A simple method to approximate gene content in large pedigree populations: application to the myostatin gene in dual-purpose Belgian Blue cattle

Gene content is the number of copies of a particular allele in a genotype of an animal. Gene content can be used to study additive gene action of candidate gene. Usually genotype data are available only for a part of population and for the rest gene contents have to be calculated based on typed relatives. Methods to calculate expected gene content for animals on large complex pedigrees are relatively complex. In this paper we proposed a practical method to calculate gene content using a linear regression. The method does not estimate genotype probabilities but these can be approximated from gene content assuming Hardy-Weinberg proportions. The approach was compared with other methods on multiple simulated data sets for real bovine pedigrees of 1 082 and 907 903 animals. Different allelic frequencies (0.4 and 0.2) and proportions of the missing genotypes (90, 70, and 50%) were considered in simulation. The simulation showed that the proposed method has similar capability to predict gene content as the iterative peeling method, however it requires less time and can be more practical for large pedigrees. The method was also applied to real data on the bovine myostatin locus on a large dual-purpose Belgian Blue pedigree of 235 133 animals. It was demonstrated that the proposed method can be easily adapted for particular pedigrees.     

Effect of dam factors on milk intake and performance of Belgian Blue suckling calves

Milk intake data were collected at 4-week intervals from Belgian Blue double-muscled (BBDM) suckling calves up to 16 weeks after birth. Dams were fed at 100%, 90%, 80% or 70% of their energy requirements during a 140-day indoor period from the beginning of December to the end of April (restriction period). Afterwards, cows were turned out on pasture (re-alimentation period). Mean calving date was 3 June, but parturitions were spread over the year. A total of 521 individual milk intake (IMI) and 120 average daily milk intake (AMI, mean of IMI) records were collected. IMI amounted to 7.0 ± 2.2 kg/day and was not affected by energy level during the indoor period. It was highest in May and June. Calves born out of multiparous cows consumed more milk than those born out of primiparous cows (7.4 v. 6.2 kg per day; P = 0.001), while intake tended to be higher in female than in male calves (7.2 v. 6.8 kg per day; P = 0.044). Dam age at calving, parity, post partum weight and body condition were only weakly correlated with AMI (r 0.4). AMI was correlated with pre-weaning live-weight gain (r = 0.807) and weaning weight (r = 0.783), with a slightly higher correlation for male than for female calves. Daily live-weight gain during the suckling period was increased by 79 g per extra kg daily milk intake on average, and by 86 and 74 g, respectively, for male and female calves. Calf solid feed intake was low, even when milk intake was low. In terms of net energy intake, milk was only substituted for solid feed by 29%. It can be concluded from the calf intake that milk production in BBDM cows is comparable with that of cows from other beef breeds, but its variation is considerable, complicating an accurate estimation.     

Identification of congenital muscular dystonia 2 associated with an inherited GlyT2 defect in Belgian Blue cattle from the United Kingdom

Two newborn Belgian Blue calves from a farm in the United Kingdom exhibited lateral recumbency, low head carriage and transient muscle spasms following tactile or auditory stimulation. DNA sequence analysis indicated that both calves were homozygous for the recessive congenital muscular dystonia type 2 (CMD2) mutation (c.809T>C, p.Leu270Pro) in SLC6A5, encoding the neuronal glycine transporter GlyT2. Further testing of animals from the index farm and a sample of Belgian Blue sires revealed an unexpectedly high frequency of CMD2 carriers. This implies that linked quantitative trait loci may be influencing the prevalence of CMD2 in the estimated 55,000 Belgian Blue cattle in the United Kingdom. We have therefore developed new inexpensive tests for the CMD2 allele that can be used to confirm diagnosis, identify carriers and guide future breeding strategy, thus avoiding animal distress/premature death and minimizing the future economic impact of this disorder.     

新生犊牛不同时间段肠道微生物菌群结构的变化

了解犊牛在不同生长发育的过程中肠道菌群的建立及肠道菌群结构的动态变化。

采集不同时间点（1 d、3 d、6 d、9 d、12 d、24 d、36 d、48 d、60 d和72 d）新生6头健康犊牛的新鲜直肠粪便样本,利用16S rDNA测序技术及生物信息学测序方法,分析犊牛不同时间点的肠道微生物结构组成及多样性。

OTUs聚类分析中共有的OTUs有413个。Alpha多样性指数显示,随着日龄的增加,物种的相对丰度逐渐增多。通过不同组间相对丰度展示了在科水平不同种类物种的相对丰度,包括厌氧环境、致病菌等方面生成了物种丰富度柱形累加图。在厌氧环境中,梭菌科随着日龄的增加逐渐增加。真菌科在48 d后逐渐出现在肠道菌群中。毛螺菌科从犊牛出生就一直存在肠道中,并逐渐增加成为优势菌群。在致病菌中,肠杆菌科随着日龄的增加逐渐减少,梭菌与瘤胃球菌不断增加。

通过分析新生犊牛肠道微生物菌群多样性的动态结构变化,对新生犊牛胃肠道微生态环境的改善和免疫调控及其生产性能具有重要的意义。

     

Genome-wide next-generation DNA and RNA sequencing reveals a mutation that perturbs splicing of the phosphatidylinositol glycan anchor biosynthesis class H gene (PIGH) and causes arthrogryposis in Belgian Blue cattle

Background

Cattle populations are characterized by regular outburst of genetic defects as a result of the extensive use of elite sires. The causative genes and mutations can nowadays be rapidly identified by means of genome-wide association studies combined with next generation DNA sequencing, provided that the causative mutations are conventional loss-of-function variants. We show in this work how the combined use of next generation DNA and RNA sequencing allows for the rapid identification of otherwise difficult to identify splice-site variants.

Results

We report the use of haplotype-based association mapping to identify a locus on bovine chromosome 10 that underlies autosomal recessive arthrogryposis in Belgian Blue Cattle. We identify 31 candidate mutations by resequencing the genome of four cases and 15 controls at ~10-fold depth. By analyzing RNA-Seq data from a carrier fetus, we observe skipping of the second exon of the PIGH gene, which we confirm by RT-PCR to be fully penetrant in tissues from affected calves. We identify - amongst the 31 candidate variants - a C-to-G transversion in the first intron of the PIGH gene (c211-10C > G) that is predicted to affect its acceptor splice-site. The resulting PIGH protein is likely to be non-functional as it lacks essential domains, and hence to cause arthrogryposis.

Conclusions

This work illustrates how the growing arsenal of genome exploration tools continues to accelerate the identification of an even broader range of disease causing mutations, therefore improving the management and control of genetic defects in livestock.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1528-y) contains supplementary material, which is available to authorized users.     

Importance of dam BW change and calf birth weight in double-muscled Belgian Blue cattle and its relationship with parity and calving interval

Factors affecting calving interval (CI) in double-muscled Belgian Blue (DMBB) beef cows were investigated with regard to the BW yield (BWY) of the cow–calf pair, using 834 CI records from 386 females with parities 1 to 6. The effect of parity and CI on BWY was also studied. Cow–calf pair BWY was defined as calf birth weight plus dam BWY per CI. CI (mean±s.e.: 404±1.9 days) was affected by parity, calving season, suckling and calf birth weight/dam weight. Primiparous cows had a shorter CI than cows with three or more calvings (P<0.05), with an intermediate CI for second-calf cows. Spring calvings resulted in a shorter CI than summer and autumn calvings, with intermediate values for winter calvings. Suckling dams had longer CIs than non-suckling dams. There were interactions (P<0.05) between calving season and suckling, and between calving season and mating system. Shortest CIs were observed for spring calvings in case of non-suckling and for summer calvings in case of suckling. Longest CIs were observed for autumn calvings in case of natural service (NS) and for winter calvings in case of artificial insemination (AI). Calf birth weight/dam weight of 6% to 10% resulted in shorter CI than a ratio of <6% (P<0.05). Body condition and mating system (NS v. AI) did not affect CI. Daily cow–calf pair BWY was affected by parity (P<0.001) and CI (P=0.013), with a tendency for an interaction (P=0.094). Daily cow–calf pair BWY did not differ for CIs of <12 to 16 months in primiparous cows and was lowest for a CI of 13 to 15 months in second-calf cows, whereas the effect of CI was more variable in older cows. Dam contribution to cow–calf pair BWY was larger than calf birth weight in first- and second-calf cows, and increased with increasing CI. Dam contribution to cow–calf pair BWY was smaller than calf birth weight in older cows, varying from 0.2 to 1.0 depending on CI. A short CI is advised for DMBB cows because of a larger BWY and more efficient nutrient utilisation.     

Design of a beneficial product for newborn calves by combining Lactobacilli,minerals, and vitamins

Diarrhea is one of the most frequent diseases affecting newborn calves in intensive systems. Several strategies were proposed to protect and improve health, such as probiotics. This work was directed to design a product containing freeze-dried bacteria, vitamins, and minerals, as well as to optimize conditions with lyoprotectors, combine strains and add vitamins, minerals, and inulin to the product. The lyoprotectors were milk, milk-whey, and actose, and products were stored for 6 months at 4°C. Combined bacteria were freeze-dried in milk and the final products were added with minerals, vitamins, and insulin. The viable cells were determined by the plate count assay and antibiotic profiles to differentiate strains. Lactobacillus johnsonii CRL1693, L. murinus CRL1695, L. mucosae CRL1696, L. salivarius CRL1702, L. amylovorus CRL1697, and Enterococcus faecium CRL1703 were evaluated. The optimal conditions were different for each strain. Milk and milk whey maintained the viability during the process and storage after 6 months for most of the strains, except for L. johnsonii. Lactose did not improve cell’s recovery. L. murinus was viable for 6 months in all the conditions, with similar results in enterococci. In strains combined before freeze-dried, the viability decreased deeply, showing that one-step process with bacteria mixtures, vitamins, and minerals were not adequate. Freeze-dried resistance depends on each strain and must be lyophilized individually.     

First investigations to refine video-based IR thermography as a non-invasive tool to monitor the body temperature of calves

In this study, a video-based infrared camera (IRC) was investigated as a tool to monitor the body temperature of calves. Body surface temperatures were measured contactless using videos from an IRC fixed at a certain location in the calf feeder. The body surface temperatures were analysed retrospectively at three larger areas: the head area (in front of the forehead), the body area (behind forehead) and the area of the entire animal. The rectal temperature served as a reference temperature and was measured with a digital thermometer at the corresponding time point. A total of nine calves (Holstein-Friesians, 8 to 35 weeks old) were examined. The average maximum temperatures of the area of the entire animal (mean±SD: 37.66±0.90°C) and the head area (37.64±0.86°C) were always higher than that of the body area (36.75±1.06°C). The temperatures of the head area and of the entire animal were very similar. However, the maximum temperatures as measured using IRC increased with an increase in calf rectal temperature. The maximum temperatures of each video picture for the entire visible body area of the calves appeared to be sufficient to measure the superficial body temperature. The advantage of the video-based IRC over conventional IR single-picture cameras is that more than one picture per animal can be analysed in a short period of time. This technique provides more data for analysis. Thus, this system shows potential as an indicator for continuous temperature measurements in calves.     

Patterns of blubber fat deposition and evaluation of body condition in growing southern right whale calves (Eubalaena australis)

Marine mammals rely on blubber mainly for energy storage, buoyancy, and streamlining. Mysticetes are born with a relatively thin fat layer that grows rapidly during nursing. However, little information on blubber deposition patterns is available for baleen whale calves. We measured blubber thickness at nine body locations in 350 southern right whale (Eubalaena australis) newborn to 4–6-month-old calves that died on the Península Valdés (Argentina) calving ground from 2003 to 2019, to document changes in blubber thickness with growth. Additionally, we looked for differences in blubber thickness and lipid content of the outer/superficial blubber in calves that died in years with high (2003, 2005, 2007–2013) and low calf mortality (2004, 2006, 2014–2019) to test whether the former were suffering from gross nutritional stress. Blubber thickness increased at all body locations with calf length. Along the cranio-caudal axis, blubber increased in the dorsal and ventral planes, but decreased laterally towards the peduncle, possibly to improve streamlining. We found no difference in blubber thickness and lipid content between high and low mortality years, suggesting that individuals were not undernourished. This is the first study to describe progressive increases in calf blubber during growth and contributes knowledge to right whale health and ontogeny.     

Effects of housing and management conditions on teat cleanliness of dairy cows in cubicle systems taking into account body dimensions of the cows

Dirty udders and teats result in a higher workload in terms of cleaning before milking and may constitute a risk for udder health. The aim of this prevalence study on 23 farms with very low within-farm variation in cubicle measures and other cubicle characteristics was to identify potentially influencing housing and management factors concerning teat and teat tip soiling. Information about udder soiling, housing and management was collected through direct recording and farmers’ interviews. Height at withers, shoulder width and diagonal body length were measured in 79% to 100% of the cows in each herd. On the basis of the 25% largest animals, compliance with recommendations for cubicle measures was calculated, which was generally rather low. Stepwise regression was used to find predictors for the percentage of dirty teats and of dirty teat tips. The final model on dirty teats explained 58.5% of the variance and contained four factors (F = 6.34, P = 0.0023). Less soiled teats were found on farms that conducted teat dipping after milking (t = −3.21, P = 0.0048), had increased daily cubicle maintenance time (t = −2.58, P = 0.0187), deep-bedded cubicles (t = −2.42, P = 0.0265) and decreasing compliance concerning cubicle length (t = 2.33, P = 0.0317). The final model on teat tip soiling explained 46.0% of the variance and contained three factors (F = 5.39, P = 0.0075). Less soiled teat tips were associated with increasing height of bedding material (t = −2.89, P = 0.0094) and decreasing compliance concerning resting length (t = 2.12, P = 0.0470). Difficult to explain was the association found between increased passage soiling and decreased teat tip dirtiness (t = −1.86, P = 0.0790). Thus, even under relatively restrictive cubicle conditions, a certain increase in teat and teat tip soiling was found with increasing cubicle length. However, at the same time, measures relating to good management may positively affect teat and teat tip cleanliness. In addition, deep-bedded cubicles yielded advantages in this regard. Both regression models for teat and teat tip cleanliness contained similar but not identical predictive variables. This indicates that dirtiness of each particular area may originate from slightly different though related causes. The low correlations found between udder, teat and teat tip cleanliness should be considered in future studies.     

Analysis of experimental data with repeated measurements

Summary. Experimental data often consist of serial measurements on subjects after a treatment. Typical questions concerning such data are: (A) Do subjects really react to treatment or are the fluctuations just random? (B) What are the numerical characteristics of the response? (C) Is the response identical in all groups? Differences between the individuals in the dynamics of the reaction make it difficult to apply standard statistical procedures. This paper proposes to answer questions (A) and (B) at the individual level, then to give an answer to (C) on the basis of this information. This kind of analysis may be useful since it can separate subjects giving response from those that do not and can identify individual response patterns and compare treatments with respect to each numerical characteristic separately. To answer question (A), a permutation test is proposed and its power is evaluated by simulation.     

First measurements of Blue intensity from Pinus peuce and Pinus heldreichii tree rings and potential for climate reconstructions

Climate changes, their regional patterns, origin, and prediction are currently one of the most important scientific challenges. Tree-rings are among the most widely used proxies for past climate variation. However tree-ring width (TRW) from certain tree species and regions often do not contain robust climate signal. Other parameters such as Maximum latewood density (MXD) of conifer tree rings are more sensitive to summer temperatures, but the measurements have high costs. A potential surrogate for MXD is blue intensity (BI), which is based on the measurements of high-resolution images of the wood. The method has been tested and applied for several species. However it has not been tested up to now for Pinus heldreichii Christ (PIHE) and Pinus peuce Griseb. (PIPE). Those species are with limited distribution on the Balkan Peninsula and due to their longevity (frequently more than 500 years with potential to more than 1000 years) may serve as one of the best proxies for past climate variation in SE Europe.We composed BI chronologies following standard procedures from PIHE and PIPE trees from subalpine locations in the Pirin Mts, Bulgaria. The correlation analysis with climate parameters revealed strong and significant positive correlations of PIHE BI (ΔBI and Latewood BI (LwBI) series) and summer temperatures. The highest were 0.74 (p < 0.05) with July-August average temperatures for the period 1933–1983. The correlation values of ΔBI remained higher than 0.6 for the whole period (1933–2014) and sub-periods both for average monthly and average maximum temperatures. LwBI values of the PIHE chronology had correlation coefficients above 0.52 (highest 0.64) with August and August-July temperatures. The PIPE BI chronologies were also positively correlated with summer temperatures, but showed lower values than PIHE with lower temporal stability. The most stable were the correlations with LwBI average August temperatures, which were above 0.52. Delta BI series displayed high correlations for the 1933–1983 period, but then decreased and this caused overall lower correlations with August temperatures. Our data shows that there is potential to develop long BI chronologies and proxy climate reconstructions from the studied species and in this way complement the knowledge of the past climate of SE Europe.     

Body measurements, hematology, and serum chemistry values of the adult Cebus apella monkey

Body measurements, hematology, and serum chemistry values were studied in 40 captured male and female Cebus apella monkeys. Some significant dimorphism with male predominance was found. Significant differences were also found for hemoglobin and red cell volume between males and females. Differential white blood cell counts indicated a marked predominance of lymphocytes and high values of gamma globulin in both sexes.     

Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacrylamide gel electrophoresis

The mitochondrial respiratory chain consists of multi-subunit protein complexes embedded in the inner membrane. Although the majority of subunits are encoded by nuclear genes and are imported into mitochondria, 13 subunits in humans are encoded by mitochondrial DNA. The coordinated assembly of subunits encoded from two genomes is a poorly understood process, with assembly pathway defects being a major determinant in mitochondrial disease. In this study, we monitored the assembly of human respiratory complexes using radiolabeled, mitochondrially encoded subunits in conjunction with Blue Native polyacrylamide gel electrophoresis. The efficiency of assembly was found to differ markedly between complexes, and intermediate complexes containing newly synthesized mitochondrial DNA-encoded subunits could be observed for complexes I, III, and IV. In particular, we detected human cytochrome b as a monomer and as a component of a novel approximately 120 kDa intermediate complex at early chase times before being totally assembled into mature complex III. Furthermore, we show that this approach is highly suited for the rapid detection of respiratory complex assembly defects in fibroblasts from patients with mitochondrial disease and, thus, has potential diagnostic applications.     

Estimation of total body volume from arm measurements in Punjabi girls aged 10–19 years

Mean values and standard deviations of total body volume, body density, arm volume, height, weight and a battery of 15 measurements of arm comprising different lengths, girths, diameters and skinfolds of 200 Punjabi girls aged 10–19 years are presented. Relationship between arm volume and total body volume in different age groups are also studied. Highly significant and positive correlations existed between arm volume and total body volume and the values of correlation coefficients r ranged between 0.78 to 0.92 in different age groups. Body volume was estimated also from arm measurements by using selective stepwise multiple linear regression analysis for each age group. Highly significant multiple correlation coefficients R were obtained for predicting total body volume from arm measurements and the values of multiple R ranged between 0.92 to 0.97 in different age groups even with a combination of just three or four arm measurements. The most commonly selected measurements up to fourth step in most of the age groups were upper arm girth and maximum forearm girth.     

Local blood flow velocities in the carotid body of fetal sheep and newborn lambs

Summary Elastically-suspended microelectrodes were used in the vascularly isolated blood-perfused carotid body of fetal and newborn lambs as well as of 6–7-day-old lambs to measure local blood flow velocities by means of hydrogen clearance. Fetal sheep (n=9) carotid bodies elicited mean local blood velocity values between 0.008 and 0.11 cm·s^–1, whereas newborn lamb carotid bodies (n=7) showed values between 0.008 and 0.067 cm·s^–1 at a perfusion pressure range between 30 and 150 mmHg. The 6–7-day-old lamb carotid bodies (n=5) were characterized by values of 0.003 and 0.049 cm·s^–1 over the same perfusion pressure range. Fetal carotid body values were statistically significantly higher than the values of the 6–7-day-old lamb carotid bodies, whereas the newborn carotid body values showed no significant difference to both other groups. The flow velocity/perfusion pressure relationship peaked at perfusion pressure values between 100 and 150 mmHg in all groups with a reduced steepness in the lamb carotid body. It is concluded that local blood flow velocities in the carotid body are similar to that in other organs, and that after birth local blood flow velocities in the carotid body decrease during the first week of life, probably induced by vasoconstriction, changed blood gas values, and/or increasing shunt flow.Abbreviations significance level - D diffusion coefficient - i.v. intravenous - n number of experiments - PCO ₂ carbon dioxide partial pressure - pH negative logarithm of hydrogen ion concentration - PH ₂ hydrogen partial pressure - Po PH₂ with perfusion - P PH₂ without perfusion - PO ₂ oxygen partial pressure - PP perfusion pressure - r radius