MULLINS, F. BOULWARE. Materials and Methods

Transcription

1 42. R B T I C U L A R T I S S U E A N D MA.ST C E L L S I N R E l A T l O N T O TENDERNESS' R. S. L. E. CROW, A. HANSARD and M. R. MULLINS, F. BOULWARE UISIANA STATE UNIVERSITY _ - -L O_ - - _ R e t i c u l a r f i b e r s of t h e endomysial a r e a of bovine s k e l e t a l muscle appear t o provide t h e framework f o r t h e sheath which surrounds i n d i v i d u a l muscle c e l l s. Paul (1963) reported t h a t t h e endomysial reticulum of b i c e s femoris muscle samples remained i n t a c t a f t e r heating. Crow, et al. observed t h a t r e t i c u l a r f i b e r s appeared t o vary i n d e n s i t y i n uncooked samples of t h e longissimus d o r s i muscle e x h i b i t i n g extreme tenderness d i f f e r e n c e s. Riley (1959) reported t h a t mast c e l l s a r e f r e e c e l l s p r e s e n t i n t h e l o o s e connective t i s s u e s of ox muscle. Crow, e t a l. (1964) observed t h a t mast c e l l s were closely a s s o c i a t e d with t h e perimysial and endomysial connective t i s s u e of bovine s k e l e t a l muscle and appeared t o be randomly distributed. (d -- This i n v e s t i g a t i o n was undertaken t o study h i s t o l o g i c a l l y, t h e r e l a t i o n s h i p of r e t i c u l a r f i b e r s and mast c e l l s t o tenderness of t h e bovine longissimus d o r s i muscle. Materials and Methods One hundred twenty carcasses from y e a r l i n g s t e e r s were surveyed f o r tenderness using shear values of t h e l o n g i s s i m s d o r s i muscle. Samples were excised from t h e a r e a of t h e 1 2 t h r i b two days post-mortem and aged an a d d i t i o n a l f i v e days a t 3 O C. From t h i s group 30 samples were s e l e c t e d. H a l f of t h e samples were taken from muscles c l a s s i f i e d as low s h e a r value (less than 10.6 kg. on a 2.5 cm. core), and t h e o t h e r f i f t e e n represented high s h e a r value muscles ( g r e a t e r than kg. on a 2. 5 cm. c o r e ). These extreme s h e a r value groups were used f o r r e l a t i n g tenderness, mast c e l l numbers and r e t i c u l a r f i b e r i n t a c t n e s s. - Shear Value Determinations. Samples removed f r o m t h e longissimus d o r s i muscle were approximately 4 cm. t h i c k. Each sample was standardized t o a thickness of 3. 2 cm. These s t e a k s were cooked i n deep f a t (135OC.) t o an i n t e r n a l temperature of 7OoC., and t h r e e 2.5 em. cores were removed and sheared using t h e Warner-Bratzler s h e a r machine. Samples, approximately 4 x 4 x 3 mm., were removed f o r r e t i c u l a r f i b e r determinations from t h e median core of t h e cooked s t e a k s a f t e r shearing. S i m i l a r samples were removed from t h e median a r e a of t h e a d j a c e n t uncooked p o r t i o n f o r r e t i c u l a r f i b e r and mast c e l l determinations. 'Published with t h e approval of t h e Director of t h e Louisiana A g r i c u l t u r a l Experiment S t a t i o n. This work w a s supported i n p a r t by t h e Eastern U t i l i z a t i o n Research and Development Division of t h e A g r i c u l t u r a l Research Service, U. S. Department of Agriculture, Philadelphia, Pennsylvania.

2 Recticular Fiber Staining Procedures. Reticular fibers were stained in both cooked and uncooked samples, using a modification of the procedure described by Soule, (1962). Samples were placed immediately into 10 percent buffered (ph ) neutral formalin, and fixed for a minimum of 72 hours. All samples were then dehydrated in graded ethyl alcohol baths, cleared in xylene and embedded for cross-sectioning in Paraplast (M.P OC.). Following sectioning at 6 microns, tissue ribbons were floated out on a water bath (50-52OC,), separated and picked up on acid clean slides coated with Payers-Albumin fixative, and allowed to dry for 12 hours. Slide-mounted sections were cleared in xylene and taken to water in graded alcohols. Staining was accomplished in one percent gold chloride for 30 minutes at 35OC., followed by dipping in distilled deionized water and transferring to a 5 percent sodium carbonate, 0.5 percent potassium hydroxide bath for 10 minutes. Slides were then transferred directly to a 5 percent potassium iodide solution for 3 minutes, followed by rinsing twice in distilled deionized water. Counter staining was carried out in a 0.25% methylene blue chloride solution for 30 seconds followed by rinsing two times in distilled deionized water. Sections were dehydrated in 95 percent ethyl alcohol, cleared in xylene and mounted in Permount. Reticular Fiber Intactness Determinations. Five fields were surveyed per section at a magnification of 4COX. A subjective score was assigned based on the relative intactness of the reticular fiber net surrounding the individual rtiuscle cells. This score ranged from one thru five with one corresgonding to zero percent intactness and 5 correspnding to 100 percent intactness. Values for each section viewed were expressed as the mean of the five observations. Mast Cell Staining Procedures. Mast cells were determined in uncooked samples, utilizing a modification of the procedure described by Smith, -- et al., Samples were fixed for 72 hours in 95 percent ethyl alcohol: 10 percent formalin (9:l) with one percent sodium acetate added. Samples were dehydrated in 2 changes each of 95 and 100 percent ethyl alcohol for one hour, followed by clearing in xylene and embedding for longitudinal sectioning in Paraplast. The embedded tissue was sectioned serially at 6 microns, and tissue ribbons were placed directly on acid clean glass slides coated with Mayers-Albumin fixture. Slides were passed over a hot plate (55OC.) and allowed to dry for 12 hours. Tissue sections were cleared in xylene and taken to 95 percent alcohol, followed by staining in 0.25 percent Toluidine Blue 0 in 70 percent acidified ethyl alcohol (ph 1.65) for 30 minutes. Sections were decolorized in two changes of 0.5 percent acid alcohol, and counterstained in one percent Eosin Y acidified with glacial acetic acid. Following dehydration in 95 and 100 percent alcohol, sections were cleared in xylene and mounted in Permount. Mast Cell Determinations. Twenty serial sections per sa ple, 6 microns thick, were placed on each slide. A net reticule (25 mm? subdivided into one mm. squares) was placed over each section to be counted. Four areas, one m. square, were observed on each section (400X magnification), and mast cells were identified and counted. The required following calculations were made as follows: 43.

3 44. L 2 mm, Length of square. W 2 m. Width of square. H 6 microns N 20, Number of s e c t i o n s. 6 x lom3mm. Depth of section. (L x W x H) 20 :Volume of t i s s u e. -3 x 2mm. x (6 x 10- )mmj 20 z h 2 1-2mm...I Duplicate samples were prepared, read and c a l c u l a t e d. equaled mm? Total t i s s u e a r e a V I :T o t a l mast c e l l numbers of d u p l i c a t e samples. MC Z W s t c e l l s p e r cubic mm Results Shear Values. Mean s h e a r values f o r t h e longissimus d o r s i muscle samples used i n t h i s study a r e shown i n t a b l e 1. The t - t e s t revealed a s i g n i f i c a n t ( P c -01)d i f f e r e n c e between high and l o w shear value groups. Table 1. Shear V a l u e,. m s t C e l l Number and R e t i c u l a r Fiber I n t a c t ness Score Means f o r Cooked and Uncooked longissimus d o r s i Nuscle. Shear Mast C e l l N value numbe rs Zc ' Tenderness group High S.Vba S.D. e Low S.Vba S.D.e 15 R e t i c u l a r Fiber I n t a c t n e s s scores Cooked Uncooked.lid d 4.ISd 0.50 Shear value expressed a s kg. of f o r c e on a 2.5 cm. core. a S.V.; b Refers t o mast c e l l s per cubic mm., c P <,.01 d P<.O5 e S.D.; Standard d e v i a t i o n uncooked t i s s u e.

4 Mast Cell Numbers. The t-test revealed no significant difference between means for mast cell numbers of the high shear and low shear value groups (table 1). Correlation coefficients, relating mast cells numbers and shear values of the high and low group were not significant (table 2). Visual observations of mast cells revealed they were in close relationship with the endomysia1 connective tissue. The cells appeared to vary in their affinity for Toluidine Blue 0 as dark stained mast cells were associated with very light stained cells in the same section. 45. Table 2. Correlation Coefficients. Comparison of Shear Values with Mast Cell Numbers and Reticular Fiber Intactness Scores. Independent Variable High shear Low shear Dependent variable" value group value group _- Mast Cell numbers Reticular Fiber intactness Uncooked 0.28 Cooked O.4gb 0.15 a d.f. 14 b P<.05 Reticular Fiber Intactness. Means for reticular fiber intactness scores are shown in table 1. Statistical analysis revealed a significant difference (P<.05) between reticular fiber intactness score means of cooked tissues between high and low shear value groups. Reticular fiber intactness score means also differed significantly (PC.05) between cooked and uncooked tissues in the low shear value group. There was a significant relationship (r z.49) between reticular fiber intactness scores of uncooked tissue and shear values within the low shear value group (table 2). Reticular fiber intactness scores were correlated (r:.54) with mast cell numbers in the high shear value group in uncooked tissues (table 3). Reticular fibers of cooked and uncooked tissue did not appear to vary in their affinity for gold chloride. However, it was observed on the high and low shear value cooked tissue sections that an unidentified substauce with a strong affinity for methylene blue was present in and around the reticular net.

5 46. Table 3. Correlation Coefficients. Comparison of Mast Cell Numbers in Uncooked Tissue with Reticular Fiber Intactness Scores. Dependent variablea Independent Variable High shear Low shear value group value group Reticular Fiber Intactness Uncooked Cooked b a d.f. 14 b P<.05 Discus s ion Reticular fiber intactness and mast cell number mean values were not significantly different between groups of uncooked tissue. When intactness and mast cell numbers were positively correlated (r.54) in the uncooked tissue samples, cooking followed by shearing did not appear to influence reticular fiber intactness in the high shear group. However, reticular fiber intactness significantly decreased (P<.05) after cooking and shearing in the low tenderness group. Intactness was not significantly related to mast cell numbers in the low shear value uncooked tissue samples. Perhaps bovine mast cell constituents may influence directly or indirectly the state of reticular fibers. However, measurement precision of mast cell numbers in the connective tissue area was not sufficient to consistently reflect the influence of their constituents. LITERATURE CITED Crow, Re E. and A. M. Mullins Demonstration of differing amounts of reticular fibers present in striated bovine muscle tissue. Presented, Louisiana Acadeqy of Sciences, Baton Rouge, La. Crow, R. E., A. M. Ulins, S. L. Hansard and R, F, Boulware sections for mast cell demonstration in bovine striated muscle. ceedings, Association of Southern Agricultural Workers 62 :133. Serial Pro- (Abstr.) Paul, P. C Influence of methods of cooking on meat tenderness. In Proceedings Meat Tenderness Symposium. Campbell Soup Company. CamdG, New Jersey. Riley, J. F The Mast Cells. E. and S. Livingstone, Ltd. London.

6 Smith, E. I?. and W. B, Atkinson Simple procedure for identification and rapid counting of mast cells in tissue sections. Science. 123 : 941. Soule, J. D Direct staining of reticular fibers with gold chloride. Stain Tech. 37: DR. CRAIG: I'd like to comnend the three speakers on their presentations and also on the general quality of the slides, I have one announcement I'd like to make before proceeding. That is, the Chairman. of the Personnel Committee, Professor Kunkle, has asked me to mention that any of you who are interested in job changes or in hiring people to check with the registration desk upstairs and the resume of the people will be there. You are asked to please not take these things away, but simply to review then on the spot. So, anybody that's looking or anybody that wants a job please go up on the second level and take care of this at your convenience. To lead the discussion of the papers which have just been presented this morning will be Dr. R. L. Henrickson of Oklahoma State University, and I will ask that when he has finished if he will handle the questions and then turn the program back to our president for the next part of the program. DR. HELXICKSOTJ: Thank you, Brad. You left the impression that I was going to make a speech. This is not planned, I assure you. I do want to thank the members who sfoke this morning for working very closely with me and the con;mittee, and for furnishing the papers which were turned over to Bill Sherman this morning before their presentations. They've responded excellently in this manner. I also want to extend my appreciation to the committee, which as you can see is noted in the back of your program, You heard this morning three dynamic speakers tell about the muscle cell, the fat cell, and the mast cell along with the reticular fibers, VJe had heard from Mr. Gillis in relation to the influence of the natura.1 variations of breed, species, sex, etc. on the size of the muscle fiber and on the shape of the muscle fiber. You heard also of the variakions that are influenced by rigor, by temperature and by tension on the muscle, regardless of whether it be artificial or whether it be natural in the carcass. &. Moody pointed out the structural variations of the muscle and their importance in fat deposition. Mr. Gillis pointed out this same point. The structure of the muscle seems to be of some importance in both cases, bk. Moody pointed out that the architecture is important from the standpoint of where the arteries are located and where the fat is deposited. Then we heard from bk. Crow in relation to the mast cells and the reticular fibers and, to some extent, the importance of the recticular fibers on tendons. I'd like at this time to open the period for questions that you might like to direct to these men. Yes?