Recent research on the br/br rabbit is summarized. In all homozygous individuals, limb amputations result from in utero haemorrhages which develop in the extremities and lead to necrosis. An inner-ear abnormality is observed in about 25% of the cases. By studying a large number of gestations and birth, we observed that homozygous crossings result in about 100% amputated animals while crossings between heterozygous and homozygous rabbits result in about 50% of the young being abnormal.

Observations of megakaryoblastic cells in 15- and 17-day foetal liver show a predominance of immature cells in 15-day br/br foetuses. This abnormality disappears on day 17. The possible role of megakaryoblastic cells in br/br rabbit amputation is discussed.

Hereditary brachydactyly was described in rabbits by Greene & Saxton (1939) and Jost, Roffi & Courtat (1969). This abnormality is characterized by limb amputations of variable severity occuring during foetal life. These result from haemorrhages which develop in the foetal extremities between the sixteenth and twenty-first days and lead to necrosis (Greene & Saxton, 1939; Inman, 1941; Jost et al. 1969). This necrosis occurs generally in the extremities of the phalanges, although the whole upper or hind limb may be amputated. At birth, cicatrization is total and the limbs are stump-like (Fig. 1).

Fig. 1

Congenital limb amputations in a new-born br/br rabbit.

Fig. 1

Congenital limb amputations in a new-born br/br rabbit.

Several treatments result in prevention of foetal limb amputations (hyperoxia and phenylhydrazine, Petter, Bourbon, Maltier & Jost, 1971, vitaminic treatments: folic acid, vitamin B12, Petter et al. 1977). These experiments confirmed the role played by blood defects in the induction of foetal abnormalities in the br/br rabbit.

The br/br rabbits were bred in our laboratory. In order to maintain fertility, frequent outcrosses with ‘Géants Normands’ were necessary. The latter were also used as controls. The animals were bred in individual cages and fed and given water ad libitum. Females were introduced into the male’s cage, and the day of mating is considered day 0 of pregnancy since ovulation is induced by mating in the rabbit. Two stages of gestation were chosen: day 15 and day 17 because they generally correspond to the appearance of limb haemorrhages in the br/br rabbit.

The experiments were performed on: fourteen 15-day foetuses from three control females ‘Géant Normand’ and twelve 15-day foetuses from three br/br rabbit females. Six 17-day control foetuses from two females and seven 17-day br/br foetuses from two females; in both cases, br/br females were mated with br/br males.

Pregnant rabbits were anaesthetized with i.v. pentobarbital (24 mg/kg) and laparatomized. The uterus was opened at each foetal site and the foetuses were removed with their adnexa. Whole foetuses were fixed in Bouin’s solution. The liver was then separated, dehydrated with ethanol, placed in isoamylacetate, embedded in paraffin, and cut in totality. Serial liver sections (5 μm) were stained with Gabe and Martoja’s trichrome.

Sections of hepatic tissue were observed under a projection microscope. As the diameter of megakaryoblastic cells is about 20–25 μm, we studied one out of five serial sections. All the cells of the megakaryoblastic line were observed and counted in each section studied. Quantitative data were based on a 100-cell count. These cells are visible among the hepatocytes and in the erythropoietic tissue. They can be easily classified into two groups: (a) Mono and binucleated cells, which are the youngest forms of megakaryoblastic cells. Their cytoplasm is coloured yellow-green and their nucleus is diploid or tetrapioid, (b) Giant cells which represent the most mature forms of megakaryoblasts and the promegakaryocytes. Their nucleus is polynucleated.

I Recent data concerning the br/br rabbit stock

Considering only the visible lesions in the limb extremities on days 19 and 20 and at birth, we have obtained the following data. By mating homozygous rabbits, we obtained 201 foetuses from 36 females and 199 presented visible limb haemorrhages; 290 young were born from 73 females, only 2 had anomalies. By mating heterozygous females with homozygous males, we obtained 622 foetuses from 110 females, 314 were abnormal and 308 normal; 804 young were born from 198 females, 338 were abnormal and 466 were normal. The relatively low number of abnormal young was probably due to the high mortality rate of the most affected foetuses at the end of gestation.

Another abnormality, occuring spontaneously in about 25% of the subjects was also observed: an internal ear syndrome, generally unilateral, evolving progressively only in homozygous individuals (Fig. 2). We have followed the evolution of this syndrome throughout the life of 12 rabbits. We have observed a lack of sensitivity of the external ear, often accompanied by ataxia. Preliminary results from histological sections of the brain indicate a lesion at the level of the trigeminic nerve nucleus.

Fig. 2

Abnormal posture in a br/br adult rabbit, probably linked with an inner-ear abnormality.

Fig. 2

Abnormal posture in a br/br adult rabbit, probably linked with an inner-ear abnormality.

II Study of the megakaryoblastic cell line in the br/br foetus

In the 15-day br/br foetuses, the mono or binucleated cells are more numerous than those of the control group. These young cells are often situated close to the vessel lumen or even in the vessels (Fig. 3). However, in 17-day br/br foetuses the frequencies of the different megakaryoblastic cells was found to be the same as in controls. In both cases, the most mature poly-nucleated cells seem to be predominant, and the observed values are similar to those in the 15-day control foetuses.

Fig. 3

Megakaryoblastic cells in a 15-day foetal rabbit liver. A, Megakaryoblastic cell in a control (note the abundance of erythropoietic cells): L.c. Liver cell; E.c., erythropoietic cell; P.m., polynucleated megakaryoblastic cell. B, Predominance of immature cells in the br/br rabbit. (Note the scarcity of erythropoietic cells): L.c., liver cell; E.c., erythropoietic cell, i.b.m., immature binucleated megakaryoblastic cells.

Fig. 3

Megakaryoblastic cells in a 15-day foetal rabbit liver. A, Megakaryoblastic cell in a control (note the abundance of erythropoietic cells): L.c. Liver cell; E.c., erythropoietic cell; P.m., polynucleated megakaryoblastic cell. B, Predominance of immature cells in the br/br rabbit. (Note the scarcity of erythropoietic cells): L.c., liver cell; E.c., erythropoietic cell, i.b.m., immature binucleated megakaryoblastic cells.

The observed abnormalities possibly result in blood platelet defects, and for this reason, we have counted the blood platelets on day 16. Five foetuses from normal ‘New Zealand’ rabbits were used as controls and compared with five foetuses from homozygous br/br rabbits. After laparotomy and uterine incision, the foetal splanchnopleura was carefully dried and a vitelline vein was punctured. The blood was collected with a Potain’s pipette. The dilution fluid consists of 1/10 procaine chlorhydrate and 9/10 NaCl (0·15% solution). An hematimeter was used for the platelet count. The results are given in Table 2.

Table 1

Megakaryoblastic cell count in the liver of control and br/br rabbit foetuses on days 15 and 17

Megakaryoblastic cell count in the liver of control and br/br rabbit foetuses on days 15 and 17
Megakaryoblastic cell count in the liver of control and br/br rabbit foetuses on days 15 and 17
Table 2

Number of blood platelets in control and br/br rabbit foetuses on day 16

Number of blood platelets in control and br/br rabbit foetuses on day 16
Number of blood platelets in control and br/br rabbit foetuses on day 16

The br/br rabbit stock has been known for more than 40 years and the genetic transmission of the abnormality has remained unchanged. The interest of this stock is due to the total penetrance of the gene with respect to limb defects. The observed lesions generally affect the limb extremities and their severity is variable. We have also observed an inner ear syndrome in the br/br rabbit, which has not been described previously.

The study of megakaryoblastic cells in the br/br rabbit foetus liver clearly shows an abnormality in their development which is especially apparent on day 15. At this stage, there is an abnormally high frequency of young cells and these lie close to the vessel lumen. It has already been shown that the br/br liver is especially poor in erythropoietic tissue at this stage and progressively becomes normal towards day 17 (Petter et al. 1977). It seems possible that a developmental defect involving all the haemopoietic cells is linked with the induction of the abnormalities. Treatment with vitamins, which increases the erythropoietic tissue and at the same time prevents limb lesions (Petter et al. 1977) may also play a preventive role in abnormal megakaryoblastic cell formation.

Although unpublished studies have shown that the platelet count is approximately the same in adult br/br rabbits and in controls, this does not exclude the possibility of temporary platelet deficiency during the critical stages in br/br foetuses. The preliminary experiments (on day 16) shown in Table 2 indicate that this indeed occurs. This deficiency could be responsible for both limb and liver haemorrhages. Haemorrhages may also destroy nervous tissue and thereby induce an inner-ear syndrome. Due to the exceptional fragility of 15-day-old foetal membranes, it was impossible to count the platelets at this stage.

This megakaryoblastic abnormality, associated with congenital limb amputations in the rabbit resembles the TAR syndrome in man discussed in detail in the survey of Hall et al. (1969). The characteristics of this human disease are usually radius aplasia and platelet deficiency at birth. A hypomegakaryocytic thrombocytopenia was observed in the children studied.

At the present time, further research is being carried out in order to better understand the haemopoietic defects observed in the br/br stock.

Greene
,
H. S. N.
&
Saxton
,
J. A.
Jr
. (
1939
).
Hereditary brachydactylya and allied abnormalities in the rabbit
.
J. exp. Med
.
69
,
301
314
.
Hall
,
J. G.
,
Levin
,
J.
,
Kuhn
,
J. P.
,
Ottenheimer
,
E. J.
,
Van Berkum
,
K. A. P.
&
McKusick
,
V. A.
(
1969
).
Thrombocytopenia with absent radius (TAR)
.
Medicine
48
,
411
439
.
Inman
,
O. R.
(
1941
).
Embryology of hereditary brachydactyly in the rabbit
.
Anat. Rec
.
79
,
483
505
.
Jost
,
A.
,
Roffi
,
J.
&
Courtat
,
M.
(
1969
).
Congenital amputation determined by the br gene and those induced by adrenalin injection in the rabbit foetus
.
In Limb Development and Deformity: Problems of Evaluation and Rehabilitation
.
Charles C. Thomas, Publisher
.
Petter
,
C.
,
Bourbon
,
J.
,
Maltier
,
J. P.
&
Jost
,
A.
(
1971
).
Prevention des amputations congénitales héréditaires du lapin par une hyperoxie maternelle
.
C.r. hebd. Séanc Acad. Sci
.,
Paris
,
273
,
2639
2642
.
Petter
,
C.
,
Bourbon
,
J.
,
Maltier
,
J. P.
&
Jost
,
A.
(
1977
).
Simultaneous prevention of blood abnormalities and hereditary congenital amputations in a brachydactylous rabbit stock
.
Teratology
,
15
,
149
158
.