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Developing the Premier Colored Huacaya Alpaca Herd in the World

Bloodtyping and DNA Fingerprinting Applied
to the Establishment of Pedigrees in Alpacas

by Maria Cecilia T Penedo

One of the traditions of animal breeding is the establishment of breed associations and registries dedicated to the promotion and advertisement of breeds, to the maintenance of pedigree records, and to the issuance of registration papers. Historically, pedigrees were based on available breeding records. Several decades ago, breed registries were quick to recognize the importance of blood-typing tests as an objective, scientific means to verify the accuracy of pedigrees and maintain studbook integrity. Cattle and horse breed registries, in particular, have relied upon blood-typing programs to validate pedigree records.

When the Alpaca Registry was formed in 1987, the membership approved a bylaw requiring blood-typing of all alpacas and verification of parentage as a prerequisite to registration. At that time, tests developed at the Veterinary Genetics Laboratory, University of California, Davis, were informative enough to warrant their use as a tool to verify parentage of alpacas and llamas. Additional developments, including the implementation of DNA fingerprinting tests in 1991, have produced a highly efficacious means to validate pedigree records, to solve problems of paternity or maternity, and to provide a system of animal identification.

Today most alpaca owners and breeders are familiar with the blood-typing process and its role in the Alpaca Registry. Over the years, several articles describing the blood-typing process and its use in checking pedigree records have appeared in industry magazines and journals. As new methods are developed and the membership of the Alpaca Registry, Inc., grows, informing members about these processes will be an important role of this journal. This article reviews concepts of blood-typing, DNA fingerprinting, and the use of genetic markers for parentage analysis.

What Is Required to Obtain a Blood-Type Record?

All alpacas considered for registration with the Alpaca Registry, Inc., are required to have a blood-type record. Newly imported alpacas are admitted to the registry only after they pass a screening process for type and if they have a blood-type record. Crias can be registered only if the parents of record are listed in the registry.

A sample of 5 to 8 cc of blood in a lavender-stopper; evacuated blood tube is required from each alpaca for blood-typing. Information about the animal being tested and its parentage is required to be provided on a sample identification form that is submitted along with the blood sample and payment to the laboratory The laboratory assigns a case number to each incoming sample and logs the information in ifie computer database.

Upon completion of the tests and parentage analyses, the laboratory forwards a blood-type report to the client and electronically transmits a copy to the registry. The blood-type report contains the information supplied in the sample identification form and a statement concerning the status of parentage verification. The statement is in the form of a "parentage qualification" or a "parentage exclusion."

What Are Blood-Typing Tests?

Blood-typing tests are a battery of laboratory assays designed to detect genetic differences in blood groups and blood proteins. Table 1 shows the systems used for blood-typing and the number and names of variants within each system. As shown in the table, the current test battery evaluates sixteen "systems," each of which is a different blood grouping or protein assay. Each letter under "Names of Variants" in the table is a "type" (blood group or protein), a "genetic marker" that is transmitted in a simple fashion from parents to offspring.

Table 1. Description of 16 Blood-Typing Systems Used for Testing Alpacas and Llamas
System Name (Full name of system)	Type of Marker	Number of Variants	Name of Variants*
A	Blood group	2	A,B
D	Blood group	2	D,-
F	Blood group	2	F,-
A1B (alpha-1B-glycoprotein)	Plasma protein	5	A,C,D,F,S
CAT(catalase)	Red-cell enzyme	2	F,S
C3(complement component)	Plasma Protein	11	1,2,3,4,5,6,7,8,9,10,11
ESD(esterase-D)	Red-cell enzyme	3	F,S,L
GC(vitamin D binding protein)	Plasma Protein	6	B,E,H,K,L,N
GPI(glucose phosphate isomerase)	Red-cell enzyme	3	F,I,S
Pa1(post-albumin 1)	Plasma Protein	2	F,S
Pa2(post-albumin 2)	Plasma Protein	3	A,B,C
PGD(phosphogluconate dehydrogenase)	Red-Cell enzyme	2	F,S
Pi1(protein inhibitor 1)	Plasma Protein	2	A,B
Pr(pre-albumin)	Plasma Protein	2	F,S
Prt(pre-transferrin)	Plasma Protein	2	F,S
TF(transferrin)	Plasma Protein	13	C,F,G,I,K,L,O,Q,R,S,T,U,X,Z

*A hyphen (-) means "absence of D (or F)" or negative (-) for these factors.


A particular technical procedure and set of reference standards is used to detect and identify a specific blood group or protein type. Blood groups are found on the surfaces of red blood cells and are detected by serological tests that use antibodies specific to each known blood group factor Protein types are detected by "gel electrophoresis," a procedure that separates the different forms of a protein based on their dis-tinctive electric charges. The genetic variation of blood groups and proteins detected by these tests is a normal occurrence and is not associated with a medical problem or genetic defect.

How Is Parentage Verification Done?

Once the blood types of an alpaca are determined, they are compared to the types of the sire and dam on record. An alpaca or llama can have two of the same or two different variants in each system. This is because genes occur in double doses, one inherited from the father and the other from the mother Basic laws of genetics establish that offspring inherit one-half of their chromosomes, and thus genes and their products, from each parent. Therefore, all blood group factors and protein variants found in a cria's blood must also be present in one or both parents. If this is the case, the blood-type report states that the indicated parents qualify. Based on this report and fulfillment of its other requirements, the animal registry can issue a registration certificate for a cria.

Quite often, a cria has blood types that are not present in either the given sire or dam. This is a case of "genetic incompatibility" and reflects an error in the cria's parentage. In the vast majority of such cases, the listed sire is the incorrect parent. A "parentage exclusion1' or "sire exclusion" is reported and a registration certificate is withheld until the parentage problem is solved. When a parentage exclusion occurs, breeders need to inform the laboratory of other males that are possible sires of the cria. Once the qualifying sire is identified, the parentage is corrected and the cria becomes eligible for registration.

A Variation on the Theme: Paternity Analysis

Alpaca breeding schemes frequently involve multiple studs and sequential breeding of females with two or more males, and birthing dates are not always an accurate indicator of sire. Blood-typing tests have been extremely useful in identifying paternity. First, blood types of the cria, dam, and potential sires are compared to determine which blood types were transmitted by the dam. Because the remaining types must be transmitted by the sire of the cria, potential fathers are then checked to determine which male possesses all the paternal blood types present in the cria's sample. An example of a paternity analysis case is shown in Table 2.

Table 2. Paternity Analysis in Alpacas
Animals Compared	GC	TF	A1B
Cria	B	TX	DS
Dam	BN	IX	D
Male 1	N	F1	F
Male 2	B	CT	D
Male 3	B	IT	DS

The cria's blood types of paternal origin are underlined. Male 3 clearly has all three critical types and thus qualifies as the sire of this cria. The other two males either lack all types (Male 1) or lack one of the types (Male 2) and are thus excluded as possible sires.

Although blood-typing tests solve most paternity problems, in some cases they cannot identify a single qualifying male and exclude all other males as possible sires. The reason is that two or more males may have all critical types needed to qualify as possible sires. DNA finger-printing is then recommended to identify the cria’s sire.

How Effective Are Blood-Typing Tests?

The strength of blood-typing tests is in the detection of errors that would otherwise be perpetuated in pedigree records. The power of the present battery of tests to detect an incorrectly identified sire or dam when the other parent is recorded accurately is about 95 percent. When both parents are incorrect, which occurs when crias are switched, the efficacy rate approaches 98 percent.

Theoretically, additional blood-typing tests can be developed to increase the efficacy rate, but based on recent research and the extensive screening for new systems that has been done to date, improvements will not likely come from the development of new blood tests. Our efforts are currently being directed to the development of DNA typing tests that are highly informative and that have efficacy rates approaching 100 percent. Such tests are already in use for cattle, horses, dogs, cats, sheep, and goats.

DNA Fingerprinting

Advances in molecular biology have produced a large assortment of laboratory procedures that allow us to detect genetic variation at its source, the DNA molecules that contain all the genetic information that turns a single-cell, fertilized egg into an alpaca or a fruit fly. Many different types of DNA tests are in use today One such test, known as "DNA fingerprinting," is so named because it generates an array of DNA bands so distinctive for each individual that it can be likened to a fingerprint. No two individuals have the same pattern unless they are identical twins. Nevertheless, all DNA bands found in an offspring must be present in one or both parents. The principles of parentage testing are the same as those involved in blood-typing.

DNA fingerprinting has been used as an ancillary test in alpaca and llama paternity cases that blood-typing tests cannot fully resolve. This extremely powerful and informative test has solved all cases in which it has been applied since its implementation in 1991. It has been particularly useful in identifying the parentage of crias conceived prior to or during the quarantine confinement required for new imports from South America.

From the standpoint of efficacy in parentage testing, DNA fingerprinting meets most of the requirements to be the method of choice. It has, however, serious limitations. Unlike blood-typing records, DNA fingerprinting profiles are difficult to store in computer databases and thus do not generate permanent records. Each time parentage verification is performed for a cria, the sire and dam need to be tested along with the cria, even if they have already been tested with other crias. This makes parentage testing by DNA fingerprinting far more expensive than blood-typing. Also, the technical procedures for DNA fingerprinting are not easily automated and are labor intensive. For these and other reasons, the test is reserved for selected cases.

Future Developments in DNA Testing

The scientific community involved in parent-age testing of domestic animals has recognized one DNA typing procedure as the method of choice to replace blood-typing tests. This procedure, "microsatellite-DNA typing," has all the necessary requirements needed for this type of work. It assays genetic variation in a battery of systems, and each system contains multiple forms. DNA results can be easily stored in com-puter databases as permanent records and re-used for parentage verification. Test procedures are easily automated, and samples are processed in batches. An added benefit is the use of hair roots in testing, thereby eliminating the need to collect blood samples.

Clearly, similar tests need to be developed for alpacas and llamas, not for the sake of utilizing state-of-the-art technology, but rather for obtain-ing the benefits they can offer. For the practical purpose of maintaining studbook integrity, a panel of twelve to fifteen informative microsatellite-DNA systems can easily obtain efficacy rates for parentage verification that approach 100 percent. From the point of view of advancing our knowledge about the genetics of South American camelids, this development will provide the necessary tools for gene mapping and for addressing other issues, such as diagnosis of genetic diseases and traits of aesthetic and economic value.

Cecilia Penedo supervises the camelid and cattle blood and DNA typing unit of the Veterinary Genetics Laboratory, School of Veterinary Medecine, University of California, Davis. A geneticist by training, she has played a leading role in the development of genetic typing tests for camelids.