Chick embryos grown in shell-less culture are calcium-deficient by 9 days and retarded in growth ... more Chick embryos grown in shell-less culture are calcium-deficient by 9 days and retarded in growth by 13 days of incubation (Dunn and Boone, Poult. Sci., 56:662-672, 1977). To determine whether addition of exogenous calcium might stimulate growth and/or survival of cultured embryos, calcium supplementation was attempted. Calcium supplementation between days 11 and 17 resulted in significant increases in both total embryo and serum calcium. Addition of shell pieces oriented with shell membranes onto the chorioallantoic membrane (CAM) resulted in significant stimulation of calcium transport by the CAM. However, growth of supplemented embryos was not increased to the same degree as were embryonic serum and total calcium levels. It is concluded that at least one factor other than calcium deficiency is responsible for retarded growth of shell-less cultured embryos.
In order to determine whether the chorionic epithelium (CE) of the chorioallantoic membrane (CAM)... more In order to determine whether the chorionic epithelium (CE) of the chorioallantoic membrane (CAM) of chick embryos maintained in shell-less culture might be suitable for studies of calcium translocation and/or tissue grafting, membranes from lo-to l&day embryos were studied histologically using Nomarski differential interference optics on living, unfixed tissue and ultrastructurally using scanning and transmission electron microscopy. Differentiation of the CAM of cultured embryos appeared similar to that in ovo. At 10 days, an electron-lucent squamous cell layer covered much of the CE. Intraepithelial capillaries were separated from the chorionic surface by a relatively thick (>5+m) cytoplasmic layer. By 12 days, dense, vacuolated, apparently degenerating cells covered much of the CE. At later stages (14 to 16 days), degenerating cells were seen infrequently and most capillaries were covered by thin (<0.2+m) cytoplasmic processes. By 18 days cultured CAM no longer appeared normal, but demonstrated local sites of squamous metaplasia. Villus-cavity and capillary-covering cells were observed in culture by 10 to 14 days of total incubation. These studies indicate that the presence and active transport of large amounts of calcium from the eggshell are unnecessary for normal differentiation of the CAM. Thinning of the cytoplasmic barrier overlying the chorionic capillaries and progressive rounding of endothelial cell nuclei indicate that vascularization of the CAM in culture follows a course similar to that in ovo.
Prostaglandins Leukotrienes and Essential Fatty Acids, Aug 1, 2012
1. The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and s... more 1. The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and shell-less culture affords a unique opportunity for direct observations over time of individual blood vessels to pharmacologic interventions. We tested a number of lipids including prostaglandins PGE 1&2 for vascular effects and signaling in the CAM. Application of PGE 1&2 induced a decrease in the diameter of large blood vessels and a concentration-dependent, localized, reversible loss of blood flow through small vessels. The loss of flow was also mimicked by misoprostol, an agonist for 3 of 4 known PGE receptors, EP 2-4 , and by U46619, a thromboxane mimetic. Selective receptor antagonists for EP 3 and thromboxane each partially blocked the response. This is a first report of the effects of prostaglandins on vasoreactivity in the CAM. Our model allows the unique ability to examine simultaneous responses of large and small vessels in real time and in vivo.
American Journal of Physiology-cell Physiology, Nov 1, 1986
By removing the shell membranes from the chorioallantoic membrane, the chorion is damaged, as vis... more By removing the shell membranes from the chorioallantoic membrane, the chorion is damaged, as visualized by electron microscopy, and rendered permeable, as evidenced by penetration of horseradish peroxidase and increased inhibition of the allantoic Na+-K+ pump by ouabain applied on the chorionic side. The short-circuit current (SCC) of this functionally isolated allantoic epithelium is augmented by nystatin, a channel-forming ionophore, when applied to the mucosal surface. Electrical parameters were determined for three age groups between 12 and 19 days of incubation. The SCC approximately doubled from the youngest (12-13 days) to the oldest (18-19 days) groups, whereas the transepithelial resistance (Re) of 700-900 omega X cm2 remained the same. Amiloride, an inhibitor of apical Na+ uptake, inhibited 98-100% of the SCC at 10(-4) M in both 15-16 and 18-19 day epithelia. In the 12- to 13-day preparation 20-25% of the SCC was insensitive to 10(-3) M amiloride. The Ki's for amiloride were similar in all preparations, at about 5 X 10(-7) M. Determination of the Hill coefficients for inhibition revealed a lower value (0.75 +/- 0.03) for the 12-13 day preparation compared with the two older preparations with coefficients not significantly different from unity. Replacing Na+ in the bathing solutions abolished the SCC of 18-19 day epithelia, whereas about 15% of the SCC remained at 12-13 days. Thus, during development, the SCC of the allantoic epithelium increases in magnitude and becomes increasingly (to 100%) amiloride-sensitive and Na+-dependent.
Journal of Experimental Zoology Part A: Ecological and Integrative Physiology
A method is described for culturing 64–70 h‐old chicken embryos and egg contents outside of the e... more A method is described for culturing 64–70 h‐old chicken embryos and egg contents outside of the eggshell through to hatching. Cultured egg contents were suspended in polymethylpentene kitchen wrap (F.O.R. wrap; Riken Fabro) supported in polyvinyl chloride tripods. Tripods were incubated in Plexiglas environmental chambers which were rocked automatically through an angle of ±20°. The concentration of CO2 was maintained at 2% throughout incubation, while that of O2 was increased from ambient to 50%, and relative humidity was decreased from 90%–92% to 83%–84% at incubation Day 9. Cultured embryos not supplemented with calcium did not hatch. The Hatch rate increased when supplemental calcium L‐lactate hydrate was increased between 250 and 350 mg. A maximal hatch rate of 54.8% was achieved when cultures were supplemented with 350 mg of calcium L‐lactate hydrate and 3.5 ml of sterile water. Adding 400 or 450 mg of calcium L‐lactate hydrate did not increase the hatch rate further. The mass...
The ultrastructure of the parathyroid glands was studied in chick embryos developing normally in ... more The ultrastructure of the parathyroid glands was studied in chick embryos developing normally in ovo or in shell‐less culture (after removal of the eggshell). Shell‐less chick embryos are significantly hypocalcemic relative to their in ovo counterparts. At 12 days of incubation, the parathyroid glands of shell‐less embryos contain more lipid and show evidence of increased protein synthetic activity relative to those grown in ovo (more rough endoplasmic reticulum, presence of some dense secretory granules). The glands from in ovo embryos do not contain secretory granules at this age. At 15 days of incubation, the in ovo glands have developed signs of protein synthetic activity similar to those of the 12‐day shell‐less embryos. However, the parathyroids of the 15‐day shell‐less embryos appear strikingly more active than at 12 days, containing stacks of concentric RER membranes and increased numbers of secretory granules. By 18 days of incubation, the ultrastructure of the glands of th...
The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology, 2005
Shell-less culture of chick chorioallantoic membrane (CAM) of developing chicken embryos is a use... more Shell-less culture of chick chorioallantoic membrane (CAM) of developing chicken embryos is a useful model to evaluate the effects of vascular agents. We assessed the response of CAM vessels to epoxyeicosatrienoic acids (EETs), derivatives of the essential fatty acid arachidonic acid, that have a number of important biological functions, including dilation of microvessels in the coronary, cerebral, renal, and mesenteric circulations. Three of four regioisomers of EETs, 14,15-, 11,12-, and 8,9-EET, induced a characteristic dose-dependent acute hyperemia within 4 min after application on 10-day-old CAMs. This response was marked in early stages of development (between days 8 and 10), but the frequency and intensity of the response were reduced after 11 days of development. Histological examination demonstrated that the hyperemia was not due to extravasation of erythrocytes. However, many capillaries were distended and contained densely packed erythrocytes as compared to uniformly arranged vessels and erythrocytes in untreated CAMs. Transmission electron microscopy showed the basal laminae surrounding capillaries remained intact, similar to those in vehicle-treated or untreated CAM tissue. The hyperemia was specific to EETs since we did not observe it to be induced by other vasodilators such as nitric oxide or prostacyclin. In conclusion, we report a novel vascular response to EETs using the CAM as an in vivo model. These lipids specifically distend a subset of capillaries in a dose-and development-dependent manner.
Abstract A technique is described whereby avian egg contents removed from the shell after 72 hour... more Abstract A technique is described whereby avian egg contents removed from the shell after 72 hours of normal incubation may be successfully cultured for extended periods of time (81% for 15.5 days), many of the embryos reaching Hamilton-Hamburger Stage 40–41 (14–15 days). Egg contents are suspended in commercial plastic wrap held in place in culture vessels consisting of modified Playtex nurser holders. Culture vessels are lowered into Kerr wide mouth quart mason jar saturation humidity chambers which have been attached to aquarium lines inside of a modified Forma model 329 CO2 incubator. Use of a 5–8% CO2 atmosphere substantially improved embryonic survivability.
Shell-less culture involves culturing chick embryos with associated yolk and albumen outside of t... more Shell-less culture involves culturing chick embryos with associated yolk and albumen outside of the eggshell and shell membranes. The technique allows direct access to and continuous observation of cultured embryos almost to the time of hatching. The plastic wrap/culture tripod technique described in this paper allows normal embryonic growth and differentiation from 48 hours (in ovo pre-incubation age) through at least 10 days of total incubation. As the duration of in ovo pre-incubation is decreased below 36 hours, there is a concomitant increase in the percentage of grossly abnormal embryos associated with decreased survival and retarded growth and differentiation. Survival of embryos pre-incubated for 72 hours through 13 and 18 days of total incubation is greater than 80% and 40%, respectively. The wrap/tripod technique allows substantially better survival and more normal development of cultured embryos than does the petri dish technique of Auerbach et al. (1974). Embryonic growt...
Abstract A photographic study of development of chick embryos cultured in vitro was conducted in ... more Abstract A photographic study of development of chick embryos cultured in vitro was conducted in order to demonstrate the relationship between embryos and extraembryonic membranes during culture. Survival of embryos in this system has been extended to 21 days of total incubation (3 days in shell plus 18 days in vitro) and development to Hamburger-Hamilton stage 45 (partial or complete yolk sac retraction). A few mature cultured embryos have “pipped” through the chorioallantoic membrane and have emitted audible peeping. Three-day embryos and egg contents were cultured in commercial plastic wrap suspended in plastic tripods. The yolk sac expanded and flattened to cover the bottom of the culture chamber by five days of total incubation. Downgrowth of the yolk sac vascular system along the plastic wrap walls of the chamber began by six days, while downgrowth of the chorioallantoic membrane (CAM) began by eight days. The CAM often completely enclosed cultured egg contents by 17 days, an event which occurs in ovo by 11 to 12 days. Lack of formation of the albumen sac resulted in inhibition of albumen uptake during the latter portion of incubation. A small number of embryos were on their right side rather than on the normal left side during the first two days of culture. Many of the right side cultured embryos experienced “compensatory torsion” onto their left side by three days of culture (six days of total incubation).
In order to assess mineral uptake by chick embryos cultured outside of the shell, growth and mine... more In order to assess mineral uptake by chick embryos cultured outside of the shell, growth and mineral (Ca, Mg, K, Na) composition of control and cultured embryos and total egg contents (embryo plus residual egg contents) were measured from 8 through 21 days of incubation. At day nine and thereafter, cultured embryos contained significantly less Ca and Mg than did control embryos. At day 11 and thereafter, ash weight of cultured embryos was significantly less than that of controls. At 13 days and thereafter, wet weight, dry weight, toe length, K and Na content of cultured embryos were significantly less than in controls. To assess relative mineral uptake of cultured embryos, mineral (Ca, Mg, K, Na) content per gram embryo dry weight was plotted against incubation age. Values for the ratio of Mg, K and Na per gram dry weight were similar for control and cultured embryos. Control values for Ca per dry weight were consistently higher than those for cultured embryos during the period from 9 to 21 days. The data suggest that Ca may not be present in sufficient quantity, even on a relative growth basis, to perform its normal physiological functions in cultured embryos.
An improved technique is described for in vitro culture of the chick embryo from three days throu... more An improved technique is described for in vitro culture of the chick embryo from three days through 21 days of total incubation (three days in shell plus 18 days in culture). Mean survival time for cultured embryos is 18 days of total incubation, and mean morphological stage is Hamburger-Hamilton stage 41 (15 days). Mean measurements of wet weight, dry weight, and right third toe length for stage 45 (partial or complete abdominal yolk sac retraction, 19-20 days) cultured embryos are comparable with corresponding means for stage 42-43 (16-17 days) embryos from control intact eggs. Possible causes of growth retardation in vitro are briefly discussed.
ABSTRACT It is shown that carboxymethyldextrans can be used to displace proteins from high-perfor... more ABSTRACT It is shown that carboxymethyldextrans can be used to displace proteins from high-performance ion-exchange columns. The high resolving power of the method is demonstrated by the total separation of the A and B genetic variants of the β-lactoglobulins, which have only 0.1 pH difference in their isoelectric points. The separation of up to 12.8 mg of the β-lactoglobulins on a 150-μl column demonstrates the potential for high capacity in displacement chromatography.
Chick embryos grown in shell-less culture are calcium-deficient by 9 days and retarded in growth ... more Chick embryos grown in shell-less culture are calcium-deficient by 9 days and retarded in growth by 13 days of incubation (Dunn and Boone, Poult. Sci., 56:662-672, 1977). To determine whether addition of exogenous calcium might stimulate growth and/or survival of cultured embryos, calcium supplementation was attempted. Calcium supplementation between days 11 and 17 resulted in significant increases in both total embryo and serum calcium. Addition of shell pieces oriented with shell membranes onto the chorioallantoic membrane (CAM) resulted in significant stimulation of calcium transport by the CAM. However, growth of supplemented embryos was not increased to the same degree as were embryonic serum and total calcium levels. It is concluded that at least one factor other than calcium deficiency is responsible for retarded growth of shell-less cultured embryos.
In order to determine whether the chorionic epithelium (CE) of the chorioallantoic membrane (CAM)... more In order to determine whether the chorionic epithelium (CE) of the chorioallantoic membrane (CAM) of chick embryos maintained in shell-less culture might be suitable for studies of calcium translocation and/or tissue grafting, membranes from lo-to l&day embryos were studied histologically using Nomarski differential interference optics on living, unfixed tissue and ultrastructurally using scanning and transmission electron microscopy. Differentiation of the CAM of cultured embryos appeared similar to that in ovo. At 10 days, an electron-lucent squamous cell layer covered much of the CE. Intraepithelial capillaries were separated from the chorionic surface by a relatively thick (>5+m) cytoplasmic layer. By 12 days, dense, vacuolated, apparently degenerating cells covered much of the CE. At later stages (14 to 16 days), degenerating cells were seen infrequently and most capillaries were covered by thin (<0.2+m) cytoplasmic processes. By 18 days cultured CAM no longer appeared normal, but demonstrated local sites of squamous metaplasia. Villus-cavity and capillary-covering cells were observed in culture by 10 to 14 days of total incubation. These studies indicate that the presence and active transport of large amounts of calcium from the eggshell are unnecessary for normal differentiation of the CAM. Thinning of the cytoplasmic barrier overlying the chorionic capillaries and progressive rounding of endothelial cell nuclei indicate that vascularization of the CAM in culture follows a course similar to that in ovo.
Prostaglandins Leukotrienes and Essential Fatty Acids, Aug 1, 2012
1. The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and s... more 1. The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and shell-less culture affords a unique opportunity for direct observations over time of individual blood vessels to pharmacologic interventions. We tested a number of lipids including prostaglandins PGE 1&2 for vascular effects and signaling in the CAM. Application of PGE 1&2 induced a decrease in the diameter of large blood vessels and a concentration-dependent, localized, reversible loss of blood flow through small vessels. The loss of flow was also mimicked by misoprostol, an agonist for 3 of 4 known PGE receptors, EP 2-4 , and by U46619, a thromboxane mimetic. Selective receptor antagonists for EP 3 and thromboxane each partially blocked the response. This is a first report of the effects of prostaglandins on vasoreactivity in the CAM. Our model allows the unique ability to examine simultaneous responses of large and small vessels in real time and in vivo.
American Journal of Physiology-cell Physiology, Nov 1, 1986
By removing the shell membranes from the chorioallantoic membrane, the chorion is damaged, as vis... more By removing the shell membranes from the chorioallantoic membrane, the chorion is damaged, as visualized by electron microscopy, and rendered permeable, as evidenced by penetration of horseradish peroxidase and increased inhibition of the allantoic Na+-K+ pump by ouabain applied on the chorionic side. The short-circuit current (SCC) of this functionally isolated allantoic epithelium is augmented by nystatin, a channel-forming ionophore, when applied to the mucosal surface. Electrical parameters were determined for three age groups between 12 and 19 days of incubation. The SCC approximately doubled from the youngest (12-13 days) to the oldest (18-19 days) groups, whereas the transepithelial resistance (Re) of 700-900 omega X cm2 remained the same. Amiloride, an inhibitor of apical Na+ uptake, inhibited 98-100% of the SCC at 10(-4) M in both 15-16 and 18-19 day epithelia. In the 12- to 13-day preparation 20-25% of the SCC was insensitive to 10(-3) M amiloride. The Ki's for amiloride were similar in all preparations, at about 5 X 10(-7) M. Determination of the Hill coefficients for inhibition revealed a lower value (0.75 +/- 0.03) for the 12-13 day preparation compared with the two older preparations with coefficients not significantly different from unity. Replacing Na+ in the bathing solutions abolished the SCC of 18-19 day epithelia, whereas about 15% of the SCC remained at 12-13 days. Thus, during development, the SCC of the allantoic epithelium increases in magnitude and becomes increasingly (to 100%) amiloride-sensitive and Na+-dependent.
Journal of Experimental Zoology Part A: Ecological and Integrative Physiology
A method is described for culturing 64–70 h‐old chicken embryos and egg contents outside of the e... more A method is described for culturing 64–70 h‐old chicken embryos and egg contents outside of the eggshell through to hatching. Cultured egg contents were suspended in polymethylpentene kitchen wrap (F.O.R. wrap; Riken Fabro) supported in polyvinyl chloride tripods. Tripods were incubated in Plexiglas environmental chambers which were rocked automatically through an angle of ±20°. The concentration of CO2 was maintained at 2% throughout incubation, while that of O2 was increased from ambient to 50%, and relative humidity was decreased from 90%–92% to 83%–84% at incubation Day 9. Cultured embryos not supplemented with calcium did not hatch. The Hatch rate increased when supplemental calcium L‐lactate hydrate was increased between 250 and 350 mg. A maximal hatch rate of 54.8% was achieved when cultures were supplemented with 350 mg of calcium L‐lactate hydrate and 3.5 ml of sterile water. Adding 400 or 450 mg of calcium L‐lactate hydrate did not increase the hatch rate further. The mass...
The ultrastructure of the parathyroid glands was studied in chick embryos developing normally in ... more The ultrastructure of the parathyroid glands was studied in chick embryos developing normally in ovo or in shell‐less culture (after removal of the eggshell). Shell‐less chick embryos are significantly hypocalcemic relative to their in ovo counterparts. At 12 days of incubation, the parathyroid glands of shell‐less embryos contain more lipid and show evidence of increased protein synthetic activity relative to those grown in ovo (more rough endoplasmic reticulum, presence of some dense secretory granules). The glands from in ovo embryos do not contain secretory granules at this age. At 15 days of incubation, the in ovo glands have developed signs of protein synthetic activity similar to those of the 12‐day shell‐less embryos. However, the parathyroids of the 15‐day shell‐less embryos appear strikingly more active than at 12 days, containing stacks of concentric RER membranes and increased numbers of secretory granules. By 18 days of incubation, the ultrastructure of the glands of th...
The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology, 2005
Shell-less culture of chick chorioallantoic membrane (CAM) of developing chicken embryos is a use... more Shell-less culture of chick chorioallantoic membrane (CAM) of developing chicken embryos is a useful model to evaluate the effects of vascular agents. We assessed the response of CAM vessels to epoxyeicosatrienoic acids (EETs), derivatives of the essential fatty acid arachidonic acid, that have a number of important biological functions, including dilation of microvessels in the coronary, cerebral, renal, and mesenteric circulations. Three of four regioisomers of EETs, 14,15-, 11,12-, and 8,9-EET, induced a characteristic dose-dependent acute hyperemia within 4 min after application on 10-day-old CAMs. This response was marked in early stages of development (between days 8 and 10), but the frequency and intensity of the response were reduced after 11 days of development. Histological examination demonstrated that the hyperemia was not due to extravasation of erythrocytes. However, many capillaries were distended and contained densely packed erythrocytes as compared to uniformly arranged vessels and erythrocytes in untreated CAMs. Transmission electron microscopy showed the basal laminae surrounding capillaries remained intact, similar to those in vehicle-treated or untreated CAM tissue. The hyperemia was specific to EETs since we did not observe it to be induced by other vasodilators such as nitric oxide or prostacyclin. In conclusion, we report a novel vascular response to EETs using the CAM as an in vivo model. These lipids specifically distend a subset of capillaries in a dose-and development-dependent manner.
Abstract A technique is described whereby avian egg contents removed from the shell after 72 hour... more Abstract A technique is described whereby avian egg contents removed from the shell after 72 hours of normal incubation may be successfully cultured for extended periods of time (81% for 15.5 days), many of the embryos reaching Hamilton-Hamburger Stage 40–41 (14–15 days). Egg contents are suspended in commercial plastic wrap held in place in culture vessels consisting of modified Playtex nurser holders. Culture vessels are lowered into Kerr wide mouth quart mason jar saturation humidity chambers which have been attached to aquarium lines inside of a modified Forma model 329 CO2 incubator. Use of a 5–8% CO2 atmosphere substantially improved embryonic survivability.
Shell-less culture involves culturing chick embryos with associated yolk and albumen outside of t... more Shell-less culture involves culturing chick embryos with associated yolk and albumen outside of the eggshell and shell membranes. The technique allows direct access to and continuous observation of cultured embryos almost to the time of hatching. The plastic wrap/culture tripod technique described in this paper allows normal embryonic growth and differentiation from 48 hours (in ovo pre-incubation age) through at least 10 days of total incubation. As the duration of in ovo pre-incubation is decreased below 36 hours, there is a concomitant increase in the percentage of grossly abnormal embryos associated with decreased survival and retarded growth and differentiation. Survival of embryos pre-incubated for 72 hours through 13 and 18 days of total incubation is greater than 80% and 40%, respectively. The wrap/tripod technique allows substantially better survival and more normal development of cultured embryos than does the petri dish technique of Auerbach et al. (1974). Embryonic growt...
Abstract A photographic study of development of chick embryos cultured in vitro was conducted in ... more Abstract A photographic study of development of chick embryos cultured in vitro was conducted in order to demonstrate the relationship between embryos and extraembryonic membranes during culture. Survival of embryos in this system has been extended to 21 days of total incubation (3 days in shell plus 18 days in vitro) and development to Hamburger-Hamilton stage 45 (partial or complete yolk sac retraction). A few mature cultured embryos have “pipped” through the chorioallantoic membrane and have emitted audible peeping. Three-day embryos and egg contents were cultured in commercial plastic wrap suspended in plastic tripods. The yolk sac expanded and flattened to cover the bottom of the culture chamber by five days of total incubation. Downgrowth of the yolk sac vascular system along the plastic wrap walls of the chamber began by six days, while downgrowth of the chorioallantoic membrane (CAM) began by eight days. The CAM often completely enclosed cultured egg contents by 17 days, an event which occurs in ovo by 11 to 12 days. Lack of formation of the albumen sac resulted in inhibition of albumen uptake during the latter portion of incubation. A small number of embryos were on their right side rather than on the normal left side during the first two days of culture. Many of the right side cultured embryos experienced “compensatory torsion” onto their left side by three days of culture (six days of total incubation).
In order to assess mineral uptake by chick embryos cultured outside of the shell, growth and mine... more In order to assess mineral uptake by chick embryos cultured outside of the shell, growth and mineral (Ca, Mg, K, Na) composition of control and cultured embryos and total egg contents (embryo plus residual egg contents) were measured from 8 through 21 days of incubation. At day nine and thereafter, cultured embryos contained significantly less Ca and Mg than did control embryos. At day 11 and thereafter, ash weight of cultured embryos was significantly less than that of controls. At 13 days and thereafter, wet weight, dry weight, toe length, K and Na content of cultured embryos were significantly less than in controls. To assess relative mineral uptake of cultured embryos, mineral (Ca, Mg, K, Na) content per gram embryo dry weight was plotted against incubation age. Values for the ratio of Mg, K and Na per gram dry weight were similar for control and cultured embryos. Control values for Ca per dry weight were consistently higher than those for cultured embryos during the period from 9 to 21 days. The data suggest that Ca may not be present in sufficient quantity, even on a relative growth basis, to perform its normal physiological functions in cultured embryos.
An improved technique is described for in vitro culture of the chick embryo from three days throu... more An improved technique is described for in vitro culture of the chick embryo from three days through 21 days of total incubation (three days in shell plus 18 days in culture). Mean survival time for cultured embryos is 18 days of total incubation, and mean morphological stage is Hamburger-Hamilton stage 41 (15 days). Mean measurements of wet weight, dry weight, and right third toe length for stage 45 (partial or complete abdominal yolk sac retraction, 19-20 days) cultured embryos are comparable with corresponding means for stage 42-43 (16-17 days) embryos from control intact eggs. Possible causes of growth retardation in vitro are briefly discussed.
ABSTRACT It is shown that carboxymethyldextrans can be used to displace proteins from high-perfor... more ABSTRACT It is shown that carboxymethyldextrans can be used to displace proteins from high-performance ion-exchange columns. The high resolving power of the method is demonstrated by the total separation of the A and B genetic variants of the β-lactoglobulins, which have only 0.1 pH difference in their isoelectric points. The separation of up to 12.8 mg of the β-lactoglobulins on a 150-μl column demonstrates the potential for high capacity in displacement chromatography.
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Papers by Bruce Dunn