Chlormequat chloride induced activation of calmodulin mediated PI3K/AKT signaling pathway led to impaired sperm quality in pubertal mice.

Chlormequat chloride (CCC), as a widely used plant growth regulator, can cause impaired sperm quality and decreased testosterone synthesis in pubertal rats, but the underlying mechanism remains unclear. The purpose of this study was to elucidate the toxicokinetics and tissue distribution of CCC, as well as the possible mechanism of CCC-induced impairment in sperm quality. The concentration of CCC reached its peak 1 h after a single dose (200 mg/kg·bw) administration in mice plasma, and a bimodal phenomenon appeared in the testes, liver, and epididymis. In vivo, 200 mg/kg CCC caused testicular damage and impaired sperm quality in pubertal mice, and the expression of p-tyrosine and GSK3α decreased in cauda epididymidis, sperm and testes. CCC also caused the down-regulation of AKAP4 and the up-regulation of calmodulin (CaM), and activated the PI3K/AKT signaling pathway in the testes. In vitro, CCC reduced the levels of p-tyrosine, AKAP4 and GSK3α, increased the level of CaM and activated the PI3K/AKT signaling pathway in GC-1 cells. CaM antagonist (W-7 hydrochloride) and PI3K inhibitor (LY294002) can effectively improve the expression of GSK3α and AKAP4 by suppressing the PI3K/AKT signaling pathway in GC-1 cells treated with CCC. It was indicated that CCC induced impairment in sperm quality might be partially related to the activation of PI3K/AKT signaling pathway mediated by CaM.

Compact shoot architecture of Osteospermum fruticosum transformed with Rhizobium rhizogenes.

KEY MESSAGE: Improved compact shoot architecture of Osteospermum fruticosum Ri lines obtained through Rhizobium rhizogenes transformation reduces the need for chemical growth retardants. Compactness is for many ornamental crops an important commercial trait that is usually obtained through the application of growth retardants. Here, we have adopted a genetic strategy to introduce compactness in the perennial shrub Cape daisy (Osteospermum fruticosum Norl.). To this end, O. fruticosum was transformed using six different wild type Rhizobium rhizogenes strains. The most effective R. rhizogenes strains Arqua1 and ATCC15834 were used to create hairy root cultures from six Cape daisy genotypes. These root cultures were regenerated to produce transgenic Ri lines, which were analyzed for compactness. Ri lines displayed the characteristic Ri phenotype, i.e., reduced plant height, increased branching, shortened internodes, shortened peduncles, and smaller flowers. Evaluation of the Ri lines under commercial production conditions showed that similar compactness was obtained as the original Cape daisy genotypes treated with growth retardant. The results suggest that the use of chemical growth retardants may be omitted or reduced in commercial production systems of Cape daisy through implementation of Ri lines in future breeding programs.

Effects of plant growth regulator application on the malting quality of barley.

BACKGROUND: Lodging can negatively affect yield and quality of barley grain. Synthetic plant growth regulators (PGRs) reduce lodging by producing shorter, thicker, and stronger stems. However, the impact of applying PGRs on malting performance of barley is not known. The objective of this work was to assess the effect of application of three PGRs (ethephon, chlormequat chloride, and trinexapac-ethyl) in combination with different seeding rates on the malting quality of barley grown in several locations and years in western Canada. RESULTS: The kernel weight in PGR-treated barley was reduced by 1.7% to 6.5% compared with the nontreated grain. Application of PGRs had no effect on the concentration of proteins and germination energy. Seeding rates significantly affected kernel weight, protein content, and germination index (GI), but no interactions between PGRs and seeding rates were observed. The smaller kernels of ethephon- and trinexapac-treated barley showed good hydration and grain modification during malting, as indicated by high levels of starch-converting enzymes, high Kolbach indices, and low levels of wort ß-glucans. Overall, the fine extract of malt from PGR-treated barley was slightly lower than that of the control malt; however, the extract reduction was statistically significant only for chlormequat- and trinexapac-treated barley. CONCLUSIONS: The application of PGRs had significant effects on kernel plumpness and kernel weight, but the effects of PGR application on the malting quality were generally small and insignificant. The decision of PGRs application on malting barley needs to be considered in combination with potential benefits of PGRs in mitigating lodging and their effects on the agronomic performance of barley. © Her Majesty the Queen in Right of Canada 2019.

Chlormequat chloride promotes rat embryonic growth and GH-IGF-1 axis.

Chlormequat chloride, a plant growth regulator, is widely applied in agriculture because it can promote sturdier growth of the crops. In this research, we found that rat embryo growth on GD11 was inhibited in vitro at 50 µg/ml but promoted in vivo at 75 mg/kg.bw by maternal oral exposure. Therefore, the concentrations of chlormequat chloride in the sera of the pregnant rats on gestation day (GD)11 were determined by a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) test to be 1.94 ± 0.023 µg/ml, 3.84 ± 0.080 µg/ml, and 7.08 ± 0.11 µg/ml, respectively, when the pregnant rats were orally exposed to chlormequat chloride at 75, 137.5, and 200 mg/kg.bw. Hence, we performed WEC tests again and confirmed that the rat embryo growth in vitro was promoted by chlormequat chloride at 5 µg/mL. The embryonic growth hormone (GH) and insulin-like growth factor 1 (IGF-1) levels were increased by chlormequat chloride both in vitro and in vivo compared with the control ones. We concluded that chlormequat chloride could elevate GH and IGF-1 levels in embryos and promote embryonic growth both in vitro and in vivo.

Proteolytic Activity in Meadow Soil after the Application of Phytohormones.

Phytohormones, similar to soil enzymes, are synthesized and secreted into the soil environment by fungi and microorganisms. Phytohormones are involved in regulating microbial community activity in the rhizosphere. This paper examines how auxins, cytokinins, ethephon and chlorocholine chloride affect the activity of native soil proteases in the organo-mineral horizon of an alpine meadow. In the meadow habitat, native soil proteases were inhibited by auxins whereas the effect of cytokinins on these enzymes was not statistically significant. A similar inhibitory effect on the activity of proteases was shown for ethephon and chlorocholine chloride, both of which also inhibited the activity of native soil proteases in the alpine meadow soil. Overall, the inhibitory effect of phytohormones on the activity of native protease activity may affect plant nutrition by retarding the nitrogen cycle in the soil. This work contributes to our understanding of the influence of substances produced by the rhizosphere that can actively participate in the activity of soil microorganisms and consequently influence the soil nitrogen cycle.

Comprehensive Transcriptome Reveals an Opposite Regulatory Effect of Plant Growth Retardants in Controlling Seedling Overgrowth between Roots and Shoots.

Seedling overgrowth always develops in undernourished plants due to biotic or abiotic stresses, which significantly decrease the yield of crops and vegetables. It is known that the plant growth retardants paclobutrazol (PBZ) and chlormequat chloride (CCC) are the most commonly used chemicals in controlling seedling height in plants by regulating the gibberellin (GA) biosynthesis pathway. However, the exact molecular regulation mechanism remains largely unknown. This study performed a comprehensive transcriptome profile to identify significantly differentially expressed genes after adding CCC and PBZ to the water culture seedling raising system for the first time. According to the obviously restrained shoots and roots, the GA biosynthesis genes were significantly decreased, as well as the endogenous GA content being reduced. Intriguingly, the GA signaling pathway genes were affected in opposite ways, increasing in roots but decreasing in shoots, especially regarding the phytochrome interacting factor SlPIF1 and the downstream genes expansins (SlEXPs), which promote cell wall remodeling. Further study found that the most down-regulated genes SlEXPA5 and SlEXPA15 were expressed specifically in shoot tissue, performing the function of repressing elongation, while the up-regulated genes SlEXPB2 and SlEXPB8 were proven to be root-specific expressed genes, which may promote horizontal elongation in roots. This research reported the comprehensive transcriptome profiling of plant growth retardants in controlling seedling overgrowth and restraining GA biosynthesis through the regulation of the GA signaling-related genes SlPIF1 and SlEXPs, with an opposite expression pattern between roots and shoots.

Improved accumulation of betulin and betulinic acid in cell suspension culture of Betula pendula roth by abiotic and biotic elicitors.

Betulin (B) and betulinic acid (BA) are two triterpenes with diverse pharmacological and physiological actions. Elicitation of Betula pendula Roth cell cultures by elicitors is an excellent strategy to increase B and BA levels. Six abiotic and biotic elicitors were studied to improve accumulation of B and BA in the cell culture of B. pendula. The B and BA production in treated cells was verified by HPLC. The results showed the maximum growth index (7) on day 3 in cells treated with 0.5 mg L-1 chlorocholine chloride (CCC). The increased accumulation of BA in the cells treated with 200 mg L-1 of chitosan was found to be 5.9 × (6.5 mg g-1 DW) higher over control cells. Treating the cells with 2 mg L-1 of CCC, after 7 days, led to 149.3× enhancement of B content (19.4 mg g-1 DW) over the controls. Production of this triterpenoid at a much shorter time with a much higher growth rate can be economic and lead to producing large amounts of B and BA for anti-cancer and HIV drugs preparation.

Evolutionary, interaction and expression analysis of floral meristem identity genes in inflorescence induction of the second crop in two-crop-a-year grape culture system.

The fruitfulness of grapevines (Vitis viniferaL.) is determined to a large extent by the differentiation of uncommitted meristems, especially in the second-crop production of some varieties, where the intermediate of inflorescence and tendril accounts for a significant proportion in two-crop-a-year grape culture system. The differentiation of uncommitted lateral meristem was reported to be regulated by a network, whose backbone was composed of several floral meristem identity genes. In the present study, the phylogenetics of grape floral meristem identity genes with their orthologues in other species, and their conserved domain and interaction networks were analysed. In addition, the effects of chlormequat chloride and pinching treatments on the expression profiles of floral meristem identity genes and content of gibberellic acid (GAs) and zeatin riboside (ZR), as well as the ratio of ZR/GAs in buds that were used to produce the second crop, and the ratio of inflorescence induction of the second crop were studied in 'Summer Black'. The present results showed that floral meristem identity genes of grape and their orthologues in one or more among Malus domastic, Citrus sinensis, heobroma cacaoT, Nicotiana tabacum, Solanum lycopersicum and Glycine hirsutum, probably originated from a common ancestor. Interaction networks of six grape-floral meristem identity genes indicated that the inflorescence induction and floral development were regulated by one more complex network, and expression profiles of genes that involved in this network could be affected by each other. Expression profiles of eight floral meristem identity genes were affected by chlormequat chloride and pinching treatments, and higher expression levels of FT, TFL1A and TFL1B, as well as lower expression levels of LFY from 3 days before full bloom to 11 days after full bloom were thought to play important roles in promoting the formation of inflorescence primordial of the second crop, and higher expression levels of CAL A, SOC1 and TFL1A at 18 days after full bloom (DAF) could promote the development of inflorescence primordial. In addition, lower ratio of ZR/GAs at 3 days before full bloom and 4 days after full bloom could promote the formation of uncommitted lateral meristems in chlormequat chloride and pinching-treated plants, and higher ratio at 11 days after full bloom was the main reason for the formation of more inflorescences after chlormequat chloride treatment.

Genome-wide identification and expression analysis of the ClTCP transcription factors in Citrullus lanatus.

BACKGROUND: The plant-specific TCP transcription factor family, which is involved in the regulation of cell growth and proliferation, performs diverse functions in multiple aspects of plant growth and development. However, no comprehensive analysis of the TCP family in watermelon (Citrullus lanatus) has been undertaken previously. RESULTS: A total of 27 watermelon TCP encoding genes distributed on nine chromosomes were identified. Phylogenetic analysis clustered the genes into 11 distinct subgroups. Furthermore, phylogenetic and structural analyses distinguished two homology classes within the ClTCP family, designated Class I and Class II. The Class II genes were differentiated into two subclasses, the CIN subclass and the CYC/TB1 subclass. The expression patterns of all members were determined by semi-quantitative PCR. The functions of two ClTCP genes, ClTCP14a and ClTCP15, in regulating plant height were confirmed by ectopic expression in Arabidopsis wild-type and ortholog mutants. CONCLUSIONS: This study represents the first genome-wide analysis of the watermelon TCP gene family, which provides valuable information for understanding the classification and functions of the TCP genes in watermelon.

Effect of chlormequat (cycocel) on the growth of ornamental cabbage and kale (Brassica oleracea) cultivars 'Kamome White' and 'Nagoya Red'.

The effect of concentration and application method of chlormequat (cycocel), a plant growth retardant, on plant height and some other traits in Brassica oleracea cultivars 'Kamome White' and 'Nagoya Red' was assessed. Plant growth retardants are commonly applied to limit stem elongation and produce a more compact plant. The experiment was done as a factorial in randomized completely blocks design (RCBD) with four replications. Plants (40 days after transplanting) were sprayed and drenched with 500, 1000 and 1500 mg l(-1) cycocel. In each experiment, control untreated plants. Data were recorded the 60 and 90 days after transplanting. Based on analysis of variance (ANOVA), the effect of different treatments and their interaction on all traits was significant at 0.05 or 0.01 level of probability. Treatment of 1500 mg I(-1) cycocel resulted in about 50 and 20% shorter plants than control plants, 60 and 90 days after transplant. The growth of Brassica oleracea cultivar 'Kamome White' and 'Nagoya Red' decreased with increased cycocel concentration. Foliar sprays of cycocel controlled plant height of both cultivars. Results indicated that the shortest plants (9.94 and 11.59 cm) were those sprayed with 1500 mg l(-1) cycocel in cultivar 'Kamome White' after 60 and 90 days, respectively. The largest number of leaves (33.94) and highest leaf diameter (9.39 cm) occurred in cv. 'Nagoya Red', when drench was used. Maximum dry matter (14.31%) accumulated in cv. 'Nagoya Red', treated with spray.

Effect of chlorocholine chlorid on phenolic acids accumulation and polyphenols formation of buckwheat plants.

BACKGROUND: Effect of chlorocholine chloride (CCC) on phenolic acids composition and polyphenols accumulation in various anatomical parts (stems, leaves and inflorescences) of common buckwheat (Fagopyrum esculentum Moench) in the early stages of vegetation period were surveyed. RESULTS: Treatment of buckwheat seeds with 2% of CCC has been increased content of total phenolics in the stems, leaves and inflorescences. On analyzing the different parts of buckwheat plants, 9 different phenolic acids - vanilic acid, ferulic acid, trans-ferulic acid, chlorogenic acid, salycilic acid, cinamic acid, p-coumaric acid, p-anisic acid, methoxycinamic acid and catechins were identified. The levels of identified phenolic acids varied not only significantly among the plant organs but also between early stages of vegetation period. Same changes as in contents of chlorogenic acid, ferulic acid, trans-ferulic acid were found for content of salycilic acid. The content of these phenolic acids has been significant increased under effect of 2% CCC treatment at the phase I (formation of buds) in the stems and at the phase II (beginning of flowering) in the leaves and then inflorescences respectively. The content of catechins as potential buckwheat antioxidants has been increased at the early stages of vegetation period after treatment with 2% CCC. CONCLUSIONS: The obtained results suggest that influence of CCC on the phenolics composition can be a result of various mechanisms of CCC uptake, transforming and/or its translocation in the buckwheat seedlings.

Effect of chlorocholine chlorid on phenolic acids accumulation and polyphenols formation of buckwheat plants

BACKGROUND: Effect of chlorocholine chloride (CCC) on phenolic acids composition and polyphenols accumulation in various anatomical parts (stems, leaves and inflorescences) of common buckwheat (Fagopyrum esculentum Moench) in the early stages of vegetation period were surveyed. RESULTS: Treatment of buckwheat seeds with 2% of CCC has been increased content of total phenolics in the stems, leaves and inflorescences. On analyzing the different parts of buckwheat plants, 9 different phenolic acids - vanilic acid, ferulic acid, trans-ferulic acid, chlorogenic acid, salycilic acid, cinamic acid, p-coumaric acid, p-anisic acid, methoxycinamic acid and catechins were identified. The levels of identified phenolic acids varied not only significantly among the plant organs but also between early stages of vegetation period. Same changes as in contents of chlorogenic acid, ferulic acid, trans-ferulic acid were found for content of salycilic acid. The content of these phenolic acids has been significant increased under effect of 2% CCC treatment at the phase I (formation of buds) in the stems and at the phase II (beginning of flowering) in the leaves and then inflorescences respectively. The content of catechins as potential buckwheat antioxidants has been increased at the early stages of vegetation period after treatment with 2% CCC. CONCLUSIONS: The obtained results suggest that influence of CCC on the phenolics composition can be a result of various mechanisms of CCC uptake, transforming and/or its translocation in the buckwheat seedlings.

[Study on rapid propagation of Scutellaria baicalensis variation].

OBJECTIVE: To establish and optimize the technology of taking root and promoting seedlings of white flower Scutellaria baicalensis test tube plantlet, and provide the theory and technology base for efficient factorization production system of white flower Scutellaria baicalensis. METHODS: Stem segments with axillary bud were cultured onto the different basic medium with different kinds and concentration of cytokinin and auxins to take root and produce strong seedling. RESULTS: The suitable culture medium for taking root of white flower Scutellaria baicalensis was 1/2 MS (all substance reduced half) + IBA 0.02 mg/L + sucrose 2%, the induction rate of root was 100%; The best medium for promoting seedling was 1/2 MS (all substance reduced half) + PP333 0.2 mg/L + IBA 0.02 mg/L + sucrose 2%, the seedling was green, the internode was normal, and its growth was vigorous and healthy. CONCLUSION: 1/2 MS (all substance reduced half) culture medium and relatively low concentration of sucrose is beneficial to inducing roots; Media adding appropriate concentration of IBA can significantly increase the root induction rate, the seedling has many stout roots; PP333 has dwarfing effect on seedlings and suitable concentration of PP333 can significantly improve the quality of the plantlets. A good technology of taking root and producing strong test tube plantlets is established.

[Effect of different plant growth regulators on yield and quality of Angelica dahurica var. formosana development].

OBJECTIVE: To investigate the effect of plant growth regulators on the growth and quality of Angelica dahurica var. formosana. METHOD: Five plant growth regulators: chlormequat chloride (CCC), Mepiquat chloride (PIX), Gibberellic acid (GA3), Paclobutrazol (PP333) and Maleic Hydrazide (MH) were sprayed in rosette stage, the effects of these plant growth regulators (PGRs) on the growth, yield and quality of A. dahurica var. formosanaw were observed. The biological traits were first measured and then imperatorin and isoimperatorin contents in roots were determined by HPLC. RESULT: Low concentration GA3 increased the yield while not influenced the premature bolting rate and the coumarin content. CONCLUSION: Spraying of GA3 (30 mg x L(-1)) could guarantee the growth and development of A. dahurica var. formosana to have a higher yield and maintain the active ingredients content in the root as well.

In vitro tuberization of Chlorophytum Borivilianum Sant & Fern (Safed musli) as influenced by sucrose, CCC and culture systems.

This study focuses on the establishment of in vitro tuberization of Chlorophytum borivilianum using solid and liquid culture systems. A high in vitro tuberization rate on solid and stationary liquid Murashige and Skoog media was observed in the presence of 60 g l⁻¹ sucrose with 950, 1,265 and 1,580 µM 2-chloroethyl-trimethylammonium chloride (CCC). Application of a higher sucrose concentration of 90 g l⁻¹ showed a negative interaction with CCC on in vitro tuber number and days to in vitro tuber induction. For economic feasibility, 950 µM CCC with 60 g l⁻¹ sucrose was chosen as the best combination for in vitro tuberization in both solid and stationary liquid media. For optimization of in vitro tuber production,a comparison between solid, stationary liquid and shake liquid culture was carried out. Liquid culture with shaking at 80 r.p.m. resulted in a >2.5-fold increase in in vitro tuber production compared with solid culture.

Efficient micropropagation and chlorocholine chloride induced stevioside production of Stevia rebaudiana Bertoni.

A promising method of micropropagation of Stevia rebaudiana Bertoni has been developed with an aim to increase the biomass, survivability of the plantlets and stevioside production, using chlorocholine chloride (CCC). Microshoots transferred to the MS medium containing different combinations CCC and IBA were found to be most effective in terms of growth pattern, hardening ability of the plantlets and stevioside content, compared to MS medium containing either IBA or CCC. Among other combinations tested, MS medium supplemented with 3 mg/l CCC and 3 mg/l IBA was found most effective in inducing significant changes like reduced shoot length, increased number of roots, higher leaf size, increased biomass and chlorophyll retaining capacity, higher survival percentage and most importantly the elevated stevioside content. Collectively, the major observations of this research indicate that application of CCC in micropropagation of S. rebaudiana Bertoni is a promising approach and has commercial prospects.

Chlorocholine chloride and paclobutrazol treatments promote carbohydrate accumulation in bulbs of Lilium Oriental hybrids 'Sorbonne'.

The present study was to test the hypothesis that the plant growth retardants chlorocholine chloride (CCC) and paclobutrazol (PBZ) could improve the carbohydrate accumulation in lily bulbs by enhancing photosynthetic capacity and changing endogenous hormones. Plants of Lilium Oriental hybrids 'Sorbonne' were treated with a foliar spray of CCC or PBZ (both at 300 mg/L) solution, at six weeks after planting (6 WAP). The morphological parameters, endogenous hormone contents (gibberellic acid (GA), abscisic acid (ABA), and indole-3-acetic acid (IAA)), and carbohydrate contents were measured from 6 to 18 WAP, at 2-week intervals. The results showed that CCC increased the biomass of leaves and stems which might produce more photoassimilates available for transportation and utilization. However, PBZ treatment suppressed vegetative growth and favored photoassimilate transportation into bulbs. A slight delay of bud and anthesis formation was observed in both treated plants. CCC and PBZ treatments substantially enhanced the sucrose contents in leaves probably due to the increase of chlorophyll contents. Treatment with CCC or PBZ decreased GA but increased IAA contents in lily bulbs which might stimulate starch accumulation and formation of new scales. Our experiment suggested that CCC or PBZ treatment is an effective method to promote carbohydrate accumulation in lily bulbs.

Epigenetic and physiological effects of gibberellin inhibitors and chemical pruners on the floral transition of azalea.

The ability to control the timing of flowering is a key strategy in planning the production of ornamental species such as azaleas; however, it requires a thorough understanding of floral transition. DNA methylation is involved in controlling the functional state of chromatin and gene expression during floral induction pathways in response to environmental and developmental signals. Plant hormone signalling is also known to regulate suites of morphogenic processes in plants and its role in flowering-time control is starting to emerge as a key controlling step. This work investigates if the gibberellin (GA) inhibitors and chemical pinching applied in improvement of azalea flowering alter the dynamics of DNA methylation or the levels of polyamines (PAs), GAs and cytokinins (CKs) during floral transition, and whether these changes could be related to the effects observed on flowering ability. DNA methylation during floral transition and endogenous content of PAs, GAs and CKs were analysed after the application of GA synthesis inhibitors (daminozide, paclobutrazol and chlormequat chloride) and a chemical pruner (fatty acids). The application of GA biosynthesis inhibitors caused alterations in levels of PAs, GAs and CKs and in global DNA methylation levels during floral transition; also, these changes in plant growth regulators and DNA methylation were correlated with flower development. DNA methylation, PA, GA and CK levels can be used as predictive markers of plant floral capacity in azalea.

Molecular cloning and function assay of a chalcone isomerase gene (GbCHI) from Ginkgo biloba.

Chalcone isomerase (CHI, EC 5.5.1.6) is one of the key enzymes in the flavonoid biosynthesis pathway catalyzing the stereospecific isomerization of chalcones into their corresponding (2S)-flavanones. In this investigation, both the cDNA sequence and the genomic sequence encoding the chalcone isomerase from Ginkgo biloba L. (designated as GbCHI) were isolated from the leaves. The GbCHI gene contained two introns and three extrons and encoded a peptide of 244 amino acids with a predicted molecular mass of 26.29 kDa and a pI of 7.76. RQPCR showed that GbCHI was expressed in a tissue-specific manner in G. biloba. Expression of GbCHI was also up-regulated by UV-B irradiation or treatment with 5-aminolevulinic acid or three plant growth regulator-ethylene, abscisic acid, and chlormequat-and these effects were consistent with analysis of the GbCHI promoter region. The recombinant protein was successfully expressed in an E.coli strain with the pET-28a vector. In vitro enzyme activity, assayed by HPLC, indicated that recombinant GbCHI protein could catalyze the formation of naringenin from 6'-hydroxychalcone. RQPCR analysis showed that CHI activity correlated with changes in transcription level of the CHI gene, GbCHI activity was also positively correlated with total flavonoid levels in ginkgo leaves, suggesting CHI as a key gene regulating flavonoid accumulation in ginkgo leaves.

The metabolite products of chlorocholine chloride (CCC) in eggs and meat of laying hens fed 15N-CCC containing diets.

An experiment was conducted to evaluate the metabolic products of chlorocholine chloride (CCC) in eggs and meat of laying hens fed a diet containing (15)N-CCC. Ten brown laying hens were randomly divided into two groups of five each. One group was offered (15)N-CCC free diet while the other group received a diet with 100 ppm (15)N-CCC for 11 days. Samples of eggs and meat from the laying hens were collected. Egg yolks and albumen were separated. Meat was collected from the breast and femur. The metabolic products of CCC were measured using ion trap electrospray ionisation mass spectrometry (ion trap-ESI-MS/MS). Determination of CCC or its metabolites in eggs and meat showed that CCC was metabolised to choline. Corresponding MS/MS spectra were obtained for m/z 104 (choline) or 105 ((15)N-choline), whereas nothing was detected at m/z 122 (CCC) or 123 ((15)N-CCC). The results from this study indicate that CCC will be metabolised in tissues of laying hens.