scholarly journals Effect of sampling time on somatic and germ cell mutations induced by acrylamide in gpt delta mice

2021 ◽  
Vol 43 (1) ◽  
Author(s):  
Soichiro Hagio ◽  
Naho Tsuji ◽  
Satoshi Furukawa ◽  
Kazuya Takeuchi ◽  
Seigo Hayashi ◽  
...  
Keyword(s):  
Gene Mutation ◽  
Germ Cells ◽  
Peripheral Blood ◽  
Day Treatment ◽  
Intraperitoneal Administration ◽  
Sampling Time ◽  
Final Treatment ◽  
Micronucleus Tests ◽  
Sampling Times ◽  
Expression Time

Abstract Background Acrylamide (AA) is a rodent carcinogen and classified by the IARC into Group 2A (probable human carcinogen). AA has been reported to induce mutations in transgenic rodent gene mutation assays (TGR assays), the extent of which is presumed to depend on exposure length and the duration of expression after exposure. In particular, it is not clear in germ cells. To investigate mutagenicity with AA in somatic and germ cells at different sampling times, we conducted TGR assays using gpt delta transgenic mice. Results The male gpt delta mice at 8 weeks of age were treated with AA at 7.5, 15 and 30 mg/kg/day by gavage for 28 days. Peripheral blood was sampled on the last day of the treatment for micronucleus tests and tissues were sampled for gene mutation assays at day 31 and day 77, those being 3 and 49 days after the final treatment (28 + 3d and 28 + 49d), respectively. Another group of mice was treated with N-Ethyl-N-nitrosourea (ENU) at 50 mg/kg/day by intraperitoneal administration for 5 consecutive days and tissues were sampled at the day 31 and day 77 (5 + 26d and 5 + 72d). Frequencies of micronucleated erythrocytes in the peripheral blood significantly increased at AA doses of 15 and 30 mg/kg/day. Two- to three-fold increases in gpt mutation frequencies (MFs) compared to vehicle control were observed in the testes and lung treated with 30 mg/kg/day of AA at both sampling time. In the sperm, the gpt MFs and G:C to T:A transversions were significantly increased at 28 + 3d, but not at 28 + 49d. ENU induced gpt mutations in these tissues were examined at both 5 + 26d and 5 + 72d. A higher mutant frequency in the ENU-treated sperm was observed at 5 + 72d than that at 5 + 26d. Conclusions The gpt MFs in the testes, sperm and lung of the AA-treated mice were determined and compared between different sampling times (3 days or 49 days following 28 day-treatment). These results suggest that spermatogonial stem cells are less sensitive to AA mutagenicity under the experimental condition. Prolonged expression time after exposure to AA to detect mutagenicity may be effective in somatic cells but not in germ cells.

scholarly journals The 28 + 28 day design is an effective sampling time for analyzing mutant frequencies in rapidly proliferating tissues of MutaMouse animals

2021 ◽  
Vol 95 (3) ◽  
pp. 1103-1116
Author(s):  
Francesco Marchetti ◽  
Gu Zhou ◽  
Danielle LeBlanc ◽  
Paul A. White ◽  
Andrew Williams ◽  
...  
Keyword(s):  
Germ Cells ◽  
False Negative ◽  
Sampling Time ◽  
Male Germ Cells ◽  
Negative Results ◽  
False Negative Results ◽  
Detection Of Mutations ◽  
The Impact ◽  
Sampling Times

AbstractThe Organisation for Economic Co-Operation and Development Test Guideline 488 (TG 488) uses transgenic rodent models to generate in vivo mutagenesis data for regulatory submission. The recommended design in TG 488, 28 consecutive daily exposures with tissue sampling three days later (28 + 3d), is optimized for rapidly proliferating tissues such as bone marrow (BM). A sampling time of 28 days (28 + 28d) is considered more appropriate for slowly proliferating tissues (e.g., liver) and male germ cells. We evaluated the impact of the sampling time on mutant frequencies (MF) in the BM of MutaMouse males exposed for 28 days to benzo[a]pyrene (BaP), procarbazine (PRC), isopropyl methanesulfonate (iPMS), or triethylenemelamine (TEM) in dose–response studies. BM samples were collected + 3d, + 28d, + 42d or + 70d post exposure and MF quantified using the lacZ assay. All chemicals significantly increased MF with maximum fold increases at 28 + 3d of 162.9, 6.6, 4.7 and 2.8 for BaP, PRC, iPMS and TEM, respectively. MF were relatively stable over the time period investigated, although they were significantly increased only at 28 + 3d and 28 + 28d for TEM. Benchmark dose (BMD) modelling generated overlapping BMD confidence intervals among the four sampling times for each chemical. These results demonstrate that the sampling time does not affect the detection of mutations for strong mutagens. However, for mutagens that produce small increases in MF, sampling times greater than 28 days may produce false-negative results. Thus, the 28 + 28d protocol represents a unifying protocol for simultaneously assessing mutations in rapidly and slowly proliferating somatic tissues and male germ cells.

scholarly journals Impact of Exposure to Low Temperature Degrees in Field Conditions on Leaf Pigments and Chlorophyll Fluorescence in Leaves of Mango Trees

2020 ◽  
Vol 10 ◽  
pp. 30-45
Author(s):  
Ali A.S. Sayed ◽  
Farouk M. Gadallah ◽  
Mohamed A. Seif El-Yazal ◽  
Gamal A. Abdel-Samad
Keyword(s):  
Chlorophyll Fluorescence ◽  
Low Temperature ◽  
Chlorophyll Concentration ◽  
Ambient Air ◽  
Sampling Time ◽  
Chlorophyll B ◽  
Physiological And Biochemical ◽  
Sampling Times ◽  
Fayoum Governorate

This experiment was conducted to found the connection between low temperature stress in vivo conditions (ambient-air temperature) and the changes in some physiological and biochemical events (leaf pigments and chlorophyll fluorescence) of mango trees in response to exposure to natural low temperature (cold). To verify this objective, 12 popular commonly mango cultivars (25 years old) which grown in private orchard in Fayoum Governorate, Egypt were selected for this study which carried out during the period from November to March of years; 2012 and 2013. The selected cultivars were: Alphonso, Baladi, Bullock's Heart, Helmand, Hindi Besennara, Mabrouka, Mestekawy, Nabeeh, Oweisi, Spates, Taimour and Zebda. Based on the obtained results, it can be stated that, chlorophyll (a) concentration in the leaves was significantly differed among the cultivars throughout the whole sampling times, in this respect, Helmand one gave the highest one while, and the highest one by sampling times was November one. The concentration of chlorophyll (b) was significant as effected by the effect of cultivars and sampling time recorded the highest value by the cultivar of Spates and December sample, respectively. Total chlorophyll concentration in the leaves reached its peak by the cultivar of Nabeeh and sampling time of December as compared to others. The both of Ewais cultivar and the sample of March showed the highest values of carotenoids concentration in the leaves. The levels of anthocyanin in leaves were significantly differed as affected by the cultivars and sampling times, indicating that the cultivar of Helmand and November sample recorded the highest values of anthocyanin in leaves. The greatest reductions in Fv/Fmratio were recorded at month of November and indicated that the reductions were in the order of Alphonso˃ Mabrouka˃Taimour˃ others. The effect of sampling time, cultivars and their interaction on Fv/Fm were significant, but small between some values of Fv/Fm.

scholarly journals BCOR Mutation and TLS-ERG Expression in Acute Myeloid Leukemia with Monoclonal Immunoglobulinemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5180-5180
Author(s):  
Jian Huang ◽  
Jingxia Jin ◽  
Shuna Luo ◽  
Xingnong Ye
Keyword(s):  
Bone Marrow ◽  
Gene Mutation ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Poor Prognosis ◽  
Plasma Cells ◽  
Fusion Gene ◽  
Next Generation Dna Sequencing ◽  
School Of Medicine ◽  
Immunofixation Electrophoresis

Acute myeloid leukemia(AML) originates from the abnormal clonal proliferation of myeloblast which often combined with clinical symptoms. Cytogenetic and molecular abnormalities are frequent in AML patience. To date, the driver genes for leukemia remain largely undiscovered. Monoclonal immunoglobulinemia is a group of diseases caused by excessive proliferation of plasma cells or immunoglobulin-producing lymphoid plasma cells and B lymphocytes. It can develop into malignant plasma cell disease. Herein, we report a AML patient was concomitant with monoclonal immunoglobulinemia, the patient was also accompanied by BCOR mutation and TLS-ERG fusion gene. A 55-year-old married female was admitted into our hospital due to repeated edema for 3 weeks. On admission, peripheral blood counts: PLT142×10^9/L, HB77g/L↓, WBC35.2×10^9/L.Bone marrow examination showed the mononuclear cell system proliferated actively, and the primitive infantile monocytes accounted for 86%. Cell morphology suggested M5b(Figure1A ). Fusion gene screening in bone marrow revealed that TLS-ERG expression. Immunophenotype of bone marrow cell:Abnormal myeloid primitive cells accounted for 96.39% of the nuclear cells,expressCD33, CD13, CD123, CD34, CD9, MPO(Figure 1D). Karyotype analysis of bone marrow cells showed in Figure 1B. Thus, AML was diagnosed. Next-generation DNA sequencing technology showed that BCOR (51.7%),PLCG1(49.9%),DIS3(48.4%),BRAF(51.6%), JAK2(45.1%) ,JAK3(49.0%) were mutated. Meanwhile, we found that Peripheral blood immunofixation electrophoresis showed that Gamma region is seen with a monoclonal light chain lambda component((Figure 1C.).Then, the patient underwent one cycle of IA(Idabisine hydrochloride 10mg d1-4, cytarabine 0.075g q12h d1-7). Twenty-five after chemotherapy onset, bone marrow examination showed that primitive and immature monocytes accounted for 3%. Chromosome become normal. Minimal residual disease(MRD):0.01%. The disease reached complete remission(CR). Peripheral blood immunofixation electrophoresis turned negative. Fusion gene detection showed that TLS-ERG turned negative. BCOR mutation was not detected by Next-generation DNA sequencing. Mutations of PLCG1,DIS3,BRAF,JAK2,JAK3 still exist. Monoclonal immunoglobulinemia and AML are both clonal diseases, but originated from different clones. This case has both malignant clones of granulocyte stem cell and malignant clones of B line, so it is worthy of discussion. By comparing CR before and after we found that while the patient's M protein turned negative, the TLS-ERG fusion gene and BCOR gene mutation also disappeared. The TLS-ERG fusion gene is formed by the rearrangement of TLS and ERG genes on chromosomes 16 and 21. The current study holds that the expression of this fusion gene indicates rapid disease progression and poor prognosis. BCOR mutations can be found in AML and often coincide with DNMT3 gene mutations, suggesting it may affect the occurrence of leukemia through epigenetics. BCOR is a newly discovered corepressor of BCL-6, which can play a supporting role when BCOR combines with DNA; when BCOR is overexpressed, it can enhance the inhibition of BCL-6. BCL-6 is highly expressed in tumor cells,it encodes transcriptional repressors which are required for the formation of germinal center and may affect apoptosis. We thinked that the monoclonal immunoglobulinemia of this patient may caused by the BCOR abnormal expression which increased the inhibitory effect of BCL-6 and affect the apoptosis of B cells, and B cells continue to secrete immunoglobulin. BCOR mutations are associated with poor prognosis. The patient with TLS-ERG fusion gene which is a poor prognosis gene.However, the BCOR gene mutation site is a non-hot spot mutation which has few clinical studies. Whether the BCOR gene mutation results in the combination of the two diseases requires further study. Acknowledgment:The research was supported by fundings of the public technology research projects of Yiwu,China (2016-S-05), the key medical discipline of Yiwu,China(Hematology,2018-2020),and the academician workstation of the Fourth Affiliated Hospital of Zhejiang University School of Medicine. Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China. Email: [email protected] Figure 1 Disclosures No relevant conflicts of interest to declare.

Comparison of Two Sampling Methods for Escherichia coli O157:H7 Detection in Feedlot Cattle

2005 ◽  
Vol 68 (8) ◽  
pp. 1724-1728 ◽  
Author(s):  
M. L. KHAITSA ◽  
M. L. BAUER ◽  
P. S. GIBBS ◽  
G. P. LARDY ◽  
D. DOETKOTT ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sampling Methods ◽  
Feedlot Cattle ◽  
Sampling Time ◽  
Escherichia Coli O157 ◽  
Confounding Factors ◽  
Rank Tests ◽  
E Coli ◽  
Signed Rank ◽  
Sampling Times

Two sampling methods (rectoanal swabs and rectal fecal grabs) were compared for their recovery of Escherichia coli O157:H7 from feedlot cattle. Samples were collected from 144 steers four times during the finishing period by swabbing the rectoanal mucosa with cotton swabs and immediately obtaining feces from the rectum of each individual steer. The number of steers with detectable E. coli O157:H7 increased from 2 of 144 (1.4%) cattle on arrival at the feedlot to 10 of 144 (6.9%) after 1 month, 76 of 143 (52.8%) after 7 months, and 30 of 143 (20.8%) at the last sampling time before slaughter. Wilcoxon signed-rank tests indicated that the two sampling methods gave different results for sampling times 3 and 4 (P < 0.05) but not for sampling time 2 (P = 0.16). Agreement between the two sampling methods was poor (kappa < 0.2) for three of the four sampling times and moderate (kappa = 0.6) for one sampling time, an indication that in this study rectoanal swabs usually were less sensitive than rectal fecal grabs for detection of E. coli O157:H7 in cattle. Overall, the herd of origin was not significantly associated with E. coli O157:H7 results, but the weight of the steers was. Further investigation is needed to determine the effects of potential confounding factors (e.g., size and type of swab, consistency of feces, site sampled, and swabbing technique) that might influence the sensitivity of swabs in recovering E. coli O157:H7 from the rectoanal mucosa of cattle.

scholarly journals Oocyte Generation in Adult Mammalian Ovaries by Putative Germ Cells in Bone Marrow and Peripheral Blood

Cell ◽  
2005 ◽  
Vol 122 (2) ◽  
pp. 303-315 ◽  
Author(s):  
Joshua Johnson ◽  
Jessamyn Bagley ◽  
Malgorzata Skaznik-Wikiel ◽  
Ho-Joon Lee ◽  
Gregor B. Adams ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Germ Cells ◽  
Peripheral Blood

316 Does sampling time matter? Relationships of circulating metabolites at various neonatal sampling times in beef calves

2016 ◽  
Vol 94 (suppl_2) ◽  
pp. 148-149
Author(s):  
J. M. Larson ◽  
B. L. Vander Ley ◽  
S. M. Bolen ◽  
N. B. Duncan ◽  
A. M. Meyer
Keyword(s):  
Sampling Time ◽  
Beef Calves ◽  
Sampling Times

Mesenchymal Stromal Cells Enhance G-CSF-Induced Mobilization Of Hematopoietic Stem- and Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2460-2460
Author(s):  
Evert-Jan F. M. de Kruijf ◽  
Ingmar van Hengel ◽  
Jorge M Perez-Galarza ◽  
Willem E. Fibbe ◽  
Melissa van Pel
Keyword(s):  
Bone Marrow ◽  
B Lymphocytes ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Calf Serum ◽  
Intraperitoneal Administration ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Hematopoietic stem- and progenitor cell (HSPC) mobilization is a property of most hematopoietic growth factors, such as Granulocyte Colony Stimulating Factor (G-CSF). Not all donors mobilize equally well and therefore the number of HSPC that are obtained following mobilization may be limited. Mesenchymal stromal cells (MSC) have the capacity to differentiate into cells of the mesodermal lineage and have immunomodulatory properties in vivo and in vitro. Here, we have investigated the effect of MSC co-administration on G-CSF-induced HSPC mobilization. MSC were obtained from bone marrow cells (bone marrow-derived) or bone fragments (bone-derived) and were expanded in alpha-MEM containing 10% fetal calf serum until sufficient cell numbers were obtained. Bone marrow or bone-derived MSC were administered intravenously for three days at a dose of 200 x103 cells per day to male C57BL/6 recipients that were simultaneously mobilized with G-CSF (10 μg per day intraperitoneally for 3 days) or PBS as a control. Co-injection of G-CSF and MSC lead to a 2-fold increase in HSPC mobilization compared to G-CSF alone (8,563 ± 3,309 vs. 4,268 ± 1,314 CFU-C per ml peripheral blood respectively; n=13, p<0.01). Administration of MSC alone did not induce HSPC mobilization (273 ± 229 CFU-C/ml blood; n=13). Furthermore, co-injection of splenocytes and G-CSF did not enhance HSPC mobilization, showing that the administration of exogeneous cells as such is not sufficient for enhancement of HSPC mobilization. It has been reported that G-CSF-induced HSPC mobilization is associated with a decrease in the number of osteal macrophages, B lymphocytes and erythroid progenitors. Administration of MSC alone induced a significant decrease in the frequency of osteal macrophages (7.9 ± 1.2 vs 6.2 ± 1.4% bone marrow cells for PBS vs. MSC respectively; n=8, p<0.05), but did not affect osteoblast numbers. Furthermore, the frequency of B lymphocytes was significantly decreased following MSC administration (29.9 ± 4.0 vs. 16.5 ± 4.9% bone marrow cells for PBS vs. MSC respectively; n=13, p<0.0001). No differences were observed in erythroid numbers following MSC administration. To investigate the mechanisms underlying these observations, the migratory capacity of luciferase transduced MSC was studied through bioluminescence imaging. Following intravenous injection, MSC were detected in the lungs, but not in other organs. In addition, no difference in MSC migration was observed between G-CSF and PBS treated mice. Moreover, intraperitoneal administration of G-CSF and MSC resulted in increased HSPC mobilization compared to G-CSF alone (10,178 ±3,039 vs. 5,158 ± 2,436 CFU-C per ml peripheral blood; n=5-12). Together, these data point to an endocrine effect of MSC on G-CSF-induced HSPC mobilization. No differences in IL-6, CXCL-12 or M-CSF levels in bone marrow extracellular fluid were observed. In conclusion, G-CSF-induced HSPC mobilization is enhanced by injection of MSC. We hypothesize that the MSC-induced partial depletion of B lymphocytes and osteal macrophages in the bone marrow are crucial factors involved in the enhancement of G-CSF-induced HSPC mobilization. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Sarpa salpa herbivory on shallow reaches of Posidonia oceanica beds

2014 ◽  
Vol 37 (1) ◽  
pp. 49-57 ◽  
Author(s):  
L. Steele ◽  
◽  
K. M. Darnelli ◽  
J. Cebrián ◽  
J. L. Sánchez-Lizaso ◽  
...  
Keyword(s):  
Spatial Variability ◽  
Leaf Area ◽  
Posidonia Oceanica ◽  
Sampling Time ◽  
Herbivorous Fish ◽  
Small Scale ◽  
Bite Size ◽  
Sarpa Salpa ◽  
Sampling Times

Here, we examined the temporal and small–scale spatial variability of grazing by the herbivorous fish Sarpa salpa on shallow beds of the temperate seagrass Posidonia oceanica. Herbivory intensity expressed as the percent of leaf area taken by fish bites was higher in September 2006 than in February 2007, and at 0.5 m than at 1.5 m during both sampling times. All S. salpa feeding at the shallow locations studied were juveniles, with bite sizes ranging from 0.03 to 0.62 cm2. Juveniles feeding at 1.5 m were larger in February 2007 than in September 2006, as evidenced by significant differences in mean bite size per shoot. However, the larger juveniles feeding at 1.5 m in February 2007 did not appear to feed as frequently as the comparatively smaller juveniles feeding at the same depth in September 2006, as suggested by significant differences in number of bites per shoot. The number of bites per shoot was also lower at 1.5 m than at 0.5 m in February 2007, although mean bite size did not differ significantly between the two depths at that sampling time. In general S. salpa juveniles did not select a particular range of leaf ages when feeding in the study locations, although the juveniles feeding at 1.5 m in September 2006 appeared to select mid–aged leaves. Fish did not show a preference for more epiphytized leaves. These results show that grazing activity by S. salpa juveniles in shallow reaches of P. oceanica meadows may vary temporally and across small changes in depth, which in turn may affect the overall intensity of herbivory on the seagrass.

scholarly journals Novel Primers and Sampling for PCR Detection of Xylella fastidiosa in Peach

2019 ◽  
Vol 109 (2) ◽  
pp. 307-317 ◽  
Author(s):  
Chunxian Chen ◽  
Clive H. Bock ◽  
Phillip M. Brannen
Keyword(s):  
Prunus Persica ◽  
Xylella Fastidiosa ◽  
Southeastern United States ◽  
Sampling Time ◽  
Pcr Detection ◽  
Detection Sensitivity ◽  
Tissue Type ◽  
Conventional Pcr ◽  
Reliable Detection ◽  
Sampling Times

Epidemics of phony peach disease (PPD), caused by Xylella fastidiosa, are of increasing concern to peach (Prunus persica) producers in the southeastern United States. Primers suitable for both conventional PCR (cPCR) and quantitative PCR (qPCR), along with optimal tissue and sampling time, are needed for comparative and reliable detection of X. fastidiosa. In this study, we developed and assessed novel primers for X. fastidiosa and for peach and compared detection of X. fastidiosa in four peach tissue types sampled at three time points using both cPCR and qPCR. Primer C06Xf-bamA was extensively tested for reliable detection of X. fastidiosa due to the more consistent intensity of the cPCR products and the marginally lower average quantification cycle (Cq) values of the qPCR products, compared with the other primers screened. Among the four peach tissue types tested, only root samples demonstrated reliable and consistent detection of X. fastidiosa; stem, petiole, and leaf samples, regardless of source trees, primers used, sampling times, or PCR methods (cPCR or qPCR), were unreliable for detection, due to insufficient quantity of DNA of X. fastidiosa in these samples based on the relative quantification assay. The Cq means and ratios were compared and statistically analyzed, to ascertain effects of source tree, tissue type, sampling time, and primer. Differences in detection sensitivity and the Cq means among sampled trees, sampling times, tested primers, and tissues (except root) were not significant or were inconsistent precluding further exploitation. In summary, these novel primers are a useful resource for detecting X. fastidiosa, and based on our results, root is the only tissue type reliable for year-round detection of X. fastidiosa in peach. Further research on potential utilization of above-ground tissues for PCR detection of X. fastidiosa are discussed.

Validation of saliva and urine use and sampling time on the doubly labelled water method to measure energy expenditure, body composition and water turnover in male and female cats

2020 ◽  
Vol 124 (4) ◽  
pp. 457-469
Author(s):  
Camila Goloni ◽  
Francine M. Peres ◽  
Igor L. S. Senhorello ◽  
Ludmilla G. Di Santo ◽  
Fernanda S. Mendonça ◽  
...  
Keyword(s):  
Body Composition ◽  
Energy Expenditure ◽  
Sampling Time ◽  
Isotope Enrichment ◽  
Water Turnover ◽  
Doubly Labelled Water ◽  
Male And Female ◽  
Before And After ◽  
Linear Elimination ◽  
Sampling Times

AbstractLess invasive protocols are necessary to study energy expenditure (EE) of cats living in homes for expressing their normal living conditions. The present study compared sampling times and the use of saliva, urine and blood to measure 2H and 18O to apply the doubly labelled water method. In the first study, four cats were used to evaluate the enrichment (2, 4, 6, 7 and 8 h) and elimination (2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 d) of 2H and 18O (subcutaneously injected). The maximum enrichment was after 5 h (R2 0·82) of injection, with an Ln linear elimination of both isotopes (P < 0·001; R2 0·99). The results of EE were similar, regardless of the sampling time used (P = 0·999). In the second study, seven male cats and seven female cats were used. Before and after isotope injection (5 h, 7 d, 10 d and 14 d), blood, saliva and urine were collected. Isotope enrichment was lower in urine (P < 0·05) and at the similar level in blood and saliva. Isotope elimination was similar for all fluids (P < 0·473). The EE calculated with blood and saliva was similar but higher for urine (P = 0·015). According to Bland–Altman statistics, blood and saliva presented low bias and high correlation (P < 0·001), but this was not observed for urine (P = 0·096). Higher EE was observed for male cats (384 (se 39) kJ/kg0·67 per d) than for female cats (337 (se 34) kJ/kg0·67 per d; P < 0·05). The sampling time for the method is flexible, and saliva can be used as a substitute for blood.

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