FIGURE 1. Day 34 equine embryo. Note the buds of the forelegs. Photo By Professor W. R. Allen, University of Cambridge.
In routine stud veterinary practice, the mare’s pregnancy is confirmed by ultrasound examinations at varying times from 13 days after ovulation. In checks at 40 days some parts of the skeleton are discernible and, by 65 days, the shape and structure of the limbs are clear.
These observations confirm that the limbs develop very early in equine pregnancies. They can be observed as small buds or outgrowths on the embryo by about 30 days of gestation, the forelimbs appearing a few days before the hind limbs (Figure 1). Precursor cells within the buds multiply and change to form the components of the limb, which include the cartilage model, on which ossification builds the bones of the limb, the muscles, their tendons and the ligaments. The shapes of the limbs, joints and hooves are readily apparent in the embryo as early as 50 days even though they contain no bone. The period between 30 and 50 days of pregnancy is critical for correct development of the embryo.
Formation Of Bones And Joints
The long bones of the limbs are derived from tubes of cartilage formed by the precursor connective tissue cells. The outer cuff is called the periosteum (Figure 2) and this layer of cells produces bone on its inner surface, which gives it shape and rigidity. Repeated absorption of this inner layer of bone coupled with further laying down by the gradually expanding periosteum increase the width of the bone shaft. In the center of each model is a primary center of ossification where cartilage is replaced by bone. At each end of the bone the cartilage develops secondary centers of ossification or epiphyses.
Between the main shaft and the epiphysis is the area called the growth plate where the bone increases its length. This whole process is called endochondral ossification and development of the individual bone’s shape is called modeling. The gross structure of a tube is mechanically the soundest that can withstand the forces of weight bearing and propulsion. The shape of the bony tube, known as the cartilage model, is determined genetically but is also molded by forces on it (movement and after birth, weight-bearing) and around it (muscle masses). Smaller bones, such as the cuboidal bones within knees or hocks, develop from cartilage models with single centers of ossification and no growth plates. The flat or plate-like bones, such as those making up the skull, do not develop from cartilage but from a matrix of connective tissue fibers (intramembranous ossification). Although their developments differ, the bone formed by endochondral and intramembranous ossification ultimately has a similar structure, the shape and density of which is varied by the forces placed upon it.
Joints also develop very early in pregnancy. As a result of limb movement they appear as spaces between the cartilaginous epiphyses and a joint capsule encloses them. Blood vessels invade the epiphyses on each side of the joint in order to ensure adequate oxygen and nutrition for the cartilage and ultimately the bone cells. Cartilage cells enlarge and bone is laid down around them but the cartilage cells lining the joint surface flatten and become the smooth white articular surface on the firm bony plate.
FIGURE 2. Diagram of long bone development in the equine fetus. (A) Cartilage model appears after 30 days; (B) Outer cuff or periosteum appears on the diaphysis by 50 days; (C) Enlargement of the diaphysis of the long bone by endochondral ossification during mid pregnancy; (D) Ossification starts in the epiphyses at each end of the bone from 250 days onward; (E) The long bone with the clear shape of the adult bone near birth. Ossified bone is shown in green.
Development Of Embryonic Limbs
The main ossification center or diaphysis of the long bones of the upper limbs, such as the humerus and radius in the forelimb and the femur and tibia in the hind limb is the earliest to contain significant amounts of calcium and be recognizable on X-ray films. The scapula and ilium or front part of the pelvis is also apparent. Next, the cannons (metacarpals and metatarsals) appear by 65 days (Figures 3 A, B). The pastern and pedal bones (phalanges) then appear as rudimentary marks, identifiable more by their position in relation to the longer bones than by their shape (Figures 4 A, B).
FIGURE 3. (A) Day 65 equine fetus. Note the distinct shapes of the limbs and hooves. (B) Radiograph of a Day 65 fetus. Note the white shadows of the bones in the skull, spine, pelvis and upper limbs indicating that ossification has started.
The epiphyses or centers of ossification at the ends of each bone are slower to ossify and only do so later in gestation (250 days onward). During the last third of gestation the fetus will double in size. Some growth plates, such as that in the distal first phalanx (long pastern bone) close and ossify during the last month. The sesamoid bones at the back of the fetlocks are also very late to ossify.
FIGURE 4. (A) Day 80 equine fetus. (B) Radiograph of Day 80 fetus. Note the appearance of bones in the lower limb or digits.
Post-Natal Bone Development
FIGURE 5. Newborn foals should be able to stand within 2 hours of birth.
Compared with other species such as people, dogs or pigs, the newborn foal has a highly developed skeleton that allows it to stand and run beside its dam within a few hours (Figure 5). The long bones of the lower limb, such as the cannons, are little shorter than they are destined to be when the animal is mature. The cannon bones only grow 20 percent more in length after birth, compared with a doubling in length of the humerus or femur in the upper limb.
The cannons have only one growth plate still apparent at birth and it is at their lower end, just above the fetlock joint. This growth plate is one of the earliest to close and it does so by 6 to 9 months of age. The long and short pastern bones also have only one growth plate, at their upper end and which closes at a similar age. In general, the growth plates lower in the limb close earlier in the animal’s life than those in the upper limb (Figures 6 A, B). Those at both ends of the forearm and gaskin (radius and tibia) close by 24–30 months old (Figures 7 A, B) and of the femur and humerus later, but usually by 36 months.
FIGURE 6. (A) Radiographs of the fetlock joint of a 10-day-old foal. Note the very open growth plates above and below the fetlock joint (arrowed) and the immaturity and incomplete ossification of the sesamoid bones. (B) Radiographs of the fetlock joint of a 7-month-old foal. Note that the growth plate in the upper long pastern bone is closing earlier than that in the lower cannon and that in the upper middle phalanx bone has closed completely (arrowed).
FIGURE 7. (A) Radiograph of the carpus and distal radial epiphysis of a yearling (arrowed). (B) Radiograph of the carpus and closed distal radial growth plate of a 3-year-old.
The growth plates at each end of a long bone do not close at the same time. Thus the lower or distal humerus closes before the upper whereas the upper or proximal radius closes before the distal radius (the growth plate just above the knee).
These timings can be varied by external factors. Physical factors include excess pressure on a growth plate as a result of overweight or longstanding lameness. The administration of hormones such as anabolic steroids, which by their nature have testosterone or male hormone effects, cause the growth plates to close earlier. Conversely, gelding will delay closure hence the tendency for some Thoroughbreds to grow tall, lean and leggy. After closure of the growth plates the bone can grow no longer but, in response to exercise, it can change its shape and strength.
Muscle And Tendon Development
FIGURE 8. A 1-month-old foal with a fracture to the distal sesamoid (arrowed).
The precursor cells in the limb buds of the embryo also start to organize and change into muscle and tendon early in pregnancy. By 50 days they are probably present and contain nerve fibers which have invaded the limb bud and are essential to muscle development and function. Fetal movement is quite marked and easily seen during ultrasound examinations at 60 days. Incidentally, fetal movement starts much later in human pregnancy, at almost mid term. Fetal muscle fibers grow in length by the addition of new muscle cells and in girth by an increase in myofibrils within the cells.
Tendons and ligaments develop from the same versatile precursor cells that produced muscle and bone in the limb buds. They turn into cells called fibroblasts and produce specialized fibers that form the connection between muscle and bone (tendon) or stabilize the joints (ligament). Their fibers develop great tensile strength and have some elasticity. The basic component of the fibers is collagen, which is laid in a crimp or zigzag pattern that allows them some elasticity and storage of energy when stretched. Tendon fibers are in parallel lines whereas those of ligaments may be crossed or in spirals according to the job required of them in holding the joint in stable alignment. Both attach to bone by a gradual transition through fibrocartilage, to mineralized cartilage to bone — a very strong and clever arrangement.
Some ligaments, such as the suspensory ligament (anatomists call it the interosseous muscle), contain significant numbers of muscle fibers but these disappear gradually during the later part of the first year of life.
It is surprising that these structures perform their function so efficiently so soon after birth when they have only been used in the weightless environment in utero. It is interesting that young foals that gallop to exhaustion sustain fractures of the sesamoid bones at the back of the fetlock joints but very rarely strain their flexor tendons or suspensory ligament (Figure 8). When older, such fractures are rare and the foal or yearling may then strain the suspensory ligament or its attachment to the sesamoid bone (a sesamoid is a bone within a tendon or ligament). This shows that flexor tendons and ligaments may have more tensile strength than bone in the newborn foal.