However, there was no correlation between the body weight and height ratio and the width ratio and CSA ratio at all levels, except for that at C1–C2. Also, there was a negative correlation between the body weight and CSA ratio at C1–C2. There were no statistical differences for the CSA of the spinal canal, the CSA of the spinal cord, and the CSA ratio between nearby levels, except for that at C1–C2. There was no statistical difference between measurements at each same level of the sagittal and transverse images. The results of this study may provide basic and morphometric information for diagnosing and researching cervical spinal diseases in small-breed dogs.Cervical spinal disease is one of the most common neurological disorders in small-breed dogs. Magnetic resonance imaging (MRI) is a common test for dogs with cervical spinal diseases. However, there is no information on cervical spinal canal and cord using MRI in normal small-breed dogs. Therefore, this study aimed to perform analyses to establish morphologic MRI reference ranges of the cervical spinal canal and cord in normal small-breed dogs. Cervical MRI examinations were taken in 20 client-owned small dogs. The height, width, and cross-sectional area (CSA) of the spinal canal and cord were measured on sagittal and transverse T2-weighted images at each vertebral body level and each intervertebral disk level from C1–C2 to C7 . The height ratio, the width ratio, and the CSA ratio were calculated. The height, width, and CSA of the spinal canal and cord increased as the dog's weight increased ( Cervical spinal disease is one of the most common neurological disorders in small-breed dogs. Cervical intervertebral disk disease (IVDD) accounts for ~15% of all intervertebral disk extrusions in small-breed dogs, with breeds such as Dachshund, beagle, and poodle representing 80% of the cases . Young sPrevious studies have evaluated the anatomic and quantitative qualities of the cervical spinal canal or spinal cord in normal dogs using radiography, computed tomography (CT), and CT myelography –10. HoweTo the authors' knowledge, there is no information on the cervical spinal canal and cord using MRI in normal small-breed dogs. Thus, in this study, we aimed to perform analyses to establish MRI reference ranges for the height, width, and cross-sectional area (CSA) of the cervical spinal canal and cord and the ratio of spinal cord size to canal in normal small-breed dogs.The study protocol was approved by the Seoul National University Institutional Animal Care and Use Committee . Cervical MRI examinations were taken in 20 client-owned small dogs presented to the Time Animal Medical Center from March 2020 to June 2021. All dogs were healthy based on physical, complete blood cell counts, serum biochemistry, and cerebrospinal fluid (CSF) analyses. None of the dogs demonstrated cervical neurological symptoms or spinal cord or disk abnormalities on cervical MRI and had no microchip in the cervical area. The dogs were normal, except for some with vision loss or presumptively idiopathic seizure. MRI scans were performed with a 1.5-T system  under general anesthesia. The anesthesia was induced in each dog with intravenous propofol  at a dose of 6 mg/kg and maintained with isoflurane  and oxygen. The dogs were positioned in ventral recumbency, with the forelimbs pulled caudally and a straight spinal alignment on the eight-channel knee coil. Sagittal and transverse T2-weighted images, with a repetition time of 3,000–4,000 ms, echo time of 100 ms, slice thickness of 2.0 mm, and gap of 0.2 mm with the phase-encoding direction being anterior–posterior, were obtained from each dog for measurements. Dorsal T2-weighted and transverse T1-weighted images were also obtained for reference of the surrounding structures.via picture archiving and the use of a communication system . The images were independently analyzed by two observers (NL and JS). Measurements followed the methods described in previous reports of dogs and humans  or Kruskal–Wallis ANOVA on ranks was used for comparisons between the measurements in all levels, and Scheffe's post-hoc tests were used to correct for multiple comparisons. A t-test was used for comparisons between measurements at each same level of the sagittal and transverse images. Intra-observer and inter-observer reproducibility was assessed using the intraclass correlation coefficient (ICC). A value close to 1 indicates excellent agreement. A p-value < 0.05 was considered statistically significant.Statistical analyses of the data were performed using SPSS software . Normally distributed data were presented as the mean ± standard deviation (SD). Pearson's correlations or Kendall's tau was used to identify the correlations between the measurements and body weight. A linear relationship between the CSA ratio of the spinal cord to the spinal canal at C1–C2 and body weight was verified by regression analysis and scatter plots. The Mann–Whitney The breeds of the dogs included in the study were Maltese (six), Pomeranian (one), toy poodle (one), Spitz (one), mixed breed (nine), and Beagles (two). Eleven dogs were male and nine were female. The mean age was 3.60 years  and the mean body weight was 8.23 kg .The measurements for each level (12 levels) and the ratio are summarized in C1–C2 showed the largest width of the spinal canal and cord and CSA of the spinal canal compared to those of other levels; thus, their roundness indices were the smallest, as well as the CSA ratio. At C2, the height of the spinal canal was the largest compared to that of the other levels, and given that the roundness index of the spinal canal was the largest, the shape was almost round . The CSA ratio at C2 was the second smallest after C1. The CSA of the spinal cord at C2–C3 was the smallest and that at C6 was the largest compared to that of the other levels. The CSA ratio at the disk level was smaller than those at nearby vertebral body levels, but there was no statistical significance. Moreover, there were no statistical differences in the CSA of the spinal canal, the CSA of the spinal cord, and the CSA ratio between nearby levels, except for those of C1–C2.p < 0.01), except for those of C1–C2. However, there was no correlation between the body weight and height ratio and the width ratio and CSA ratio at all levels, except for those of C1–C2. There was a negative correlation between the body weight and the CSA ratio at C1–C2 (p = 0.047)  . There wIn previous studies, the ratio of the spinal cord area to the spinal canal area was greater in small dogs compared to that of large dogs , 9, 17. In the present study, the height, width, and CSA of the spinal canal and cord showed a tendency to increase as the body weight increases, similar to that in previous studies , 17. HowDogs ≤5 kg showed a relatively larger cord size at C1–C2 than did dogs >5 kg. Although dogs with an apparent anomaly of the cranio-cervical junction (CCJ) and SM were excluded in this study, most of the dogs ≤5 kg were toy-breed dogs that are predisposed to these conditions . In the To the authors' knowledge, there are no well-defined guidelines to grade the severity of spinal compression on MRI. In previous studies and in the veterinary literature, the spinal cord compression ratio was obtained by measuring the percentage of the CSA of the spinal canal that is occupied by the compression material or the ratio of the CSA of the spinal cord at the point of maximal compression to the CSA of the closest spinal cord without compression –26. HoweT2-weighted sagittal images are routinely acquired prior to planning transverse images as a guide. However, it has been reported that the evaluation of sagittal images in IVDD was not 100% accurate . Given tMost measurements were in near-perfect agreement between intra- and inter-observers at the vertebral body levels, except for that of the transverse images at the disk levels. Given that it was difficult to discriminate the margin of the CSF due to epidural fat in some images , we measA limitation of this study was that the number of dogs was small; therefore, we had to use a non-parametric test in some statistical analyses. Another limitation is that a relatively large number of mixed-breed dogs and a relatively low variety of breeds were included in this study. Further studies with larger groups and more representative small-breed dogs are needed. There were also fewer dogs ≤5 kg that showed a larger tendency of relative cord size at C1–C2. Given that there are significant morphological differences in the cervical region like the vertebral body and disk length between dog breeds , it cannIn conclusion, this study provides the height of the spinal canal and cord on the midsagittal T2-weighted images, the height, width, and CSA of the spinal canal and cord on the transverse T2-weighted images, and the height ratio, width ratio, height-to-width ratio, and CSA ratio from C1–C2 to C7 in normal small-breed dogs. In particular, the CSA ratio is useful for evaluating the degree of spinal cord compression, and dogs ≤5 kg that showed a relatively larger cord size at C1–C2 need future studies on the relationship between the morphological characteristics of the spinal cord at C1–C2 and common diseases in this region. These measurements may serve as a morphometric baseline for diagnosing and researching cervical spinal diseases in small-breed dogs.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by Seoul National University Institutional Animal Care and Use Committee . Written informed consent was obtained from the owners for the participation of their animals in this study.NL and JY contributed to the conception and design of the study. NL and JS organized the database and wrote sections of the manuscript. NL performed the statistical analysis and wrote the first draft of the manuscript. All authors contributed to manuscript revision and read and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Rt). We used data from the Johns Hopkins University Centre for Systems Science and Engineering Coronavirus repository. Day 1 was taken from the first assumed local transmission of COVID-19. We estimated Rt by using the EpiEstim package and plotted the epidemic curve and Rt. Then, we extracted the mean Rt at day 1, day 5 and day 10 for all MCO phases and compared the differences. The Rt values peaked around day 43, which was shortly before the start of MCO 1. The means for Rt at day 1, day 5, and day 10 for all MCOs ranged between 0.665 and 1.147. The average Rt gradually decreased in MCO 1 and MCO 2. Although spikes in the number of confirmed cases were observed when restrictions were gradually relaxed in the later MCO phases, the situation remained under control with Rt values being stabilised to below unity level .To curb the spread of SARS-CoV-2 virus (COVID-19) in Malaysia, the government imposed a nationwide movement control order (MCO) from 18 March 2020 to 3 May 2020. It was enforced in four phases . In this paper, we propose an initiative to assess the impact of MCO by using time-varying reproduction number ( To combat the coronavirus disease 2019 (COVID-19) pandemic, the Malaysian government initiated MCOs, effective on 18 March 2020, with the aim to aggressively contain the outbreak and slow down the transmission rate. This strategy was proclaimed under the Prevention and Control of Infectious Diseases Act 1988 Act 342) and the Police Act 1967. Offenders violating MCO directives could face a maximum of RM 1000 fine and/or six months in jail or both if convicted [42 and thVarious non-pharmacological interventions had been implemented during MCO 1 that were continued in MCO 2; a gradual relaxation of rules occurred in subsequent MCO phases . During On top of the four MCO phases that were implemented nationwide , the MalRt, defined as the average number of secondary cases from a partially susceptible population per infectious case [With the increasing number of cases and the introduction of multiple non-pharmaceutical interventions (NPIs) by most countries, assessing and monitoring the transmissibility of the disease as a measurement of the effectiveness of control measures is necessary. Time-varying ous case , is a unR0, whereas the reproduction number after mitigation starts is named Rt; both can be used to measure the transmissibility of the infection. A value of Rt which is more than 1 means that the infection is spreading, with additional new cases being generated at an exponential rate. A value of Rt which is less than 1 means that the spread of infection is decreasing. Theoretically, we need information about generation time, which is defined as the period between the infection of the index and the next case. However, this information is usually difficult to ascertain. Therefore, information regarding the serial interval  distribution in the data is used instead [The basic reproductive number before mitigation starts is named  instead ,9,10,11.R0) for COVID-19 was 3.38 (standard deviation (SD) = 1.40) and a range of 1.90–6.49 [R0 was estimated to be 2.2  [A recent systematic review reported that the basic reproduction (.90–6.49 . An earl1.4–3.9) .Rt to assess and explain transmissibility dynamics and epidemic progression [Rt reflects, although with limitation, the spread of infection. In the case of COVID-19, NPI has been thought to reduce the spread through isolation and contact tracing. Both reduce the time during which cases are infectious in the community, thereby reducing Rt [Rt below 1, then on average, the number of new cases reported can decline, which can become apparent after a delay of approximately one incubation period plus time to case detection and reporting, following the implementation of the control measure  [Several studies have indicated the feasibility of using gression ,12,13. Rucing Rt ,13,14. IRt may indicate the impact of MCO. The numbers can be quantified to indicate the effectiveness of the impact of MCO on the spread of COVID-19. This indication, in return, can provide critical information to help epidemiologists and public health workers strategize their COVID-19 control programme.The relationship between MCO and Rt. Specifically, using the incidence number of confirmed COVID-19 cases between 4 February 2020  until 16 May 2020 (day 102), we (a) measure the Rt of SARS-CoV-2 at day 1, day 5 and day 10 after the initiation of each of the four MCOs and (b) quantify the impact of each MCO by measuring the difference in the Rt between the beginning of MCO (day 1) and day 5 and between day 5 and day 10 of each MCO.The general objective of the study is to use the daily incidence data for confirmed COVID-19 cases in Malaysia to quantify time-varying effective https://github.com/RamiKrispin/coronavirus/tree/master/csv (accessed on 10 September 2020). The owner of this GitHub repository compiled the COVID-19 data retrieved from the Johns Hopkins University Centre for Systems Science and Engineering (JHU CCSE) coronavirus repository (https://github.com/CSSEGISandData/COVID-19) (accessed on 10 September 2020).We downloaded the COVID-19 data by using the R software from this link: The downloaded data are in the time series format and contain variable date, province, country, latitude, longitude, type and case. For the analysis, we selected observations to only include data from Malaysia and type equals “confirmed,” which refers to confirmed COVID-19 cases. The variable case is the variable that contains the number of cases reported every day.We considered the first day of our data to be 4 February 2020, because we assumed that was the earliest evidence of local COVID-19 transmission as reported by the Ministry of Health (MOH), Malaysia. We included data up to 16 May 2020, that is, day 102.https://github.com/RamiKrispin/coronavirus/tree/master/csv (accessed on 10 September 2020) and are in an aggregated format. The original source of data (raw data) came from the JHU CCSE coronavirus repository (https://github.com/CSSEGISandData/COVID-19) (accessed on 10 September 2020).The study did not require ethical approval because we used data that are publicly available at The duration of different MCO levels  was deciestimate-R function) allows us to estimate the instantaneous and case Rt for COVID-19 by using (a) a time series of COVID-19 incidence and (b) the distribution of the COVID-19 serial interval (time between symptoms onset in a primary case and symptoms onset in the secondary case of COVID-19) [Data were analysed using the R IDE software version 4.0.2  . We emplOVID-19) ,13.estimate-R function, for estimating the Malaysia COVID-19 instantaneous or time-varying Rt, we initially used the values of a mean of 7.5 days and SD of 3.4 days to represent the serial intervals (SIs) [Rt. We assumed for our analysis that the COVID-19 SI follows a normal distribution. The EpiEstim package uses the Poisson likelihood to calculate the instantaneous Rt by using the sliding window of seven days [Rt on sliding weekly windows. We uploaded the complete R codes for the analyses at https://github.com/drkamarul/R0_MYS/blob/master/repro_covid19_malaysia.md (accessed on 10 September 2020). In the ls (SIs)  to estimven days ,12,13. TRt values at day 1, day 5 and day 10 for each of the four MCOs (the Rt at day 10 in MCO 4 was taken the third day of CMCO). To see the trend of the mean Rt over 10 days for each MCO, we calculated the difference of the mean Rt between (a) day 5 and day 1 and (b) day 10 and day 5; we also plotted the Rt values  by using the line plots.We also extracted the mean We analysed the daily Malaysia COVID-19 incidence data starting from 4 February 2020 (day 1) to 17 May 2020 (day 102). The incidence data reported by the MOH, Malaysia refer to the day when a patient was detected positive by the RT-PCR test. In the data, the variable date corresponds to the date that the test became positive and not the onset of the symptoms. All COVID-19 tests in Malaysia are performed by the COVID-19 gazetted laboratories using the RT-PCR antigen test.Rt from day 1 (4 February 2020) to day 100 (14 May 2020). The plot of Rt in Rt in Rt values decreased gradually after day 43, remained above 1 until day 65 and after which reduced to below 1. In comparison with the epidemic curve in The transmissibility of COVID-19 in Malaysia was estimated on the basis of Rt values soon after the second peak indicates reduced transmissibility. With Rt values greater than 1, the number of confirmed cases continued to increase, as illustrated in Rt values appeared to start before the imposition of MCO. The sensitivity of the Rt values to the choice of parameter values  might have introduced some level of uncertainty in the exact timing of the second peak. A further investigation is obviously needed to verify the accuracy of this part of the results.The decrease in Rt values from day 44 to day 58. The strict measures which continue during MCO 2 managed to bring down Rt values to below 1 after day 65. As Rt values dipped below 1, the number of confirmed cases in Stricter measures imposed during MCO 1 had a significant impact on the fast reduction in Rt values remained below 1 for most of MCO 3, indicating that the transmission of the disease was under control. As the MCO entered phase 4 (MCO 4), the further relaxation of restrictions brought a few spikes in the number of cases. Nevertheless, the transmissibility remained well under control because the Rt values had already reduced significantly and hovered around 1 or less by then.Rt estimated from the second SI , instantaneous mean Rt values for day 1, day 5 and day 10 for each MCO phase are calculated based on seven-day rolling average and the results are illustrated in Rt values.Using the Rt values is elucidated further. From day 1 to day 10 of MCO 1, a progressive drop in the instantaneous mean is observed and subsequently, from day 1 to day 10 of MCO 2 a similar monotonic decrease persists. It is important to bear in mind that during MCO 1 and MCO 2, the entire country was under an almost complete lock down with very restricted movement. The strict measures are reflected in the continuous decrease in the instantaneous mean Rt values and by day 5 of MCO 2, the value had decreased to below the threshold value of 1. In this case, we can see that the instantaneous mean Rt values provide a stronger indicator of the impact of strict MCO that manage to bring down the mean Rt value from 1.23 to below 1 in about two weeks.In this illustration, the connection between the different phases of MCO and Rt values is detected. However, the rise in values do not persist in MCO 4 where we observe that between day 1 and day 10 in MCO 4, the instantaneous mean Rt declines and arrives at fairly steady values (from 0.8 to 0.9). At this point, it is unclear whether the rise in values is due to the relaxation of MCO directives during MCO 3 which saw some sectors were allowed to operate . Upon scrutiny of the timeline of COVID-19 in Malaysia, we learned that a large cluster was detected during this period which involves students returning from Temboro, Magetan, Indonesia with 43 cases  and later 72 cases from the same cluster  [Rt, but the effect is not significant enough to influence the long-term behaviour. The fact that the instantaneous mean Rt declines soon after and the 95% percentile remains mostly below the threshold value of 1 throughout MCO 3 and MCO 4 goes to show that the pandemic has been tamed sufficiently.During the first 10 days of MCO 3, a steady increase in the instantaneous mean , MCO 4) . This clRt [The MCO enforced by the Malaysian government is an example of an NPI, which is the intervention proposed strongly by epidemiologists and infectious disease modellers to halt the transmissibility of SARS-CoV-2; NPI aims to prevent and/or control SARS-CoV-2 transmission in the community ,19. UntiRt ,13,14,20Rt values as well as the instantaneous mean Rt values on day 1, day 5 and day 10 after the initiation of each of the MCO phases.The Malaysian government initiated MCOs from 18 March 2020 to 3 May 2020. It had three types: MCO, CMCO and RMCO. The MCO was also implemented in four phases  nationwide, with the main objective to stop or curb the spread of the SARS-CoV-2 virus. In order to quantify the impact of MCO during the four different phases, we have analysed the daily incidence data to compute time-varying effective Rt values do correspond with the timeline of the pandemic and the level of strict measures imposed during each phase of the MCOThe overall trend of . The Rt values peaked around day 43, which was shortly before the start of MCO 1. A marked decline in Rt values was observed during MCO 1, and the reduction in transmissibility is reflected in a reduction in the rate of increase in daily cases. Thus, we can say that measures taken during MCO 1 were successful in taming the exponential growth of the number of infections. The Rt values started to fall below the threshold value of 1 during MCO 2, at which time the number of daily cases began to show a downward trend. Rt values remained below 1 throughout MCO 3. The relaxation of certain restrictions during MCO 3 and MCO 4 might have allowed a few spikes of confirmed cases and this is indicated by a slight increase in Rt values, but the Rt values remained stable and hovered around 1 and slightly below that.Rt values computed for day 1, day 5 and day 10 of each phase of the MCO provide further insight into the suitability of Rt values as a quantifiable indicator to the impact of each MCO phaseThe instantaneous mean . From day 1 to day 10 of MCO 1, the instantaneous mean Rt reduces progressively and monotonically and this behaviour persists until day 10 of MCO 2. The continuous decrease in the mean Rt is a reflection of the strict measures imposed during MCO 1 and MCO 2. The impact of strict MCOs managed to bring down the mean Rt value from 1.23 to below 1 in about three weeks where at this level of Rt, the spread of infection is expected to be under control. The relaxation of the level of restrictions during MCO 3 and MCO 4 is also reflected in the instantaneous mean Rt, where a steady rise of the values is picked during MCO 3.Rt values with respect to the choice of parameter values.Sensitivity of  Our results reveal the sensitivity of Rt values with respect to the choice of parameter values, namely, the mean and SD. This is particularly a concern, especially at the start of the pandemic where two significant peaks of Rt values were observed. A change in mean and SD values modified the profile of these peaks rather abruptly. Our hypothesis is that the sensitivity is due to the small sample size problem where data are insufficient to capture the essential features of the dynamical system. This hypothesis is supported further by the wider 95% percentiles for values observed on day 1 until day 45. Although reasons exist to believe that these peaks represent the relevant historical events of the pandemic , whereas peak 2 can be tied up with the wave of infection originated from a mass gathering at Sri Petaling, Kuala Lumpur, which took place between 27 February 2020 and 1 March 2020), the accuracy of computed Rt values and the time they occurred should be investigated further.Sensitivity to outliers. In the presence of outliers, the instantaneous mean Rt values can be affected. This is observed in our analyses of the mean values during MCO 3 and MCO 4 and the associated timeline of the pandemic in Malaysia. The outlier is a cluster of cases involving students returning from Temboro, Magetan, Indonesia which is not a contributing factor to the community transmission during MCO 3. During the first 10 days of MCO 3, a steady increase in the instantaneous mean Rt values was detected, peaked around the first day of MCO 4, and then decreased to steady values between 0.8 and 0.9. It is possible that the rise of cases may also be due to the relaxation of certain measures during MCO 3, however, the rising trend does not persist in MCO 4. This gives the indication that the effects of outliers on Rt are possibly short-lived and do not contribute significantly to the baseline value associated with community transmission that is directly impacted by MCO measures. This supposition is further strengthened by the fact that the 95% percentile of the instantaneous mean Rt remain mostly below the threshold value of 1 throughout MCO 3 and MCO 4.The main results can be summarized as follows:Rt values and a close scrutiny of the timeline of COVID-19 in Malaysia that the impact of the MCO phases is quantifiable by the Rt values. From our Rt calculation, we are able to highlight the importance of the strict measures imposed in MCO 1 and MCO 2 in bringing the Rt values down to below the threshold value of 1 in a matter of weeks. The continuous steady decrease in Rt values is indicative of the role of MCO 1 and MCO 2 in slowing down the spread of the pandemic. This factor is crucial for developing countries, such as Malaysia, which face problems of limited medical resources , to ensure that the surge in the number of people requiring medical care due to COVID-19 at any particular time is reduced, and the healthcare system does not exceed its capacity.We have shown in our analyses of the Rt values have reached below the threshold value of 1 before introducing any relaxation of MCO measures. Once certain restrictions are lifted, there are bound to be spikes in the number of cases which will cause Rt values to increase. However, if Rt values have reached below 1, the situation can still be kept under control.Another useful discovery from our analyses is the importance of making sure that Rt can be further improved.Our results do provide an indication of the success of the NPI enforced by the Malaysian government during the period between 18 March 2020 to 3 May 2020. However, we would like to express caution that the insights from our analysis also depend on data that come from the MOH, Malaysia that include laboratory testing and contact tracing strategies. For instance, the number of confirmed cases can often be determined by the speed of the notifications of COVID-19 tests, the accuracy of the test and the maximum number of COVID-19 tests performed daily by the MOH. In simple words, with additional tests conducted, new cases may be recorded. Still, the quality of the data can be better than before, and the predictability of the pandemic using quantities such as Timely track and trace process is undoubtedly one of the strategies in curbing the pandemic, and Malaysia’s MySejahtera apps play a vital role, which has reached almost 60% of the Malaysian population . Given tRt. In our analysis, we believe the effect of the low per capita test on our Rt calculation is negligible because Rt is more dependent on the trend of confirmed COVID-19. Our analysis assumes that there was not much individual variation in the COVID-19 infection in Malaysia despite a few COVID-19 outbreaks in South Korea, USA and Poland, indicative of the presence of super-spreaders. The variation of individual infection is measured by a metric, known as K . The presence of a super-spreader will significantly increase the size of the COVID-19 cluster in a short time interval. The K value is more are critical in the late stages of the epidemic when the virus is almost eradicated, unlike the focus of this paper, where the interest was on the early part of the epidemic [On 2 March 2020, the estimated COVID-19 test for Malaysia was less than 0.01 per 1000 people (while for South Korea it was 0.23 per 1000 people), and it gradually increased to 0.28 per 1000 people on 23 May 2020 (0.21 per 1000 people for South Korea) . Though epidemic ,27.Rt. The ideal parameter required to estimate Rt is the generation time. However, in most studies, the generation time is impossible to ascertain. The next best parameter is the SI of the infection. Unfortunately, we do not have data about infectors and infectees to estimate the SI. A few studies have shown that using laboratory notifications can still provide a useful reference to the transmissibility of the SAR-Cov-2.The main source of limitation is using the date of a notification of a positive COVID-19 test to construct the epidemic curve and to estimate Rt estimates reflect the average value for the country. However, different states and even different districts within a state may likely have different Rt values because the response to MCOs, the population density and the bulk of infections vary. This study utilises the number of confirmed cases, although the exact number of infected cases can be larger if asymptomatic cases are considered. We estimate that 5–80% of cases are asymptomatic.The Rt peaked earlier (between day 25 and day 35) which is before the start of MCO and thus may reflect the response from the community and the government. Strict MCO 1 and MCO 2 correspond to a gradual decrease in Rt, but relaxed MCO 3 and MCO 4 correspond to a slight increase in Rt before it plateaued below 1.In Malaysia, the daily confirmed case data indicate that the epidemic curve for COVID-19 reached a peak between day 40 and day 70. However, the time-varying


In this paper, properties of concretes incorporating recycling waste and corrosion susceptibility of reinforcing steel bars were studied. It was established that fineness of ground granulated blast furnace slag (GGBFS) and fly ash (FA) and their simultaneous combination have an influence on the kinetics of strength development of Portland cements and concretes. The compressive strength of concrete containing 10% by mass of GGBFS and 10% by mass of FA even exceeds the compressive strength of control concrete by 6.5% and concrete containing 20% by mass of GGBFS by 8.8% after 56 days of hardening. The formation of the extra amount of ettringite, calcium hydrosilicates as well as hydroaluminosilicates causes tightening of a cement matrix of concrete, reducing its water absorption, and improving its resistance to freezing and thawing damage. Environmental problems are becoming more and more serious from year to year. Despite the remarkable development of scientific and technological progress, some industrial processes result in the significant production of waste, which cannot be landfilled completely and present a big problem for both the producer and the environment . The wasThe cementing materials and products on their basis have played a very important role in the development of construction for many years. The Portland cements and concretes are the most used materials in the construction industry ,3. The tGGBFS is rather valuable waste and can be recycled in environment processes, agriculture, and the construction industry. Ground granulated blast furnace slag has begun to be used widely since the discovery of its latent hydraulic properties. Many researchers have been conducting lots of experiments to determine the properties of cements and concretes containing GGBFS and fly ash ,19,20,21FA is a by-product of electricity generating plants. It is used both in the technology of cement and concrete production ,27. It hSanytsky et al.  and Li eFernández et al.  also poiThe use of GGBFS and FA may also have an influence on the initial pH of the cementitious system and the protective passive layer, which is formed on the reinforcing steel bars due to the alkaline environment in concrete . In the According to Yeau and Kim , the corThis is why the aim of this research was to establish the effect of fineness of GGBFS and unactivated fly ash on the strength of Portland cements and properties of concretes as well as the level of Portland cement replacement without the reduction of compressive strength in comparison with the control cement and concrete without SCMs and to study the behavior of reinforcing steel bars in concretes incorporating such recycling waste.2/kg, respectively. The commercially available lignosulfonate based plasticizer  with a specific gravity of 1.18 and solid content of 35% was used in the research. The properties of Portland cement are presented in Portland cement CEM I 42.5 , ground granulated blast-furnace slag , and unactivated fly ash (Class F) derived from the Burshtyn power plant  were used in this study as supplementary cementitious materials. The specific surfaces of GGBFS and unactivated FA were 310, 380, 510 and 310, 510 mThe Portland cements of the second type CEM II, especially CEM II/A-S, is very popular in the construction market. The content of additions can range between 6–20% by mass in this cement according to EN 197-1. This is why the maximum amount of Portland cement (20% by mass) was replaced with GGBFS in the first stage of the research.The properties of Portland cements and aggregates were determined according to Ukrainian standards ,45,46,47The specific surface of Portland cement, GGBFS and FA was determined using the Tovarov device . The Vicat apparatus  was used to determine the setting time. The specific surfaces of GGBFS and fly ashes and residues in sieve 008 (the mesh opening size was 0.08 mm) are shown in The chemical composition of Portland cement, ground granulated blast-furnace slag and fly ash, which was determined by x-ray spectrometer ARL 9800 XP , is shown in Fly ash in comparison with Portland cement and GGBFS is characterized by a higher amount of silica, alumina, and iron oxides, which provide its pozzolanic activity.The morphology and chemical composition of the main mineral components of the studied materials were determined using scanning electron microscope SEM FEI Quanta 250 FEG, equipped with EDS .3 were molded according to 51], respTwelve specimens (six basic and six control specimens) of each concrete composition were molded and cured 28 days according to the Ukrainian standard (at the temperature of 20 ± 3 °C and the relative humidity of 95 ± 5 °C) to determine the freeze–thaw resistance of concretes. Then, the accelerated method was used according to  to deter3, S = 800 kg/m3, G (5–10 mm) = 1150 kg/m3, W = 175 kg/m3, plasticizer . The consistency class of fine-grained concrete mix was S1. Twenty percent by mass of Portland cement was replaced with 10% by mass of GGBFS and 10% by mass of FA in a reference test fine-grained concrete. Nine similar specimens (prisms of 40 × 40 × 160 mm3) of each mixture were molded. A smooth steel bar (working electrode) with the diameter of 6 mm and length of 120 mm was placed in the center of the mold and secured to prevent movement during filling and compaction. Before making the specimens, steel bars were cleaned using emery paper and then degreased with acetone. All specimens were cured 28 days at the temperature of 20 ± 3 °C and relative humidity of 95 ± 5 °C. Three specimens were tested after 28 days of hardening in the above-mentioned conditions. The other six specimens of each mix-proportion were kept in conditions of daily alternating wetting and drying . Three specimens were tested after three and six months. Before measurement, specimens were saturated with drinking water by boiling them for 3 h. Before the beginning of the test, concrete was chipped from one end of the beam specimen, exposing the reinforcing bar by 20 mm ± 10 mm. Then the working, reference, and counter electrodes were connected to the polarization circuit. Measurements of the magnitude of the current in microamperes were carried out in concrete specimens in (60 ± 5) min after turning on the potentiostat. The output of the potentiostat was raised, and the current recorded when passing the range from the steady-state potential to plus 1000 mV for 60 min. For each test specimen, the surface area of the working electrode, which was in direct contact with the concrete, was calculated. Using the calculated area of the rebar, the current density was determined.The effect of Portland cement replacement with 10% by mass of GGBFS and 10% by mass of FA on the corrosion susceptibility of reinforcing steel in concrete was studied by the potentiostatic electro-chemical test according to . ConcretThe strength of building materials is an important characteristic, which defines their quality. This study focused mainly on the influence of SCMs on the compressive strength of Portland cements and properties of concretes as well as the corrosion susceptibility of reinforcing steel bars. It is a well-known fact that there are two schemes that show how GGBFS can be added to Portland cement. It can be mixed with constituents of cement by intergrinding or after previous separate grinding to the appropriate specific surface. The second way was used as it is more effective. It allows for the achievement of a more complete realization of potential hydraulic properties of GGBFS, because the size of the slag particles remained rather coarse after intergrinding of clinker and GGBFS due to the poor grindability of the GGBFS. Therefore, the Portland cement and GGBFS, ground to the appropriate specific surface previously, were mixed in the proportions that are shown in 2/kg was 34, 21 and 16% lower than the control mortar (C) after 2, 7, and 28 days of hardening, respectively. The increase of fineness of GGBFS caused gradual growth of strength at both early and later ages in comparison with CS310. The compressive strength of mortar CS380 was reduced by 6% after 28 days of hardening, while CS500 almost reached the compressive strength of the control mortar. However, GGBFS with a specific surface of 380 m2/kg was used for further research, because the process of slag grinding up to 500 m2/kg would be a rather expensive and simultaneously time consuming process. Moreover, GGBRS is becoming more and more expensive on the market year by year, and scientists are looking for SCMs to replace it.As can be seen from 2/kg and 510 m2/kg was used in the research. The Portland cement CEM I 42.5 was replaced with 5% (CF5(310), CF5(510)), 10% (CF10(310), CF10(510)) and 15% (CF15(310), CF15(510)) by mass of FA. The obtained results are presented in Widespread by-products such as fly ash, which needs to be utilized, were used to improve the properties of cements. The specific surface of fly ash can be different and range widely at the thermal power plant. The unactivated fly ash with specific surfaces of 310 m2/kg was rather coarse and 5% by mass of Portland cement could only be replaced without the compressive strength reduction. At the same time, the usage of fly ash with a higher specific surface (510 m2/kg) made it possible to increase the replacement level of Portland cement without the reduction of strength after 28 days. The results in 2/kg was used, but the replacement level could be increased to 10% by mass.A reduction in compressive strength was observed in mortars CF10(310) and CF15(310). Nicoara et al.  observed2/kg (CS10(380)F10(510)) was studied. The mortar incorporating 20% by mass of GGBFS (CS20(380)) was used for comparison. The above-mentioned mortars containing the same amount of SCMs (20% by mass) was studied and compared with control mortar C. The results in The literature review and analysis of the obtained results show the necessity of combining GGBFS and FA. Compressive strength of mortars containing 10 and 15% by mass of GGBFS as well as 5 and 10% by mass of FA with specific surfaces of 310 (CS15(380)F5(310)) and 510 mHowever, the kinetics of the strength gain of mortars in which Portland cement was replaced with both GGBFS and fly ash was lower for up to two days, but due to the synergetic effect of the combination of these two by-products, accelerated, leveled CS10(380)F10(510), and even exceeded CS15(380)F5(310) in the compressive strength of control mortar at later ages (28 days). The existence of this effect was also confirmed by many researchers ,34,55.2O3 and SiO2. The above-mentioned hydration products appear in the vacant space of cement matrix and also on the fly ash surface F10(510) to freezing and thawing damage is obtained due to the improvement of the pore structure. The higher amount of secondary CSH gel obtained due to the pozzolanic reaction results in a decrease in the capillary porosity of concrete incorporating GGBFS and FA, which confirms the efficiency of the simultaneous combination of these SCMs.The compressive strength reduction of designed concretes is reported in 2 at 1 V for RCC and RTC, respectively. The content of calcium hydroxide in RCC was lower because 20% by mass of Portland cement was replaced with SCMs and the pozzolanic reaction also consumed some amount of Ca(OH)2, reducing its content in hardened concrete, and likely a thinner protective passive layer formed on the rebars. Over time, the current density deference of compared concretes was reduced to 7.1 µA/cm2 after 90 days and even almost levels after 180 days at a potential of 1 V due to the formation of the tighter structure of concretes incorporating GGBFS and FA. Polder [The replacement of cement with SCMs in concretes also results in the reduction of the clinker and as a result, calcium hydroxide, which is produced during Portland cement hydration and is responsible for creating a protective passive layer on the rebars due to the alkaline environment. This is why the corrosion susceptibility of reinforcing steel bars in such concretes was studied. As can be seen from . Polder  also obs2/kg resulted in the growth of Portland cement strength by 47 and 18% at 2 and 28 days of hardening, respectively.The increase of fineness of GGBFS from 310 to 500 mThe kinetics of strength development of Portland cements and concretes incorporating GGBFS and FA is lower at the early age of structure formation, but accelerates at later ages due to the pozzolanic reaction that takes place.The synergetic effect of the compressive strength development was revealed in Portland cement and concrete containing granulated blast-furnace slag and fly ash.The replacement level of clinker in cements and Portland cement in concretes with GGBFS and FA depends on their specific surface.The obtained results show the efficiency of unactivated fly ash usage for the cement and concrete production and the necessity of permanent control of fly ash fineness to determine the replacement level of the clinker in a cement and a cement in a concrete.Simultaneous use of supplementary cementitious materials of different nature activity such as GGBFS (latent hydraulic) and FA (pozzolanic) allows for the improvement in the microstructure of concretes due to the formation of an extra amount of ettringite and tobermorite-like low-basic calcium hydrosilicates as well as hydroaluminosilicates in the non-clinker part of cement paste. Unactivated fly ash also allows the particle size distribution to be optimized in cements and concretes to archive the prolong time of cement hydration, which results in obtaining a more refined and compact structure over time due to its role both as a mineral addition with pozzolanic properties and as a microfiller. As a result, the durability and the stability of rebars in such concretes increase, which conforms to the strategy of sustainable development in the construction.Incorporation of cement replacement materials such as GGBFS and FA in concretes results in the reduction of water absorption from 5.5% to 4.7% and, as a result, in the increase of resistance to freezing and thawing damage, improving its durability. The decrease in compressive strength reduction was observed from 30.2 for concrete C to 24.1% for concrete CS10(380)F10(510) after 100 freeze–thaw cycles.Concretes containing 20% by mass of SCMs will have a better corrosion resistance in comparison with the concrete on the basis of Portland cement CEM I. Steel reinforcement protection does not worsen in concretes incorporating 10% by mass of GGBFS and 10% by mass of FA due to the low Portland cement substitution rate and their tighter microstructure. The obtained results concerning the corrosion susceptibility of reinforcing steel bars in concrete incorporating GGBFS and FA will be taken into account in the future developments of this study in the real reinforced concrete elements.The experimental work was done to evaluate the influence of a granulated blast-furnace slag and fly ash on the properties of Portland cements and concretes as well as the corrosion susceptibility of the reinforcing steel bars. The above-mentioned recycling waste was used for a partial replacement of binding materials in this article. The following conclusions can be drawn from the study:


The proportion of seronegative women in the age group 40–49 was significantly higher than of seronegative men: 22 [18–26]% and 11 [6–18]% respectively . The revealed gender characteristics that affect persistence of measles immunity may be important in personalization of vaccinal prevention for men and women.Individuals without a protective antibody level are susceptible to measles infection. There are differences in the persistence of antibodies after vaccination and infection, while the impact of gender on this process has not been sufficiently studied. Measles Ig G antibodies were measured in 1742 employees of a large hospital facility—403 men and 1339 women aged from 25 to 67 years; 15% participants had antibody levels less than the protective threshold of ≥0.18 IU/mL. Significant differences were found in the age group 40–49, where the level of IgG antibodies to measles among men was higher than among women  vs. 0.70 IU/mL  respectively, (U = 3.2,  Before active universal vaccination, the incidence of measles was very high and ranked first among airborne infections among children of early age. Mass immunization has led to a sharp decline in the incidence, lack of periodicity, and seasonality ,2. At prFrom the published data, it is known that there is an existing gender difference in the formation of post-vaccinal immunity for administration of some vaccines. For example, after vaccination against tick-borne encephalitis, a significantly more expressed immune response in the dynamics of virus-neutralizing antibodies to virus strains accumulation was revealed among women of all age groups in comparison with men . AccordiThe aim of the study was to evaluate the intensity of humoral immunity to measles among men and women working in a large hospital complex.A single-step analytical study was carried out as part of a program to improve the work on measles prevention conducted in the entities of the Russian Federation, with an analysis of the reasons for growth of the proportion of seronegative people and comparison of the results of seromonitoring with data of age-related measles incidence .In the USSR and then in the Russian Federation, mass vaccination of the population with one dose of the vaccine against measles was started in 1968, and since 1987, revaccination has been carried out with the administration of a booster dose. Currently, according to the national immunization schedule of the Russian Federation, measles vaccination is carried out for children aged 12–15 months with mandatory revaccination at 6–6.5 years. Also, immunization is recommended for all people under 35 years who have not suffered from measles, have not been vaccinated at all, or have been vaccinated once and who have no information about previous vaccinations against measles; in the case of an unfavorable epidemiological situation that has been observed in recent years, people under 55 years of age are vaccinated.After obtaining signed informed consent, the levels of IgG antibodies to measles were measured in 1742 employees (403 men and 1339 women aged 25 to 67 years) of a large hospital complex in Moscow in 2018. Respondents were divided by age and sex into four groups: up to 39 years old, 40–49 years old, 50–59 years old, and 60 years and older in agreement with the epidemiologist, due to the convenience of identifying groups with a difference of 10 years. Blood for study was taken during working hours in the morning in compliance with the antiseptic rules and ethical standards. No acute respiratory infections or exacerbations of concomitant diseases were reported among the staff at the time of examination.Medical records of each employee contained information confirming vaccination against measles performed in the past, since this is a prerequisite condition for working in a medical facility.1.known vaccination history;2.presence of medical documents on vaccination and revaccination against measles;3.only employees of the medical organization participated in the study.The inclusion criteria were:4.immunosuppressive therapy, systemic use or inhalation of corticosteroids in high doses , radiotherapy, cytotoxic drugs or nonsteroid anti-inflammatory drugs;5.HIV infection , hepatitis B (acute form) and hepatitis C (acute form);6.therapy containing immunoglobulins and other donor blood products within 90 days prior to the study participation;7.any vaccine administration within 30 days prior to inclusion in the study;8.contraindications to introduction of the measles vaccine (according to the instruction);9.the accession of acute respiratory infections during the first days after vaccination.Since the production of postvaccinal antibodies and the duration of their maintenance can be influenced by various factors and health conditions of respondents, non-inclusion criteria were:For measles prevention in the Russian Federation, both mono- and combined with the antigen of mumps home-produced vaccines are used. The monoprepararion is a live measles cultural vaccine prepared from the vaccine strain L-16 (Leningrad-16) or its cloned variant-Moscow-5 grown on the culture of Japanese quail embrio cells . Since 2001, associated mumps-measles vaccine has been used, which is produced in the same way as monovalent vaccine .Among foreign preparations, Priorix  and MMR II ) were registered in Russia, which were rarely used and mostly in private clinical practice. Comparative data of the study of safety and immunogenicity of domestic and foreign measles vaccines did not reveal significant differences ,18,19.Measles virus IgG antibodies were measured by enzyme-linked immunosorbent assay (ELISA), using kit «Vector-best IgG–measles» (Russia) in a licensed laboratory. According to enclosed regulatory and technical documentation for the quantitation of IgG antibodies to measles virus, the result of the analysis was considered negative if antibodies concentration in the sample was less than 0.12 IU/mL; it was positive if the concentration was equal or more than 0.18 IU/mL. Samples with doubtful concentrations of IgG antibodies to measles virus (in the range 0.12–0.17 IU/mL) were interpreted as negative, because this level of antibodies cannot be considered significantly protective.p < 0.001 and W = 0.88, p < 0.001, respectively). The Box-Tidwell linearity test revealed that the relationship between age and IgG levels (taking into account the respondent’s sex) was statistically significantly different from linear . Therefore, the local polynomial regression (LOESS regression) with the calculation of 95% confidence interval was used for the regression analysis; the assessment was carried out using the bootstrap method based on 9999 cycles . In order to compare the level of antibodies depending on the sex of the respondents, Mann-Whitney test was applied. A comparison of the level of antibodies between age groups within the same sex was carried out by the Mann-Whitney test with Holm correction. A comparison of the proportion of seronegative respondents depending on sex was carried out by the Chi-Square criterion, depending on the age group within one sex, by the Chi-Square criterion with Holm correction. In the case of cells with expected frequencies less than 5%, Fisher’s exact test was applied. If there were statistically significant differences, the relative risk and its 95% confidence interval was calculated. Descriptive statistics of quantitative data were represented by median and interquartile ranges of the quantitative proportions of respondents with considered characteristics in the group, with an indication of 95% confidence interval calculated by the Clopper-Pearson method. The absolute number of respondents with a trait from the total group size (n N) was also indicated. All calculations were performed in the free computing environment R (v.3.6.0).A preliminary analysis of the data showed that the distribution of IgG antibodies to measles virus was different from normal among both men and women ), nor in the part of seronegative respondents (13 [10–17]% (54/403) among men and 16 [14–18]% (214/1339) among women ).The analysis of the laboratory results of the study in which 1742 people aged 25–67 years (403 men and 1339 women) were involved, showed that the median of IgG antibodies level to measles virus in samples was 1.2 (0.4–3.2) IU/ mL; the proportion of seronegative respondents was 15 [14–17]%. Without taking into account the age of the respondents between men and women, no statistically significant differences were found neither in the level of IgG antibodies  IU/mL in the group of men and 1.3  IU/mL in the group of women  with the use of bootstrap; the analysis was carried out separately for men and women .From At the age of 40–49 years old, the level of IgG antibodies to measles virus among men is statistically significantly higher than among women; and at the age of 60 years and older, on the contrary, the level of antibodies is higher among women in comparison with men. Antibody levels increase with age in both men and women. However, if among men after 50 years old the growth rate slows down, at the age of 60 years and older compared to the age group of 50–59 years it does not change, then among women an increase in antibodies is observed in each age period.Furthermore, we consider the proportion of seronegative respondents depending on sex in each selected age group .2 = 7.0, p = 0.001).In the age group 40–49 years old, the proportion of seronegative women is statistically significantly higher than the proportion of seronegative men: 22 [18–26]% and 11 [6–18]%, respectively (χp = 0.16 according to Fisher).It should also be noted that a statistically significant decrease in the proportion of seronegative respondents among men is observed after the age of 40 years (with further stabilization) and among women after the age of 50 years (with a further decrease). Despite the differences in dynamics, the proportion of seronegative respondents among men and women over the age of 60 did not differ statistically: 5.4 [1.1–15]% and 2.0 [0.5–4.7]%, respectively  and the risk of measles outbreaks remains. However, it is interesting that there are differences in the levels of protective antibodies among men and women; the latter being more likely to work in health facilities (1339 women and 403 men in our study). In the analysis of sex differences on the detection of the level of IgG antibodies and the proportions of seronegative employees without taking into account the age of respondents, there were no statistically significant differences between men and women. However, in the distribution of health workers by age groups, there were differences in the levels of IgG antibodies to the measles virus: Among men aged 40–49, the level was statistically significantly higher than among women. Consequently, the relative risk of lack of protective IgG levels among women aged 40–49 is 2 [1–3.6] times higher than among men. At the age of 60 and older, in contrast, the level of antibodies is higher among women in comparison with men. It should be noted that with an age of 60 years and older, the proportion of people with low antibody levels decreases and the proportion of people with average levels of antibodies to measles virus increases among both sexes . Of cour2 = 7.0, p = 0.001).As for the proportion of seronegative to the measles virus persons, they are more often detected among women of the age group 40–49, making up 22%, compared to 11% in men of the same age  does not mean a complete loss of measles immunity, since the response of memory B-cells can quickly increase the synthesis of protective antibodies ; howeverOf interest are the results of the works of a number of authors, which are devoted to the study of sex differences in the formation of the immune response after vaccination against vaccine-preventable infections. Existing regression models show that the two most significant predictors of antibody production and effectiveness of vaccination are age at the time of vaccination and female sex . For exaThus, it can be assumed that sex differences of immune response can contribute to the difference in the pathogenesis of infectious diseases among men and women, and in the development of reactions to vaccine preparations. Sex differences in response to vaccination are observed among different age groups, ranging from infants to the elderly. In some articles, a higher level of post-vaccination antibodies to vaccine-preventable infections in women is associated not only with sex steroids (associated with estradiol) throughout life, but also with the expression of a X-linked gene located on the X chromosome, Tlr7» ,35,36. BThe study revealed that due to the start of mass vaccination against measles of the population of the Russian Federation in 1968, nowadays specific immunity at the protective level in adults, in particular among employees of a large hospital complex (aged 25–67), is detected in 85% of cases regardless of gender, and the lack of protection to the measles virus is noted in 15% of employees. However, among women aged 40–49, IgG antibody levels are recorded in values lower, and the proportion of seronegative to the measles virus is higher than among men. Significant differences are observed in the age group 60 years and older, in which, on the contrary, the level of IgG antibodies is higher among women in comparison with men. Despite the duration of preservation of protective antibodies, men have revealed a pronounced dynamics of their decline, regardless of the nature of their formation—vaccination or infection (before the era of vaccination 1968).


Studies comparing methodologies for fatty acids are very important, since they can influence the quality and final quantification of the lipid fraction. Objective—to compare different extraction methods for total lipids and to evaluate the effect of these methodologies on the quantitative composition of fatty acids in milk of lactating ewes raised in tropical pastures. The methodologies used were simple direct transesterification, using the HPLC grade organic solvent n-hexane, Bligh; Dyer  of chloroform-methanol-water). The fatty acid methyl esters were separated by gas chromatography coupled with a flame ionization detector (GC-FID). The results show that there was no significant difference (p <0.05) in the total lipid content between the extraction methods. However, the Bligh’s method; Dyer obtained the best yield of lipids to be extracted from ewe’s milk, since the volume with 15 mL of the extraction solution was able to identify 87% of the chromatographic peaks. It was also observed that ewe’s milk has a higher percentage of palmitic, stearic and oleic fatty acids, with percentages of 20.1%, 15.5% and 33.1%, respectively. Therefore, an extraction mixture with a volume of 15 mL used in this study may be an alternative to perform the extraction of milk fat from high lactating ewes in tropical pastures as a routine method, as it expresses the best lipid content of this food. Ewe’s milk has a chemical composition comprising proteins of high biological value and essential fatty acids, besides their mineral and vitamin content, which qualifies as a food of high nutritional value, also represents great importance in the diet for its characteristics of hypo allergenicity and digestibility because of the decreased fat cells .However, ruminant milk fat contains high levels of saturated fatty acids (SFA), approximately 2/3 of total milk fatty acids .In this way, studies have been developed to evaluate the effects of the lipid fraction of milk and its derivatives on human health, since the determination of lipids is important for biochemical, physiological and nutritional studies . The extTherefore, methodological comparison studies for fatty acids are very important, because some samples require special care to get the lipid fraction, given that factors such as co-extraction of non-fatty component lipids and unwanted oxidation may influence the quality and final quantification of the lipid fraction .In this sense, information on the nutritional quality of ewe’s milk is presented as a contribution to the search for alternatives for sheep farming and the strengthening of this production chain, since the literature on cow’s milk is constantly updated, whereas a few studies have been reported for ewes’ milk .Therefore the aim of in this study compares different extraction methods for total lipids and evaluates the effect of these methodologies on the quantitative composition of fatty acids in milk of lactating ewes reared in tropical pastures, with the hypothesis of validating the technique that best expresses the total lipid content of milk from lactating ewes reared in tropical pastures, by providing a method that ensures the efficiency and reproducibility of the results, since this generates subsidies for standardization of method in several laboratories.https://dx.doi.org/10.17504/protocols.io.b3xsqpne, and is included for printing as The protocol described in this peer-reviewed article is published on protocols.io, The milk samples used in this research were collected in the Sheep Farming Sector of the Federal University of Mato Grosso do Sul, at the School Farm of the Escola da Faculty of Veterinary Medicine and Animal Science. This experiment was carried out according to the ethical principles established by the National Experimental Control Council (CONCEA) and was approved by the Ethics Committee on Animal Use (CEUA) of the Federal University of Mato Grosso do Sul (UFMS—Protocol nº 1.119 / 2019).The experimental trial occurred during the lactation phase 60 days), and the ewes were milked from the seventh to the 60th postpartum day, using the direct collection method 0 days, a, with a Total lipid extractions were performed according to the direct transesterification method (method 1), using the organic solvent n-hexane grade HPLC. Since method 1 was evaluated with extraction and derivatization in a single step, that is, directly (in situ), the other methods  were performed with extraction and derivatization in different phases, using the  method, 2SO4, 10g NH4Cl and 300 mL of methanol) was added, repeating the water bath process for 2 min and cooling at room temperature. Then, 2 mL of the saturated NaCl aqueous solution was added and agitated thoroughly 4 mL of hexane was added and vortexes again for 30 s. The solution was then centrifuged for 5 min at 2000 rpm and the supernatant removed, for it was where the formed fatty acid methyl esters (FAMEs) were found. After the procedure, the sample was used to analyze the FA profile by gas chromatography coupled to a flame ionization detector (GC-FID).Total lipid extractions were performed in duplicate, and for a first direct attempt at extraction and derivatization (extraction method 1 –using HPLC grade n-hexane), the following steps were performed: in a 15 mL centrifuge tube with screw cap and Teflon septum, 1 mL of sample ewes’ milk was added to 2 mL of 0.5 M NaOH solution in methanol (chromatography grade), kept under heating in a water bath (60 C) for 5 min, and cooled to reach room temperature. Subsequently, 3 mL of the esterifying solution  was added, repeating the same heating method in the water bath at the same temperature, but left only 2 min, and cooled at room temperature again.After completion of the extraction process, the samples of methods 2, 3 and 4  were submitted to the derivatization process according to . Into thThen 2 mL of a saturated NaCl solution was added, and the content was agitated thoroughly 4 mL of hexane was used to extract the fatty acid methyl esters. The sample was then centrifuged for 5 min at 2000 rpm and an aliquot of the supernatant was removed and placed in a 2 mL bottle with a lid for further analysis by GC-FID. A summary of the experimental procedures for the extraction of lipids from ewe’s milk is shown in The FAMEs were separated and determined by a gas chromatograph  equipped with a fused silica capillary column  and flame ionization detector . The carThe results were subjected to analysis of variance (ANOVA), and a comparison of treatment means was done by the Tukey test, adopting a significance level of 5%. Statistical analyses were performed using SAS Statistical Analysis Systems  version On the other hand, when comparing method 4 of this work with previous studies. It is possible to confirm that the ewes’ milk matrix has the FAs C16:0 , C18:0 (Stearic acid) and C18:1n9c (Oleic acid) as major components in their composition .Although there are analytical methods for extracting lipids recommended for each class of food, these are under constant technical evaluation because of the need to consider the cost, extraction efficiency, toxicity, availability and quality of the final product . FurtherThus, based on the methodologies described by previous studies , 14–16, In this sense, the experimental conditions for the extraction of lipids were based on different methods to determine the proportions of hexane and chloroform–methanol that would yield the greatest quantitative extraction of lipids.In the in situ method, in which there is a single step of direct extraction and esterification of lipids, we observed the identification of 15 FAs, with oleic acid in the greatest proportion. However, this method identified an average of 53.47% of chromatographic peaks; this is because non-polar solvents  do not have the same efficiency in extracting lipids as polar solvents . A seconAs for the different dilutions  chloroform–methanol–water extraction solution) used in this test, it was observed that the percentage of identifying FAs increased with the volume of the extraction solvent. Thus, the method that used 15 mL of extraction solution identified on average 87.21% of the chromatographic peaks and presented the lowest percentage of unidentified compounds (12.79%).This good yield observed for total lipids, provided by the method of Bligh and Dyer can be explained by the wide range of polarity presented by the mixture of solvents used. Thus, an advantage of the use of chloroform and methanol is that they are more polar than n-hexane and, thus, there is a more efficient extraction of polar and non-polar lipids .GC-FID is the most widely used separation and determination method, as it is easy to maintain and cheap (when compared to other detectors), and FAMEs can easily be identified and detected. The identification of FAMEs is made based on the elution order and retention time of these analysts .However, GC-FID has a disadvantage, in that it is limited to volatile and thermally stable samples, so for non-volatile analysts, a previous derivatization step is necessary in order to transform them into volatile compounds . FurtherBased on this perspective, after the extraction process, samples from methods 2, 3 and 4 were subjected to derivatization, with the intention of the triglycerides being transformed into FAMEs. The programming was started at a temperature lower than the boiling point of the most volatile component present in the sample, after the heating rate of 10°C/min, and the second temperature level of 140°C assisted in the elution of fatty acid esters with an intermediate boiling point, that is, not as volatile as the previous ones. The final temperature used was 250°C to allow methyl fatty acid esters with a higher boiling point to be eluted .In this way, through GC-FID it was possible to determine the chromatographic profile of the constituent fatty acids in sheep’s milk: about 68.65% of the total FAs found in the milk was palmitic acid 20.05%, 15.46% stearic acid and 33.14% oleic acid. The total of SFAs represented 50.15% of the total of FAs, while the UFA content was 37.02%. These results agree with other authors  who inveWhen comparing method 4 (15 mL of extracting mixture) in this work with previous studies, it was possible to observe that the analysed milk samples had a greater level of UFAs. Similarly, the results obtained demonstrate that oleic acid (C18:1n9c) presented a higher percentage when compared to literature values.The aim of this study was to compare different extraction methods for total lipids and evaluate the effect of these methodologies on the quantitative composition of FAs  in milk from lactating sheep raised in tropical pastures and provide a standardized protocol and easy-to- for extracting total lipids from the milk of lactating ewes raised in tropical pastures.The methodology chosen for extracting the lipid fraction from the milk matrix of lactating ewes raised in tropical pastures may affect the yield of extracted lipids. In this study, the solvents used in the extractions influenced the extraction efficiency. Thus, based on the results of this work, when the lipid fractions were extracted, the use of 15 mL of extraction mixture was the one that presented the best results, which demonstrates the efficiency of the Bligh and Dyer methodology, since the extracts obtained with hexane presented a lower yield of total lipids in the analysed samples.S1 File(PDF)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


K-means clustering algorithm has high quality and short time for CT image segmentation. The overall incidence rate of complications in minimally invasive group was 10%, lower than that in conservative group (25%) (P < 0.05), and the length of hospitalization in minimally invasive group was longer than that in conservative group (P < 0.05). The hematoma volume of minimally invasive group was 16.5 ± 2.4 mL on the 3rd day after operation, and that of conservative group was 27.4 ± 1.8 mL. There was significant difference between the two groups (P < 0.05). In addition, CT showed that the hematoma reduction degree of minimally invasive group was higher than that of conservative group, and the hematoma dissipation rate was higher than that of conservative group on the 3rd and 7th day (P < 0.05). The good MRS score in minimally invasive group was 3.15 times that in conservative group, and the good ADL score was 1.6 times that in conservative group, and there was significant difference in the total score between the two groups (P < 0.05). Minimally invasive puncture drainage is better than conservative treatment in the clearance of hematoma, which is conducive to the recovery of neurological function and daily life of patients with cerebral hemorrhage and is of great help to the prognosis of patients.The objective of this study was to explore the effect of minimally invasive puncture drainage under unsupervised learning algorithm and conservative treatment on the prognosis of patients with cerebral hemorrhage. Fifty patients with cerebral hemorrhage were selected as the research objects. The CT images of patients were segmented by unsupervised learning algorithm, and the application value of unsupervised learning algorithm on CT images of patients with cerebral hemorrhage was evaluated. According to the treatment wishes of the patients themselves and their authorizers, they were divided into 30 patients with cerebral hemorrhage in the minimally invasive group and 20 patients with cerebral hemorrhage in the conservative group. The incidence rate of complications of cerebral hemorrhage, the length of hospitalization of the two groups, hematoma volume at admission, 3 days and 7 days after operation, and the hematoma dissipation rate on the 3rd and 7th day after operation were used as the evaluation index of therapeutic effect. MRS and ADL scores were used as prognostic indicators. The results show that  The definition of cerebral hemorrhage refers to the cerebral parenchymal hemorrhage caused by nontrauma. It is a common brain disease in neurology and belongs to stroke subtype . CerebraAlthough the traditional craniotomy evacuation of hematoma can remove hematoma, but because the trauma is relatively large, more postoperative complications occur to patients with subsequent secondary injury , 9. The K-means clustering algorithm is one of the most classical clustering algorithms. The K-means clustering algorithm can segment the lesion of the imaging image. Through the reasonable mining of the image data and the correct analysis of the lesion area, the three-dimensional framework of the medical image of the lesion or the feature area can be reconstructed to assist clinicians in formulating more accurate treatment plans [With the rapid development of computer technology and medical information, unsupervised learning algorithm plays a great role in clinical application in helping imaging doctors to mine more in-depth and detailed imaging information of patients. It can deeply analyze image data and has been applied to the prediction of disease incidence and prognosis. Unsupervised learning algorithm is generally divided into clustering algorithm and dimension reduction algorithm, including clustering algorithm, hierarchical clustering algorithm, density-based clustering kernel function algorithm, density-based clustering deep neural network algorithm, principal component analysis, independent component analysis, distributed domain embedded algorithm, and other algorithms. However, the nt plans .K-means clustering algorithm in CT images of patients with cerebral hemorrhage was evaluated. The effects of the two groups on the improvement of cerebral hemorrhage and the prognosis of patients on the 3rd and 7th day after admission were compared, so as to provide reference for doctors to choose reasonable treatment methods for patients in clinical treatment.Based on this, the patients with cerebral hemorrhage were grouped by minimally invasive puncture drainage and conventional conservative treatment through retrospective analysis. The application value of P > 0.05), which can be used for subsequent comparison.Chinese Guidelines for the Diagnosis and Treatment of Cerebral Hemorrhage, and the diagnosis was confirmed as cerebral hemorrhage by imaging examination; (3) all patients had a history of hypertension; (4) all patients received treatment for the first time at admission, and the onset time was less than 24 hours; (5) the degree of disease can accept conventional conservative treatment; (6) the maximum amount of brain bleeding was 30 mL; (7) no liver, kidney, and other important organ dysfunction diseases were present.  Inclusion criteria: (1) patients in the two groups were aged from 26 to 59 years; (2) the diagnosis was confirmed as cerebral hemorrhage according to the 2019 Edition of   Exclusion criteria: (1) the patients were aged under 26 or over 60 years; (2) the presence of serious coagulation dysfunction or long-term use of anticoagulant drugs; (3) there was another secondary cerebral hemorrhage, such as cerebral hemorrhage caused by trauma, aneurysm, brain tumor, and stroke; (4) the patient's pathological data and imaging data within 24 hours of hemorrhage were incomplete.50 patients with cerebral hemorrhage admitted to the Department of Neurosurgery of XX Hospital from January 2020 to July 2021 were selected as the research subjects. According to the patient himself and the patient's authorization, 50 patients were randomly divided into minimally invasive group and conservative group. In the minimally invasive group, there were 30 cases, 18 males and 12 females, aged between 26 and 59 years, with an average age of 42.5 years. In the conservative group, there were 20 cases, 11 male patients, 9 female patients, aged between 29 to 60 years, with an average of 44.5 years old. Consciousness level standard was divided into fully awake, drowsy or hazy, and drowsy. There was no significant difference between the two groups in general data, including age, gender, onset time of cerebral hemorrhage, history of hypertension, bleeding volume, and consciousness status classification  the incidence of complications in two groups of patients, as this study specifically refers to intracranial infection, pulmonary infection, and bleeding; (2) length of hospital stay of the two groups; 3) the hematoma dissipation rates of the two groups on the 3rd and 7th postoperative days calculated as shown in  the hema can be uAll patients were followed up by telephone and regular outpatient. The prognosis was evaluated by modified Rankin Scale (MRS) and activities of daily living (ADL). The MRS score level and ADL score level are scored according to s), and the independent t test is used for the comparison between the two groups. The counting data are expressed in percentage (%). When P < 0.05, it is considered that the difference is significant and statistically significant.All data of this study are analyzed by SPSS20.0 statistical software. The measurement data are expressed as mean ± standard deviation (P > 0.05), as shown in In this study, 50 patients with cerebral hemorrhage were selected as the research object. There were 30 cases in the minimally invasive puncture drainage group and 20 cases in the conventional conservative treatment group. According to the factors that may affect the prognosis of cerebral hemorrhage, the factors of the two groups were compared. The results showed that there was no significant difference between the two groups . The incidence of complications in the minimally invasive group was 10%, lower than 25% in the conservative group (P < 0.05), but the independent incidence rate of complications was not significant.The length of stay in the minimally invasive group was 10 ± 1.2 days and that in the conservative group was 18 ± 2.4 days as shown in P < 0.05). The hematoma dissipation rates of the two groups on the 3rd and 7th day after operation were compared. According to the figure, the hematoma dissipation rate in the minimally invasive group was 48.9% on the 3rd day after operation, the hematoma clearance rate on the 7th day was 83.6%, the hematoma dissipation rate in the conservative treatment group was 16.6% on the 3rd day, and the hematoma dissipation rate on the 7th day was 33.5%. The hematoma dissipation rate in the minimally invasive group was higher than that in the conservative treatment group on the 3rd and 7th day, and the difference was statistically significant (P < 0.05). Moreover, through CT scanning, it was found that the hematoma volume of minimally invasive group and conservative group was significantly smaller than that of admission on the third day after operation, but the reduction degree of minimally invasive group was higher than that of conservative group, as shown in The volume of craniocerebral hematoma was compared between the two groups at admission, the third day after operation, and the seventh day after operation. The results are shown in P < 0.05).The prognosis of the two groups was evaluated by MRS score and ADL score. In this study, MRS score is ≤2, ADL score is grade I-II (70∼100), and the prognosis is good. The results are shown in In China, cerebral hemorrhage patients account for about 48% of cardiovascular and cerebrovascular disease cases. Because of the rapid onset of cerebral hemorrhage and the critical condition at the time of onset, the annual mortality of cerebrovascular patients in China has reached 55% –15. EpidK-means clustering algorithm is commonly used in clustering algorithm. It is a clustering algorithm based on Euclidean distance. The basic principle is to use Euclidean distance to cluster the samples in the space as the cluster center and update the values of each center step by step through continuous iterative method, so as to output the best results. It has been found that the fuzzy K-means clustering algorithm can effectively divide the gray matter, cerebrospinal fluid, and white matter in the magnetic resonance brain image [K-means clustering algorithm has high quality and short average segmentation time for CT images of patients with cerebral hemorrhage.At present, many researches apply intelligent algorithms to medical image processing. For example, Hu et al.  realizedin image . It is fThe main pathogenesis of cerebral hemorrhage is the formation of hematoma in the brain. Hematoma will always exist in the brain. The sterile products of hematoma have toxic reactions to brain tissue. Toxic substances induce changes in the elasticity of cerebral vascular wall, resulting in cell edema and elevated intracranial pressure. The existence of hematoma bleeding points will destroy normal neural function. This study found that the hematoma volume of minimally invasive group was significantly smaller than that of conservative group on the third day after operation. Through CT examination, the hematoma volume of minimally invasive group and conservative group was significantly smaller than that of admission on the third day after operation, but the reduction degree of minimally invasive group was higher than that of conservative group. The hematoma dissipation rate of minimally invasive group on the third day after operation was 48.9%, and the hematoma clearance rate on the seventh day was 83.6%. Both results were higher than that of conservative treatment group at 16.6% on the third day and 33.5% on the seventh day.P < 0.05). The total incidence rate of complications in the minimally invasive group was 10%, lower than that in the conservative group (25%) (P < 0.05). Based on this, it is suggested that minimally invasive puncture drainage can effectively and quickly remove craniocerebral hematoma, alleviate the increase of intracranial pressure caused by hematoma, and reduce the occurrence of complications [For the treatment of cerebral hemorrhage, conservative drug treatment and minimally invasive hematoma debridement are often used in clinics. For elderly patients with cerebral hemorrhage, traditional craniotomy is not the best treatment. Compared with conservative drug treatment, minimally invasive puncture drainage has better therapeutic effect, is suitable for most patients, and has a better prognosis . The stuications .P < 0.05). It is proved that, compared with conservative treatment, minimally invasive surgery can significantly reduce the incidence rate of complications, shorten the length of stay, reduce the volume of hematoma, accelerate the evacuation of hematoma, improve the neurological function recovery and daily living ability of patients with cerebral hemorrhage, and greatly help the prognosis of patients with cerebral hemorrhage.This study found that the proportion of patients with good MRS score prognosis in the minimally invasive group was 3.15 times that in the conservative group. The ADL score of minimally invasive group was 1.6 times that of conservative group, and there was significant difference in the total score between the two groups (Minimally invasive puncture drainage is better than conservative treatment in the clearance of hematoma, which is conducive to the recovery of neurological function and daily life of patients with cerebral hemorrhage and is of great help to the prognosis of patients.