NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
March 1955] E. S. E. HAFEZ AND M. A. M. KAMAL 41
III Quantitative analysis
(a) Total nitrogen: For this a modified ' Micro-Kjeldahl' (modification of
Parnas and Vagnar) apparatus was used.
(b) Creatinine nitrogen : The colorimetric method of Folin 'Micro' modifica-
tion was carried out [Hawk, et al., 1947]. It is based on comparison
against standard creatinine solutions yielding colour reactions by means
of Jaffe reaction. The quantity of creatinine was calculated as follows :
Reading of st andar d/ Readi ng of unknown+ mg. of creatinine in standard = mg.
of creatinine in volume analysed
(c) Amino nitrogen : The technique used here is that given by Pope and
Stevens [1939] and adopted by Kamal [1951].
RESULTS
The amniotic fluid was clear, colourless and slightly heavier than water. In
a few cases, it was more or less opaque and showed reddish or yellowish colour.
In no case it contained meconium (i.e. there was no foetal diarrhoea). The allantoic
fluid was clear and had the appearance and smell of urine and was slightly heavier
than water.
The specific gravity of the amniotic fluid fluctuated from 1.0040 to 1.0090 ;
there was no trend for any increase or decrease throughout pregnancy. The allan-
toic fluid had a specific gravity ranging from 1.0060 to 1.0120, with an apparent
increase as pregnancy advanced (Table II). The reducing sugars were either not
detected or were present in small amounts in the amniotic fluid. In one case (No. 14)
reducing sugars were detected in the amnion in good amounts; this might have been a
pathological case. In most of the cases the allantoic fluid contained small quantities
of the reducing sugars. The presence of glycogen was doubtful in the amniotic
fluid whilst the allantoic fluid had no glycogen at all. Acetone bodies were not
detected at all in both the placental fluids. Creatinine was not detected, by the
qualitative tests, in the amniotic fluid. The allantoic fluid contained creatinine
which increased in amount with advance in gestation.
Amino acids of benzene ring (tyrosine, phenylalanine and tryptophane) and
glycoproteins were not detected in the amniotic fluid whilst the peptide linkaged
amino acids, cystine and cysteine were detected in very slight amounts. During
stage P V (mid-pregnancy) there were detectable amounts of amino acids of benzene
ring in the amniotic fluid. The allantoic fluid contained larger quantities of tyrosine
than of other amino acids of benzene ring or of glycoprotein. There was a trend for
the former amino acids to be more abundant during late pregnancy than during
early one. (Table II).
It is of great interest to note that the amniotic fluid contained no amino-nitrogen.
The creatinine-nitrogen content in the amniotic fluid varied from 0.43 to 0.78 mg.
per cent, there was no significant trend of increase or decrease throughout pregnancy.
During early pregnancy (stage P I) the allantoic fluid contained 58 mg. per cent
total-nitrogen, 2.25 mg. per cent amino-nitrogen and 2.25 mg. per cent creatinine-
nitrogen. These values increased gradually throughout pregnancy. The total-
nitrogen, amino-nitrogen and creatinine-nitrogen content of the allantoic fluid during
42
PHYSIO-CHEMICAL INVESTIGATION ON PLACENTAL FLUIDS [Vol. XXV, Part I
mid-pregnancy were 169, 44.19 and 15.38 mg. per cent, respectively (Table III).
The percentage of amino-N. to total-N. in the allantoic fluid varied from 3.69 to 26.15
(Table IV). The percentage of creatinine-N. in the same fluid ranged from 3.88
to 10.09, it tended to be constant after early pregnancy.
DISCUSSION
The gestation period in the Egyptian buffalo is 317 [Ragab and Asker, 1951]
compared to about 286 days in cattle [reviewed by Asdell, 1946]. Table I
which shows the developmental changes in the diameter and weight of buffalo
foetuses, were compared with similar figures given for cattle by Hammond [1927].
It is concluded that the present data concerning the placental fluids (five stages of
pregnancy P I, II, III, IV and V) only include the first half of pregnancy. The
source of physiological amniotic fluid has not been fully determined. Some believe
that it is due to a secretion by the amnion itself while according to some it is at least
partly composed of urine. Williams [1951] suggests that it is secreted from the
foetal skin. The allantoic fluid appears to be derived chiefly from the foetal kidneys.
There is anatomical evidence that the urethra and arachus offer ample outlet for
any fluid contents within the urinary bladder [Hafez, 1955].
TABLE II
Physio-chemical properties of placental fluids in Egyptian buffaloes
Fluid
Amniotic
Allantoic
Age of
pre-
gnancy
PI
P II
P III(*)
P IV
P V
PI
P II
P III
P IV
P V
Specific
gravity
at (25"C)
1.0090
1.0080
1.0070
1.0050
1.0040
1.0060
1.0080
1.0070
1.0120
1.0100
Reduc-
ing
sugars
—
—
×
—
±
+
+
++
±
+
Glyco-
gen
—
±
—
±
—
—
—
—
—
—
Acetone
bodies
—
—
—
—
—
—
—
—
—
—
Creati-
nine
—
—
—
—
—
+
+
++
+++
++
Amino-acids
B
±
±
±
+
++
+
++
++
+++
++
X
—
—
—
—
—
+
+
++
+
±
Mi
+
+
+
+
++
+
+++
++
++
+++
A
..
—
—
—
—
±
±
+
+
±
S
±
±
+
±
+
+
+
++
+++
+
Mo
—
—
+
—
—
±
±
+
+
+++
B—Peptide linkage
X—Tyrosine, phenylalanine, tryptophane
Mi—Tyrosine
A-Tryptophane
S—Cystline and cystine
Mo—Glyeoprotein
(*) In one case there were plenty of reducing sugars
— no colour reaction
± slight colour reaction
+ moderate colour reaction
++ perfect colour reaction
+++ deep colour reaction
March 1955] E. S. E. HAFEZ AND M. A. M. KAMAL 43
TABLE III
Micro-determination of total, amino and creatinine-nitrogen in placental fluids in
Egyptian buffaloes (Average figures, calculated as mg.(100 ml.)).
Stage of preg-
nancy
P I
P II
P III
P IV
P V
Amniotic fluid
Total N
(n.d.)
(n.d.)
(n.d.)
45
80
Amino-N
..
..
..
..
..
Creatinine-N
0.43
0.78
0.50
0.74
0.45
Allantoic fluid
Total N
58
85
119
132
169
Amino-N
2.25
3.14
10.16
19.05
44.19
Creati-
nine-N
2.25
5.21
11.11
13.32
15.38
(*) See Table I
(n.d.) not determined due to the small volume of sample
TABLE IV
Per cent amino and creatinine N to total N in the allantoic fluid of the buffalo
Per cent of total N
Amino-N
Creatinine-N
Stage of pregnancy
P I
3.88
3.88
P II
3.69
6.13
P III
8.54
9.34
P IV
14.43
10.09
P V
26.15
9.10
It has been believed that the ' liquor amnii' has a certain nutritive value for
the foetus. Data represented in Table II show that there are but little amounts of
reducing sugars, glycogen and most tested amino acids. It would appear that the
amniotic fluid has perhaps no nutritive value at least during the first half of preg-
nancy. It seems that the foetus acquires the capacity of swallowing during the
second half of pregnancy. The abundance of the amino-acids in the allantoic fluid is
of great interest. It is suggested that some of the amino-acids circulating from the
mother to the foetus during the first half of pregnancy are not wholly metabolised
and someof these amino-acids escape and appear in the allantoic fluid. How far this
escape of amino-acids occur during the second half of pregnancy remains to be seen.
Table III shows that the ' liquor amnii' contains no amino-nitrogen but slight
amounts of creatinine-nitrogen (this is not detected by qualitative colour reactions).
The gradual increase of total nitrogen and its fractions in the allantoic fluid shows
the increase of protein metabolic activity throughout the first half of pregnancy.
March 1955] E. S. E. HAFEZ AND M. A. M. KAMAL 45
8. Total and creatinine-N were much higher in the allantoic than in the amniotic
fluid. No amino-nitrogen was detected in the amniotic fluid. Total amino and
creatinine-N of the allantoic fluid increased significantly with advanced pregnancy.
9. The physio-chemical properties of placental fluids are discussed in relation
to the origin and function of the fluids, to evolutionary changes (species differences)
in duration of pregnancy and to placental physiology.
REFERENCES
Amroso, E. C. (1952). Placentation (In Marshall's Physiology of Reproduction), Third edition. Edited
by Parkes, A. S. Longmans, Green & Co., London & N. Y.
Asdell, S. A. (1946). Patterns of Mammalian Reproduction, Comstock, Ithaca, N. Y.
Bergmann, R. (1921). Arch. Tierheilk., 47, 292
Cloette, J. H. L. (1939). Onderstepoort J. vet. Sci., 13, 417.
Draper, R. L. (1920). Anat. Rec., 28, 369
Hafez, E. S. E. (1952). Indian J. vet. Sci., 22, 257
Hammond, J. (1927). The Physiology of Reproduction in the Cow, University Press, Cambridge
Hammond, J. (1953). Trans. Dynamics of Development, Moscow, 10, 93.
Huggett, A. St. G. and Hammond, J. (1952). Physiology of Placenta (In Marshall's Physiology of Re-
production) Third Edition, Edited by Parkes, A.S. Longmans, Green & Co. London & N. Y.
Hawk, Ph. B., Bernard, L. and Summerson, W. H. (1947). Practical Physiological Chemistry, Twelfth
edition. Blokiston Co., Philadelphia
Ibsen, H. L. (1928). J. exp. Biol., 51, 51.
Kamal, M. A. M. (1951). Nitrogen Fractions in Trifolium alexandrinum, under conditions of different
phosphotic levels with reference to P H value, Cairo University, Faculty of Agriculture, Ph. D.
Thesis
Malan, A. P. and Curson, H. H. (1937). Onderstepoort J. vet. Sci., 8, 417
Minot, C. S. (1892). Human Embryology, New York
Needham, J. (1931). Chemical Embryology, University Press, Cambridge
Ragab, M. T. and Asker, A. A. (1951). Indian J. J. vet. Sci. 21, 519
Pope, C. G. and Stevens, M. F. (1947) Biochem. J., 33, 1070
Weeth, H. J. and Herman, H. A. (1952) Mon. Agric. Exp. Sta. Res. Bull. No. 501
Williams, W. L. (1951). Veterinary Obstetrics, Fourth edition, New York.
FACTORS INFLUENCING THE TITRATABLE ACIDITY OF
COW AND BUFFALO MILK
By A. A. BADR, Ibrahim Pasha University, Faculty of Agriculture, Cairo, Egypt
(Received for publication on 10 October 1953)
(With one text-figure)
W HILE there have been numerous methods of expressing the titratable acidity
of milk such as those recommended by Richmond and Huisk (1920) 0.1 N.
Baryta, Storch [Richmond and Miller, 1920] by using a saturated solution of lime-
water and Soxhlet-Henkel [Davies, 1939] by using 0.25 N. NaOH, the most widely
used and accurate method is that of expressing the acidity as percentage of lactic
acid by using N/9 NaOH for titration. The calculation is then avoided since the
equivalent of lactic acid is 90 and every ml. of N/9 alkali used for 10 ml. of milk
corresponds to 0.1 per cent. According to Rogers [1928] and Davies [1939] what
is measured in the titration of fresh milk is the amount of alkali necessary to shift
the protein and other buffer systems from initial state of equilibrium somewhere
near pH 6.6 to that pH value at which phenolphthalein shows the colour arbitrarily
selected as indicating the hypothetical 'end point'.
In several text books the acidity of fresh milk has been reported as ranging
from 0.10 to 0.14 per cent. Preliminary studies with cow and buffalo milk
in Egypt indicated that such low values are not typical of any large number of cows.
This observation led to the organization of a plan for a detailed study of the titratable
acidity of fresh milk from individual cows and buffaloes of the dairy herd of the
Faculty of Agriculture at Giza.
Factors studied as possibly connected with acidity of cow and buffalo milk
were, 1. species, 2. individuality of the cow or the buffalo, 3. stage of lactation, 4.
variations from month to month, 5. variations from day to day, 6. variations from
milking to milking, 7. variations from quarter to quarter of the same udder, and
8. the effect of dilution on the titratable acidity of buffalo milk. The acidity
of the milk from each cow or buffalo, was determined weekly over a fourteen-month
period. More frequent determinations were made with selected groups of cows
and buffaloes for shorter periods of time in conducting certain phases of the investiga-
tion.
PROCEDURE
The technique used by different investigators in determining the titratable
acidity of milk has varied widely. This fact undoubtedly is an important factor
in the variable results which have appeared in the literature. Sommer and Menos
[1931] have shown that the addition of an equal volume of water to a sample of
47
112 AR/54
9
48
TITRATABLE ACIDITY OF COW AND BUFFALO MILK
[Vol. XXV, Part I,
milk lowers its titratable acidity approximately by 0.02 per cent, and the addition
of nine volumes of water lowers it by 0.06 per cent. Burgwald and Bachtel [1933]
found that an increase in the amount or concentration of indicator lowered the
results of acidity determinations. This observation has been verified by Caulfield
and Riddell [1936]. They found that when replicate 18 gram samples of whole
milk were titrated with varying amounts (3 drops to 2.0 c.c.) of one per cent
solution of phenolphthalein indicator, the following results were obtained: three
drops of indicator, 0.172 per cent acid ; 0.5 c.c. 0.159 per cent acid ; 1.0 c.c. 0.154
per cent acid; 1.5 c.c. 0.151 per cent acid; and with 2.0 c.c. of indicator, 0.149
per cent acid.
It is evident that a carefully standardized procedure for determining the titra-
table acidity of whole milk must be employed if comparable results are to
be obtained. The following procedure was used in the present investigation:
10 c.c. of milk was taken for the test and put in a flat white porcelain dish and
10 c.c. in a similar dish for comparison of colour. Three drops of one per cent
solution of phenolphthalein in alcohol was added to the first dish only, while to the
control dish about 1.5 c.c. of N/10 solution hydroxide was added for fresh milk.
Tenth-normal sodium hydroxide was carefully added from a standard burette to the
first dish, the additions being made drop by drop until a faint pink colour was
observed on comparing the two dishes. The results were expressed as lactic acid in
all the cases.
In the determination of acidity at weekly intervals a representative sample
of milk of each cow or buffalo was obtained at the time of evening and morning milk-
ings separately. The acidity was determined immediately after milking in these
samples. When it was necessary to obtain samples from both the milkings, the
same procedure was followed except that the samples collected at the morning
milking were held in ice water for a period not exceeding five hours before the
acidity determination was made.
In studying the influence of lactation on acidity, milk samples were obtained
from two groups, the first group of 10 cows and the second of 15 buffaloes for the
first seven successive milkings following parturition and at less frequent intervals
thereafter. Hydrogen-ion determinations were also made by means of the quin-
hydrone electrode against a saturated calomel electrode at 25°C.
RESULTS
Variations due to species of herd
Practically most of the milk sold in raw or manufactured form in Egypt is
from the buffalo. It is worthy to note that in Egypt, dairy herds whether of
cows or buffaloes, do not constitute pure breeds.
The average acidity of the milk for the Egyptian cows and buffaloes varied
as follows: For cows at morning milking, 0.192 per cent and at evening milking,
0.201 per cent; for buffaloes at morning milking, 0.177 per cent and at evening
milking 0.166 per cent (Table I).
March 1955]
A.
A.
BADR
49
TABLE I
Average titratable acidity of milks of various Egyptian mammals
Species
Buffalo
Cow
M. milkings
E. milkings
M. milkings and E. milkings
M. milkings
E. milkings
M. milkings and E. milkings
Goat: M. milkings
Sheep (Rahmani): M. milkings
Sheep (Ossimi): M. milkings
Horse: M. milkings
Ass: M. milkings
No. of
Samples
1225
781
2006
517
489
1006
35
12
12
27
27
Average
acidity
(Per cent)
0.1767±0.003
0.1656± 0.004
0.1725±0.003
0.1920±0.005
0.2009±0.006
0.1959±0.005
0.1833±0.017
0.1967±0.040
0.2009±0.039
0.0424±0.005
0.0404±0.005
Co-
efficient
of
variation
(Per cent)
15.14
17.74
16.07
16.56
13.85
15.07
17.21
22.93
17.41
42.92
39.11
M. Milkings=Morning milkings
E. milkings = Evening milkings
A glance at Table I shows the following:
(a) In general, the average titratable acidity of cow milk is higher than that
of buffalo milk.
(b) Average titratable acidity of evening milkings is higher than that of
morning milkings in cow milk. In buffalo milk the reverse is true.
(c) There are no significant differences in the average acidity between cow
milk and the milk of goat and sheep, whereas the average acidity
is very low in horse and ass milk.
(d) Coefficients of variation of the Egyptian cow and buffalo milkings are very
high when compared with those found by Caulfield and Riddel [1936]
in the case of the European cow breeds. They found that the co-
efficients of variation of acidity ranged only from 4.0 per cent for
Holstein breed to 4.7 per cent for Guernsey breed and advocated
that these figures show that the data were grouped quite closely
around the main values for each of the different breeds. These great
variations in the case of Egyptian cows and buffaloes are principally
due to differing inheritance as the animals are not of pure breeds.
Variations due to individuality of the cow or the buffalo
While it has been pointed out that the acidity of fresh milk from individual
cows varies widely, little data have been presented to indicate the frequency of
such variations. The acidity of milk from individual cows and buffaloes in the
9A
50
TITRATABLE ACIDITY OF COW AND BUFFALO MILK
[Vol. XXV, Part I,
present study (exclusive samples obtained at the very beginning or end of the lacta-
tion period) varied from 0.08 to 0.26 per cent. These values are in general agree-
ment with those from Henkel [1907] who examined 10,000 or more samples of
individual cow milk and found them varying from 0.1237 to 0.2025 per cent.
McInerney [1920] reported acidity values ranging from 0.10 to 0.22 based upon the
examination of the milk from 42 cows. Sommer and Hart [1921] found the acidity
in 86 tests on individual samples from 31 cows ranging from 0.102 to 0.257 per cent.
TABLE II
Distribution of 2006 and 1006 acidity determinations of freshly drawn milk from
individual buffaloes and cows respectively
Interval per cent
0.08—0.0999
0.10—0.1199
0.12—0.1399
0.14—0.1599
0.16—0.1799
0.18—0.19999
0.20—0.2199
0.22—0.2399
0.24—0.2599
0.26
TOTAL
Per cent of determinations falling within the interval indicated
Buffalo milk samples taken at
Morning
0.00
1.31
0.08
14.36
25.64
31.84
15.02
6.05
1.71
0.00
100.01
Evening
1.20
3.84
7.69
20.49
26.63
25.10
12.29
2.30
0.39
0.26
100.01
Morning
and
evening
0.40
2.30
5.48
16.74
26.03
29.21
13.96
4.58
1.20
0.10
100.00
Cow milk samples taken at
Morning
0.39
1.93
4.64
5.80
13.75
20.31
29.93
17.41
5.42
0.39
100.00
Evening
0.00
0.40
2.05
4.09
10.84
15.33
37.83
10.45
8.58
0.41
99.98
Morning
and
evening
0.20
1.20
3.38
4.97
12.23
17.89
33.80
18.89
6.96
0.40
100.02
When the results on 2006 and 1006 milk samples of buffaloes and cows respec-
tively were grouped in a frequency distribution table according to the percentage
of acid present, it was found that approximately 85 per cent of the samples of
buffalo milk and cow milk fell within the following ranges: Cow milk, 0.16 to 0.23;
buffalo milk, 0.14 to 0.21 per cent. For the two species 8 to 9 per cent of the
samples analysed fell below 0.14 per cent in buffalo milk and below 0.16 per cent
in cow milk only while 6 to 7 per cent of the samples were over 0.21 per cent in
buffalo milk and over 0.23 per cent in cow milk. These data indicate that while
the acidity of milk from individual buffaloes or cows varies widely, the percentage
of samples falling near either extreme is limited.
Variations due to lactation period
Colostrum milk from 10 cows and 15 buffaloes was found to be high in acidity
Table IV). Milk samples from these two groups averaged 0.42 and 0.47 per cent
March 1955]
A.
A.
BADR
51
Fig. 1. Acidity in milk during the lactation period
March 1955]
A.
A.
BADR
53
TABLE III
Acidity and pH values of milk samples following parturition
Items
Number of cows
Average acidity (per cent)
Coefficient of variation
(per cent)
Average pH values
Coefficient of variation
(per cent)
Number of buffaloes
Average acidity (per cent)
Coefficient of variation
(per cent)
Average pH values
Coefficient of
variation
(per cent)
Number of milkings after parturition
1
10
0.42
25
6.28
12
15
0.47
11
6.21
7
2
10
0.33
19
3.36
12
15
0.38
10
6.28
8
3
10
0.26
22
6.35
10
15
0.33
9
6.29
9
4
10
0.27
22
3.36
10
15
0.33
8
6.30
10
5
10
0.28
17
6.36
12
15
0.31
10
6.35
11
6
10
0.27
17
6.37
10
15
0.30
9
6.37
8
7
10
0.26
22
6.38
10
15
0.29
8
6.37
9
8
10
0.26
19
6.39
8
15
0.28
7
6.38
7
9
10
0.23
22
6.42
8
15
0.26
8
6.39
8
10
10
0.22
25
6.45
7
15
0.25
6
6.42
6
54
TITRATABLE ACIDITY OF COW AND BUFFALO MILK
[Vol. XXV, Part I,
buffaloes in these two groups showed very little change, whereas in other instan-
ces, a pronounced decline occurred. If a cow or a buffalo is showing good average
production near the end of a normal lactation period, the decline in acidity does
not appear to be so pronounced as when milk production is low. Data taken from
two groups of cows and two groups of buffaloes during the last month of lactation
substantiate this statement. One group of seven cows with an average daily
production of 5.2 pounds showed an average acidity of 0.122 per cent whereas,
in a second group of five cows producing 9.8 pounds daily, the acidity of milk
averaged 0.155 per cent. One group of five buffaloes with an average daily
production of 6.3 pounds showed an average acidity of 0.116 per cent.
whereas in a second group of 10 buffaloes producing 10.2 pounds daily, the
acidity of milk averaged 0.146 per cent.
TABLE IV
Acidity and pH values of milk samples after calving
Items
Number of cows
Average acidity
(per cent)
Coeficient of variation
(per cent)
Average pH values
Coefficient of variation
(per cent)
Number of buffaloes
Average acidity
(per cent)
Coefficient of variation
(per cent)
Average pH values
Coefficient of variation
(per cent)
Days after parturition
7
10
0.22
25
6.46
12
15
0.21
16
6.48
10
14
10
0.21
22
6.47
15
15
0.20
18
6.50
11
21
10
0.20
18
6.48
10
15
0.18
16
6.54
9
28
10
0.20
19
6.50
8
15
0.18
18
6.57
7
35
10
0.19
18
6.52
8
15
0.17
16
6.58
6
42
10
0.17
15
6.54
9
15
0.16
14
6.58
8
49
10
0.18
18
6.55
8
15
0.16
15
6.59
7
57
10
0.17
17
6.58
6
15
017
15
6.61
5
Variations in acidity from month to month
The average acidity of milk from cows and buffaloes showed some variations
from one month to the next over a 14-month period. The monthly average
acidity of milk varied as follows: Cow milk, 0.141 to 0.26 per cent; buffalo
milk, 0.135 to 0.280 per cent.
Variations in the average monthly acidity in cow and buffalo milk can be attri-
buted primarily to differences in the stage of lactation and to the number and indivi-
duality of the cows and buffaloes represented in any one month. Several cows or
buffaloes freshening in a particular month will result in an increase in the average
acidity for that month. Likewise, if several cows or buffaloes are in advanced