PROTOCOL 1 FOR COLLECTION OF BOVINE TESTIS MATERIAL

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PROTOCOL FOR COLLECTION OF BOVINE TESTIS MATERIAL 
 
1. Collection of bovine testis 
1.1. Collect the bovine testis with attached epididymis at the slaughterhouse and put it on ice in a 
thermal bag. 
1.2. Transport the whole testis on ice to the laboratory. 
1.1. At the lab, wash the surface of each testis/epididymis with physiological saline (0.9% NaCl) 
supplemented with 1% antibiotic solution (10,000 IU penicillin, 10,000 μg/mL streptomycin) or 
with 50 µg/mL gentamicin. 
1.3. Remove the epididymis from the testis and dissect it in different segments (Caput, corpus and 
cauda) as illustrated in the figure 1. 
 
 
2. Collection of epididymal fluid 
Use only epididymides with sperm reservoir, as evaluate by the presence of swollen tubule in the 
distal cauda portion. 
 
2.1 Caput and Corpus fluid/sperm recovery by float-up method 
2.1.1. Put the dissected caput or corpus epididymis in a Petri dish 
2.1.2. Cut a few tubules with a scalpel in the distal part of the caput epididymis or in the 
central part of the corpus epididymis. 
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2.1.3. Recover the fluid with spermatozoa by applying pressure with hands on the caput or 
corpus epididymis. 
2.1.4. Apply this procedure carefully to avoid blood or tissue contamination. 
2.1.5. Transfer the fluid containing spermatozoa to a 2 mL eppendorf tube. 
 
2.2 Caudal fluid/sperm recovery by retrograde flushing method 
2.2.1. Put the isolated cauda epididymis and ductus deferens in a Petri dish. 
2.2.2. Cannulate the lumen of the ductus deferens with a blunted 22G needle. 
2.2.3. Couple a syringe loaded with air in the 22G needle. 
2.2.4. Flush the intraluminal fluid containing spermatozoa in a retrograde direction from 
ductus deferens through the cauda epididymis by applying air pressure with the syringe. 
2.2.5. Transfer the fluid containing spermatozoa to a 2 mL eppendorf tube. 
 
2.3. Epididymal fluid and spermatozoa recovery 
2.3.1. Centrifuge the epididymal fluid containing spermatozoa at 100 X g for 1 min at 4 °C to 
sediment any tissue fragments and contaminants that may be present within the sample 
2.3.2. Transfer the resulting supernatant to a new 2 mL eppendorf tube and centrifuge the 
tube with epididymal fluid containing spermatozoa at 1000 X g for 20 min at 4 °C to 
remove spermatozoa (or 1500 X g for 10 minutes). 
2.3.2.1. Transfer the supernatant fluid to a new 2 mL eppendorf tube and centrifuge the 
eppendorf at 4000 X g for 20 minutes at 4 °C to remove the any cellular 
2.3.2.2. Wash the sperm pellet in 2 mL PBS at 1000 X g for 20 min at 4 °C. Collect the 
resulting sperm pellet and store it at -80 °C 
2.3.3. Transfer the supernatant fluid resulting from the item 2.3.2.1 to a new 2 mL eppendorf 
tube and Centrifuge the eppendorf at 4000 X g for 20 minutes at 4 °C to remove the any 
cellular. 
2.3.4. Collect the supernatant fluid and store it at -80 °C 
 
2.4. Storage of testis and epididymal tissue 
Prepare the samples for further protein, protease, mRNA and histology analysis 
 
 
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2.4.1. For protein/protease/mRNA 
2.4.1.1. Using a scalpel cut the testis/epididymis in small pieces. (approximately 30 g) 
2.4.1.2. Wash the testis/epididymis pieces in PBS and put in a 50 mL tube 
2.4.1.3. Snap freeze the sample in liquid nitrogen 
2.4.1.4. Store the samples at -80 °C 
 
2.4.2. For Histology 
2.4.2.1. Using a scalpel cut the testis/epididymis in small pieces (no thicker than 10 mm). 
2.4.2.2. Immerse the dissected tissues in the fixative solution (4% formaldehyde in PBS) 
- For complete fixation, the volume of the fixative solution should be 50 times 
larger than the volume of the tissue 
2.4.2.3. Fix the tissue for 24 hour at room temperature. 
2.4.2.4. Wash the tissue 3 times in PBS for 5 minute each. 
2.4.2.5. Place the tissue in 70% ethanol and store at 4 °C.