NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
ACCURACY OF A SIMPLE METHOD FOR ESTIMATING
THE PERCENTAGE OF ABNORMAL SPERMATOZOA
IN SEMEN SAMPLES
By S. S. PRABHU and U. D. SHARMA, Animal Genetics Division, Indian Veterinary
Research Institute, Izatnagar, Uttar Pradesh
(Received for publication on 23 May 1953)
F OR the assessment of potential fertility of semen samples used in artificial
breeding, microscopic examination of stained spermatozoa is still the routine
procedure followed in all advanced artificial insemination laboratories. Details of
the various methods in vogue are given by Anderson [1945]. Salisbury, Willet and
Seligman [1942], who tested four methods of making semen smears, found that the
most satisfactory one consisted of drawing a drop of diluted semen between two
microscope slides, drying and clearing in chlorazine and staining in carbol-fuchsin
and gentian voilet. Gunn, et al. [1942] have obtained uniformly satisfactory results
with a modified method of Cary and Hotchkiss [1935] using Mayer's acid haemalum
and eosin. Most of these methods involve considerable labour and time; a certain
amount of skill in preparing the smears and later, in staining them is also essential.
Besides, due to the processing at the various stages of its preparation, an inevitable
increase in certain types of abnormal spermatozoa may occur. Mercier and Salisbury
[1947], who studied the effects of the methods of making, clearing and staining
smears on the proportion of tailless heads and on the proportion of abnormally
formed spermatozoa in fresh and stored semen of fertile bulls, observed that (a) the
'pulling' and 'drop' methods of making smears gave the same result when the
smears were of uniform thickness, (b) fixing smears by heat or by albumen did not
influence the proportion of tailless heads or true abnormals, (c) clearing of smears
with one per cent chlorazine was responsible for producing most of the tailless
heads and (d) with semen of fertile bulls, the staining of thin uncleared smears
eliminated most of the tailless heads.
At artificial insemination field stations elaborate laboratory facilities are not
always available. To meet the special needs of the persons entrusted with such
work it is necessary to place in their hands a method which, while requiring a
minimum of laboratory equipment, may at the same time be expected to give results
of about equal precision as of the elaborate tests. Such a method has been experi-
mented upon by us; in this paper its results are compared with those got by one
of the more elaborate laboratory methods. Incidentally, the comparison also
reveals the relative merits of the two methods of processing of sperm—one simple
and the other complex on the estimates of percentage of morphologically abnormal
spermatozoa.
MATERIAL AND METHOD
The simple method tried consisted in mounting the eosin stained solution of
diluted semen prepared for density estimation, on a slide and placing a cover slip
283
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ESTIMATING ABNORMAL SPERMATOZOA
[Vol. XXIII, Part IV,
on it, as is done in temporary mounts for rapid examination. To prevent the
evaporation of the liquid under the coverslip, it was completely sealed with a thick
side coating of vaseline. Thus protected, the slide can be examined at leisure. In
the second method smears were prepared as described by Gunn, et al. [1942], i.e.
smear was fixed in methyl-alcohol, washed in water, stained with Mayer's acid
haemalum, counterstained with acidified saturated alcohol solution of eosin, washed
in water and dried. For each ejaculate, two slides per method were made, and at
least 300 spermatozoa were examined per slide under high dry power objective.
The head, midpiece and tail abnormalities were classified separately. In all 52
first ejaculates and 53 second ejaculates from nine different buffalo bulls were
studied.
RESULTS
An overall distribution of the different types of abnormalities observed in the
slides prepared by the two methods is given in Table I. The total number of
spermatozoa observed are of the same order, being 67,628 in the first case and
67,745 in the second. In view of this, the actual number of the different types of
abnormalities will indicate their relative frequency of incidence, and comparisons
between methods were made on the basis of these numbers only.
TABLE I
Distribution of the various kinds of morphological sperm abnormalities
found by the two methods
Method
I (Temporary
mount)
II (Permanent
mount)
I (Temporary
mount)
II (Permanent
mount)
I (Temporary
mount)
II (Permanent
mount)
Various kinds of sperm abnormalities
(i) Head
Double
1
1
Micro
85
110
Mega
24
10
Pointed
39
43
Degenerate
215
258
Circular
6
27
Total
370
449
(ii) Mid-piece
Double
2
5
Broken
3
78
Beaded
449
424
Thick
52
65
Filiform
7
12
Total
513
584
(iii) Tail
Double
1
1
Beaded
49
49
Bent
1324
1868
Coiled
1468
1395
Tailless
96
239
Rudi-
mentary
288
169
Broken
at base
30
80
Total
3256
3796
Total sperm observed for I (Temporary mount) method = 67,628
Total sperm observed for II (Permanent mount) method = 67,745
December, 1953]
S.
S.
PRABHU
AND
U.
D.
SHARMA
285
TABLE II
The bull-wise average estimates of morphologically abnormal sperm
Bull number
Method
Abnormality per cent
(i) Head
(ii) Mid-piece
(iii) Tail
(iv) Total
(i) Head
(ii) Mid-piece
(iii) Tail
(iv) Total
17
I
II
4
I
II
30
I
II
19
I
II
21
I
IT
16
I
II
20
I
II
31
I
II
33
I
II
I
Ejaculate
0.41
0.54
3.87
4.82
0.53
0.58
5.11
6.28
0.64
0.66
4.40
5.70
0.78
0.90
5.97
7.65
0.42
0.80
4.86
6.08
0.54
0.66
5.41
6.61
0.62
0.68
6.03
7.33
0.63
0.71
5.30
6.64
0.58
0.78
4.76
6.12
0.70
1.05
5.93
7.68
0.50
0.82
1.91
3.23
0.52
0.68
3.91
5.11
0.90
1.11
9.08
11.09
1.04
1.12
8.56
10.72
0.42
0.84
5.42
6.68
0.62
0.76
5.28
6.66
0.50
0.87
4.32
5.69
0.72
0.78
5.63
7.13
II
Ejaculate
0.68
0.47
3.67
4.82
0.54
0.79
4.30
5.63
0.50
0.70
4.25
5.45
0.70
0.89
5.87
7.46
0.41
0.72
4.99
6.12
0.53
0.80
5.83
7.16
0.39
0.65
5.54
6.58
0.65
1.08
5.27
7.00
0.62
0.92
4.57
6.11
0.75
0.96
5.15
0.86
0.32
0.77
1.81
2.90
0.58
0.66
3.30
4.54
0.77
0.95
7.72
9.44
0.89
1.07
8.15
10.11
0.53
0.68
4.21
5.42
0.72
0.91
4.99
6.62
0.51
0.67
4.16
5.34
0.49
0.79
4.95
6.23
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ESTIMATING ABNORMAL SPERMATOZOA
[Vol. XXIII, Part IV,
The figures presented in Table II are bull-wise averages got with the two
methods. These were tested by the technique of analysis of variance [Snedecor,
1940]. Results are discussed below:
(i) Head abnormality. The actual percentage varied from 0.3 to 1.0. It
formed the smallest portion of the total morphological abnormalities
of the sperm observed. On the whole, the abnormality was higher
in the second than in the first method. The difference was statistically
highly significant.
(ii) Mid-piece abnormality. The actual percentage varied from 0.46 to 1.42
and on an average was slightly higher than (i) above. On the whole,
the abnormality was higher in the second than in the first method.
The difference, after eliminating the variation due to bulls and
ejaculates, was found to be not significant.
(iii) Tail abnormality. These form the largest portion of the total abnormality
observed and varied from 1.91-9.07 per cent. On the whole, the
percentage was higher in the second than in the first method. The
difference was statistically significant. In addition with different
bulls significantly different percentages were obtained.
(iv) Total abnormality. The percentage varied from 3.00 to 11.09. The
second method gave significantly higher percentage than the first.
CONCLUSION
The second method gave, on an average, significantly higher estimates of
morphologically abnormal sperm than the first method. This was so particularly
in case of head and tail abnormalities. As stated earlier, the second method involves
a more elaborate processing of sperm than the first. The higher percentage of
abnormals thus appears to be due in large measure to the additional manipulations
to which the sperm were subjected. This will be seen by the larger number of
broken mid-piece, degenerate head, bent and tailless abnormalities shown by the
second method (Table I). In view of this, it may reasonably be concluded that
the first method gives a better estimate of the percentage of morphologically
abnormal sperm actually present than the second. Besides, it is the easier of the
two methods to learn and practice, requiring minimum laboratory facilities. As
such it can conveniently be adopted by all workers in the field for routine semen
examination without sacrificing accuracy.
SUMMARY
1. Two methods of making semen smears for estimating the proportion of
morphologically abnormal spermatozoa in semen samples were compared. For
the first method, temporary mounts made with eosin stained semen samples were
used, while for the second, permanent mounts prepared as described by Gunn,
et al. [1942] were used. Two slides per method per ejaculate and 52 first and 53
second ejaculates obtained in quick succession were studied. Per slide about 300
spermatozoa were counted.
December, 1953] S. S. PRABHU AND U. D. SHARMA 287
2. Results showed that the first method gave, on an average, a significanty
lower proportion of abnormal spermatozoa than the second method. This was
attributed to the comparatively fewer manipulations to which the speraatozoa
were subjected. It has been shown that the first method, being the easier one of
the two, can be used in place of the second one at field artificial insemination centres
for routine semen examinations without sacrificing accuracy of estimates.
ACKNOWLEDGEMENT
The authors are indebted to Dr P. Bhattacharya, Officer-in-Charge of the
Animal Genetics Section, for his keen interest in this work, and to Messrs, A. R. Roy
and V. N. Amble of the Statistical Section, Indian Council of Agricultural Research,
New Delhi, for their help and advice in the statistical treatment of the data.
REFERENCES
Anderson, J. (1945). Semen of animals and its use for artificial insemination. Imp. Bur. Anim.
Breed. Genetics, 96-97
Cary, W. H. and Hotchkiss, R. S. (1935). Cornell Vet. 25:79
Gunn, R. M. C., Sanders, R. N., and Granger, W. (1942). Bull. Coun. Sci. Industr. Res. Austr
148
Mercier, E. and Salisbury, G. W. (1947). J. Anim. Sci. 6:60-66
Salisbury, G. W., Willett, E. T. and Seligman, J. (1942). J. Anim. Sci. 1:199-205
Snedecor, George, W. (1940). Statistical Methods, XIII +422 pp. (The Collegiate Press, Inc.
Iowa)
PADDY-CUM-FISH CULTURE
PILOT STUDIES CONDUCTED AT HESSERAGHATTA AND
VISWESWARAYA CANAL FARMS IN MYSORE STATE
By H. D. R. IYENGAR, M.Sc., PH.D. (Washington), Fisheries Officer, Department
of Animal Husbandry, Government of Mysore, Bangalore
( Received for publication on 22 April 1953)
(With three text-figures)
O NE of the several items of fishery development work is the practice of rearing
fish in paddy fields. The vast stretches of water 6 in. to 8 in. deep that are
maintained in the paddy fields are rich in fish food, hence form excellent nurseries
for fish. The idea of paddy-cum-fish culture is new only to India, for it is extensively
practised in several other countries of the Indo-Pacific region, Japan, China, Indo-
nesia, etc. The credit for popularising such an enterprise should go to the Chinese,
who have been pioneers in fresh water fish culture. The Chairman of the Sub-
Committee on fish culture of the Indo-Pacific Fisheries Council, F.A.O. states
(F.A.O./IPFC 1951) that fish are reared simultaneously with paddy in over 4,00,000
hectares* of paddy fields in the Indo-Pacific area. Malaya leads in this with 3,00,000
hectares of paddy fields under fish-culture. Indonesia is second with 62,000 hectares,
while Japan has 18,200 hectares, Indo-China and Ceylon have 15,000 hectares each
under paddy-cum-fish culture.
In India several attempts have been made to popularise the practice of paddy-
cum-fish culture. The available literature on paddy-cum-fish culture is limited to
brief reports and notes.
Hora [1949] states that in an acre along with a paddy crop, a fish crop of
100 to 300 lb. could be raised. In China, it has been shown that paddy yield
increased by 10 to 15 per cent by practising fish culture in rice fields. The fish to
an extent control the spread of the stem borer and also the propagation of mosquito
larvae. In Dutch East Indies, fish culture in paddy fields has been characterised
as the 'highest form of pisciculture.' Experiments conducted by the Directorate
of Fisheries, Bengal during 1945-46 showed an increase in paddy yield of 150 lb.
per acre. The F. A. O. Rice Conference at Baguio, in March 1948, recommended
the adoption of this practice in all suitable places.
Hora [1951] while commenting on the paper by Hofstede and Ardiwinata
[1950] on the compilation of data on fish culture in irrigated rice fields in West
Java draws the attention of the public on the importance of fish culture in paddy
fields. The possibilities of growing fish should on no account be underestimated
as the advantages derived are many. Fish production in paddy-fields has been
classified into four categories:
(a) Catch on irrigated rice fields which represents a wild crop.
* One acre = 0.40468 hectare
289
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PADDY-CUM-FISH CULTURE
[Vol. XXIII, Part IV,
(b) Fish culture as a secondary crop, which represents the raising of fish as
an alternate crop.
(c) Fish culture between harvest and planting in fields where there is perennial
irrigation, thus using the fields continuously.
(d) Mixed paddy cultivations and fish culture.
In this system, fish is reared as a catch crop during longer or shorter period in
the paddy fields while the paddy is actually growing. Also the farmer may use the
paddy fields as a nursery for young fish.
In Indonesia, there is an extension service which gives practical demonstration
on the utility of paddy-cum-fish culture and results of these experiments are given in
the paper published by Hofstede and Ardiwinata. According to them, it is quite
practicable to have fish culture in paddy fields and they recommend carp as an ideal
fish for such an enterprise. As the tillering action of fish produces more stems, the
fish control the growth of weeds and the manurial value of fish excreta will result in
an increase of paddy yield which is shown to be 6.3 per cent.
In Mysore State the first attempt at paddy-cum-fish culture was made in Nagar
taluka of Shimoga district in the paddy fields of a private land owner when a few
hundred 1 in. size fingerlings of Ophicephalus striatus were released into a paddy
plot. Even though the experiment was not completed due to certain difficulties
experienced by the landowner on the field, his reactions to this novel venture of
paddy-cum-fish culture was expressed in a letter to the Fisheries Officer, wherein
the landowner says:
"I am glad to report that the small fishes let out in one of my fields of paddy
at Nagar a few months ago are thriving well and attained a size of about
3 in.
In my opinion the scheme of paddy-cum-fish culture can be made a success
in the Malnad parts provided the fishes are let out in the fields immediately
after the paddy seedlings are transplanted so that at least a period of
four months may be allowed for them to grow. I would suggest fishes
of bigger size than those used at Nagar should be used.
As far as I could see only two difficulties are met with. When the rains are
heavy, it is almost impossible in the Malnad parts to avoid the flooding
of fields with the result that the fishes are likely to escape with the
flowing water. Secondly crabs which are in abundance in the paddy
fields bore opening across the mud bunds surrounding the fields and it is
through these opening made by the crabs that the impounded fish find an
exit to get out of the fields."
These points raised by the cultivator are noteworthy and should be provided
with an answer.
In Mysore, the Policy Committee for Agriculture at its sixteenth meeting of
the Mysore Economic Conference held on 11 April, 1949 recommended to Govern-
ment a Scheme for the introduction of paddy-cum-fish culture as a very promising
method of short term food production. In the meanwhile the Fisheries Division of
the Department of Animal Husbandry conducted two pilot experiments in the selec-
ted paddy plots at the Hesserghatta and Visweswaraya Canal farms respectively.
December, 1953]
H.
D.
R.
IYENGAR
291
Fig. 1. Paddy-cum-fish culture
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PADDY-CUM-FISH-CULTURE
[Vol. XXIII, Part IV,
Results of these two experiments are reported for record, while further research is in
progress.
EXPERIMENT No. I
(Conducted at Hesserghatta Grass Farm, from 19 December, 1949 to 21 April, 1951)
An area of 1/4 acre was selected for the experiment on paddy-cum-fish culture.
This plot was divided into small plots of 169 sq. yards each. The bund around these
plots was raised to 1½ ft. Only one plot of 169 sq. yards in the area was selected for
fish culture, the remaining plots were kept as control plots. The water inlet
and outlet were properly screened to prevent the escape of fish fingerlings. A
depression of 2 sq. ft. and 1½ feet deep was dug up near the water inlet to act as
retreats for the fish which were attracted to the water flow at the inlet.
The depth of water maintained in the paddy plots was 4 in. to 6 in., thus a
total volume of 507 cubic feet water was always maintained in each paddy plot.
No special manure was used in the preparation of the paddy field.
One thousand and twenty fingerlings of Ophicephalus striatus of an average
size of 1.84 in. and an average weight of 0.87 gm. were released into one of the
selected plots on 19 December, 1949. The paddy seedlings were 12 in. high and
6 in. apart at the time of fish release.
A daily record of the water temperature in the paddy plot was kept. Every
month, 100 fish were taken and their average length and weights were recorded
(Table I). The experiment lasted 122 days. At the conclusion of the experi-
ment, i.e. on 21 April, 1950, only 193 fish were collected. They measured on an
average, 3.20 in. in length and weighed 9.85 gm. (Fig. 1).
TABLE I
Data on the rearing of 1,020 Ophicephalus striatus fingerlings in a paddy plot at
Hesserghatta Farm
Date
19-12-1949
19-1-1950
20-2-1950
25-3-1950
21-4-1950
Average
length of
paddy
seedling
in inches
12"
16"
24"
40"
42"
Average
length of
fish
fingerlings
in inches
1.84"
1.94"
2.24"
2.70"
3.20"
Gain
in
length
..
0.10
0.30
0.46
0.50
Per-
centage
gain
..
5.43
15.46
20.53
19.52
Average
weight in
gm.
0.87
1.36
3.31
4.05
9.85
Gain
in
weight
..
0.49
0.95
1.74
5.85
Per-
centage
gain
..
56.3
70.0
75.0
143.2
Monthly
average
water
temperature
in centigrade
..
19.4ºC
22.6ºC
27.6ºC
32.3ºC
Water temperature and rate of growth
The water temperature in the paddy plot during the month of December was
19.4ºC., this gradually increased to 32.3ºC. at the conclusion of the experiment,
December, 1953]
H.
D.
R.
IYENGAR
293
Fig. 2. Pddy-cum-fish culture
294
PADDY-CUM-FISH
CULTURE
[Vol. XXIII, Part IV,
Weight of fish in grammes. Scale : Two divisions represent one gramme.
Fig. 3. Paddy-cum-fish culture
December, 1953]
H.
D.
R.
IYENGAR
295
Figure 2 shows the correlation of water temperature with the average growth in
length and weight of the fish.
The water in the Hesserghatta plot is not very fertile and may be classified as
belonging to the Desmid type [Soong, 1947], being predominant in desmid popula-
tion and low in fertility.
Hora [1947] records a growth of 8 in. to 10 in. in five months for 1 in. to 2 in.
carp fingerlings introduced in paddy fields while Gopinath [1950] reports a growth
of 8 in. to 9 in. of Trichopodus trichopterus, an anabantid fish, in the paddy fields of
Malaya. Here, in Hesserghatta experimental paddy plot, 1.84 in. fingerlings of
Ophicephalus striatus have grown to 3.20 in. in 122 days, while their gain in weight
is also not any spectacular (Table I). This record of 1.37 in. of gain in length and
8.98 gm. gain in weight is not very encouraging (Fig. 3). This is due to the low
fertility of the soil and water in the paddy plots. Computing the weight of fish
produced in 169 sq. yards, it is estimated that 109.4 lb. of fish could be produced
along with an acre of paddy. This is in conformity with Hora's [1949] estimates of
100 to 300 lb. of fish production in an acre of paddy.
Paddy yield
Hora [1947] reports an increase of 10 per cent in paddy yield from paddy-cum-
fish culture. At Hesserghatta on harvesting, 90 seers of paddy was collected in
the paddy-cum-fish culture lot and 84 seers from the control lot thus recording an
increase of 7.2 per cent in paddy yield in the plot where fish was also reared.
Fish survival
Out of the 1,020 fish fingerlings introduced, only 193 survived recording a
survival rate of 18.1 per cent. The loss of 81.9 per cent of the fish, besides natural
causes, was also due to predation and escapement.
EXPERIMENT No. II
(Conducted at Visweswaraya Canal Farm from 23 September, 1951 to 28 November,
1951)
An area of 726 sq. yards was selected. This was divided into two plots of
363 sq. yards each. One of the plots was used for fish culture, while the other one
was used as a control lot. Bunds were raised to 1½ ft., screens were provided to the
water inlet and outlet and a small depression was dug up near the water inlet.
The depth of water in the plots was maintained between 6 in. to 9 in. The
plot held a total volume of 1,906 cubic feet of water. The water temperature
ranged from 23.3ºC. to 24.4ºC.
Special manure mixture of 6 2/3 maunds of groundnut cake, two maunds of super-
phosphate and four maunds of ammonium sulphate per acre was used in preparing
the paddy plots. The paddy plant used, was the special deep water paddy.
1,100 fingerlings of Ophicephalus striatus of an average of 1.95 in. and of an
average weight of 1.07 gm. were released into one of the selected paddy plots on
December, 1953]
H.
D.
R.
IYENGAR
297
The author is grateful to Dr P. M. Narainswamy-Naidu, Director of Animal
Husbandry Services, Government of Mysore and to Dr S. L. Hora, Director, Zoolo-
gical Survey of India, for their continued interest and guidance.
REFERENCES
Chacko, P. I. and Ganapati, S. V. (1952). Fish culture in paddy fields. Indian Comm. J.
Dasen, O. F. Paddy-cum-fish culture. A memorandum on food production, Lebanon Mission
Road, Tanjore
F. A. O. Indo-Pacific Fisheries Council. (1951). Preliminary report. Indo-Paoific fisheries
Council, 3rd meeting February 1951, Madras, India. 75, App. 2 and Table 7
Gopinath, K. (1950). Fresh water fish farming in the Malay archipelago. Zool. Soc. India.
2, 2
Hickling, G. F. (1948). Fish farming in the middle and far east. Nature, London. 161, 748-
751
Hora, S. L. (1943). Fish farmer's calender (Department of Fisheries, Government of Bengal),
Amritbazar Patrika, Puja number
—(1947).
Agriculture, Animal Husbandry and fisheries as correlated Industries.
Indian Fmg. VIII, 8, 391-394
—
(1949). Fisheries resources of West-Bengal and their utilization. Sci. and Cult.
XV, 5, 176-180
—
(1948). Indian Fmg. IX, 2
—
(1951). Fish culture in rice fields. Curr. Sci. XX, 7, 171-173
—
(1951). Fish culture in rice fields. Curr. Sci. 20, 171-172
—
(1952). Paddy-cum-fish culture in relation to public health. Curr. Sci. 21, 5, 135
Hofstode, A. F. and Ardiwinata, R. O. (1950). Compiling statistical data on fish culture in
irrigated rice fields in West Java, Landbouw. 12, 469-94
Indian Fisheries. (1951). Report on Indian fisheries by the Ministry of Agriculture, Government
of India
Mitter, S. C. (1934). A recovery plan for Bengal, Calcutta
Nicholson, F. (1907). Notes on fisheries in Japan, Bull. Madras Fish. Dep. 2, 86
Soong-Ming-Kong. (1947). Preliminary report on Sepat Siam investigations in Krian. Paper
No. 9 S. E. Asia fish. conf. Dep. Fish., Malaya