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A technique for muscle biopsy sampling on pigs to assess intramuscular fat
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Meat Science 32 (19923 123-129
A Technique for Muscle Biopsy Sampling on Pigs to Assess Intramuscular Fat H. Vill6fl G. Maes, b R. Geersfl V. Goedseelsfl G. Parduynsfl J. Van Baelfl S. Janssens" & P. Dekempeneeff ~Laboratory for Agricultural Buildings Research, K. U. Leuven, Kard. Mercierlaan 92, B-3001, Heverlee, Belgium bLaboratory for Physical Chemistry, CLaboratory for Radiochemistry, K. U. Leuven, Celestijnenlaan 200 F, Bo3001 Heverlee, Belgium (Received 18 April 1991; revised version accepted 10 July t99b: accepted 20 July 1991)
ABSTRACT The objective of the research was to develop a rapid, convenient method/br sampling the muscle of live young pigs. A biopsy needle has been developed. It consists of two parts, a hollow outer cylinder and an inner stick. From female piglets (10-25 kg) a biopo, sample of 108 +_42 mg was takenJi'om the biceps femoris. No injections or mobility problems were observed. The sample can be usedjbr further i.m.fat anah,sis. The mean fat content in the biceps femoris is 2.24 +_1.12%. This i.m. fat content was measured by extraction in a Soxhlet apparatus and was determined by a Fourier transform ln./i'ared (FTIR) method. Fatty acid composition was determined by gas liquid chromatography.
INTRODUCTION Body samples of muscle tissue are often needed to predict meat quality, for example i.m. fat content, in live animals. Several biopsy techniques have been described in the literature. A needle biopsy technique to obtain fat and muscle samples has been described by Lundstr6m et al. (1973). However, the sample volume as obtained by this technique was insufficient for analysis of the fat content of 123 Meat Science 0309-1740/92/$05.00 (~ 1992 Elsevier Science Publishers Lid, England. Printed in Great Britain
124
H. ViHk et al.
the muscle tissue. Other possibilities are the shotbiopsy described by Von Lore Sch6berlein (1975) or the koffler-tong (Schmidt et al., 1972), a device used in nose surgery for humans. Using those instruments did not result in a larger sample volume. The sample can also be excised with an electric cautery knife (Moser et al., 1972). With this method, the blood vessels are cauterized, thereby preventing or restricting any bleeding. All these methods, however, are too expensive for field application. Different methods have been investigated for cattle. Harris & Bennett (1970) used a muscle tissue extractor for taking muscle biopsy, but the incision length required was 5-7-6.4cm, so that it could not be used for young pigs. MATERIALS A N D M E T H O D The instrument
The biopsy needle which has been developed consists of two parts. There is a hollow outer cylinder with a hook at the end: both the edge of the cylinder and the hook are sharpened. The inner part is a rod, which is used for pushing the sample out of the cylinder. The diameters of the outer cylinder and of the inner cylinder are respectively 4.8 and 4.0mm. The needle is shown in Fig. 1. Animals
Female pigs of 10-25 kg were used for taking a biopsy sample. The piglets originated from a homozygous, halothane-positive line (Belgian
~"4.3 ~'12
~7.----------------------------------------------------I I _~ L
120 ~4.0
B
L_ F"
121
v ~
Fig. 1. The biopsy needle (dimensions in/~m).
~'12
Muscle biopsy sampling on pigs
125
Landrace--line 2), a homozygous halothane-negative line (Landrace--line 1, Large White--line 4) and a heterozygous line (Belgian Landrace x Large White--line 3). The pigs were provided by Seghers Hybrid (Buggenhout, Belgium). The experimental animals were housed in standardized environmental conditions (Geers et al., 1990). Food and water were available ad libitum.
Muscle biopsy The biopsy was taken from the biceps femoris. First the hair was shaved locally. The shaved area and the biopsy needle were disinfected with a solution of alcohol and hibitane. The pigs were anaesthetized with an i.m. injection of azoperone (2mg/kg) and an i.p. injection of medomidate (10 mg/kg). It was necessary to anaesthetize the piglets otherwise they moved too much: local anaesthesia was not sufficient. The biopsy method is applied only to piglets from 10 to 25 kg. An incision, of approximately ~cm length, was made by means of a scalpel. The biopsy needle was inserted in the cut twice. After the first attempt the muscle and skin tissue were pushed out. The second time the needle was twisted, to cut the muscle sample. The wound was dressed and disinfected. Finally, the wound was covered with a small piece of sterile cotton. The procedure required about 1 min. Samples were stored at - 18°C before further analysis. After regaining consciousness, the pigs were placed in pens together with non-experimental pigs.
Fat analysis The i.m. fat content was measured by extraction in a Soxhlet apparatus with petroleum ether. Each sample to be analysed was weighed and the percentage of moisture was determined by the ratio of the difference between weights of fresh and dried (18h, 102°C) muscle and the weight of fresh muscle. The sample was then digested with HC1 (37%) for 1 h. With this pretreatment, the fatty acids are better separated from proteins and the total fat content can be determined, although it is possible that the fatty acids may change in structure (Deymi6 et al., 1981). The solution was then filtered. After drying the filters for 24 h at 60°C, they were inserted into the extraction cups and the soluble material was extracted by petroleum ether in a twostage process, followed by a solvent recovery cycle of 1 h (Association of Official Analytical Chemists, 1980). Finally, for the gravimetric technique, the extraction cups were dried and weighed, to determine the i.m. fat content. The fat content was then dissolved in 5 ml of diethylether and the content of fat determined by a Fourier transform IR (FTIR) method, using Briiker
126
H. Villb et al.
14.0-
12.010.0} 8.0.~ 6.0-
o e s ~ =z.29x+o. =
4.02.00.0
0
1 0.5
2 1.5
3 2.5
4 3.5
mg/ml Fig. 2.
Calibration curve expressing fat content as absorption (FTIR) vs gravimetry (mg/ml).
IFS-66 apparatus. (The cell thickness was 1"5 mm: spectra between 4000 and 450cm -x were received from a Fourier transformation of 10 interferograms. The resolution was 2 c m - 1 and the accuracy of the wavenumber was 0"01 c m - 1.) Extraction of fat from the muscle is necessary to avoid interaction with proteins on F T I R spectroscopy, as the C = O bands of lipids and the amide of protein and peptide have the same wavelengths (Jackson et al., 1989). Diethylether is used for dilution of samples to allow analysis by IR spectroscopy. It is a suitable solvent as it dissolves fatty acids. The wavelength band from 1750 c m - t was chosen, over which the fatty acid absorption is integrated for quantitative analysis (Hellgeth et al., 1986). After each sample the cell was thoroughly rinsed with diethylether and dried with a vacuum pump. Because F T I R spectrometers are computerized, with all data in digital format, area integration is straightforward. After inspection of the whole spectrum an integration from 1700 to 1770cmwas taken. A linear calibration line was fitted between the gravimetric method and the F T I R method as follows. Fat extracted from a commercial piece of pigmeat was divided in different concentrations. The fat content of each fraction was measured at 1750 c m - 1 and a linear fit (r 2 = 0"9939) was made (Fig. 2). The fatty acid composition was determined using a Shimadzu (GC-15A type) gas-liquid c h r o m a t o g r a p h y device.
Muscle biopsy sampling on pigs
127
RESULTS A N D DISCUSSION
Condition of pigs The biopsy was accomplished successfully on live pigs of 10-25 kg. No infections or mobility problems were observed. Collected quantity and quality of muscle tissue A sample of 108 + 42 mg (n = 79) was taken in approximately 1 rain. In a first experiment, one biopsy sample was taken, but the mean contents of i.m. fat, as well as the standard deviations (11.05 + 12.09%, n = 21), were large when compared with results in literature. It was concluded that the first sample contained too much s.c. fat or interfascicular fat. As a consequence, the first sample was discarded in the following experiment and only the sample obtained in the second attempt was used for further fat analysis. The percentage of i.m. fat extracted in the second attempt (2-4 4- 1.21%, n = 49) was comparable with the fat percentage (1.4%) found in the hic~7~s ./~,moris of female crossbred Large White and Pi~train pigs (LeseigneurMeynier & Gandemer, 1990) and with the fat percentage (1 3• ,/ , ) i n the longissimus dorsi of sows (Warriss et al., 1990). The data were discarded if the fat content was excessive when compared with results in the literature. It is feasible that, together with lean muscle tissue, i.m. fat might also be taken with the biopsy procedure. Pieces of i.m. fat could not be detected, or removed, because the samples were too small (and sometimes too much blood was present). Taking two biopsy samples would give a more accurate prediction of the real i.m. fat content, but the piglets were too small to do this. Probably, the large standard deviation is a result of this limitation to only one sample per piglet. TABLE I Comparison of Fat Content Determined b.~, FT1R and Gravimelric Methods
g/at per IO0~ tJ'c.sh nlU,~cle
Line Line Line Line
I 2 3 4
Mean
FTIR
n
G r a r m u , tric
n
1-74 +0-65 1.48 _+0-66 2.48+ 1-71
I1 13 14
1-77_+0.79 2.07_+0.98 ~-1~+ 1.37 2.34+ 1.01
8 18 20 7
1.81 _+0.98
38
2.07_+ 111
53
128
H. VillO et al. TABLE 2
Fatty Acid Composition from i.m. Fat % Fatty acid Fatty acM
14 15 16 16:1 16:4 17 18 18:I 18:2 18:3 20 : I
Samph, I
Sample 2
Sample 3
Sample 4
Mean
SD
2.53 0.47 25-9 2.32 1.47 0"38 10.3 20"1 5"99 0'48 0"46
3.1 1.05 26.7 2-62 2.69
2.97 0.63 30.22 2.91
2.78 0.42 32.04 2.5
10.7 15-1 6"81 0"78
12.28 20'47 5"65
0.51 12.60 21"61 2"37
2.84 0.64 28.74 2-59 2.08 0.45 11.48 19"33 5"21 0'63 0"46
0-22 0.25 2.48 0.21 0"61 0"07 0'97 2"5 1"69 0"15 0
Literature value"
1.5 22.1 3 11.9 45 10 0"8
Leseigneur-Meynier& Gandemer (1990).
In the future, i.m. fat will be measured with 1H-NMR spectroscopy by means of an imaging procedure, by which a volume free of i.m. fat can be chosen. One piglet can also be measured several times, and at several locations by this method, allowing the calculation of a mean value. Table 1 shows that the standard deviation for F T I R is smaller than that for the gravimetric method, and the mean value is closer to literature data. FT1R is obviously a more accurate method than gravimetry. The weight of the extracted fat is probably too small relative to the weight of the recovery cups, preventing the use of a sufficiently accurate balance. Fatty acid composition was determined from four samples (see Table 2). The composition of fatty acids in the b i c e p s f e m o r i s is comparable with the results of Leseigneur-Meynier & G a n d e m e r (1990), except for oleic and linoleic acid. Further analyses should determine the influence of race, line or nutrition (Wood & Enser, 1989). Also, the possibility of destruction of fatty acids, as a result of pretreatment with HC1 (Deymi6 et al., 1981), will be investigated.
CONCLUSION The results show that the a m o u n t of muscle tissue obtained by the biopsy method described above is sufficient for further fat analysis. The presence of
Muscle biopsy sampling on pigs
129
small amounts of i.m. fat cannot be controlled in the biopsy, and they may influence the results. In comparison with the gravimetric method, F T I R proved to be more accurate in determining the i.m. fat content of the small samples available.
ACKNOWLEDGEMENTS Funding was provided by the European Community (DGXII, ECLAIRprogramme), and R. Geers and G. Maes were supported by the N.F.W.O., Belgium. The authors thank Professor R. De Schrijver and Mr D. Vermeulen for the fatty acid analyses. Mr J. Vogeleer made the biopsy needle.
REFERENCES Association of Official Analytical Chemists (AOAC)(1980). Official Methods of Analysis, 13th edition, AOAC, Washington, p. 376. Deymi6, B., Multon, J. L. & Simon, D. (1981). Techniques d'Analyse et de Controle dans les Industries Agroalimentaires 4. Technique et documentation; Apria, Paris, p. 175. Geers, R., Goedseels, V., Parduyns, G., Nijns, P. & Wouters, P. (1990). J. Agric. Eng. Res., 45, 149. Harris, R. M. & Bennett, J. A. (1970). J. Anita. Sci., 31,451. Hellgeth, J. W., Jordan, J. W., Taylor, L. T. & Ashraf Khorassani, M. (1986). J. Chromatogr. Sci., 24, 183. Jackson, M., Haris, P. I. & Chapman, D. (1989). J. Mol. Struct., 214, 329. Leseigneur-Meynier, A. & Gandemer, G. (1990). Meat Sci., 29, 229. Lundstr6m, K., Asp-Malmfors, B. & Hansson, J. (1973). Swedish J. Agric. Res., 3, 211. Moser, B.d., Peo, E. R., Zimmerman, D. R. & Cunningham, P. J. (1972). J. Anita. Sci., 34, 752. Schmidt, G. R., Zuidam, L. & Sybesma, W. (1972). J. Anita. Sci., 34, 25. Von Lore Sch6berlein (1975). Mh. Vet.-Med., 31,457. Warriss, P. D., Brown, S. N., Franklin, J. G. & Kestin, S. C. (1990). Meat Sci., 28, 21. Wood, J. D. & Enser, M. (1989). 40th Ann. Meeting Study Commissions, EAAP, Dublin, 27-31 August, p. 1.
~
Meat Science 32 (19923 123-129
A Technique for Muscle Biopsy Sampling on Pigs to Assess Intramuscular Fat H. Vill6fl G. Maes, b R. Geersfl V. Goedseelsfl G. Parduynsfl J. Van Baelfl S. Janssens" & P. Dekempeneeff ~Laboratory for Agricultural Buildings Research, K. U. Leuven, Kard. Mercierlaan 92, B-3001, Heverlee, Belgium bLaboratory for Physical Chemistry, CLaboratory for Radiochemistry, K. U. Leuven, Celestijnenlaan 200 F, Bo3001 Heverlee, Belgium (Received 18 April 1991; revised version accepted 10 July t99b: accepted 20 July 1991)
ABSTRACT The objective of the research was to develop a rapid, convenient method/br sampling the muscle of live young pigs. A biopsy needle has been developed. It consists of two parts, a hollow outer cylinder and an inner stick. From female piglets (10-25 kg) a biopo, sample of 108 +_42 mg was takenJi'om the biceps femoris. No injections or mobility problems were observed. The sample can be usedjbr further i.m.fat anah,sis. The mean fat content in the biceps femoris is 2.24 +_1.12%. This i.m. fat content was measured by extraction in a Soxhlet apparatus and was determined by a Fourier transform ln./i'ared (FTIR) method. Fatty acid composition was determined by gas liquid chromatography.
INTRODUCTION Body samples of muscle tissue are often needed to predict meat quality, for example i.m. fat content, in live animals. Several biopsy techniques have been described in the literature. A needle biopsy technique to obtain fat and muscle samples has been described by Lundstr6m et al. (1973). However, the sample volume as obtained by this technique was insufficient for analysis of the fat content of 123 Meat Science 0309-1740/92/$05.00 (~ 1992 Elsevier Science Publishers Lid, England. Printed in Great Britain
124
H. ViHk et al.
the muscle tissue. Other possibilities are the shotbiopsy described by Von Lore Sch6berlein (1975) or the koffler-tong (Schmidt et al., 1972), a device used in nose surgery for humans. Using those instruments did not result in a larger sample volume. The sample can also be excised with an electric cautery knife (Moser et al., 1972). With this method, the blood vessels are cauterized, thereby preventing or restricting any bleeding. All these methods, however, are too expensive for field application. Different methods have been investigated for cattle. Harris & Bennett (1970) used a muscle tissue extractor for taking muscle biopsy, but the incision length required was 5-7-6.4cm, so that it could not be used for young pigs. MATERIALS A N D M E T H O D The instrument
The biopsy needle which has been developed consists of two parts. There is a hollow outer cylinder with a hook at the end: both the edge of the cylinder and the hook are sharpened. The inner part is a rod, which is used for pushing the sample out of the cylinder. The diameters of the outer cylinder and of the inner cylinder are respectively 4.8 and 4.0mm. The needle is shown in Fig. 1. Animals
Female pigs of 10-25 kg were used for taking a biopsy sample. The piglets originated from a homozygous, halothane-positive line (Belgian
~"4.3 ~'12
~7.----------------------------------------------------I I _~ L
120 ~4.0
B
L_ F"
121
v ~
Fig. 1. The biopsy needle (dimensions in/~m).
~'12
Muscle biopsy sampling on pigs
125
Landrace--line 2), a homozygous halothane-negative line (Landrace--line 1, Large White--line 4) and a heterozygous line (Belgian Landrace x Large White--line 3). The pigs were provided by Seghers Hybrid (Buggenhout, Belgium). The experimental animals were housed in standardized environmental conditions (Geers et al., 1990). Food and water were available ad libitum.
Muscle biopsy The biopsy was taken from the biceps femoris. First the hair was shaved locally. The shaved area and the biopsy needle were disinfected with a solution of alcohol and hibitane. The pigs were anaesthetized with an i.m. injection of azoperone (2mg/kg) and an i.p. injection of medomidate (10 mg/kg). It was necessary to anaesthetize the piglets otherwise they moved too much: local anaesthesia was not sufficient. The biopsy method is applied only to piglets from 10 to 25 kg. An incision, of approximately ~cm length, was made by means of a scalpel. The biopsy needle was inserted in the cut twice. After the first attempt the muscle and skin tissue were pushed out. The second time the needle was twisted, to cut the muscle sample. The wound was dressed and disinfected. Finally, the wound was covered with a small piece of sterile cotton. The procedure required about 1 min. Samples were stored at - 18°C before further analysis. After regaining consciousness, the pigs were placed in pens together with non-experimental pigs.
Fat analysis The i.m. fat content was measured by extraction in a Soxhlet apparatus with petroleum ether. Each sample to be analysed was weighed and the percentage of moisture was determined by the ratio of the difference between weights of fresh and dried (18h, 102°C) muscle and the weight of fresh muscle. The sample was then digested with HC1 (37%) for 1 h. With this pretreatment, the fatty acids are better separated from proteins and the total fat content can be determined, although it is possible that the fatty acids may change in structure (Deymi6 et al., 1981). The solution was then filtered. After drying the filters for 24 h at 60°C, they were inserted into the extraction cups and the soluble material was extracted by petroleum ether in a twostage process, followed by a solvent recovery cycle of 1 h (Association of Official Analytical Chemists, 1980). Finally, for the gravimetric technique, the extraction cups were dried and weighed, to determine the i.m. fat content. The fat content was then dissolved in 5 ml of diethylether and the content of fat determined by a Fourier transform IR (FTIR) method, using Briiker
126
H. Villb et al.
14.0-
12.010.0} 8.0.~ 6.0-
o e s ~ =z.29x+o. =
4.02.00.0
0
1 0.5
2 1.5
3 2.5
4 3.5
mg/ml Fig. 2.
Calibration curve expressing fat content as absorption (FTIR) vs gravimetry (mg/ml).
IFS-66 apparatus. (The cell thickness was 1"5 mm: spectra between 4000 and 450cm -x were received from a Fourier transformation of 10 interferograms. The resolution was 2 c m - 1 and the accuracy of the wavenumber was 0"01 c m - 1.) Extraction of fat from the muscle is necessary to avoid interaction with proteins on F T I R spectroscopy, as the C = O bands of lipids and the amide of protein and peptide have the same wavelengths (Jackson et al., 1989). Diethylether is used for dilution of samples to allow analysis by IR spectroscopy. It is a suitable solvent as it dissolves fatty acids. The wavelength band from 1750 c m - t was chosen, over which the fatty acid absorption is integrated for quantitative analysis (Hellgeth et al., 1986). After each sample the cell was thoroughly rinsed with diethylether and dried with a vacuum pump. Because F T I R spectrometers are computerized, with all data in digital format, area integration is straightforward. After inspection of the whole spectrum an integration from 1700 to 1770cmwas taken. A linear calibration line was fitted between the gravimetric method and the F T I R method as follows. Fat extracted from a commercial piece of pigmeat was divided in different concentrations. The fat content of each fraction was measured at 1750 c m - 1 and a linear fit (r 2 = 0"9939) was made (Fig. 2). The fatty acid composition was determined using a Shimadzu (GC-15A type) gas-liquid c h r o m a t o g r a p h y device.
Muscle biopsy sampling on pigs
127
RESULTS A N D DISCUSSION
Condition of pigs The biopsy was accomplished successfully on live pigs of 10-25 kg. No infections or mobility problems were observed. Collected quantity and quality of muscle tissue A sample of 108 + 42 mg (n = 79) was taken in approximately 1 rain. In a first experiment, one biopsy sample was taken, but the mean contents of i.m. fat, as well as the standard deviations (11.05 + 12.09%, n = 21), were large when compared with results in literature. It was concluded that the first sample contained too much s.c. fat or interfascicular fat. As a consequence, the first sample was discarded in the following experiment and only the sample obtained in the second attempt was used for further fat analysis. The percentage of i.m. fat extracted in the second attempt (2-4 4- 1.21%, n = 49) was comparable with the fat percentage (1.4%) found in the hic~7~s ./~,moris of female crossbred Large White and Pi~train pigs (LeseigneurMeynier & Gandemer, 1990) and with the fat percentage (1 3• ,/ , ) i n the longissimus dorsi of sows (Warriss et al., 1990). The data were discarded if the fat content was excessive when compared with results in the literature. It is feasible that, together with lean muscle tissue, i.m. fat might also be taken with the biopsy procedure. Pieces of i.m. fat could not be detected, or removed, because the samples were too small (and sometimes too much blood was present). Taking two biopsy samples would give a more accurate prediction of the real i.m. fat content, but the piglets were too small to do this. Probably, the large standard deviation is a result of this limitation to only one sample per piglet. TABLE I Comparison of Fat Content Determined b.~, FT1R and Gravimelric Methods
g/at per IO0~ tJ'c.sh nlU,~cle
Line Line Line Line
I 2 3 4
Mean
FTIR
n
G r a r m u , tric
n
1-74 +0-65 1.48 _+0-66 2.48+ 1-71
I1 13 14
1-77_+0.79 2.07_+0.98 ~-1~+ 1.37 2.34+ 1.01
8 18 20 7
1.81 _+0.98
38
2.07_+ 111
53
128
H. VillO et al. TABLE 2
Fatty Acid Composition from i.m. Fat % Fatty acid Fatty acM
14 15 16 16:1 16:4 17 18 18:I 18:2 18:3 20 : I
Samph, I
Sample 2
Sample 3
Sample 4
Mean
SD
2.53 0.47 25-9 2.32 1.47 0"38 10.3 20"1 5"99 0'48 0"46
3.1 1.05 26.7 2-62 2.69
2.97 0.63 30.22 2.91
2.78 0.42 32.04 2.5
10.7 15-1 6"81 0"78
12.28 20'47 5"65
0.51 12.60 21"61 2"37
2.84 0.64 28.74 2-59 2.08 0.45 11.48 19"33 5"21 0'63 0"46
0-22 0.25 2.48 0.21 0"61 0"07 0'97 2"5 1"69 0"15 0
Literature value"
1.5 22.1 3 11.9 45 10 0"8
Leseigneur-Meynier& Gandemer (1990).
In the future, i.m. fat will be measured with 1H-NMR spectroscopy by means of an imaging procedure, by which a volume free of i.m. fat can be chosen. One piglet can also be measured several times, and at several locations by this method, allowing the calculation of a mean value. Table 1 shows that the standard deviation for F T I R is smaller than that for the gravimetric method, and the mean value is closer to literature data. FT1R is obviously a more accurate method than gravimetry. The weight of the extracted fat is probably too small relative to the weight of the recovery cups, preventing the use of a sufficiently accurate balance. Fatty acid composition was determined from four samples (see Table 2). The composition of fatty acids in the b i c e p s f e m o r i s is comparable with the results of Leseigneur-Meynier & G a n d e m e r (1990), except for oleic and linoleic acid. Further analyses should determine the influence of race, line or nutrition (Wood & Enser, 1989). Also, the possibility of destruction of fatty acids, as a result of pretreatment with HC1 (Deymi6 et al., 1981), will be investigated.
CONCLUSION The results show that the a m o u n t of muscle tissue obtained by the biopsy method described above is sufficient for further fat analysis. The presence of
Muscle biopsy sampling on pigs
129
small amounts of i.m. fat cannot be controlled in the biopsy, and they may influence the results. In comparison with the gravimetric method, F T I R proved to be more accurate in determining the i.m. fat content of the small samples available.
ACKNOWLEDGEMENTS Funding was provided by the European Community (DGXII, ECLAIRprogramme), and R. Geers and G. Maes were supported by the N.F.W.O., Belgium. The authors thank Professor R. De Schrijver and Mr D. Vermeulen for the fatty acid analyses. Mr J. Vogeleer made the biopsy needle.
REFERENCES Association of Official Analytical Chemists (AOAC)(1980). Official Methods of Analysis, 13th edition, AOAC, Washington, p. 376. Deymi6, B., Multon, J. L. & Simon, D. (1981). Techniques d'Analyse et de Controle dans les Industries Agroalimentaires 4. Technique et documentation; Apria, Paris, p. 175. Geers, R., Goedseels, V., Parduyns, G., Nijns, P. & Wouters, P. (1990). J. Agric. Eng. Res., 45, 149. Harris, R. M. & Bennett, J. A. (1970). J. Anita. Sci., 31,451. Hellgeth, J. W., Jordan, J. W., Taylor, L. T. & Ashraf Khorassani, M. (1986). J. Chromatogr. Sci., 24, 183. Jackson, M., Haris, P. I. & Chapman, D. (1989). J. Mol. Struct., 214, 329. Leseigneur-Meynier, A. & Gandemer, G. (1990). Meat Sci., 29, 229. Lundstr6m, K., Asp-Malmfors, B. & Hansson, J. (1973). Swedish J. Agric. Res., 3, 211. Moser, B.d., Peo, E. R., Zimmerman, D. R. & Cunningham, P. J. (1972). J. Anita. Sci., 34, 752. Schmidt, G. R., Zuidam, L. & Sybesma, W. (1972). J. Anita. Sci., 34, 25. Von Lore Sch6berlein (1975). Mh. Vet.-Med., 31,457. Warriss, P. D., Brown, S. N., Franklin, J. G. & Kestin, S. C. (1990). Meat Sci., 28, 21. Wood, J. D. & Enser, M. (1989). 40th Ann. Meeting Study Commissions, EAAP, Dublin, 27-31 August, p. 1.