FSH secretion in vivo and in vitro in the quail, while Krishnan et al. (1993) failed to detect any stimulatory effect of LHRH-I. The reasons for this discrepancy are not apparent. A primary role for FSH is related to granulosa cell differentiation and the induction of steroidogenesis in prehierarchal follicle granulosa cells (see above). Although recombinant FSH is capable of inducing ovulation in mammals, this apparently has not been tested in avian species. 4. Progesterone Highest concentrations of plasma progesterone are found 6–4 hr before ovulation and coincide with the preovulatory LH peak (Figure 6). This increase is predominantly the result of progesterone secretion by the largest preovulatory follicle (Etches, 1990). Systemic and intraventricular injection of progesterone can induce both a preovulatory surge of LH and premature ovulation, while administration of progester-

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one antiserum prior to the preovulatory surge of progesterone can block ovulation. Evidence suggests that the preovulatory LH surge is preceded by an increase in progesterone in view of the findings that: (1) in the absence of the normal preovulatory rise in progesterone (blocked by the steroidogenesis inhibitor, aminoglutethimide), there is no preovulatory increase in plasma LH; and (2) intramuscular injection of progesterone in aminoglutethimide-treated hens induces a normal preovulatory LH surge in the absence of any increase in plasma testosterone or estrogen ( Johnson and van Tienhoven, 1984). Moreover, results from a study of hypophysectomized hens suggest that progesterone, in the absence of preovulatory gonadotropin, can induce ovulation (Nakada et al., 1994). Progesterone circulates in the blood bound to a highaffinity corticoid-binding globulin or to albumin and other 웂-globulins. Progesterone-specific receptors have been demonstrated in the hypothalamus and pituitary of the hen and the number of receptors is influenced by the reproductive state. In the oviduct, progesterone receptors are present in surface epithelial cells, tubular gland cells, stromal fibroblasts, and smooth muscle cells in the arterial walls and myometrium (Yoshimura and Bahr, 1991a). These findings provide evidence for a role of progesterone in the production of avidin, contraction of the myometrium, and shell formation. Moreover, progesterone receptors are expressed within granulosa, theca, and germinal epithelial cells of preovulatory follicles (Isola et al., 1987; Yoshimura and Bahr, 1991b). The localization of such receptors associated with the stigma region provides a physiological mechanism by which progesterone may mediate a direct effect on ovulation. 5. Androgens Peak preovulatory concentrations of testosterone occur 10–6 hr prior to ovulation, whereas highest levels of 5움-dihydrotestosterone (DHT) occur approximately 6 hr before ovulation (Figure 6). The increase in testosterone at this time is the result of secretion from at least the four largest follicles. The role of androgens in ovulation is, as yet, unclear. Intraventricular injections of testosterone fail to release LH or induce premature ovulation, and peripheral injections of testosterone that generally result in unphysiologically high circulating concentrations of testosterone are required to stimulate LH secretion and induce ovulation. Moreover, ovulation can occur in the absence of any preovulatory increase in plasma testosterone ( Johnson and van Tienhoven, 1984). On the other hand, in vivo administration of antitestosterone serum effectively blocks ovulation, while tes-

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tosterone treatment induces ovulation in vitro (Tanaka and Inoue, 1990). Both granulosa and theca cells of prehierarchal and hierarchal follicles express an androgen receptor, and androgens regulate steroidogenesis and plasminogen activator activity via a paracrine/autocrine action (Tilly and Johnson, 1987; Lee and Bahr, 1990; Yoshimura et al., 1993b). As in the male, androgens are responsible for the growth and coloring of the comb and wattles at the time of sexual maturation and synergize with estrogen to induce medullary ossification. In addition, androgens induce protein synthesis in the oviduct of estrogenprimed birds; however, the mechanism has been less intensively studied than that for progesterone.

6. Estrogens Both estradiol-17웁 and estrone show highest plasma concentrations 6–4 hr before ovulation with a smaller, more inconsistent rise in estrogens 23–18 hr before ovulation (Figure 6). Preovulatory follicle secretion of estradiol increases in each of the four largest follicles 6–3 hr prior to ovulation and is greatest in the third- and fourthlargest follicles. Overall, however, the majority of estrogen produced by the ovary originates from prehierarchal follicles. Like testosterone, estrogens are unlikely to be involved in the direct induction of LH secretion or ovulation, as injection of estradiol in laying hens either inhibits or has no effect on ovulation or LH secretion. Moreover, ovulation can occur in the absence of a preovulatory increase in plasma estrogens. Estrogens, together with progesterone, are required for priming of the hypothalamus and pituitary in order that progesterone can induce LH release (Wilson and Sharp, 1976). Ovarian estrogens have a variety of additional functions related to reproduction, including the regulation of calcium metabolism for shell formation (Etches, 1987), induction of its own receptor in the oviduct (Moen and Palmiter, 1980), and induction of progesterone receptor expression in the ovary and reproductive tract (Pageaux et al., 1983; Isola et al., 1987). Treatment of immature Japanese quail and young female chickens with estradiol enhances growth of the oviduct and promotes the formation of tubular secretory glands and epithelial differentiation. Moreover, estradiol induces the synthesis of ovalbumin, conalbumin, ovomucoid, and lysozyme in the oviduct (Palmiter, 1972) and vitellogenin in the liver (Deely et al., 1975). Secondary sex characteristics, such as color and shape of plumage and sexual behavior, are also under the control of estrogens. Sexual differentiation of the female brain is under the influence of estrogen, as indicated by

the finding that treatment of female quail embryo with an antiestrogen results in behavioral masculinization.

7. Corticosterone Plasma concentrations of corticosterone display both a daily (photoperiod-related) rhythm and a peak coincident with oviposition. The role of corticoids on the ovary and in the ovulatory process is unclear, as both facilitory and inhibitory actions have been described. While there is some evidence to suggest that the adrenal gland, via corticosterone secretion, regulates the timing of the preovulatory LH surge (Wilson and Cunningham, 1980), there is no ovulation-related increase in circulating corticosterone. Injection of corticosterone, deoxycorticosterone, or ACTH induces premature ovulation; however, it is unlikely to act at the level of the hypothalamus and/or pituitary to directly induce LH release (Etches et al., 1984a,b). On the other hand, infusion of corticosterone blocks the photoperiod-induced rise in LH as well as the gonadotropic effects of eCG on follicle growth (Petitte and Etches, 1988, 1989). In several wild species seasonal increases of plasma corticosterone are associated with egg laying (e.g., White-crowned sparrow, European starling, Western meadowlark) or with brooding behavior (e.g., Pied flycatcher), whereas in others (e.g., Canada goose) there is little change or a decrease in corticosterone during the breeding season. Absolute plasma levels of corticosterone can be misleading, however, as the capacity of the corticosteroid binding globulin may also fluctuate with season (Wingfield and Farner, 1993).

8. Prolactin It is well accepted that prolactin is associated with parental behavior and may also play a role in osmoregulation, especially in marine birds. Prolactin secretion from the anterior pituitary is primarily under the stimulatory control of vasoactive intestinal peptide (see Chapter 14). Circulating levels of prolactin increase at the onset of egg laying in many species of birds, including the ruffed grouse (Etches et al., 1979), bantam fowl (Sharp et al., 1979), turkey hen (Proudman and Opel, 1981), Japanese quail (Goldsmith and Hall, 1980), pied flycatcher (Silverin and Goldsmith, 1990), and song sparrow (Wingfield and Goldsmith, 1990). During the ovulation–oviposition cycle of the domestic hen, prolactin is highest approximately 10 hr and lowest 6 hr prior to ovulation, though it is unlikely that prolactin plays a direct role in the ovulatory process. Prolactin levels are also elevated during the summer months in response to increasing photoperiod.


Expression of prolactin receptor mRNA and prolactin binding has been detected in crop-sac mucosa, liver, brain, ovary, and oviduct (Tanaka et al., 1992). Recent data indicate that prolonged elevated prolactin levels (as occurs during incubation; see below) have an antisteroidogenic effect on the ovary, in part via inhibition of steroidogenic enzyme gene expression (Tabibzadeh et al., 1995), and act on the neuroendocrine system to reduce hypothalamic GnRH levels and inhibit LH secretion (Rozenboim et al., 1993). 9. Other Factors Preovulatory follicles produce prostaglandins, and the secretion of prostaglandin (PG) F2움 by the F1 follicle is greatest around the time of ovulation. Although prostaglandins do not affect granulosa cell steroid production, both PGE1 and PGE2 enhance follicle plasminogen activator activity (Tilly and Johnson, 1987). This latter action might suggest a role for PGs in mediating the enzymatic rupture of the stigma. In addition, PGF2움, PGE2, acetylcholine, and oxytocin increase the contraction of smooth muscles in the connective tissue wall of the follicle in vitro and may, in combination with proteolytic enzymes, play a role in the rupture of the stigma at ovulation (Yoshimura et al., 1983). Nevertheless, it is clear that PGs are not obligatory for ovulation to occur as neither the administration of prostaglandins nor the injection of indomethacin into the follicle affects the timing of ovulation. Administration of serotonin (5-hydroxytryptamine) has been reported to block ovulation, but this result is probably mediated via its inhibitory effect on LH release. The catecholamine-synthesis inhibitor, 움methylmetatyrosine and the 움-adrenergic blocking agents phentolamine and dibenzyline block ovulation when injected intrafollicularly, whereas anti-웁-adrenergic agents are ineffective. Subsequent administration of EPI or NE overcomes the blocking effect of phentolamine, while only EPI overcomes the effect of 움methylmetatyrosine, and neither catecholamine could reverse the blocking effect of dibenzyline. While NE has no effect on basal or LH-stimulated progesterone production in granulosa cells, preculture with NE for 2 days enhances LH-responsiveness via 움-adrenergic receptors (Chotesangasa et al., 1988). Nevertheless, it remains to be demonstrated that catecholamines play a physiologic role in the process of ovulation.

B. Hormones during Sexual Maturation and Molt Investigation of the changes in gonadotropin concentrations between the time of hatching and the onset of lay in the domestic hen shows that there is an early LH

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peak at about 1 week of age and a prepubertal LH rise beginning approximately 15 weeks posthatch that is highest about 3 weeks prior to sexual maturity. Ovarian steroidogenesis can be demonstrated as early as day 3.5 of incubation. Plasma estradiol increases from less than 100 pg/ml 6 weeks before lay to a peak of about 350 pg/ml 2–3 weeks before lay; it then decreases to basal concentrations (100–150 pg/ml) at the time of first lay. Plasma progesterone concentrations are low (0.1– 0.5 ng/ml) until about 1 week before laying, at which time baseline levels increase to 0.4-0.6 ng/ml (Williams and Sharp, 1977). The hen’s ovary is unresponsive to stimulation by either mammalian gonadotropins or avian pituitary extracts until 16–18 weeks of age. In contrast, the pituitary becomes less responsive to exogenous LHRH as the onset of puberty approaches (Wilson and Sharp, 1975); this change is probably the result of negative feedback by increasing steroid secretion by the ovary.

C. Effect of Light on the Ovary and Ovulation Photoperiod influences the reproductive activity of birds relative to both seasonal changes (signaling the onset and termination of breeding seasons in temperate latitudes) and daily changes (entraining the time of ovulation and/or oviposition). Ovarian development and egg laying appear to be most stimulated by increasing photoperiod, as normally occurs during the spring in the northern hemisphere. A notable exception is the Emperor penguin (Aptenodytes forsteri), which breeds during the short photoperiod of antarctic winters (Groscolas et al., 1986). Migratory species such as the junco or white-crowned sparrow, as well as the domestic turkey, fail to show sexual development when held under constant short-day conditions. In contrast, domestic fowl raised under a continuous photoperiod of either 6 hr of light or 22 hr of light will reach sexual maturity at about 21 weeks of age. Even in this species, however, a progressively increasing photoperiod from hatch to 18 weeks of age advances the onset of egg laying by 2–3 weeks when compared to birds raised under a constant photoperiod (Morris, 1978). Maximal photostimulation is generally considered to occur with a photoperiod of 12–14 hr of light; however, normal egg production can be obtained with a photoperiod between 12 and 18 hr, and the domestic hen will continue to lay (although more sporadically) even if kept in continuous darkness. Moreover, an intermittent photoperiod consisting of a total of 10 hr of light in the pattern 8 hr light (L):10 hr dark (D):2 hr L:4 hr D has proven as effective in maintaining egg production as a 14L:10D photoperiod.

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D. Photorefractoriness The seasonal termination of reproduction in birds is an adaptive mechanism to insure that the newly hatched young are raised under optimal conditions. For instance, many species that breed in northern latitudes end the nesting phase (and undergo spontaneous gonadal regression) early enough in the summer to insure sufficient time for development of fledglings prior to fall migration. A common (but not universal) mechanism responsible for ending the breeding season is photorefractoriness; this state is characterized by the inability of the reproductive system to respond to the otherwise stimulatory effects of increasing daylength. Photorefractoriness is often initiated by long days, and the number of long days required for induction is dependent upon day length. In the White-crowned sparrow (Zonotrichia leucophrys gambelii), gonads will remain regressed for several years when continuously maintained on long days, and recovery of photosensitivity occurs only after birds are exposed to short photoperiods for 40–60 days. Considerable attention has been focused on the hypothalamus as the anatomical location of photorefractoriness. For example, there is evidence for increased hypothalamic sensitivity to the negative feedback effects of sex steroids as the breeding season progresses and for a decline in hypothalamic concentrations of LHRH. By comparison, neither the gonads nor the anterior pituitary appear to become refractory. For additional information and references, see Wingfield and Farner (1993).

E. Molt Molt refers to the orderly replacement of feathers and is accompanied by the total regression of the reproductive organs and the cessation of lay. In wild, temperate species a prenuptial molt may occur prior to the breeding season, while a postnuptial molt occurs between the end of the reproductive season and the onset of autumn or the autumn migration. In the domestic fowl, molt occurs after approximately 1 year of egg production. The physiologic mechanisms that cause molt remain obscure; however, the pituitary, adrenal, and particularly the thyroid glands have been suggested as mediators ( Jallageas and Assenmacher, 1985). Forced molt in domestic fowl can be induced by restriction of feed and water, decreased day length, or by administration of prolactin or large doses of progesterone. In addition, injection of thyroxine induces molt in the domestic hen and promotes feather growth in redheaded buntings (Pati and Pathak, 1986), while thyroidectomy retards molt in the spotted munia and redheaded bunting. This relationship is not consistent among birds, however, as

thyroidectomy of juvenile mallard ducks fails to alter timing of the molt to breeding plumage. Circulating concentrations of plasma prolactin, growth hormone, LH, and ovarian steroids are lower in molting females than in laying hens (Scanes et al., 1979b; Hoshino et al., 1988). In turkey hens, plasma prolactin levels during molt are less than half those found in laying hens (Proudman and Opel, 1981). On the other hand, plasma levels of corticosterone, testosterone, and triiodothyronine (T3), but not thyroxine (T4) increase during the molting period. The elevation in plasma thyroxine may not only play a role in the induction of molt, but may also be required for thermoregulation during the period of heavy feather loss.

V. OVIPOSITION Expulsion of the egg (oviposition) involves the relaxation of abdominal muscles and sphincter between the shell gland and vagina and the muscular contraction of the shell gland. The majority of research to elucidate the mechanisms controlling oviposition has been performed in the quail and domestic hen and has implicated neurohypophyseal hormones, prostaglandins, and hormones of the pre- and postovulatory follicles.

A. Neurohypophyseal Hormones Both oxytocin and arginine vasotocin induce premature oviposition in hens. While arginine vasotocin activity in the blood of the laying hen was reported to be highest during oviposition, removal of the neurohypophysis failed to affect the pattern of timing of oviposition. On the other hand, it is possible that an additional source of arginine vasotocin is the ovary itself (Saito et al., 1990). Finally, there is evidence that the ovipositioninducing actions of oxytocin may be mediated via prostaglandins, as administration of indomethacin, a prostaglandin synthesis inhibitor, blocks oxytocin-induced premature oviposition.

B. Prostaglandins There is much experimental data suggesting that prostaglandins are directly involved in oviposition. Exogenous administration of PGF2움 stimulates shell gland contractility, relaxes the vagina, and induces premature oviposition (Shimada and Asai, 1979), while treatment with indomethacin or aspirin depresses the peak of prostaglandins in the plasma and in pre- and postovulatory follicles, suppresses uterine contractility, and delays oviposition (Hertelendy and Biellier, 1978). In addition, injection of PGE1 induces premature oviposition in

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chickens and Japanese quail, while passive immunization with PGE1 antiserum delays oviposition in the hen. Further evidence to suggest that prostaglandins mediate the oviposition-inducing activity of arginine vasotocin is provided by the finding that plasma prostaglandins are significantly elevated at the time of arginine vasotocininduced premature oviposition and that arginine vasotocin stimulates the biosynthesis and release of prostaglandin from the shell gland (Rzasa, 1984). Arginine vasotocin administered to hen shell gland muscle in vitro increases tissue content of prostaglandins and stimulates shell gland muscle contractility, while concomitant treatment with indomethacin blocks the production of prostaglandins and decreases the rate of muscle contraction. Endogenous concentrations of prostaglandins E and F increase in the largest preovulatory follicle beginning 6–4 hr prior to the expected time of ovulation (or oviposition). In contrast, postovulatory levels of prostaglandin F increase about 100-fold approximately 24 hr after that ovulation or 2 hr before the next expected oviposition (Day and Nalbandov, 1977). Increased prostaglandin production and secretion from one or more of these sources is reflected by significantly elevated plasma concentrations of the prostaglandin metabolite 13,14dihydro-15-keto prostaglandin F2움, coincident with the time of oviposition. TGF움 and TGF웁 are among factors that have been shown to regulate granulosa cell PG production (Li et al., 1994). Administration of the prostaglandin precursor, arachidonic acid, or prostaglandin F2움 (PGF2움) increases intraluminal pressure of the infundibulum, magnum, isthmus, and shell gland. Substance P, PGF2움, and arginine vasotocin stimulate shell gland contractility in vitro (Olson et al., 1978; De Saedeleer et al., 1989), while PGF2움 has been demonstrated to be effective in vivo (Shimada and Asai, 1979). Specific binding sites for PGF2움 and arginine vasotocin have been identified and characterized in the membranes of shell gland muscle (Toth et al., 1979; Takahashi et al., 1992). In contrast, prostaglandins of the E series (PGE1, PGE2) suppress spontaneous motility and decrease intraluminal pressure in the vagina (Wechsung and Houvenaghel, 1978). These observations suggested that ovum transport through the oviduct and expulsion of the egg from the shell gland may involve prostaglandin-stimulated contractility of oviduct smooth muscle.

C. Postovulatory Follicle Removal of the most recent postovulatory follicle results in a 1- to 7-day delay in oviposition; this effect has been attributed to the removal of factors produced by the granulosa cell layer (Gilbert et al., 1978); the factor(s) may be a peptide, possibly analagous or identi-

cal to the neurohypophyseal hormones or prostaglandins.

D. Preovulatory Follicle A role for the preovulatory follicle in the process of oviposition was first suggested by Fraps (1942), who found that a premature midsequence ovulation induced by the injection of LH was accompanied by premature expulsion of the egg within the oviduct. Soon after, Rothchild and Fraps (1944) demonstrated that removal of the most mature preovulatory follicle resulted in the delayed oviposition of the egg in the shell gland, although the delay was not as long as that found after removal of the postovulatory follicle.

E. Other Factors As mentioned above, plasma concentrations of corticosterone increase dramatically at the time of oviposition. Premature oviposition (induced with vasopressin) or delayed oviposition (resulting from the removal of the postovulatory follicle) failed to alter the timing of the corticosterone peak from when the egg would normally have been laid; an additional increase in corticosterone occurs at the time of vasopressin-induced oviposition (Beuving and Vonder, 1981). Nevertheless, these results do not necessarily indicate a causative relationship between corticosterone secretion and the induction of oviposition. Despite the fact that the shell gland contractility is influenced by epinephrine and norepinephrine, there is no evidence that these catecholamines influence the timing of oviposition. Many other factors, including acetylcholine, histamine, pentobarbital, and lithium, induce premature oviposition, but the mechanisms and physiological relevance of their actions are unclear. For additional information, see Shimada and Saito (1989).

F. Broodiness and Nesting Behavior Broody behavior consists of termination of egg production, the incubation of eggs, and care of the young. The onset of incubation occurs coincident with complete regression of the ovary and accessory reproductive tissues such as the oviduct and comb. In present-day commercial egg-producing hens, broodiness is virtually nonexistent, but is still present in the Bantam hen and broiler-breeder and is common in turkeys. Broody behavior is often accompanied by the development of an incubation (or brood) patch, and the increased vascularity, edema, and thickening of the epidermis occurs under the regulation of estrogen and prolactin. Induction of incubation behavior in turkey hens can be initiated by

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administration of prolactin, and active immunization against prolactin reduced the incidence or delayed the onset of broodiness in Bantam hens (Youngren et al., 1991; March et al., 1994). Incubation activity stimulates the development of the crop sac in the pigeon; proliferation of this gland is under the direct control of prolactin. In the ring dove, endogenous estrogen production, stimulated by FSH secretion, is thought to induce the female to build nests. In turn, the initiation of nestbuilding activity in the male is dependent on the hormonal environment and behavior of the female. In general, behavioral broodiness in the female is preceded by decreases in circulating concentrations of LH, progesterone, testosterone, and estradiol, while growth hormone tends to be lower only while hens are caring for the young (Harvey et al., 1981). As discussed above, concentrations of prolactin are frequently increased during egg laying and around the onset of incubation and remain elevated throughout incubation. Among species in which the male assumes the predominant role of incubation, levels of prolactin are higher in the male than in the female (Oring et al., 1988). For further information, see El Halawani et al. (1988).

weight. However, variations do occur in the lipid, amino acid and carbohydrate content of albumen and yolk, with precocial species (e.g., from the orders Anseriformes and Galliformes) having a higher energy density than altricial species (e.g., the pigeon Columba livia and pelican Pelecanus occidentalis) (Roca et al., 1984; Bucher, 1987).

A. Yolk Yolk serves to provide lipids and many of the proteins required for embryonic growth. The final composition of yolk in the hen’s egg consists of approximately 33% lipid (by wet weight) compared to 17% protein. The greatest proportion of yolk is water (48%) with lesser amounts of free carbohydrates (0.2%) and inorganic elements (1%). Of the major classes of lipids, approximately 70% are triacylglycerols, 25% are phospholipids, and 5% cholesterol and cholesterol esters. Yolk is usually deposited in concentric bands which result from a nonuniform pattern of daily feed intake.

B. Albumen There are four distinct layers of albumen in the fully formed egg: (1) the chalaziferous (inner thick) layer, attached to the yolk; (2) the inner thin (liquid) layer; (3) the outer thick layer; and (4) the outer thin (fluid) layer. Approximately one-fourth of the total albumen by weight is found in the outer thin layer and slightly greater than one-half in the outer thick layer. The inner layer represents 16.8% of the total albumen, while the chalaziferous layer plus chalazae 2.7% of the total (Table 2). The function of the egg albumen layer is likely to prevent invasion of microorganisms into the yolk (see below), as well as to serve as a source of water, protein, and minerals to the embryo during development. The

VI. COMPOSITION AND FORMATION OF YOLK, ALBUMEN, ORGANIC MATRIX, AND SHELL Components of the egg include the yolk, albumen, the organic matrix, and the crystalline shell. These components have been reviewed by a number of workers, and the reader is referred to the following literature for more details: Gilbert (197la,b), Simkiss and Taylor (1971), Parsons (1982), and Burley and Vadehra (1989). There appears to be a remarkable uniformity among many species of birds as to the density of yolk and albumen and the proportion of egg yolk relative to egg

TABLE 2 Composition of the Hen’s Egga Albumen

Weight (g) Water (%) Solids (%)

Yolk

Outer

18.7 48.7 51.3

7.6 88.8 11.2

Middle

Inner

Chalaziferous

Shell

18.9 87.6 12.4

5.5 86.4 13.6

0.9 84.3 15.7

6.2 1.6 98.4

All layers

Proteins (%) Carbohydrates (%) Fats (%) Minerals (%) a

From Romanoff and Romanoff (1949).

Yolk

Albumen

Shell

16.0 1.0 32.6 1.1

10.6 0.9 Trace 0.6

3.3 — 0.03 95.10


chalazae are two fiberlike structures at each end of the egg permit limited rotation but little lateral displacement of the yolk. In the chicken, the initial albumen layer is deposited by the caudal region of the infundibulum. However, the majority of albumen proteins are deposited by tubular gland cells of the magnum, while avidin is synthesized by the goblet cells. The discrete layers of albumen are the result of either its consecutive deposition by different regions of the magnum or changes that occur during plumping within the shell gland and with movement of the egg through the oviduct. The major proteins found in albumen include: (1) ovalbumen, 54%; (2) ovotransferrin (conalbumin), 13%; (3) ovomucoid, 11%; (4) ovoglobulins (G2 and G3), 8%; (5) lysozyme, 3.5%; and (6) 움- and 웁-ovomucin, 1.5–3.0%. There are also several characteristic proteins present in lesser concentrations, including avidin, flavoprotein- and thiamine-binding proteins; ovomacroglobulin; and cystatin. Ovalbumin serves as a source of amino acids for the embryo during development and, based on its homology to the serpin family of protease inhibitors, may also suppress enzyme activity in the egg. Ovotransferrin acts primarily as an iron-chelator, thereby preventing bacterial growth within the egg. Ovomucoid is a serine protease inhibitor and represents the major inhibitor of protease (primarily trypsin) activity in the egg; target enzymes may be produced by invading microorganisms or, alternatively, ovomucoid may serve to modulate the enzymatic degradation of albumin during embryo development. Alpha-ovomucin (a glycoprotein) and 웁-ovomucin (60% carbohydrate by weight) are insoluble, fibrous proteins that are responsible for the gel-like qualities of egg white, particularly in the thick layers. Ovomucins block invasion of microorganisms and may express antiviral properties. The main biologic function egg albumen lysozyme is lytic activity against Gram-negative bacterial cell walls. Several egg proteins are known for their ability to bind specific substrates (e.g., ovotransferrin binds iron, copper and zinc; flavoprotein, riboflavin; and avidin, biotin), and others act as protease inhibitors (ovomucoid, ovoinhibitor, cystatin, ovomacroglobulin). Avidin is a progesterone-dependent secretory protein; it’s biologic role in the hen egg is likely related to its ability to decrease biotin levels, thus inhibiting bacterial growth. For more detailed information on albumen synthesis and properties, see Burley and Vadehra (1989).

C. Organic Matrix The organic fraction of the eggshell consists of shell membranes, the mammillary cores, the shell matrix, and the cuticle. Although these components constitute only

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a small fraction of the entire eggshell, their integrity is critical to its formation and strength. 1. Shell Membranes Shell membranes, organized into an inner (앑50 to 70-애m thick) and outer (앑15 to 25-애m thick) membrane, are produced by the isthmus region of the oviduct. The membranes lie adjacent to one another except at one end where they part to form the air cell. There is some question as to whether epithelial cells or tubular gland cells of the isthmus are responsible for the secretion of the membranes. The membranes consist of a meshwork of protein fibers, cross-linked by disulfide and lysine-derived bonds, with small fibrous protuberances of uncertain function. The membranes are composed in part of collagen (10%), inasmuch as both hydroxyproline and hydroxylysine can be identified. The remainder of the fibrous component is composed of protein (70–75%) and glycoprotein (Candlish, 1972). The membranes are semipermeable and permit the passage of gases, water, and crystalloids but not albumen; there is no relationship between thickness of the membranes and thickness of the shell, but membrane thickness does decrease with the age of the hen. 2. Mammillary Cores The mammillary cores, which are projections from the outer membrane surface (Figure 7), are proposed as the initial sites of calcification. They are composed largely of protein, but also contain carbohydrate and mucopolysaccharides and are thought to be formed by the epithelial cells of the isthmus. The mammillary cores represent the greatest proportion of organic material in the eggshell. 3. Shell Matrix The organic shell matrix is a series of layers of protein plus acid mucopolysaccharide on which calcification takes place. It represents approximately 2% of the total organic composition of the eggshell. The matrix plus calcified crystals make up the palisade layer of the shell. The innermost region of the shell has a greater density of matrix compared to the outer regions. Both calciumbinding protein and carbonic anhydrase have been identified within the matrix. Deposition of the matrix occurs soon after the egg reaches the shell gland. 4. Cuticle The outermost surface of the egg is often (but not always) covered by a thin waxy cuticle composed of protein, polysaccharide, and lipid. The cuticle may be

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FIGURE 7 Diagram of section through the shell and membranes of a hen’s egg, showing crystalline structure and organic material that remains after calcium carbonate has been dissolved. (Adapted from How eggs breathe, H. Rahn, A. Ar, and C. Paganelli. Copyright 䉷 1979 by Scientific American, Inc. All rights reserved.)

unevenly distributed over the entire surface, ranging in depth from 0.5 to 12.8 mm; the average dry weight of the cuticle in a 60 g chicken egg is 12 mg (Parsons, 1982). Its function is likely to protect the egg from water evaporation and microbial invasion, although it is unlikely to add to the structural integrity of the shell. The cuticle, when present, is formed during the last 30 min prior to oviposition.

D. Layers of Crystallization The calcified portion of the shell can be arbitrarily divided into the mammillary knob layer, the palisade layer, and the outer surface crystal layer (see Figure 7). Collectively, these layers represent the major portion of the avian egg shell, are largely responsible for its mechanical strength, and consist of approximately 97% inorganic material. While calcium is the predominant cation, the shell also sequesters a significant amount of magnesium in the form of magnesium carbonate; manganese is likely required for the development of the mammillae because of its role in the synthesis of mucopolysaccharides (Leach and Gross, 1983).

brane. Formation of the mammillary knobs occurs in the shell gland during the first 5 hr of calcification. There is some disagreement as to whether or not the density of mammillary knobs is related to the structural integrity of the eggshell. The passage of water during plumping stretches the shell membranes and increases the distance between the tips of the mammillae. Crystals that form laterally grow and eventually abut with crystals from other mammillae, whereas those that grow outward may extend to the shell surface. At some points the crystals do not grow completely together, leaving pores with a diameter of 0.3–0.9 애m (Figure 7). 2. Palisade Layer The crystallized palisade (or spongy) layer (approximately 200 애m thick) is composed mainly of crystalline calcium carbonate in the form of calcite and represents the greatest portion of the shell. Calcification of this layer is initiated 5–6 hr after the entrance of the egg into the shell gland, during the process of plumping. The palisade layer is arranged as columns situated directly over mammillary knobs that are perpendicular to the surface (Silyn-Roberts and Sharp, 1986).

1. Mammillary Knob Layer Outward crystallization of the mammillary cores results in the formation of the mammillary knob layer. In addition, calcium deposits radiate from the bottom of the mammillary cores to penetrate the outer shell mem-

3. Surface Crystal Layer The outermost layer of calcification is designated the surface crystal layer. The crystalline structure is more dense than that of the palisade region and lies perpen-


dicular to the shell surface. The overall thickness of this layer ranges from 3 to 8 애m. 4. Respiration via the Eggshell Shell pores of the hen egg are simple funnel-shaped openings that arise at the shell surface and protrude through to the mammillary knob layer. In most species, however, pores traverse the shell radially and branch longitudinally along the axis of the egg. The pores are the result of areas of incomplete crystallization. The number of pores is generally related to metabolic demand prior to the initiation of lung function, the number of pores per unit area decreasing with increasing egg weight (Tullett, 1978). The function of eggshell pores is to serve as a mechanism of chemical communication between the air cell of the egg and the external environment during embryo development (Tullett, 1984; Rahn and Paganelli, 1990). The exchange of oxygen, carbon dioxide, and water occurs predominantly via passive diffusion, and a 79 gm turkey egg is estimated to exchange the equivalent of 27 l of gas (oxygen, carbon dioxide and water vapor) during the 28-day incubation period (Paganelli et al., 1987). The average diffusive water loss during incubation for 117 species of birds is 15%; the loss is determined by the difference in water vapor pressure between the egg and nest environment (see Chapter 21). Inner and outer shell membranes play a minor role in limiting the flow of oxygen, carbon dioxide, and water vapor, while the major resistance to passage of these substances comes from the inner end of the pore. Therefore, air-cell gas tensions are determined predominantly by the number and size of the pores and the thickness of the shell, relative to the metabolic rate of the embryo. The total area of pores on a turkey egg with a surface area of 90 cm2 is calculated to be 2.2 mm2 (Paganelli et al., 1987). Furthermore, experimental data suggest that a change in environmental conditions may be reflected by an alteration in shell conductance (see Tullett, 1978).

E. Calcium Metabolism The shell gland transports 2–2.5 g of calcium within a period of 15 hr for the calcification of a single egg. A hen that lays 280 eggs in a production year will use a quantity of calcium for the purpose of shell formation corresponding to 30 times the calcium content of the entire body. A detailed description concerning mechanisms of absorption and changes in uptake by the intestine relative to age and egg production are beyond the scope of the present discussion, and the reader is referred to Bar et al. (1977), Gilbert (1983), and Etches (1987).

589

1. Sources of Eggshell Calcium Calcium for eggshell formation is provided via the blood following absorption from the intestine (duodenum and upper jejunum) or resorption from bone (principally medullary bone, but also cortical bone under conditions of calcium deficiency). Resorption of calcium from bone is regulated by both parathyroid hormone and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), while absorption through the gut is facilitated by 1,25 (OH)2D3. The relative importance of the two organs as sources of calcium depends on the concentration of dietary calcium. Hens consume approximately 25% more feed on days when eggshell formation occurs than on days when it does not. If concentrations of calcium in the food are 3.6% or higher, most of the eggshell calcium is derived directly from the intestine. If the concentration is 2.0%, bone supplies 30–40% of the shell calcium, and on calcium-free diets, the skeleton is the principal source. However, it is likely that these relationships vary depending on the time of day. When hens are provided constant free access to feed, much of the daily intake occurs early in the photoperiod; the remainder is consumed at the end of the photoperiod. However, much of the shell is formed during the night, when generally no calcium is consumed and when the calcium content of the digestive tract is gradually decreasing. Therefore, medullary bone is the primary source of shell calcium during the latter hours of darkness. Blood calcium circulates in two forms, as nondiffusible protein-bound calcium and as diffusible ionized calcium. Nondiffusible calcium is bound by plasma calcium-binding proteins vitellogenin and albumin. Estrogen treatment increases total plasma calcium, in part by stimulating the production of blood calcium-binding proteins. Similarly, total plasma calcium increases several weeks before laying, and the increase is attributable to an increase in the protein-bound, but not in the diffusible fraction. During the ovulation–oviposition cycle, concentrations of ionized calcium peak (0.057 mg/ml) 4 hr after oviposition, then decrease significantly during the period of shell calcification (minimum of 0.049 mg/ ml). On the other hand, plasma concentrations of total calcium (ionized plus inionized) fluctuate only minimally (between 0.2 and 0.26 mg/ml) during the ovulation–oviposition cycle. Feeding a calcium-deficient diet to laying hens causes a significant decrease in plasma ionized calcium concentrations, a significant decrease or complete cessation of lay, and regression of the ovary within 6 to 9 days. However, birds on the same diet injected with crude chicken pituitary extracts continued to lay for a more prolonged period, despite a comparable decrease in ionized calcium (Luck and Scanes, 1979a). Basal con-

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centrations of LH in birds fed a calcium-deficient diet (0.2% Ca) are significantly lower compared to birds on a calcium-rich diet (3.0% Ca), but between the two groups the LH-releasing activity of exogenously administered LHRH is similar (Luck and Scanes, 1979b). These results suggest that pituitary LH synthesis and release and the ability of the ovary to respond to gonadotropins are resistant to a calcium deficiency. Mongin and Sauveur (1974) determined that voluntary calcium consumption by laying hens increases at the time the egg enters the shell gland and given a choice between a calcium-deficient diet (1.0% Ca) and a diet supplemented with calcium-rich oyster shell, hens will preferentially consume the calcium-rich diet during the period of eggshell calcification. For further information, see Etches (1987). 2. Vitamin D Metabolism Vitamin D plays an important role in the regulation of calcium metabolism via the metabolically active metabolite 1,25-(OH)2D3. Conversion to this form occurs by the 1-hydroxylation of 25-hydroxyvitamin D3 (25OH-D3) in the kidney under the hormonal control of estradiol and PTH (Soares, 1984). For instance, renal 1-hydroxylase activity increases just prior to the initiation of egg laying, at a time corresponding with an increase in circulating estrogen and total plasma calcium. In addition, 1-hydroxylase activity during the ovulation–oviposition cycle increases at the time of ovulation. The increase in activity is followed by an increase in circulating concentrations of 1,25-(OH)2D3 4 hr after ovulation, and elevated levels persist until 10 hr after ovulation or after the initiation of eggshell formation (Castillo et al., 1979). For additional information, see Norman (1987). 3. Calcium Mobilization from Medullary Bone In the long bones (e.g., femur and tibia), medullary bone lines the endosteal surface and grows with a system of interconnecting spicules that may completely fill the narrow spaces. Medullary bone forms in female birds during the final 10 days before egg laying under the influence of estrogen and testosterone; it can be readily induced in intact male birds by administration of estrogen or in castrated males by estrogen and testosterone. Gonadal steroids apparently act directly on the cells in the medullary cavity and independently of calcium intake. In addition, it is unlikely that 1,25-(OH)2D3 mediates the transfer of calcium to medullary bone at this time, as the appearance of this bone occurs 1–2 weeks prior to the increase in renal 1-hydroxylase activity and the elevation of plasma total calcium.

During the ovulation–oviposition cycle, periods of intense medullary bone formation alternate with periods of severe bone depletion. Hens fed a high-calcium diet are generally able to replenish the calcium lost from medullary bone during shell calcification when shell formation is not taking place, but on a low-calcium diet the cortical bone of the femur is eroded, while medullary bone is maintained in a fairly constant amount. Under these conditions the new medullary bone that forms is only partially calcified, and an increase in the number of osteoblasts is indicative of a more rapid turnover rate. 4. Calcium Absorption and Secretion by the Shell Gland The mechanism of calcium transfer across the intestine and shell gland, as well as the hormonal regulation of the process, is poorly understood. As discussed above, the initiation of egg production is associated with increased 1,25-(OH)2D3 synthesis and accumulation in the intestine and shell gland, and calcium transport as well as concentrations of the calcium binding protein, calbindin-D28K, increase in both organ systems during egg production. Calbindin is expressed in tubular gland cells of the shell gland and in the distal portion of the isthmus, but not in the proximal isthmus or magnum (Wasserman et al., 1991). Calbindin synthesis in the intestine is regulated primarily by 1,25-(OH)2D3, yet its production in the shell gland apparently requires ovarian steroids and other, as-yet-unidentified factors in addition to 1,25(OH)2D3 (Bar et al., 1990). While levels of calbindin mRNA increase during the ovulatory cycle at a time coincident with shell calcification there is little to no change in tissue levels of calbindin protein; such results indicate posttranslational control of calbindin levels (Nys et al., 1989). Calcium secretion from the shell gland increases approximately 7 hr after ovulation, reaches a maximum as the shell is being formed, and decreases to the basal secretion rate after shell formation is complete but before the expulsion of the egg (Eastin and Spaziani, 1978a). The presence of an egg within the shell gland is not likely to be the major stimulus for the initiation of calcium secretion but appears to be more closely related to ovulation. The hormonal signal(s) that affects changes in the rate of calcium secretion is unknown, although estrogen involvement has been suggested (Eastin and Spaziani, 1978a). Similarly, termination of a high rate of calcium secretion is not related to egg removal, as calcium deposition decreases fully 2 hr before eggs are laid. Movement of calcium across the shell gland, which may occur by both diffusion and active transport (Eastin and Spaziani, 1978b), involves expenditure of metabolic energy. In the aged hen, calcium


deposition and egg shell weight and density are related to shell gland levels of calbindin (Bar et al., 1992). 5. Carbonate Formation and Deposition The mineral content of the eggshell consists of about 97–98% calcium carbonate while the remainder consists of magnesium carbonate and tricalcium phosphate. Carbonate in the shell is derived from blood bicarbonate or is synthesized from CO2 by carbonic anhydrase located in oviduct tissue and the shell. In addition, luminal carbonate ion content is supplemented by bicarbonate in fluid flowing from the magnum to the shell gland. The secretion from the shell gland of HCO⫺3 , like that of calcium, is accomplished by active transport (Eastin and Spaziani, 1978b) and may be influenced by luminal HCO⫺3 concentrations. The net amount of calcium secretion is functionally linked to HCO⫺3 production and to luminal HCO⫺3 concentrations. For excellent reviews of carbonate/bicarbonate production and calcium– bicarbonate interactions and secretion, see Simkiss (1967) and Eastin and Spaziani (1978b).

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Barber D. L., Sanders, E. J., Aebersold, R., and Schneider, W. J. (1991). The receptor for yolk lipoprotein deposition in the chicken oocyte. J. Biol. Chem. 266, 18761–18770. Beuving, G., and Vonder, G. M. A. (1981). The influence of ovulation and oviposition on corticosterone levels in the plasma of laying hens. Gen. Comp. Endocrinol. 44, 382–388. Breen, P. C., and DeBruyn, P. P. H. (1969). The fine structure of the secretory cells of the uterus (shell gland) of the chicken. J. Morphol. 128, 35–66. Bucher T. L. (1987). Patterns in the mass-independent energetics of avian development. J. Exp. Zool. 1, (Suppl.), 139–150. Burke, W. H., Ogasawara, F. X., and Fuqua, C. L. (1972). A study of the ultrastructure of the uterovaginal sperm-storage glands of the hen, Gallus domesticus, in relation to a mechanism for the release of spermatozoa. J. Reprod. Fertil. 29, 29–36. Burley, R. W., and Vadehra, D. V. (1989). ‘‘The Avian Egg.’’ Wiley, New York. Calvo, F. O., Wang, S.-C., and Bahr, J. M. (1981). LH–stimulable adenylyl cyclase activity during the ovulatory cycle in granulosa cells of the three largest follicles and the postovulatory follicle of the domestic hen (Gallus domesticus). Biol. Reprod. 25, 805–812. Candlish, J. K. (1972). The role of the shell membranes in the functional integrity of the egg. In ‘‘Egg Formation and Production’’ (B. M. Freeman and P. E. Lake, eds.). British Poultry Science Ltd., Edinburgh. Castillo, L., Tanaka, Y., Wineland, M. J., Jowsey, J. O., and DeLuca, H. F. (1979). Production of 1,25-dihydroxyvitamin D3 and formation of medullary bone in the egg–laying hen. Endocrinology 104, 1598–1601. Chalana, R. K., and Guraya, S. S. (1978). Histophysiological studies on the postovulatory follicles of the fowl ovary. Poult. Sci. 57, 814–817. Chotesangasa, R., Kamiyoshi, M., Tanaka, K., Tomogane, H., and Yokoyama, A. (1988). Enhancement of the response of hen granulosa cells to LH with norepinephrine in vitro. Comp. Biochem. Physiol. C 90, 225–229. Conkright, M. D., and Asem, E. K. (1995). Intracrine role of progesterone in FSH– and cAMP–induced fibronectin production and deposition by chicken granulosa cells: Influence of follicular development. Endocrinology 136, 2641–2651. Crossley, J., Ferrando, G., and Eiler, H. (1980). Distribution of adrenergic receptors in the domestic fowl oviduct. Poult. Sci. 59, 2331– 2335. Dahl, E. (1970a). Studies on the fine structure of ovarian interstitial tissue. 2. The ultrastructure of the thecal gland of the domestic fowl. Z. Zellforsch. Mikrosk. Anat. 109, 195–211. Dahl, E. (1970b). Studies of the fine structure of ovarian interstitial tissue. 3. The innervation of the thecal gland of the domestic fowl. Z. Zellforsch. Mikrosk. Anat. 109, 212–226. Day, S. L., and Nalbandov, A. V. (1977). Presence of prostaglandin F (PGF) in hen follicles and its physiological role in ovulation and oviposition. Biol. Reprod. 16, 486–494. De Saedeleer, V., Wechsung, E., and Houvenaghel, A. (1989). Influence of substance P on in vitro motility of the oviduct and intestine in the domestic hen. Anim. Reprod. Sci. 21, 293–300. Deely, R. G., Mullinix, K. P., Wetekam, W., Kronenberg, H. M., Meyers, M., Eldridge, J. D., and Goldberger. R. F. (1975). Vitellogenin synthesis in the avian liver. Vitellogenin is the precursor of the egg yolk phosphoproteins. J. Biol. Chem. 250, 9060–9066. Dick, H. R., Culbert, J., Wells, J. W., Gilbert, A. B., and Davidson, M. F. (1978). Steroid hormones in the postovulatory follicle of the domestic fowl (Gallus domesticus). J. Reprod. Fertil. 53, 103–107. Donham, R. S., Dane, C. W., and Farner, D. S. (1976). Plasma luteinizing hormone and the development of ovarian follicles after loss

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