BIRDS
ARE ONE OF THE MOST DIVERSE GROUPS OF ANIMALS ON EARTH, WITH OVER 10,000
SPECIES KNOWN TO SCIENCE. ONE OF THE MOST IMPORTANT ASPECTS OF BIRD BIOLOGY IS
THE ABILITY TO ACCURATELY DETERMINE ITS SEX. WHILE SOME SPECIES OF BIRDS HAVE
OBVIOUS PHYSICAL DIFFERENCES BETWEEN MALES AND FEMALES, SUCH AS DIFFERENCES IN
SIZE OR COLORATION, MANY SPECIES LOOK VERY SIMILAR AND REQUIRE MORE ADVANCED
METHODS TO DETERMINE THEIR SEX.
BIRD DNA SEXING IS A
PROCESS THAT ENABLES BIRD BREEDERS, AVIAN RESEARCHERS, AND BIRD ENTHUSIASTS TO
DETERMINE THE SEX OF THEIR BIRDS WITH ACCURACY. IN THE PAST, THE PROCESS OF
SEXING BIRDS WAS DIFFICULT, ESPECIALLY FOR SPECIES THAT DO NOT HAVE ANY
EXTERNAL SEXUAL DIMORPHISM OR THOSE THAT DO NOT EXHIBIT SEXUAL BEHAVIOR.
HOWEVER, ADVANCEMENTS IN DNA TECHNOLOGY HAVE MADE BIRD DNA SEXING EASY AND
ACCESSIBLE. IN THIS ARTICLE, WE WILL DISCUSS THE PROCEDURE FOR BIRD DNA SEXING
FROM START TO FINISH.
NOTE: BIRDS HAVE A SEX-DETERMINATION SYSTEM THAT IS DIFFERENT
FROM MAMMALS. WHILE MAMMALS HAVE A PAIR OF SEX CHROMOSOMES, XX FOR FEMALES AND
XY FOR MALES, BIRDS HAVE A PAIR OF Z AND W CHROMOSOMES, WITH FEMALES HAVING A
PAIR OF ZW CHROMOSOMES AND MALES HAVING A PAIR OF ZZ CHROMOSOMES.
AVIAN DNA SEXING METHODS
THERE ARE DIFFERENT
METHODS FOR SEXING THE DNA OF BIRDS, BUT THE TWO MOST COMMONLY USED TECHNIQUES
ARE POLYMERASE CHAIN REACTION (PCR) AND FLUORESCENT IN SITU HYBRIDIZATION
(FISH).
PCR AMPLIFIES SPECIFIC
REGIONS OF THE SEX CHROMOSOMES, WHICH CAN THEN BE VISUALIZED AND ANALYZED TO
DETERMINE THE SEX OF THE BIRD. FISH, ON THE OTHER HAND, INVOLVES LABELING DNA
PROBES WITH FLUORESCENT MOLECULES THAT BIND TO SPECIFIC REGIONS OF THE SEX
CHROMOSOMES. THE PROBES ARE THEN VISUALIZED UNDER A MICROSCOPE TO DETERMINE THE
SEX OF THE BIRD.
BOTH METHODS ARE HIGHLY
ACCURATE, AND THE CHOICE OF TECHNIQUE MAY DEPEND ON THE LABORATORY'S
PREFERENCE, THE TYPE OF BIRD BEING SEXED, AND THE AVAILABILITY OF RESOURCES.
OTHER METHODS, SUCH AS RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP),
SINGLE-STRAND CONFORMATION POLYMORPHISM (SSCP), AND ALLELE-SPECIFIC
OLIGONUCLEOTIDE (ASO) HAVE ALSO BEEN USED IN THE PAST, BUT ARE LESS COMMONLY
USED TODAY.
AVIAN DNA SEXING SAMPLES
THERE ARE SEVERAL
DIFFERENT METHODS FOR COLLECTING DNA SAMPLES FROM BIRDS FOR SEXING PURPOSES,
INCLUDING FEATHERS, BLOOD, EGGSHELL, FECAL MATTER, AND A SWAB OF THE INSIDE OF
THE BIRD'S MOUTH. EACH METHOD HAS ITS OWN ADVANTAGES AND DISADVANTAGES, AND THE
CHOICE OF METHOD WILL DEPEND ON THE SPECIES OF BIRD, THE AGE OF THE BIRD, AND
THE RESOURCES AVAILABLE.
FEATHER
DNA SEXING: FEATHER DNA SEXING IS A NON-INVASIVE
METHOD FOR COLLECTING DNA SAMPLES FROM BIRDS. THIS METHOD INVOLVES PLUCKING A
FEW FEATHERS FROM THE BIRD, TYPICALLY FROM THE CHEST OR BACK, AND SENDING THEM
TO A LABORATORY FOR ANALYSIS. FEATHER DNA SEXING WORKS BECAUSE THE FEATHER
SHAFT CONTAINS A SMALL AMOUNT OF BLOOD, WHICH CONTAINS DNA THAT CAN BE USED TO
DETERMINE THE BIRD'S SEX. FEATHER DNA SEXING CAN BE PERFORMED ON BIRDS OF ANY
AGE, AND IS PARTICULARLY USEFUL FOR SPECIES THAT ARE DIFFICULT TO SEX VISUALLY,
SUCH AS MANY SPECIES OF PARROTS AND RAPTORS.
BLOOD DNA
SEXING: BLOOD DNA SEXING IS ANOTHER COMMON METHOD FOR
COLLECTING DNA SAMPLES FROM BIRDS. THIS METHOD INVOLVES COLLECTING A SMALL
AMOUNT OF BLOOD FROM THE BIRD'S VEIN, TYPICALLY FROM THE WING OR LEG. BLOOD DNA
SEXING IS HIGHLY ACCURATE AND CAN BE USED ON BIRDS OF ANY AGE. HOWEVER, THIS
METHOD REQUIRES A TRAINED INDIVIDUAL TO PERFORM THE BLOOD DRAW, WHICH CAN BE
STRESSFUL FOR THE BIRD. BLOOD DNA SEXING IS PARTICULARLY USEFUL FOR SPECIES
THAT ARE DIFFICULT TO SEX VISUALLY OR FOR YOUNGER BIRDS THAT MAY NOT HAVE
DEVELOPED EXTERNAL SEXUAL CHARACTERISTICS.
EGGSHELL
DNA SEXING: EGGSHELL DNA SEXING IS A NEWER METHOD FOR
COLLECTING DNA SAMPLES FROM BIRDS. THIS METHOD INVOLVES COLLECTING THE MEMBRANE
THAT LINES THE INSIDE OF AN EGGSHELL. THIS MEMBRANE CONTAINS DNA FROM THE
DEVELOPING EMBRYO, WHICH CAN BE USED TO DETERMINE THE SEX OF THE BIRD. EGGSHELL
DNA SEXING IS NON-INVASIVE AND CAN BE USED ON HATCHED EGGS, MAKING IT PARTICULARLY
USEFUL FOR SPECIES THAT ARE DIFFICULT TO SEX VISUALLY OR ARE TOO YOUNG TO SEX
BY OTHER METHODS.
FECAL DNA
SEXING: FECAL DNA SEXING IS A NON-INVASIVE METHOD FOR
COLLECTING DNA SAMPLES FROM BIRDS. THIS METHOD INVOLVES COLLECTING A SMALL
AMOUNT OF FECAL MATTER FROM THE BIRD. FECAL DNA SEXING IS LESS ACCURATE THAN
OTHER METHODS AND IS MOST COMMONLY USED FOR LARGER BIRDS, SUCH AS RAPTORS,
WHERE COLLECTING BLOOD OR FEATHERS IS MORE DIFFICULT. HOWEVER, THIS METHOD IS
STILL USEFUL FOR SEXING BIRDS THAT CANNOT BE SEXED BY OTHER METHODS.
SWAB DNA
SEXING: SWAB DNA SEXING IS A NON-INVASIVE METHOD FOR
COLLECTING DNA SAMPLES FROM BIRDS. THIS METHOD INVOLVES COLLECTING A SWAB OF
THE INSIDE OF THE BIRD'S MOUTH, TYPICALLY FROM THE CHEEK OR THROAT. SWAB DNA
SEXING IS MOST COMMONLY USED FOR SMALLER BIRDS, SUCH AS SONGBIRDS, AND CAN BE
PERFORMED BY A TRAINED INDIVIDUAL. THIS METHOD IS LESS ACCURATE THAN OTHER
METHODS BUT IS STILL USEFUL FOR SEXING BIRDS THAT CANNOT BE SEXED BY OTHER
METHODS.
AVIAN DNA SEXING IDENTIFICATION
METHODS
SEX IDENTIFICATION OF
BIRDS CAN BE DIFFICULT AS THEY LACK EXTERNAL GENITALIA. THEREFORE, RESEARCHERS
AND BREEDERS USE DNA-BASED METHODS TO DETERMINE THE SEX OF BIRDS ACCURATELY.
TWO POPULAR METHODS FOR BIRD SEXING ARE POLYMERASE CHAIN REACTION (PCR) AND
FLUORESCENT IN SITU HYBRIDIZATION (FISH). EXPLAINS THE PROTOCOLS USED TO
IDENTIFY THE SEX OF BIRDS USING PCR AND FISH.
POLYMERASE
CHAIN REACTION (PCR)
PCR IS A TECHNIQUE USED
TO AMPLIFY DNA SEQUENCES. IT IS A POWERFUL TOOL THAT ENABLES THE IDENTIFICATION
AND QUANTIFICATION OF DNA SEQUENCES. IN BIRD DNA SEXING, PCR CAN BE USED TO
AMPLIFY A REGION OF THE SEX CHROMOSOMES THAT IS UNIQUE TO EITHER THE MALE OR
FEMALE SEX.
ONE OF THE MOST COMMON PCR-BASED METHODS FOR BIRD SEXING IS BASED ON THE CHROMODOMAIN-HELICASE-DNA-BINDING PROTEIN 1 (CHD1) GENE. THE CHD1 GENE IS LOCATED ON THE W CHROMOSOME, AND PCR PRIMERS ARE DESIGNED TO AMPLIFY A REGION OF THIS GENE THAT IS UNIQUE TO THE W CHROMOSOME. THE PCR PRODUCT IS THEN RUN ON AN AGAROSE GEL, AND THE PRESENCE OR ABSENCE OF A BAND INDICATES THE SEX OF THE BIRD. FEMALES WILL HAVE A BAND, WHILE MALES WILL NOT.
ANOTHER PCR-BASED METHOD FOR BIRD SEXING INVOLVES THE USE OF PRIMERS THAT AMPLIFY A REGION OF THE SEX-LINKED GENE THAT IS ONLY PRESENT IN MALES OR FEMALES. FOR EXAMPLE, PRIMERS CAN BE DESIGNED TO AMPLIFY A REGION OF THE Z CHROMOSOME-SPECIFIC GENE ZINC FINGER PROTEIN (ZFX) OR THE W CHROMOSOME-SPECIFIC GENE ZINC FINGER PROTEIN (ZFY). THE AMPLIFIED PRODUCTS CAN THEN BE ANALYZED USING GEL ELECTROPHORESIS OR DNA SEQUENCING TO DETERMINE THE SEX OF THE BIRD.
ADVANTAGES:
PCR-BASED METHODS ARE HIGHLY SPECIFIC AND SENSITIVE, ENABLING THE DETECTION OF
SMALL AMOUNTS OF DNA. THEY ARE ALSO RELATIVELY FAST AND CAN BE PERFORMED USING
SMALL AMOUNTS OF DNA, WHICH IS PARTICULARLY USEFUL IN CASES WHERE SAMPLE
AVAILABILITY IS LIMITED. ADDITIONALLY, PCR-BASED METHODS ARE COST-EFFECTIVE AND
CAN BE USED FOR HIGH-THROUGHPUT SEXING OF LARGE NUMBERS OF BIRDS.
LIMITATIONS:
ONE
OF THE LIMITATIONS OF PCR-BASED METHODS FOR BIRD SEXING IS THAT THEY CAN ONLY
BE USED TO DETERMINE THE SEX OF BIRDS WITH CONVENTIONAL SEX CHROMOSOMES (I.E.,
ZW OR XY). BIRDS WITH UNUSUAL SEX CHROMOSOME SYSTEMS, SUCH AS ZZ/ZW OR MULTIPLE
SEX CHROMOSOMES, MAY REQUIRE ALTERNATIVE METHODS FOR SEX DETERMINATION.
FLUORESCENT
IN SITU HYBRIDIZATION (FISH)
FISH USES FLUORESCENTLY LABELED DNA PROBES THAT BIND TO SPECIFIC DNA
SEQUENCES WITHIN CHROMOSOMES. IN BIRD SEXING, FISH PROBES ARE DESIGNED TO
HYBRIDIZE TO THE SEX CHROMOSOMES, ALLOWING FOR THE IDENTIFICATION OF THE
SEX-SPECIFIC CHROMOSOME.
ONE OF THE MOST COMMON FISH-BASED METHODS FOR BIRD SEXING USES PROBES
THAT HYBRIDIZE TO THE CHROMODOMAIN-HELICASE-DNA-BINDING PROTEIN 1 (CHD1) GENE.
THE CHD1 GENE IS LOCATED ON THE W CHROMOSOME IN BIRDS, AND PROBES ARE DESIGNED
TO HYBRIDIZE TO THIS GENE. THE W CHROMOSOME-SPECIFIC PROBE IS LABELED WITH A
RED FLUOROPHORE, WHILE A GREEN FLUOROPHORE IS USED TO LABEL A PROBE THAT
HYBRIDIZES TO A REGION OF THE AUTOSOMES. THE HYBRIDIZATION OF BOTH PROBES TO
THE CHROMOSOMES RESULTS IN RED AND GREEN SIGNALS IN FEMALE BIRDS, INDICATING
THE PRESENCE OF THE W CHROMOSOME, WHILE MALE BIRDS WILL ONLY EXHIBIT GREEN
SIGNALS, INDICATING THE ABSENCE OF THE W CHROMOSOME.
ANOTHER FISH-BASED METHOD FOR BIRD SEXING INVOLVES THE USE OF PROBES
THAT HYBRIDIZE TO THE SEX CHROMOSOME-SPECIFIC GENES ZFX AND ZFY. PROBES FOR THE
Z CHROMOSOME-SPECIFIC GENE ZFX ARE LABELED WITH A RED FLUOROPHORE, WHILE PROBES
FOR THE W CHROMOSOME-SPECIFIC GENE ZFY ARE LABELED WITH A GREEN FLUOROPHORE.
THE HYBRIDIZATION OF BOTH PROBES TO THE CHROMOSOMES RESULTS IN RED AND GREEN
SIGNALS IN MALE BIRDS, INDICATING THE PRESENCE OF THE Z CHROMOSOME, WHILE
FEMALE BIRDS WILL ONLY EXHIBIT RED SIGNALS, INDICATING THE ABSENCE OF THE Z
CHROMOSOME.
ADVANTAGES: FISH-BASED METHODS FOR BIRD SEXING ARE
ADVANTAGEOUS BECAUSE THEY CAN DETECT SEX CHROMOSOME ABNORMALITIES AND
MOSAICISM, WHICH CANNOT BE DETECTED BY PCR-BASED METHODS. ADDITIONALLY, FISH
CAN BE PERFORMED ON A VARIETY OF SAMPLE TYPES, INCLUDING FEATHERS, BLOOD, AND
EGGSHELLS.
LIMITATIONS: THERE
ARE SOME LIMITATIONS TO FISH-BASED METHODS FOR BIRD SEXING. FISH REQUIRES
SPECIALIZED EQUIPMENT AND EXPERTISE, AND THE INTERPRETATION OF FISH RESULTS CAN
BE SUBJECTIVE. ADDITIONALLY, FISH CAN BE TIME-CONSUMING AND EXPENSIVE, MAKING
IT LESS PRACTICAL FOR ROUTINE BIRD SEXING COMPARED TO PCR-BASED METHODS.
AVIAN DNA SEXING RESULT
AFTER THE SAMPLE HAS
BEEN COLLECTED, IT IS SENT TO A LABORATORY FOR ANALYSIS. THE LABORATORY
TECHNICIANS EXTRACT DNA FROM THE SAMPLE AND ANALYZE SPECIFIC DNA MARKERS THAT
ARE ASSOCIATED WITH THE BIRD'S SEX CHROMOSOMES. THESE MARKERS ARE UNIQUE TO
MALE OR FEMALE BIRDS AND CAN BE USED TO IDENTIFY THE BIRD'S SEX WITH A HIGH
DEGREE OF ACCURACY.
ONCE THE LABORATORY HAS
IDENTIFIED THE BIRD'S SEX, THEY WILL SEND THE RESULTS BACK TO THE BIRD OWNER.
THE RESULTS WILL INDICATE WHETHER THE BIRD IS MALE OR FEMALE. THE OWNER CAN USE
THIS INFORMATION TO DETERMINE THE BIRD'S BREEDING POTENTIAL, PAIR THEM WITH
COMPATIBLE MATES, AND PLAN FOR THEIR FUTURE CARE.
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