Morphological classification of ganglion cells in the central retina of chicks.

Classification of retinal ganglion cells (RGCs) in the chick central retina was studied by retrograde labeling of carbocyanine dye (DiI) and intracellular filling with Lucifer Yellow. Ganglion cells were divided into 4 groups, Group Ic/Is, Group IIc/IIs, Group IIIs, Group IVc, according to sizes of somal area and dendritic field and dendritic branching pattern. Group I cells had small somal area and small dendritic field. They were further divided into 2 subgroups by complexity (subgroup Ic) and simplicity (subgroup Is) of the dendritic arborization. Group II cells had medium-sized soma and dendritic field. They were also divided into subgroup IIc and IIs by the same definitions as those of subgroup Ic and Is. Group IIIs had medium-sized soma, large and simple dendritic arborization. Group IVc in which all cells had large soma, showed large and complex dendritic arborization. Cell populations of each group were 51.8% (subgroup Ic), 21.1% (subgroup Is), 6.2% (subgroup IIc), 14.6% (subgroup IIs), 4.2% (Group IIIs), and 2.1% (Group IVc). Subgroup Ic cells, which were very similar to beta-cells in the mammalian central area, represented about a half of the ganglion cell population. Cells in subgroup Is and IIs, which were not reported in the mammalian retina, were found in the chick central retina in relatively high population (35.7%). Morphological features of chick RGCs in the central retina were considered in comparison with those of other vertebrates.     

Dimensional differences among the groups of retinal ganglion cells according to the retinal zones in chicks

The populations of retinal ganglion cell (RGC) groups (Groups I, II, III, IV) were similar each other between the central and intermediate zones, but the population in the peripheral zone were clearly different from those in the central and intermediate zones due to increase of Group III and IV cells and decrease of Group I cells. The dimensions of somal area and dendritic field of Group I cells increased very gradually toward the peripheral zone, but those of other three Groups grew steeply in the peripheral zone. The correlation index between somal area and dendritic field of RGCs showed high coefficient in the central (r=0.73) and intermediate (r=0.77) zones, but lowered clearly in the peripheral zone (r=0.64) due to increase of Group III cells, which showed nonlinear relation between somal area and dendritic field.     

Distribution and cytoarchitecture of sympathetic neurons innervating the pineal gland in chick: a CTB-HRP study

The neurons in bilateral superior cervical ganglia (SCG) innervating the chick pineal gland were labelled by using the technique of retrograde axonal labelling with cholera toxin B subunit linked to horseradish peroxidase (CTB-HRP). To our results, perikarya of these sympathetic neurons distributed from rostral to caudal in the SCG, and mainly localized in the opposite side of the paravertebral trunk. The fibres of these neurons were collected by the cephalic carotid nerve. According to the sizes of somal area and dendritic field, these sympathetic neurons projecting to the pineal gland were classified into four major groups: group I cells (52.4%) with a small somal area (303.5 μm² on average) and narrow dendritic field (3767.8 μm² on average), group II cells (39.0%) with a middle-sized somal area (473.3 μm²) and middle-sized dendritic field (7522.2 μm²), group III cells (6.4%) with a middle-sized somal area (473.4 μm²) and wide dendritic field (13 104.4 μm²), and group IV cells (2.2%) with a large somal area (940.7 μm²) and wide dendritic field (14 553.2 μm²). Of these pineal projecting neurons, most took on a lesser dendritic field. The neurons with small or middle-sized dendritic field from group I and II were about 91.4% of the total neurons labelled with CTB-HRP, and the neurons with wide dendritic field from group III and IV were less with 8.6%.     

Retrograde tracing with fluorescent microspheres reveals bifurcating projections from central retina to tectum and thalamus in chicks

The goal of this study is to demonstrate the dual-projection pattern of retinal ganglion cells (RGCs) projecting to the tectum and visual thalamus in chick using retrograde fluorescent tracers and also to define the morphological properties of these RGCs with dual projections by intracellular injection of Lucifer Yellow (LY) combined with immunohistochemistry. Thirty-two chicks received double injections of green and red fluorescent microspheres into their thalamus and tectum in the same side. In the central retina, most of the labelled RGCs were tec-RGCs (RGCs projecting to the tectum), a quarter was tha-RGCs (RGCs projecting to the thalamus), and almost all of the tha-RGCs were double-labelled RGCs. An intracellular injection of LY into the double-labelled RGCs showed all six groups of RGCs without specific populations in each group (J. Comp. Neurol., 2004, 469: 360). These dendritic patterns were mostly mono- and bistrata, which extended horizontally in the deeper part of the inner plexiform layer.     

Changes in the distribution of labeled retinal ganglion cells after an implant of DiI into the optic nerve in the chick embryos

Changes in the distribution of retinal ganglion cells (RGCs) were studied using the retrograde labeling of DiI in chicks and chick embryos. The small retinal area filled with labeled RGCs was observed in the retinal fundus on E8. The labeled retinal area expanded radially toward the peripheral retina as the retina grew, and finally occupied a whole retina by P1. The temporal retina was labeled more rapidly than in the nasal retina. The observed-increasing rate of the labeled area was corrected with the growing rate of the retina. Consequently, the corrected-increasing rate of the labeled area was estimated to be about 390% between E8 and E11, and 20-50% after E11. This means that spreading speed of the maturated RGCs lowered until 1/10-1/20 after E11.     

Quantitative analysis of cells in the ganglion cell layer of the chick retina: developmental changes in cell density and cell size

Changes in cell density and size in the ganglion cell layer (GCL) of the retina were studied in chick embryos and post-hatching chicks. The total number of cells in the GCL increased from 3.64 million at embryonic day 8 (E8) to the maximal 7.85 million at E14. After E14, the number of cells decreased to 6.08 million at post-hatching day 1 (P1) and 4.87 million at P8. Cell density in the GCL decreased unevenly according to retinal regions; cell density in the presumptive central area (pCA) of P8-chicks decreased to approximately 45% of that in E8-embryos. Densities of the nasal peripheral retina (NP) and temporal peripheral retina (TP) of P8-chicks decreased to 23 and 18% of E8-embryos, respectively. Differentiation of the central (44,000 cells/mm(2) in pCA) - peripheral (28,000 cells/mm(2) in TP) gradient in cell density was formed by E8. The presumptive dorsal area (pDA) was shaped by E11, but became obscure with age. Although ganglion cell sizes were basically uniform at E8, differentiation occurred with the appearance of larger ganglion cells after E14. Mean size of retinal ganglion cells increased 2.8-fold in the pCA and 3.8-fold in the TP between E8 and P8, accompanying a similar scale of decreases in cell densities.     

The developmental study on lamination of the optic tectum in relation to the retinotectal projection in chicks and chick embryos

The tectal lamination was investigated in the central part of the chick embryonic tectum. Two and 5 layers were observed above the neuroepithelium (NE) on embryonic day 6 (E6) and E8, respectively. Optic fibers extended on the surface of the tectum by E8. On E10-11, the outer tectum was composed of 2 layers, that is, a fibrous layer forming the optic fiber layer on the tectal surface and a cellular layer showing the gradient of cell density. In the inner tectum, the lamination was almost completed. On E12-13, the outer tectal layers, which showed the gradient of cell density, was divided into dark and light cellular layers. The dark cellular layer was divided into 2 layers on E14-15 and further into 4 layers (layer C-F in chick) on E18. On the other hand, the light cellular layer did not change until E18, but finally, it was divided into 2 layers (layer A and B in chick) by E20. Optic fibers reached the bottom of the outer tectum by E14 showing different densities of terminals. Stratification by optic fibers was going to step into the final stage on E18. On E20, laminations according to cytoarchitectural features and the optic fiber terminals were substantially completed. In the tectum affected by destruction of the contralateral embryonic eye (E4), some cellular layers were incompletely discriminated by differences of cell density.     

Regional specialization of ganglion cell layer of the chick retina

Specialization of the ganglion cell layer (GCL) was studied by Nissl-staining and axonal tract-tracing methods in chicks and chick embryos. The changes in the retinal area and the cell number in the GCL produced a disparity in the cell density that occurred through the two different processes, cell generation (before embryonic days 10-14, E10-14) and cell loss (after E10-14). One high-density area was found in the retinal fundus on E8 (presumptive central area, pCA) and its density decreased toward the peripheral retina. Another high-density area was found in the dorsal retina on E11 (presumptive dorsal area, pDA). Cell densities of the pCA and the pDA on E11 decreased gradually to 25-30% by P1, and after that they further decreased to 40-60% by P30. The pCA was still identified on P30, but the pDA became very obscure by this age. In contrast, ganglion cell sizes increased 5-7 times in the pCA and pDA from E8 to P30, and increased 12 times in the temporal periphery. The present study suggests that the center-peripheral gradient of cell density results from lager scale of cell genesis in the pCA, but not from lager scale of cell loss in the peripheral retina. However, obscuration of the pDA results from equalization of cell density in cellular degeneration processes.     

用数学模型拟合鸡胚胸腺生长发育的研究

观察成都白羽鸡不同胚龄的鸡胚胸腺,结果,鸡胚胸腺在受精卵孵化到第9天时才开始形成.14胚龄时,胸腺长度为20胚龄的50%.18胚龄时,胸腺重量为20胚龄的50%.鸡胚胸腺长度与胚龄关系呈逻辑斯蒂S曲线生长,胸腺的重量呈指数增长模型.两者与胚体的关系分别呈对数函数和幂函数增长.建议采用逻辑斯蒂S曲线生长和指数增长模型分别拟合胸腺的长度、重量与胚龄的关系.     

Effects of Broiler Strain,Dietary Nonphytate Phosphorus,and Phytase Supplementation on Chick Performance and Tibia Ash

Three experiments were conducted to study the effects of broiler strain and phytase supplementation on chick nonphytate P (NPP) requirements for growth, feed intake, and tibia ash. The first experiment compared the NPP requirements for 8- to 22-d-old chicks from 2 broiler strains, Ross 308 and 708, that have been selected for differences in early weight gain and performance. The second experiment utilized similar 8- to 22-d-old Ross 308 and 708 chicks but also compared the effects of dietary fungal phytase supplementation (600 U/kg) on broiler NPP requirements. The third experiment utilized a younger starting age, 5 to 23 d old, for Ross 308 and 708 chicks with and without phytase supplementation. Minor differences in chick growth did not affect chick NPP requirements in Experiments 1 and 3, but a substantial and unexplained reduction of growth of the Ross 708 chicks in Experiment 2 resulted in a lower NPP requirement for chick growth and feed intake but not for tibia ash. As expected, supplementation of diets with fungal phytase did result in decreased NPP requirements for growth, feed intake, and tibia ash in both strains used in Experiment 3.     

通过雏鸡MDV攻毒试验选择抗马立克氏病亲本

对F0、F1、F2亲本经继代No.614血型基因选择后繁殖的F2、F3雏鸡,10日龄以马立克氏病强毒（MDV）攻击至60日龄的死亡率,其父母亲对No.614抗血清呈阳性反应的试验组显著低于其父母亲对此抗血清呈阴性反应的对照组。试验组25个家系中,有14个家系的F2雏鸡死亡率显著低于对照组;F3雏鸡有21个家系的死亡率低于对照组,其中8个家系具有统计意义。肿瘤发生率,以肾脏和腺胃最高,肺脏和神经组织最低。综合血型抗原检测和MDV攻毒结果,从试验组25个家系中选留强MDV抗性家系留种繁殖,继代选育。     

缓释复方免疫增强剂对雏鸡免疫功能各项

将40只14日龄蛋用雏鸡随机分成2组,每组20只,A组为对照组,颈部皮下注射新城疫疫苗0.2ml/只,B组为试验组,在颈部皮下注射新城疫疫苗的同时,每只雏鸡投服缓释复方免疫增强剂1粒（0.25 mg左右）,在试验后10、20、30和40 d时检测的新城疫HI抗体效价、T淋巴细胞ERFC形成率、外周血T淋巴细胞ANAE阳性率,试验组显著高于对照组（P＜0.01）,对免疫器官相对重量变化具有明显的促生长作用,对机体的囊体比和脾体比有一定的提高,但差异不显著（P＞0.05）。由此可见,研制的缓释复方免疫增强剂的免疫增强效果明显,持续时间长,同时还可促进机体的生长发育,是一种效果好、副作用小的新型免疫增强剂。     

Characterization of pathogenic Escherichia coli isolated from humans in Austria: phenotypes, toxin gene types and epidemiology

One hundred and ten clinical Escherichia coli isolates of serovar O157 (n = 102) and O26 (n = 8) were characterized for the presence of putative virulence genes by PCR. All but one of these isolates contained the eae gene. The EHEC-hly gene could be detected in all E. coli O157 and in 50% of E. coli O26 isolates. Forty-five (40.9%) of the 110 E. coli were positive for both stx(1) and stx(2) genes, 2 (1.8%) isolates were positive for stx(1) and 57 isolates (51.8%) were positive for stx(2) only. Among the 102 stx(2) positive isolates, 14 (13.7%) E. coli O157 contained also the stx(2c) variant gene. No other stx(2) variant was identified. Six clinical isolates (five E. coli O157:H7 and one E. coli O26) did not contain stx genes. Ten non-pathogenic E. coli isolates which were amplified as controls didn't contain any stx and eae gene but two of the ten strains contained the EHEC-hly gene. By their growth on chromogenic media, all but two of 50 E. coli O157 could be differentiated from eight E. coli O26 and 10 non-pathogenic E. coli. Sixty-one of the O157:H7 isolates were further subjected to pulsed-field gel electrophoresis (PFGE) which identified 49 distinguishable patterns. In five cases where contact infection among family members was suspected, indistinguishable PFGE patterns confirmed the epidemiological relatedness of the isolates. Moreover, two PFGE clusters were identified which comprised five and three strains, respectively. These findings indicate the occurrence of both family and diffuse outbreaks of E. coli O157 infections in Austria during recent years and demonstrate the need for molecular subtyping of these pathogens.     

Interaction of low-pathogenicity reoviruses and low levels of infection with several coccidial species

Various combinations of reoviruses and coccidia were studied to see if interactions would occur. Two reoviruses were used: virus 2035, a moderate to low pathogen, and virus 2177, a nonpathogen. Coccidia used were Eimeria acervulina, E. mitis, and E. maxima at dosages of 10(3) or 10(4) sporulated oocysts/chick and E. brunetti at 10(4) sporulated oocysts/chick. In Hubbard-Hubbard cockerels, a combination of virus 2035 and E. acervulina (10(4) oocysts/chick) or E. maxima (10(3) oocysts/chick) significantly (P less than or equal to 0.05) increased the frequency of stunting (% of chicks with body weight less than 80% of controls) and further depressed weight gain over that seen with either virus or coccidia alone. Conversely, virus 2177 ameliorated the same effects in Shaver-Arbor Acre cockerels given 10(4) oocysts/chick of E. mitis or E. maxima. The interaction could not be attributed to changes in the degree of coccidial infection based on oocyst production. Reovirus did not generally change the effect of coccidia on levels of plasma pigment and plasma protein. In Hubbard-Hubbard cockerels, coccidia-induced effects were not ameliorated by virus 2177, suggesting that breed difference in interaction can be expected.     

Expression of hLAMP‐1‐Positive Particles During Early Heart Development in the Chick

Heart development requires coordinated activity of various factors, the disturbance of which can lead to congenital heart defects. Heart lectin‐associated matrix protein‐1 (hLAMP‐1) is a matrix protein expressed within Hensen's node at Hamburger–Hamilton (HH) stage 4, in the lateral mesoderm by HH stages 5–6 and enhanced within the left pre‐cardiac field at HH stage 7. At HH stages 15–16, hLAMP‐1 expression is observed in the atrioventricular canal and the outflow tract. Also, the role of hLAMP‐1 in induction of mesenchyme formation in chick heart has been well documented. To further elucidate the role of this molecule in heart development, we examined its expression patterns during HH stages 8–14 in the chick. In this regard, we immunostained sections of the heart during HH stages 8–14 with antibodies specific to hLAMP‐1. Our results showed prominent expression of hLAMP‐1‐positive particles in the extracellular matrix associated with the pre‐cardiac mesoderm, the endoderm, ectoderm as well as neuroectoderm at HH stages 8–9. After formation of the linear heart tube at HH stage 10, the expression of hLAMP‐1‐stained particles disappears in those regions of original contact between the endoderm and heart forming fields due to rupture of the dorsal mesocardium while their expression becomes confined to the arterial and venous poles of the heart tube. This expression pattern is maintained until HH stage 14. This expression pattern suggests that hLAMP‐1 may be involved in the formation of the endocardial tube.     

Efficacy of an E. coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs

Four chick trials and one pig trial were conducted to investigate the phosphorus-releasing efficacy oftwo commercial phytase enzymes (Natuphos and Ronozyme) and an experimental E. coli phytase enzyme (ECP) when added to corn-soybean meal diets containing no supplemental inorganic P (iP). In the 13- or 14-d chick trials, three or four graded levels of iP (0, 0.05,0.10,0.15%) from KH2PO4 were added to the basal diet to construct standard curves from which bioavailable P release could be calculated for the phytase treatments. In all cases, phytase supplementation levels were based on an assessment of phytase premix activity (i.e., P release from Na phytate at pH 5.5). Linear (P < 0.01) responses in tibia ash and weight gain resulted from iP supplementation in all assays. In the first chick trial, supplementation of 500 phytase units (FTU)/kg of ECP resulted in superior (P < 0.01) weight gain and tibia ash values compared with 500 FTU/kg of Natuphos. Results of the second chick trial revealed P-release values of 0.032 and 0.028% for 500 FTU/kg Natuphos and Ronozyme, respectively, and these were lower (P < 0.01) than the 0.125% P-release value for 500 FTU/kg of ECP. Tibia ash responded quadratically (P < 0.05) in response to graded levels of ECP up to 1,500 FTU/kg in the third chick trial. Combining Natuphos with either Ronozyme or ECP in Chick Trial 4 revealed no synergism between phytases with different initiation sites of P removal. The pig trial involved 10 individually fed weanling pigs per diet, and and phytase enzymes were supplemented to provide 400 FTU/kg in diets containing 0.60% Ca. Based on the linear regression of fibula ash on supplemental iP intake (r2 = 0.87), P-release values were 0.081% for Natuphos, 0.043% for Ronozyme, and 0.108% for ECP. These trials revealed an advantage of the E. coli phytase over the commercial phytases in young chicks.     

胚胎及新生期蛋鸡和肉鸡垂体生长激素基因表达的比较研究

本文采用Northern杂交分析法研究了14胚龄、18胚龄、1日龄、5日龄及10日龄肉鸡和蛋鸡垂体生长激素的基因表达，同时采用放射免疫分析法（RIA）测定了垂体内生长激素的含量。结果表明：14胚龄时在肉鸡和蛋鸡垂体总RNA中均未出生长激素mRNA(GH mRNA)。从18胚龄开始可检测出一条0.8kb的GHmRNA，并且垂体GHmRNA水平的发育性变化在品种间呈现不同的规律：蛋鸡从18胚龄到10日龄垂体GHmRNA水平不断升高，日龄间差异显著（P＜0.05）。肉鸡从18胚龄到出壳1日龄，GHmRNA水平有较大幅度的升高，但从1日龄至10日龄维持在1日龄时的水平。18胚龄肉鸡垂体GH mRNA水平显著高于蛋鸡（P＜0.05），并在1日龄和5日龄均维持在较高水平，与生长速度呈正相关；而10日龄时垂体HGmRNA水平的品种差异发生逆转，蛋鸡GHmRNA水平反而高于肉鸡。垂体HG含量的发育性变化趋势与垂体HGmRNA水平相一致，10日龄时蛋鸡垂体GH含量显著高于肉鸡（P＜0.05），与生长速度呈相反的趋。     

Changes in the muscle proteome after compensatory growth in pigs

Sixteen female pigs (Duroc x Landrace x Large White) were divided into 2 groups, which had either free access to the diet (control group) or were feed-restricted from d 28 to 80 and then had free access to the diet (compensatory growth group). The sensory analysis showed that the pigs exhibiting compensatory growth produced meat with increased tenderness compared with control pigs (P < 0.05). To gain further knowledge of the influence of compensatory growth on meat tenderness, the sarcoplasmic protein fraction of muscle tissue was studied at the time of slaughter and 48 h postmortem using proteome analysis. At slaughter, 7 different proteins were found to be affected by compensatory growth: HSC70, HSP27, enolase 3, glycerol-3-phosphate dehydrogenase, aldehyde dehydrogenase E2, aldehyde dehydrogenase E3, and biphosphoglycerate mutase. The HSC70 and HSP27 both belong to the heat shock family and are known to play a role during muscle development. Hence, they may be affected by compensatory growth and increased protein turnover. Forty-eight hours after slaughter, 8 different proteins were found to be affected by compensatory growth: myosin light chain (MLC) II, MLC III, sulfite oxidase, chloride intracellular channel 1, 14-3-3 protein gamma, elongin B, and phosphohistidine phosphatase 14. The changes observed on MLC II and MLC III could be a consequence of enzymatic cleavage in the neck region of the globular myosin head domain that causes the release of MLC II and MLC III from the actomyosin complex. It has previously been hypothesized that compensatory growth results in an increased postmortem proteolysis; thus it was presumed that the intensity of some protein fragments would be affected by compensatory growth. However, the peptides that were found to be affected at 48 h postmortem were all full-length proteins. The 14-3-3 protein gamma has been proposed to play a role in the contraction of muscle during rigor and may thereby have an effect on meat tenderness. This study reveals some very interesting changes in the muscle proteome affected by compensatory growth, which may be useful in understanding the relationship among compensatory growth, protein turnover, and meat tenderness.     

Early Morphological Differentiation of the Bipolar Neurons in the Chick Retina A Golgi Analysis

We report a histogenetic study of the bipolar cells of the chick embryo retina between days 5 and 9, using the Golgi technique. On day 8, at the level of the developing outer plexiform layer, small delicate spines appear on the outer processes of the bipolar cells, which represents the commencement of their dendritic ramification. At a later staige in development che detachment of Landolt's club from the outer limiting membrane is observed in some cells The inner ramifications of the bipolar cell, at the inner plexiform layer, appear later than those of the outer process.     

Effect of vitamin E and selenium on iron utilization in neonatal pigs.

Supplying adequate iron (Fe) to neonatal pigs to support normal growth and hematological and antioxidant status, while preventing iron toxicity, is a challenge for producers. Three experiments were conducted to determine the effect of frequency and route of Fe administration with or without vitamin E (E) and selenium (Se) on growth, Fe, and antioxidant status of neonatal pigs. In Exp. 1, 12 pigs from dams with reduced E status were fed a semipurified diet without added Fe from d 3 to d 14 of age. At d 6 of age, pigs received the following i.m. injections: 1) FE, 1 mL containing 200 mg of Fe (iron dextran); 2) FEE, treatment FE plus 1 mL containing 300 IU of vitamin E (d-alpha tocopherol); or 3) FESEE, 1.03 mL containing 200 mg of Fe (iron dextran), .15 mg of Se (sodium selenite), and 15 IU of vitamin E (d-alpha tocopherol). Pigs were weighed daily and blood was collected at 3, 7, and 14 d of age. From d 8 to 14, growth was depressed (P < .05) in pigs injected with FESEE. At 14 d of age, pigs injected with FE or FEE had increased (P < .05) hemoglobin (Hb) concentration. Ceruloplasmin activity (CP) was greater (P < .05) at d 7 of age than at d 3 or 14 regardless of treatment. In Exp. 2, 3-d-old pigs (n = 94) received the following: 1) FE, 200 mg Fe (iron dextran) i.m.; (2) FEE, treatment FE plus 300 IU vitamin E i.m.; 3) EFE, 300 IU vitamin E i.m. followed by 200 mg Fe (iron dextran) i.m. 24 h later; or 4) OFE, 100 mg Fe and 10 mg Cu orally. On d 21 of age, one-half of the pigs in each treatment received a second dose of their respective treatment. Blood samples (n = 60) were obtained on d 3 and 21 of age. Pigs injected with FE, FEE, or EFE had greater (P < .05) Hb at d 21 than pigs given OFE. Copper/zinc superoxide dismutase (Cu/ZnSOD) activity was greater (P < .05) at d 21 with OFE than with the other treatments. At 65 d of age, ADG did not differ among treatments. In Exp. 3, pigs (n = 150, in three farrowing groups) were injected with 200 mg of Fe (iron dextran) on d 1 or d 1 and 14. Blood samples were obtained on d 7 and 21 of age. Hemoglobin concentration on d 21 was improved equally by both treatments. Catalase and Cu/ZnSOD activities were increased (P < .05) on d 21 of the experiment compared with d 7 regardless of treatment. Growth was not affected by injection frequency. Results from these experiments indicate that one Fe injection (200 mg) for pigs from sows fed adequate vitamin E will result in adequate growth and hemoglobin concentration with today's improved genetics.