scholarly journals Genetic variation determination and interbreed differentiation of two ukrainian dairy cattle breeds using microsatellite loci of DNA

2018 ◽  
Vol 5 (1) ◽  
pp. 51-56
Author(s):  
A. Shelyov ◽  
K. Kopylov ◽  
S. Kramarenko ◽  
O. Kramarenko

Aim. The aim of our work was to investigate the interbreed differentiation of Ukrainian Red-and-Motley and Black-and-White dairy cows based on the results of the analysis of allelic and genotypic polymorphism of DNA microsatellites. Methods. Genotyping of 88 DNA samples of two most numerous dairy cattle breeds in Ukraine – Ukrainian Red-and-Motley and Black-and-White dairy cows – was conducted by 10 loci, recommended by the International Society for Animal Genetics (ISAG). The analysis of allelic and genotypic polymorphism was performed using parametric and non-parametric methods. Results. Informative value of DNA microsatellites as markers of genetic processes, which take place in the populations of domestic animals, was assessed. Conclusions. The investigated breeds demonstrate a reliable level of genetic differentiation with a high level of similarity.

2017 ◽  
Vol 4 (1) ◽  
pp. 74-78 ◽  
Author(s):  
A. Shelyov ◽  
K. Kopylov ◽  
S. Kramarenko ◽  
O. Kramarenko

Aim. The aim of our research was to analyze the population-genetic processes in different dairy breeds using highly polymorphic molecular and genetic markers (DNA microsatellites). Methods. We used 10 loci recommended by the International Society for Animal Genetics (ISAG) to analyze 88 DNA samples of the two most abundant dairy breeds of cattle in Ukraine – Ukrainian red-motley dairy and Ukrainian black-and-white dairy breeds. Using formulae that related the expected linkage disequilibrium (LD) to the effective population size (Ne), Ne was estimated. Results. The work presents the results of the study on genetic processes in the populations of Ukrainian red-and-motley breed using 10 microsatellite loci of DNA. It was shown that, being highly polymorphic multilocus genetic systems, microsatellites of DNA are highly informative markers of populationgenetic processes, occurring in the populations of cattle. Conclusions. The studied populations of Ukrainian dairy cattle breeds are impacted by population-genetic and genetic-automatic processes. In particular, the effect of the latter on Ukrainian red-and-motley dairy breed was noted. These animals had notable signifi cant loss of rare alleles and the manifestation effect of “bottle neck”. The values obtained testify to a low level of inbredness in these populations. The effective population size in the studied Ukrainian dairy cattle breeds was estimated in the approximate range of 397–555 heads which testifi ed to a favorable condition of the population of Ukrainian red-and-motley dairy cattle and a critical condition of the Ukrainian black-and-white dairy breed.


2016 ◽  
Vol 52 ◽  
pp. 6-12 ◽  
Author(s):  
M. V. Gladiy ◽  
G. S. Kovalenko ◽  
S. V. Priyma ◽  
G. A. Holyosa ◽  
A. V. Tuchyk ◽  
...  

The main goal of dairy breeds selection should be improving breeding and productive qualities of animals under modern conditions. The majority of farms, using native breeds to produce milk, has created optimal conditions for keeping and feeding, selection and matching, growing of replacements etc. Further improvement of created native dairy breeds for economically useful traits occurs at total use of purebred Holstein bulls (semen) of foreign selection. In order to realistically assess milk productivity (milk yield, fat content in milk and fat yield) of Ukrainian Black-and-White and Red-and-White Dairy cows should be conducted a comparative analysis of Holstein cows under the same conditions of feeding and keeping. It was established that Ukrainian Red-and-White Dairy cows were characterized by the highest milk yields for 305 days of all lactations, taken into account, the among three investigated breeds. Their milk yield during the first lactation was 5933 kg of milk, during the second – 6393 kg, the third – 6391 kg and during higher lactation – 6650 kg. Ukrainian Black-and-White Dairy cows were second by milk yield (except for the second lactation), during the first lactation – 5932 kg of milk, the third – 6462 kg and higher – 6541 kg, and Holstein cows were third, during the first lactation – 5794 kg of milk, the second – 6381 kg, the third – 6335 kg and higher – 6469 kg. The fat content was almost the same and varied within 3.49-3.58% in milk of Ukrainian Red-and-White Dairy cattle, 3.50-3.60% in milk of Ukrainian Black-and-White Dairy cattle and 3.50-3.56% in Holsteins’ milk. The difference between the breeds was within 0.01-0.04%. All the investigated breeds had predominance in fat yield for three lactations over standards of these breeds: Ukrainian Red-and-White Dairy cows from 75.1 to 93.4 kg, Ukrainian Black-and-White Dairy cows – 75.1-89.0 kg respectively and Holstein cows – 41.9-60.2 kg. It was found different level of positive correlation between milk yield and fat yield in all the cases and high correlation (r = 0.604-0.921, P < 0.001) in five cases (41.7%) Negative correlation coefficients indicate that selection of animals to higher milk yield in the herd will decrease the second trait – fat content in milk. Positive and highly significant correlation between milk yield and fat yield indicates that selection of cows in the herd to higher milk yields will increase fat yield. It was revealed that bulls were among the factors impacted the milk productivity (milk yield, fat content, fat yield) of three investigated breeds. So, the force (η²x) of father’s impact on milk yield was15.4-47.9%, fat content – 22.0-43.4% and fat yield – 14.9-47.7% taking into account a lactation and a breed. The force of lines impact (η²x) was second; it was on milk yield 6.1-24.5%, fat content – 4.1-17.1 and fat yield – 5.8-23.5%. The force of breeds impact (η²x) was last; it was on milk yield 0.3-2.9%, fat content – 0.2-0.3% and fat yield – 0.6-2.7%. So, the comparative studies of milk productivity of Ukrainian Red-and-White and Black-and-White Dairy cattle with Holsteins indicate that under similar conditions of feeding and keeping, these native breeds can compete with Holstein cattle. The milk yield for 305 days of higher lactation was 6650 kg of milk in Ukrainian Red-and-White Dairy cows, 6541 kg in Ukrainian Black-and-White Dairy cows and 6469 kg in Holsteins. It was found the inverse correlation r = -0.025-0.316 between milk yield and fat content in milk in most cases. Selection and matching of animals in the herd should be carried out simultaneously on these traits. It was found positive repeatability of milk yields between the first and second, the third and higher lactations (rs = 0.036-0.741), indicating the reliability of forecasting increase in milk productivity during the next lactations in all herd. Bulls have the greatest impact (η²x) on milk productivity among the factors taken into account: milk yield – 15.4-47.9%, fat content in milk – 22.0-43.4% and fat yield – 14.9-47.7%.


2018 ◽  
Vol 51 ◽  
pp. 112-118
Author(s):  
V. V. Machulnyi

Milk yield of cows in breeding herds, Cherkassy region, for last completed lactation averaged 6442 kg of milk with 3.62 % fat content. Currently the question of interconnection of cows’ milk production and reproductive capacity is especially important, because the significant increase in milk yields makes minimize calves. The successful conduct of breeding had set the goal to detect the best options for combining milk production and reproductive ability of animals.To characterize the level of milk production including reproductive ability of cows we used indicator of daily average milk yield per one day of periods between calving (PBC). The best service period can be considered period of 51-90 days, that allows you to get one calf per cow per year (average PBC is 351 days) and maintain high milk production (6431 ± 39.6 kg during 305 days of lactation). With this service period all indicators of milk production and reproductive ability are at high level. Reduced service period below recommended levels will reduce the duration of lactation and milk production, and increasing service period will increase the duration of lactation with a simultaneous reduction in milk production for 305 days. Herewith deterioration of reproductive ability is marked and manifested in the increase of the insemination index, decline in the reproductive capacity, increase of periods between calving and as a result, reduce of calves and culling barren cows.Analysis of genealogical structure of the studied herds points to low diversity (Ukrainian Black-and-White dairy cows belong to 6 lines and Ukrainian Red-and-White dairy cows – to 8 ones) with high affinity (by Chief 1427381.62 to R. Sovereign 198998 bloodline, by Starbuck 352790.79 to Elevation 1491007 and Ideal1013415 bloodlines).Genealogical structure of the herds is saturated a lot of bulls genealogical groups of Holstein cattle: Chief 1427381 – 11 bulls in the array of Ukrainian Black-and-White dairy breed and 5 ones in Ukrainian Red-and-White dairy breed, Starbuck 352790.79 – 10 sires in the array of Ukrainian Black-and-White dairy cattle and 3 ones in Ukrainian Red-and-White dairy breed, Elevation 1491007.65 – 4 sires in the array of Ukrainian Black-and-White dairy cattle and 3ones in Ukrainian Red-and-White dairy breed.Currently the sires of Chief 1427381.62 (7712-3,76-289,9-3,14-242,1), Starbuck 352790 (7857-3,87-295,4-3,07-241,2), Jocko Besne 694028588.94(6687-3,88-259,5-3,03-202,6), and Bell 1667366 bloodlines (61341-3,95-242,3-3,02-185,2) among sires of Ukrainian Black-and-White dairy cattle and the sires of Cavalier 1620273.72 (7285-3,98-289,9-3,08-224,4), Chief 1427381.62 (7522-3,76-282,8-3,00-225,7),Starbuck 392405 (7667-3,87-296,7-3,00-230,0), and Elevation 1491007.65 bloodlines (7544-3,80-286,7-3,5-230,0) among sires of Ukrainian Red-and-White dairy breed are classified to a highly productive and promising for breeding and wider use by productive qualities.After the first insemination the lowest proportion of fertilized cows is among offspring belonging to Chief 1427381 bloodline which is only 38 % among both breeds. However, the milk yield of breeding stock belonging to this bloodline is 7712 kg with 3.76 % fat content for Ukrainian Black-and-White dairy cows and 7522 kg of milk with 3.76 % fat content for Ukrainian Red-and-White dairy cows. Daughters belonging to Elevation1491007, Valiant 1650414, and Hanover 1629391 bloodlines with yields of 4566-5478 kg of milk per lactation had a part of fertilized cows, after the first insemination, at the level of 41-49 %. Fertilization after the first insemination above 50% was in daughters belonging to Cavalier 1620273.72 (52%), Starbuck 392405(50-55 %), Bell 1667366(56%) and Jocko Besne 694028588.94 bloodlines (64 %). The level of performance of these animals for milk yield ranged from 6134 kg to 7857 kg.Heritability coefficients of milk yield and duration of service period were low (0.118-0.289). A positive correlation can be explained by the fact that extension of service period (to a certain level) leads to growing number of milking days (duration of lactation) and defers a decrease in productivity of cows by physiological reasons, namely as a result of pregnancy. Low rates of heritability indicate little genetic diversity of traits and the significant influence of physiological and environmental factors in their formation.As Ukrainian Black-and-White and Ukrainian Red-and-White dairy breeds have a high proportion of heredity of Holstein breed which has genetically large fetus, there is need to study the characteristics of nature passing calving in cows of these breeds. Itis found the number of first-calf heifers which needed help was 52.4%. Animals calve alone in 47.6 % of all cases. Calving was without complications if the live weight of calves not exceeding 6-7% of mother’s weight. The cows calved without help of staff exceeded by 11.7% of the width of the pelvis in the buttocks, 7.5 % of the width of the ilium, and 7.1 % of its oblique length compared with animals of same age.


2016 ◽  
Vol 52 ◽  
pp. 31-36
Author(s):  
V. S. Kozyr ◽  
A. D. Hekkiyev

It was proved that features of lactation curves of cows should be considered at developing breeding programs in dairy cattle breeding, contributing to an objective assessment of a genotype and thus, use of genetic and mathematical methods would increase probability of predicting performance for dairy herd.


Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1680
Author(s):  
Nattakan Ariyaraphong ◽  
Nararat Laopichienpong ◽  
Worapong Singchat ◽  
Thitipong Panthum ◽  
Syed Farhan Ahmad ◽  
...  

Domestication and artificial selection lead to the development of genetically divergent cattle breeds or hybrids that exhibit specific patterns of genetic diversity and population structure. Recently developed mitochondrial markers have allowed investigation of cattle diversity worldwide; however, an extensive study on the population-level genetic diversity and demography of dairy cattle in Thailand is still needed. Mitochondrial D-loop sequences were obtained from 179 individuals (hybrids of Bos taurus and B. indicus) sampled from nine different provinces. Fifty-one haplotypes, of which most were classified in haplogroup “I”, were found across all nine populations. All sampled populations showed severely reduced degrees of genetic differentiation, and low nucleotide diversity was observed in populations from central Thailand. Populations that originated from adjacent geographical areas tended to show high gene flow, as revealed by patterns of weak network structuring. Mismatch distribution analysis was suggestive of a stable population, with the recent occurrence of a slight expansion event. The results provide insights into the origins and the genetic relationships among local Thai cattle breeds and will be useful for guiding management of cattle breeding in Thailand.


Biologija ◽  
2021 ◽  
Vol 67 (3) ◽  
Author(s):  
Romualdas Lapickis ◽  
Loreta Griciuvienė ◽  
Asta Aleksandravičienė ◽  
Indrė Lipatova ◽  
Algimantas Paulauskas

Large numbers of different scientific studies are conducted to preserve the breeds and improve the existing ones by introducing the variability of the plate genes, which best shows the phenotypic characteristics that can improve the health of dairy cattle and the quality of their production. The main purpose of this study was to perform an analysis of genetic variability of Lithuanian cattle breeds. Three subpopulation groups were studied: Lithuanian Black and White (95 individuals), Lithuanian Red (49), and Lithuanian White and Red (48). Bovine genetic material was genotyped using a total of 11 fluorescent microsatellite primers to estimate genetic variability. All loci presented a high degree of polymorphism and a total of 292 different alleles (Na) were detected. Thirty-two private alleles were detected in all evaluated subpopulations. After completing pairwise population assignment, which is based on the distribution of allelic frequencies, three populations showed the tendency to group into three separate clusters. However, the performed Principal Coordinates Analysis (PCoA), which is based on genetic distances, showed no anticipated clear grouping. Bayesian structure analysis revealed three genetic clusters. Analysis of FST (0.001–0.027) and Nei genetic distance (0.029–0.084) revealed that the genetic diversity of inter subpopulation in cattle groups was estimated to be lower than the genetic diversity of intra subpopulation.


2019 ◽  
Vol 86 (4) ◽  
pp. 436-439 ◽  
Author(s):  
Ivan Holko ◽  
Vladimír Tančin ◽  
Martina Vršková ◽  
Kristína Tvarožková

AbstractThe data presented in this research communication represent the first comprehensive report on the prevalence of udder pathogens and their resistance in Slovak dairy cattle breeds. A total of 633 milk samples from 42 herds were tested. The most frequently isolated pathogens were coagulase negative staphylococci CNS or non-aureus staphylococci (NAS) followed by Escherichia (E.) coli, Staphylococcus (S.) aureus, Streptococcus (Str.) uberis and Streptococcus (Str.) agalactiae. Generally, isolated pathogens showed common resistance to aminoglycosides (streptomycin, neomycin), which are the most commonly used antibiotics to treat mastitis in Slovakia.


2018 ◽  
Vol 51 ◽  
pp. 205-213 ◽  
Author(s):  
T. Suprovich ◽  
Т. Karchevska ◽  
R. Kolinchuk ◽  
V. Mizyk

The main objective of research "BoLA and disease" is the need to develop approaches and obtain reliable criteria which would allow to judge about animal genetic predisposition to the disease and about change of its immunological status in the development of pathological process. Genes of class II of main histocompatibility complex have the greatest association to diseases. Now 54 alleles of BoLA-DRB3.2 have been described by PCR-RFLP. The high level of allelic diversity of the gene is caused by necessity of tying a wide range of foreign antigens, which leads to the possibility of its use as a marker for various diseases of cattle. This article presents the results of detecting alleles of BoLA-DRB3.2 gene, which have the expressed relationship with the disease of Ukrainian Black-and-White Dairy cows on necrobacteriosis and can be used as DNA markers of this disease. Diagnosis of necrobacteriosis was set at the basis of clinical, pathological and epizootic data and laboratory results. The blood samples were taken from 114 cows, 43 of which had the disease. Spectrum of alleles of exon 2 of BoLA-DRB3 gene was studied by PCR. 54 alleles were determined in total. Alleles, which have a close relationship with susceptibility or resistance to necrobacteriosis and can be used as DNA markers, were established on indicators of frequency and relative risk (RR) with test on Pearson criterion (χ2). 32 alleles were determined in the experimental group of animals. There were seven alleles with a frequency greater than 5%. The most often determined allele of BoLA-DRB3.2 was *24. It is present in 18% of the animals. And often determined alleles were *22 (7,9%) and *28 (7,5%). Limit higher than 5% was for alleles *08 and * 09 (6,1%), *03 and *16 (5,3%). The lowest frequency of detection was for alleles *06, *25, *31 and *41 (0,4%). Alleles of BoLA-DRB3.2*24 (16,9%), *22 (10,6%), *28 (8,5%), *03 (7,7%), *08 and *10 (6,3%) were often determined in the group of healthy cows. Alleles *06, *14, *19, *25 and *51 weren’t determined in this group. The animals with necrobacteriosis had often alleles *24 (19,8%), *16 (12,8%), *23 (8,1%), *8, *10 and *28 (5,8%). Alleles *01, *11, *21, *31 and *41 weren’t in general. In the three experimental groups 8 alleles were determined with a frequency of over 5% (all herd, healthy and diseased animals respectively). There are four alleles among them presented in all three samples: *08, *10, *24 and *28. Two "informative" alleles (*03 and *22) were found in every the 20th animal simultaneously in two groups of cows – healthy animals and in the total sample. Also two "informative" alleles *16 and *23 were simultaneously in the diseased cows and in the total sample. 11 alleles have significant association with susceptibility or resistance to necrobacteriosis on criterion of relative risk. There are 4 alleles *16 (24,1%), * 18 (5,25%), *25 (5,04%) and *23 (4,41%), indicating the relationship with disease (RR ≥ 2). Four alleles of BoLA-DRB3.2 are significant on criterion χ2 and have a sufficient test of validity for the studied biological objects. Allele *16 shows a very high level test of validity P = 0,999 (χ2 = 16,6). Three alleles *03 (4,93), *23 (4,86) and *22 (4,03) have a minimum acceptable test of validity for χ2 for P = 0,95. 8 alleles: *3 (-7,7), *21 (- ,44), *36 (-3,87), *22 (-3,57), *12 (-3,18), *1 and *11 (-3,13) and *26 (- 2,51) indicate neсrobaсteriosis resistance (RR ≤ -2). Allele would be associated with the disease if the condition performed RR ≥ 2 і χ2> 3,8. There are two such alleles: *16 (RR = 24,1; χ2 = 16,6), *23 (RR = 4,41; χ2 = 4,86). Also "negative" alleles on risk of disease manifest are *18 (5,25) and *25 (5,08), but with insufficient validity of Pearson criterion (respectively 2,45 and 1,66). Allele would be associated with the resistance to disease if the condition performed RR ≤ -2 і χ2> 3,8. There are 2 alleles associated with resistance to necrobacteriosis: *03 (RR = -7,7; χ2 = 4,93) and *22 (RR = -3,57; χ2 = 4,03). Also six alleles (* 01, * 11, * 12, * 21, * 26 and * 36) detected resistance to neсrobacteriosis on high level of relative risk, but with insufficient validity. It should be noted, that allele BoLA-DRB3.2*22, which proved to be a "positive" marker of resistance to necrobacteriosis, has a strong correlation with resistance to mastitis in cows of Ukrainian Black-and-White Dairy (RR = -2,52; χ2 = 5,02) and Ukrainian Red-and-White Dairy breeds (RR = -4,66; χ2 = 11,11) in previous studies. The study of the distribution of alleles of exon 2 of BoLA-DRB3 gene at the Ukrainian Black-and-White Dairy cows, which were healthy and diseased by necrobacteriosis, revealed the alleles which had a close relationship with penchant to this disease (* 16 and * 23) and two alleles associated with resistance (* 03 and * 22). Given the fact that the research was conducted directly on animal blood DNA the detected alleles BoLA-DRB3 should be used as DNA markers in the analysis of susceptibility or resistance to necrobacteriosis of cows.


1967 ◽  
Vol 9 (4) ◽  
pp. 491-500 ◽  
Author(s):  
Alan Jamieson ◽  
Alan Robertson

1. An analysis has been made of the association between transferrin genotype and progeny test for both milk yield and fat content in a sample of 879 bulls of five dairy cattle breeds, the majority used in artificial insemination in Great Britain. Although significant effects were found in only one of the ten analyses when the breeds were considered separately, a significant effect on milk yield was found when the results from all breeds were combined. A significant effect was not found for fat content, although the effects of the allele substitutions were in the opposite direction to that on milk yield in five cases out of six.2. Although our results were in the same direction as those of Ashton, the effects were smaller. It was estimated that the locus accounted for 1·1% of the genetic variation in yield and 0·4% of the genetic variation in fat content.3. Some information based on the production records of 178 experimental cows with known genotypes was also analysed. For milk yield, the pattern of effects was similar to that in the sire data though the differences were much larger. The variance removed by fitting constants was not statistically significant for milk yield or fat content. It was estimated that the sire data contained some fifty times as much information as did the cow data.


2017 ◽  
Vol 54 ◽  
pp. 19-29 ◽  
Author(s):  
N. P. Babik ◽  
Ye. I. Fedorovych ◽  
V. V. Fedorovych

Productive longevity is a very important feature, which is determined by a combination of genotype factors in specific environments. It is known that in recent decades to improve of productive qualities of domestic dairy cattle, the Holstein breeders of different breeds are used. Such measures, although allowed to increase the milk productivity of cows significantly, however, led to reduction in the duration of their productive use. Therefore, the purpose of our research was to study the duration and effectiveness of lifetime use of dairy cows, depending on the country of origin of their father. The research was conducted on animals of Holstein (n = 2902), Ukrainian Black-and-White (n = 14876) and Ukrainian Red-and-White (n = 2176) breeding in various regions of Ukraine. It was established that the controllable number of Holstein cows came from 116 bulls from five different countries. Cows originated from the bulls of Hungarian selection had the longest life and lactation. Cows that came from Canadian bulls had the best life-long yields and life-long number of milk fat. Cows from French bulls were characterized by the worst indicators of productive longevity. Among the descendants of the German bulls, the daughters of the following bulls: Lord 661287, Trend 2761400782690, Bg. Rodeo 27642626161 and Ingo 27677179331, American selection – daughters of B. Astronomer 2160438 and Bob Hubby 2109267, Canadian – daughters of Rock 373840409 and H. R. Artist 6284191, Hungarian – daughters of V.Vilmos 3101733688 and E.Samba 3035115974 and French – daughters of Brico 5794006324 had the best indicators of the duration and efficiency of lifelong use. The studied population of cows of Ukrainian Black-and-White dairy breed came from 167 bulls from eight countries. The largest number of daughters was obtained from domestic bulls (34.6%) and German (29.5%) selection. However, cows of Russian selection had the highest rates of longevity, productive use, lactation, the number of lactations per life, life expectancy and lifetime of milk fat. Daughter Valentina 373840175, Matadora 373840109, Piclenda 373880102 and Tigris 373880127 were lactured more than 4 lactations and had lifelong yield of over 25,000 kg, among the descendants of Russian bulls. Of the descendants of the domestic selection the daughters of the Abrykos 5806 lactated in an average of 6.2 lactation, and their lifetime yields were 38,401 kg. By the number of lactations per life and life-long yields of the daughters of the German bulls, the leader was Presbyter 27621490100, the American – Bob Hubby 2109267 and T. D. Fiasco 1709950, the Canadian – B. Goldgate 6387868 and B. Regensy 394223, the Netherlands – G. Tristan 3021652032 and Baneliai 243931215, Hungarian – E. Samba 3035115974 and P. Selvichar 3023006464. The controlled population of cows of Ukrainian Red-and-White dairy breed came from 69 bulls from five countries. More than 50% of cows of this breed are derived from bulls of German selection. However, the longest duration of productive use and the greatest lifelong yields had daughters of bulls from Canada and Ukrainian selection. Among the descendants of the domestic bulls, according to the indicators of lifetime productivity, the daughters of Khlor 2052 differ markedly. They lacted in average 7.7 lactations, and lifetime longevity was 41,000 kg. Among the descendants of Canadian bulls, the best of their productive use and their lifelong yields had daughters of V. Texel 393522, V.N.Dan 5510544 and Inhibitor 402151, German – Roman 660886883 and Tumpi 112367468, and American – Addikshn 17143107. The influence of the father's country origin on the longevity, productive use, lactation and the number of lactations per life, depending on the breed and the indicator, was within the range of 1.4–17.1, on the indicators of lifetime productivity – within 1.5–9.7%, and the influence of the father on these indicators was, respectively, 9.9–19.9 and 11.4–29.3%.


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