October 24, 2007

DNA Testing in Equines

By Michal Prochazka, MD, Founder & Director, Pet DNA Services of Az, Tempe, Arizona (www.petdnaservicesaz.com). PDSAz is offering tests in horses (colors, spotting, selected heritable disorders), and bird sexing.

Over the last couple of decades, advances in molecular genetics made it possible for DNA testing to become the method of choice for a variety of applications, ranging from human medicine and forensics to our hobbies involving pets and domestic animals. In this article, I have attempted to present a simple overview of the use of DNA tests in horses.

When talking about genetics and DNA testing, one cannot avoid the use of specialized vocabulary, which I will explain briefly, before discussing the applications of this technology in our equine friends.

Practically every cell in the body contains DNA, which is the “blueprint” code of instructions determining the appearance and function of an organism. The DNA is arranged in rod-like structures called chromosomes located in the nucleus. Chromosomal segments carrying discrete codes for specific traits and functions are called genes, and with the exception of the sex chromosomes all of the other chromosomes (and genes) in animals and humans are present in pairs, with each parent contributing one half of the material. In general, both copies of a gene are identical, but some carry discrete differences (mutations, variants), some of which can result in visible differences – for example a variation in the coat color. Such alternate variant forms of the same gene are called alleles, and an individual is referred to as a homozygote when both alleles in a pair are the same, whereas an individual carrying two different alleles of the same gene is called a heterozygote.

Depending on their biological properties, two alleles at any gene can interact in different ways. An allele is dominant when in a heterozygote carrier only this allele will show an effect. Furthermore, its effect is the same, regardless whether the individual has one or two copies of such allele (= is either a heterozygote or a homozygote for it). Conversely, an allele which is masked by its dominant counterpart is called recessive, and the effect can only be visible in a homozygote. A special situation is that of incomplete dominance, when one allele shows a certain effect in a heterozygote, but the expression is more pronounced when the carrier is a homozygote (a typical example in horses is the Cream Dilution).

The combination of alleles at one or more genes is referred to as genotype, while the visible manifestation of their effects is called phenotype. Due to interactions between genes and alleles, a phenotype usually does not provide full information about the underlying genotype.

The molecular differences between DNA alleles are tested with the use of sophisticated laboratory technology. The majority of routine laboratory DNA tests involve the Polymerase Chain Reaction (PCR), which allows for selective amplification of a DNA segment of interest in a test tube, thus making it accessible for further analysis to find out what alleles an individual may carry within this particular piece. As practically all tissues and cells from the same individual contain identical DNA, one can use a variety of sources for the testing. The material typically used in horses and other equines is pulled mane or tail hair, or blood. The accuracy of DNA testing is not dependent on age, and one can obtain reliable results already in newborn foals.

DNA testing in horses can be used for various purposes. One of the common applications, familiar to owners of registered horses is DNA “profiling”. In principle this technology is the same as DNA “fingerprinting” used in human forensics and paternity testing, which utilizes highly informative sets of genetic markers to differentiate with a great precision between individuals. Such tests are used very accurately to verify parentage, and identity of animals. DNA testing is now a mandatory requirement for registration by several US registries, with the main purpose of preserving the integrity of horse breeds.

Another important application is testing for the presence of mutations causing heritable disorders. Some examples include HYPP (Hyperkalemic Periodic Paralysis) and HERDA (Hereditary Regional Dermal Aplasia) which are carried in some lines of Quarter horses and related breeds, or SCID (Severe Combined Immunodeficiency) found in Arabians. These tests allow for an early and reliable identification of carriers, thus providing the option to either exclude such horses from breeding, or to avoid mating of two carriers to prevent production of affected offspring.

Further application of DNA testing became possible with the identification of some of the genes involved in coat color differences, or development of spotting patterns. It has been estimated that there are at least 15 separate genes contributing to different colors and white patterns in horses, and several of them have been already identified and reported in scientific literature.

To make the best use of DNA testing for color, one first needs to understand the concept of base colors. Like most animals, the horse has two pigments - black (eumelanin), and red (pheomelanin). The difference between the production of red alone, and red plus black together is controlled by alleles at the Extension gene. The dominant “E” allele determines the production of the black pigment in addition to red, whereas the recessive “e” allele (also called the Red Factor) permits the expression of red only. Consequently, e/e homozygotes have only red pigmented hair (chestnut, sorrel), while horses heterozygous or homozygous for the dominant allele (E/e or E/E) produce both red and black pigment. In the presence of the E allele, the body distribution of black pigmented hair is further controlled by alleles of the Agouti gene. A horse carrying the dominant “A” allele (genotypically either A/a or A/A) will have the black hair restricted to the points (mane, tail, legs) and will be phenotypically a bay. In contrast, the recessive allele “a” does not restrict the black hair, and a homozygote (a/a) will have this color distributed evenly and will be phenotypically black. Although not formally proven yet, there is considerable evidence for a third Agouti allele (called At) which appears to be responsible for the seal brown color in E/E or E/e horses. The current Agouti test (specific for “a”) cannot differentiate between the bay A allele and At, and we at PDSAz are conducting a study to further explore the genetic basis of this color.

In principle there are two base colors in horses: black, and red. However, for practical purposes we usually recognize three base colors (bay, black, and red), which are produced by the interactions of the known alleles at the Extension gene and Agouti (I am purposely leaving out the brown color, as it has not been scientifically proven yet). Depending on the alleles in the parents, two horses of the same color (= phenotypically identical) can differ in their genotypes at the involved genes: there are four different possible genotypes in a bay horse (E/E A/A; E/E A/a; E/e A/A; E/e A/a ), three in a chestnut (e/e A/A; e/e A/a; e/e a/a), an two possibilities in a black horse (E/E a/a; E/e a/a). Although the Agouti has no obvious effect in red-based (e/e) horses, it is important to keep in mind that such horses always carry two Agouti alleles, which can become relevant if they are crossed with a bay or black partner. Accurate knowledge of these genotypes is very helpful to predict which colors any given horse can (or cannot) produce, and at what frequency.

The variety of colors and patterns which occur in horses are produced by interactions of one of the base colors with additional modifying genes and alleles. Most of them are dominant and include several dilution genes (such as Cream, Dun, Silver, Champagne, Pearl), or genes determining the presence of white hair, which can be distributed diffusely throughout the coat (Grey, Roan), or as distinctive spots (Tobiano, Overo, Sabino, Appaloosa). As these colors and patterns are caused by alleles in separate genes, combinations of two or more of them can occur in the same horse.

Regarding white spotting, I believe that it is worth mentioning the most consequential mutation which is the Lethal White Overo (LWO) causing the Lethal White Foal Syndrome in LWO/LWO homozygotes. In carriers (= heterozygotes carrying one LWO allele), this mutation if frequently associated with the frame overo pattern, but the expression can vary widely, ranging from a loud overo to a solid colored individual. LWO mutation is found across various breeds - not just Paints and Pintos. If there is the slightest chance of a horse carrying LWO, it is a good practice to routinely test for LWO, to avoid unintentional mating of two carriers, which would have a 25% calculated risk of producing an affected white LWO/LWO homozygote.

While the primary focus of horse breeders is the improvement of conformation and performance, the diversity of coat colors and spotting patterns can also play an important role. From the described facts it is clear that DNA testing combined with the understanding of basic genetic rules can enable breeders to make informed decisions about which horses to mate to maximize the production of desired colors and patterns. Furthermore, quite often is DNA testing critical to determine the true color of a horse, thus facilitating a correct registration.
Although the focus of this article is genetic testing in horses, color and spot breeding is also becoming popular in donkeys. Our laboratory is currently involved in a study with the goal to develop DNA tests for donkeys in the foreseeable future.
(- A few questions to clarify things:)

1. At the moment, can DNA testing determine a donkey breed? Or just parentage?
DNA testing in donkeys or horses can determine parentage, but not breed.

2. Can DNA testing determine a horse breed, or again, just parentage?
Please see my answer to Question #1.

3. A bit off topic, can DNA testing determine a dog's breed?
Yes. There is a company in California which can test for dog breeds (http://www.mmigenomics.com/products2a.html)

4. What does the word "Agouti" mean?
Agouti is a rodent living in South America, which has a typical hair color pattern that is also found in many other animals (mice, rats, rabbits etc). The hair has dark and light banding, which is controlled by a gene that has been named Agouti. This gene modifies where the black pigment shows up. In the mice, for example, it gets switched on/off during hair growth, and therefore the so called grey (wild-type) mice have a part of the hair that contains the yellow/brown pigment (Agouti is on), which alternates with black (Agouti off).
This gene is found in all mammals. There are two variants of this same gene in horses (A, and a) which are responsible for the difference between bay (black hair only in the mane, tail, and legs), and solid black color.
BTW - It is my understanding that some of the gene names we are using in pets and life stock were taken originally after the gene names in laboratory mice. These include for example Agouti, or Extension (black and red factor).

5. A few of our reader's have had DNA testing done to determine the parentage of foals. How accurate is the DNA testing?
Parentage testing is very accurate in horses (although there could be differences between different labs). Because donkeys' DNA is very similar to horse, the horse parentage tests will also work in donkeys, but I am not aware of any info about how accurate it may be.

6. Have you found that the Lethal White gene in horses also occurs in donkeys?
We have not looked specifically at that yet.

7. Eventually, will we be able to color test our donkeys to ensure the correct color on registration?
This is the main goal of our study, to develop tests to correctly define colors in donkeys, and also predict what colors can individual donkeys produce in their offspring. We are conducting the study in collaboration with Leah Patton from ADMS.

8. What are some of the differences so far that you've encountered between horse DNA for color and donkey DNA for color?
Our study started just recently, and we do not have enough data yet to make any reliable statements about this.

9. I have been told in the past that all donkeys have a cross and dun stripe, although it doesn't show on all donkeys, do you know if that is true? Or will that be one of the questions your researching now?
Leah Patton and I have been talking about this. Right now, we are focusing on genes which determine the base colors (brown, black, and sorrel/red), Depending on how the study progresses and if we can get some funding, we would consider in the future looking into the genetics of donkey markings (cross vs no cross; light points vs no light points).

Michal Prochazka, MD
Founder & Director
Pet DNA Services of Az
(Equine testing; Avian sexing)

If anyone wants to help with color research on donkeys:
ADMS does not provide any funding for this project. Any donations should say that it's for the donkey color research, and should be made to Pet DNA Services of Az, and mailed to PO Box 7809, Chandler, AZ 85246.

Posted by Tanya Tourjee at 08:55 PM | Comments (0)

Anatomical Differences in Donkeys

We all know the differences between donkeys and horses by sight. But, the differences that are easily seen, like ears, are not the only differences to be aware of.


Donkeys have a short, upright mane, usually no forelock and a broomstick kind of tail. Donkeys are not just a “poor man’s” horse. A donkey reacts totally differently than a horse, has a different outlook on life, and different nutritional requirements. For those of us who are familiar with donkeys, we understand that they are a smart, affectionate, unique equine, well worthy of their own respect and appreciation. Obviously, some training methods commonly used for horses won’t work very well for donkeys. That will be another article. For now, let’s just cover some of the major anatomical differences we have in our donkeys.

One of the biggest concerns with donkeys is castration. A normal castration of a horse, the vet clamps the blood vessels until the bleeding stops. Then, he allows the incision site to stay open and drain. Unfortunately, donkeys seem to bleed more than horses, and some can be heavy bleeders. It is recommended that the vet use ligation (this is when the vet places a stitch in the blood vessel to help stop bleeding) in donkeys when castrating. If your vet isn’t aware of the differences in donkeys, a frank discussion with him/her before scheduling the surgery is in order. There are some places that you can find reference materials on the internet that you can print copies of and give to your vet (a few reference sites will be listed at the end of this article). Occasionally, a vet will not take what your saying to heart, our advice to this is to find a vet who will. There have been way too many close calls to leave it up to chance, and a few deaths have been reported.

Another common difference is gestation time. Gestation for horses is generally about 11 months. For donkeys, gestation is usually about 12 months, but anything from 11 ½ to 13 months is normal. Live, healthy twins are also more common in donkeys than horses, by about 100 times. Care still must be taken, and many breeders will have jennets known to have twins ultra sounded early in their pregnancy, and one of the twins pinched off, to give mom and the other twin a better chance of surviving.

Donkeys also have a longer life span than horses, and can live easily in excess of 45 years. Horses average lifespan is 25-30 years.

To find the jugular vein in a donkey is a bit more difficult than in a horse. Blood used for tests such as coggins, or drugs that need to be administered intravenously, such as tranquilizers, are usually drawn or given through the jugular vein. The muscle that covers this area in donkeys is much thicker, and usually hides the middle third of the jugular vein. The jugular farrow in donkeys is obscured by this muscling in comparison to horses. Most vets will use the upper one third or lower one third of the jugular farrow in a donkey.

The nasolacrimal duct is found in a different location compared to horses. In a donkey, the duct is found on the flare of the nostril. In a horse, this duct is found on the floor of the nostril.

Chromosome differences are usually pretty well known. A donkey has 62 chromosomes and horses have 64. For this reason, almost all mules are sterile, since they end up with 63 chromosomes.

In donkeys, the pelvis tips down more vertically than in horses. This is especially helpful to know when a vet is conducting a pelvic exam, or if dystocia occurs.

Also, a jennets cervix is longer and narrower than that of a mare. There is also a large protrusion from the cervix into the vagina. And, dorsal and ventral folds in the vaginal passage that can hinder passage to the cervix.

Male donkeys, or jacks, also have some notable differences. Some jacks have teats on their sheath. Jacks are also notably larger in reproductive areas than horses.

Donkeys do not have chestnuts on their hind legs. They do have ergots on their front legs, which are mainly just a flat pad.

Donkeys hooves are also smaller than horses, and tend to be boxier and more upright.

One primary difference between donkeys and horses is their larynx. Vocal folds and laryngeal saccules were found to be different then horses and account for the braying sound that a donkey makes. One other thing to be aware of when a vet is tubing a donkey, the nasal passage is very narrow, and there is also a recess between the openings of the guttural pouches. For this reason, when passing a tube, a smaller diameter tube is better to be used. Donkeys are known for excessive bleeding during this procedure, and a smaller tube may help the bleeding problem.

There are many other differences between horses and donkeys, such as vertebrae, that are too lengthy and confusing to go into for this article. Just be aware that if the vet needs to give an anesthesia shot in the spine for any reason, it differs from horses and the vet should be aware of this fact.
Of course, there are many other differences besides the physical ones mentioned here. Donkeys are known to be stoic, what many call stubborn is just self preservation, and donkey behavior must be understood to manage them effectively. Subtle differences in behavior or attitude may indicate severe problems.

It’s good to know your animals, it helps with the vet, the farrier, and anyone who may come into contact with them. If you treat a donkey like a horse, you will be disappointed with the results.

Suggested reading:
The Definitive Donkey- A Textbook on the Modern Ass. Hutchins, Betsy and Paul. Hee Haw Book Service, 1999.
The Professional Handbook of the Donkey. Svendsen, Elisabeth D.. Whittet Books, 1997.

Posted by Tanya Tourjee at 08:48 PM | Comments (0)

On the Donkey page: Colors in the Donkey

By Leah Patton
Office Manager, ADMS

Colors in the donkey range from the gray shades of gray-dun to brown, a rare bay (though not as red-toned as in horses) , black, light-faced roan (both red and gray), variants of sorrel (Registry term - RED), the blue-eyed Ivory (also called cream or white-phase), Frosted/spotted White, and a unique Spotted pattern. True horse pinto, horse aging gray, horse appaloosa, palomino and buckskin do not occur in the donkey.
The more unusual colors are the Dappled Roan, where the face and legs are light and the body is marked with "reverse" dapples (dark spots on a light background, as opposed to the horse dapple where the dapples themselves are light on dark), frosted gray (with light faces and legs and some white hairs in the coat) the pink-skinned, blue-eyed Ivory white, and the frosted spotted white. The frosted spotted is an apparent combination of a graying or roan with the spotted pattern, and can throw either more FSW, spotted, or frosty roan colts. The animals are best defined as a spotted animal where the skin is spotted but the color does not necessarily show through on the coat (it has roaned or "grayed"; out) . Frosted spotted white (FSW) can be identified from Ivory white by checking the skin around the eyes and muzzle. Ivory (creams) will have blue eyes and true pink skin, while FSW will have dark eyes, dark "eyeliner" and dark spotting on the skin.

Another unusual variant of the spotting line is the "tyger spot" pattern. These donkeys vary from the typical large spots over the ears, eyes, and topline. The body will be covered with small round spots resembling the appaloosa type.

Registry color terms

Dk Brown
Very Dark Brown
Black w/Cross
White * (FSW or BEW)
+ Spotted (ie, Gray and white spotted, Brown and white Spotted, Red/Gray and white spotted).

Note - no Mammoth donkey are ever recorded as having been Blue Eyed White (Ivory).

Russet is a descriptive term only, until such time that there is a genetic determination of the workings of russet. They are listed as Red (Frosty roan, black trim, russet pattern) or Brown (red tones in coat, frosty roan, black trim, russet pattern).

Posted by Tanya Tourjee at 08:35 PM | Comments (0)