NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
236
OVARIAN ACTIVITY IN PREGNANT BUFFALOES
[Vol. XXV, Part III,
MATERIALS AND METHODS
The material for this study was constituted of ovaries from non-pregnant and
pregnant Egyptian buffaloes. Twenty-four pairs of ovaries from non-pregnant
buffaloes represented different phases of the oestrus cycle. The phase of the oestrus
cycle was determined by the macroscopic appearance of the ovaries. Large graafian
follicles or a pale corpus luteum denoted late dioestrus orprooestrus small pinkish;
corpus luteum denoted early dioestrus whilst large protruding and veined corpus
luteum periodicum denoted mid-dioestrus.
The exact date of conception was not known but the approximate age of
pregnancy was determined by the weight and diameter of the foetus (the diameter
being more accurate than weight in determining the age of pregnancy). In the
majority of cases, the previous history of animals was not known. In certain cases,
such as the animals from the Animal Breeding Research Farm, however, the history
of the buffalo-cows was available. Fifty pairs of ovaries from pregnant buffaloes re-
presented different phases of recent, early and mid-pregnancy and this study
covers these different stages. They were classified into five stages (P-I, P-II, P-III,
P-IV and P-V) according to the weight and diameter of the foetus (Table I).
The animals were slaughtered during April and May 1953, at Cairo slaughter-
house, the reproductive organs were so removed that the ovaries and ovarian bursa
were maintained intact. They were sent to the laboratory for dissection and
examination. Immature animals and pathological conditions were excluded from
this study. The side of the most recent ovulation was recorded and the site of the
foetus in one of the two uterine horns was also noted in the gravid genitalia. The
ovaries were weighed and examined macroscopically and described.
As the thickness of the ovary varied to a great extent with the size and position
of the corpus luteum and the degree of prominence of the rupture point, this was
not recorded. A thin central longitudinal section was made through the ovaries
with a sharp razor. Notations were made on the internal structure with particular
TABLE I
Different phases of pregnancy under investigation
Phase of
pregnancy
P-I
P-II
P-III
P-IV
P-V
No. of
cases
0
16
18
6
4
Foetus
Weight (gm.)
Range
0.1—1.0
1.8—9.5
28—171
202—350
932—1870
Mean
0.7
4.2
97.5
282.3
1541.7
Diameter (gm.)
Range
0.2—2.0
1.6—5.9
8.9—16.0
16.6—21.0
26.3—40.0
Mean
1.2
3.5
12.9
19.1
32.2
September 1955]
E.
S.
E.
HAFEZ
237
Fig. 1. Histograms showing the weight of ovaries of pregnant buffaloes at different stages of pregnancy. Each column represents the weight
of two ovaries at a certain age of gestation, the white area represents the C. L. ovary and the black area the N. C. L. ovary.
238
OVARIAN ACTIVITY IN PREGNANT BUFFALOES
[Vol. XXV, Part III,
Fig. 2. Diameters of corpus luteum and graafian follicle in C.L. and N.C.L. Ovaries.
240
OVARIAN ACTIVITY IN PREGNANT BUFFALOES
[Vol. XXV, Part III,
TABLE II
Weight of ovaries during pregnancy and non-pregnancy
Reproduc-
tive phase
Pregnancy
Non-
pregnancy
Particulars
P-I
P-II
P-III
P-IV
P-V
Early-dioestrus
(and metaoestrus)
Mid-dioestrus
Late-dioestrus
(and proestrus)
No. of
cases
6
16
18
6
4
6
10
8
Wt. of C. L. ovary
Range
4.2—6.8
4.3—6.2
3.2—7.3
5.1—5.5
5.3—6.0
5.2—7.7
3.2—6.0
3.5—5.0
Mean
5.7
5.3
5.5
5.4
5.6
5.8
5.2
4.3
Wt. of N. C. L.
ovary in gm.
Range
2.6—4.3
2.7—5.6
2.4—6.8
1.4—2.6
2.5—3.6
4.1—4.9
3.3—4.9
3.1—4.2
Mean
3.9
3.9
3.5
2.2
2.7
4.6
4.1
3.5
*Wt. of two
ovaries
in gm.
9.6
9.2
9.0
7.6
8.3
10.4
9.3
7.8
*Wt. Ratio
C. L. ovary/
N. C. L. ovary
146
136
157
245
207
126
127
123
The most recent ovulation was in the right ovary in 74 per cent of the pregnant cases and 75 per cent of the
non-pregnant cases.
* Mean values.
Corpus luteum
One well-developed functioning corpus luteum was found in all the pregnant
cases. In recent stages of pregnancy, it was soft in consistency, oval in shape and
not always even in contour. It protruded somewhat beyond the surface of the
ovary and had an orange red colour. In stage P-II and P-III of pregnancy it tended
to sink in the ovarian stroma, became round in shape and had a pink red colour.
During later stages of pregnancy, it was chocolate in colour and was covered by
the epithelium from the tunica albuginea and from the peritoneum. In general,
the shape of the corpus luteum was mainly dependent on the pressure condition in
the ovary whilst its colour was associated somewhat with the degree of blood vessel
organization. The corpus luteum was sometimes triangular when follicles developed
on two sides of it. The morphology of the corpus luteum is represented in Plate VI,
B. In some cases there was a small hollow longitudinal centre in the corpus luteum,
which did not contain any fluid.
The average diameter of the corpus luteum of pregnancy ranged from 1.9 to
2.2 cm. while it varied from 1.0 to 1.5 cm. during non-pregnancy. The average
diameter of the corpus luteum of pregnancy (irrespective of the stage of pregnancy)
is significantly bigger than that during non-pregnancy (irrespective of the oestrus
cycle). The relative size of the corpus luteum may be estimated indirectly by the
percentage ratio of diameter of corpus luteum to weight of C. L. ovary. Its size
did not vary throughout early to mid-pregnancy, while it varied with the stage of
the oestrus cycle (Table III).
PLATE VI]
Indian J. vet. Sci., Vol. XXV, Part III
A. Pairs of ovaries from pregnant (P) and non-pregnant (N.P.)
buffaloes. Note the two in each pair are unlike. The
corpus luteum is protruding from the ovarian surface.
B. The morphology of corpus luteum of pregnancy in buffaloes
(painted with Indian ink).
C. Luteal scars (coloured blue or brown in the ovaries) were
more abundant during pregnancy (P) than during non-preg-
nancy (N.P.) in the buffalo.
September 1955]
E.
S.
E.
HAFEZ
241
Graafian follicles
During pregnancy the C. L. ovary contained one or two follicles of relatively
large size in 41 per cent of the cases. The N. C. L. ovary contained from one to
three follicles of relatively large size in 97 per cent of the cases. Whenever developing
follicles were observed they were in different sizes showing waves of growth; also,
they were all filled with liquor folliculi. A large portion of the growing follicles
were on the surface of the ovary. The average diameter of the largest follicles in
the N. C. L. ovary during pregnancy varied from 0.8 to 0.9 cm., while it ranged from
0.8 to 1.3 cm. during non-pregnancy. The average diameter was unchanged
throughout recent to mid-pregnancy whilst it was correlated with the stage of the
oestrus cycle during non-pregnancy. The follicle diameter was significantly bigger
TABLE III
Size of most recent corpus luteum during pregnancy and non-pregnancy
Reproduc-
tive phase
Pregnancy
Non-
pregnancy
Particulars
P-I
P-II
P-III
P-IV
P-V
Early-dioestrus (and
metaoestrus)
Mid-dioestrus
Late-dioestrus (and
proestrus)
No. of
cases
6
16
18
6
4
6
10
8
Mean wt. of
C. L. ovary
(gm.)
5.7
5.3
5.5
5.4
5.6
5.8
6.2
4.3
Diameter of most recent
C. L. (cm.)
Range
1.6—2.1
1.6—2.2
1.5—2.1
2.0—2.3
1.6—2.0
1.1—1.3
1.2—1.7
0.9—1.0
Mean
1.9
1.9
1.9
2.2
1.9
1.2
1.5
1.0
Ratio of C. L.
diameter to
ovary weight
×100
33
36
35
41
34
18
29
23
during non-pregnancy than during pregnancy. The average diameter of the next
largest follicle in the N. C. L. ovary during pregnancy varied from 0.4 to 0.5 cm.
while it was some 0.1 cm. during non-pregnancy. The average diameter was not
correlated with the stage of pregnancy or oestrus cycle. The diameter of the next
largest follicle was significantly bigger during pregnancy than during non-pregnancy
(the largest follicles being bigger during non-pregnancy) (Table IV).
In the C. L. ovary, the largest follicles varied from 0.2 to 0.4 cm. during
pregnancy and 0.5 to 0.7 cm. during non-pregnancy. The next largest follicles
were some 0.1 cm. irrespective of the reproductive phase. Fig. 2 shows that the
diameter of the corpus luteum in the C. L. ovary and that of the largest graafian
follicle in the N. C. L. ovary do not change from recent to mid-pregnancy.
242
OVARIAN ACTIVITY IN PREGNANT BUFFALOES
[Vol. XXV, Part III,
TABLE IV
Size of graafian follicles during pregnancy and non-pregnancy
Reproduc-
tive phase
Pregnancy
Non-
pregnancy
Particular
P-I
P-II
P-III
P-IV
P-V
Early - dioestrus
(and inetaocs-
trus)
Mid-dioestnis
Late - dioestrus
(and proestrus)
Follicle diameter in C. L. ovary
Largest
Range
Cm.
0.4—1.2
0.6—1.4
0.8—1.2
0.4—1.0
0.8—1.0
0.6—0.9
0.9—1.8
1.2—2.0
Mean
Cm.
0.9
0.8
0.9
0.8
0.9
0.8
1.3
1.8
Next largest
Range
Cm.
0.3—0.6
0.4—0.5
0.4—0.8
0.4—0.8
0.4
0.1 or less
0.1 or less
0.1—0.2
Mean
Cm.
0.4
0.5
0.5
0.5
0.4
0.1
0.1
0.1
Follicle diameter in N. C. L. ovary
Largest
Range
Cm.
0.1—0.4
0.1—0.9
0.1—0.4
0.1—0.7
0.1—0.6
0.2—0.6
0.4—0.9
0.3—0.9
Mean
Cm.
0.3
0.4
0.2
0.4
0.2
0.5
0.7
0.7
Next largest
Range
Cm.
0.1 or less
0.1—0.2
0.1 or less
0.1—0.2
0.1—0.2
0.1 or less
0.1—0.2
0.1 or less
Mean
Cm.
0.1
0.1
0.1
0.1
0.1
0.1
01
0.1
During pregnancy, the developing follieles degenerated before becoming mature.
In these undischarged follicles, the liquor folliculi was absorbed, the epithelium
broke down and the ovum became flattened and shrivelled. The cavity eventually
became filled in by a loose ingrowth of connective tissue from the thecal wall. In
later stages, the ovum had disappeared as well as the epithelial cells. Granules of
black, blue, brown or yellow pigment were seen in the thecal wall. The luteal
scars were abundant in the N. C. L. ovary and more frequent during pregnancy
than during non-pregnancy (Plate VI, C).
DISCUSSION
The location of the ovaries underneath the pregnant uterus makes it difficult
to palpate the former per rectum. The form of the ovary is mainly dependent on
the presence of corpus luteum. The alternating action between the two ovaries is
not significant in the buffalo since the frequency of corpora lutea is more in the
right ovary than in the left. This is possibly due to the presence of the rumen on
the left side of the abdominal cavity which might hinder the blood flow to this side
as compared with the right side. Consequently the conception is more frequent
in the right uterine horn since there is no ova migration. The percentage weight
ratio of C. L. ovary to N. C. L. ovary is higher during pregnancy than during non-
pregnancy. This is due to a slight absolute increase in the weight of the C. L.
ovary and a slight decrease in the other, with the advancing pregnancy. This is a
valuable anatomical criterion to differentiate ovaries from pregnant and non-
pregnant animals. Voss [1950] has found in non-pregnant sheep a relationship
between the ovarian weight on one hand and the live-weight or the body length on
the other hand. This does not seem to apply to pregnant animals or to different
species.
September 1955] M. R. DHANDA, J. M. LALL AND M. M. SINGH 247
Pathogenicity of Salmonella abortus equi to mice
In the absence of a suitable potency test to assess the value of Salmonella abortus
equi vaccine in preventing abortion in mares, it was considered desirable that some
method whereby the antigenic value of the vaccine could be judged should be
evolved. A reference to the available literature did not show any record of the
effect of artificial inoculation of Salmonella abortus equi into laboratory animals.
Since abortion is one of the symptoms exhibited by mares, infected with this orga-
nism, it would be reasonable to assume that any protection afforded against the
infection would also result in a corresponding abatement of symptoms generally
attributed to this organism including abortion. It was, therefore, decided to study
the pathogenic effect of the three strains of Salmonella abortus equi (S. 128, S. 129
and S. 130) used in the preparation of mare abortion vaccine at this Institute in
white mice, and, if found pathogenic, to see whether the vaccine when administered
to mice would afford any protection against the pathogenic effect. The strains
had been isolated from natural cases of abortion in mares at Coleyana in the year
1946.
To begin with, it was found that subcutaneous inoculation of a fairly large dose
of young culture suspensions from each strain (agar wash) would kill white mice in
a day or two and in each case the organisms could be isolated from the heart blood
of the mice after death. When repeated in a comparatively smaller dose of 1 c.c.
(24-hour-old broth culture), death occurred of bacillaemia in one to five days, the
organisms being again recovered from the heart blood in each case.
To determine the M.L.D. of each strain, groups of two mice were inoculated
subcutaneously with ten-fold serial dilutions representing 10- 6 to 10- 2 of a 24-hour-old
broth culture of each strain. As the course of the disease was noted to be pretty
long when small doses of cultures were used for artificial infection, the mice were
kept under observation for one month. The results are presented in Table I.
TABLE I
Effect of different strains in varying doses on mice
Strain
S. 128
Dose
10- 6
10- 5
10- 4
10- 3
10- 2
No. of
mice
2
2
2
2
2
Results
S D 24 (other
cause)
S D 9
S D 15
S D 13
S D 6
Cultural examination of heart
blood of dead mice
Not examined
+
+
+
248
STUDIES ON SALMONELLA ABORTUS
[Vol. XXV, Part III,
TABLE I—(contd.)
Effect of different strains in varying doses on mice
Strain
S. 129
S. 130
Dose
10- 6
10- 5
10- 4
10- 3
10- 2
10- 6
10- 5
10- 4
10- 3
10- 2
No. of
mice
2
2
2
2
2
2
2
2
2
2
Results
S S
S D 22
S D 21
S D 17
D 7 D 7
S S
S D 21
S D 9
D 12 D 16
S S
Cultural examination of heart
blood of dead mice
+
+
+
+
++
+
+
+
++
S=Survival
D=Died with the figure indicating the number of days after which death occurred
+= Positive culture
The above results indicated that the pathogenicity of these strains for white
mice was not of a very high order and that the dose required to produce lethal effect
would be fairly high. However, the tests were repeated using higher concentration
of the cultures with larger numbers of mice and the results are shown in Table II.
It was concluded from these experiments that each of the three strains is certainly
lethal when used in 1 c.c. dose of undiluted 24-hour-old broth culture, the higher
dilutions proving ineffective in a certain percentage of the mice in each case.
September 1955] M. R. DHANDA, J. M. LALL AND M. M. SINGH 249
TABLE II
Effect of higher concentrations of strains on mice
Strain
S. 128
S. 129
S. 130
Dose
10- 2
10- 1
Undiluted
1 c.c.
10- 2
10- 1
Undiluted
1 c.c.
10- 2
10- 1
Undiluted
1 c.c.
No. of mice
used
4
4
4
4
4
4
4
4
4
Results
SS D 6 D 9
D 6 D 7 D 7 D 9
D 1 D 2 D 2 D 2
S S S D 6
S S S D 7
D 2 D 2 D 2 D 3
S S S S
S S D 2 D 6
D 1 D2 D4 D 6
Result of cultural examination
of heart blood of dead mice
++
++++
++++
+
+
++++
++
++++
S=Survived an observation period of one month
D=Died with the figure indicating the number of days after which the death occurred
+=Positive culture
In order to see whether any exo or endotoxins lethal to white mice were liberated
by these cultures, and also if the vaccine as was previously used for immunising mares
would prove antigenic in white mice and whether inoculated mice would withstand
a challenge with live cultures, experiments were undertaken to study the effect
of (i) filtrates of broth cultures, (ii) centrifuged and washed bacilli killed by heating
at 56°C for 30 minutes and (iii) cultures killed by heating at 56°C for 30 minutes.
Effect of filtrate of broth cultures
(a) Filtrate of unheated culture : Twenty-four-hour-old broth culture of each
of the three strains was passed through Seitz filter and the bacteria-free filtrate was
inoculated subcutaneously into groups of four mice in doses of 2 c.c. to see evidence
of any exotoxins liberated by these cultures. No untoward effect was noticed in
the mice over a period of observation of a fortnight, except in the case of two mice
inoculated with culture filtrate of strain S. 130, both of which were found to have
died of infection with haemolytic streptococci which on further study were found
to belong to group G.
(b) Filtrates of cultures heated at 56°C for 30 minutes : Twenty-four-hour-old
broth culture of each strain heated at 56°C for 30 minutes and filtered through
Seitz filter was given subcutaneously in a dose of 2 ml. each into groups of eight
250
STUDIES ON SALMONELLA ABORTUS
[Vol. XXV, Part III,
white mice. It was interesting to note that the filtrate proved toxic to white mice
as per results shown in Table III, thereby showing that endotoxins could be easily
liberated from the organisms by the effect of heat and set free in the surrounding
fluid, although the amount of toxin liberated was not complete as judged by the
results shown in Tables III and IV.
TABLE III
Effect of filtrates of cultures heated at 56°C for 30 minutes
Strain
S. 128
S. 129
S. 130
No. of mice
used
8
8
8
Results
D 5 D 15 D 16 S S S S S
D 1 S S S S S S S
D 1 D 2 D 3 D 1 D 4 S S S
P. M. notes
Organs congested, cultures
sterile
Cultures sterile
Organs congested, cultures
sterile
S=Survived; D=Died with figure indicating the number of days after which the death
occurred.
Effect of centrifuged, washed and heat-killed bacilli
Broth cultures of each strain centrifuged and washed twice with N.S.S. and
resuspended in N.S.S. were strandardised to match B.W. opacity tube No. 4 and
then killed by immersion in a water bath at 56°C for 30 minutes. This suspension
was injected subcutaneously in a dose of 2 ml. each into groups of eight mice. The
results are shown in Table IV from which it is observed that killed cultures, washed
free of all aggressions, etc., if any, proved lethal although the time taken was
comparatively longer than that with live cultures indicating clearly the presence
of a potent endotoxin.
TABLE IV
Effect of washed and heat-killed bacilli
Strain
S. 128
S. 129
S. 130
No. of mice
8
8
8
Results
D 4 D 6 D 7 D 8 D 16 D 17 D S S
D 1 D 3 D 3 D 3 D 6 D 7 D 7 S
D 3 D 4 D 6 D 8 D 9 D 29 S S
P. M. Notes
Organs congested,
cultures
sterile
do.
do.
S=Survived; D=Died with the figure indicating the number of days after which the death
occurred.
September 1955] M. R. DHANDA, J. M. LALL AND M. M. SINGH 251
Effect of heat-killed whole cultures
Twenty-four-hour-old broth cultures were heated at 56°C for 30 minutes and
injected subcutaneously into groups of four mice each in doses of 1 ml. The results
as shown in Table V indicated that in the dose in which these cultures were used,
they were lethal to some of the mice and, therefore, for the purpose of vaccination
the dose would have to be suitably adjusted.
TABLE V
Effect of heat-killed whole cultures on white mice
Strain
S. 128
S. 129
S. 130
No. of mice
used
4
4
4
Results
D 8 S S S
D 2 D 4 S S
D 2 D 5 D 9 S
P. M. notes
Cultures sterile
do
do
The experiment was repeated on groups of 12 mice by using a suspension of
cultures containing equal parts of each of the three strains in a dose of 0.5 c.c. sub-
cutaneously into each mouse. This dose was well tolerated with all the 12 mice
used in the experiment surviving, and it was decided to use this as a vaccinating
dose for mice to be later challenged with the live cultures.
Comparative tests on the protective values of the 'hcat-killed-phenol-preserved-vaccine'
and the new 'formalin-detoxicated-alum-precipilated-vaccine '.
Having fixed a safe dose for immunising mice, experiments were carried out to
study the comparative protective value in white mice of the old heat-killed vaccine
and the new vaccine. For this purpose groups of eight mice were vaccinated with
the two products and after an interval of 13 days they were challenged with the
mixture of live culture containing all the three strains used for the preparation of
the vaccine. The challenge dose was fixed at 0.2 ml. of the mixture on the basis of
an experiment on mice in which it was found that a dose of 0.5 ml. killed all the four
mice in one to six days and that of 0.2 ml. killed them within seven days. The
latter dose was used in the challenge experiments. The results of the immunity
test are shown in Table VI.
252
STUDIES ON SALMONELLA ABORTUS
[Vol. XXV, Part III,
TABLE VI
Results of immunity tests
No. of
mice
used
8
8
8
Type of vaccine
and dose
Heat-killed pheno-
lized 0.5 ml.
Formal
alum
vaccine.
Unvaccinated
(Controls)
No. of mice
challenged
8
8
8
Challenge dose
0.2 ml. of mixed
culture
13
days
after
vaccination.
„
„
Results of challenge
after 13 days
D 5 D 6 D 7 D 11
D 7
D 1 D 3 D 2 D 6 D 6 D 6 D 7 D 7
Survival
percentage
50
85.5
D=Dcad with the figure indicating the number of days after which the death occurred
NOTE: Positive cutlures were obtained from the heart blood of each mouse dying after challenge
The results clearly showed the superiority of the formalin-killed alum-precipita-
ted vaccine over the heat-killed vaccine in protecting mice against a certain lethal
dose of the culture which had killed all the eight mice in the control group.
In order to get more reliable and statistically significant data, the experiment
was repeated on groups containing 16 mice in the two vaccinated groups and four in
the control groups and the results are shown in Table VII. It was noted, however,
that the heat-killed vaccine did not prove quite safe for mice as five out of 16 mice
died of shock soon after vaccination, although this phenomenon was not exhibited
by mice used earlier. The results of challenge in the second experiment (Table VII)
confirmed the results of the previous experiments exhibiting beyond doubt that
formol-killed alum-precipitated vaccine afforded a definite protection to mice
against infection with artificial inoculation of cultures in a dose certainly lethal
to unprotected mice and that the product was superior in this respect to the heat-
killed vaccine.
TABLE VII
Results of challenge with both the vaccines
No. of
mice
used
16
16
4
Type of
vaccine and
dose
Heat
killed
phenolized
0.5 ml.
Formol alum
vaccine.
No. of
challenge
11
14
4
Nature of
challenge
0.2 ml. of-24-
hour-old-broth-
culture
con-
taining equal
parts of the
three strains.
do
do
Results
D 2 D 11 D 12 D 12 D 12
D 6 D 9
D 2 D 2 D 2 D 2
Survival
percentage
54.5
85.7
nil
Remarks
Five mice died
immediately
following vacci-
nation.
Two mice died
of other causes
before challenge.
D=Died with the figure indicating the number of days after which the death occurred
NOTE: Positive cultures were obtained from the heart blood of each mouse dying after challenge