Genetic-statistical analysis of growth in selected and unselected mouse lines

The growth of males sampled from two mouse lines long-term selected for over 86 generations on body weight (DU6) or on protein amount (DU6P) was analysed from birth till 120 days of age and compared to the growth of an unselected control line (DUKs). Animals from the selected lines are already approximately 40 to 50% heavier at birth than the controls. This divergence increases to about 210 to 240% at the 120 day of age. With birth weights of 2.2 and 2.4 g and weights of 78 and 89 g at the 120 day these selection lines are the heaviest known mouse lines.

The fit of three modified non-linear growth functions (Gompertz function, Logistic function, Richards function) was compared and the effect of three different data inputs elucidated. The modification was undertaken to use parameters having a direct biological meaning, for example: A: theoretical final body weight, B: maximum weight gain, C: age at maximum weight gain, D (only Richards function): determines the position of the inflection point in relation to the final weight. All three models fit the observed data very well (r² = 0.949–0.998), with a slight advantage for the Richards function. There were no substantial effects of the data input (averages, single values, fitting a curve for every animal with subsequent averaging the parameters).

The high growth of the selected mice is connected with very substantial changes in the final weight and in the maximum weight gain, whereas the changes of the age at the point of inflection were, although partially significant, relatively small and dependent on the model used.     

QTL analysis of body weight and conformation score in commercial broiler chickens using variance component and half-sib analyses

The aim of the study was to investigate quantitative trait loci (QTL) in previously identified regions of chicken chromosomes 1, 4 and 5 relating to 40-day body weights and conformation scores. Half-sib (HS) and variance component analyses were implemented and compared using QTL Express software. Data were from a two-generation design and consisted of 100 dam families nested in 46 sire families with trait values for 2,708 offspring. Chicken chromosome 4 showed nominal significance for QTL affecting body weight and conformation, and linkage was confirmed for both traits on chromosome 5. Results varied according to method of analysis and with common parent in the HS method.     

Genetic parameters for feed efficiency in Romane rams and responses to single-generation selection

Feeding costs represent one of the highest expenditures in animal production systems. Breeding efficient animals that express their growth potential while eating less is therefore a major objective for breeders. We estimated the genetic parameters for feed intake, feed efficiency traits (residual feed intake (RFI) and feed conversion ratio (FCR)), growth and body composition traits in the Romane meat sheep breed. In these traits, selection responses to single-generation divergent selection on RFI were evaluated. From 2009 to 2016, a total of 951 male lambs were tested for 8 weeks starting from 3 months of age. They were weighed at the beginning and at the end of the testing period. Backfat thickness and muscle depth were recorded at the end of the testing period through ultrasound measurements. Feed intake was continuously recorded over the testing period using the automatic concentrate feeders. The heritability of RFI was estimated at 0.45 ± 0.08, which was higher than the heritability of FCR (0.30 ± 0.08). No significant genetic correlations were observed between RFI and growth traits. A favourable low negative genetic correlation was estimated between RFI and muscle depth (−0.30 ± 0.15), though additional data are needed to confirm these results. The selection of low RFI sires based on their breeding values led to the production of lambs eating significantly less concentrate (3% decrease in the average daily feed intake), but with the same growth as lambs from sires selected based on high RFI breeding values. We concluded that in meat sheep, RFI is a heritable trait that is genetically independent of post-weaning growth and body composition traits. A one-generation divergent selection based on RFI breeding values highlighted that substantial gains in feeding costs can be expected in selection schemes for meat sheep breeds.     

Phenotypic variation during cloning procedures: analysis of the growth behavior of clonal cell lines

The production of recombinant protein from mammalian cells is a key feature of the biotechnology industry. However, the generation of recombinant mammalian cell lines is still largely performed on an empirical basis and there are many potential areas for enhancement. We have shown previously that despite two rounds of limiting dilution cloning (LDC) of recombinant cell lines, there remained a high degree of heterogeneity in the resulting cell lines. We suggested that a rapid phenotypic drift occurred with these cells. It was unclear if this was a consequence of the added burden of production of a recombinant protein, the selection procedures, or merely an inherent feature of cell growth in culture. To address this, we have subjected untransfected (parental) cells to three successive rounds of LDC and monitored the growth properties of the resultant cells. The results show that despite repeated rounds of cloning, it was not possible to obtain phenotypically similar cell lines. We also demonstrated that this phenotypic drift is not due to gross changes in the protein p27, a key regulators of the cell cycle. Although cells with a range of growth properties were observed even after three rounds of cloning, the variation in growth patterns between cell lines decreased after cloning. Hence, we suggest that by cloning it may be possible to generate untransfected cells, which have particular growth properties. Starting with a well-defined population of parental cells may aid in the subsequent generation of tranfectants with desired growth properties.     

Marker-assisted analysis of three grain yield QTL in barley (Hordeum vulgare L.) using near isogenic lines

Three previously identified grain yield quantitative trait loci (QTL) on chromosomes 2S(2HS), 3C(3HC) and 5L(1HL), designated QTL-2S, QTL-3 and QTL-5L, respectively, were evaluated for their potential to increase yields of high-quality malting barley without disturbing their favorable malting quality profile. QTL mapping of yield related traits was performed and near-isogenic lines (NILs) were developed. QTL for plant height, head shattering, seed weight and number of rachis nodes/spike were detected in the QTL-3 region. NILs developed by introgressing QTL-3 from the high-yielding cv. Steptoe to the superior malting quality, moderate-yielding cv. Morex acquired reduced height, lodging and head shattering features of Steptoe without major changes in malting quality. The yield of NILs, measured by minimizing the losses due to lodging and head shattering, did not exceed that of Morex. Steptoe NILs, with the Morex QTL-2S region, flowered 10 days later than Steptoe but the grain yield was not changed. None of the 3 QTL studied altered the measured yield of the recipient genotype, per se, although QTL 2S and QTL-3 affected yield-related traits. We conclude that these yield QTL must interact with other genes for full expression. Alternatively, they affect the harvestable yield through reduced lodging, head shattering, and/or altered flowering time.     

一种鉴定小鼠胚胎质量的双重荧光染色方法

尝试改进并建立一种较可靠的、适合小鼠胚胎质量参考鉴定的双重荧光染色(细胞计数)方法.与传统方法相比,本实验中抗羊脾细胞抗血清的最佳稀释度为1∶5,作用时间为30 min.豚鼠补体最佳稀释度为1∶5,两种分染染料H33342和PI的工作浓度均降低至5.3 μg/ml便可着色,染色时间延长至90 min,加大了染料工作液中柠檬酸钠的浓度,并在计数观察前增加压片这一步骤.染色过程中应注意避免血清物质对抗体补体的不良影响以及避光和快速的操作.双重荧光染色后小鼠胚胎内细胞团(ICM)细胞着色为蓝色,滋养层(TE)细胞着色为粉红色,数目清晰可辨.此方法可以作为一种简洁有效的小鼠胚胎质量鉴别参考方法.     

Analysis of response to 20 generations of selection for body composition in mice: fit to infinitesimal model assumptions

Data were analysed from a divergent selection experiment for an indicator of body composition in the mouse, the ratio of gonadal fat pad to body weight (GFPR). Lines were selected for 20 generations for fat (F), lean (L) or were unselected (C), with three replicates of each. Selection was within full-sib families, 16 families per replicate for the first seven generations, eight subsequently. At generation 20, GFPR in the F lines was twice and in the L lines half that of C. A log transformation removed both asymmetry of response and heterogeneity of variance among lines, and so was used throughout. Estimates of genetic variance and heritability (approximately 50%) obtained using REML with an animal model were very similar, whether estimated from the first few generations of selection, or from all 20 generations, or from late generations having fitted pedigree. The estimates were also similar when estimated from selected or control lines. Estimates from REML also agreed with estimates of realised heritability. The results all accord with expectations under the infinitesimal model, despite the four-fold changes in mean. Relaxed selection lines, derived from generation 20, showed little regression in fatness after 40 generations without selection.     

An improved protocol for the isolation and cultivation of embryonic mouse myocytes

In vitro cultures of cardiomyocytes have proven to be a useful tool for toxicological, pharmacological, and developmental studies, as well as for the study of the cellular and molecular mechanisms responsible for proper myocyte function. One deficient area of research is that of myocyte proliferation. Cardiomyocyte proliferation dramatically diminishes soon after birth and has a very limited occurrence within the adult heart, thus limiting the use of adult cells for proliferation studies. An improved understanding of the requirements for myocyte proliferation will allow for the development of better approaches to repair damaged heart tissue. Here, we provide a protocol for the reliable isolation of embryonic mouse myocytes. These myocytes behave similarly to those in vivo, including their ability to proliferate, providing an ideal system for the study of cardiomyocyte proliferation.     

Phenotypic responses of mouse mammary tumours and normal mammary epithelium cultured in collagen gels: Correlation with tumour type and progression

Mammary tumours in female BR6/Icrf mice and the corresponding contralateral normal mammary glands were disaggregated with collagenase and the epithelial structures released ('organoids') separated from other cellular components by filtration. The organoids were established in primary culture in a collagen matrix and the outgrowths obtained were studied by light microscopy and time-lapse cinemicroscopy. The pattern of three-dimensional outgrowths produced was found to be specific to the original tissue. Organoids from normal tissue formed a characteristic outgrowth designated Pattern A. Normal tissue from pregnant mice formed an additional characteristic outgrowth (Pattern A') which has not been described previously. Pregnancy-dependent tumours produced a distinctive phenotypic outgrowth designated Pattern D, whereas pregnancy-independent tumours gave a different distinctive Pattern B as well as a unique specific outgrowth designated Pattern C. Outgrowths of Pattern D from a pregnancy-dependent tumour were removed from culture and implanted into a syngeneic female mouse. Tumours arising in the host were found to be pregnancy-independent and showed phenotypic outgrowths in subsequent culture of pregnancy-independent Patterns B and C. The results show that the type of outgrowths in these cultures correlates with the biology of the tissue in vivo and that changes in tumour progression in vivo are accompanied by alterations in phenotypic outgrowths in culture.     

Mode of Infant Feeding and Achieved Growth in Adolescence: Early Feeding Patterns in Relation to Growth and Body Composition in Adolescence

TULLDAHL, JENNY, KJELL PETTERSSON, SUSAN W. ANDERSSON, AND LENA HULTHÉN. Mode of infant feeding and achieved growth in adolescence: Early feeding patterns in relation to growth and body composition in adolescence. Obes Res. Objective: Feeding mode in infancy and differences in childhood growth have been studied in several longitudinal studies, but few studies have followed children up to adolescent age. There is evidence that formula-fed infants weigh more and are taller than their breast-fed counterparts, and indications that this difference may sustain. Research Methods and Procedures: We have studied the relations between length of breast-feeding, growth, and body composition in a group of 781 representatively chosen adolescents. Data on feeding pattern in infancy and on weight and height from birth up to 18 years were collected. We studied the relation between high body mass index (BMI) (defined as ≤85^th percentile) in adolescence and length of breast-feeding. Results: Girls who were not breast-fed or breast-fed for less than 3 months had a significantly higher height curve than girls exclusively breast-fed for more then 3 months. There were tendencies towards higher values of adipose tissue measured by skinfolds in girls breast-fed for 3 months or less. Short duration of exclusive breast-feeding was associated with higher BMI (p<0. 04). In a subgroup of 194 adolescents, body composition was measured with dual energy X-ray. Both boys and girls who were exclusively breast-fed for more than 3 months were leaner and showed a trend towards lower skinfold values. Conclusion: These results are important to include in the debate about optimal feeding in infancy. Regarding breastfeeding as a standard, our results imply that formula fed infants may be at risk for overfeeding, which might lead to overweight, even up to adolescent age.     

Evolution of starvation resistance in Drosophila melanogaster: measurement of direct and correlated responses to artificial selection

Laboratory selection for resistance to starvation has been conducted under relatively controlled conditions to investigate direct and correlated responses to artificial selection. With regard to starvation resistance, there are three physiological routes by which the trait can evolve: resource accumulation, energy conservation and starvation tolerance. A majority of energetic compounds and macromolecules including triglycerides, trehalose and other sugars, and soluble protein increased in abundance as a result of selection. Movement was additionally investigated with selected males moving less than control males and selected females exhibiting a similar response to selection. Results obtained from this study supported two of the possible evolutionary mechanisms for adaptation to starvation: energy compound storage and conservation. If the response to selection is based on an evolutionarily conserved pattern of genetic correlations (elevated lipid, elevated sugars and reduced movement), then the response to selection is medically relevant and the genetic architecture should be investigated in depth.     

Sexual size dimorphism in caecilian amphibians: analysis, review and directions for future research

Sexual dimorphism, widespread in the animal kingdom, describes differences between the sexes in size, shape and many other traits. Sexual size dimorphism (SSD) plays a significant role in understanding life history evolution and mating systems. The snakelike morphology of limbless caecilian amphibians lacking obvious secondary sexual characters (in contrast to frogs and salamanders) impedes accurate intrasexual comparisons. In this study, sexual size dimorphism in the oviparous caecilian Ichthyophis cf. kohtaoensis, a phylogenetically basal caecilian, was analysed. Females were larger in all body and head characters tested. However, when adjusted to body size (total length), females differed only in their cloacal shape. Clutch volume was positively correlated to female body size, thus female fecundity increased with body size supporting the hypothesis of a fecundity-selected SSD in the oviparous Ichthyophis cf. kohtaoensis. A review of the present SSD data for caecilians shows that many species are monomorphic for body size but show dimorphism in head size, while other species demonstrate female-biased SSD. Male-biased SSD has not been reported for caecilians. To understand life history evolution in caecilians, further studies on the reproductive biology of other taxa are urgently needed, in particular for rhinatrematids and uraeotyphlids. New data will allow phylogenetically controlled comparative analyses to fully explore the pattern of SSD among caecilian lineages.     

Quantitative analysis of mid-gestation mouse aggregation chimaeras: non-random composition of the placenta

Mouse chimaeras were produced by aggregating eight-cell embryos from two different F₂ matings, abbreviated to AF₂ and BF₂ respectively: (C57BL/ OIa.AKR-Gpi-1s ^a, c/Ws female × BALB/c male)F₂ and (C57BL/Ws female × CBA/Ca male)F₂. Quantitative electrophoresis of glucose phosphate isomerase (GPI-1) was used to estimate the proportions of the two cell populations in different tissues of the 12 day chimàeric conceptuses, with the % GPI-1A indicating the percentage of cells derived from the AF₂ embryos. The % GPI-1A was found to be highly positively correlated within the primitive ectoderm lineage (between the fetus, amnion and yolk sac mesoderm) and within the primitive endoderm lineage (between the yolk sac endoderm and the parietal endoderm) but no correlation (either positive or negative) was seen between the two lineages. This confirms the results of a previous,study of chimaeras made between partially congenic strains and suggests the original conclusions have general validity. The % GPI-1A in the placenta was corrected for the expected contribution of maternal GPI-1, based on control experiments involving transfer of homozygous Gpi-1s ^b /Gpi-1s ^b embryos to the uteri of Gpi-1s ^a /Gpi-1s ^a pseudopregnant females. The corrected % GPI- lA in the placenta was positively correlated with that in each of the three primitive ectoderm derivatives. This suggests either (1) exchange of cells between the polar trophectoderm and the underlying part of the inner cell mass that forms the primitive ectoderm or (2) cells are incompletely mixed in the chimaeric blastocyst and patches of AF₂ and BF₂ cells straddle the boundary between the polar trophectoderm and the underlying primitive ectoderm. The second explanation does not imply the existence of shared developmental lineages between trophectoderm and primitive ectoderm in non-chimaeric embryos. Unlike that of any other tissue, the distribution of placental GPI-1A was U-shaped; in 17/28 placenta samples the proportion of the minor component was 10% or less. This suggests that the placental trophoblast is derived from a small number of coherenct clones of polar trophectoderm cells (either a small number of polar trophectoderm cells or a larger number if the two cell populations are not finely intermingled). Thus, although as a population the placentas of chimaeric conceptuses are balanced with respect to the % GPI-1A (mean close to 50%), individually most placentas are extremely unbalanced in their chimaeric composition (< 10% or > 90% GPI-IA). This non-random composition of the chimaeric placentas is in contrast to the widely held assumption that the distribution of cells in chimaeric conceptuses is normally random. Correspondence to: J.D. West     

Distribution of lymphatic vessels in mouse thymus: immunofluorescence analysis

Thymic blood and lymphatic vessels in humans and laboratory animals have been investigated in morphological studies. However, occasionally a clear distinction between blood vessels and lymphatic vessels cannot be made from morphological characteristics of the vasculature. To visualize thymic lymphatics in normal adult BALB/c mice, we used antibodies against specific markers of lymphatic endothelial cells. Expression of vascular endothelial growth factor receptor–3 (VEGFR–3) was detected throughout the thymus, i.e., the capsule, cortex, and medulla. Most thymic lymphatics were present in capillaries of ~20 μm in caliber. The plexuses of lymphatic capillaries were occasionally detectable. Lymphatic vessels were frequently adjacent to CD31–positive blood vessels, and some lymphatic vessels were seen in the immediate vicinity of or within the perivascular spaces around postcapillary venules. The identity of VEGFR–3–positive vessels as lymphatics was further confirmed by staining with additional markers: LYVE–1, Prox–1, neuropilin–2, and secondary lymphoid tissue chemokine (SLC). The distributions of LYVE–1 were similar to those of VEGFR–3. Most lymphatic vessels were also identified by Prox–1. Neuropilin–2 was restricted to lymphatic vessels in the thymus. The most abundant expression of SLC in the thymus was in medullar epithelial cells; SLC was also expressed in lymphatic vessels and blood vessels. Thus, lymphatic endothelium in mouse thymus was characterized by positive staining with antibodies to VEGFR–3, LYVE–1, Prox–1, neuropilin–2, or SLC, but not with an antibody to CD31. Our results suggest the presence of lymphatic capillary networks throughout the thymus.     

Influence of copper on the early post-implantation mouse embryo: An in vivo and in vitro study

Summary Female mice were injected intravenously with copper sulphate on either the 7th day (early egg cylinder stage of development), the 8th day (late egg cylinder stage), or the 9th day (early somite stage of development), and examined on the 10th day of gestation. Injection on the 7th day was found to be embryo-lethal; when females were injected on the 8th day, the majority of the surviving embryos exhibited anomalies of the neural tube and/or the heart, while injection on the 9th day resulted in a very low incidence of anomalies. The most common malformations seen on the 10th day involved failure of closure of the neural tube in the head region of the embryo, and various types of anomalies of cardiac rotation and shape. When additional females injected on the 8th day were examined on the 12th day, a high proportion of the fetuses examined had developed exencephaly.A further group of embryos from untreated females were explanted on the 9th day and cultured in vitro in various concentrations of copper sulphate. The lowest levels tested had little obvious effect on neural tube closure. Intermediate doses resulted in, retarded and anomalous embryonic development, while the highest levels employed resulted in neural tube and cardiac anomalies similar to those produced in vivo.The results demonstrate both the direct toxic effect of copper on embryonic development and that the stage of embryonic development at the time of exposure determines both the nature and the extent of the effect.     

Bupropion for Weight Loss: An Investigation of Efficacy and Tolerability in Overweight and Obese Women

Objective: On the basis of the clinical observations that bupropion facilitated weight loss, we investigated the efficacy and tolerability of this drug in overweight and obese adult women. Research Methods and Procedures: A total of 50 overweight and obese (body mass index: 28.0 to 52.6 kg/m²) women were included. The core component of the study was a randomized, double‐blind, placebo‐controlled comparison for 8 weeks. Bupropion or placebo was started at 100 mg/d with gradual dose increase to a maximum of 200 mg twice daily. All subjects were prescribed a 1600 kcal/d balanced diet and compliance was monitored with food diaries. Responders continued the same treatment in a double‐blind manner for an additional 16 weeks to a total of 24 weeks. There was additional single‐blind follow‐up treatment for a total of 2 years. Results: Subjects receiving bupropion achieved greater mean weight loss (last‐observation‐carried‐forward analysis) over the first 8 weeks of the study (p = 0.0001): 4.9% ± 3.4% (n = 25) for bupropion treatment compared with 1.3% ± 2.4% (n = 25) for placebo treatment. For those who completed the 8 weeks, the comparison was 6.2% ± 3.1% (n = 18) vs. 1.6% ± 2.9% (n = 13), respectively(p = 0.0002), with 12 of 18 of the bupropion subjects (67%) losing over 5% of baseline body weight compared with 2 of 13 in the placebo group (15%; p = 0.0094). In the continuation phase, 14 bupropion responders who completed 24 weeks achieved weight loss of 12.9% ± 5.6% with fat accounting for 73.5% ± 3.7% of the weight lost and no change in bone mineral density as assessed by DXA. Bupropion was generally well‐tolerated in this sample. Discussion: Bupropion was more effective than placebo in achieving weight loss at 8 weeks in overweight and obese adult women in this preliminary study. Initial responders to bupropion benefited further in the continuation phase.     

Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: I—Comparative fundamental cryobiology of multiple mouse embryonic stem cell lines and the implications for embryonic stem cell cryopreservation protocols

The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols.