University of Rhode Island DigitalCommons@URI Senior Honors Projects Honors Program at the University of Rhode Island 2007 A Comparison of Pyronin Y-Methyl Green Stain and Methylene Blue Stain for Somatic Cell Count in Sheep Milk Emily Mirek University of Rhode Island, EmilyMirek@yahoo.com Stacey O Donnell University of Rhode Island, sknurtserge@sbcglobal.net Follow this and additional works at: http://digitalcommons.uri.edu/srhonorsprog Part of the Dairy Science Commons, Public Health Commons, and the Veterinary Medicine Commons Recommended Citation Mirek, Emily and O Donnell, Stacey, "A Comparison of Pyronin Y-Methyl Green Stain and Methylene Blue Stain for Somatic Cell Count in Sheep Milk" (2007). Senior Honors Projects. Paper 49. http://digitalcommons.uri.edu/srhonorsprog/49http://digitalcommons.uri.edu/srhonorsprog/49 This Article is brought to you for free and open access by the Honors Program at the University of Rhode Island at DigitalCommons@URI. It has been accepted for inclusion in Senior Honors Projects by an authorized administrator of DigitalCommons@URI. For more information, please contact digitalcommons@etal.uri.edu.
A Comparison of Pyronin Y-Methyl Green Stain and Methylene Blue Stain for Somatic Cell Count in Sheep Milk Emily Mirek and Stacey O Donnell Faculty Sponsor: Dr. Katherine Petersson, Department of Animal & Veterinary Science Dairy Sheep Produce up to 1,100 lbs per lactation depending on breed Profit In Wisconsin the producer receives ~$55.00/100 lbs of milk Popular for cheese Feta and other gourmet cheeses Also used for yogurt, ice cream and soap
Regulations Milk is highly regulated because it is consumed by people SCC is used as an indicator of milk quality Standards were set up for cows Modified for goats Sheep are similar to goats, but regulated as cows Procedures for SCC strictly controlled Training at RI State Lab in Providence and NY State Lab in Albany Somatic Cell Count Somatic cells are primarily white blood cells WBCs are indicators of infection USDA Guidelines Cows: 750,000 cells per ml Sheep: 750,000 cells per ml Goats: 1,000,000 cells per ml
Secretory Systems Merocrine Cows Milk only released Less particles Apocrine Sheep and Goats Milk released along with part of cell More cytoplasmic particles Secretory Systems
Objectives Compare Blue Stain, Green Stain, and machine counting Show that sheep need different regulations Observe effect of lactation stage on SCC Recommend changes to national regulations Stains Blue Stain aka Cow Stain Stains cells and cytoplasmic particles Green Stain aka Goat Stain Stains only nuclear material
Machine Count Fossamatic 500 Dyes nuclear DNA Cell electrical impulse is recorded Standardized to cow s milk Not 100% reliable DMSCC used to confirm for goats Testing conducted at Dairy One, Inc., Ithaca, NY Samples Samples from 60 Friesian crossbreed sheep at varying stages of lactation 14 to 199 Days Mailed from Chatham Farm in NY 13 year old sheep dairy Over 1,000 sheep Produce gourmet cheese and yogurt Samples sent off to Dairy One for machine counting and part was kept for staining
Methods Pipette 10 µl of milk onto each circle on slide Spread and dry sample Stains Stain slides with either blue or green stain
Blue Stain Blue stain Methylene Blue Procedure Methylene Blue 2 3 changes of water Dip Green Stain Green stain Pyronin Y-Methyl Green Procedure Carnoy s Fixative 5 50% Ethanol 1 30% Ethanol 1 Water 1 Pyronin Y-Methyl Green 6 Butanol Dip Xylene Dip
Direct Microscopic Counting Cells are counted in a straight horizontal line SCC for whole circle is then calculated Statistical Analysis Correlation analyses were conducted to determine relationship between 3 counting procedures (SAS Inc., Cary, NC) General Linear Models were utilized to analyze the effect of days in lactation (SAS Inc., Cary, NC) Significance was defined at P < 0.05
Results Correlation Analysis Blue Stain vs Machine Counting R = 0.914 Green Stain vs Machine Counting R = 0.953 Blue Stain vs Green Stain R = 0.979 450,000 Blue Stain vs Machine Count Somatic Cell Count 400,000 350,000 300,000 250,000 200,000 150,000 Blue Stain vs Machine Count P < 0.05 Days in lactation NS, Blue increases by 1 unit, machine by.21 Blue Stain Machine Count 100,000 50,000 0 14 17 21 25 28 32 37 39 41 48 49 51 53 61 66 67 74 146 168 173 180 185 187 189 192 194 199 Days in Lactation
350,000 Green Stain vs Machine Count Somatic Cell Count 300,000 250,000 200,000 150,000 100,000 Green Stain vs Machine Count P < 0.05 Days in lactation NS, Green increases 1 unit, machine by.254 Green Stain Machine Count 50,000 0 14 17 21 25 28 32 37 39 41 48 49 51 53 61 66 67 74 146 168 173 180 185 187 189 192 194 199 Days in Lactation 450,000 Blue Stain vs Green Stain Somatic Cell Count 400,000 350,000 300,000 250,000 200,000 150,000 Blue Stain vs Green Stain P < 0.05 Days in lactation P < 0.05 Blue increases by 1 unit, Green by.84 Blue Stain Green Stain 100,000 50,000 0 14 17 21 25 28 32 37 39 41 48 49 51 53 61 66 67 74 146 168 173 180 185 187 189 192 194 199 Days in Lactation
Results Green Stain Yields lower SCC than blue More difficult and time consuming More expensive Cracking and peeling occurs fairly often Results Days in lactation affected relationship between Blue Stain and Green Stain Probably due to cytoplasmic particles in later lactation Did not account for all variation though Machine not accurate, but correlated Increased and decreased with direct counts Not standardized to sheep milk Clumping (counts clumps as one) Individual machines may vary
Conclusions Green stain may be more accurate Shows promise Blue stain produces higher counts Perhaps due to particles Machine counting not accurate for sheep milk More testing needed More samples Further into lactation Implications Preliminary results could inspire more studies Change national regulations Approve Green Stain Allow producers to milk longer Increase profits
Recommendations for Future Studies Evaluate individual ewes over entire lactation Investigate optimization of machine counts for sheep milk Search for less complicated substitute for Green Stain Works Cited Gonzalo, C., J. Baro, J. Carriedo, and F. Primitivo. 1992. Use of the Fossomatic Method to Determine Somatic Cell Counts in Sheep Milk. Journal of Dairy Science. 76:115-119 Gonzalo, C. J.R. Martínez, J.A. Carriedo, and F. San Primitivo. 2003. Fossomatic Cell-Counting on Ewe Milk: Comparison with Direct Microscopy and Study of Variation Factors. Journal of Dairy Science. 86: 138-145 Haenlein, G. and L.Hinckley. 1997. Goat Milk Somatic Cell Count Situation in the United States. University of Delaware and the University of Connecticut. Haenlein, G. 1997. Producing Quality Goat Milk. University of Delaware.