Layer Of Egg Research Articles

The effect of dietary crude protein on the fertility of male broiler breeders

Reports on the influence of dietary crude protein on male broiler breeder fertility are not consistent, and therefore an experiment was conducted to determine the effect of three isoenergetic diets containing 10.5%, 12.6% and 15% crude protein, respectively, on Ross broiler breeder male fertility from 26 to 41 weeks of age. Feed allocation was the same for each treatment, and was done according to breeder recommendations. Fertility was assessed by determining the number of sperm trapped in the outer perivitelline layer of eggs laid after artificial insemination with a fixed volume of semen collected from 12 males per treatment. Semen concentration, motility and morphology were also determined. Crude protein intake had a significant effect on the rate of decline in fertility post insemination. This resulted in a longer predicted length of the fertile period over all ages when eggs were fertilised with sperm obtained from males that received 12.6% CP diets (14.5 d) than males that received 10.5% and 15% CP (7 and 8.6 d respectively). There was, however, no treatment effect on the measures of live sperm with normal motility or morphology.Keywords: Cockerel, insemination, perivitelline membrane, nutrition, reproduction

Diet exposure to technical hexabromocyclododecane (HBCD) affects testes and circulating testosterone and thyroxine levels in American kestrels (Falco sparverius)

Hexabromocyclododecane (HBCD) is a high-production-volume, brominated flame-retardant that is used in items such as polystyrene foams. HBCD has been detected in the environment, wildlife tissues and in humans globally with some of the highest recorded levels in predatory birds. This study examined the effects of exposure to environmentally relevant levels of HBCD on the reproductive physiology of captive male American kestrels (Falco sparverius), a predatory bird. Two sets of males were used: one group not housed with females (unpaired: nc=12, nHBCD=10) and the second group housed with females (breeding: nc=10, nHBCD=20). All treatment birds were exposed to 0.51μg HBCD/g kestrel/day technical HBCD, and controls to safflower oil only, injected into their food during seasonal testicular development. Unpaired males were exposed for 3 weeks and euthanized for testicular analysis. Breeding males were exposed for 3 weeks prior to pairing and throughout the courtship period. The HBCD-exposed unpaired males had heavier testes (p≤0.017) and a trend towards more seminiferous tubules containing elongated spermatids (p=0.052). There was also a moderate increase in plasma testosterone concentrations (p=0.056) compared to controls. In breeding males, testosterone levels increased during courtship to culminate in higher levels than controls by the time the first egg was laid (p=0.010) and circulating free and total T4 was reduced throughout. The number of sperm cells reaching the perivitelline layer of the first egg for breeding males did not differ between the two groups. This study is the first report that HBCD exposure at environmentally relevant levels alters reproductive physiology in male birds and suggests that birds may be more sensitive to HBCD than mammals.

Embryonic Exposure to the Polybrominated Diphenyl Ether Mixture, DE-71, Affects Testes and Circulating Testosterone Concentrations in Adult American Kestrels (Falco sparverius)

Polybrominated diphenyl ethers (PBDEs) are additive flame retardants that are environmentally persistent and bioaccumulative. The developmental effects of in ovo exposure to environmentally relevant levels of the PBDE technical mixture, DE-71, on male reproductive physiology in captive American kestrels (Falco sparverius) was determined. Males were exposed in ovo by direct maternal transfer to DE-71 at three mean concentrations of 289 ng/g ww (low exposure), 1131 ng/g ww (high-exposure), or background levels of 3 ng/g ww (control). As adults, males were paired with unexposed females for breeding and, 1 year later, sacrificed for testes evaluation. While breeding, high-exposure males demonstrated a trend of reduced circulating testosterone levels when their female mate commenced egg laying when compared with controls (p = 0.056). No differences in circulating free T₄ or T₃ were detected. Sperm numbers were elevated on the perivitelline layer of the first egg of both high- and low-exposure males when compared with controls (p = 0.021). High-exposure males had a higher gonadosomatic index (p = 0.046) and heavier right testis than controls (p = 0.034) with a similar trend for their left testis (p = 0.055). High-exposure males had more seminiferous tubules containing lumen than controls (p = 0.030), and in proportion to the total number of tubules, low-exposure males had more tubules containing lumen than did controls (p = 0.016). Only high-exposure males had fewer than half of tubules containing final spermatids (43%). The results of the present study demonstrate that embryonic exposure to technical DE-71 affects the reproductive tract of adult male kestrels.

SHORT COMMUNICATION: THE EFFECT OF TANNIN SOLUTION IN PERFORMING SUCCESFULL EGG MICROINJECTION ON AFRICAN CATFISH (Clarias gariepinus)

In order to successfully perform a gene transfer activity using microinjection, an individual egg is needed. The common standard operating procedure to collect the individual egg is by applying chemical solution to remove the sticky layer covering the egg surface. Among several available solutions, tannin is one of alternative substances that can be used in performing egg microinjection on African catfish. In this study, the effect of different dosages of tannin solution to remove the sticky layer of eggs was observed. The study was conducted at the Laboratory of Fish Breeding and Genetics, Bogor Agricultural University. Prior to microinjection, the eggs were treated with different dosages of tannin solution right after fertilization. There were four different levels of dosage used as the treatments i.e.: 0.3, 0.5, 0.7, and 0.9 ppm. After the treatments, each individual egg was injected using DNA pmâactin-hrGFP plasmid with a concentration of 20 μg/mL. The results showed that the dosage of 0.5 ppm of tannin was the most effective treatment compared to the other dosages. This dosage produced 71.4% hatching rate and 57.1% transgenic embryos respectively.

Structural Memory in the Contractile Ring Makes the Duration of Cytokinesis Independent of Cell Size

Cytokinesis is accomplished by constriction of a cortical contractile ring. We show that during the early embryonic divisions in C. elegans, ring constriction occurs in two phases--an initial phase at a constant rate followed by a second phase during which the constriction rate decreases in proportion to ring perimeter. Cytokinesis completes in the same amount of time, despite the reduction in cell size during successive divisions, due to a strict proportionality between initial ring size and the constant constriction rate. During closure, the myosin motor in the ring decreases in proportion to perimeter without turning over. We propose a "contractile unit" model to explain how the ring retains a structural memory of its initial size as it disassembles. The scalability of constriction may facilitate coordination of mitotic events and cytokinesis when cell size, and hence the distance traversed by the ring, varies during embryogenesis and in other contexts.

Vitrification of the inner perivitelline layer of chicken eggs for use in the sperm-egg interaction assay

The sperm-egg interaction assay is a good predictor of the fertilizing potential of rooster semen; the ability of chicken sperm to interact with the egg can be assessed by counting the number of holes in the inner perivitelline layer (IPVL) of a freshly laid egg. Atlhough isolated IPVL can be stored for up to 24 h, preservation of IPVL for prolonged intervals in liquid nitrogen would facilitate the sperm-egg interaction assay. The objective of this study was to adapt the technique of vitrifying swine oocytes for use with the IPVL. Our hypothesis was that vitrification would not alter the ability of the membrane to bind sperm; therefore, there would be no difference between vitrified and fresh IVPL in the number of hydrolysis holes made by sperm. Our hypothesis was supported; there were no differences in the mean ± SEM number of holes made by the same sample of sperm in vitrified and in fresh membranes (146.0 ± 17.7 holes/mm 2 IPVL and 159.5 ± 17.7 holes/mm 2 IPVL, respectively, P > 0.05; n = 123 IVPLs tested). Furthermore, 80% of frozen-thawed membranes were recovered intact. Because vitrification did not significantly change the ability of membranes to bind sperm, vitrified membranes can be safely used for the sperm-egg interaction assay. Vitrified IVPL would ensure availability for sperm evaluation and facilitate wide distribution of IPVL, enabling assays to be conducted even in the absence of facilities or expertise to prepare membranes.

VMO-II Mediates the Binding of the Chalaziferous Layer with the Vitelline Membrane in Quail Eggs

Electron microscopic observations have revealed an electron density gradient in the chalaziferous layer of avian egg. The density is highest in the innermost sub-layer, which is bound with the vitelline membrane, and is thus traditionally referred to as the vitelline membrane outer layer (VMO). After high-concentration salt treatment, two proteins, VMO-I and VMO-II, are liberated from the egg envelopes of quail (Coturnix japonica), which results in the separation of the chalaziferous layer from the vitelline membrane. VMO-I and VMO-II were purified through gel-filtration and subjected to antisera produced in rabbits. The molecular sizes of these two proteins were estimated to be 9 to 15kDa for VMO-II and 18kDa for VMO-I. Their amino acid sequences were very similar to those of chickens. Immunofluorescent and immunoelectron micrographs showed that VMO-II was produced by the luminal epithelium and VMO-I by both the luminal and glandular epithelia of the infundibulum. Immunogold particles for the labeling of both proteins were distributed throughout the chalaziferous layer, as well as in the chalazae, with a density gradient similar to that mentioned above. In salt-treated envelopes, immunogold labeling was completely absent when stained with anti-VMO-II antiserum and weak with anti-VMO-I antiserum. Immunohistochemical studies revealed that purified VMO-II bound with vitelline membranes of salt-treated envelopes, and ligand blotting tests revealed that ZP1 and ZP3 were the molecules bound by VMO-II; the binding was inhibited by various kinds of sugars. VMO-II might play a role in the binding of the chalaziferous layer with the vitelline membrane, a process that leads to the anchoring of the chalaza cord.

Eggshell structure, mode of development and growth rate in birds

The shell of an egg contributes to successful embryogenesis in many ways, such as through protection, respiration and water exchange. The shell is also the major source of calcium for the development of high-calcium consuming organs, e.g. the skeleton, muscles and brain. Some studies show, moreover, that growth rate may play a fundamental role in the pattern of skeletal development in birds: the faster the growth the less ossified the skeleton is at hatching. We predicted, therefore, that slow (precocial) and fast (altricial) growing bird species should lay eggs encased in shells with different structures adapted to support different rates of calcium removal by developing embryos. We tested this prediction by comparing the fine structure of the inner eggshell surface (mammillary layer) from 36 bird species belonging to 18 orders ranging from Struthioniformes to Passeriformes. Using scanning electron microscopy, we compared the mammillary layer of both non-incubated eggs and eggs at the time of hatching, i.e., before and after embryonic development and the accompanying calcium removal. The results were consistent with the prediction, i.e., the number of mammillary tips per unit of surface area was associated with mode of development and growth rate. The number was higher, and calcium removal was also more extensive, in shells from precocial bird species than in shells from altricial bird species.

Unhatched eggs: methods for discriminating between infertility and early embryo mortality

Unhatched eggs are a common phenomenon in birds and are often referred to as being ‘infertile’, which (confusingly) can mean at least two things: (1) that the ovum has not been fertilized or (2) that the embryo has died during development. These two broad categories of hatching failure can be difficult to distinguish, particularly in the early stages of embryo development. We describe methods to distinguish between infertility (due to insufficient sperm) and early embryo mortality in passerine eggs using the Zebra Finch Taeniopygia guttata as a model. We also describe how we successfully adapted these methods for use on eggs from a wild species, the Tree Sparrow Passer montanus, collected after the incubation period, and show that sperm can be visualized on the perivitelline layer of unhatched eggs even several weeks after laying.

Sperm proteasomes are responsible for the acrosome reaction and sperm penetration of the vitelline envelope during fertilization of the sea urchin Pseudocentrotus depressus

The roles of sperm proteasomes in fertilization were investigated in the sea urchin Pseudocentrotus depressus. Two proteasome inhibitors, MG-132 and MG-115, inhibited fertilization at 100 μM, whereas chymostatin and leupeptin showed no inhibition. Among three proteasome substrates, Z-Leu-Leu-Glu-MCA showed the strongest inhibition toward fertilization. MG-132 inhibited the egg-jelly-induced, but not ionomycin-induced, acrosome reaction. In addition, MG-132, but not E-64-d, inhibited fertilization of dejellied eggs by acrosome-reacted sperm. MG-132 showed no significant inhibition toward the binding of reacted sperm to the vitelline layer. Proteasomes were detected by Western blotting in the acrosomal contents, which are partially released upon exocytosis. We also found that the inhibition pattern of the caspase-like activity of the proteasome in the acrosomal contents by chymostatin and proteasome inhibitors coincided well with their inhibitory abilities toward fertilization. Furthermore, the vitelline layer of unfertilized eggs appears to be ubiquitinated as revealed by immunocytochemistry and Western blotting. Extracellular ATP, required for the degradation of ubiquitinated proteins by the proteasome, was also necessary for fertilization. These results indicate that the sperm proteasome plays a key role not only in the acrosome reaction but also in sperm penetration through the vitelline envelope, most probably as a lysin, during sea urchin fertilization.

Signal transduction mechanisms regulating ion fluxes in the sea urchin sperm

AbstractIon permeability changes involving ion channels and transporters are essential for fertilization, since they are key elements in sperm‐egg signaling and environmental sensing. Changes modulated by external factors and components from the outer layer of the homologous egg, like the sperm‐activating peptides and the fucose‐sulfate glycoconjugate, trigger complex signaling systems in the sperm that modulate how sperm swim, find the egg and fertilize it. These signaling systems depend on plasma membrane ion permeability and involve an alteration of second messenger levels, changes of membrane potential and intracellular Ca2+, Na+ and pH, along with changes in sperm morphology in the case of the acrosome reaction (AR). New procedures to elucidate the signaling pathways implicated in sperm ion transport have expanded the opportunities to dissect sperm‐egg signaling revealing how sperm ion channels participate in activation, motility, chemotaxis, and the AR. The sperm signaling pathways involve a large variety of ion channels and transporters, which are discretely localized and finely orchestrated to play specific roles that define the elaborate performance of sperm and allow a successful fertilization. In this review we will focus on the signaling molecules involved in the final aim of the sperm, to reach and fertilize the egg.

A new pipefish, Stigmatopora narinosa (Syngnathidae) from South Australia

2 Inshore Fish Group, www.ifg.bioteck.org Abstract Browne, R.K. and Smith, K. 2007. A new pipefi sh, Stigmatopora narinosa (Syngnathidae) from South Australia. Memoirs of Museum Victoria 64: 1-6. A new species of pipefi sh, Stigmatopora narinosa sp. nov. (Teleostei, Syngnathidae) is described from Gulf St. Vincent and Spencer Gulf, South Australia. S. narinosa shares with the other three described Stigmatopora species a similar fi n placement, fully enclosed brood pouch, superior and inferior trunk and tail ridges continuous, a lateral trunk ridge ending midlaterally. S. narinosa, S. nigra Kaup, 1853, and S. argus Richardson, 1840, have long prehensile tails and all Stigmatopora lack caudal fi ns. S. narinosa is most similar in meristics to S. nigra in having the dorsal fi n initiating on about the fi fth to seventh trunk ring and the lateral trunk ridge terminating across the second tail ring. In other Stigmatopora species the dorsal fi n originates on the ninth to thirteenth trunk ring, the lateral trunk ridge terminates between the eighth to thirty-second tail ring, S. narinosa is distinguished from sympatric S. nigra in having nine (range, 8.2-9.8), rather than six sub-dorsal tail rings (range, 4.8-7.1), a greater number of sub-dorsal tail and total rings, a greater number of dorsal-fi n rays, a shorter laterally fl attened and dorsally elevated snout, a distinct banded pattern in both live and preserved specimens, a larger brood number and a double layer of eggs in the brood pouch. The brood pouch is under the anterior portion of the tail and extends for 15-18 rings from the anal ring; pouch plates are absent or vestigial, and the folds of the semi-pouch enclosure meet on the ventral midline. The eggs are deposited in up to two layers, one lining the dorsum of the pouch and the other separated by a membranous partition, and the brood of up to 98 eggs is larger than the maximum number of 41 found in S. nigra and S. argus. S. narinosa young at birth are approximately 18 mm, S. nigra 13 mm, and S. argus 32 mm total length. S. narinosa has a very restricted known range and habitat, inhabiting patchy open beds of sea-grasses with brown algae on sandy rubble substrate between 1 m and 5 m depth over less than 200 km of coastline.

Dam and Sire Effects on Sperm Penetration of the Perivitelline Layer of Eggs Produced by Two Strains of Commercial Turkeys

The reproductive performance of 2 commercial turkey breeder lines was examined using reciprocal crosses between sires and dams of each line. One line had been selected using artificial inseminations performed at biweekly intervals, whereas the second line had been selected using inseminations performed at weekly intervals. The hypothesis was proposed that sires and dams of the 2 lines differ because of different abilities for sperm to penetrate the inner perivitelline layer (IPVL) and fertilize eggs.Fertilized eggs to hatch poults for the experiment were obtained from the primary breeders and were incubated using conditions recommended by the industry. Hens (n = 72) and toms (n = 15) from each strain were identified and reared in preparation for a reproductive cycle using commercially accepted standards. Beginning just prior to the onset of egg production and at weekly intervals thereafter, half of the hens were inseminated with semen from males of the same line, whereas the remaining half received semen from the opposite line. Eggs were collected from the pens daily and set in incubators to determine fertility and embryo survival. At biweekly intervals 3 eggs per pen were used for counting sperm penetration holes in the IPVL. Data were collected for fertility, hatchability, and time that embryos died for each of the pens. Dam and sire affected IPVL penetration independently. A dam by sire interaction influenced fertility, whereas hatchability was affected only by dam. Thus, it is concluded that selection of dam and sires for commercial breeders alters IPVL sperm penetration ability of hens as well as egg-binding ability of sires.

Influence of Hen Age and Number of Inseminated Sperm on the Number of Holes Hydrolyzed in the Inner Perivitelline Layer of Turkey Eggs

Eggs from young turkey breeder hens have a higher rate of early embryonic mortality (EEM) than eggs from older hens. Preliminary field data indicated that increased sperm concentration decreased the incidence of EEM in eggs from young hens. Possible explanations for decreased EEM following insemination with more concentrated sperm may include altered sperm binding and hydrolyzing of the inner perivitelline layer (IPVL) of eggs from hens of different ages. The current study examined differences in the number of sperm penetration (SP) holes hydrolyzed in the IPVL in hens at 2 different ages when inseminated with 25, 50, 100, 200, 400, or 800 million viable sperm. Hens (12/treatment) were inseminated on d 14 and 21 after photostimulation (32 and 33 wk of age) and were inseminated again at 12 and 13 wk of egg production (44 and 45 wk of age). The SP holes hydrolyzed in the IPVL were counted in the 1,098 eggs produced in the 3 wk following each insemination period. The number of SP holes hydrolyzed in the IPVL was significantly (P ≤ 0.05) greater in younger hens than older hens. Furthermore, the number of SP holes was significantly greater (P ≤ 0.01) with the 400 and 800 million insemination doses as compared with the other 4 insemination doses. There was no interaction between hen age and sperm insemination dose. In conclusion, the absence of an interaction between hen age and insemination dose suggests that factors other than numbers of sperm binding to the IPVL influence EEM. These factors may be a combination of oviduct influences and IPVL properties that change as the hen ages.

High Concentration of Progesterone is Located in the Outer Layer of Japanese Quail Eggs (<I>Coturnix Coturnix Japonica</I>)

High Concentration of Progesterone is Located in the Outer Layer of Japanese Quail Eggs (<I>Coturnix Coturnix Japonica</I>)

Allurin, a 21 kD sperm chemoattractant, is rapidly released from the outermost jelly layer of theXenopus egg by diffusion and medium convection

Allurin, a 21 kD protein from Xenopus laevis egg jelly, has been demonstrated to attract sperm by video microscopy and by quantitative chemotaxis chamber assays. Here, we use immunocytochemistry to demonstrate that this sperm chemoattractant is located in the outermost layer of egg jelly (J3) and is rapidly released into the surrounding medium. SDS-PAGE analysis and Western blotting confirm the appearance of allurin in the medium within 1.5 min and separation of proteins in the medium by anion exchange FPLC, shows that nearly half of the allurin released over a 12-hr period is discharged in the first 5 min. The kinetics of allurin release from J3 and its appearance in the medium were quantitatively accounted for, by computer simulation of mathematical diffusion and convection models. Comparison of simulation data to quantitative measurements of allurin appearance in the medium suggests that allurin, although larger than most chemoattractants, is effectively dispersed by a combination of diffusion and medium mixing at the jelly surface during spawning. Our model further predicts that the innermost jelly layer, J1, is less permeable to allurin than the other layers, allowing it to act as a "reflector" to speed up allurin discharge.

Sperm supply and egg fertilization in the ostrich (Struthio camelus).

We used egg break-out and spermatozoa trapped in the perivitelline layer of eggs to test the hypothesis that sperm supply and egg fertilization rate are high in the ostrich. Egg fertilization status was determined at break-out by the appearance of the germinal disc (GD) and then the perivitelline layer overlying the GD region was collected to count sperm (SpermOPVL) under fluorescence following staining with 4',6'-diamidino-2-phenyindole (DAPI). The study was carried out on commercial ostrich farms over two laying seasons. In the first year, 229 eggs from nine randomly chosen ostrich pens comprising pairs, trios (two females, one male) and larger groups were collected for 1 week of every month of laying. Eggs contained 253 +/- 18 SpermOPVL/mm2 of the GD (mean +/- SEM; range 0-1330). Egg fertilization rate averaged 89.4 +/- 3.4% and varied from 78.6 to 98.2% between pens. Month had no effect on sperm supply or egg fertilization status. Eggs from paired birds (sex ratio 1 : 1) had less sperm in the GD than the eggs from pens with a higher sex ratio. In the second year, 150 eggs from seven pens, each containing only one male and either one, two or three females, were studied for 2 weeks at the beginning (winter), middle (spring) and end (summer) of laying. Eggs contained 364 +/- 45 SpermOPVL/mm2 of the GD (range 0-2880). Season had no effect on sperm supply or egg fertilization. The number of SpermOPVL varied between pens, assumed to be due to variation between individual males. The number of SpermOPVL increased as the sex ratio increased only when very high-ranking males were excluded from the analysis. Egg fertilization rate was 94.4 +/- 3.1% but varied from 64.0 to 100% between pens. Egg fertilization was not affected by season or sex ratio. Low fertilization rates were observed in two pens and appeared related to the lack of synchrony between timing of laying and sperm production in the first, and lack of mating in the second pen. We conclude that ostrich flocks generally have high rates of egg fertilization and any infertility is associated with lack of sperm supply.

Identification of the Sea Urchin 350-kDa Sperm-binding Protein as a New Sialic Acid-binding Lectin That Belongs to the Heat Shock Protein 110 Family: IMPLICATION OF ITS BINDING TO GANGLIOSIDES IN SPERM LIPID RAFTS IN FERTILIZATION

The 350-kDa sperm-binding protein (SBP), a species-specific sperm-binding protein, is localized in the vitelline layer of sea urchin eggs. In this study, we have shown for the first time that sperm gangliosides are ligands for the intact glycosylated SBP. Using recombinant fragments of the SBP, the N-terminal heat shock protein 110-like domain was shown to be responsible for the binding. The intact SBP could bind various gangliosides, and the binding was sialidase-sensitive and inhibited by sialyllactose, thus indicating that it is the sialic acid-binding protein. Calcium and magnesium ions were not required but they did enhance the binding activity of SBP. The observation that bacterially expressed recombinant SBP and the sialidase-treated intact glycosylated SBP lost divalent cation-dependent enhancement of binding activity suggests that the sialylated carbohydrate moieties of the SBP may be involved in this property. Furthermore, the SBP was shown to bind sperm lipid rafts, in which gangliosides are enriched, and this binding was lost upon sialidase treatment of the lipid rafts. Finally, liposomes containing the ganglioside specifically inhibited fertilization. Taken together, these results allow us to identify SBP as a member of a new class of sialic acid-binding lectin belonging to the Hsp110 family, and indicate that SBP may be involved in interaction of sperm with the vitelline layer of the egg.

Repeated inseminations required for natural fertility in a wild bird population.

In most bird species, pairs copulate many times before egg laying. The exact function of repeated inseminations (i.e. successful copulations) is unknown, but several suggestions have been made. We tested the hypothesis that repeated inseminations are required to ensure fertilization of eggs, by using an experimental method where free-ranging male collared flycatchers (Ficedula albicollis) were prevented from inseminating their mates. We show that egg fertility was lower when females had not copulated during the studied part of their fertile period. By counting sperm on the inner perivitelline layer of eggs, we estimated that a minimum of 86 sperm must reach the site of fertilization to ensure average fertility. Using the timing of inseminations and the numbers of sperm on successive eggs, we show that repeated copulations are necessary to achieve an average rate of fertilization of a single clutch. Our results thus provide evidence that repeated inseminations function to ensure fertilization success. We discuss possible constraints on sperm production and utilization that may have contributed to this pattern.

Use of Nonsettable Eggs to Evaluate Turkey Hen Fertility

The use of nonsettable eggs (checked, cracked) to perform fresh egg breakouts to estimate true fertility and to assess the rate of sperm penetration of the perivitelline layer overlying the germinal disc was evaluated. Germinal discs and the perivitelline layer overlying the germinal disc were accessible for assessments. The stage of blastodermal development positively correlated (r = 0.65, P < 0.0001) with eggshell thickness. It was also determined that the perivitelline layer of nonsettable eggs could be isolated and stained to determine the presence or absence of sperm holes. True fertility of nonsettable eggs (checked and cracked only) and settable eggs was 90 and 95%, respectively. It was concluded that checked and cracked eggs could be used to estimate the true fertility of a flock.