Biochar is a carbon rich product made from the biomass pyrolysis process. Recently, biochar addition in anaerobic digestion processes has attracted attention for its possible functions to act as pH stabilizing agent, microbial carrier, and interspecies electron transfer. In this study, the effects of rice husk biochar addition in sorghum anaerobic digestion were investigated in batch tests. Under high F/M (food to microorganism) ratio conditions, obvious pH decreases and volatile fatty acids (VFA) accumulation was observed. Addition of 15 g/L biochar was found effective to increase the sorghum maximum methane production rate by 25 % and shorten the lag phase time by 44 %. Further biochar concentration increase showed little effects. Alkalinity increase brought by biochar addition contributed to the performance enhancement. Moreover, the promotive effects of biochar addition on degradation of acetic acid, propionic and butyric acid were not obvious under neutral pH conditions. Organic matters and fertility deficiency of the soil after decontamination were the main problems faced by these areas. For fertility recovery and efficiently use of sterile land, a feasible solution is to cultivate energy crops which could serve as green manure as well as energy resources. Among lots of crops, sorghum is characterized with high biomass yield and desirable chemical composition to be converted to bioenergy . The rhiAnaerobic digestion is one of the most efficient treatment approaches for different kinds of waste biomass such as sewage sludge, livestock manure, food waste and agricultural waste ,4. NowadHigh lignocellulose biomass such as sorghum is formed of recalcitrant structure consisting cellulose, hemicellulose and lignin, which become an obstacle of hydrolysis process during anaerobic digestion . DiffereHowever, the application of biochar addition on high lignocellulose biomass is rarely reported. In this study, the effects of F/M ratios on sorghum anaerobic digestion, and the role of biochar addition under high F/M ratio were investigated by batch experiments.22.1Harvested sorghum was firstly dried at 50 ℃ for 2 days and then crushed to the size of less than 1 cm before the experiments. The characteristics of the prepared sorghum are shown in 2.24OAc (pH 7) and determined by inductivity coupled plasma optical emission spectrometer (ICP-OES).The biochar was produced by rice husk pyrolysis  under 600 ℃ and the main characteristics of the biochar used are shown in 2.3Batch experiments were conducted to investigate the roles of biochar addition in high loading sorghum fermentation. Different F/M ratios were tested to simulate different loading conditions and the effects of biochar addition were investigated under high loading conditions.2, CH4 and CO2 were analyzed by a gas chromatography  equipped with TCD detector. The GC column used was a stainless packed column . Liquid samples were taken for pH and VFA analysis. pH was analyzed by a compact pH meter  and VFA was analyzed by a gas chromatography  equipped with FID detector. A capillary column  was used for VFA detection. Each test was conducted in duplicate.Firstly, to evaluate the effects of substrate induced inhibition on sorghum anaerobic digestion, batch experiments were conducted at F/M ratios of 0.1, 0.3, 0.6, 0.9, 1.2, 1.8, 2.4. For each test, 50 mL of seed sludge, corresponding amount of sorghum at set F/M ratio based on VS concentrations, and 30 mL of deoxygenated water were added in serum vials. Then the serum vials were sealed with rubber stoppers and aluminum caps. The head space of the serum vials was purged with pure nitrogen gas to obtain anaerobic condition. The vials were set at a shaker (120 rpm) and cultivated in a thermostat (35℃). The gas production volume was determined by syringe and gas components of NAfter previous experiments, a F/M ratio high enough to induce inhibition was determined. Different biochar addition concentrations were selected to verify its effects on sorghum anaerobic digestion. The experiments were conducted following the same process described above except for extra biochar addition. The biochar addition concentration was set at 0, 5, 10, 15, 20 g/L based on the seed sludge volume. The main VFAs detected during the high F/M ratio tests were then used directly as substrate to investigate the role of biochar addition in degradation of these VFAs by batch tests. For each vial, 50 mL of seed sludge, and 30 mL of VFA solutions were added and operated as the same procedure described above. The pH of the VFA solutions were adjusted to 7.0–7.5 by adding 1 M NaOH solutions. The concentration of VFAs in the vials were set at 2000 mg-COD/L. Biogas production, pH and VFA concentration variation were monitored until the biogas production almost stopped.2.40 represents the methane production potential (NmL), Rmax represents the maximum methane production rate (NmL/gVSadded/d), t0 represents the lag phase time (days) and t represents the reaction time (days).Gompertz equation was widely used in the evaluation of biomass anaerobic digestion for its capability to determine the methane production potential, methane production rate and lag phase time before the rapid methane production . In this33.12 > 0.93. With the increase of F/M ratio from 0.1 to 2.4, the methane production potential increased linearly from 18.5 to 540 NmL-CH4/gVS/d , which indicated the sorghum VS degradability to be around 45 %. The VS reduction of lignocellulose biomass such as sorghum was relatively low because of the recalcitrant structure and lignin content. Nevertheless, the methane yield and VS reduction could also vary as a result of different between substrate and inoculum. Generally, methane yield of 200–330 NmL/gVS with VS reduction rate of around 50 % from sorghum was reported in the previous studies [By using eq. (4), the methane production potential from complete sorghum degradation is around 504 N mL/gVS. Due to the actual methane production potential simulated by Gompertz equation, methane production potential for different groups was almost the same  while the lag phase decreased from 2.67 to 1.49 days (44 % decreased). However, further increase of biochar concentration from 15 to 20 g/L showed almost no effect on both Rmax and lag phase time  functions between acidogenesis and methanogenesis microbes to relieve the VFA accumulation ,27. But During anaerobic digestion of sorghum, acetic acid, propionic acid, and butyric acid were the main VFAs detected to accumulate under high organic loadings. Effects of biochar addition on the conversion of these VFAs were tested in batch tests . Unlike 2. The methane production almost finished in 2.7 days and acetic acid concentration decreased to 0 at the same time. Almost no difference was observed between the groups with and without biochar addition. Propionic and butyric acids are commonly considered difficult to be oxidized because of thermodynamically unfavourability without the syntrophic hydrogen consuming of methanogens [Methane production from acetic acid degradation was the fastest in the beginning as methanogens could directly convert acetic acid to methane and COhanogens . MoreoveGeobacter species and Methanosaeta, which need to be cultivated after an acclimation period of biochar addition. In the condition of anaerobic digestion system under proper pH condition and with healthy methanogens community, promotive effects of further biochar addition on VFA conversion were limited.The role of biochar as stabilizing agent for anaerobic digestion has been widely reported . The pH 4•Lag phase increased significantly along with F/M ratio increase and F/M ratio higher than 1.2 induced obvious pH decrease and VFA accumulation.•The biochar addition of 15 g/L was found effective to increase the sorghum maximum methane production rate by 25 % and shorten the lag phase time by 44 %. Further biochar concentration increase showed little effects.•The promotive effects of biochar addition on degradation of acetic acid, propionic and butyric acid were limited under neutral pH conditions.In this study, the role of rice husk biochar addition on sorghum anaerobic digestion under high loading conditions was investigated and key findings could be summarized as follows:Haiyuan Ma: Writing - original draft, Writing - review & editing, Investigation. Yong Hu: Methodology. Takuro Kobayashi: Funding acquisition, Supervision. Kai-Qin Xu: Supervision, Writing - review & editing.We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.


Acipenser dabryanus) trained with natural and formula diets at both the prerelease and postrelease stages. The results showed that (1) the gut microbial communities of the individuals trained with a natural diet  and formula diet  evolved to the optimal status for their corresponding diet provisions, (2) the individuals in the natural diet group paid a lower cost  during diet transformation and release into the natural environment than did the individuals in the formula diet group, and (3) the gut microbes in the natural diet group better supported postrelease fitness and survival than did the gut microbes in the formula diet group. The results indicated that better prerelease diet training with more appropriate training diets and times could improve the reintroduction of Yangtze sturgeon by adjusting the prerelease gut microbial community. Because a relationship between diet (preference) and gut microbes is common in animals from insects (such as Drosophila melanogaster) to mammals (such as Homo sapiens), our hypothesis verified by the case study on Yangtze sturgeon applies to other animals. We therefore encourage future studies to identify optimal training diets and times for each species to best adjust its prerelease gut microbial community and then improve its postrelease fitness and survival in reintroduction projects.As one of the most important tool for biodiversity restoration and endangered species conservation, reintroduction has been implemented worldwide. In reintroduction projects, prerelease conditioning could effectively increase postrelease fitness and survival by improving animals’ adaptation to transformation from artificial to natural environments. However, how early-life diet training affects individuals’ adaptation, fitness, and survival after release remains largely unknown. We hypothesized that early-life diet training would adjust the host’s gut microbial community, the gut microbial community would influence the host’s diet preference, and the host’s diet preference would impact its adaptation to diet provision transformation and then determine postrelease fitness and survival. To verify this hypothesis, we investigated the growth characteristics and gut microbes of Yangtze sturgeon ( With increasing human activities, biodiversity has decreased globally , which hReintroduction projects require viable source populations for release, derived from either wild or captive-bred populations . HoweverTraining captive-bred prerelease animals with natural diets could result in increased postrelease survival . WhitesiAcipenser dabryanus) is a critically endangered endemic species and a flagship species in the Yangtze River , when the embryonic yolk was completely absorbed. Before feeding, the earthworms were soaked in 2–5 ppm potassium permanganate for 3–5 min, irradiated with UV light for 0.5 h and then minced to an appropriate size for the larvae. When the larvae grew to a full length of 25–35 mm (approximately 23 dph), one group  continued to feed on earthworms, while the other group  was gradually transitioned to an artificial formula diet. The duration of the transition period from earthworms to the artificial formula diet was approximately 15 days. Under laboratory rearing conditions, the two treatment groups were fed four times daily , fully aerated tap water was used for circulation, and the water temperature and dissolved oxygen content were controlled at 19 ± 0.5°C and 6.5–8.5 mg/L, respectively. Because captive-bred juveniles of Yangtze sturgeon were released in the same year as part of the reintroduction project, 60 individuals of each group were selected randomly, marked, and then released into a natural pond at 7 months post hatching (mph) and recaptured 2 months later .We randomly sampled individuals from each group five times: larval stage (1 mph), early juvenile stage (2 and 3 mph), and juvenile stage (7 and 9 mph). At the larval stage, the formula diet group was transitioned from earthworms to the formula diet. We measured the body length and weight of the sampled individuals. All sampled individuals were euthanized with an overdose of MS 222  before dissection, and then, approximately 0.5 g of the hindgut contents was extracted aseptically. The contents from multiple individuals were pooled into one sample, especially for the small individual samples. At least three replicates per group were collected at each sampling time, and the samples were flash-frozen in liquid nitrogen and then stored at −80°C until DNA extraction. The experimental procedure is conceptually outlined in ® Stool DNA Kit  according to the manufacturer’s protocols. Then, the final DNA concentration and purity were determined by a NanoDrop 2000 UV-vis spectrophotometer , and DNA quality was checked by 1% agarose gel electrophoresis. The V3–V4 hypervariable regions of the bacterial 16S rRNA gene were amplified with the primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) by using a PCR thermocycler system . The PCRs were conducted using the following program: 3 min of denaturation at 95°C; 27 cycles of 30 s at 95°C, 30 s for annealing at 55°C, and 45 s for elongation at 72°C; and a final extension at 72°C for 10 min. PCRs were performed in triplicate 20 μl mixtures containing 4 μl of 5 × FastPfu Buffer, 2 μl of 2.5 mM dNTPs, 0.8 μl of each primer (5 μM), 0.4 μl of FastPfu Polymerase, and 10 ng of template DNA. The resulting PCR products were extracted from a 2% agarose gel, further purified using an AxyPrep DNA Gel Extraction Kit  and quantified using QuantiFluorTM-ST  according to the manufacturer’s protocol.Microbial DNA was extracted from gut samples using an E.Z.N.A.1, and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by the RDP Classifier algorithm2 against the Silva (SSU128) 16S rRNA database using a confidence threshold of 70%. The raw data have been deposited in the CNSA3 of CNGBdb with accession number CNP0000907.Purified amplicons were pooled in equimolar amounts and subjected to paired-end sequencing (2 × 300 bp) on an Illumina MiSeq platform  according to standard protocols by Majorbio Bio-Pharm Technology Co., Ltd. . Raw fastq files were demultiplexed, quality-filtered by Trimmomatic, and merged by FLASH. Operational taxonomic units (OTUs) were clustered with a 97% similarity cutoff using UPARSE (version 7.1)The samples of both groups, including individuals living in the artificial environment and recaptured individuals, from five stages  were delineated into 10 subgroups. Alpha diversity analysis, including analysis of the Chao richness index, Shannon diversity index, and Simpson diversity index, was conducted to reveal the variation in all gut microbial samples. Beta diversity analysis, including hierarchical clustering, sample distances, and non-metric multidimensional scaling (NMDS) analysis, was conducted to reveal the similarity of samples based on their gut microbial communities. The gut microbial community of each subgroup was analyzed at the family level. Analysis of the common OTUs, species, genera, and families between adjacent developmental stages of each group was performed to examine the drift of gut microbial communities with diet training by the natural and formula diets. Analysis of the common OTUs, species, genera, and families between the two groups at each developmental stage was performed to examine the gut microbial community divergence between the natural diet group and the formula diet group. Analysis of the common OTUs, species, genera, and families between each stage and the recaptured stage of each group was performed to examine the fitness of gut microbial communities for release. Then, based on these data sets, (1) lines of best fit for fitness at the OTU, species, genus, and family levels were generated for each group using a polynomial fitting method, and (2) the drift of gut microbial communities was outlined.P < 0.01 and P < 0.05). Then, these significant differences vanished as the individuals of the formula diet group adapted to the formula diet at the juvenile stage (7 and 9 mph) (P > 0.05). After living in the natural environment for two months (9 mph), the recaptured individuals of both groups exhibited significantly lower growth characteristics than the individuals living in the artificial environment. The recaptured individuals of the natural diet group showed significantly higher growth characteristics than the recaptured individuals of the formula diet group (P < 0.05).The growth characteristics of the two groups are shown in Moreover, after 2 months of 120 individuals being released into a natural pond, 37 individuals with natural diet group labels and 32 individuals with formula diet group labels were recaptured. The recapture rate of the natural diet group was 61.67%, and that of the formula diet group was 53.33%. In other words, the survival rate of the natural diet group was 61.67%, and that of the formula diet group was 53.33%.A total of 1,406,433 clean sequences were obtained from 38 samples of the two groups, and the average length of these sequences was 437.70 bp. A total of 2165 kinds of bacterial OTUs were detected  among these sequences, which belonged to 39 phyla, 84 classes, 192 orders, 351 families, 738 genera, and 1202 species  of the gut microbial communities for release in both the natural and formula diet groups declined continually from 1 to 3 mph, followed by some recovery at 7 mph . The fitThe continually increasing divergence of gut microbial communities between the natural diet group and the formula diet group from 1 to 7 mph in the artificial environment  indicateDiet training changed the gut microbial communities of Yangtze sturgeon individuals. It is well known that diet impacts the types of microorganisms that colonize the gut . GeneralDrift of the gut microbial communities influenced Yangtze sturgeons’ preference for a natural diet or formula diet. Gut microbes can impact hosts’ diet preferences and then shape host fitness-related behavior . In the Prerelease diet preference impacted the postrelease survival rates and fitness of Yangtze sturgeon. After more than five months of adjustment to diet transformation, at 7 mph, there was no significant difference in growth characteristics between the natural diet group and the formula diet group , which iTo adapt to the natural environment, the formula diet group paid a higher cost for transforming the prerelease gut microbial community into a new optimal assemblage than did the natural diet group. As the gut microbial community impacted host diet preference, to match the diet provision in the natural environment, the gut microbial communities of released individuals needed to gradually evolve into a new optimal assemblage from the prerelease gut microbial community. Because the natural diet was more similar to the diet provision in the natural environment than was the formula diet, the similarity (coverage) of gut microbial communities between prerelease individuals and recaptured surviving individuals was higher in the natural diet group than in the formula diet group . This reBetter diet training is possible if better diets are chosen with suitable training times. Because the diet provisions in the artificial environment and the natural environment were different, after being released into the natural environment, all individuals needed to adjust their gut microbial communities and diet preferences in order to adapt to the new diet provision. Then, there was an obvious difference in the gut microbial community between prerelease individuals and recaptured individuals . The posDrosophila melanogaster) to mammals (such as Homo sapiens)  , our hypStarvation is a significant source of mortality in captive-bred carnivores after release, and prerelease diet training could improve short-term postrelease survival. Understanding the mechanism of diet training effects is crucial for successful reintroduction projects. In this manuscript, we explore the mechanism by which diet training influences short-term postrelease fitness and survival. Diet training adjusts host gut microbes, which then impact host diet preference. Prerelease diet preference impacts host postrelease fitness, which then impacts postrelease survival rates. Thus, we propose that better diet training leads to a more suitable gut microbial community for release, followed by better postrelease fitness and ultimately better reintroduction effects. Better diet training of a prerelease source population requires (1) the use of better diet assemblages that are similar to the diet provision in natural environments and (2) a more appropriate training time that is long enough to ensure that the host gut microbes form a suitable assemblage for the natural environment.https://db.cngb.org/cnsa/) of CNGBdb with accession number CNP0000907.The datasets generated for this study can be found in the CNSA (The animal study was conscientiously abide by the ethical principles of animal welfare in People’s Republic of China and Hubei Province, accepted the supervision and inspection of the Animal Experimental Ethical Committee of Laboratory Animal Centre, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences.HY contributed to the conceptualization, formal analysis, visualization, writing the original draft, and reviewing and editing the manuscript. XL worked on the methodology, investigation, data curation, formal analysis, conceptualization, funding acquisition, project administration, supervision, validation, and reviewing and editing the manuscript. HD was responsible for the conceptualization, funding acquisition, project administration, supervision, validation, and reviewing and editing the manuscript. JL and JW carried out the investigation. QW  was responsible for the project administration, resources, supervision, and validation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Supplemental Digital Content is available in the text Development of white matter hyperintensities (WMH) in the brain is associated with blood thrombogenicity in recently menopausal women. This study examined the influence of menopausal hormone treatments (MHTs) on this association.Measures of blood thrombogenicity were examined in women of the Kronos Early Estrogen Prevention Study (n = 95) who had brain magnetic resonance imaging before and during the 48 months of randomization to transdermal 17β-estradiol (n = 30), oral conjugated equine estrogen (n = 29) both with progesterone for 12 days per month or placebo pills and patch (n = 36). Principal components (PCs) analysis was used to reduce the dimensionality of 14 markers of platelet activation and blood thrombogenicity. The first 5 PCs were assessed for association with treatment and changes in WMH. Within-person slopes were obtained to capture the extent of WMH change for each woman.P = 0.044). The partial effect of PC1, representing an average of six thrombogenicity variables  on WMH was significant (P = 0.003). PC3, reflecting a contrast of platelet microaggregates and adenosine triphosphate secretion versus total platelet count, differed across groups (P = 0.006) with higher scores in the oral conjugated equine estrogen group. The global association between PCs and WMH increase, however, did not differ significantly by MHT (P = 0.207 for interaction between MHT and PC's).WMH increased in all groups over the 48 months (In recently menopausal women, the type of MHT did not significantly influence the association of markers of blood thrombogenicity with development of WMH in the brain. Women were excluded if they had a coronary artery calcium score of more than 50 Agatston Units, smoked over 10 cigarettes per day, had body mass index more than 35 kg/m2, had a history of cardiovascular disease, or had low-density lipoprotein (LDL) cholesterol higher than 190 mg/dL, triglycerides higher than 400 mg/dL, diagnosis of diabetes, uncontrolled hypertension (systolic blood pressure >150 mm Hg and/or diastolic blood pressure >95 mm Hg) or current or recent (6 months) use of cholesterol lowering medications . Women were randomized to: oCEE ; transdermal tE2 ; or PBO pills and patch. Micronized progesterone was given orally  for 12 consecutive days each month to both active MHT groups.11 The study was approved by the Mayo Clinic Institutional Review Board and all participants gave written informed consent.Women enrolled in an ancillary MRI study of the KEEPS (NCT000154180) at Mayo Clinic were eligible for this study. KEEPS was a double-blind, placebo-controlled study to determine the effects of two different hormonal treatments on progression of atherosclerosis defined by increases in carotid intima-medial thickness in recently menopausal women.17Women underwent MRI at baseline before randomization, and at 18, 36, and 48 months for measurement of WMH volumes on fluid-attenuated inversion recovery sequences as previously described.18 Platelet activation using this technique and as measured by surface expression of P-selectin and fibrinogen receptors is less than 5%.19 Collections occurred at baseline before randomization to study treatments and at each study time point.18 Total cholesterol, LDL cholesterol, and high-density lipoprotein cholesterol, triglycerides, blood glucose, and 17β-estradiol were measured by Kronos Science Laboratories  and the Mayo Clinic Department of Laboratory Medicine and Pathology .Women were asked to refrain from aspirin 2 weeks before blood collection. Fasting venous blood was collected into a syringe through a 19 gauge butterfly needle and dispensed into plastic tubes containing anticoagulants needed for each assay and maintained at 33°C until processed within 30 minutes.20 Expression of activated platelet membrane P-selectin and glycoprotein IIb/IIIa complex binding to PAC-1 antibody (an indirect measure of the expression of membrane fibrinogen receptor) was measured by validated flow cytometry techniques.21Platelet count was determined by Coulter counter, as previously described.22Total numbers of thrombogenic (phosphatidylserine-positive defined by annexin V binding) MV and other MV staining positive for selected cell-specific markers were measured using fluorophore conjugated recombinant proteins and antibodies by flow cytometry.Data reduction methods were used to achieve some degree of parsimony in these analyses so that the complexity could be reasonably supported by the sample size. Analysis of longitudinal WMH data was performed with a two-stage approach to first characterize the time-response profile and then assess differences in profiles across treatment. The goal of the first stage is to adequately summarize the repeat measurements into single WMH responses per person. For this, slope coefficients were computed using least squares regression in fitting each woman's time-response data with a linear equation. To minimize the impact of skewed values, we took the logarithm of WMH, with a constant of 1 added to the raw value, before modeling. One-sample Wilcoxon signed-rank tests for zero slopes were used to test for a significant change in WMH over 48 months separately in each group. The slopes were then assessed for treatment difference using the proportional odds ordinal logistic model, which provides a generalization of the Kruskal-Wallis test for pairwise testing. The results of the two-stage approach were compared with generalized least squares in which all the serial log-transformed data were analyzed jointly in a single model, and the correlation of the repeat measurements taken into account.P values are computed from the likelihood ratio χ2 statistic for the model that is due to the individual or multiple variables of interest, with the exception of pairwise treatment comparisons which are based on approximate Wald χ2 statistics.For each of the 14 cellular activation markers, repeat measurements on the same person were averaged across visits, and the resulting mean values were converted to normal scores based on their ranks. Scored dimensions of platelet reactivity and MVs were derived using principal component (PC) analysis on the 14 transformed variables. The multiple PCs were carried forward and analyzed for association with three-level treatment in multinomial logistic regression model (an extension of the binary logistic model for >2 unordered outcome categories), and for association with WMH response in a proportional odd ordinal logistic regression model while adjusting for randomized treatment group. 12 did not differ across treatment group assignments except for smoking status , with pairwise comparisons indicating greater increases in oCEE than in PBO (P = 0.011). Results from repeated measures modeling  were in reasonable agreement with the summary measure analysis, supporting the validity of the simpler two-stage approach, and with differences between oCEE and PBO reported in the original analysis (Tables 1 and 3 of reference 17).Baseline characteristics of the 95 participants, a subset of the 118 KEEPS participants at the Mayo Clinic site for whom WMH data were availableThe average of each of the 14 intravascular cellular activation markers (6 platelet reactivity variables and 8 MV variables) over treatment follow-up is shown by treatment group inTable P = 0.059), with partial tests revealing an overall group difference for PC3 (P = 0.006). PC3 represents a contrast of platelet microaggregates, adenosine triphosphate secretion, basal expression of P-selectin, and fibrinogen receptor complex (PAC-1 binding) versus total platelet count and numbers of leukocyte-derived MV. The composite scores in oCEE were marginally to significantly higher compared to other groups .A global test of association with treatment for these five PCs approached significance (P = 0.104). However, of the individual components PC1 reflecting MV positive for expression of tissue factor, intracellular adhesion molecule 1 and vascular cell adhesion protein 1, and MVs derived from leukocytes and monocytes showed the most prominent effect (P = 0.003). This finding indicates that, after controlling for treatment, the higher the composite score for PC1 the greater the rate of increase in WMH. Also from this model, the association between treatment group and WMH increase persisted after adjustment for PC variables (P = 0.009). Based on the global test of interaction on 10 degrees of freedom, there was no evidence (P = 0.204) that the overall association between PCs and WMH increase differed by MHT 27 and cerebral microvasculature affecting development of WMH.Factors influencing generalized inflammation and, indirectly, blood thrombogenicity, include conventional cardiovascular risk factors such as age, blood pressure, hyperlipidemia, insulin resistance, and life-style choices such as diet, activity, and smoking. In KEEPS, the conventional cardiovascular risk factors such as body mass index, blood pressure, triglycerides, HDL and LDL, glucose, and smoking status, however, did not associate with WMH at 48 months, which is consistent with other studies.P < 0.05 level. The PC3 that represented a contrast of platelet reactivity measures, however, differed significantly in the oCEE compared to the tE2 or PBO groups. This result is consistent with previous findings of significant differences in platelet functions between tE2 and oCEE groups.15 Effects of various genetic variants on the responses to treatment might also have masked potential treatment effects as genetic variants associated with metabolism and uptake of estrogen, with innate immunity, and with APOE ε4 was observed for MHT effects measured by differences in chronological age for onset of menopause, in carotid artery intima-medial thickness, and deposition of β-amyloid in the brain.32 In spite of these effects, after controlling for treatment, the overall association of the five PCs with increase in WMH reflected the strong positive correlation between PC1 score and WMH increase. Taken together these results suggest that both MHT and the composite of the MV measurements explaining PC1 show an independent effect on development of WMH.In the present study, the overall association between treatment and the five PCs describing a number of cellular activity measures did not reach statistical significance at the There are several limitations of this study that should be considered. First, the results may not be applicable to the general population as the KEEPS enrolled recently menopausal women within a relatively narrow age range. In addition, these women were predominantly white, healthy, educated, and most were nonsmokers. The advantage of this homogenous population is that the findings may, however, reflect general physiological processes that are not confounded by manageable cardiovascular risk factors. Second, the influences of the MHT used in KEEPS on development of the WMH may not apply to other doses or formulations of MHT used in other studies. Third, the overall association between PCs and WMH increase did not apparently differ by MHT. However, the relatively small sample in our study may have limited the power to detect such a difference.33 Activation of blood platelets, endothelium, and monocytes associated with development of WMH are most likely multifactorial including synergistic effects of conventional risk factors such as age, blood pressure, and components of metabolic syndrome. In addition, other potential sources of platelet and cellular activation such as effects of natural menopausal aging processes, adverse pregnancy histories, commensal infections, and comorbid inflammatory conditions and behaviors could have additive effects. Specific mechanisms by which these activated cells and MV affect cerebral microvascular function leading to formation of WMH remain to be determined.The findings of the present study are consistent with those of other investigations that implicate thrombogenicity of the blood and inflammation as contributors to development of WMH.


With optimal hydration, most of the tested OMA lines, compared to oat cv. Bingo, showed higher pigment content, and some of them were characterized by increased values of selected CF parameters. Although 14 days of drought caused a decrease of chlorophylls and carotenoids, only slight changes in CF parameters were observed, which can indicate proper photosynthetic efficiency in most of examined OMA lines compared to oat cv. Bingo. The obtained data revealed that expected changes in hybrid functioning depend more on the specific maize chromosome and its interaction with the oat genome rather than the number of retained chromosomes. OMA lines not only constitute a powerful tool for maize genomics but also are a source of valuable variation in plant breeding, and can help us to understand plant susceptibility to drought. Our research confirms more efficient functioning of hybrid photosynthetic apparatus than oat cv. Bingo, therefore contributes to raising new questions in the fields of plant physiology and biochemistry. Due to the fact that the oat genome is not fully sequenced yet, the mechanism of enhanced photosynthetic efficiency in OMA lines requires further research.The oat × maize chromosome addition (OMA) lines, as hybrids between C3 and C4 plants, can potentially help us understand the process of C4 photosynthesis. However, photosynthesis is often affected by adverse environmental conditions, including drought stress. Therefore, to assess the functioning of the photosynthetic apparatus in OMA lines under drought stress, the chlorophyll content and chlorophyll  Avena sativa L. 2n = 6x = 42) and maize (Zea mays L. 2n = 2x = 20) are the most remotely related plant species of which we are aware that can be sexually hybridized and produce stable fertile partial hybrids [loci on maize chromosome 9 encoding this trait are differentially silenced depending on their interaction with the oat genome or that they interact differently with specific oat alleles. Their study showed that C4 plants also had larger parenchymal bundle sheath cells that contain more chloroplasts than those of C3. OMA lines possessing maize chromosome 1, 2, 3, 7, 8 and 9 had significantly larger cell areas than the oat parental lines. Moreover, the presence of maize chromosome(s) often leads to morphological  and physiological  abnormalities, the nature of which depends on the specific maize chromosome and oat genotype [Oat . In the present study we analyzed the chlorophyll content and chlorophyll Grande-1  amplified with primers specific for the maize retrotransposon  X97604; ). CopiesAvena sativa L. chromosomes (Genomic in situ hybridization (GISH) with maize gDNA as a probe was performed on OMA lines in order to verify the number of added chromosomes A–E. Oat omosomes F. All ofn = 46 (42 + Zm1″ + Zm2″)) (n = 44 (42 + Zm2″)) (n = 43 (42 + Zm3′)) (n = 44 (42 + Zm5″)) (The number of detected maize chromosomes differed between the OMA lines . Identif+ Zm2″)) A. Line I+ Zm2″)) B. Line I+ Zm3′)) C. Line I+ Zm5″)) D and lin+ Zm5″)) E.a, b, carotenoids) and selected chlorophyll a fluorescence parameters.The functioning of the photosynthetic apparatus was assessed on the basis of the total chlorophyll content (SPAD), the content of photosynthetic pigments (chlorophyll a fluorescence parameters (v/Fm) and energy dissipated from PSII (DI0/CS).The analysis of variance showed significant differences between tested oat plants with optimal water supply in terms of the content of total chlorophyll and photosynthetic pigments, as well as most of the chlorophyll rameters . There wa (Chla), chlorophyll b (Chlb), and carotenoids (Car). Among examined OMA lines, three of them had greater pigment content: line II, IV (with two preserved maize chromosomes) and V (with preserved maize chromatin). Line I (with four preserved maize chromosomes) showed intermediate values between cv. Bingo and other OMAs.The chlorophyll concentration in SPAD units showed differences between tested lines . The higa fluorescence were selected (v/Fm (a fluorescence induction curve (Area) (0/CS) (0/CS) (0/CS) (0)  B, which e (Area) D, as wel) (0/CS) E. These ) (0/CS) F while s) (0/CS) G, althou/CS) (0) H. Line Ib and Car, while term of measurements had no impact on Chla/b ratio. The interaction of the genotype with the treatment turned out to be significant only in Chla/b ratio. The interaction of treatment with term and the interaction of genotype with treatment and with term were significant for all pigments.The analysis of variance showed significant differences in the relative water content in leaves (LRWC) only in terms of applied treatment (control or drought), regardless of plant genotype . HoweverTo verify the impact of drought stress level on tested plants, the leaf relative water content (LRWC) was measured. The applied 14-day drought caused a decrease in the LRWC value in most of the tested lines as well as cv. Bingo . CompareThe chlorophyll concentration in SPAD units did not show significant differences in the majority of the tested oat lines regardless of the plant developmental phase in which the SPAD readings were taken . Howevera and Chlb content was observed in lines I and II by approx. 60% and 50%, respectively, and in line V by approx. 40%. Similar trends were shown in the content of Car., whereas 14-day drought treatment caused a decline by 20% in the Chl content in the leaves of two tested lines IV and V, which was maintained during further plant growth only in line V. In turn, in line II a decrease in the content of photosynthetic pigments was noted only in the kernel milk maturity phase and it declined by about 47% of Chla, 58% of Chlb, and 35% of Car. In this phase, a significant decrease in the above-mentioned pigments was also observed in line V and it oscillated around 20% compared to the control conditions.The content of photosynthetic pigments  did not a fluorescence parameters showed significant differences between tested oat plants under drought conditions in terms of both single traits and their interaction  was observed, as well as a decrease in thermal energy dissipation in the PSII reaction centers (DI0/CS). However, drought had less of an impact on the electron transport through PSII (ET0/CS), which may indicate a certain adaptability of the tested lines to adverse conditions. In addition, in most cases there were no significant changes in the number of active reaction centers in the excited leaf fragment (RC/CS0), while their amount increased in the relaxed state (RC/CSm). The only exception from this rule was line I in the panicle ejection phase.The values of chlorophyll fluorescence parameters during plant development were characterized by some variation between the tested lines. An increase in the F drought . Furthera fluorescence parameters could distinguish the treatment of the plants as they are all together compared for all the parameters. Acute angle flanked by the parameters means positive correlation, whereas an obtuse angle is negative, and right angle means no correlation. Both PCs  accounted for 72.67% of the total variation of the data set for chlorophyll a fluorescence parameters and 84.03% for photosynthetic pigments  indicated that the measured chlorophyll pigments A,B. PCA rameters A and 48.rameters B. The serameters A and phorameters B, respeca fluorescence parameters and photosynthetic pigments grouped tightly examined hybrids and cv. Bingo , number of side tillers  and the term of entering subsequent development phases . Among tested OMA lines only line I showed in a bushy type of growth, without a clearly formed main shoot . Moreovecis element in the alien maize chromosome [In oat and maize crosses, some maize chromosomes are not extruded during embryogenesis, but are ultimately stabilized and act as oat chromosomes in mitosis . Studiesromosome . Gene exromosome . A. sativa cv. Bingo. It is worth stressing that in the case of most of the analyzed CF parameters, OMA lines were characterized by higher values, which may confirm more efficient functioning of their photosynthetic apparatus. Their suggested predominance can be also reflected in the higher pigment content  compared to oat cv. Bingo. Furthermore, results for OMA lines also exceed the values obtained for doubled haploids (DH) of oat [n = 44 (42 + Zm5″)), showed higher values of most CF parameters. Interestingly, line II, which also comprises two maize chromosomes (2n = 44 (42 + Zm2″)), was characterized by lower photosynthetic efficiency compared to line IV. Therefore, an important issue seems to be not only the number of retained chromosomes but also the kind of introduced maize chromosomes as well as oat genome. Moreover, line I with four retained maize chromosomes (2n = 46 (42 + Zm1″ + Zm2″)) turned out to be less efficient in the context of the functioning of the photosynthetic apparatus. In our opinion, these results can confirm that preserved maize chromosomes can positively affect the photosynthetic apparatus but this effect relies not only on the amount but also on the number of retained maize chromosome. Nevertheless, we are aware that a greater number of repetitions would provide even more reliable results and possibly reveal differences that were not statistically significant in the conducted experiment. Therefore, further studies including additional OMA lines are planned to gather more insightful information on the hybrids’ functioning. Despite the OMA lines being valuable genetic materials, they are also used in the examination of expression of the C4 photosynthetic system ,9,10. Th) of oat . The greDrought is the most severe stress and the main cause of significant losses in growth and productivity of crop plants , inducinUnder drought stress, LRWC is proposed as a more important indicator of water status than other water potential parameters . A signiDespite the observed reduction in LRWC values we did not note such a clear response in the chlorophyll content under drought conditions. Chl degradation is listed as one of the consequences of drought stress that may result from sustained photoinhibition and photo-bleaching , even thb is greater than that of Chla which directly translates into Chla/b value [a/b occurred despite no significant changes in the content of individual Chl. Similar results were obtained in wheat, where a slight increase in Chla/b ratio in drought tolerant cultivars under water deficit conditions was observed [It is known that drought stress causes not only substantial damage to photosynthetic pigments, but also leads to deterioration of thylakoid membranes ,39. Thus/b value . In our observed . a fluorescence may be used as a direct indicator of photosynthetic activity giving a valuable insight into the exploitation of the excitation energy by PSII, and indirectly by other protein complexes of the thylakoid membranes [Chl absorbed light energy which is then transformed into Chl fluorescence (CF) . In vivoembranes . CF deliembranes . Therefoembranes ,53,54,55embranes . CF-modum, which probably may indicate a more efficient functioning of the photosynthetic apparatus in drought conditions despite the observed decrease in chlorophyll content in drought conditions. Our results revealed some changes in the PI parameter which is sensitive to changes in either antenna properties, trapping efficiency or electron transport beyond QA. The Fv/Fm ratio is the most commonly used CF parameter characterizing the maximum quantum yield of PSII, which in most higher plants under physiological conditions ranges from 0.78 to 0.84. In the presented study, its values were within the range indicated by the literature, regardless of the conditions of plant growth and the date of taken measurements. Furthermore, in oat DH lines after 14 days of drought, Fv/Fm did not differ significantly [v/Fm was not linked to an increase in yield as Liang et al. [v/Fm alteration as a symptom of adaptive or developmental changes. The direction of changes seems to depend on the level of stress because Souza et al. [v/Fm occurred only in case of severe drought stress.The presented results seem to indicate a different response to drought stress of examined lines, allowing us to divide them, based on the similarity of the value and direction of changes in CF parameters, into two groups: (a) line III and V, in which hardly any significant changes in most parameters were shown, and (b) other hybrid lines and cv. Bingo, characterized by a similar response to applied drought stress . Nevertheless, plants of the V line differed from line III with a higher PI and RC/CSficantly . In mostg et al.  reportedg et al. ,58. Its g et al.  which coa et al.  proved t0/CS and RC/CS0 showed only slight changes during plant development. An exception was line I, in which these values decreased in drought at panicle ejection phase. Therefore, reduced ET0/CS indicates that active reaction centers (RCs) are converted into inactive RCs, reducing the efficiency of trapping and a decline in PSII activity [0/CS, RC/CSm, as well as ABS/CS, TR0/CS, and PI, indicate better functioning of photosynthetic apparatus. The observed decrease of the ABS/CS ratio under drought stress in our experiment was possibly due to inactivation of some PSII RCs or a decrease in antenna size. In some of the tested OMA lines we observed an increase of RC/CSm which provides information about the number of active reaction centers. In our study most of the tested lines showed higher performance index (PI) values in drought condition which occurred with a simultaneous decrease of energy dissipated from PSII (DI0/CS). A similar relationship was observed in the oat DH lines [0/CS). Area, ETactivity . Accordiactivity  higher vDH lines . The relAccording to the literature data, OMA lines can be screened for the effects of individual maize chromosomes on photosynthesis in oat ,9,63. SiMorphological observations of the tested plants revealed some changes within OMA lines. Line I with four maize chromosomes manifested in bushy shoots and poor grain yield. A similar observation was obtained by Riera-Lizarazu et al.  who descAvena sativa L.) cv. Bingo and 5 selected OMA lines, DC 06011-6 × POB 722 (I), STH 9511 × Bingo (II), STH 8-50 × Canyon (III), STH 9110 × Contender (IV) and STH 93-61 × DC 2112/07 (V), which differed in the number of maize chromosomes added to the oat genome. The 10 plants per line/cultivar were grown in pots under an open-air vegetation tunnel protected from rain by gardening foil during the summer–autumn period (July–September). Plants were grown in conditions close to natural daylight and air temperature. The average temperatures recorded in these three months were 25 °C (32–19 °C), 20 °C (30–10 °C) and 15 °C (25–5 °C), while the air humidity was as follows: 70%, 75%, 80%. Oat seeds of cv. Bingo and the OMA lines were planted individually into 3 dm3 pots filled with soil composed of horticultural soil (loamy soil (85%) and loam (15%)) and sand (1:1 v/v). Drought stress was caused by withholding the watering of the soil as described by Marcińska et al. [The objects of research in this study were oat plants , Kraków, Poland. The experiments was conducted at The DNA extraction  and PCR analyses  were performed according to Skrzypek et al. . Two GraThe chromosome preparations were made using the methodology described by Jenkins and Hasterok . The rooFor GISH experiments, maize total genomic DNA (gDNA) was extracted from young leaves using standard C-TAB procedure and labelled with digoxygenin-11-dUTP  by nick-translation method according to the manufacturer instructions. In some experiments, 25S rDNA sequence labelled with tetramethylrhodamine-5-dUTP by nick translation was used as an additional chromosome marker. The GISH procedure followed the protocol described in Skrzypek et al.  with mod2. DNA isolation was performed using DNeasy Plant Mini Kit . The concentration of DNA was measured at 260 nm by NanoDrop 2000c  and its quality was checked on agarose gel. The added maize chromosomes in the tested OMA lines were identified with specific for maize SSR markers chosen from the Maize Genome Database (https://www.maizegdb.org) and described by [The total genomic DNA of OMA lines, oat cv. Bingo, and maize cv. Waza was extracted from ca. 0.8 g of fresh tissue frozen with liquid Nribed by . Leaf relative water content (LRWC) was determined on the 14th day of drought according to Ober et al.  using thThe chlorophyll concentration indicator in SPAD units was determined by a photometric method using a SPAD 502 device  based on the leaf greenness index. By measuring the wavelength of 650 nm, the amount of light absorbed by the chlorophyll was determined. In addition, light was measured at a wavelength of 940 nm which is absorbed by the rest of the structure. These values were used to correct the result calculated by the microprocessor and given in contractual units, called SPAD readings (with maximum at 60 units). SPAD readings are directly proportional to the chlorophyll content. The chlorophyll concentration indicator in SPAD units was measured on four terms: on the 1st, 14th day of drought, panicle ejection phase (PE), and kernel milk maturity phase (MM). The measurements were taken in the middle part of the second or third fully developed leaf (counting from the youngest leaf) from each independent plant.a and b and carotenoids were calculated using the following formula:a = chlorophyll a, Chlb = chlorophyll b, Car = carotenoids, A664 = absorbance at 664 nm, A648 = absorbance at 648 nm; A470 = absorbance at 470 nm. The concentrations was expressed in micrograms of a given pigment in 1 mL of extract.The content of photosynthetic pigments was determined on the 1st, 14th day of drought, panicle ejection phase, and kernel milk maturity phase by a modified spectrophotometric method according to Lichtenthaler and Wellburn . Leaf saa fluorescence kinetics parameters were conducted using a Handy PEA fluorometer . The measurements were carried out on the same leaves on which the chlorophyll concentration indicator in SPAD unit had been measured previously on the four terms mentioned above. The leaves’ fragments were shaded with clips for about 20 min and then the measurement was taken. It was initiated by illuminating the leaf fragment with saturated light (intensity of 90 units = 5400 μmol (quantum) m2s−1, illumination time 0.9 s) to determine the maximum fluorescence (Fm) and calculate the maximum photochemical potential (Fv/Fm). Then, actinic light was switched on automatically (25 units = 1500 μmol (quantum) m−2s−1) for a period of 270 s. When it was switched off, the leaf light was emitted by a diode emitting light in the far red range of about 15 W m−2. On the basis of chlorophyll a fluorescence measurements, the following parameters were calculated and analyzed: Fv/Fm—maximum photochemical efficiency of PSII; Area—area over the chlorophyll a fluorescence induction curve; PI—overall performance index of PSII photochemistry; ABS/CS—light energy absorption; TR0/CS—excitation energy trapped in PSII reaction centers; ET0/CS—energy used for electron transport; DI0/CS—energy dissipated from PSII; RC/CS0—number of active reaction centers in the excited leaf fragment; RC/CSm—number of active reaction centers in the relaxation state of leaf fragment.The measurements of the chlorophyll To estimate the impact of drought on plants, morphological observations were carried out and selected components of yield  were assessed.The results presented in figures constitute the mean values ± SE based on 5 plants as replicates per treatment. One-way and multivariate analysis of variance and Duncan’s multiple range test at the 0.05 probability level were used to determine the significance of differences between lines/cultivars, which were marked with different letters. Student’s t-test at the 0.05 probability level was also used to compare the average values for each line/cultivar. Principal component analysis (PCA) was done and presented as biplot. Data were analyzed using STATISTICA v. 13.1 software .Our studies have revealed the complexity of interspecific hybrid functioning and some of the consequences of preserving alien chromosome(s). Obtained results of CF parameters and pigment content can indicate that OMA lines may predominate oat cultivar in terms of photosynthetic apparatus functioning both in optimal and adverse conditions. We postulate that the functioning of the photosynthetic apparatus in OMA lines depends not only on the number of preserved maize chromosomes but on their interaction with a given oat genome. Although the whole oat genome sequence is currently unavailable, additional studies of the effects of maize chromosomes on oat transcriptome in OMA lines should be continued to explore the mechanism underlying their adjustment to adverse environmental conditions. Possibly, generating crop hybrids may contribute to solving the problem of further production level enhancement, which currently is rather limited. As ways to increase the yield potential, an improvement of photosynthesis and ascension of photosynthetic utilization are mentioned .


The availability of (co)variance components and genetics parameter estimatesfor traits included in a selection program is crucial since the estimatedbreeding values of the selected traits are computed based on the available(co)variance components and genetics parameters. The present study aimed toestimate (co)variance components and genetics parameters for linear traitsrelated to foot/leg and udder  in Holstein cattle in Indonesia. Linear traits were measured(instead of scored) on 310 lactating Holstein cows raised in the NationalBreeding Centre for Dairy Cattle and Forage of Indonesia (BBPTUHPTBaturraden). These were nearly all cows in lactation owned by the centre atthe time of study. Lactating cows which were not measured during study werethose which are technically difficult to handle. The Average InformationRestricted Maximum Likelihood (AI-REML) method of the DMU program was used to estimate the(co)variances and genetics parameters of the considered linear traits. Afour-multivariate animal model was employed by including farm (fixed), animal(random), and age (covariate) effects in the model of analysis. Thephenotypic means (standard deviation) for rear legs set, foot angle, udderdepth, and teat length were 139.70 (6.03), 50.65 (5.04), 10.67 (6.19), and5.27 (0.96), respectively. The results showed that the estimated heritability( BBPTUHPT Baturraden, which is themain government institution for genetics improvement of Indonesian dairycattle, serves the country to rear, produce, and improve genetically dairycattle, specifically Holstein cattle. The centre has focused its geneticsimprovement program on milk yield and its component traits. Inclusion oflinear traits or other traits in its genetics improvement program was notconsidered until recently.Milk yield and its components of dairy cows in general have been successfullyincreased through both genetics and management. Milk yield per lactation hasincreased dramatically in the last 40 years and some cows were reported to beable to produce more than 20 000 kg per lactation . The milk yield increase of 5997 kg from 1957 to 2007 is attributedmostly (56 %) to genetics improvement . Many studies haveshown that selecting dairy cows focusing merely on milk yield (and itscomponents) and ignoring other traits, especially linear traits, has resultedin declined performances in animals' health and reproduction performancesdespite the increased production traits.Rauw et al. (1998) reviewed quite extensively the opposite relationshipbetween high-production cows and health performance. The different directionrelationship between high-production cows and health expenses has beenreported from a classical selection study . Ingvartsen etal. (2003) reported the evidence of genetics correlation between milk yieldand the incidence of ketosis, ovarian cyst, mastitis, and lameness. Healthperformances also experienced a similar trend along with the successfulincrease in milk production due to the selection program. The relationshipbetween milk yield and risk of infection of mastitis is clear and if theanimals continue to be selected for high milk yield, the situation willworsen . Milk yield of previous lactation isassociated with retained placenta, mastitis, and milk fever .Lucy (2001) has shown the negative association between the successfulincrease in milk yield and reproductive performances over the last decades.Similar to what has happened in the US, first conception rate has decreasedin Ireland  and the United Kingdom .Despite an increased milk yield from 7800 kg to 10 200 kg within theperiod of 1991–2000, pregnancy rate and cyclicity have declined by up to6 % and 7.6 %, respectively, and incidence of inactive ovariesincreased by up to 8 % . This phenomenon wasaddressed to the presence of undesirable genetic correlation between milkyield and fertility traits  or due to both management andthe negative genetic correlation between those traits .Linear traits are correlated with some economically important traits inlivestock and, to some extent, the correlation is genetic. Selecting animalsbased upon a particular trait will affect the mean of the correlated traits.This is critical especially when the genetics correlation is in the oppositedirection, since the improvement in one trait will decrease the performanceof the other traits. The International Committee on Animal Recording  has recommended recording linear traits on a breeding program since thegenetics correlation is present between linear traits and production traits.Selection of traits that need to be recorded in a genetics improvementprogram needs to be done because it is inefficient and expensive to recordall traits included in the breeding objective. Genetics improvement of atrait can be achieved by including traits that are genetically correlated andeasier/cheaper to record in the selection program. Therefore the informationabout the (co)variance components and genetics parameters of alternativetraits of a population is important. (Co)variance components and parametersgenetics of linear traits of Indonesian Holstein cattle, to the best of ourknowledge, have not been reported. Thus the objective of this present paperwas to estimate the necessary genetics parameters of linear traits forHolstein cows in Indonesia.22.1Linear traits related to foot/leg and udder, including rear legs set, footangle, udder depth, and teat length, were reported in the study. Thecorresponding linear traits of Holstein cattle in Indonesia were measuredduring the months of July and August 2017 at the National Breeding Centre forDairy Cattle and Forage of Baturraden, Indonesia. As many as 310 lactatingdairy cows were measured for their linear traits. The udder depth trait wasmeasured about an hour before milking in the afternoon; animals are milkedtwice daily: early morning and afternoon. All animals were reared in twoseparated farms, namely Tegalsari and Limpakuwus farms. Animal age andpedigree data were obtained from the database available in BBPTUHPTBaturraden. Animals with an unknown date of birth were assigned as datamissing for the age variable and unknown parents were also assigned asmissing. All animals are reared under an intensive system in a free stallbarn where grass and concentrate are provided twice daily and water isprovided ad libitum.®. Foot angle was measured with aconventional angular measure; teat length was measured with a standard tapemeasure; udder depth was measured with a calliper. Rear legs set and footangle were both measured in angle degree unit  definition. Lack of certified classifiers for typing/scoring for theselinear traits inspired the author to measure the traits instead of scoringthem. Rear legs set, foot angles, and teat length were measured for both leftand right parts. Rear legs set was measured with the digital angle level ofNankai2.2The model used in the (co)variance analysis included farm (fixed), animalgenetics additive (random), and animal age (random-fixed covariate) effects.Multivariate genetics analysis including four traits at a time was chosen byemploying the equal design model for all traits being analysed. Themathematical model can be written as in Eq. (1):The standard errors of heritabilities and correlations between random effectsincluding genetic correlations are computed based upon a Taylor seriesapproximation .3It is quite a challenge to have reliable estimates of heritability and(co)variance components in developing countries since the pedigree data ofthe population are not well documented. Pedigree information as the basis ofthe variance structure of additive genetics of the animals is critical. TheDMU software allows the formation of a numerator relationship matrix inversewithout considering the inbreeding coefficient (option 2). Selecting option 2in DMU will command the program to create an approximation of the trueinverse of the additive relationship matrix. Phenotypic means and standarddeviation of the RLS, FA, UD, and TL are presented in Table 2. In the presentstudy, linear traits were measured instead of scored/classified, as opposedto most studies reported in the literature, for instance Toghiani Pozveh etal. (2009), Mazza et al. (2015), Wiggans et al. (2006), and Zink etal. (2012), and therefore comparing directly estimated parameters of thepresent study with those in the literature is not possible except for theratio between estimates such as correlations and heritabilities. Comparingthe estimated parameters with other studies was done cautiously after theestimated parameters obtained were converted into values of the 1–9 scalescoring system. However, phenotypic means of the observed traits werecompared with the 1–9 scale scoring system to get a general overview of thepopulation parameters under study compared to other studies.Table 2 shows that the phenotypic means of the observed linear traits areclosed to the intermediate score in a 1–9 scale scoring system according tothe reference scale of the World Holstein Friesian Federation .The intermediate scores of RLS, FA, UD, and TL according to the WHFF (2005)are 4–6, 4–6, 5, and 4–6, respectively. This indicates that the lineartraits of Indonesian Holstein cows have not undergone selection. The observedphenotypic means of the present study of RLS and FA were slightly higher thanthose reported by Toghiani (2011), whereas UD is about the same. Toghiani(2011) reported that the phenotypic means (standard deviation) of RLS, FA,and UD scored in a 1–9 scale scoring system are 5.4 An additive genetic coefficient of variation (CV) of 13 % has beenreported by Berry et al. (2004) for udder depth in primiparous dairy cows,whereas Fernández et al. (1997) reported a coefficient of variation of34.48 % for udder depth in dairy ewes. The extremely high phenotypiccoefficient of variation obtained from the present study, especially udderdepth (58.01 %), might indicate that the source of variation originatingfrom non-genetic factors, such as the field technician who measured thevariable, is dominant. Measuring udder depth of dairy cows kept in a freestall barn is quite challenging. Obtaining a large coefficient of variationof field data is possible and has been reported in the literature. Forinstance, Royal et al. (2002) reported a phenotypic coefficient of variationof commencement luteal activity (CLA) of 63 %. Samoré et al. (2012)reported CVs of 57 % and 62 % for somatic cell scores of Brown Swissand Holstein Friesian cattle, respectively.3.1The estimated genetic and phenotypic (co)variances of the present study arepresented in Tables 3 and 4. The two tables show that (co)variances, bothgenetically and phenotypically, were observed in the population, and theyvaried in magnitude. Comparing the magnitude of (co)variances obtained in thepresent study with results from studies using the 1–9 scale scoring system,for instance, is irrelevant and thus avoided. The discussion is thereforeemphasized on the variance ratio (i.e. heritabilities and correlations). Theestimated heritabilities along with the standard errors are shown in Table 5.It is shown in Table 5 that rear legs set, foot angle, udder depth, and teatlength of dairy cattle in Indonesia are heritable traits, though themagnitudes of the estimates varied. The estimated heritabilities ranged from0.147 for TL to 0.334 for RLS. These results were different compared to thosereported in the literature. For example, Pérez-Cabal and Alenda (2002)reported heritabilities for RLS, FA, and UD of 0.17, 0.11, and 0.24,respectively. Dal Zotto et al. (2007) reported heritabilities of 0.14, 0.23,and 0.32 for RLS, UD, and TL, respectively, on Brown Swiss cattle. Estimatedheritabilities of 0.19, 0.16, 0.30, and 0.33 for RLS, FA, UD, and TL,respectively, were reported by Pryce et al. (2000). From a study of Holsteincattle in Brazil, Campos et al. (2015) reported heritability estimates of0.21, 0.09, 0.25, and 0.38 for RLS, FA, UD, and TL, respectively. Theheritability estimates could be different from study to study since theestimates are a function of additive genetic and non-genetic variances. Thehigher heritability estimate reflects the higher proportion of additivegenetic variance to the total variance. Results of the present study showthat the linear traits under study are heritable and a significant selectionresponse might be obtained when included in a selection program. The standarderror of all heritability estimates of the present study are slightly larger(0.066–0.181) than those reported in the literature, except for udder depth,which has a standard error for heritability of 0.066. For instance, Pryce etal. (2000), Pérez-Cabal and Alenda (2002), Dal Zotto et al. (2007), andCampos et al. (2015) reported standard errors of heritabilities of 0.01–0.06for the corresponding linear traits using the restricted maximum likelihood(REML) method. The standard error of heritability estimates of the presentstudy could probably be reduced by increasing sampling size of the study bymeasuring new lactating animals. In addition, the smaller standard errorcould possibly be obtained by improving the information accuracy of animals'pedigree such as animals' date of birth so that the true numerator additiverelationship matrix of the animals can be computed by including theinbreeding coefficient. In the present study, the variance structure ofanimals' additive genetic was approximated by excluding the inbreedingcoefficient by allowing the DMU software to run with option 2 .3.2RLS, FA, UD, and TL were correlated with each other both genetically andphenotypically, though the magnitude was variable, ranging from The magnitude and direction of both genetic and phenotypic correlations amongRLS, FA, UD, and TL reported in the literature are not consistent, forinstance Toghiani (2011), Berry et al. (2004), and Němcová etal. (2011), where the same method as in the present study (the restrictedmaximum likelihood method) was used in the genetic analysis. Results of thepresent study were similar regardless of the direction, with those reportedby Berry et al. (2004), Němcová et al. (2011), and Toghiani (2011)for some pairs of correlations, for example the genetic correlation ofRLS–UD (4Some conclusions can be drawn from the present study. Rear legs set, footangle, udder depth, and teat length are heritable traits. Rear legs set, footangle, udder depth, and teat length are correlated and the geneticcorrelations are stronger than those of phenotypic ones. This suggests thatrear legs set, foot angle, udder depth, and teat length could be included inthe selection program. Including the studied traits in the selection programshould be done with proper weights so that an optimum selection response canbe achieved.