Alfalfa as a single dietary source for molt induction in laying hens

Molting is a process by which a hen's reproductive tract is rejuvenated prior to the beginning of a laying cycle. This process is often artificially induced in commercial settings in order to extend the productive life of a flock of hens. The most common method for the induction of molt is feed withdrawal for a period of several days. It has been noted that feed withdrawal, while effective in inducing molt and allowing an adequate reproductive rest period for the hen, may cause deleterious effects on the animal. This has prompted the investigation of alternatives to feed deprivation for the induction of molt in commercial laying hens. This study involved feeding alfalfa to hens to assess its ability to induce molt. Results show that alfalfa meal and alfalfa pelleted diets were equally effective as feed withdrawal in causing ovary weight regression in birds. Molted hens induced by alfalfa diets exhibited postmolt levels of egg production over a twelve week period that were similar to that of hens molted by feed withdrawal. The postmolt eggs laid by hens molted by alfalfa were of comparable quality to eggs from feed deprived hens. Alfalfa, a fibrous feed with low metabolizable energy, may be provided to hens on an ad libitum basis for an effective molt induction that retains comparable egg quality and production.     

Relationships among age, eggshell thickness and vitamin D metabolism and its expression in the laying hen.

Hens forming uncalcified shells synthesized less 1,25-hydroxycholecalciferol (1,25(OH)2D3) and less duodenal and eggshell gland (ESG) calbindin than normal laying hens. Hens forming thin shells had lower intestinal and ESG calbindin and its mRNA. Reducing ESG calcium (Ca2+) transport by the carbonic anhydrase inhibitor acetazolamide, but not by dietary Ca2+ restriction, reduced ESG calbindin and its mRNA. Two sub-populations of hens characterized by shell thickness (ST) maintained this characteristic throughout the whole production period. The differences between the two sub-populations increased with age. In old laying hens, the two sub-populations responded differently to dietary Ca2+ restriction and to exogenous 1,25(OH)2D3. Those forming a thin shell responded to 1,25(OH)2D3 by a significant improvement in ST. The results suggest that: (a) the mechanism responsible for Ca2+ transport to the egg shell consists of a vitamin D-dependent absorption of Ca2+ and a multi-factor-dependent transfer of Ca2+ to the shell; (b) both steps are, most likely, calbindin-mediated; however, the induction of calbindin gene expression in the ESG is predominantly calcium-dependent; and (c) the apparent defect in vitamin D metabolism or its expression in old hens is typical of, or even exclusive, to thin-shell-forming hens.     

Using a feed-grade zinc propionate to achieve molt induction in laying hens and retain postmolt egg production and quality

A commercial-feed-grade form of zinc propionate was examined as a potential feed amendment at a concentration of 1% zinc to induce molt in 90-wk-old hens. Dietary treatments consisted, of 4 treatment groups of 28 birds each randomly assigned to either (1) molted conventionally by feed withdrawal, (2) 1% zinc as Zn acetate, (3) 1% zinc as Zn propionate, or (4) nonmolted control for 9 d. Ovary weights of hens fed Zn acetate or Zn propionate were not significantly different from each other, but hens fed Zn acetate or Zn propionate were significantly (p<0.05) lighter than the ovary weight of nonmolted control hens. Zinc concentrations in the kidney and liver were significantly (p<0.05) increased in both Zn acetate- and Zn propionate-molted hens when compared to either nonmolted control-fed hens or feed-withdrawal molted hens. Over the entire 3-mo postmolt period, there were no significant differences in interior or exterior egg qualities among the four treatments Egg production of hens fed Zn acetate was significantly lower than feed-withdrawal hens, Zn propionate-fed hens, or nonmolted control hens (p<0.05). The data of the current study demonstrated that feeding a feed grade of Zn propionate (1% Zn)-supplemented diet can induce molt and retain postmolt egg quality and production comparable to hens molted by feed withdrawal.     

Potential of alfalfa as an alternative molt induction diet for laying hens: egg quality and consumer acceptability

Dietary molt induction to initiate additional egg laying cycles in commercial laying hen flocks is a wide spread practice in the United States. Feed deprivation is the most commonly used method but this practice has generated several concerns which has lead to research for viable alternative approaches. From a management standpoint a single ingredient molting diet consisting of high fiber-low energy represents an easily adaptable diet for large laying hen production units. Alfalfa meal is readily available in most commercial locations and possesses many of the desirable properties of an ideal laying hen molt diet. In the current study hens at a commercial laying facility were molted by both alfalfa and feed deprivation. After the hens had reentered post-molt commercial egg production, eggs were examined for egg quality performance. Egg shell strength, albumen height, yolk height, weight, length, and yolk color were all tested using various mechanical techniques. The eggs were also sampled for testing by consumer sensory panels that assessed the desirability of the eggs' color and flavor/texture. Eggs laid by hens molted by alfalfa had a significantly lower (p<0.05) "a*" level of colorimetry. Eggs laid by hens molted with alfalfa also exhibited significantly higher (p<0.05) egg weights and length. In the consumer sensory test, there was no significant difference (p>0.05) in color or flavor/texture scores in eggs from either feed deprived or alfalfa molted hens. The consumer sensory and mechanical quality attributes indicates that alfalfa shows promise as an alternative molt induction diet by providing a single diet option for extending egg production into a second egg laying cycle.     

Dietary thyroxine induces molt in chickens (Gallus gallus domesticus)

Thyroxine increases during a molt in wild and captive birds, and thyroidectomy prevents induction of molt. This trial examined the effect of dietary thyroxine on molt induction molt in chickens (laying hens, 59 weeks of age). In a completely randomized design (n=15 hens/replication; 6 replications/treatment), hens were randomly assigned to either a traditional molting program consisting of feed withdrawal (FWD), or to diets containing 40 mg thyroxine/kg diet (HT), 20 mg thyroxine/kg diet (LT), or 40 mg thyroxine from thyroactive iodinated casein/kg diet (TIC). The molting treatment lasted 7-13 d, until egg production reached 0%. After molt induction, birds had ad libitum access to the same diet, until egg production was re-initiated and maximized ( approximately 56 d). All treatments induced molt, based upon cessation of egg laying and regression of ovary and oviduct. Birds on FWD treatment lost more body weight during the molting period, but gained more after molt compared to thyroxine treatments (P<0.01 for each), although all body weights were similar when egg production was maximized. Data demonstrate that oral thyroxine, in purified or non-purified form, induces a molt and may enhance animal well-being by reducing the need for FWD.     

Immunological cell and serum metabolite response of 60-week-old commercial laying hens to an alfalfa meal molt diet

The practice of induced molting involves the restriction of light, feed removal and optionally water for 5-14 days. However, there is growing concern regarding feed removal and animal welfare issues. With this in mind, alternative diets have been developed to produce similar molting effects as that of feed deprivation. Alfalfa, which largely consists of insoluble fiber, can be used as a molting diet. In this study, heterophil and lymphocyte counts, serum chemistry, and organ weight parameters were evaluated in hens that were deprived of feed or fed alfalfa during a nine day induced molt. Full-fed hens were used as the control. Blood serum parameters assessed included calcium, magnesium, glucose, total protein, ketone bodies, uric acid, and cholesterol. White blood cells were counted and categorized by cell type. On the ninth day of the trial, the hens were euthanized and the liver, spleen, heart, intestine, pancreas, ovary, oviduct, and kidney were collected and weighed. On day 8 birds molted with alfalfa or by feed deprivation had significantly higher (P<0.05) levels of ketone bodies and cholesterol and lower levels of calcium, and magnesium compared to the full-fed hens while birds molted by feed deprivation exhibited significantly lower levels of uric acid. Birds molted by both methods exhibited significant reductions in ovary, oviduct, liver and pancreas weights and increased spleen weights when compared to the non-molted hens. On days 0, 2, and 6 there were no significant differences (P>0.05) in either heterophil or lymphocyte percentages. However, heterophil percentages were higher in feed withdrawal birds than full-fed birds on day 4 but lymphocyte percentages were higher in full-fed birds compared to feed withdrawal birds. On day 8 of the induced molt lymphocyte percentages were higher from full-fed birds when compared to feed withdrawal birds but no significant differences were detectable for heterophil percentages. Based on reproductive organ weight loss and changes in serum and immunological responses of birds during molt, it appears that alfalfa meal can be an effective molt induction alternative.     

Effect of estrogen on egg production,shell quality and calcium metabolism in molted hens

Force-molted White Leghorn laying hens were implanted with 0.25, 0.5, 1 and 3 Compudose 200 pellets (24 mg 17β estradiol/pellet). Plasma estradiol increased with increasing E₂ dosages in a linear manner and decreased over time in a quadratic manner (P < 0.01). E₂ treatment had a nonlinear effect on total plasma calcium. Oviduct weight, shell thickness and egg weight were not significantly affected by exogenous estradiol whereas tibial bone ash percentage was increased at only one dose (P < 0.05:0.5 pellet group). Physiological supplementation with estradiol does not improve shell quality.     

Organ weight and serum triglyceride responses of older (80 week) commercial laying hens fed an alfalfa meal molt diet

Since one of the costs in the commercial egg industry is that of replacement pullets, commercial egg layer managers have opted to induce molt older hens in order to extend their productive life for additional egg laying cycles. Conventional molt induction involves the complete removal of feed for several days. However, this management practice can lead to deleterious physiological responses by the hen and subsequent susceptibility to infection by pathogens. Consequently less stressful molting regimens involving the feeding of low energy diets such as alfalfa have been developed. In this study, 80 week old laying hens that were deprived of feed or fed alfalfa meal during a nine day induced molt. Full fed hens were used as the control. On day 8 serum triglycerides were quantified and on day 9 hens were euthanized and the liver, spleen, heart, intestine, pancreas, ovary, and kidney were collected and weighed. Intestinal weight were highest in the non-molted hens, lower in the hens fed alfalfa, and lower still in the hens deprived of feed. Molted hens exhibited reduced weights of liver, heart, ovary, and pancreas compared to the non-molted hens. Serum triglycerides were highest in the non-molted hens, less in feed deprived hens, and the lowest in alfalfa fed hens. These results suggest that a comparable molt could be achieved with feeding alfalfa meal to 80 week hens compared to feed deprivation.     

Effect of melengestrol acetate as an alternative to induce molting in hens on the expression of yolk proteins and turnover of oviductal epithelium

Inducing hens to molt increases egg quality, egg production and extends the productive life of hens. It has been previously demonstrated that melengestrol acetate (MGA), an orally active progestin, decreased gonadotropic support for the ovary, which decreased the steroidogenic support for the oviduct and resulted in the cessation of lay. Estradiol produced by the theca cells of small follicles stimulates the production of the yolk proteins vitellogenin II and apolipoprotein II by the liver and supports the oviductal epithelial cells. The objective of the present experiment was to determine gene expression for yolk proteins and oviductal epithelial cell turn-over in response to a MGA-induced molt. Hy-Line W-36 laying hens were fed either 0 or 8mg MGA per day for 28 days in a balanced diet and then returned to a standard layer ration until day 36. Four birds per treatment on days 1, 8, 16, 28 and 36 were euthanized and the liver was removed and snap frozen in liquid nitrogen until RNA was extracted. Expression of vitellogenin II and apolipoprotein II genes was determined using real-time RT-PCR. A portion of the magnum was removed to determine proliferation and programmed cell death for secretory and ciliated luminal epithelium. Vitellogenin II and apolipoprotein II gene expression was reduced in hens fed 8mg MGA compared to those fed 0mg MGA. There was no effect of day on gene expression of either yolk protein. Cell proliferation was increased in the ciliated epithelial cells of the oviduct in hens receiving 8mg MGA compared to those receiving 0mg. However, programmed cell death of the ciliated epithelial cells was not different between controls and MGA treatment. Programmed cell death and proliferation increased in the secretory epithelial cells in hens receiving 8mg MGA compared to controls. Therefore, utilizing MGA as an alternative method to induce molt results in some, but not all, of the physiological changes previously described for hens molted by feed withdrawal. These findings lead us to suggest that some of the observed physiological changes resulting from feed withdrawal are required to increase egg quality and egg production following molt and other changes are not necessary, but are just a result of nutrient deprivation.     

Variation in the ovarian and plasma progesterone and estradiol levels of the domestic hen during a pause in laying

Little is known about the physiological events occurring in the chicken ovary during a pause in laying, therefore the aim of the present study was to examine changes in sex steroid concentration in the follicle wall and blood plasma during cessation of egg laying. The experiment was performed on laying Isa Brown hens. Control hens were fed ad libitum whereas the experimental ones were subjected to a pause in laying by complete food deprivation for 5 days and water deprivation on 3 day followed by feeding every second day up to 9 day and then ad libitum. Blood samples were taken from the wing vein each day. The hens were decapitated on day 3, 6, 9, and 16. The ovary was isolated and the following follicles were dissected: white (1-2; 2-4; 4-6; 6-8 mm) and yellow preovulatory ones (F1-F3). Progesterone and estradiol were measured in the follicle wall and blood plasma by RIA methods. The hens stopped egg laying on day 4 and began egg restoration on day 14 of the experiment. Cessation of egg laying was preceded by a decrease in estradiol and progesterone levels in the ovary as well as in the blood plasma. The plasma level of these steroids began to increase 7 days before the start of egg restoration. Autopsy of the ovary showed that the atrophy of the chicken yellow preovulatory follicles during the pause in laying was accompanied by a significant increase in the total number of white follicles.     

DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE) AS A RAPID METHOD FOR ASSESSING GASTROINTESTINAL TRACT MICROFLORA RESPONSES IN LAYING HENS FED SIMILAR ZINC MOLT INDUCTION DIETS

Induced molting through feed withdrawal can change the microenvironment of crop and ceca sufficiently to allow them to be the sites of Salmonella colonization in the chicken intestine. This study compares the denaturing gradient gel electrophoresis (DGGE) profiles of microbial crop and cecal communities among molted hens fed similar zinc acetate or zinc propionate amended molt diets to hens either undergoing feed withdrawal or hens full fed and not molted. Dendrograms of DGGE amplicon patterns indicated over 85% similarity of cecal communities between zinc acetate fed hens and zinc propionate fed hens and over 60% similarity of crop communities between zinc acetate fed hens and zinc propionate fed hens. Rapid comparison of complex gastrointestinal microflora profiles in laying hens fed similar diets is possible using DGGE.     

Changes in plasma thyroid hormones, luteinizing hormone (LH), estradiol, progesterone and corticosterone of laying hens during a forced molt

1. Circulating concentrations of iodothyronines, luteinizing hormone(LH), estradiol(E2), progesterone and corticosterone were measured in hens before, during, and after a forced molt induced by fasting. 2. Corticosterone increased at the onset of molt, peaked at the maximal molt and returned to pre- and post-molt levels. LH, E2 and progesterone declined during the molt, and the decline was coincident with the cessation of egg production. 3. Thyroxine(T4), triiodothyronine (T3) and reverse triiodothyronine(rT3) increased during the molt. The increases of T4 and T3 were not abolished even if the forced molt was conducted in mild weather.     

Effects of Zinc Oxide Nanoparticles on Performance,Egg Quality,Tissue Zinc Content,Bone Parameters,and Antioxidative Status in Laying Hens

The objective of this study was to observe the effects of dietary supplementation with zinc oxide nanoparticles (ZnO-NPs) on performance, egg quality, tissue Zn content, bone parameters, superoxide dismutase activity (SOD), and egg malondialdehyde (MDA) content in laying hens. A total of 288 laying hens at 64 weeks of age were randomly assigned to 4 treatments, 6 replicates, with 12 birds each. Experimental diets included the based diet (without Zn supplementation), and basal diet supplemented with 40, 80, and 120 mg Zn/kg from ZnO-NPs. Feed intake and egg mass were significantly higher in the 40 and 80 ZnO-NPs groups than the other groups. The birds in the 80 ZnO-NPs group had significantly higher egg product than the 120 ZnO-NPs and control groups. Egg shell thickness and shell strength significantly increased in the 40 and 80 ZnO-NPs groups. Moreover, Haugh unit significantly improved in the groups supplemented with ZnO-NPs, compared to the control group. Bone-breaking strength was significantly greater in the 80 ZnO-NPs group than the 120 ZnO-NPs and control groups. Also, ash weight was significantly greater in the 40 and 80 ZnO-NPs groups than the control group. There were significant differences among the groups in the Zn content in plasma, tibia, liver, pancreas, and egg. Relative to the control group, ZnO-NPs supplementation significantly increased the activities of SOD in the liver, pancreas, and plasma. The MDA content in egg was significantly reduced in the groups supplemented with ZnO-NPs. In conclusion, this study demonstrates that ZnO-NPs as dietary supplementation can improve the performance of laying hens, and levels of 40 to 80 ZnO-NPs are the optimal concentrations.

     

Effects of phytase supplementation on production performance,egg and bone quality,plasma biochemistry and mineral excretion of layers fed varying levels of phosphorus

Excessive fecal excretion of phosphorus (P) has increasingly become an environmental issue due to oversupply of P in layer rations, and thus it is imperative to minimize safety margins for P to ensure the sustainability of the egg industry. In this study, a 12-week feeding trial (22 to 34 weeks of age) was conducted to evaluate the effects of phytase supplementation on production performance, plasma biochemistry, egg and bone quality and P excretion of laying hens fed various levels of non-phytate P (NPP). Forty-eight Lohmann white laying hens were randomly allocated to one of six corn–soybean meal–oat-based diets: diets containing 2.0, 2.5 or 3.0 g/kg NPP without phytase, and diets containing 1.0, 1.5 or 2.0 g/kg NPP with phytase (1 000 U/kg diet) where phytase inclusion was expected to provide 1.0 g/kg NPP to laying hens, thus making the phytase-unsupplemented treatment served as a control for the phytase-supplemented treatment accordingly. Productive performance was recorded during the experimental period. Blood and egg samples were collected, and digestibility studies were conducted at weeks 6 and 12 of the experiment. Bone mineralization was evaluated at the end of the experiment. Egg weight and egg production, feed consumption, BW and feed conversion ratio of laying hens fed lower NPP diets supplemented with phytase were comparable to those of hens fed high NPP phytase-unsupplemented controls. Eggshell thickness, specific gravity, Haugh units, tibia bone mineral density, tibia ash percent, plasma P and other biochemical parameters were not significantly different among dietary treatments. Total P intake, excretion and retention were affected by diet (P < 0.001), but its deposition in eggs was not significantly different. Contrast analysis further showed that total P excretion of phytase present vs phytase absent was averagely reduced by 40.4 mg/hen per day (P < 0.01). Moreover, total P excretion was linearly (P < 0.01) reduced with lowering dietary NPP, and this relationship was similar regardless of whether phytase was supplemented or not. The results from this study indicated that NPP levels in laying hen diets could be reduced to 1.0 g/kg (excluding the portion of NPP released by phytase) with the inclusion of phytase, without negative effects on production performance and health of the hens, thereby diminishing P excretion into environment.     

Decreased granulosa cell luteinizing hormone sensitivity and altered thecal estradiol concentration in the aged hen, Gallus domesticus

Few studies have examined the effect of age on the ovulation cycle of the hen. Our aim was to determine if changes in the ovary account for the decrease in egg production with age. Young hens (28-38 wk of age) laying at least 20 eggs per sequence and old hens (53-63 wk of age) laying 3-6 eggs per sequence were used. We determined luteinizing hormone (LH) sensitivity of the ovary of young and old hens by measuring LH stimulable adenylyl cyclase (AC) activity of the granulosa layer. We also measured theca- and granulosa-layer weights and steroid concentrations of these layers and of the serum in young and old hens. Mean basal AC activity (pg/min/mg protein) for the largest (F1) and second largest (F2) follicles from young and old hens did not differ. A significant dose-response relationship to LH was present in all groups, and AC responsiveness to increasing doses of LH was greater in the F1 and F2 follicles of young hens than in the same follicles of old hens. The F4 and F5 follicles of young hens had a significantly greater estradiol (E2) concentration (pg/mg theca protein) compared to old hens, while the E2 concentration in the F2 follicle was greater in old hens. The theca layer of the F1 follicle of old hens weighed significantly more than that of young hens, whereas the theca layer of the F3, F4 and F5 follicles from young hens weighed more than those of old hens.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Effects of Melatonin on the Physical Properties of Bones and Egg Shells in the Laying Hen

Laying hens often experience unbalanced calcium utilization which can cause deficiencies in bone and egg mineralization. Because melatonin has been shown to affect bone mineralization in other animals, we examined whether treating hens with melatonin would affect eggshell thickness and improve skeletal performance, thereby reducing skeletal and egg shell defects. Birds were given a diet containing either low (30 µg/kg), medium (300 µg/kg), or high (3 mg/kg) concentrations of melatonin, or control feed through approximately one laying cycle. We examined the weight, length, and strength of egg, femur, tibia, and keel. Hens treated with a high concentration of melatonin showed significant strengthening in their femur and tibia, as measured by maximum force sustained and breaking force, compared to controls. Egg weights from hens treated with melatonin were significantly greater than those from hens that were not treated with melatonin. Conversely, egg shell mass of hens treated with melatonin was significantly lower than those of hens not treated with melatonin. Our data suggest that melatonin may affect the allocation of calcium to bone at the expense of egg shell mineralization.     

Steroidogenic activity of chicken ovary during pause in egg laying

The present study was designed (i) to assess the changes in the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and cytochrome P450 aromatase (P450arom) in the ovaries of hens which are subjected to a pause in egg laying by fasting, and (ii) relate these changes with progesterone (P(4)) and estradiol (E(2)) production in the ovary. Hy-Line Brown laying hens (n=90) were fasted for 5 days with water deprivation only on day 3 and subsequently fed every second day up to day 13 and then ad libitum. Birds were euthanized (n=18) on day 0, 3, 6, 9 and 16 of the experiment. The activities of 3beta-HSD and P450arom were evaluated in stroma with cortical follicles (<1mm) and in the wall of white non-hierarchical (1-8 mm) and yellow hierarchical follicles (>8 mm) by histochemical and immunohistochemical method, respectively. Ovarian P(4) and E(2) were measured radioimmunologically. Hens stopped egg laying on day 4 of the experiment and pause in egg laying lasted up to day 12. The hens then began to gradually resume egg laying and on day 16 all hens laid eggs. It was found that during the pause in egg laying: (i) the activity of 3beta-HSD in stroma and normal white follicles was slightly decreased while P450arom activity was significantly increased; (ii) in yellow hierarchical follicles which became atretic and regressed, activity of both enzymes were markedly decreased; (iii) ovarian P(4) production dramatically decreased, whereas ovarian E(2) production after an initial decrease significantly increased. In white atretic follicles the activity of 3beta-HSD and P450arom was very weak during the whole experiment. In conclusion, the present results indicate that during a pause in egg laying white follicles become resistant to atresia.     

Effect of growth hormone on steroid content,proliferation and apoptosis in the chicken ovary during sexual maturation

The present study was undertaken to examine in vivo the effect of growth hormone (GH) on progesterone and estradiol levels and on cell proliferation and apoptosis in the chicken ovary during sexual maturation. Hy-Line chickens (10 weeks old) were injected three times a week with 200 μg recombinant chicken GH (cGH) per kilogram body weight until sexual maturity. GH treatment significantly increased ovarian weight at 16 weeks of age, i.e., ∼1 week before onset of egg laying. The progesterone content in the ovary just before and at the time of sexual maturity and the estradiol content before onset of egg laying were also elevated after cGH injections. The highest number of proliferating (positive for proliferating cell nuclear antigen) and apoptotic (positive for terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labeling) cells was found in the ovarian stroma and white follicles (>1-4 mm diameter), whereas the lowest number of these cells was detected in yellow (>8-30 mm) follicles. Administration of cGH significantly stimulated cell proliferation and inhibited cell apoptosis in the ovarian stroma and small ovarian follicles. The number of ovarian follicles and the weight of the ovary prior to the first oviposition were also higher in cGH-injected hens. Thus, prior to and after the onset of egg laying, GH participates in the growth, maturation and hormonal activity of ovarian follicles in the chicken, via the regulation of steroidogenesis, proliferation and apoptosis processes.     

Influence of egg production on zinc, copper and iron metabolism in the turkey hen (Meleagris gallopavo)

1. Serum concentrations of zinc, copper, iron, calcium and protein increased significantly with the onset of egg production in turkey hens. 2. Hepatic concentrations of zinc, copper and iron declined significantly with the onset of egg production, whereas the total hepatic content of zinc and copper increased significantly. 3. When plasma from laying and nonlaying hens was subjected to gel-permeation column chromatography, a high molecular weight protein fraction was detected in laying but not in nonlaying plasma which bound significant amounts of zinc, copper and iron and which coeluted with purified vitellogenin. 4. Vitellogenin purified from the plasma of laying hens is a metalloprotein which upon analysis was found to contain 0.54, 0.09 and 0.72 micrograms of zinc, copper and iron, respectively, per mg of protein.