‘ANNEX VIMETHODS OF ANALYSIS FOR THE DETERMINATION OF CONSTITUENTS OF ANIMAL ORIGIN FOR THE OFFICIAL CONTROL OF FEED 1.PURPOSE AND SCOPEThe determination of constituents of animal origin in feed shall be performed by light microscopy or polymerase chain reaction (PCR) in accordance with the provisions laid down in this Annex.These two methods make it possible to detect the presence of constituents of animal origin in feed materials and compound feed. However, they do not make it possible to calculate the amount of such constituents in feed materials and compound feed. Both methods have a limit of detection below 0,1 % (w/w).The PCR method makes it possible to identify the taxonomic group of constituents of animal origin present in feed materials and compound feed.These methods shall apply for the control of the application of the prohibitions laid down in Article 7(1) and Annex IV to Regulation (EC) No 999/2001 and in Article 11(1) of Regulation (EC) No 1069/2009.Depending on the type of feed being tested, these methods may be used, within one single operational protocol, either on their own or combined together in accordance with the standard operating procedures (SOP) established by the EU reference laboratory for animal proteins in feedingstuffs (EURL-AP) and published on its websitenote.2.METHODS2.1.Light microscopy 2.1.1.PrincipleThe constituents of animal origin which may be present in feed materials and compound feed sent for analysis are identified on the basis of typical and microscopically identifiable characteristics like muscle fibres and other meat particles, cartilage, bones, horn, hair, bristles, blood, feathers, egg shells, fish bones and scales. 2.1.2.Reagents and equipment 2.1.2.1.Reagents 2.1.2.1.1.Concentrating agent 2.1.2.1.1.1.Tetrachloroethylene (specific gravity 1,62) 2.1.2.1.2.Staining reagent 2.1.2.1.2.1.Alizarin Red solution (dilute 2,5 ml 1M hydrochloric acid in 100 ml water and add 200 mg Alizarin Red to this solution) 2.1.2.1.3.Mounting media 2.1.2.1.3.1.Lye (NaOH 2,5 % w/v or KOH 2,5 % w/v) 2.1.2.1.3.2.Glycerol (undiluted, viscosity: 1490 cP) 2.1.2.1.3.3.Norland ® Optical Adhesive 65 (viscosity: 1200 cP) or a resin with equivalent properties for permanent slide preparation 2.1.2.1.4.Mounting media with staining properties 2.1.2.1.4.1.Lugol solution (dissolve 2 g potassium iodide in 100 ml water and add 1 g iodine while frequently shaking) 2.1.2.1.4.2.Cystine reagent (2 g lead acetate, 10 g NaOH/100 ml water) 2.1.2.1.4.3.Fehling’s reagent (prepared before use from equals parts (1/1) of two stock solutions A and B. Solution A: dissolve 6,9 g copper (II) sulphate pentahydrate in 100 ml water. Solution B: dissolve 34,6 g potassium sodium tartrate tetrahydrate and 12 g NaOH in 100 ml water) 2.1.2.1.4.4.Tetramethylbenzidine/Hydrogen peroxide. (dissolve 1 g 3,3’,5,5’ tetramethylbenzidine (TMB) in 100 ml glacial acetic acid and 150 ml water. Before use, mix 4 parts of this TMB solution with 1 part 3 % hydrogen peroxide) 2.1.2.1.5.Rinsing agents 2.1.2.1.5.1.Ethanol ≥ 96 % (technical grade) 2.1.2.1.5.2.Acetone (technical grade) 2.1.2.1.6.Bleaching reagent 2.1.2.1.6.1.Commercial sodium hypochlorite solution (9 - 14 % active chlorine) 2.1.2.2.Equipment 2.1.2.2.1.Analytical balance with an accuracy of 0,001 g 2.1.2.2.2.Grinding equipment: mill - or mortar 2.1.2.2.3.Sieves with square meshes of 0,25 mm and 1 mm width 2.1.2.2.4.Conical glass separation funnel with a content of 250 ml with Teflon or ground glass stopcock at the base of the cone. Stopcock opening diameter shall be ≥ 4mm. Alternatively, a conical bottomed settling beaker may be used provided the laboratory has demonstrated that detection levels are equivalent to that obtained using the conical glass separation funnel.Separation funnel 2.1.2.2.5.Stereomicroscope covering at least a 6,5× to 40× final magnification range 2.1.2.2.6.Compound microscope covering at least a 100× to 400× final magnification range with transmitted light bright field. Polarised light and differential interferential contrast can additionally be used 2.1.2.2.7.Standard laboratory glassware 2.1.2.2.8.Equipment for slide preparation: classical microscope slides, hollow slides, coverslips (20 × 20 mm), tweezers, fine spatula2.1.3.Sampling and sample preparation 2.1.3.1.SamplingA representative sample, taken in accordance with the provisions laid down in Annex I shall be used. 2.1.3.2.Precautions to be takenIn order to avoid laboratory cross-contamination, all reusable equipment shall be carefully cleaned before use. Separation funnel pieces shall be disassembled before cleaning. Separation funnel pieces and glassware shall be pre-washed manually and then washed in a washing machine. Sieves shall be cleaned by using a brush with stiff synthetic hairs. A final cleaning of sieves with acetone and compressed air is recommended after sieving of fatty material like fishmeal. 2.1.3.3.Preparation of samples other than fat or oil 2.1.3.3.1.Sample drying: samples with a moisture content > 14 % shall be dried prior to handling. 2.1.3.3.2.Sample pre-sieving: it is recommended to pre-sieve at 1 mm pelleted feeds and kernels and to subsequently prepare and analyse the two resulting fractions as distinct samples. 2.1.3.3.3.Sub-sampling and grinding: at least 50 g of the sample shall be sub-sampled for analysis and subsequently ground. 2.1.3.3.4.Extraction and preparation of the sediment: a portion of 10 g (accurate to 0,01 g) of the ground sub-sample shall be transferred into the separation funnel or conical bottomed settling beaker and 50 ml of tetrachloroethylene shall be added. The portion transferred into the funnel shall be limited to 3 g in case of fishmeal or other pure animal products, mineral ingredients or premixes which generate more than 10 % of sediment. The mixture shall be vigorously shaken for at least 30 s and at least 50 ml more of tetrachloroethylene shall be added cautiously while washing down the inside surface of the funnel to remove any adhering particles. The resulting mixture shall be left to stand for at least 5 minutes before the sediment is separated off by opening the stopcock.If a conical bottomed settling beaker is used then the mixture shall be vigorously stirred for at least 15 s and any particles adhering to the side of the beaker shall be carefully washed down the inside surface with at least 10 ml of clean tetrachloroethylene. The mixture shall be left to stand for 3 minutes and then stirred again for 15 seconds and any particles adhering to the side of the beaker shall be carefully washed down the inside surface with at least 10 ml of clean tetrachloroethylene. The resulting mixture shall be left to stand for at least 5 minutes and then the liquid fraction is removed and discarded by careful decanting, taking care not to lose any of the sediment.The sediment shall be dried and subsequently weighed (accurate to 0,001 g). If more than 5 % of the sediment consists of particles > 0,50 mm, it shall be sieved at 0,25 mm and the two resulting fractions shall be examined. 2.1.3.3.5.Extraction and preparation of the flotate: after recovery of the sediment with the method described above, two phases should remain in the separation funnel: a liquid one consisting of tetrachloroethylene and a solid one made of floating material. This solid phase is the flotate and shall be recovered by pouring off completely tetrachloroethylene from the funnel by opening the stopcock. By inverting the separation funnel, the flotate shall be transferred into a large Petri dish and air dried in a fumehood. If more than 5 % of the flotate consists of particles > 0,50 mm, it shall be sieved at 0,25 mm and the two resulting fractions shall be examined. 2.1.3.3.6.Preparation of raw material: a portion of at least 5 g of the ground sub-sample shall be prepared. If more than 5 % of the material consists of particles > 0,50 mm, it shall be sieved at 0,25 mm and the two resulting fractions shall be examined.2.1.3.4.Preparation of samples consisting of fat or oilThe following protocol shall be followed for the preparation of samples consisting of fat or oil: 2.1.3.5.Use of staining reagentsIn order to facilitate the correct identification of the constituents of animal origin, the operator may use staining reagents during the sample preparation in accordance with guidelines issued by the EURL-AP and published on its website.In case Alizarin Red solution is used to colour the sediment, the following protocol shall apply:2.1.4.Microscopic examination 2.1.4.1.Slide preparationMicroscopic slides shall be prepared from the sediment and, depending of operator’s choice, from either the flotate or the raw material. In case sieving has been used during the sample preparation, the two resulting fractions (the fine and the coarse one) shall be prepared. Test portions of fractions spread on slides shall be representative of the whole fraction.A sufficient number of slides shall be prepared in order to ensure that a complete examination protocol as laid down in point 2.1.4.2 can be carried-out.Microscopic slides shall be mounted with the adequate mounting medium in accordance with the SOP established by the EURL-AP and published on its website. The slides shall be covered with coverslips. 2.1.4.2.Observation protocols for the detection of animal particles in compound feed and feed materialThe prepared microscopic slides shall be observed in accordance with the observation protocols laid down in diagram 1 for compound feed and feed materials other than pure fishmeal, or in diagram 2 for pure fishmeal.The microscopic observations shall be conducted using the compound microscope on the sediment and, depending of the operator’s choice, either on the flotate or on the raw material. The stereomicroscope may be used in addition to the compound microscope for the coarse fractions. Each slide shall be screened entirely at various magnifications.The minimum numbers of slides to be observed at each step of the observation protocol shall be strictly respected unless the entire fraction material does not permit to reach the stipulated slide number. No more than 6 slides per determination shall be observed.In order to facilitate the identification of the particles’ nature and origin, the operator may use support tools like decision support systems, image libraries and reference samples.Diagram 1Observation protocol for the detection of animal particles in compound feed and feed material other than fishmealYESGrinding process of sample insures 95 % of particles ≤ 0.50 mmNOSieving on 0.25 mm mesh3 slides from sediment + 1 slide from flotate or raw material3 slides from < 0.25 mm sediment + 1 slide < 0.25 mm flotate or raw material> 5 animal particles of same nature detected?YESsample declared according to point 2.1.5.3 for FISHrepetition of analysis according to point 2.1.4.3YES> 5 animal particles of same nature detected?NO> 5Number of fish particles?≤ 5NO1 slide from sedimentFISH1 slide from > 0.25 mm sediment> 5 animal particles of same nature detected?YESnature of animal particles?YES> 5 animal particles of same nature detected?NOTERRESTRIALNO1 slide from flotate or raw material> 5Number of terrestrial particles?≤ 51 slide from > 0.25 mm flotate or raw materialanimal particles detected?YESsample declared according to point 2.1.5.3 for TERRESTRIAL ANIMALrepetition of analysis according to point 2.1.4.3YESanimal particles detected?NOsample declared according to point 2.1.5.1NODiagram 2Observation protocol for the detection of animal particles in fishmealYESGrinding process insures 95% of particles ≤ 0.50 mmNOSieving on 0.25 mm mesh3 slides from sediment3 slides from < 0.25 mm sediment> 5 terrestrial particles detected?YESYES> 5 terrestrial particles detected?NOsample declared according to point 2.1.5.3 for TERRESTRIAL ANIMALNO2 slides from sediment> 52 slides from > 0.25 mm sediment> 5 terrestrial particles detected?YESNumber of terrestrial particles?YES> 5 terrestrial particles detected?NO≤ 5NO1 slide from flotate or raw materialrepetition of analysis according to point 2.1.4.31 slide from < 0.25 mm flotate or raw materialterrestrial particles detected?YESYESterrestrial particles detected?NOsample declared according to point 2.1.5.1 for TERRESTRIAL ANIMALNO 2.1.4.3.Number of determinationsIf following a first determination carried out in accordance with the observation protocol laid down in diagram 1 or diagram 2 as relevant, no animal particle of a given nature (i.e. terrestrial animal or fish) is detected, no additional determination is necessary and the result of the analysis shall be reported using the terminology laid down in point 2.1.5.1.If, following a first determination carried out in accordance with the observation protocols laid down in diagram 1 or in diagram 2 as relevant, the total number of animal particles of a given nature (i.e. terrestrial animal or fish) detected ranges from 1 to 5, a second determination shall be performed from a new 50 g sub-sample. If, following this second determination, the number of animal particles of this given nature detected ranges from 0 to 5, the result of the analysis shall be reported using the terminology laid down in point 2.1.5.2., else a third determination shall be carried out from a new 50 g sub-sample. Nevertheless, if following the first and the second determination, the sum of the particles of a given nature detected over the two determinations is higher than 15, no additional determination is necessary and the result of the analysis shall be directly reported using the terminology laid down in point 2.1.5.3. If, following the third determination, the sum of the animal particles of a given nature detected over the three determinations is higher than 15, the result of the analysis shall be reported using the terminology laid down in point 2.1.5.3. Otherwise, the result of the analysis shall be reported using the terminology laid down in point 2.1.5.2.If following a first determination carried out in accordance with the observation protocols laid down in diagram 1 or in diagram 2 as relevant, more than 5 animal particles of a given nature (i.e. terrestrial animal or fish) are detected, the result of the analysis shall be reported using the terminology laid down in point 2.1.5.3.2.1.5.Expression of the resultsWhen reporting the results, the laboratory shall indicate on which type of material the analysis has been carried-out (sediment, flotate or raw material) and how many determinations have been carried-out.The laboratory report shall at least contain information on the presence of constituents derived from terrestrial animals and from fish.The different situations shall be reported in the following ways.2.1.5.1.No animal particle of a given nature detected:2.1.5.2.Between 1 and 5 animal particles of a given nature detected on average:Or, as relevant,In case of sample pre-sieving, the laboratory report shall mention in which fraction (sieved fraction, pelleted fraction or kernels) the animal particles have been detected insofar as the detection of animal particles only in the sieved fraction may be the sign of an environmental contamination.2.1.5.3.More than 5 animal particles of a given nature detected on averageOr, as relevant,In case of sample pre-sieving, the laboratory report shall mention in which fraction (sieved fraction, pelleted fraction or kernels) the animal particles have been detected insofar as the detection of animal particles only in the sieved fraction may be the sign of an environmental contamination.2.2.PCR 2.2.1.PrincipleDeoxyribonucleic acid (DNA) fragments of animal origin which may be present in feed materials and compound feed are detected by a genetic amplification technique through PCR, targeting species-specific DNA sequences.The PCR method first requires a DNA extraction step. The amplification step shall be applied afterwards to the so-obtained DNA extract, in order to detect the animal species targeted by the assay. 2.2.2.Reagents and equipment 2.2.2.1.Reagents 2.2.2.1.1.Reagents for DNA extraction stepOnly reagents approved by the EURL-AP and published on its website shall be used. 2.2.2.1.2.Reagents for genetic amplification step 2.2.2.1.2.1.Primers and probesOnly primers and probes with sequences of oligonucleotides validated by the EURL-AP shall be usednote. 2.2.2.1.2.2.Master MixOnly Master Mix solutions which do not contain reagents susceptible to lead to false results due to presence of animal DNA shall be usednote. 2.2.2.1.2.3.Decontamination reagents 2.2.2.1.2.3.1.Hydrochloric acid solution (0,1 N) 2.2.2.1.2.3.2.Bleach (solution of sodium hypochlorite at 0,15 % of active chlorine) 2.2.2.1.2.3.3.Non-corrosive reagents for decontaminating costly devices like analytical balances (e.g. DNA EraseTM of MP Biomedicals) 2.2.2.2.Equipment 2.2.2.2.1.Analytical balance with an accuracy of 0,001 g 2.2.2.2.2.Grinding equipment 2.2.2.2.3.Thermocycler enabling real-time PCR 2.2.2.2.4.Microcentrifuge for microfuge tubes 2.2.2.2.5.Set of micropipettes allowing to pipet from 1 μl up to 1000 μl 2.2.2.2.6.Standard molecular biology plastic-ware: microfuge tubes, filtered plastic tips for micropipettes, plates suitable for the thermocycler. 2.2.2.2.7.Freezers to store samples and reagents 2.2.3.Sampling and sample preparation 2.2.3.1.SamplingA representative sample, taken in accordance with the provisions laid down in Annex I, shall be used. 2.2.3.2.Sample preparationThe preparation of laboratory samples up to DNA extraction shall comply with the requirements set out in Annex II. At least 50 g of the sample shall be sub-sampled for analysis and subsequently ground.The sample preparation shall be performed in a room different from the ones dedicated to DNA extraction and to genetic amplification reactions as described by ISO 24276.Two test portions of at least 100 mg each shall be prepared.2.2.4.DNA extractionThe DNA extraction shall be performed on each test portion prepared using the SOP established by the EURL-AP and published on its website.Two extraction controls shall be prepared for each extraction series as described by ISO 24276.2.2.5.Genetic amplificationThe genetic amplification shall be performed using the methods validated for each species requiring identification. These methods are laid down in the SOP established by the EURL-AP and published on its website. Each DNA extract shall be analysed at least at two different dilutions in order to evaluate inhibition.Two amplification controls shall be prepared per species target as described by ISO 24276.2.2.6.Interpretation and expression of resultsWhen reporting the results, the laboratory shall indicate at least the weight of the test portions used, the extraction technique used, the number of determinations carried-out and the limit of detection of the method.Results shall not be interpreted and reported if the positive DNA extraction control and the positive DNA target controls do not provide positive results for the target under assay while the amplification reagent control is negative.In case results from the two test portions are not consistent, at least the genetic amplification step shall be repeated. If the laboratory suspects that the DNA extracts can be the cause of the inconsistency, a new DNA extraction and a subsequent genetic amplification shall be performed before interpreting the results.The final expression of the results shall be based on the integration and the interpretation of the results of the two test portions in accordance with the SOP established by the EURL-AP and published on its website. 2.2.6.1.Negative resultA negative result shall be reported as follows:No DNA from X was detected in the submitted sample (with X being the animal species or group of animal species that is targeted by the assay). 2.2.6.2.Positive resultA positive result shall be reported as follows:DNA from X was detected in the submitted sample (with X being the animal species or group of animal species that is targeted by the assay).’