COBISS: 1.08 Agris category code: L01, U10 PREDICTION OF FEEDING SYSTEM IN IBERIAN PIGS WITH THREE ANALYTICAL METHODS BY MEANS OF DISCRIMINANT ANALYSIS Juan M. GARCÍA CASCO Carmen DELGADO-CHAVERO 2, Andrés PAREDES 2, Ana I. REY 3 abstract Subcutaneous fat samples from Iberian pigs were analysed by means of three analytical methods developed to predict the feeding system during the last fattening period (bellota, recebo, campo and cebo). These three methods were: (1) quantification of palmitic, stearic, oleic and linoleic fatty acids methyl esters by gas chromatography, (2) quantification of alpha and gamma tocopherol by high-performance liquid chromatography and (3) quantification of 13C/12C ratio by gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS). The results obtained by these methods over three season's samples were used to perform a discriminant analysis in order to predict the feeding system during the fourth season, considered as the problem data. The results showed the capacity of the discriminant analysis for distinguishing between the feeding regime based in the consumption of acorn and pastures and that based in mixed diets (90% of success rate). However, differentiation of feeding systems in the case of mixed diet and natural resources during any time of the fattening phase seems not to be feasible. Key words: Iberian pig / fatty acids / isotopes / tocopherol / feeding systems / discriminant analysis 1 introduction Over the last years several analytical methods have been developed by different research groups to predict the feeding system during the last fattening period of the Iberian pigs (Garcia Casco et al., 2013). The feeding system is a major issue in this breed because it determines the quality standard of the dry-cured products (hams, shoulders and loins) and, therefore, the price of the products as well as the price of the slaughtered animals. At present, four feeding systems are recognized with a legal status: - extensive system with feeding based on acorn and pastures, known as bellota (B), - extensive system similar to bellota and a final period of feeding based on mixed diets (recebo, R), - extensive system with feeding based on mixed di- ets and natural resources (access to grass; campo, CA) - intensive system based exclusively on mixed diets (cebo, CE) The quality standard of dry-cured products is assumed to range from superior organoleptic quality of bellota to the lowest of cebo. In the present study the feasibility of classifying fat samples from four Iberian pig feeding systems using a combination of three analytical methods was evaluated. The methods were: quantification of palmitic, stearic, oleic and linoleic fatty acid methyl esters by gas chro-matography (Ordonez et al., 1996), quantification of alpha and gamma tocopherol by high-performance liquid chromatography (Rey et al., 2006) and quantification of the 13C/12C isotope ratio by GC-C-IRMS (Delgado-Chavero et al., 2013). Hence, the aim of this work was to use data obtained by the application of the three analyti- 1 Centro de I+D en Cerdo Ibérico, INIA, 06300 Zafra, Spain, e-mail: garcia.juan@inia.es 2 Asoc. Interprof. Cerdo Ibérico (ASICI), 06300 Zafra, Spain 3 Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain cal methods over three seasons (period from November to February with acorn and pastures feeding in the Mediterranean holm oak and cork woodland, López Bote, 1998) to predict the feeding system of Iberian pig on fat samples collected during the fourth season by means of discriminant analysis. 2 MATERIAL AND METHODS The fatty acid percentages (palmitic, stearic, oleic and linoleic), alpha and gamma tocopherol concentrations (^g per g of fresh matter) and 13C/12C isotope ratio of the fatty acids methyl esters (expressed as 8 %o units, excess of heavy isotope in a sample referred to the international reference PDB, Recio et al., 2013) were quantified in subcutaneous fat samples from Iberian pigs fattened over three seasons (S1-S3). Table 1 shows the total number of analysed samples according to the feeding system categories. Table 1: Total number of batches and samples analysed over the four seasons First three seasons 4th S1 S2 S3 TOTAL (S4) Batches 8 8 22 38 22 Feeding System Bellota 57 52 135 244 101 Recebo 40 50 72 162 73 Campo 72 74 47 193 72 Cebo 31 24 80 135 50 Total 200 200 334 734 296 A discriminant analysis was performed using the analytical results in 749 samples over the first three seasons (version 9.3, SAS Institute Inc., Cary, NC) and the linear discriminant functions were used to predict the feeding system in those samples collected over the fourth season (S4, Table 1). 3 RESULTS AND DIScUSSION Means of the variables for each feeding system over the first three seasons are presented in Table 2, with the Duncan's multiple range test on the feeding type effect. The extensive feeding system based on acorn and pastures (B) showed higher values of oleic acid and Y-tocopherol, whereas palmitic acid, stearic acid, and S-values for the four fatty acids were lower. On the opposite, subcutane- Table 2: Means of fatty acid percentages, S-values of the fatty acids methyl esters and concentration of a- and y-tocopherol (p/g) over seasons 1 to 3 for each feeding system Bellota Recebo Campo Cebo Palmitic 19.99d 20.46c 21.62b 22.75a Stearic 9.12d 9.64c 11.24b 12.06a Oleic 55.63a 53.84b 51.60c 50.65d Linoeic 9.08b 9.61a 8.96b 7.86c 813C C16:0 -26.40d -25.10c -24.37a -24.79b 813C C18:0 -23.82c -22.53b -21.89a -22.29b 813C C18:1 -27.53d -26.07c -24.91a -25.18b 813C C18:2 -33.06d -31.75b -30.94a -32.38c a-tocopherol 9.89a 10.36a 9.22b 5.83c y-tocopherol 1.60a 0.99b 0.43c 0.40c S % units, excess of heavy isotope in a sample referred to the international reference PDB; Different letters in superscript following values indicate statistical significance ous fat from pigs fed on mixed diet in intensive conditions (CE) was more saturated and had lower concentrations of a-tocopherol. Table 3 shows mean values for the measured variables obtained in the fourth season according to the feeding system. Fatty acids, S values and tocopherols showed a pattern similar as observed in Table 2. The results of the discriminant analysis with all the variables are presented in Table 4. For the group B, 74% of the fat samples were assigned to the correct feeding system, however, 26% were considered as Recebo (R). Therefore none of the samples were assigned to the pro- Table 3: Means of fatty acid percentages, S-values of the fatty acids methyl esters and concentration of alpha and gamma tocopherol (p/g) in the season 4 for each feeding system Bellota Recebo Campo Cebo Palmitic 19.36d 19.88c 22.06b 23.17a Stearic 8.58c 8.90c 11.04b 12.19a Oleic 56.97a 56.41a 51.32b 50.40c Linoeic 8.82a 8.41b 8.91a 7.69c 813C C16:0, -26.75d -25.71b -24.95a -26.28c 813C C18:0 -23.88d -22.71b -22.14a -23.48c 813C C18:1 -27.30d -26.22c -24.33a -25.63b 813C C18:2 -32.76c -32.34b -30.80a -33.52d a-tocopherol 1.68a 1.11b 0.59c 0.29d y-tocopherol 14.30b 15.77a 10.29dc 6.78d S % units, excess of heavy isotope in a sample referred to the international reference PDB; Different letters in superscript following values indicate statistical significance Table 4: Classification (%) in four feeding categories of the fourth season samples based on the linear discriminant analysis obtained over the first three seasons samples % of observations of Classified as Bellota Recebo Campo Cebo Bellota 74.3 25.7 0.0 0.0 Recebo 20.6 61.6 16.4 1.4 Campo 2.8 11.1 83.3 2.8 Cebo 0.0 4.0 26.0 70.0 duction system based on mixed diets. On the other hand, samples from pigs fed in intensive conditions (CE) with 70% correct classification, showed 4% of samples wrong classified as R and 26% as CA. Table 5: Classification (%) in two feeding categories of the fourth season samples based on the linear discriminant analysis obtained over the first three seasons samples Classified as % of observations of Bellota and Recebo Campo and Cebo Bellota and Recebo 88.51 11.49 Campo and Cebo 8.20 91.80 The higher number of misinterpretation was obtained for the group fed in extensive conditions and supplemented with a mixed diet (R) that were assigned to B (21%) or CA (16%) groups, giving a 62% of success rate. The extensive system based on mixed diets and access to grass (CA) had 83% of correct classification showing 11% of fat samples classified as R. The total percentage of samples assigned to the right category was 73%. When two feeding systems were considered (Table 5), pig feeding mainly based on acorn and pasture (B and R) vs. those based-mixed diets (CE and CA), the prediction accuracy improved to 89% (B and R) and 92% (CA and CE). 4 REFERENCES Delgado-Chavero C., Zapata-Márquez E., García-Casco J.M., Paredes-Torronteras A. 2013. Statistical model for classifying the feeding system of Iberian pigs through Gas Chromatography (GC-FID) and Isotope Ratio Mass Spectrometry (GC-C-IRMS). Grasas y Aceites, 64, 2 (special issue): 157-165 García Casco J.M., Muñoz M., González E. 2013. Predictive ability of feeding system by several analytical methods in Iberian pig. Grasas y Aceites, 64, 2 (special issue): 191-200 López-Bote C. 1998. Sustained utilisation of the Iberian pig breed. Meat Science, 49: S17-S27 Ordóñez J.A., López M.O., Hierro E., Cambero M.I., De la Hoz L. 1996. Efecto de la dieta de cerdos Ibéricos sobre la composición en ácidos grasos del tejido adiposo y muscular. 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