GENETIC SAMPLES COLLECTION

-> Collection of samles

-> Description of samples

-> Preservation and storage of biological material during transport

-> Address of the genetics laboratory

Collection of samples:

What kind of biological material is proper for genetic research?

– blood

– hair with bulbs

– soft tissues

– skin samples collected with the use of darts

– bones and cartilage

Rules of peripheral blood samples collecting

1. peripheral blood should be collected by a veterinarian at the occasion of immobilizing or death of animal

2.  blood must be collected into sterile test tubes contained anticoagulant (EDTA-K2; EDTA-K3)

3. blood should be collected in quantities of 4 ml up to 8 ml (test tube should not be filled above the marked (yellow)    line)

4. after securing the tube with a stopper, test tube must be repeatedly reversed „up and down” to mix the blood with anticoagulant sprayed on the walls of the tube, the tube should not be shaken!

5. collected blood can be stored in the refrigerator – no longer than 2-3 days

6. if you need longer storage, freeze the test tube with blood (temperature: – 20 ° C)

 <- 7. EACH TEST TUBE MUST BE NUMBERED WITH A WATERPROOF PEN.

<- 8. EACH NUMBERED TEST TUBE MUST BE DESCRIBED. SEE INFORMATION BELOW!

Rules of hair with bulbs samples collecting

1. in enclosed breeding centers hair can be collected (pulled out) by caretakers (breeders) during treatment procedures or feeding of animals; a bunch of hair with bulbs should be pull out (not cut!) (bunch: 10-20 hair)

2. in free and semi-free living herds hair could be collected in winter from places where individuals were lying and resting in the night; such hair should be removed carefully from the frozen ground (to avoid break off the bulbs)

3. hair should be placed in an envelope (wet hair sholud be first dryed)

4. envelopes should be stored  in a dry, airy place

<- 5. EACH ENVELOPE MUST BE NUMBERED WITH A WATERPROOF PEN.

<- 6. EACH NUMBERED ENVELOPE MUST BE DESCRIBED. SEE INFORMATION BELOW!

 Rules of soft tissues samples collecting

1. soft tissues of liver, kidney, heart or muscles should be taken by a veterinarian; in the absence of mentioned tissues a sample of skin should be taken

2. proper size of tissue sample is approx. 5 square centimeters (size of a box of matches)

3. all tissues should be taken when wearing gloves (when samples are taken from different individuals, each time should be used: new gloves, clean knife/clean scalpel)

4. each tissue sample should be inserted in a separate ziplock bag

5. each ziplock bag with sample should be marked with a waterproof pen

6. tissue samples should be stored frozen

7. when it is not possible to freeze the sample, the tissue can be placed in a jar with ethanol (40%)

<-  8. EACH ZIPLOCK BAG MUST BE NUMBERED WITH A WATERPROOF PEN.

<- 9. EACH NUMBERED ZIPLOCK BAG MUST BE DESCRIBED. SEE INFORMATION BELOW!

Rules of bones and cartilage samples collecting

In the case of finding the remains of the animal, when it is not possible to take of blood and soft tissues we collect bones and cartilage samples. Found bones should not be boiled or washed with the use of detergent!

1. bone fragments (containing the cancellous bone, typically found at the ends of long bones, proximal to joints and within the interior of vertebrae) and teeth should be taken.

2. each tissue sample should be inserted in a separate ziplock bag

3. hard tissue taken from the remains of the animal (septic) must be stored frozen (- 70 ° C)

4. bone samples taken from the archives or dry remains  should be stored in dry and airy places

<-  5. EACH ZIPLOCK BAG MUST BE NUMBERED WITH A WATERPROOF PEN.

<-  6. EACH NUMBERED ZIPLOCK BAG MUST BE DESCRIBED. SEE INFORMATION BELOW!

Rules of skin samples collecting with the use of darts

Kit for tissue sampling with the use of biopsy needles comprises:

[1] dart

[2] stabilizer

[3] plastic syringe

[4] metal adapter (connector)

[5, 6, 7] sterile set: [5] metal needle biopsy with a side opening;[6] metal nail ; [7]   metal pin to move the contents within the needle

[8] plastic covers for darts

[9]  test tubes for tissue storage (2 ml) (not shown in the picture)

[10] ziplock bags predicted for dirty needles with samples (not shown in the picture)
[11] ziplock bags branderd „DIRTY” predicted for nails, pins and needles after use (without samples) (not shown)

Procedure

1. dart should be take from a plastic cover and described with the number / name / symbol of wisent

2. dart should be ballaseted with water (3ml) with the use of syringe with an adapter (through its connection to the hole (from the opposite side than pink brush)) [A-1.2]; after ballasting with water DO NOT PRESS THE DART!

3. after removing the syringe, nail should be put to the same hole (plugging the hole) [B] then needle biopsy should be imposed

4. before inserting needle it should be ensured that the hole inside needle is way; if not – DO NOT BLOW WITH MOUTH! The side opening in the needle is used for discharging water from dart.

5. dart should be placed in the shotgun; the pressure may be increased (1-2 bars) compared to standard shots; than the shot should be done!

6’a. after finding the dart, the needle shoul be carefully removed  and inserted into a sterile ziplock bag – described with the number / name / symbol of  wisent; nail should be inserted into ziplock bag described „DIRTY”; in this form the needle with sample may be transported to the room

6’b. after finding the dart, the wohole dart (with all elements and sample inside) could be also taken to the room

7. (in the room) the needle should be pushed through using a sterile pin [C] and the entire content of the needle should be put on a piece of paper under the lamp (40W) for 30 minutes [D]; the dried piece of tissue should be inserted into a test tube (2 ml), which should be described with the number / name / symbol of wisent

Important notes

I: each set of a needle, a nail and a pin can be used ONLY ONCE

II: after using all the needles should be together with pins, nails and dried-SAMPLES send them to the Department of Genetics and Animal Breeding (WULS)

III: dart prepared to shoot (with water and a needle) can be stored no longer than TWO DAYS (with the cover put on it)

Description of samples

Each sample should be described in a proper way. Description should contain:

• the number of the sample (the same as the one included on the test tube or ziplock bag)
• circumstances of sample taking (intravitally – transport;  intravitally – collars given, post mortem – veterinary elimination, post mortem – found body; etc.)
• date of sample taking
• the place of origin of the animal and / or place of sample taking
• pedigree number, registration number and / or other identification data of the animal (collar number, chip number etc.)
• sex of the animal
• age of the animal
• body mass of the animal
• any additional information

Preservation and storage of biological material intended for genetic research

The best way to protect the biological material during the transport to laboratory is placing the sample into a styrofoam box equipped with frozen cooling inserts (contributions are filled with a substance long-lasting the low temperatures).

In the case of glass test tubes you can wrap them with the bubble wrap to prevent damages.

Tissues in alcohol should be sent in a tightly closed canisters to prevent damages.

Address of the genetics laboratory

Samples should be sent to the address:

Katedra Genetyki i Ogólnej Hodowli Zwierząt

Wydział Nauk o Zwierzętach

Szkoła Główna Gospodarstwa Wiejskiego w Warszawie

Ciszewskiego 8

02-786 Warszawa, POLSKA (POLAND)

 Based on:

 Wojciechowska M. (2015) „Pobranie, zabezpieczanie i przechowywanie materiału biologicznego przeznaczonego do badań genetycznych„, http://www.zubry.sggw.pl/pobieranie-materialu-biologicznego-do-badan-genetycznych/

Nowak Z., Gruszczyńska J. (2007) „Wybrane techniki i metody analizy DNA„. Warszawa, Wydawnictwo SGGW

Pictures of equipment: Marlena Wojciechowska

Picture of wisent: Janusz Sochacki