Totals of 5219, 2130 and 2981 fibre samples, from the three breeds respectively, were taken prior to shearing at 32.5 ± 01.25 months of age from four body sites; shoulder, side-portion, abdomen and leg. Breed effect was significant (P < 0.01) for most hair and cashmere properties. IMCG and F1 hair lengths were longer (P < 0.001) with less variable lengths than DBG. Shoulder hair diameters of the three breeds were not different (P > 0.05) but biggest of the side-portion and abdomen sites of DBG were bigger (P > 0.01), however, the biggest (P < 0.001) hair diameter was recorded for the leg site of F1 and the smallest (P > 0.01) for IMCG. IMCG recorded the longest value for cashmere lengths followed by DBG, while F1 recorded the lowest (P = 0.001), whilst F1 recorded the biggest (P = 0.001) diameter whereas no differences existed between parents' breeds. The cortical cell lengths of IMCG and DBG were 94.57 and 86.85 μm without significant difference detected between breeds. Differences between hair length and diameter for body sites of the studied goat breeds were significant (P < 0.01) but between whiteness, cashmere diameter and diameter of cortical cells were not. Sex had no significant effect on all hair/cashmere properties. Quality characteristics of cashmere fibres from IMCG and F1 were better (P < 0.001) than from DBG. Leg hair diameter, curl recovery rate and cashmere diameter were superior in the crossbred F1 compared to pure breed parents, and DBG was superior to IMCG for fibre elasticity and intensity traits. FGF-5 gene was detected as a candidate gene for hair and cashmere traits in IMCG breed. Whilst, KIT gene was found to be associated with coat colour in the studied breeds. Extra investigations to examine more cashmere goat breeds and crosses are needed to discover genetic variability in cashmere production locally and worldwide.The aim of this study was to determine the properties and quality characteristics of hair and cashmere fibres of three goat breeds raised in Southwest China, namely; Dazu black goat , Inner Mongolia cashmere goat  and their first cross (F Hair/fibre is an important product from goats kept in some of the most marginal inhospitable agriculture areas of the world. There are three significant fibre types produced from goats; cashmere, mohair, and hair. The fourth is cashgora, a hybrid type, with intermediate characteristics between cashmere and mohair. The important physiological difference between mohair and cashmere production lies in the response of fibre growth to changing levels of nutrition. The yield and diameter of cashmere fibres are highly unresponsive to changing nutritional status. Also, cashmere growth is remarkably insensitive to the nutritional influence, and may successfully be produced in harsh continental climates5.Throughout many domestication events, several goat breeds have stepped into the luxury fibre production field6. The hair/fibre of the cashmere goats consists of two types; (1) fibre covering the goat body, known as guard hair or outer coating coarse fibre and (2) down fibre or undercoat cashmere, soft and fine fibre protects goats from cold8. In south Asia, cashmere is called "pashmina" a Persian word that means "fine wool"9. The guard hair, could be short or long, but straight, coarse and distinguishable from the undercoat cashmere fibres. Cashmere fine fibres must be separated from guard hair before being sold or processed. In the industry, this step is known as ''Dehairing'', and should take place before fibre dyeing and converting into textile yarn, fabrics then clothes8.The cashmere, is a fibre collected from cashmere, pashmina and several other goat breeds. It is stronger, finer, softer, lighter, and approximately three times more insulating than sheep wool10, has an estimated population of more than 1,230,000 heads of goats that produce around 10,000 tons of cashmere presenting 40% of world production11.China, one of the largest cashmere producing countries7, while cashmere wool is one of the most expensive fibres commercially produced. Cashmere is harvested once a year, yielding about 260 g of fibre/head9.Goat hair/cashmere is used for the manufacture of coarse cloth, tent fabric and rope13. Although cashmere breeds raised around and in the Tibetan mountains have extremely unique soft fibres14, cashmere could be produced from other breeds/strains, in other locations around the world. For example; in Asia and Europe, cashmere goats are geographically found between 50°–120° east longitude and 35°–55° north latitude. These breeds are adapted to cold regions and raised in areas at more than 1000 m above sea level7. In general, goat breeds in China are distributed in five eco-economical regions; Northern pasture, Qinghai-Tibetan high land pasture, Agriculture-Animal husbandry integrated region and Northern and Southern Agricultural regions7.Cashmere goats are economically important livestock sector distributed mainly in remote semi-arid areas of Asia especially in north China, Mongolia and recently in southwest China15.Several cashmere breeds have been moved from different regions of China to Southwest China including Inner Mongolia cashmere goat (IMCG) to be raised with the local Dazu black goat (DBG) breed which is native of Chongqing. It is characterized by high adaptability and sound reproductive performance, therefore, is used extensively for breeding purposes16. Several investigations confirmed that; the value of Chinese cashmere fibre is related primarily to its small fibre diameter and is influenced by secondary characteristics, such as lustre, colour, elasticity, intensity, whiteness and fineness, in addition to yield and proportion of down fibres in total fleece17.In fact, Chinese cashmere as being the best fibres is utilized in making luxury tricot19, it would be wonderful to investigate trait like that and its genetic background, besides the properties and quality characteristics of hair and cashmere fibres in the goats. Many genes like; FGF-520, KIT, IGFBP-7 and PLOD321 have been reported to be correlated with different hair/cashmere traits, such as; type, appearance, length, diameter, coat colour, etc. in several goat breeds.Also, the coat colour plays a necessary role in the adaptation, dodge predators in harsh environments and indirect effects on the productive traitsFew investigations are available on the differentiation of hair/cashmere properties and quality characteristics over the body sites of cashmere goats and their crosses and yet, research on the down-fibre characteristics of native goat breeds in China are still limited. To the best of our knowledge, no comprehensive studies have examined differences in hair and cashmere characteristics between different genotypes/breeds or their association with genetic reasonings.1). The impact of crossing, and FGF-5 and KIT (rs647214940 site) genes on fibre properties and coat colour, respectively, were appraised.So, this study is a part of a comprehensive investigation for characterization of several goat breeds raised in Southwest China. Evaluation of morphological traits, physiological variables and fibre properties, involving the use of whole-genome sequencing (WGS) and genome-wide association studies (GWAS) are being undertaken with a special focus on the coat colour of IMCG, DBG and their first cross (FP < 0.001) for all hair and cashmere properties and quality characteristics, except; curl degree, elongation at break cortical cell dimensions (length and diameter) which was different at (P = 0.033), (P = 0.032), (P = 0.148) and (P = 0.559), respectively, but no effects were detected for sex or interaction between breed and sex (P > 0.05) (Supplementary File P < 0.001) of IMCG and F1  between means of hair length of different goat breeds, IMCG produced the longest (P = 0.001) hair followed by F1 (P < 0.01) concomitantly with less variability than in DBG which recorded the lowest hair length. The same trend was found on hair length of all body sites (P < 0.001). Shoulder and leg hair lengths were longer than of abdomen and side-portion in IMCG and F1, comparing to DBG breed (P < 0.001) (Table There were highly significant differences P < 0.00 between P = 0.004) were found between means of hair diameter of all studied breeds, but for shoulder site, no significant differences (P > 0.05) were observed. The highest (P < 0.001) value for leg hair diameter was recorded for F1, but the lowest was for IMCG (P < 0.01). For the side-portion site, the highest (P < 0.001) hair diameters were obtained on DBG and the other two breeds had similar diameters (P > 0.05). For abdomen hair, parents IMCG and DBG had similarly high diameters (P < 0.05) while F1 had the smallest (P < 0.001), , Fig. .Figure 2P = 0.001) between means of cashmere length of different goat breeds, IMCG produced the longest (P = 0.001) cashmere length followed by DBG concomitantly with less variability than in F1 which recorded the lowest cashmere length. Shoulder and leg cashmere lengths were longer than of abdomen and side-portion in all studied breed (P < 0.05) (Table There were highly significant differences (5) Table .Figure 31. The difference between IMCG and DBG was not significant (P > 0.05), while F1 was significantly higher than both parents breeds (P < 0.01) though closer to DBG. Moreover, there are significant differences in cashmere diameter were detected among various body sites within each breed  were obtained between breeds for most quality traits of the studied breeds, while no differences (P > 0.05) were found between different body sites and both sexes within each breed  than those of DBG and F1, whilst F1 recorded the lowest means. All cashmere fibre elasticity variables were different (P < 0.03) among breeds. As for curl degree, the highest value was observed on DBG, followed by F1 and IMCG which were not significantly different. The highest curl recovery rate was recorded for F1 followed by DBG then IMCG but no significant differences were found between F1 and DBG. With regard to curl modulus, differences among all breeds were significant (P = 0.002), with the lowest value obtained for F1 and the highest for DBG.Means of cashmere natural length and straight length of IMCG breed were higher (P < 0.001) among breeds except elongation at break which was different at (P = 0.032). IMCG recorded the highest elongation at break (P = 0.032), elongation % and EYS1.5 (P < 0.001). While DBG recorded the highest values for intensity traits of breaking strength, work, strength and yield point (P < 0.001). For all cashmere intensity traits, F1 won intermediate positions between the values of both parents breeds. As for cashmere whiteness, the differentiation indicated significant (P = 0.019) variability among breeds, while no significant differences were found between body sites within each breed. The highest degree of whiteness was observed on IMCG, followed by F1, while the lowest degree was obtained on DBG.All cashmere intensity traits were significantly different (P = 0.148) between the two parents breeds (94.57 vs 86.85 μm), whilst for diameters (5.86 vs 5.35 μm) were not significant. However, the abdomen site of IMCG showed the highest cortical cell length (P = 0.045) and diameter (P = 0.543) among all body sites , g. 92124227A>T (rs665580959) and g. 92111741A>T (rs672199363), and 4 mutations were absent from goat dbSNP-database, namely; g.92124199C>T, g.92124134G>C, g.92124131A>G and g.92124086T>G within the exon sequence of the FGF-5 gene. Only the candidate mutation (g.92124199G>C) could lead to a ''serine'' to ''leucine'' transition at the 224th position (p.S224L)*  and DBG (n = 6) parents; previously sequenced by some of the current authors, averaged 6.02-folds coverage which produced raw data of 192.74 Giga base (Gb) then cleaned to reach 191.57 Gb, after filtration and quality check by removing the low-quality and paired reads. The two parents breeds contained 294 and 155 candidate regions, respectively. The top region was located on chromosome 2 of IMCG (113850001:113900001) bp while was located on chromosome 8 (41890001:41950001) bp for DBG. Thus, 176 and 239 selective sweep regions involving 106 and 135 candidate genes were detected in IMCG and DBG respectivelyKIT gene/rs647214940 site (199 bp) revealed the presence of one genotype (AA) for IMCG, three genotypes for DBG (AA = 0.1290), (AG = 0.2903) and (GG = 0.5807), and two genotypes for F1 (AA = 0.4118) and (AG = 0.5882), (Table 765glutamic (Glu)/765glycine (Gly) (off white to light brown coat colour) in F1, while was 765Glu/765Glu (white coat colour) in IMCG, and was 765Gly/765Gly (black coat colour) in DBG 25, Krygyzstan (5.00 cm)16 Gorno Altai cashmere goat (4.50 cm)26 and hair goats in Turkey (4.40 cm)16. On the contrary to IMCG and F1, hair length values obtained on DBG were humble to those of Chinese hair goats (4.89 cm), Kilis goats (5.30 cm)27, but similar to some Chinese hair goats (3.80 cm), and higher than those of Norduz goats (2.44 cm)28.The hair length obtained on parents breeds was similar to those reported by Guan et al.1 had a diameter of 86.38 μm, much closer to that of IMCG which indicated that crossing affected hair diameter of F1 noticeably towards the finess hair. There is a general agreement that hair diameter is a highly heritable trait. Estimates of heritability of fibre diameter included values of 0.4129, 0.51, 0.61 and 0.9917, indicating a good opportunity for progress by selection and breeding programs towards production of fine hair. Hair diameters of all body sites of F1 were closer to or even higher than that of fine hair of IMCG except leg site which was the lowest but still higher than that of DBG. These results revealed that hair diameters of all studied breeds were thicker than those of hair goat cashmere (14.30 μm)26, Turkish hair goats (16.00–18.20 μm), Kilis goats (16.20 μm), Savannah goats (16.00–18.50 μm), Boer goats (16.20–18.50 μm)26, Gorno Altai goat (18.50–19.00 μm) and Van-Turkish hair goats (17.32–17.94 µm)16.The hair diameter of DBG (92.42 μm) was much higher than that of IMCG (84.60 μm) but F26, Turkish hair goats (2.80 cm)30, Van-Turkish hair goats (2.50–2.96 cm)16, South African Boer (2.00–3.10 cm), Savannah goats (2.00–3.10 cm)26, Anatolian native goat (3.30–6.10 cm)25 and Norduz goat (2.44 cm)28, while were lower than those of Chinese cashmere (4.89 cm), Kilis goats (5.30 cm)27 and Krygyzstan cashmere goat (5.00 cm)16. The cashmere length of IMCG (4.55 cm) was similar to that of Gorno Altai cashmere goat (4.50 cm)26. On the contrary to IMCG and DBG, the fibre length values obtained on F1 were lower than those of Boer goats, Savannah goats (3.10 cm)26 and Anatolian goat (3.30 cm)25.The current cashmere length values for parents breeds were higher than those of South African domestic goats (1.5–3.0 cm)1 possessed a value of 16.27 μm, being the highest while no differences existed between DBG and IMCG , cashmere hair goat (14.30 μm), Chinese cashmere goats (14.50–15.50 μm)16, (14.63–15.75 μm)29 and South-African domestic goats (14.00–16.50 μm). While, the cashmere diameter values obtained on F1 were similar to Turkish hair goats (16.00–18.20 μm), Kilis goats (16.20 μm), Savannah goats (16.00–18.50 μm), Boer goats (16.20–18.50 μm)26 and Cashmere goats (16.50–17.50 μm)26, but were lower than those of Gorno Altai goats (18.50–19.00 μm)16, Iranian and Afghanistan cashmere goats (17–18.50 μm)26 and Van-Turkish hair goats (17.32–17.94 µm)16. Worth mentioning, cashmere fibre diameters of more than 16.5 μm is generally utilized in a fabric weave mixing with wool31. The diameter of the fibre for processed cashmere should be less than 19 μm. A value among 14–19 μm on average is considered appropriate16.As for cashmere diameter, F32 measured the effect of age on the length of hair and cashmere, and cashmere diameter in Chinese Alashan Left-Banner White-Cashmere goat, and they reported that all of the above parameters were significantly influenced by the age, but in the current study, the experimental goats from different three breeds were randomly selected to be of the same age (32.50 ± 01.25 months) to measure the effectiveness of the breed (genotype).On the contrary, another study by Pallotti et al.1 (78.07%) compared to IMCG (74.49%) and DBG (77.12%). These values were also higher than those found by Tuncer16 for Turkish goats in the Central districts of Van (31.42%), Başkale (25.63%) and Özalp (28.84%) and were also higher than Turkish goat down fibre cashmere (33.00–38.80%)33 and Australian cashmere (50%)34.Cashmere fibre elasticity measured as curl recovery rate was high for F1 being 15.64, 40.07 and 33.99 cN/dT, respectively were lower than those found on Turkish hair goat (45.60–46.60 cN/dT)16, Australian cashmere (110.20 cN/dT)34 and Anatolian native goat (< 120 cN/dT)25. However, DBG and F1 cashmere strengths contrary to IMCG were higher than those reported by Mehmet et al. for hair goats (25.00 cN/dT)33.Cashmere fibre strengths of IMCG, DBG and F1 which again was intermediate between parents (31.28) whilst the lowest was obtained on DBG (26.98).The cashmere whiteness measured colourimetrically as the degree to which the cashmere surface is white. IMCG recorded the highest degree of whiteness (34.98) followed by F1 (28.98 cN) was very close to that of the high DBG value (32.55 cN) and much higher than that of IMCG parent (11.51 cN). The elongation values for DBG (25.34%) and F1 (27.02%) were lower than that for IMCG and that was similarly reported by Göktepe et al.25 on Anatolian goat breeds. Surprisingly, sex had no effect on all hair and cashmere properties but this agrees with the results of Mehmet et al. on Turkish hair goat33.Comparing between breeds, the cashmere yield point of F35. In this regard, Satlow36 made a comprehensive comparison between several morphological investigations and concluded that Mongolian cashmere fibre was bilateral and the amount of para material in the wool was more than in cashmere. On the contrary to the findings of Roberts37 cashmere (49.6% para and 50.4% ortho) was different from lambs-wool of similar fibre diameter (34.8% para and 65.2% ortho).The cortical cells appear under the microscope as spindle-shaped and is aligned parallel to the fibre axis Fig. . General38. In accordance to this, goats in the current study were under the same management, nutrition level and conditions, therefore, the values of cortical cell dimensions of IMCG and DBG were not different (P > 0.1) but were higher than those reported by McGregor and Liu38. McGregor and Quispe39 studied the dimensions of fibre cortical cells of Peruvian alpaca  and reported the values of (42.50–57.30 μm) and (4.50–4.90 μm) for length and diameter.The current cortical cell dimensions of IMCG (94.57 μm) being longer than that of DBG (86.85 μm), while lacking difference in diameters, were different from those of McGregor and Liu who reported that the cashmere goats produced longer (50.8 μm) and thicker (4.57 μm) cortical cells with high levels of nutrition, but shorter (34.5 μm) and thinner (4.14 μm) cells with low levels of nutrition40. In the present study, IMCG and F1 were light coat colour, while DBG had dark coat colour. The dark coat colour of animals forms an adaptive mechanism necessary for the regulation of temperature, especially in the cold seasons. Light coat colour is advantageous under intensive radiation environments because of its reflectance property41. The coat colour influences radiant heat loss exerting an effect on the animal weight and some productive traits in different livestock species, especially under tropical environments19.Coat colour is one of the most important qualitative traits to distinguish between farm animals42. Worth mentioning, the pigmentation of the coat colour is genetically regulated by several genes that control the packaging of pigment granules, the amount, pigment-cell number, and type, also, the transfer of pigments to surrounding-cells40.The variability in goat coat colour is an indication of the indiscriminate crossing existing between different goat breeds. Also, the variability in the morphological characteristics may reflect an adaptability advantage to different ecological systems under which goats are raised including vegetation features pertaining to those zonesFGF-5, KIT, OXTR, PRKCD, IGFBP-7, SGK-3, and ROCK-143. Li et al. identified 135 genomic regions that were associated with hair/cashmere properties within the cashmere goat populations. These selected genomic regions contained genes, which are potentially involved in the production of the hair and cashmere fibre, such as FGF-5, IGFBP-7 and ROCK-143. Hair/cashmere growth was supposedly regulated by the hairless, hypertrichosis, hair over-growth, FGF-5, FOXI-3, Sox-9, Trps-1 and ABCA-5 genes44. However, natural long fibre resulted from the regulator FGF-5 gene, in several species of mammals44. Moreover, He et al. reported that the expression of FGF-5s and a mRNA alternative splicing of this gene, probably act as the main cause of increasing the cashmere fibre length45.Several genes have been reported to be correlated with different hair/cashmere properties, in many goat breeds. There are candidate genes related to cashmere fibre properties which were identified in many genomic regions in the selective sweep, involving; 1 cross in comparison to DBG. In a previous study, that was carried out by some of the current authors, on selected individuals of parents breeds22, the genome sequence of IMCG breed was found to contain 294 candidate regions, involving 176 selective sweep regions and 106 candidate genes including FGF-5 which had 3 synonymous mutations while 4 other mutations were absent which may lead to differentiation in the hair/cashmere characteristics, especially length22. According to several investigations, FGF-5 gene has a high effect on hair/cashmere formation and natural hair growth variation49 (Supplementary File 50 reported that FGF-5 gene can produce a full-length protein (FGF-5) and a short form (FGF5s) through mRNA alternative splicing. In the current study, this gene is located on goat chromosome 6 (92103281–92124341), with two transcripts (FGF-5 and FGF-5S), FGF-5 transcript contains 3 exons, while FGF-5s contains 2 exons (Supplementary File 51. Editing of FGF-5 gene in goat embryos resulted in an increased number of secondary hair follicles and longer fibre length which would suggest greater cashmere production20. A mutation in FGF-5 was able to grow secondary hair-follicles and produce longer fibres in Cashmere breeds43.Hair/cashmere length was a characteristic trait in IMCG and its F18. Crossing between IMCG and DBG produced F1 progeny having light coat colour because white is dominant over black. The mutation site rs647214940 (KIT gene) of IMCG may have descended from the white coat colour of ancient cashmere through mutations. The alleles at this locus are either A or G, and therefore three genotypes: AA, AG or GG may exist. Among them, the dominant allele A is for the white coat, and allele G is the recessive white coat. There is a differentiation in the rs647214940 site (KIT gene), where three genotypes are in DBG, but two only in F1, while only one was found. This coincides with the difference in coat colour for the three breeds.Moreover, coat colour plays an important role in the adaptive mechanism through the regulation of temperature especially in severe cold or extremely hot areas. According to Odubote coat colour influences radiant heat loss exerting an effect on animal economic traits in many species, especially goats in tropical environmentsKIT gene plays a key role in different colour patterns in the goat, pig, cat, cow, mouse, horse, rabbit, dog and camel7, and was found to be associated with white coat in Holstein cattle52. Nazari-Ghadikolaei et al. detecting a significant association with coat colour in mohair goats, reported that KIT and PDGFRA genes on chromosome 6 were found to be related to light coat colour, while ASIP, AHCY, RALY and ITCH genes were found to be associated with dark coat colour on chromosome 1353.In this regard, CUX-1 and PLOD-3 play a necessary role in type and formation of hair/cashmere. The CUX-1 is associated with wavy hair and curly whiskers in mouse54. The PLOD-3 play a role in the formation of hair or in their texture in Angora and Ankara goat breeds21. The keratin (KRT) and keratin-associated protein (KRTAP) family genes located on chromosomes 1 and 5 were highlighted as candidate genes potentially responsible for diameter and kemp traits in Angora goats and have been previously confirmed in sheep56. In Rayini goat breed there were several regions on chromosomes 2, 5 and 13 related to Cashmere yield57. However, the genes related to fibre properties in the studied goat breeds have not been extensively investigated yet, therefore more investigations are needed in this area.On the other hand, Salo et al. and Sansregret et al. confirmed that there are 24 genes, including; 1  goats, respectively, at a rate of 6–12 samples per site for each animal, to assess the hair/cashmere properties and quality characteristics. The experimental goats from the three breeds were adults (32.50 ± 01.25 months) randomly selected from the respective groups of contemporaries born within 2–5 weeks. All goats were raised under the same conditions of feeding, weather and management. The F1 goats were produced from crossing between IMCG does with normal estrus and DBG bucks using artificial mating.Goats used in this investigation were kept under a semi-intensive system, in the Dazu Black farm, Southwest University, Beibei, Chongqing, China . Totals of 52192369) ♂, 2850♀), 2130  and 2981  hair/cashmere samples have been collected from individual goat body sites; shoulder (A), side-portion (B), abdomen (C), and leg (D) of DBG , IMCG  and FThe hair fibre traits of concern were hair length and hair diameter, while the cashmere fibre traits were: cashmere length, cashmere diameter and cashmere quality measured as; natural length, straight length, cashmere elasticity; curl degree (J), curl recovery rate (JW/%), curl modulus (JD/%), cashmere intensity; elongation at break (mm), breaking strength (cN), work (cN*mm), strength (cN/dT), EYS1.5 (mm) ''Elongation corresponding to 1.5 times the yielding stress in a stress–strain curve'', yield point (cN), elongation (%), cashmere whiteness, coat colour and finally measurements of cortical cells of the samples. All these traits were measured professionally from the four different body sites of both sexes. The units and definitions used in the measurements are shown in Supplementary File https://www.news-medical.net/cellSens), according to Peterson and Gherardi58. Naturally, spiky and coarse hair of outer-coat (hair) upheld among the undercoat cashmere fibres were removed manually by the dehairing process, to measure both separately.The guard hair (outer coating coarse) and down-fibres of cashmere were collected manually from individual goat body sites in spring (April and May) using a fleece comb and electric scissors . Fibre length was measured using a metal ruler, and fibre diameter using (400 ×) digital microscope  with CellSens Standard software (http://en.czsgddkj.com/products/926.html) was used to estimate the mechanical properties of fibres, and fibre crimp elasticity tester device ''YG362A''  was used to measure other quality traits of the studied goat breeds.Cashmere quality testing was carried out at Southwest University  and Gansu Agricultural University . Electronic single fibre strength tester device ''YG001B''  to examine the coat colour for the progeny.Data on coat colour have been collected professionally by visual assessment for the three studied breeds. Also, 20 IMCG does with normal estrus were selected for artificial mating with 5 DBG bucks to produce the first cross FCortical cells were measured according to the following steps; (1) few fibres of each sample were placed on a glass slide. (2) A drop of concentrated (95%) sulphuric acid has been dripped on the sample and covered with a coverslip. (3) After 2–3 min, the coverslips were pressed and ground gently with tweezers. (4) Cortical cells were, then isolated and observed under the digital microscope (400 ×) with CellSens Standard software. (5) The CellSens Standard software was used for real-time observation and measurement of the length and diameter of the cortical cells. This test was done only on the pure breeds (IMCG and DBG parents).1  experimental goats with DNA isolation-kit . The DNA samples were separated by electrophoresis on 1.0–1.2% agarose in 0.5 × TBE buffer according to Sambrook et al.59 which contained 0.5 μg/ml ethidium-bromide. The electrophoresis run was performed using apparatus with power-supply and visualized by ultraviolet transilluminator and Gel-documentation system . The purity and integrity of DNA were appropriate, the OD260/280 was 1.82, and the average values of degree, volume and mass were 70.47 ng/μL, 29.5 μL and 2.14 ng, respectively.Total genomic DNA was isolated from blood samples of randomly selected IMCG , DBG  and FKIT gene (199 bp fragment from exon 16) was carried out using Primer Premier 5.0 software (https://macdownload.informer.com/advice/Primer_Premier_5.html), as follows; F: 5′-TGCCTGCAAGT TCACATCAG-3′ and R: 5′-AAAGCTCAGCAAGTCCTCCA-3′, that synthesized by . The amplification was performed using , ten p.mol of the primer and 80–100 ng of each sample of genomic DNA were processed under the following PCR amplification conditions; 34 cycles of denaturation (94 °C/1 min), annealing (57.8 °C/1 min), extension (72 °C/1 min) and a final extension step at 72 °C/2 min. The amplification was carried out using a Thermo-cycler T100 .The specificity of the PCR primer targeting KIT gene by DNA Sequencer . The resulted sequences were analyzed using ExPASy program (http://web.expasy.org/translate) and MEGA 6.0 V4. The genetic indices of the three studied populations; polymorphism information-content (PIC) and gene-heterozygosity (He) were calculated according to Nei’s methods61.The sequence analysis was carried out for 62 has been used for testing the homogeneity of variance for all studied factors for each trait and the results were not significant at all. The same results have been obtained by Levene's Test63 which is widely considered to be the standard homogeneity of variance test.All data were tested for normality with the Shapiro–Wilk test using PROC-UNIVARIATE procedure of SAS 9.0  and results indicated that all data were distributed normally. Also, Bartlett's testij is an observation on each studied trait, μ is the mean, and Bi is the fixed effect of ith (i = 1:3) breed, Sj is the fixed effect of the jth sex (j = 1:2), BSij is the interaction between breed and sex and eijk is the residual error. Significances among means were tested using least significant differences .The dependent variables were tested using the GLM procedure of SAS 9.0  according to the following model:https://www.aaalac.org/about/Ag_Guide_3rd_ed.pdf. Also, the study was carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org).All animals and sampling procedures in this experiment were supervised and approved by the institutional Animal Care and Use Committee of Southwest University, College of Animal Science and Technology, China . All procedures and experimental protocols were in accordance with the Guide for the Care and Use of Agricultural Animals in Research and Teaching, Federation of Animal Science Societies  1 hair/cashmere were analysed and reported for the first time. The present results indicated that high hair length was obtained for IMCG followed by F1 with significant difference from DBG, on contrary to hair diameter. IMCG and F1 recorded the highest of most cashmere quality traits. FGF-5 was detected as a candidate gene in IMCG, with significant effects on several of hair/cashmere traits. While, KIT gene was found to be associated with coat colour. More trials to detect hair/cashmere genes in different breeds and test their effects on hair/cashmere production and benefits to the industry are necessary.Superior properties of cashmere fibres made it known as “fibres of kings”, presenting a valuable high-quality source for textile products in China. In this respect, the importance, characteristics and genetic background of the cashmere fibres especially ''down-fibres'' of IMCG and its first cross was highlighted in the present work. The characteristics of FSupplementary Information 1.Supplementary Information 2.


CYP2U1 c.947A > T p., a known cause for SPG56. However, they were also homozygous for the novel variant CLCN2 c.607G > T, p.(Gly203Cys), classified as a variant of unknown significance. Testing of additional family members revealed homozygosity for both variants in an additional brother, whom we initially considered unaffected. Both male CLCN2 carriers were infertile, and review of the literature revealed one reported case with azoospermia, however the brother had no overt signs of SPG56. His testicular biopsy revealed incomplete maturation arrest in spermatogenesis; clinically we found mild memory impairment and hand tremor and MRI showed similar changes as his siblings. We consider CLCN2 c.607G > T pathogenic because of the neuroradiological and clinical findings, including azoospermia.Two rare autosomal recessive neurological disorders, leukoencephalopathy with ataxia and spastic paraplegia 56 (SPG56), were found in members of the same family. Two siblings presented with spastic paraplegia, cognitive impairment, bladder and bowel dysfunction and gait ataxia; their consanguineous parents were unaffected. Ophthalmological examination revealed chorioretinopathy. Brain MRI showed T2 hyperintensities and T1 hypointensities in the internal capsules, cerebral peduncles, pyramidal tracts and middle cerebellar peduncles. Both affected siblings were homozygous for Considerable workup may be required to determine the pathogenicity of novel variants, and to unambiguously associate phenotype with genotype. In very rare disorders, highly specific clinical or biomarker combinations provide sufficient evidence for a variant’s pathogenicity. Phenotypic variation of monogenic disorders described in the literature may be attributed to a second co-occurring monogenic disorder, especially in consanguineous families. SPG56 may have reduced penetrance. Variable additional clinical features may include intellectual disability, visual problems, ataxia and peripheral neuropathy. There are more than 70 genetic types of HSP II:2) was a 24-year-old woman born after uneventful pregnancy and normal early developmental milestones. At the age of 6 months bilateral lower extremity weakness and spasticity were noted. She became only able to walk with support but subsequently required a wheelchair. On neurological examination at age 7 years, she was alert and oriented, speech, eye movements and other cranial nerves were normal. There was leg spasticity but normal muscle tone in upper extremities. She was unable to walk. At the age of 7 brain MRI showed T2-weighted hyperintensities and T1-weighted hypointensities in the internal capsules, cerebral peduncles, pyramidal tracts and middle cerebellar peduncles. MRI of the spine showed mid-thoracic syringohydromyelia  was a 21-year-old man with who developed bilateral lower extremity spasticity, gait ataxia and mild cognitive problems at 6 years of age. At the age of 7 brain MRI showed changes similar to (II:2) MRI images, with white matter hyperintensities on T2-weighted images  revealed a known homozygous variant for CYP2U1 NM _183075.2c.947A > T p. rs397514513 on chromosome 4q25. This variant was absent from gnomAD v3.1.1 but identified, in heterozygous carriers, in 3 of 993 exomes from the Greater Middle East Variome Project and with a variant frequency of 0.0023 in Saudi Arabian case series . It had previously been identified in two Saudi Arabian families with autosomal recessive spastic paraplegia 56 CLCN2 NM_004366.5c.607G > T p.(Gly203Cys) on chromosome 3q27.1. This variant was absent from gnomAD v3.1.1, from greater than 2,500 exomes in the Greater Middle East Variome Project that were analyzed at this position, and from a Saudi Arabian NGS dataset (Prof. Alkuraya). CLCN2 encodes for the ClC-2 plasma membrane chloride channel which plays an important role in regulating ions and water homeostasis in the brain. Homozygous or compound heterozygous mutations in CLCN2 are known causes of Leukoencephalopathy with ataxia , a rare autosomal recessive disease that has so far been reported in 18 individuals , was homozygous. He provided informed consent for us to access to his medical records, and it turned out that he had undergone assessment in an infertility clinic. A testicular biopsy had shown a cessation of spermatogenesis at the spermatocyte level with no obvious mature sperm cells  variant causes CLCN2-related leukoencephalopathy in family members II:1, II:2 and II:4, but that all three also carried homozygous likely pathogenic CYP2U1 variants and that II:2 and II:4 had the full clinical phenotype of SPG56. We have no explanation for the lack of overt SPG56 findings in II:1, but recent work has implicated folate deficiency in SPG56 The clinical and radiological features of II:1, II:2, and II:4 were well compatible with CLCN2 c.607 > T pathogenicity revealed PM2, PM3 , PP1, PP3, PP4, resulting in a classification of “likely pathogenic”, albeit closer to “variant of uncertain significance” CLCN2-related leukoencephalopathy in humans and present the first testicular biopsy that shows that the mechanism behind the azoospermia is incomplete maturation arrest. Our detailed ophthalmological examination showed a phenotype that agrees well with previous descriptions of findings associated with mutations in the CYP2U1 and CLCN2 genes Formal probabilistic reevaluation of Next generation sequencing (NGS)-based testing can identify the genetic cause in 12.1 of 61.8 % of patients for ataxia/spastic paraplegia A relatively large body of literature has previously described “expansions of the phenotype” of known monogenic disorders. Although a certain amount of phenotypic variability may be observed in genetically defined human disorders, some of the more unusual phenotypes might also be caused by the co-occurrence, in an individual or family, of a second genetic disorder, especially in consanguineous families. Considerable multidisciplinary clinical and genetic workup can be required to prove or refute the pathogenicity of novel variants, and to delineate core phenotypes for genetic disorders, especially for monogenic recessive disorders in children of consanguineous parents.Wejdan Almasoudi: Conceptualization, Data curation, Investigation, Methodology, Project administration, Writing – original draft. Christer Nilsson: Investigation, Methodology, Writing – review & editing. Ulrika Kjellström: Investigation, Methodology, Visualization, Writing – review & editing. Kevin Sandeman: Investigation, Methodology, Visualization, Writing – review & editing. Andreas Puschmann: Conceptualization, Funding acquisition, Methodology, Supervision, Visualization, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Quantitative δ44/42Ca changes associated with Ca flux across body compartment barriers relative to the dietary Ca and the correlation of δ44/42CaSerum with bone histology are unknown. We analyzed Ca and δ44/42Ca by mass-spectrometry in rats after two weeks of standard-Ca-diet (0.5%) and after four subsequent weeks of standard- and of low-Ca-diet (0.25%). In animals on a low-Ca-diet net Ca gain was 61 ± 3% and femur Ca content 68 ± 41% of standard-Ca-diet, bone mineralized area per section area was 68 ± 15% compared to standard-Ca-diet. δ44/42Ca was similar in the diets, and decreased in feces and urine and increased in serum in animals on low-Ca-diet. δ44/42CaBone was higher in animals on low-Ca-diet, lower in the diaphysis than the metaphysis and epiphysis, and unaffected by gender. Independent of diet, δ44/42CaBone was similar in the femora and ribs. At the time of sacrifice, δ44/42CaSerum inversely correlated with intestinal Ca uptake and histological bone mineralization markers, but not with Ca content and bone mineral density by µCT. In conclusion, δ44/42CaBone was bone site specific, but mechanical stress and gender independent. Low-Ca-diet induced marked changes in feces, serum and urine δ44/42Ca in growing rats. δ44/42CaSerum inversely correlated with markers of bone mineralization.Serum calcium isotopes (δ The bone matrix, defined as bone without cells, consists of organic components, mainly collagen type 1, glycoproteins and proteoglycans and of inorganic structures, mainly hydroxyapatite . The bonCurrently, bone formation and resorption can only be detected indirectly, i.e., by dual X-ray absorptiometry (DXA), by quantitative bone histomorphometry, which is rarely performed in clinical routine and by determination of serum bone markers reflecting osteoblast and osteoclast activity. However, up to now there is no biochemical parameter quantitatively reflecting the bone Ca mineralization process.44Ca, 0.65% is 42Ca and 96.9% is 40Ca [44/42CaSerum and δ44/42CaUrine) has been proposed as a tool to quantify Ca fluxes between the different compartments of the body [44Ca) and relatively light isotopes (42Ca) compared to the ratio of a well-known international standard material such as NIST SRM 915a  and is reported as δ44/42Ca per mil (‰) deviation relative to this standard [Of the naturally occurring Ca, 2.1% is the body . As a restandard .44/42CaSerum and δ44/42CaUrine occurs within 10 days [44/42CaSerum. The Ca isotope composition of the subsequently formed new bone also decreases to a value lower than before. Continued bone Ca loss exceeding bone Ca gain results in lighter δ44/42Ca ratio of both the bones and the serum; serum values are then indicative for bone Ca loss and eventually osteoporosis. This concept has been derived from several studies [44/42CaSerum ratios below a well-defined threshold value indicating equilibrium between Ca input and output to the bones [44/42CaSerum ratios shifts towards values above the defined threshold. The urine, enriched in the remaining 44Ca, is excreted with increasing δ44/42CaUrine ratios [44/42Ca fractionation steps between the different body compartments  and the impact of dietary Ca supply are uncertain. It is unknown whether δ44/42Ca differs between specific sites within one bone and between weight bearing and non-weight bearing bones and between genders. Similarly, the reflection of bone histology mineralization indices by δ44/42CaSerum has not yet been assessed in vivo. To address these crucial questions, we studied the Ca and δ44/42Ca kinetics in serum, urine and feces and the resulting bone Ca content and δ44/42Ca in young, growing rats on a standard and a Ca deficient diet.Skulan and DePaolo demonstrated that mineralized tissue is enriched in light Ca isotopes compared to blood; light isotopes in serum are reduced with bone formation . In vert 10 days . In the  studies ,12,13,14he bones . During e ratios ,12,15. Wp = 0.23), and group 2 and 3 during weeks three to six (p = 0.40)  . At sacr = 0.40) .p = 0.24). Final body length was 45.4 ± 0.9 cm in group 2 and 45.9 ± 1.0 cm in group 3 (p = 0.15). Daily food intake, urine and fecal excretions were similar throughout the study between groups (p = 0.39). During weeks three to six, mean serum Ca concentrations were 2.73 ± 0.10 mmol/L in group 2 and 2.65 ± 0.04 mmol/L in group 3 (p = 0.25). Serum creatinine, alkaline phosphatase and phosphate were normal throughout the study period, largely unaffected by diet —Ca excretion (feces + urine). Intestinal Ca absorption is given in absolute terms as Ca uptake (food + water)—fecal Ca excretion as well as the relative intestinal Ca absorption rate . Net Ca gains were comparable between the three groups during the first two weeks. However, animals on a low-Ca-diet had a 39 ± 2% lower Ca gain in weeks three to four and 39 ± 4% lower in weeks five to six (both p < 0.0001). Compared to the animals on a standard-Ca-diet, animals on a low-Ca-diet in total had a 61 ± 3% lower Ca gain during weeks three to six. The lower dietary Ca content was partially compensated by a higher relative intestinal Ca absorption rate as compared to animals on standard-Ca-diet of 24 ± 4%  and 22 ± 8%  and a reduced urinary Ca excretion of 16 ± 29%  and 62 ± 35% .Compared to the mean baseline femur Ca content measured after two weeks in group 1, femur Ca content increased by 27.0 ± 16 mg in the low-Ca-diet group over four weeks and by 39.6 ± 15 mg in the standard-Ca-diet group, i.e., the increase was only 68 ± 41% of the increase in femur Ca content in animals on standard-Ca-diet . After fp = 0.0002). Mean bone volume gain compared to baseline  was 31 ± 23% lower with a low-Ca-diet (p = 0.0002). This was due to a smaller increase in cortical bone volume (p < 0.0001), while the cancellous bone volume was similar . The differences in bone density were distinct in the diaphysis, metaphysis and epiphysis, with the highest bone density values in the diaphysis. Taking the changes in tibia volumes into account, the mean increase in bone density × tibia volume (∆ Hounsfield Units × mm3 tibia volume) in animals on low-Ca-diet was 73 ± 25% of the increase in animals on a standard-Ca-diet , mirroring the differences in net total body Ca uptake during the four weeks of intervention .δver time . During 44/42CaSerum did not increase from the end of the baseline period (week two) until week 6 (p = 0.22), while in animals on low-Ca-diet δ44/42CaSerum increased (p = 0.007) and reached significantly higher values at week five (0.64 ± 0.07‰) and week six (0.63 ± 0.07‰) as compared to the δ44/42CaSerum at week two (p = 0.01/0.04). δ44/42CaSerum at sacrifice did not correlate with the bone Ca content.In animals on standard-Ca-diet, δp = 0.80) and declined in the low-Ca-diet group during week three to week six (p = 0.01 vs standard-Ca-diet group) and was accompanied by higher absolute and relative intestinal Ca absorption , slightly declined in the animals on standard-Ca-diet during the subsequent four weeks (p = 0.001), and markedly declined in animals on low-Ca-diet (p < 0.0001). δ44/42CaFeces was much lower during week three to six in animals on low versus standard-Ca-diet . Below an absorption rate of about 95%, almost all δ44/42CaFeces values were lower than their diet in animals on low-Ca-diet and inversely correlated from weeks three to six with relative intestinal Ca absorption  and even more in animals when switched to a low-Ca-diet for 4 weeks (p < 0.0001), along with a reduced urinary Ca excretion rate . In animals on standard-Ca-diet, mean δ44/42CaUrine inversely correlated with mean δ44/42CaSerum, but not in animals on low-Ca-diet , i.e., relative more 44Ca accumulated in the growing bone.Weight-bearing femora and non-weight-bearing ribs were homogenized and δ44Ca in the serum , but not in animals on standard-Ca-diet . δ44/42CaSerum at time of sacrifice correlated with osteoid area/bone area , and negatively with tibia bone area/tissue area , and mineralized area/total area , but not with tibia bone mineral density as measured by µCT , absolute femur Ca content  and the gain in femur Ca during weeks three to six . In animals on a standard-Ca-diet (group 1 and 2) δ44/42CaSerum at time of sacrifice correlated with tibia osteoid area/bone area , but not with bone area/tissue area , mineralized area/total area , tibia bone mineral density as measured by µCT , absolute femur Ca content  and the gain in femur Ca during weeks three to six . At the standardized measurement points of the diaphysis, metaphysis and epiphysis, µCT BMD findings inversely correlated with δ44/42CaBone obtained from the same bone site in all animals , and in animals on standard-Ca-diet  and low-Ca-diet .Mean weekly δsorption . Overall44/42CaSerum values have been suggested as a novel biomarker of bone metabolism reflecting the actual bone calcification process, based on studies in various animal models [42Ca is preferentially incorporated into the bone, whereas 44Ca becomes enriched in the blood and is excreted in the urine [44/42CaSerum and δ44/42CaUrine increase during bone formation and decreased with bone resorption and age, with the highest values in adolescent boys [44/42CaSerum positively correlates with serum biomarkers of bone formation and negatively correlates with bone resorption markers and cortical bone mineral density [42Ca across a proximal tubular epithelial cell barrier, whereas the fractionation process across endothelial cells and enterocytes cells was low [44/42CaSerum and δ44/42CaUrine as a biomarker of bone Ca balance. An understanding of the fractionation processes at the compartment transitions is relevant in Ca homeostasis and is needed for mathematical modelling of Ca balances in vivo. Moreover, it has not yet been shown in how far δ44/42Ca is a biomarker of histological indices of bone calcification and actual bone Ca content.δl models  and in hl models ,18,19,20he urine ,18,19,20ent boys  and the ent boys . δ44/42C density . In vitr was low . Taken t44/42Ca kinetics in the different body compartments. Body weight and length gain were comparable in rats on standard- and low-Ca-diet and as expected from the literature [We studied young, rapidly growing rats, which undergo major, rapid changes in bone morphology and Ca content and allow for a sensitive analysis of associated δterature . We infe44/42CaDiet value of the food used in our study was about 0.58‰, independent of the Ca content. This is higher than the δ44/42Ca of dairy and non-dairy products in the European diet, which were estimated to be approximately −0.58‰ and −0.31‰, respectively [44/42Ca was calculated in children based on a prospective three-day food diary to be −0.46‰ (−0.49‰ to −0.41‰) [44/42Ca of the animal food is due to the inorganic origin of Ca products used in this study . In contrast, the δ44/42Ca value is about 1‰ lower in biological non-dairy and lowest in dairy foods, based on the Ca isotope fractionation process in the food chain [44/42CaSerum values in the rats were about 0.50‰, which is higher than usually observed in humans, where values range from about –1‰ in postmenopausal women to up to 0.3‰ in children [44/42CaSerum and height Z-score and higher δ44/42CaSerum values at pubertal Tanner stage 4 than 5, i.e., during the pubertal growth spurt. These observations reflect the higher Ca accumulation rates during rapid longitudinal growth. Independent of the dietary Ca intake, our young rats grew 5–6 cm within four weeks, corresponding to a linearly extrapolated  average annual growth of 60–72 cm per year, which is much faster than the average annual growth in humans during the pubertal growth spurt of about 5–10 cm. The high Ca deposition rate in newly formed bone, with preferential deposition of isotopically lighter 42Ca, together with the high δ44/42CaDiet readily explains the high δ44/42CaSerum in the rapidly growing rats.The δectively ,25. In a −0.41‰) . The higchildren  due to t44/42CaFeces. δ44/42CaFeces was about two times greater than δ44/42CaDiet and the δ44/42CaSerum, suggesting the preferential intestinal uptake of 42Ca. In animals with reduced dietary Ca supply, however, more than 90% of the dietary Ca was absorbed, fecal Ca concentrations were low, and fecal δ44/42Ca decreased to values below δ44/42CaDiet and δ44/42CaSerum. Lowering of the δ44/42CaFeces values cannot be explained by kinetic isotope fractionation because isotope values should become increasingly higher with increasing absorption rate—other mechanisms have to be considered. The absorption of inorganic nutrients  mainly takes place in the upper small intestine by the active or diffusive process, respectively. The Ca uptake rate depends on the daily Ca supply and is highest when the Ca intake is low [44/42CaFeces in Ca deficient Göttingen minipigs compared to animals with standard Ca supply. Heuser et al. estimated the Ca isotope composition of the digestive fluids to be considerably lower than the corresponding δ44/42CaDiet and δ44/42CaSerum. These observations are in full support of our findings here, suggesting that the digestive fluids are isotopically low in order to reconcile the kinetic fractionation process with the measured δ44/42CaFeces. Following this hypothesis, a similar inference must then be made for the mucus cells, however, no such data are presently available. Following this approach, the conceptual model must take into account that at high intestinal Ca uptake during low Ca supply conditions, the Ca originating from digestive fluids and mucus cells dominates at the end of the gastrointestinal tract and the formation of the Ca isotope composition of the feces.The first compartmental border to consider in Ca homeostasis is the intestinal epithelium, which regulates the uptake of dietary Ca. The intestinal Ca uptake was associated with major changes in δe is low ,27. It ie is low ,29,30. T44/42Ca are different routes of calcium transport across the intestinal cell barrier. Dietary Ca is absorbed via the paracellular and transcellular route. The active, ATP dependent transcellular Ca uptake preferentially occurs in the duodenum and upper jejunum [42Ca isotope should more rapidly cross the epithelial barrier following the physiological laws of diffusion provided by the Stokes-Einstein equation and Fick’s first law of diffusion [42Ca isotope selectivity [44Ca is absorbed, increases δ44/42CaSerum and reduces fecal δ44/42Ca, and contributes to the low fecal δ44/42Ca. Thus, intestinal Ca uptakes may be associated with the preferential uptake of 42Ca unless the dietary Ca supply is low and compensatory energy dependent, non-isotope selective Ca uptake mechanisms are active.Another factor potentially contributing to the marked changes in fecal δ jejunum ,27, whil jejunum ,32,33,34iffusion . In animectivity . In this44/42CaUrine values were two- to three-fold higher in animals on a standard-Ca-diet than δ44/42CaSerum values and were inversely correlated. This suggests the preferential tubular reabsorption of 42Ca and the enrichment of 44Ca in the urine, similar to recently reported findings in humans [44/42CaUrine ratios and the latter were not correlated with δ44/42CaSerum. Renal tubular Ca reabsorption and δ44/42Ca handling follows similar principles as intestinal Ca uptake. Ca is freely filtered across the glomerular barrier, only a small fraction (∼1–2%) of filtered Ca is excreted under normal physiological conditions in healthy kidneys [44/42Ca with reduced urinary Ca excretion may be related with relatively higher tubular uptake of heavy Ca isotopes via the energy dependent Ca reabsorption via the transcellular route, with reduced Ca isotope selectivity.δn humans . Rats on kidneys . Seventy kidneys ,37. The 44/42Ca isotope fractionation, i.e., with preferential kinetic deposition of the lighter 42Ca isotope, similar to other biomineralization processes [44/42Ca gradient between bone and serum (δ44/42CaBone—δ44/42CaSerum) in the rats was −0.44 ± 0.07‰ at week 2 and −0.36 ± 0.06‰ and −0.36 ± 0.08‰ with a standard- and low-Ca-diet, respectively. This is in the range of findings recently demonstrated in vitro [44/42Ca is similar in weight-bearing and non-weight-bearing bones, independent of the age and the gender of the animals and the dietary Ca supply. Thus, conclusions drawn based on δ44/42Ca from serum and urine should apply independent of the mechanical stress, as different bones are exposed. Within the bones, δ44/42Ca distribution, however, differs, with lower ratios in the diaphysis, independent of the gender and the dietary Ca supply. The bone Ca isotope ratios inversely correlate with bone mineral density assessed by µCT at the different bone sites, with the highest HU counts in the diaphysis in all groups, the site of the largest mineralization, followed by fairly similar values in the metaphysis and epiphysis. Although the growth plate between the epiphysis and metaphysis is the site of Ca deposition during growth, the site of the highest amount of mineralization is the cortical bone of the diaphysis. Our findings support the preferential deposition of light Ca isotopes with bone mineralization as reported previously [Bone mineralization, i.e., extracellular matrix calcification, is associated with δrocesses . The δ44in vitro  and in vin vitro ,19. In aeviously .44/42CaSerum with bone histomorphological parameters of bone mineralization, using methods applied in the field of renal osteodystrophy, which also include the quantification of non-mineralized osteoid. The unmineralized osteoid is deposited by the osteoblasts, and serves as a ‘scaffold’ for the later mineralization. A recent study demonstrated a positive correlation of bone mineral density with δ44/42Ca of the metaphyseal tibia in rats with type 1 diabetes, but not in rats with chronic kidney disease and healthy rats [44/42CaSerum inversely correlated with the mineralized bone area per total bone area and the bone area per tissue area and positively with the amount of non-mineralized osteoid area in the bone, i.e., the higher the δ44/42CaSerum, the lower the bone mineralization activity. δ44/42CaSerum negatively correlated with the intestinal uptake of Ca, i.e., the lower the intestinal Ca uptake the higher the δ44/42CaSerum. A low-Ca-diet resulted in subtotal intestinal Ca uptake and a δ44/42CaFeces even below δ44/42CaSerum, a finding distinct from the δ44/42CaFeces in rats on a standard-Ca-diet, even though a subgroup of these rats had similarly high relative intestinal Ca uptake as rats on a low-Ca-diet. In the same direction, urinary Ca losses and δ44/42CaUrine were reduced in the animals on a low-Ca-diet, and δ44/42CaSerum increased. As illustrated in 44/42CaSerum with bone mineralization in the entire group of rats was based on the rats on the low-Ca-diet. No correlations of δ44/42CaSerum with histological markers of bone mineralization could be demonstrated in the animals on a standard-Ca-diet. Thus, the major changes in Ca isotope balances in the subgroup of animals on a low-Ca-diet may have overcome the effect of preferential deposition of the light 42Ca during bone mineralization. At all bone sites studied, diaphysis, metaphysis and the epiphysis of the tibia, and in the ribs and femur  were housed at the Interfaculty Biomedical Facility (IBF) at Heidelberg University with a daily cycle of 12 h light and 12 h darkness in groups of three or four according to the recommendations of GV-SOLAS. Tap water was supplied ad libitum. The room temperature was maintained at 22 ± 2 °C with a relative humidity of 50–60%. The animals were allowed seven days of adaptation before the onset of experiments at an age of five to six weeks. Animals were assigned to the three experimental groups using weight-ordered randomization. For anesthesia, 100 mg/kg body weight ketamine and 5 mg/kg body weight xylazine were injected intraperitoneally. The study was approved by the respective authorities .p = 0.38). Group 1 consisted of 12 male and 6 female SD rats who received a standard-diet ad libitum for two weeks to obtain baseline values for comparison with results in group 2 and 3. In group 1, six female rats were studied and compared to male rats  consisted of 5000 mg/kg Ca (=0.5%), 3000 mg/kg P (=0.3%), 15 µg/kg Vitamin D3, 50.5 g/kg fat, 178.9 g/kg protein. The low-Ca-diet only differed in the Ca content .A study scheme is given in ale rats  but not Body weight was measured weekly. Body length was measured at sacrifice during anesthesia. Blood was sampled weekly from the tail vein, and also spot urine and spot feces samples were collected. After two, four and six weeks (prior to sacrifice), rats were placed in a metabolic cage  for 24 h urine and feces collection. Spot and 24 h urine and feces collections alternated weekly. Since food intake varied from day to day within and between animals, but without any trend over time in any group, mean food intake per animal was calculated by the total amount consumed over the diet-specific intervention periods: group 1 over the two weeks of standard-diet, group 2 over the six weeks of standard-diet, and group three over the two weeks of standard-diet followed by four weeks of the low-Ca-diet. At sacrifice, blood sampling was performed in anesthetized rats by heart puncture and urine collection was performed by bladder puncture. The tibia and femora of both hind legs and the right caudal rib were collected and the adjacent soft tissue was meticulously removed. Bones samples were stored in 70% ethanol at 4 °C for histological analyzes, native bone at room temperature and −25 °C, respectively for Ca studies.Urine, food and fecal Ca content were measured by mass spectrometry. Measured food Ca content was as given by the manufacturer. The water intake was estimated based on previously studied age related fluid intake , since v3 at 60 °C for 24–48 h and water was added to increase the final volume to 5 mL. The clear digests were then diluted 1/5000 in a 0.1% La(NO3)3 solution and applied to the F-AAS instrument. Results are expressed as Ca per wet weighted tissue (mg/g). One animal each from group 2 and one from group 3 was excluded from the Ca content analyses due to technical issues.Total Ca analysis was performed using flame atomic absorption spectrometry (F-AAS). Femur samples were weighed, destroyed in 500 µL HNOComputed tomography (CT) imaging was performed using a micro-CT scanner . The scanning parameters were an X-ray voltage of 60 kV with an anode current of 250 µA and an exposure time of 1000 ms. 360 rotation steps were performed resulting in a total rotation of 360°. The field of view was set to 31.12 mm × 42.10 mm and the binning factor was 1. Under med-high magnification the effective pixel size was 14.30 μm and the protocol was Hounsfield Unit (HU) calibrated. HU were standardized relative to distilled water and air with cylinder size adapted to the specimen size. For image reconstruction, a downsample factor of 2 was used. Analysis of the reconstructed images was performed using the vendor software package Inveon™ Research Workplace (IRW) software 2.2.Measurements were performed in the right tibiae using predefined regions of interest (ROI), i.e., in the middle of the tibia (=diaphysis), 3/4 of the distance between the measurement point of the diaphysis and the proximal end of the bone (=metaphysis) and 3/4 of the distance between the measuring point of metaphysis and the proximal end of the bone (=epiphysis) as well as for the whole tibia. The thresholds for determining the volumes and density values were defined as follows: “whole bone” 675–5900 HU, “cortical bone” 1800–5900 HU, “cancellous bone” 675–1800 HU.The method for quantitative histomorphometry of bone has been described before ,45. AfteAll samples were processed and compared with one procedural blank and one of each internationally available reference materials  as well as in-house standards . Before isotope ratio measurements were taken, the samples had to be pre-treated. Blanks, reference materials, in-house standards and samples were always treated the same way.3, ~14 mol/L) overnight on the hot plate at 120 °C. Bones were pre-treated for approx. 4 h in a 4% sodium hypochlorite solution (NaClO) to remove organics and were afterwards rinsed several times with ultrapure water and dried at 105 °C. Precleaned bone samples were then either drilled at predefined spots . In the case of liquid materials, 250 µL for serum samples and SERA-1, 1 mL for urine samples and AK-1, 60 µL for IAPSO seawater standard, 60 µL for NIST SRM 1486 of a stock solution prepared by dissolving the powder of SRM 1486 were used. In the case of solid samples like feces and food, we weighed an amount of the 10–50 mg sample and dissolved it in a 15 mL PFA beaker with 5mL of nitric acid  and 2 mL H2O2 (30%) in PFA tubes, and were then digested in a laboratory microwave (CEM MARS6) following a defined heating program. Next, sample solutions were transferred to 15 mL PFA beakers and dried down at 120 °C and redissolved in 1 mL of 2 mol/L HNO3, and the Ca concentration of these solutions was determined using Q-ICP-MS .Dissolved/liquid samples were mixed with a mixture of 8 mL of concentrated nitric acid  following a defined procedure . The col40Ar1H2 on 42Ca) an APEX IR  sample introduction system was used. All measurements were performed in medium resolution  on the interference-free plateau of the low mass side of the peaks. This was achieved by choosing an appropriate center cup mass which was verified on a daily basis [44/42Ca in parts per mill (‰).Ca isotope measurements were performed on a MC-ICP-MS  at the mass spectrometer facilities of the GEOMAR Helmholtz Center for Ocean Research Kiel, Germany. The mass spectrometer was equipped with nine Faraday cups, of which eight are moveable. The mass spectrometer was set up to measure masses 42, 43, 43.5 and 44 simultaneously. In order to suppress interfering Ca- and Ar-hydrides  that can interfere with the measurement of 42Ca+, 43Ca+ and 44Ca+, as they have the same mass to charge ratio by measuring 87Sr++ [44/42Ca—2·δ43/42Ca| > 0.2‰. A sample measurement (average of 4 single measurements) was rejected when the average intensity was outside a 70–130% intensity window compared to the average intensity of the NIST SRM 915a measurements from the same batch. A complete session was rejected when more than one of the measured reference materials deviated >0.2‰ from the literature value or the data did not fall along the mass-dependent fractionation line. δ44/42Ca of international standard materials were in line with published values.Further tests confirmed that there is negligible isotope fractionation during purification (<0.01‰). Although nearly all Sr was removed from the samples during chemical preparation, we corrected samples for doubly charged Sr  following Coplen (2011):The Ca isotopic composition was reported as δ44Ca/42Ca)Serum values are lower than the (44Ca/42Ca)NIST SRM 915a values. Values are positive when (44Ca/42Ca)Sample values are greater than the (44Ca/42Ca)NIST SRM 915a value. Note that the sign of the δ44/42Ca-notation also points into the direction of fractionation. Negative δ44/42Ca values indicate enriched 42Ca, positive values indicate 44Ca enrichment.Based on the notation above, Ca isotope values turn negative, because  and comparison of median values between two/three groups was conducted using a two-sided Mann-Whitney, respectively. For quantification of linear correlations, a Pearson correlation coefficient was applied. The significance level p for α was set at <0.05.For statistical analysis Graph Pad Prism 9.0  and Microsoft Excel  were used. Normal distribution was assessed graphically and by Shapiro-Wilk test. Normally distributed data were reported as means and standard deviation (SD). Outliers were defined as values beyond 2SD. Means were tested with an unpaired two-sided Student’s 44/42Ca in the diet, feces urine and in bone revealed several important findings. δ44/42CaBone is lower in the diaphysis, the site of the highest Ca deposition, than in the epiphysis and metaphysis, independent of the dietary Ca supply. Bone distribution of δ44/42Ca is independent of the mechanical stress the bone is exposed to, and is similar in female and male rats. Dietary Ca restriction in rapidly growing rats results in subtotal intestinal Ca absorption, a marked reduction in δ44/42Ca in feces and urine, and in an increase in δ44/42CaSerum, suggesting dietary Ca dependent differences in intestinal and renal tubular Ca isotope balances. δ44/42CaSerum inversely correlates with the intestinal Ca uptake and at time of sacrifice inversely with histological features of bone mineralization, but not with dietary Ca dependent bone Ca content and bone Ca gain. Our findings deserve validation in humans and inform future mathematical multi-compartmental modelling of δ44/42Ca.In summary, our longitudinal analysis of total body Ca balance and δ


Agrobacterium tumefaciens underlies the pathogenesis of crown gall disease and is characterized by tumor-like gall formation on the stems and roots of a wide variety of economically important plant species. The bacterium initiates infection by colonizing and forming biofilms on plant surfaces, and thus, novel compounds are required to prevent its growth and biofilm formation. In this study, we investigated the ability of tannic acid, which is ubiquitously present in woody plants, to specifically inhibit the growth and biofilm formation of A. tumefaciens. Tannic acid showed antibacterial activity and significantly reduced the biofilm formation on polystyrene and on the roots of Raphanus sativus as determined by 3D bright-field and scanning electron microscopy (SEM) images. Furthermore, tannic acid dose-dependently reduced the virulence features of A. tumefaciens, which are swimming motility, exopolysaccharide production, protease production, and cell surface hydrophobicity. Transcriptional analysis of cells (Abs600 nm = 1.0) incubated with tannic acid for 24 h at 30 °C showed tannic acid most significantly downregulated the exoR gene, which is required for adhesion to surfaces. Tannic acid at 100 or 200 µg/mL limited the iron supply to A. tumefaciens and similarly reduced the biofilm formation to that performed by 0.1 mM EDTA. Notably, tannic acid did not significantly affect R. sativus germination even at 400 µg/mL. The findings of this study suggest that tannic acid has the potential to prevent growth and biofilm formation by A. tumefaciens and thus infections resulting from A. tumefaciens colonization. Agrobacterium tumefaciens is a soil-borne, Gram-negative bacterium that thrives in the rhizosphere and readily forms biofilms and thus colonizes plant surfaces [A. tumefaciens exerts its pathogenicity by transferring T-DNA from its tumor-inducing plasmid to host plant cells [A. tumefaciens are responsible for the neoplastic condition of plants referred to as crown gall [A. tumefaciens is extensive, and it has been reported to cause crown gall disease in 643 plants belonging to 331 genera [A. tumefaciens infections result in huge financial losses due to reduced crop production [Biofilm formation on plant surfaces is of considerable importance, as in some cases, biofilms can promote plant growth, whereas in others, they can lead to infections ,2. Agrobsurfaces . A. tument cells ,5,6. Stuown gall . The hos1 genera . Not sur1 genera , and thuoduction ,10.A. tumefaciens is a pervasive component of soil microbiota [A. tumefaciens efficiently locomotes using multiple flagella positioned around a single pole [A. tumefaciens, called the two-component regulatory system, activates the virulence genes required for the transfer of Ti-plasmid to the host [A. tumefaciens from free-floating planktonic cells to surface adherent biofilms is regulated by the production of exopolysaccharides (EPS), which plays key roles in surface attachment and confer structural stability.crobiota  that colcrobiota . Infectigle pole . The Virthe host . The traAgrobacterium radiobacter  offers an alternative means of disease control by inhibiting leucyl tRNA synthetase in A. tumefaciens and thus its ability to synthesize proteins [A. tumefaciens, but it may also target other beneficial organisms; additionally, the genetically modified organisms like A. radiobacter are not permitted for usage in certain countries [A. tumefaciens biofilm formation to successfully control crown gall disease.The agents used to control bacteria-induced plant diseases are usually based on antibiotics that are expensive and have limited bioavailabilities . Agrocinproteins ,16. Thisountries . Thus, nA. tumefaciens encounters tannic acid. In addition, tannic acid has been used traditionally to treat diarrhea and skin burns [Pseudomonas aeruginosa (at 100 µg/mL) and antibiofilm effects against Staphylococcus aureus (at 20 µg/mL) [Polyphenols protect plants from pathogen attack and bacterial toxins , and tanin burns  and has in burns , and it in burns ,22. Mechin burns . Tannic 0 µg/mL) ,24, and 0 µg/mL) ,26. In a0 µg/mL) . Further0 µg/mL) .A. tumefaciens growth and biofilms have not been explored, which prompted us to assess the ability of tannic acid to inhibit A. tumefaciens planktonic growth and biofilm formation, the major virulent traits of A. tumefaciens , and its surface colonization of roots. In addition, we examined the effects of tannic acid on the expressions of 11 biofilm and virulence-related genes in A. tumefaciens.Nonetheless, the effects of tannic acid on A. tumefaciens strain GV2260 was used for all experiments. A single colony of A. tumefaciens was inoculated in LB medium and incubated for 24 h at 250 rpm and 30 °C. Shake cultures were prepared under similar growth conditions in flasks. Experiments were performed using at least two independent cultures (prepared from two independent colonies) in triplicate.Tannic acid (Product Code 403040) was purchased from Sigma-Aldrich . A. tumefaciens against tannic acid was analyzed using a crystal violet assay as previously described [A. tumefaciens was diluted with LB at 1:50. Tannic acid was then added in the concentration range 10–400 µg/mL, and 300 µL of these mixtures was pipetted into a 96-well plate and incubated at 30 °C for 48 h. The plate was then stained with 0.1% crystal violet for 20 min, washed with sterile water, and air-dried. Crystal violet retained by biofilms was dissolved with 300 µL of 95% ethanol, and biofilm formation was quantified by measuring absorbance (λmax) at 570 nm [The antibiofilm susceptibility of escribed . Brieflyt 570 nm .A. tumefaciens in the presence of tannic acid was used to investigate the ability of tannic acid to inhibit planktonic cell growth following our earlier described method [A. tumefaciens was diluted with LB at 1:100, and tannic acid was added to final concentrations of 10, 20, 25, 50, 75, 100, 150, 200, or 400 µg/mL. A 300 µL aliquot of each of these cultures was then added to a 96-well plate and incubated at 30 °C for 24 h under static conditions. OD values were recorded at 620 nm every 2 h during this 24 h period, and growth curves were plotted. To determine colony-forming units (CFUs), a 24 h culture of A. tumefaciens was diluted with LB (1:100), and tannic acid was added (at 10–400 µg/mL) and cultured at 250 rpm for 24 h at 30 °C. Cultures were then diluted and 100 µL of each dilution was spread onto LB agar plates, which were incubated for two days at 30 °C. Colonies were then counted.Time-dependent growth of d method . After c4·7H2O). An earlier method [A. tumefaciens in LB. Various concentrations of EDTA  were checked on growth and biofilm formation of A. tumefaciens in 96-well plates under the same incubation conditions as described earlier (2+ (FeSO4·7H2O) in 96-well plates. In a similar manner, two concentrations of tannic acid (100 and 200 µg/mL) were tested in two different experiments with varying concentrations of FeSO4·7H2O (0.01–1 mM), and the growth and biofilm formation were checked after 48 h at 30 °C.To check the role of ten galloyl groups of tannic acid in creating iron-limiting conditions and thus inhibition of growth and biofilms, 100 and 200 µg/mL of tannic acid were used with various concentrations of an iron source  of 24 h cultures were placed at the centers of agar plates and incubated at 30 °C for 72 h. Swimming diameters were recorded at every 48 h and 72 h during incubation [Swimming motilities of A. tumefaciens cells were estimated using a standard phenol-sulfuric acid assay, as previously described [A. tumefaciens cells (OD600 nm = 0.5) were grown in the presence or absence of tannic acid at different concentrations in 1.5 mL microfuge tubes at 250 rpm for 24 h at 30 °C. After incubation, tubes were centrifuged for 10 min at 13,000 rpm, and supernatants were collected and diluted with chilled ethanol at 1:3. The tubes were then allowed to stand at 4°C overnight, and precipitates obtained were dissolved in 1 mL of distilled water. Phenol (5%) and concentrated sulfuric acid in the ratio of 1:5 was added to the dissolved EPS and incubated for 30 min at room temperature. Absorbances were recorded at 490 nm.Amounts of EPS released by escribed . A. tumeA. tumefaciens cells (OD600 nm = 0.5) were grown for 24 h in the presence of various concentrations of tannic acid in 1.5 mL microfuge tubes at 30 °C and 250 rpm. Tubes were then centrifuged for 10 min at 13,000 rpm. The pellets obtained were washed three times and resuspended in 1 mL of sterile PBS, and OD was measured at 600 nm (denoted A0). Xylene (250 µL) was added to suspensions, vortexed vigorously, and left undisturbed for 30 min. Aqueous phases were then carefully removed, and absorbances (Ai) were measured at 600 nm. Percent hydrophobicities were then calculated using the following formula [ formula :Percent A. tumefaciens was grown with 25, 50, 75, 100, 150, or 200 µg/mL of tannic acid at 250 rpm for 24 h at 30 °C. Microfuge tubes were centrifuged for 10 min at 13,000 rpm, and supernatant (100 µL) was collected in fresh microfuge tubes, treated with 100 µL of azocasein for 30 min at 37°C, and then with 600 µL of trichloroacetic acid (10%) to halt proteolysis. The tubes were then cooled at −20 °C for 30 min, and 700 µL of 1 M NaOH was added. Absorbances were recorded at 440 nm.Bacterial exoprotease production was quantified as previously described  in the pA. tumefaciens were formed on 3 × 3 mm nitrocellulose membranes in a 96-well plate. Bacterial culture diluted with LB (1:50) and treated with 200 µg/mL of tannic acid was pipetted into wells and allowed to grow for 48 h at 30 °C. To investigate biofilm development on roots and the effects of tannic acid, surface-sterilized seeds of R. sativus were placed on soft agar plates (0.7% agar + 0.86 g/L Murashige and Skoog plant growth medium) and grown for four days. At least five seedlings from control and treated groups were placed in 6-well plates containing 200 µg/mL of tannic acid in inoculum of A. tumefaciens (1:50 dilution) and incubated at 30 °C for 48 h. Roots were then gently rinsed once with PBS. Before SEM imaging, samples (nitrocellulose membranes and roots) were processed as follows: (i) they were fixed in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde for 30 min at room temperature and overnight at 4 °C, and (ii) samples were dehydrated using an ethanol series , (iii) preserved in isoamyl acetate, (iv) critical point dried and sputter coated with gold or platinum, and (v) visualized at different magnifications using a S-4200 Hitachi Field Emission (FE) SEM microscope  at 15 kV.The biofilms of A. tumefaciens and checked after 3 weeks (incubation at 25 °C) for the development of tumors. Potatoes were surface cleaned with tap water thoroughly to remove all the dirt, followed by surface cleaning with ethanol (70%). Potatoes were cut to obtain flat cubes. Thick slices were made using the sterile knife and immersed in NaOCl (6%) for 15 min. Potato discs (1 cm in diameter) were cut from the middle part of cubes using a sterile cork borer. Discs were rinsed thrice in sterile distilled water. For infection, A. tumefaciens culture (OD600 nm = 1.0) was exposed to tannic acid (200 µg/mL) in PBS and vortexed. A 50 µL of the cell suspension was added to the surface of each potato disc and placed in 6-well tissue culture plates. Plates were sealed and incubated at 25 °C for three weeks in the dark. The numbers of A. tumefaciens induced tumors that appeared on potato surfaces were counted, and percent tumor induction was calculated.An earlier described method for potato disc tumor formation was followed  with sliR. sativus seeds were rinsed with distilled water three times and once with 95% ethanol and surface disinfected by emersion in 3% sodium hypochlorite for 10 min. Seeds (n = 10) were then placed on agar plates containing different concentrations of tannic acid (25–400 µg/mL), 0.7% agar, and 0.86 g/L Murashige and Skoog plant growth medium [h medium . Plates A. tumefaciens was grown in the presence or absence of tannic acid. A. tumefaciens culture in 25 mL LB having an OD of 1.0 at 600 nm was added with 200 µg/mL of tannic acid and kept at 250 rpm for 24 h at 30 °C, then 700 µL of RNase inhibitor  was added with gentle mixing [celA, cheA, exoR, phoB), virulence , motility , and stress response . Gene functions and primer sequences are given in To determine whether tannic acid treatment affected the expression levels of genes related to biofilm, stress, motility, and virulence, e mixing . Culturep ≤ 0.05 were considered significant as per Student’s t-test. All statistical analyses were performed, and graphs were plotted on SigmaPlot Windows Version 14.0 (Build 14.0.0.124), Systat Software, Inc., Chicago, IL, USA. Asterisks in figures indicate significant changes versus nontreated controls.Two independent bacterial colonies were used for experiments in triplicates, and results are presented as means ± standard deviations. Values of A. tumefaciens biofilm formation  (2SO4·7H2O (0.01–1 mM) was supplied with 1 mM EDTA in LB, the growth and biofilm formation started to resume at 0.02 mM Fe2SO4·7H2O and increased in an iron concentration-dependent manner  was used instead of EDTA (1 mM), it substantially reduced the biofilm in the absence of external iron supply  significantly reduced the biofilm formation and growth of O4·7H2O) A. When Ft manner A. This sn supply B,C. The nic acid B, while nic acid C. At ≥0.A. tumefaciens using a phenol-sulfuric acid assay  lost their ability to induce tumors on potato slices. The average reduction in tumor formation was 78% by tannic acid over nontreated control (R. sativus seed germination (R. sativus seeds germinated on soft agar plates (Tannic acid (200 µg/mL) exposed cells of  control . Tannic mination . R. satir plates A containr plates B.celA, cheA, exoR, phoB), stress response , motility , and virulence , and thus, we examined the effects of tannic acid at 200 µg/mL on the expressions of these genes  at concentrations of ≥30 µg/mL [A. tumefaciens  or 2,2′-dipyridyl (DIP) at 20–1000 µM for iron chelation, and the relative biofilm formation was inhibited dose-dependently; however, the addition of 11–1100 µM FeSO4 resumed the biofilm formation under the constant 200 µM concentration of either EDDHA or DIP [A. tumefaciens infection [A. tumefaciens. In our study, tannic acid at 100 and 200 µg/mL acted as the iron chelator similar to the EDTA (1 mM)  . Tannic efaciens B,C growiA. tumefaciens. For example, varying levels of tannins released by roots of sorghum genotypes [A. tumefaciens inoculation efficiencies [R. sativus  to tomato roots infected with A. tumefaciens C58 derivative C107-KmR RifR. These chemical or biological combinations significantly reduced the LogCFU g−1 of A. tumefaciens C58 in root colonization and relative abundance [Tannic acid when present in the medium also inhibits the plant genetic transformation by enotypes  have beeciencies , which c sativus G–L and io slices . This rebundance .dnaK gene, which oversees bacterial stress response to tannic acid [A. tumefaciens is a prerequisite for wounded plant infection and tumor genesis, and cheV protein (encoded by the chvE gene) causes a sugar-induced increase in the expression of vir genes [chvE mutation can create an imbalance between sugar utilization and consumption, which suggests the gene plays a major role in the control of A. tumefaciens virulence [chvE (responsible for sugar-induced increase in virulence), which suggested tannic acid reduced virulence. In addition, tannic acid targeting may be dependent on strain type; for example, tannic acid significantly repressed biofilm formations by methicillin-sensitive or resistant S. aureus by targeting the ica operon and several other regulatory genes [exoR gene by >15-fold  and plant root surfaces and suppresses virulence traits such as EPS secretion, protease activity, cell surface hydrophobicity, and swimming motility. Tannic acid remarkably inhibited biofilm formation on the surfaces of R. sativus roots without affecting R. sativus seed germination. One of the possible mechanisms of antibacterial and subsequent biofilm inhibition could be the chelation of iron by tannic acid due to the presence of galloyl groups. The other mechanism could be decreased expressions of dnaK and chvE, but significant downregulation of exoR, which codes for periplasmic succinoglycan-regulatory protein (a prerequisite of A. tumefaciens motility). This study improves the understanding of the interaction between tannic acid and A. tumefaciens. The results from this in vitro study are promising and pave the way for further research to assess the antipathogenic applications of tannic acid in agricultural settings with other plant species. Though pure tannic acid may inhibit the growth of other nontarget microbes; however, the possibility is low since some plants also release exudates having different tannins and still recruit beneficial microbes to the rhizosphere. Based on the results obtained, we propose that tannic acid be assessed for use in anti-Agrobacterium formulations under field conditions.This study demonstrates the polyphenol tannic acid efficiently prevents planktonic growth and biofilm formation of