The method of chicken whole embryo culture using the eggshell windowing, surrogate eggshell and ex ovo culture system.

1. The unique accessibility of the avian embryo have made them an ideal model for the study of development and genome editing. Chicken whole embryo culture has provided important insights into toxicity tests, gene manipulation, clarifying gene functions, cell transplantation and cell tracking. 2. A simple technique for chicken manipulation is eggshell windowing, without or with seal, the latter having demonstrated some improvement in hatching rates. 3. Likewise, a surrogate eggshell system provides an accessible model for manipulation during chicken and quail development, with a higher hatchability compared to the simple windowing method. 4. The development of the chicken ex ovo culture systems in a synthetic environment as an efficient technique for imaging and microsurgery applications has enabled the study of important events of live chicken embryos at a specific time point. 5. This short review illustrates recent applications of well-designed whole embryo culture systems as a robust model for research into numerous biological mechanism, drug discovery, gene manipulating and production of functional proteins.

The evolution of chicken stem cell culture methods.

1. The avian embryo is an excellent model for studying embryology and the production of pharmaceutical proteins in transgenic chickens. Furthermore, chicken stem cells have the potential for proliferation and differentiation and emerged as an attractive tool for various cell-based technologies. 2. The objective of these studies is the derivation and culture of these stem cells is the production of transgenic birds for recombinant biomaterials and vaccine manufacture, drug and cytotoxicity testing, as well as to gain insight into basic science, including cell tracking. 3. Despite similarities among the established chicken stem cell lines, fundamental differences have been reported between their culture conditions and applications. Recent conventional protocols used for expansion and culture of chicken stem cells mostly depend on feeder cells, serum-containing media and static culture. 4. Utilising chicken stem cells for generation of cell-based transgenic birds and a variety of vaccines requires large-scale cell production. However, scaling up the conventional adherent chicken stem cells is challenging and labour intensive. Development of a suspension cell culture process for chicken embryonic stem cells (cESCs), chicken primordial germ cells (PGCs) and chicken induced pluripotent stem cells (ciPSCs) will be an important advance for increasing the growth kinetics of these cells. 6. This review describes various approaches and suggestions to achieve optimal cell growth for defined chicken stem cells cultures and use in future manufacturing applications.

Chemoprevention of spontaneous ovarian cancer in the domestic hen.

The hen is an attractive animal model for in vivo testing of agents that thwart ovarian carcinogenesis because ovarian cancer in the domestic hen features clinical and molecular alterations that are similar to ovarian cancer in humans, including a high incidence of p53 mutations. The objective of the study was to test the potential ovarian cancer chemopreventive effect of the p53 stabilizing compound CP-31398 on hens that spontaneously present the ovarian cancer phenotype. Beginning at 79 wk of age, 576 egg-laying hens (Gallus domesticus) were randomized to diets containing different amounts of CP-31398 for 94 wk, 5 d, comprising a control group (C) (n = 144), which was fed a diet containing 0 ppm (mg/kg) of CP-31398; a low-dose treatment (LDT) group (n = 144), which was fed a diet containing 100 ppm of CP-31398; a moderate-dose treatment (MDT) group (n = 144) which was fed a diet containing 200 ppm of CP-31398; and a high-dose treatment (HDT) group (n = 144), which was fed a diet containing 300 ppm of CP-31398. Hens were killed at 174 wk of age to determine the incidence of ovarian and oviductal adenocarcinomas. Whereas the incidence of localized and metastatic ovarian cancers in the MDT and HDT groups was significantly lower (up to 77%) compared to levels in the C and LDT groups (P < 0.05), the incidence of oviductal cancer was unaffected by CP-31398. CP-31398 appears to be an effective tool for chemoprevention against ovarian malignancies, but does not appear to affect oviductal malignancies.

The effect of hyperammonemia on myostatin and myogenic regulatory factor gene expression in broiler embryos.

Myogenesis is facilitated by four myogenic regulatory factors and is significantly inhibited by myostatin. The objective of the current study was to examine embryonic gene regulation of myostatin/myogenic regulatory factors, and subsequent manipulations of protein synthesis, in broiler embryos under induced hyperammonemia. Broiler eggs were injected with ammonium acetate solution four times over 48 h beginning on either embryonic day (ED) 15 or 17. Serum ammonia concentration was significantly higher (P<0.05) in ammonium acetate injected embryos for both ED17 and ED19 collected samples when compared with sham-injected controls. Expression of mRNA, extracted from pectoralis major of experimental and control embryos, was measured using real-time quantitative PCR for myostatin, myogenic regulatory factors myogenic factor 5, myogenic determination factor 1, myogenin, myogenic regulatory factor 4 and paired box 7. A significantly lower (P<0.01) myostatin expression was accompanied by a higher serum ammonia concentration in both ED17 and ED19 collected samples. Myogenic factor 5 expression was higher (P<0.05) in ED17 collected samples administered ammonium acetate. In both ED17 and ED19 collected samples, myogenic regulatory factor 4 was lower (P⩽0.05) in ammonium acetate injected embryos. No significant difference was seen in myogenic determination factor 1, myogenin or paired box 7 expression between treatment groups for either age of sample collection. In addition, there was no significant difference in BrdU staining of histological samples taken from treated and control embryos. Myostatin protein levels were evaluated by Western blot analysis, and also showed lower myostatin expression (P<0.05). Overall, it appears possible to inhibit myostatin expression through hyperammonemia, which is expected to have a positive effect on embryonic myogenesis and postnatal muscle growth.

Differential expression of genes characterizing myofibre phenotype.

Skeletal muscle is composed of metabolically heterogeneous myofibres that exhibit high plasticity at both the morphological and transcriptional levels. The objective of this study was to employ microarray analysis to elucidate the differential gene expression between the tonic-'red' anterior latissimus dorsi (ALD) muscle, the phasic-'white' posterior latissimus dorsi (PLD) and 'mixed'-phenotype biceps femoris (BF) in 1-week-and 19-week-old male turkeys. A total of 170 differentially expressed genes were identified in the muscle samples analysed (P < 0.05). Gene GO analysis software was utilized to identify top gene networks and metabolic pathways involving differentially expressed genes. Quantitative real-time PCR for selected genes (BAT2D, CLU, EGFR and LEPROT) was utilized to validate the microarray data. The largest differences were observed between ALD and PLD muscles, in which 32 genes were over-expressed and 82 genes were under-expressed in ALD1-PLD1 comparison, and 70 genes were over-expressed and 70 under-expressed in ALD19-PLD19 comparison. The largest number of genes over-expressed in ALD muscles, as compared to other muscles, code for extracellular matrix proteins such as dystroglycan and collagen. The gene analysis revealed that phenotypically 'red' BF muscle has high expression of glycolytic genes usually associated with the 'white' muscle phenotype. Muscle-specific differences were observed in expression levels of genes coding for proteins involved in mRNA processing and translation regulation, proteosomal degradation, apoptosis and insulin resistance. The current findings may have large implications in muscle-type-related disorders and improvement of muscle quality in agricultural species.

Fiber phenotype and coenzyme Q10 content in Turkey skeletal muscles.

Phenotypical differences between muscle fibers are associated with a source of cellular energy. Coenzyme Q(10) (CoQ(10)) is a major component of the mitochondrial oxidative phosphorylation process, and it significantly contributes to the production of cellular energy in the form of ATP. The objective of this study was to determine the relationship between whole-tissue CoQ(10) content, mitochondrial CoQ(10) content, mitochondrial protein, and muscle phenotype in turkeys. Four specialized muscles (anterior latissimus dorsi, ALD; posterior latissimus dorsi, PLD; pectoralis major, PM, and biceps femoris, BF) were evaluated in 9- and 20-week-old turkey toms. The amount of muscle mitochondrial protein was determined using the Bradford assay and CoQ(10) content was measured using HPLC-UV. The amount of mitochondrial protein relative to total protein was significantly lower (p < 0.05) at 9 compared to 20 weeks of age. All ALD fibers stained positive for anti-slow (S35) MyHC antibody. The PLD and PM muscle fibers revealed no staining for slow myosin heavy chain (S35 MyHC), whereas half of BF muscle fibers exhibited staining for S35 MyHC at 9 weeks and 70% at 20 weeks of age. The succinate dehydrogenase (SDH) staining data revealed that SDH significantly increases (p < 0.05) in ALD and BF muscles and significantly decreases (p < 0.05) in PLD and PM muscles with age. The study reveals age-related decreases in mitochondrial CoQ(10) content in muscles with fast/glycolytic profile, and demonstrates that muscles with a slow/oxidative phenotypic profile contain a higher proportion of CoQ(10) than muscles with a fast/glycolytic phenotypic profile.

Apoptosis and macrophage infiltration occur simultaneously and present a potential sign of muscle injury in skeletal muscle of nutritionally compromised, early post-hatch turkeys.

Physical stress and malnutrition may cause elimination of myonuclei and produce inflammatory response in muscle. The objective of this study was to histochemically determine the association of apoptosis and/or macrophage infiltration with changes in muscle satellite cell mitotic activity in pectoralis thoracicus muscle of early post-hatch turkey toms. Feed-deprived birds and birds provided with three different levels of crude protein and amino acids (0.88 NRC, 1.00 NRC, and 1.12 NRC) were used in this model. The number of apoptotic nuclei was significantly elevated (P<0.05) and presence of macrophage infiltration was readily detectable in feed-deprived and 0.88 NRC treatment groups 72 h and 96 h post-hatch suggesting potential muscle injury and/or muscle remodeling. The number of apoptotic nuclei was the same (P>0.05), and there was no detectable macrophage infiltration present in birds placed on 1.00 NRC and 1.12 NRC diet 72 h, 96 h, and 120 h post-hatch. At 120 h post-hatch, feed-deprived and 0.88 NRC birds were characterized by no detectable levels of macrophage infiltration and a significant drop (P<0.05) in apoptotic nuclei. Understanding mechanisms that correlate early nutrition with skeletal muscle growth and development may present a useful tool in optimizing muscle health and improving meat quality and yield.

Broiler embryo bone development is influenced by incubator temperature, oxygen concentration and eggshell conductance at the plateau stage in oxygen consumption.

1. Four experiments were conducted to evaluate the effects of temperature (TEM) and oxygen (O(2)) concentrations during the last 4 d of incubation on bone development. Fertile eggs from two strains were obtained that either exhibited Low or High eggshell conductance (G). 2. Four experimental cabinets provided either four TEM (36, 37, 38 or 39 degrees C) or four O(2) concentrations (17, 19, 21 or 23% O(2)). Data were analysed as a 2 x 2 factorial design. In the fourth experiment, two temperatures (36 and 39 degrees C), two O(2) concentrations (17 and 23%) and the same Low and High G strains were evaluated in a 2 x 2 x 2 factorial design. 3. Body weights (BW) and residual yolks were obtained, both legs were dissected. Femur, tibia and shank weights, length and thickness were recorded. Relative asymmetry (RA) of each leg section was calculated. 4. The results indicated that elevated TEM during incubation increased RA between the two legs, mainly in the Low G strain. Chickens at the lowest O(2) concentrations had lighter and shorter tibias, lighter shanks, and increased RA of femur length compared to chickens in the 23% O(2). In the fourth experiment no interactions were observed between O(2) and TEM. High TEM depressed BW of Low G broilers, but no significant effect of treatments was observed on BW of High G broilers. Nevertheless, the high TEM or low O(2) independently caused reduced femur and tibia weights and length, shank length and thickness, and both low O(2) and high TEM together increased RA in shank weight. 5. These results suggest that late incubation conditions affect long bone development in broilers.

The effect of early dietary amino acid levels on muscle satellite cell dynamics in turkeys.

Understanding the relationship between nutrition and satellite cell activity will be beneficial in obtaining optimal muscle growth and meat production. The objective of this study was to evaluate the effect of early post-hatch levels of dietary amino acids+/-0.88 NRC, 1.00 NRC, and 1.12 NRC), and feed deprivation on the satellite cell mitotic activity, pectoralis thoracicus muscle weight, and body weight of male turkeys (Meleagris gallopavo). Birds from each treatment were injected with 5-bromo-2'-deoxyuridine (BrdU) to label mitotically active cells. The right pectoralis thoracicus was harvested 1 h after BrdU injection for immunohistochemical and myofiber diameter analysis. On the third day post-hatch, satellite cell mitotic activity was the highest (P<0.05) in the 0.88 NRC amino acid treatment group and the lowest (P<0.05) in the feed-deprived group. On the fourth day post-hatch, feed-deprived birds exhibited the lowest (P<0.05) satellite cell mitotic activity and muscle weight. At 140 days of age, there were no significant differences (P>0.05) between treatments in body weight or pectoralis thoracicus muscle weight. Research evaluating species-related differences in apoptotic events and in genes regulating cell proliferation may be necessary to devise feeding strategies aimed at obtaining optimal pectoralis thoracicus muscle yield at market age.

Production of chick germline chimeras from fluorescence-activated cell-sorted gonocytes.

Modification of the chicken germline has been difficult, because it has been challenging to fractionate sufficient numbers of primordial germ cells for manipulation and implantation into developing embryos. A technique to enrich cell suspensions for primordial germ cells, using fluorescence-activated cell sorting (FACS), has recently been developed. The objective of the current study was to demonstrate that the FACS-enriched early embryonic gonocytes could fully participate in development of the germline. Therefore, cells were disassociated from stage 27 gonads, incubated with mouse anti-stage-specific embryonic antigen-1, which was detected with goat-antimouse IgM-fluorescein isothiocyanate, and the fluorescently labeled cells were sorted from the unlabeled cells using FACS. The isolated gonocyte population was injected into the blastoderm of unincubated stage X embryos, the germinal crescent of 3-d embryos, and into the circulation of stage 17 embryos that were pretreated with busulfan. Barred Plymouth Rock gonocytes were implanted exclusively into recipient White Leghorn embryos, and White Leghorn gonocytes were implanted exclusively into Barred Plymouth Rock recipient embryos. Embryos were cultured until hatch, and male putative chimeras were reared to sexual maturity. Germline chimerism was evaluated by observing feather color of the progeny. All injection methods resulted in germline chimeras demonstrating that FACS-sorted gonocytes can fully participate in development. Moreover, it was demonstrated that gonocytes isolated from stage 27 embryonic gonads can be introduced into embryos at an earlier stage of development, and the introduced gonocytes can fully participate in germline development.

Culture of chicken embryos in surrogate eggshells.

The chick embryo is a classical model to study embryonic development. However, most researchers have not studied the effect of embryonic manipulation on chick hatchability. The objective of this study was to determine the effect of egg orientation and type of sealing film on the hatchability of cultured embryos. Windows were made in the small end of recipient surrogate chicken eggshells, and donor embryos were placed into the recipient eggshell for the first 3 d of incubation. Survival over the first 3 d was maximized (P < 0.05) when windowed eggs sealed with Saran Wrap were positioned with the window-end down compared with window-end up. Three-day-old cultured embryos were transferred into recipient turkey eggshells, sealed with cling film, and cultured until hatch. Water weight loss of the surrogate eggshell cultures regardless of cling film type was not significantly different from control intact eggs. The embryos cultured in turkey eggshells and sealed with Handi Wrap exhibited higher hatchability (75% +/- 10.2%) than cultures sealed with Saran Wrap (45.2% +/- 13.8%). Hatchability of control intact eggs (86.4% +/- 5.3%) was not significantly (P > 0.05) different from the hatchability of eggs sealed with Handi Wrap, which suggested that Handi Wrap was an excellent sealant for chick embryos cultured after 3 d of incubation.

Early post-hatch fasting induces satellite cell self-renewal.

Early post-hatch satellite cell kinetics are an important aspect of muscle development, and understanding the interplay between fasting and muscle development will lead to improvements in muscle mass following an illness, and optimal meat production. The objective of this experiment was to test the influence of immediate post-hatch fasting on satellite cells in the poult. Male Nicholas poults (Meleagris gallopavo) were placed into two treatments: a fed treatment with immediate access to feed and water upon placement and a fasted treatment without access to feed and water for the first three days post-hatch. 5-bromo-2'-deoxyuridine (BrdU) was injected intra-abdominally in all poults to label mitotically active satellite cells. The pectoralis thoracicus muscle was harvested two hours following the BrdU injection. Immunohistochemistry for BrdU, Pax7, Bcl-2, Pax7 with BrdU, and determining myofiber cross-sectional area along with computer-based image analysis was used to study muscle development. Fed poults had higher body masses throughout the experiment (P< or =0.01), and they had higher pectoralis thoracicus muscle mass (P< or =0.01) at ten days of age than the fasted poults. Fed poults had higher satellite cell mitotic activity at three days and four days of age (P< or =0.01) compared to the fasted poults. However, Pax7 labeling index was higher in the fasted poults (P< or =0.01) at three days, four days, and five days post-hatch than the fed group. Similarly Bcl-2 labeling was higher in the fasted than in the fed group at three days post-hatch. Therefore, fasting depleted proliferating satellite cells indicated by the lower BrdU labeling in the fasted poults compared to the fed poults, and conserved the satellite cell proliferative reserve indicated by the higher level of Pax7 labeling for the fasted poults compared to the fed poults.

The effect of early nutrition on satellite cell dynamics in the young turkey.

Early posthatch satellite cell mitotic activity is an important aspect of muscle development. An understanding of the interplay between nutrition and satellite cell mitotic activity will lead to more efficient meat production. The objective of this study was to test the influence of the leucine metabolite, beta-hydroxy beta-methylbutyrate (HMB), and feed deprivation on muscle development in the early posthatch poult. Male Nicholas poults were placed on 1 of 4 treatments: immediately fed a starter diet with 0.1% HMB (IF-HMB), immediately fed a starter diet containing 0.1% Solka-Floc for a control (IF-No HMB), feed and water withheld for 48 h immediately posthatch and then fed the HMB diet (WF-HMB), and feed and water withheld for 48 h immediately posthatch and then fed the control starter diet (WF-No HMB). 5-bromo-2'-deoxyuridine (BrdU) was injected intra-abdominally into all poults to label mitotically active satellite cells. The pectoralis thoracicus was harvested 2 h after the BrdU injection. Immunohistochemistry for BrdU, Pax7, and laminin along with computer-based image analysis was used to study muscle development. IF-HMB poults had higher body weights (P < 0.01) at 48 h and 1 wk of age and had higher satellite cell mitotic activity at 48 h of age (P < 0.01) compared with the IF-No HMB and WF poults. Therefore, dietary supplementation of HMB may have an anabolic effect on early posthatch muscle.

Isolation of chicken primordial germ cells using fluorescence-activated cell sorting.

Presently, it is difficult to undertake germ line modification of the chicken with primordial germ cells (PGC) because it has been difficult to efficiently fractionate the PGC from the total somatic cell population. The objective of this study was to develop a method that allows isolation of an enriched population of viable PGC from embryonic blood and embryonic gonadal tissue. Blood was harvested from early chick embryos (stages 13 to 15), and cells were liberated from the gonads of stage 27 chick embryos. Subsequently, viable PGC were labeled with anti-stage-specific embryonic antigen-1 (SSEA-1), which was detected with goat-anti-mouse IgM-fluorescein isothiocyanate. Fluorescently labeled cells were sorted from the unlabeled cells using fluorescence-activated cell sorting (FACS), and the identities of the PGC were confirmed using periodic acid-Schiff (PAS) staining or anti-embryonic mouse antigen-1 (EMA-1) staining followed by microscopic evaluation. Finally, PGC were sorted from somatic cells of sex-identified embryos. Less than 0.1% of the blood cell population was collected as SSEA-1-positive cells. Similarly, approximately 2% of the gonadal cell population were collected as SSEA-1-positive cells. Therefore, fewer (-1,000 to 9,000) PGC were recovered from each isolate. Placing the sorted SSEA-1-positive cells on a glass slide from a microcentrifuge tube resulted in a recovery rate of 53 to 73% relative to the number detected by FACS. Furthermore, the proportions of sorted cells that stained with PAS or anti-EMA-1 following sorting were 92+/-4% PAS positive and 94+/-1% anti-EMA-1 positive. Finally, the sorted SSEA-1-positive cells were maintained in vitro to demonstrate their viability after sorting. It was demonstrated that it is possible to label blood and gonadal chicken PGC with SSEA-1 and subsequently to sort viable SSEA-1-positive PGC from somatic cells.

Effects of posthatch feed deprivation on heparan sulfate proteoglycan, syndecan-1, and glypican expression: implications for muscle growth potential in chickens.

The heparan sulfate proteoglycans, syndecan-1 and glypican-1 (glypican), are low affinity receptors for fibroblast growth factor 2 (FGF2). Because FGF2 stimulates skeletal muscle cell proliferation but inhibits differentiation, changes in FGF2 signaling due to early posthatch feed deprivation may play a significant role in modulating muscle growth. To study the effect of early posthatch feed deprivation in chickens on heparan sulfate proteoglycan relative protein concentration, syndecan-1 expression, and glypican mRNA expression, pectoralis major muscle tissue was isolated from pretreatment d 0 chicks and chicks fed or feed deprived for 3 d, and after d 3 feeding was resumed in the feed-deprived birds until d 7. Heparan sulfate proteoglycan protein concentration was measured by ELISA analysis and was significantly decreased in the feed-deprived birds beginning at d 2 (P < 0.05). The expression of syndecan-1 and glypican was measured by semi-quantitative reverse transcription PCR. Syndecan-1 expression was unaffected by feed withdrawal and refeeding (P > 0.05). Glypican mRNA expression was decreased in the muscle tissue from feed-deprived birds at d 3 (P < 0.05), but by d 7, after initiating feeding on d 4, it was significantly elevated compared with in muscle tissue from chicks maintained on feed (P < 0.05). The results from the present study demonstrate that the heparan sulfate proteoglycan protein concentration and syndecan-1 and glypican mRNA expressions are differentially affected by early posthatch feed deprivation, which may alter signaling events associated with muscle growth.

Development of transgenic chickens expressing bacterial beta-galactosidase.

Replication-defective retroviral vectors are efficient vehicles for the delivery of exogenous genes, and they may be used in the generation of transgenic animals. The replication-defective retroviral SNTZ vector carrying the lacZ gene with a nuclear localized signal was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the eggs were allowed to hatch, and the chickens were screened for the lacZ gene by using the polymerase chain reaction. Eight of 15 male chickens that survived to sexual maturity contained the lacZ gene in their semen. Subsequently, these males were mated with wild-type female chickens. From one of the eight lacZ-positive G(0) males, two lacZ-positive male chickens were produced from a total of 224 G(1) progeny for a germline transmission rate of 0.89%. Both G(1) male chickens carrying the lacZ gene were mated with wild-type female chickens and 46.5% of the G(2) progeny contained the lacZ gene, which is consistent with the expected Mendelian 50% ratio for a heterozygous dominant allele. The product of the lacZ gene, nuclear localized beta-galactosidase, was expressed in primary myoblast cultures derived from G(2) chickens, and it was also expressed in whole G(2) chicken embryos.

The effect of early posthatch nutrition on satellite cell mitotic activity.

Myofiber growth is dependent upon the contribution of new nuclei from the mitotically active satellite cell population. The objective of this study was to examine satellite cell mitotic activity in conjunction with different nutritional paradigms during the early posthatch period. Turkey poults were provided a standard turkey starter diet; the starter diet top-dressed with a hydrated low-fat, highly digestible protein and carbohydrate nutritional hatchling supplement, Oasis; the starter diet top-dressed with Solka-floc dyed green; or no food for the first 3 d posthatch. All birds were fed a standard starter diet during the experimental period. 5-Bromo-2'-deoxyuridine (BrdU) was continuously infused into all treatments (n = 5 all groups) between hatch and 3 d of age. A second group of identically treated poults housed in separate pens (n = 3 to 5) was continuously infused with BrdU between 2 and 9 d of age. Mitotically active satellite cells were identified in the pectoralis thoracicus and quantitated using BrdU immunohistochemistry in combination with computer-based image analysis. Satellite cell mitotic activity was significantly higher (P < or = 0.05) in the birds fed a standard starter diet compared to all other treatments at 3 d posthatch. However, there were no (P > or = 0.05) differences in satellite cell mitotic activity among treatments at 9 d posthatch. The results of the current study suggest that any improvements in meat yield through early nutritional supplementation do not appear to occur through a satellite cell pathway and that there is no compensatory response in the satellite cell population following refeeding after early posthatch starvation.

The effect of early posthatch starvation on calpain mRNA levels.

The calpain system is a family of calcium activated proteases that degrade myofibrillar protein. Male broiler chickens (Ross) were provided a standard starter diet top-dressed with Oasis((R)) nutritional supplement (fed; Novus International, St. Louis, MO, USA), or they were not provided any feed (starved) for the first 3 days posthatch. Subsequently, the standard starter diet was provided to all chickens between 3 and 7 days posthatch. RNA was extracted from the Pectoralis thoracicus, and skeletal muscle-specific n-calpain-1 (p94) calpain, mu-calpain, and m-calpain expression was evaluated using quantitative Northern analysis. Early posthatch starvation did not (P>0.05) affect calpain mRNA levels on each day examined. Similarly, there were no (P>0.05) changes in mu-calpain or m-calpain mRNA levels between 0 and 7 days posthatch in fed birds. However, p94 calpain mRNA levels were significantly (P<0.05) lower at 7 days posthatch compared to 0 or 2 days posthatch. Therefore, in the early posthatch chicken, it appears that the calpain system may not be affected by the presence of oral nutrition, and that there is an age-related downregulation of p94 calpain mRNA expression.

Muscle regeneration during hindlimb unloading results in a reduction in muscle size after reloading.

The hindlimb-unloading model was used to study the ability of muscle injured in a weightless environment to recover after reloading. Satellite cell mitotic activity and DNA unit size were determined in injured and intact soleus muscles from hindlimb-unloaded and age-matched weight-bearing rats at the conclusion of 28 days of hindlimb unloading, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-unloaded rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb unloading, but they were the same (P > 0.05) as those of weight-bearing rats 2 and 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, number of nuclei per millimeter, and DNA unit size were significantly (P < 0.05) smaller for the injured soleus muscles from hindlimb-unloaded rats than for the soleus muscles from weight-bearing rats at each recovery time. Satellite cell mitotic activity was significantly (P < 0.05) higher in the injured soleus muscles from hindlimb-unloaded rats than from weight-bearing rats 2 wk after reloading, but it was the same (P > 0.05) as in the injured soleus muscles from weight-bearing rats 9 wk after reloading. The injured soleus muscles from hindlimb-unloaded rats failed to achieve weight-bearing muscle size 9 wk after reloading, because incomplete compensation for the decrease in myonuclear accretion and DNA unit size expansion occurred during the unloading period.

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells.

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.