How Were Mules Created and Why Are They Sterile?

Mules are the offspring of a male donkey (called a jack) and a female horse (called a mare), and they are sterile because their two parent species carry different numbers of chromosomes that cannot pair up properly during reproduction. Horses have 64 chromosomes, donkeys have 62, and the resulting mule ends up with 63, an odd number that disrupts the cell division needed to produce eggs or sperm. The story behind this hybrid is more layered than that summary suggests, touching on millions of years of evolutionary divergence, rare exceptions that puzzle geneticists, and a set of traits that made the mule one of the most valued working animals in human history.

How the Cross Works

A mule is produced when a male donkey mates with a female horse. The reverse cross, a male horse mating with a female donkey, produces a different hybrid called a hinny. Both end up with 63 chromosomes, but they are not identical animals. The mother’s uterine environment shapes fetal development in ways that go beyond genetics. A horse mare provides a larger womb and more resources during pregnancy, so mules tend to be bigger and more robust than hinnies. Hinnies are also harder to produce because conception rates are lower when a stallion breeds a jenny (female donkey), partly because of differences in how the two species’ placentas interact with the uterine lining.

Equids as a group are remarkably flexible when it comes to interbreeding. Mares can even carry embryos from completely different equid species to term, including Przewalski’s horse and Grant’s zebra, through embryo transfer. This ability seems to depend on specialized invasive placental cells that help the foreign fetus attach and survive inside the uterus despite being genetically foreign to the mother.¹ The immunological tolerance involved is unusual among mammals, and it explains why the horse-donkey cross succeeds so readily in the first place.

Millions of Years of Divergence

Horses and donkeys look similar enough that you might expect them to be close relatives, but they have been on separate evolutionary paths for a long time. One genomic study placed the split at roughly 4 million years ago, while another analysis using thousands of single-copy genes estimated the divergence at somewhere between about 8 and 15 million years ago.²³ The range reflects different methods and molecular clocks, but the takeaway is clear: these species have been evolving independently for millions of years. During that time, chromosomes were rearranged, fused, and restructured differently in each lineage. The donkey ended up with 62 chromosomes, the horse with 64. The number difference alone does not fully explain sterility, though. It is the structural differences between the chromosomes, not just the count, that create the real problem during reproduction.

Why 63 Chromosomes Cause Sterility

When any animal produces eggs or sperm, its cells go through a special type of division called meiosis, during which chromosomes line up in matching pairs. Each pair then splits apart so the resulting egg or sperm cell gets one chromosome from each pair. In a horse, 64 chromosomes form 32 tidy pairs. In a donkey, 62 chromosomes form 31 pairs. But in a mule, with 63 chromosomes inherited from two different species, there is always one chromosome left without a partner. Worse, many of the chromosomes that do try to pair up are structurally mismatched because the horse and donkey versions differ in shape. The donkey’s set has more metacentric chromosomes (those with the attachment point near the middle), while the horse’s set has more with the attachment point off to one side.⁴

These structural mismatches are thought to cause what geneticists call synaptic failure: the chromosomes simply cannot lock together properly during meiosis. Without proper pairing, the cell’s quality-control machinery halts the process. In male mules and hinnies, sperm production stalls almost entirely at an early stage of meiosis called pachytene. Detailed studies of mule and hinny testes found that the vast majority of germ cells break down at this stage, though a small number of mature sperm cells were recovered from at least one hinny.⁵ Those rare sperm are generally considered nonfunctional, and no verified case of a male mule fathering offspring has ever been documented.

Why Males Are Always Sterile but Females Occasionally Are Not

There is a longstanding pattern across the animal kingdom: when two species hybridize, the sex that carries two different sex chromosomes (XY in mammals) tends to be more severely affected. This observation, known as Haldane’s rule, predicts that male hybrids will be sterile or inviable more often than females. The mule fits this pattern well. Male mules are invariably sterile, and there is evidence that the high mutation rate of genes on the Y chromosome contributes to this vulnerability.⁶

Female mules, by contrast, are sterile in the vast majority of cases, but not always. Verified cases of fertile female mules exist, and they are among the most fascinating anomalies in genetics. In China, where mules have been bred extensively for farm work, both a fertile female mule and a fertile female hinny were confirmed through chromosomal analysis. Each mated with a donkey and produced a filly foal, and the offspring had unique hybrid karyotypes distinct from either parent type.⁷ In another documented case, a female mule produced a male foal sired by a jack donkey, and chromosomal testing confirmed the foal was karyotypically a mule.⁸

How do these rare females manage to produce viable eggs when the chromosomal math should prevent it? Research on the gene Prdm9, which plays a key role in directing where chromosomes recombine during meiosis, found that neither the odd chromosome number nor differences in Prdm9 structure fully explain mule sterility. In one studied fertile mule and her offspring, both parents had the same number of functional domains in their Prdm9 protein, even though the sequences were chimeric mixtures of horse and donkey versions.⁹ The implication is that the causes of mule sterility are more complex than any single gene or simple chromosome-count problem. Some female mules may simply get lucky during meiosis, with their mismatched chromosomes sorting out well enough in a given egg cell to produce a viable embryo. But this remains poorly understood, and fertile female mules are rare enough that systematic study is difficult.

Hybrid Vigor and Why People Kept Breeding Mules Anyway

If mules cannot reproduce, why did humans bother creating them for thousands of years? The answer is hybrid vigor, the phenomenon where a cross between two genetically distinct parents produces offspring that outperform both in certain traits. Mules inherited the horse’s size and speed along with the donkey’s endurance, sure-footedness, and resistance to disease. The combination made them ideal draft and pack animals, especially in rough terrain.

This hybrid advantage shows up at the cellular level. Research comparing fibroblasts (basic connective-tissue cells) from mules, horses, and donkeys found measurable differences in cell proliferation, resistance to cell death, and energy metabolism, with the mule cells showing characteristics distinct from either parent species.¹⁰ Even during embryonic development, mule conceptuses appear to develop faster than horse embryos at comparable stages, with earlier formation of structures like limb buds, brain vesicles, and heart compartments.¹¹

Transcriptome analysis has pointed to specific molecular pathways that might underlie the mule’s famous stamina. Genes involved in muscle contraction and neuronal signaling were found to be expressed differently in mules compared to their parent species, with particular genes potentially contributing to muscular endurance and cognitive function.¹² This last point connects to something anyone who has worked with mules knows intuitively: they are smart, often described as more cautious and deliberate than horses.

Are Mules Actually Smarter Than Horses?

“Stubborn as a mule” is one of the most persistent clichés in English, but mule handlers often argue the reputation is undeserved. What looks like stubbornness may actually be a stronger sense of self-preservation. A horse in a panic might run blindly into danger; a mule is more likely to freeze and assess the situation. This behavioral difference has long been attributed to the donkey side of the family tree, and there is some experimental support for the idea.

In a spatial cognition test where animals had to find a detour path around a barrier, the barrier was moved after the animals learned the first route. Horses (and dogs, tested for comparison) committed the classic error of returning to the original, now-blocked path, performing below chance levels. Mules and donkeys, on the other hand, did not fall into this trap as readily. By the second trial at the new location, donkeys were already successfully choosing the correct direction at above-chance levels.¹³ All equid groups eventually figured it out, but the pattern was consistent with mules and donkeys being less prone to perseverative errors, the tendency to repeat a previously rewarded behavior even after it stops working. Whether that counts as “smarter” depends on how you define intelligence, but it does support the folk wisdom that mules are more adaptable problem-solvers than horses in certain situations.

Genomic Imprinting and the Mule-Hinny Difference

Mules and hinnies share the same total genetic material, 50 percent horse and 50 percent donkey, but they are not identical. One reason is genomic imprinting, a process in which certain genes are expressed differently depending on whether they were inherited from the mother or the father. In the placenta especially, this matters. RNA sequencing of trophoblast tissue (the cells that form the placental interface with the mother) from mule and hinny conceptuses identified a core group of 15 imprinted genes, of which 10 were expressed from the father’s copy. An additional 78 candidate imprinted genes also showed a paternal bias.¹⁴

Since the father’s species differs between mules (donkey father) and hinnies (horse father), these imprinted genes are expressed differently in the two hybrids, leading to real differences in placental anatomy and physiology.¹⁵ The placenta is the first organ to develop and the one that determines how much nutrition the fetus receives, so imprinting differences there have cascading effects on the animal’s size, growth rate, and overall vigor. This is part of why mules (with a horse mother’s large uterus and a donkey father’s imprinting pattern) tend to be bigger and more robust than hinnies.

Mules in Archaeology and Identification Challenges

Mule breeding has an ancient pedigree. Historical records from Mesopotamia, classical Greece, and Rome describe mules as essential working animals. But identifying mule remains in the archaeological record is surprisingly difficult. Mule bones fall on a size spectrum between horse and donkey, and distinguishing them by shape alone is unreliable. A study of equid remains from Roman-era Switzerland put this problem into sharp relief: of 12 animals initially identified as mules based on bone measurements, shotgun DNA sequencing revealed that 11 were actually horses. Meanwhile, one animal classified as a horse turned out to be a female mule.¹⁶ The finding is a caution against relying solely on physical measurements and suggests that historical estimates of mule populations in ancient societies could be significantly off.

Veterinary Care Is Not the Same as for Horses

One practical consequence of the mule’s hybrid biology is that veterinary treatment cannot simply be copied from horse protocols. Donkeys and mules metabolize many drugs differently than horses, generally at a faster rate, which means doses that work well in a horse may wear off too quickly or prove insufficient in a mule.¹⁷ A study comparing the sedative xylazine in mules versus Haflinger horses demonstrated this directly. Both groups received the same dose per kilogram, but the mules showed good sedation for only the first 10 minutes and insufficient sedation by 30 minutes, while the horses remained well-sedated for about twice as long.¹⁸

This difference is not just an inconvenience. A mule undergoing dental work or surgery that receives a horse-standard dose of sedation could wake up mid-procedure. Mules also have a higher cellular water content than horses, which affects how drugs distribute through the body. Because species-specific pharmacological studies are still sparse for mules, veterinarians treating them often have to rely on close monitoring and clinical judgment rather than published dose charts. It is one of the less glamorous consequences of being a hybrid: the medical knowledge base for your species barely exists, because you are not really a species at all.

The Immunology of Mule Pregnancy

Carrying a mule fetus is immunologically unusual for the mare. The fetus is half donkey, meaning it carries proteins from a different species that the mare’s immune system could potentially attack. Early in pregnancy, specialized cells from the mule embryo’s placenta invade the mare’s uterine lining and form structures called endometrial cups, which secrete hormones critical for maintaining the pregnancy. Research on mule pregnancies showed that the rate of hormone production by these cups depends on the fetus’s genotype rather than the mother’s. Interestingly, the immune cell accumulation around the cups does not scale with how genetically different the fetus is from the mother, suggesting the system has evolved tolerance mechanisms that are not strictly proportional to foreign-ness.¹⁹

This tolerance is part of what makes equids such prolific hybridizers. Most mammalian species would reject a fetus that genetically foreign. The equine placenta’s invasive trophoblast cells seem to play an active role in suppressing or redirecting the maternal immune response, creating a privileged environment for the developing hybrid. Understanding how this works has implications beyond mule breeding: it offers a natural model for studying immune tolerance during pregnancy more broadly, including in human medicine where maternal immune rejection of the fetus is a factor in some pregnancy complications.