I M P A I R E D R E P R O D U C T I O N I N T H E R A B B I T FED

SUPPLEMENTED DIETS CONTAINING

SOYBEAN HAY
K. A. KENDALL, R. L. HAYS, AND G. D. ROLLERI
University o] Illinois ~

R E D reproduction as well as evidence of vascular breakdown

I MhasP A Ibeen
reported in the rabbit fed diets containing soybean hay
(Kendall et al. 1950). These abnormalities were not observed in what
were considered normal New Zealand White female rabbits whose diets
contained a roughage other than soybean hay namely: lespedeza, alfalfa
or timothy hay. Litter sizes of females fed diets containing the latter
roughages averaged 8.4 young per litter with no evidence of abnormal
body function either in the females or in their young. This was in
contrast to the performance of females in a series of studies fed a diet
containing soybean hay, whose litter size was 5.8 young per pregnancy
and was frequently accompanied by vascular breakdown in different
parts of the body, as well as evidence of fetal mortalffy during the
latter half of the gestation period.
In the earlier report (Kendall et al., 1950) a number of questions
were raised as to the possible cause or causes for the observed syndrome
in the rabbit fed a soybean hay-wheat diet. A number of the questions
raised grew out of the cited studies in which nutritional deficiencies
have been reported when the soybean or its fractions have been fed
to other animals. The close similarity of this syndrome to other cited
syndromes in which impaired reproduction had been associated with
certain vitamin deficiencies, was called to the reader's attention. Therefore, the purpose of this report is to summarize the results of a number
of studies that were conducted with the idea of attempting to answer
some of the questions raised earlier by testing the effects of administering certain supplements, namely: d-1 methionine, fl-carotene, vitamin
A acetate, a-tocopherol, 2 methyl-naphthoquinone, vitamin B~2, choline,
bonemeal and progesterone as well as the feeding of autoclaved soybean
hay upon the possible alleviation of a sterility syndrome which occurs
in the rabbit fed diets containing soybean hay.
1 Department of Dairy Science, Urbana, Illinois.

860

K . A . KENDALL, R. L. HAYS, AND G. D. ROLLER][

Experimental
Three series of experiments were conducted and are designated as
experimental series 1, 2 and 3. In all three series, the basic diet fed
consisted of mow-cured~ finely ground, soybean hay 49.5 parts, mixed
with finely ground wheat 49.5 parts and sodium chloride 1 part. The
composition of this diet as fed as determined by proximate analysis
(AOAC 1945) was dry matter 88.5 percent, total protein (nitrogen x
6.25) 15.1 percent, ether extract 3.1 percent, crude fiber 13.1 percent,
nitrogen free extract 51.5 percent and ash 5.7 percent. The carotene
content of the soybean hay as fed averaged approximately 20r per gram
of hay.
In planning these investigations it was considered unnecessary to
include roughages other than soybean hay in the diets to serve as normal
controls. Such an assumption was believed reasonable since the first
positive evidence of impaired reproduction was observed in ten New
Zealand White females whose diet contained soybean hay and whose
litters averaged 3.2 young per mating. This same group of females
had previously reproduced normally, when fed the same diet except
that it contained lespedeza instead of soybean hay, with an average of
8.9 living young per mating. Further justification for omitting roughages
other than soybean hay from the diets for control purposes was supported by the fact that when alfalfa replaced soybean hay in the diet
the litter size for 14 females of similar breeding was 8.0 young per
mating.
Two breeds of rabbits were used, namely, the New Zealand White
and Dutch breeds whose average weights were 3.5 kg. and 2 kg.
respectively. The animals were housed in individual cages and were fed
ad libitum. Fresh water was available to the animals at all times. The
animals were placed on the diet on the same day they were mated to
fertile males.

Experimental series 1.
In this series a number of supplements were administered or fed to
the mated females in addition to the above described basic soybean
hay-wheat diet. Each supplement tested was fed or administered to
five females with the exception of one group (vitamin B12 group)
which included seven females. The following supplements were given
at the indicated rates or were included in the diet at the indicated level:
d-1 methionine added to make up 0.3 percent of the diet; choline

REPRODUCTION AND DIETS CONTAINING SOYBEAN I-tAY

861

chloride 0.3 percent; steamed bone meal 1.0 percent; carotene (90
percent fl 10 percent a) in cottonseed oil administered orally each day,
200 9 per animal; vitamin A acetate in cottonseed oil administered
orally each day, 200 ~- per animal, a-tocopherol, orally administered,
at the rate of 4 mg. per kg. body weight; progesterone, 2 rag., were
injected subcutaneously per animal daily; 2 methyl-naphthoquinone,
2 rag. per animal daily; vitamin B12 100 ~- was given orally two times
per week. The females in this series were permitted to kindle at 31
days.
During the course of gestation observations were made for any
abnormal symptoms: hemorrhage, abortion, or voiding of partially
resorbed fetuses.
Experimental series 2.

In this series of experiments, two levels of a-tocopherol were administered daily to mated females receiving the basic diet of soybean
hay and wheat. In one experiment 10 rag. of a-tocopherol were given
orally to one group of seven females, to another similar group 10 rag.
of a-tocopherol were injected subcutaneously. Seven females receiving
only the basic diet were used as controls. To another group of six
females a level of 60 rag. of a-tocopherol were given orally each day.
A similar group of six were used as controls in this experiment.
On the 28th day after mating the animals were sacrificed and
autopsied. Observations were made of the number of living young;
the number of resorption sites; the number of corpora lutea as well as
any abnormalities which were evident.
In some experiments of this series only blood plasma samples were
taken for vitamin A and tocopherol analysis. In other experiments blood
plasma and liver samples were taken for vitamin A analysis. Vitamin
A in blood plasma was determined by Kimble's (1939) method modified by using activated glycerol dichlorohydrin for developing the color
(Sobel and Werbin, 1946). For determining liver vitamin A the methods
of Gallup et al. (1946) and Sobel and Werbin (1946) were used. Vitamin A values were obtained through the use of a Coleman Spectrophotometer, Model 14. Blood plasma tocopherol was determined by the
method of Quaife et al. (1944).
Experimental series 3.

This series was conducted for the purpose of studying the influence
of heating the hay by autoclaving upon the alleviation of the sterility

862

K.A.

KENDALL,R. L. HAYS, AND G. D. ROLLERI

symptoms. This was done with the idea that any possible enzymes, if
present in the hay and were factors in the syndrome, would be inactivated through the action of heating. An attempt to accomplish this
was made by autoclaving soybean hay for 30 minutes at 120~ and at
a pressure of 15 pounds. The autoclaved hay was used in compounding
a diet of the same proportions as the basic diet. Five groups of five
females each were used in this series. Groups 1 and 2 received a diet
containing the autoclaved soybean hay. Groups 3, 4 and 5 received the
basic diet. In addition groups 2 and 5 received 50 mcg. of fl carotene
daily per kg. of body weight. Group 4 received 80 mcg. of vitamin A.
On the 28th day following mating by sacrificing and autopsying the
females, observations were made of the number of living young, the
number of sites of resorption, the number of corpora lutea. Plasma and
liver samples were taken for vitamin A analysis.
Results
In tables 1, 2 and 3 the reproduction performance of the mated
females is presented.
Under the conditions of these studies d-1 methionine failed to prevent
the onset of hemorrhage. Two females out of five in this group homerrhaged severely. The number of young born for this group (table 1)
was well below normal of the two breeds used in this study. Normal
average litter sizes per mating for the Dutch and New Zealand White
breeds of rabbits as observed in this laboratory have been approximately
6.1 and 8.3 young per mating respectively for the Dutch and New
Zealand White breeds.
Although those females receiving carotene in this series failed to show
evidence of hemorrhaging, the number of young born was below normal
(table 1) as was also true when a-tocopherol was used as a supplement
to the basic diet.
Progesterone when administered at the indicated levels, was ineffective
in promoting normal litter size (table 1) even though no hemorrhage
was observed in this group.
Further evidence was shown in this study (table 1) that the cause
for hemorrhage was probably not due to a lack of vitamin K since in
this group of five females, 2 methyl-naphthoquinone appeared to be
without effect in controlling hemorrhage.
When the basic diet was supplemented with either choline or steamed
bone meal, females continued to abort fetuses as well as to show evidence of high fetal mortality having occurred during the latter part

REPRODUCTION

DIETS

AND

CONTAINING

SOYBEAN

HAY

863

of the gestation period as observed at autopsy on the 28th day following

mating.
The observations made in this series of studies indicate that the
symptoms typical for the syndrome, namely, hemorrhage emanating
from the reproduction tract, fetal resorption and abortion, are not
specific for a single breed.
TABLE
AS

1. T H E

REPRODUCTIVE

ASSOCIATED

WITH

PERFORMANCE

FEEDING

HAY-WHEAT

OF

FEMALE

SUPPLEMENTED

RABBITS

SOYBEAN

DIET

Females
Supplement
Dx

No supplement

Young carried
Known
28 days or to term
pregN Z 2 nant. Living Dead Corpora

5a

Series IA
26
3

--

Abnormal
symptoms observed
during gestation

cases

severe

hemorrhage.

1 female died 22nd clay of

preg.
d-1 methionine

fl-carotel~e
m-tocogherol

4
4.
4

1
I
i

4a
4a
3a

13
25
I7

2
2
0

~
---

2 cases of severe hemorrhage

None
1 female voided tissue rem-

nants 28th day

2 methyl4

38

20

--

Progesterone
Vitamin Ba2

naphthoquinone

4
0

1
7

4a
58

14
22

2
24

~
--

I abortion at 14 days followed

by severe hemorrhage
None
2 cases severe hemorrhage

No supplement
Choline
Steamed bone meal

0
0
0

5
5
5

44
44
44

39
37
39

1 abortion 27th day

1 abortion 26th day
1 abortion 27th day

Series I B
12
10
6

17
25-]17

1 Dutch breed.
New Zealand White breed.
3 31 days after mating.
Sacrificed 28 days after mating.

The results of the second series of experiments where higher levels

of a-tocopherol than those which were administered in experimental
series 1, indicate that the diet was not improved as indicated by the
observed reproductive performance. The high mortality rate in the
young observed on the 28th day after mating where 10 rag. of a-tocopherol was fed (table 2) suggests the possibility of a higher concentration of the syndrome including factor(s) in this particular lot of
soybean hay. Feeding 60 rag. a-tocopherol daily per animal failed to
promote an improved reproductive performance over that of the controls.
In the experimental series 3, where autoclaved soybean hay replaced
the regularly used soybean hay, the results indicate that autoclaved

864

K.A.

KENDALL,

R. L. HAYS, AND G. D. ROLLERI

hay did not improve the reproductive performance over that observed
where the non-autoclaved hay was included in the diet.
In many of the autopsy studies light colored placentae were observed.
In some instances some fetuses in litters were considerably smaller than
litter mates.
Discussion.

The evidence shows that the sterility syndrome does affect reproduction after implantation has occurred. The numbers of implantations per
TABLE

2. T H E

REPRODUCTIVE
RESPONSE TO INCREASED
OF a-TOCOPHEROL
SUPPLEMENTATION
Females showing
evidence of pregnancy

a tocopherol
Administered
None
Injected (10 mg.)
Oral (10 mg.)
Oral (60 rag.)

Females
At
mated 28 days
13
7
7
6

11
7
4
5

Living
young

Dead
young

53
0
12
31

35
24
20
8

LEVELS

Vitamin A
Plasma
Liver
Corpora "y/I00 ml. "V/gram
73
18
39
47

38
29
26
60.3

24.42

Tocopherol
plasma
m g / 1 0 0 ml.
.4958
.427
.778

38.3

1 All females were of New ZeMand White breed.

2 Average liver vitamin A values for 6 animals.
8 Average for 7 animals.

mating or per individual frequently have been equal to those of normal

reproductive performance. The appearance of small fetuses in some
instances and the presence of light colored placental membranes suggest
a possible lowered vascularity within the placenta which may have
resulted in a lowered nutrient supply thus affecting the developing
fetuses to a varying degree. This raises the question as to whether the
syndrome may be due in part to a partial failure in placental function
which may involve some endocrine relationships. That threshold levels
of impaired placental function may have existed are suggested by the
presence of varying numbers of fetuses, some small and some normal
size. In addition the presence of varying numbers of sites of resorption
ranging in age from highly advanced stages of resorption to fetuses
having died within 24 hours before the time of autopsy offers further
evidence that possible threshold levels of impaired placental function
may have occurred.
It is apparent that the syndrome was not prevented by supplementing
the soybean hay-wheat diet at the indicated levels with d-I methionine,

REPRODUCTION

AND

DIETS

CONTAINING

SOYBEAN

865

HAY

Carotene, a-tocopherol, vitamin A, 2 methyl-naphthoquinone, progesterone, vitamin B12, choline or bone meal.
It appears that vitamin A levels in the plasma and liver as well as
levels of vitamin E in plasma, though somewhat variable between
individual animals, for the most part were normal. That the syndrome
appears in animals having normal levels of these vitamins is evidence
T A B L E 3. R E P R O D U C T I V E P E R F O R M A N C E AS A S S O C I A T E D W I T H
AUTOCLAVED AND NON-AUTOCLAVED SOYBEAN
HAY IN THE DIET
Females showing
evidence of pregnancy at 28 days
Vitamin A
Dietary
factor or
factors
Autoclaved hay
Autoclaved bay
plus carotene
Basic diet
Basic diet
plus vitamin A
Basic diet
plus carotene

Females 1
Mated

PregNo.
Site
nancy living
of reAborting positive young sorption

No.
Corpora

Plasma
"//100 ml.

Liver
,y/gram

28

58

50.0

39,0

5
5

2~
1~

5
5

10
23

22
25

47
50

51,0
31.5

34.2
34.3

30

14

55

44.4

40.5

26

11

51

38.7

60.0

One animal of the D u t c h breed and 4 animals of the New Zealand White breeds were included
in each group.
One case of severe hemorrhage in each group.

which discounts the possibility, of any reduction in vitamin levels in

females due to carotene destruction in the hay curing process or in the
gastrointestinal tract of the animals during digestion.
It does not seem likely that a lack of dietary provitamin A in the
soybean hay fed rabbit would be a factor contributing to the syndrome's
appearance. This assumption seems reasonable on the basis of evidence
gained in other studies in this laboratory where normal reproduction
has been observed in normal female rabbits when placed on a carotene
deficient diet at mating time and. thus fed without vitamin A supplementation throughout pregnancy.
The fact that the administration of progesterone at the rate of 2
mg. daily appeared to be ineffective in alleviating reproductive failure
does not preclude the possibility that higher levels of progesterone administered may have an alleviating effect. The effects of administering
higher levels of progesterone upon the syndrome's alleviation are now
being studied.
The failure of the animals to carry their young to term is suggestive
of impaired nutrition for normal growth of the fetus. This may be due

866

K.A.

KENDALL, R. L. HAYS, AND G. D. ROLLERI

to abnormal physiological function of the placenta. Whether the presence of a toxic factor is contributing to the sterility condition is not
evident from these studies. Studies are now in progress to test the
effects of feeding certain fractions of the h a y as they m a y relate to the
syndrome in an a t t e m p t to isolate more definitely the factor or factors
that m a y be contributing to the failure in reproduction.
Summary
The results of three series of experiments in which further study
of the sterility syndrome in the rabbit fed soybean h a y are reported,
Supplementing the soybean hay-wheat diet at the indicated levels
with d-l methionine, carotene (fl 90 percent and a I0 percent) a-tocopherol, 2 methyl-naphthoquinone, vitamin A, progesterone, vitamin
B12, choline or steamed bone meal failed to alleviate the sterility symptoms or improve the reproductive performance of females over t h a t of
females receiving no supplements.
Plasma and liver vitamin A levels in the females fed the soybean
hay-wheat diet appeared to be normal without carotene or vitamin A
supplementation. Plasma tocopherol levels also appeared normal. These
findings suggest that this syndrome is not directly associated with the
levels of these vitamins even though the nature and incidence of the
syndrome parallels in m a n y respects that of the vitamin A or E deficient
animal.
T h e feeding of soybean hay, autoclaved at 120~
for 30 minutes,
was ineffective in alleviating the syndrome. This would suggest that
the causative factor or factors are p r o b a b l y not enzymic in nature.
Literature

Cited

Association of Official Agricultural Chemists. 1945. Official and Tentative Methods

of Analysts. 6th ed., Washington, D. C.
Gallup, Wallis D. and J. A. Hoeffer. 1946. Determination of vitamin A in liver.
Ind. Eng. Chem. Anal. Ed. 18:288.
Kendall, K. A., G. W. Salisbury and N. L. VanDemark. 1950. Sterility in the
rabbit associated with soybean hay feeding. Jour. Nutr. 42:487.
Kimble, M. S. 1939. The photocolorimetric determination of vitamin A and
carotene in human plasma. Jour. Lab. Clin. Med., 24:1055-1065.
Moore, L. A. and R. Ely. 1941. Extraction of carotene from p~ant material. Ind.
Eng. Chem. Anal. Ed. 13:600.
Quaife, Mary Louise and P. L. Harris. 1944. The chemical estimation of tocopherols
in blood plasma. Jour. Biol. Chem. 156:499.
Sobel, Albert E. and Harold Werbin. 1946. Activated glycerol dichlorohydrin a
new colorimetric reagent for vitamin A. Ind. Eng. Chem. 18:570.