Aust. J. BioI. Sci., 1974, 27, 423-5
Fertility following Inseminations
with Frozen-Thawed Reconcentrated
and Unconcentrated Ram Semen
D. Visser and S. Salamon
Department of Animal Husbandry, University of Sydney, Sydney, N.S.W. 2006.
Abstract
Fertility results are presented for ram semen that was pellet-frozen in tris-glucose-yolk-g1ycerol
diluent. Lambing following insemination with concentrated and unconcentrated inseminates was
35·0% (49/140) and 38·6% (56/145) respectively. Increasing the number of motile spermatozoa and
double insemination improved the fertility.
Introduction
It has been reported that, when using ram semen frozen in a raffinose-citrate-yolk
diluent, reconcentration of the thawed semen by centrifugation was necessary before
insemination in order to establish an adequate cervical population of spermatozoa
and to obtain satisfactory fertility (Lightfoot and Salamon 1970; Salamon and
Lightfoot 1970). Subsequently, Visser and Salamon (1973) used semen frozen in a
tris-based diluent and found that unconcentrated thawed semen may also be used
successfully for insemination. The practical advantages of omitting reconcentration
of the thawed semen before insemination motivated further investigation and the
results are presented in this paper.
Methods
The main purpose of the experiment was to examine the fertility following insemination with
reconcentrated or unconcentrated frozen-thawed semen. In addition, two inseminating doses were
used for each type of semen and single and double inseminations were also performed.
Semen was collected from five Merino rams by artificial vagina and ejaculates of good initial
motility were pooled. Half of the pooled semen was diluted 1 : 4 (semen: diluent v/v) with 300 mM
tris-27 . 75 mM glucose-94· 7 mM citric acid (Salamon and Visser 1972) and the other half at a ratio of
1 : 2 with 360 mM tris-33 . 3 mM glucose-113 . 7 mM citric acid in order to obtain the same amount of
ingredients in the diluted semen for both dilution rates. The diluted semen in each case contained
12% (v/v) egg yolk and 4% (v/v) glycerol. Further processing and freezing of semen was done as
described previously (Visser and Salamon 1973).
Reconcentrated semen was obtained by thawing the frozen pellets (pre-freezing dilution 1 : 4) in
test tubes containing tris (300 mM)-fructose (55·5 mM)-dtric acid (94·7 mM) and held in a water-bath
at 3rC (thawing dilution 1 : 2, pellets: thawing solution v/v). The thawed semen was centrifuged,
at 1000 g for 10 min and the supernatant removed to obtain a cell concentration of 1 ·8 X 109 motile
spermatozoa per millilitre. Volumes of 0·05 and 0·10 ml containing 90x 10 6 and 180x 106 motile
spermatozoa were inseminated. For unconcentrated inseminates, the pellets (1 : 2 dilution prefreezing) were thawed in dry test tubes at 3rc and volumes of 0·125 and o· 250 m1 containing 90 and
180 x 106 motile spermatozoa were used.
Mature Merino ewes were inseminated at the second oestrus after synchronization with intravaginal sponges (Robinson 1965). Oestrous ewes were detected by vasectomized rams previously
424
D. Visser and S. Salamon
tested by electroejaculation. The ewes were drafted twice daily (OSOO and ISOO h) and inseminations
were carried out 12-14 h (single) or 12-14 and 23-25 h (double) after drafting. The oestrous animals
were allocated randomly into treatment groups and single and double inseminations with both types
and doses of inseminate were performed on each day.
Results and Discussion
The lambing results are presented in Table 1. Fertility was similar for reconcentrated and unconcentrated thawed semen. The X2 analysis of data showed a significant
effect in favour of both higher number of spermatozoa (90xI06: 44/144,30·6%;
180xI06: 61/141, 43·3%; P < 0'05) and double insemination (P < 0·01). It
should be pointed out, however, that both these 'subtreatments' were confounded in
the experiment and a clear picture of the effect of each could not be gained. The apparTable 1. Lambing results following insemination with frozen-thawed reconcentrated and unconcentrated
semen
No. of
motile spermi
inseminate
Single insemination:
No. of ewes
No.
Percentage
inseminated
lambing
1ambing
Double insemination:
No. of ewes
No.
Percentage
inseminated
lambing
lambing
Mean
percentage
lambing
Reconcentrated semen
90x 106
ISO X 106
36
37
11
Totals and
means
73
IS
7
19·4
29·7
34
33
12
19
35·3
57·6
27·1
42·9
24·7
67
31
46·3
35·0
Unconcentrated semen
90 X 10 6
ISO X 10 6
39
3S
12
13
30'S
34·2
35
33
13
IS
37·1
54·5
33·9
43·7
Totals and
means
77
25
32'5
6S
31
45·6
3S'6
150
43
2S'7
135
62
45·9
36'S
Overall totals
and means
Table 2.
Lambing results in relation to the number of motile spermatozoa
inseminated
No. of motile sperm
No. of ewes lambing/
ewes inseminated
Percentage
lambing
19/75
24/75
25·3
32·0
25/69
37/66
36'2
56'1
Deposited by one insemination
90 x 106
ISO X 106
Deposited by two inseminations
ISOx 106
360 X 106
ent benefit of double insemination can be accounted for on the basis of increased
number of spermatozoa. Table 2 shows that fertility improved with the increase in
total number of motile spermatozoa deposited by either one or two inseminations
12-25 h after detection of oestrus, which can be considered as being within the optimum time range (Salamon and Lightfoot 1970; Salamon 1971, 1972; Schindler and
Amir 1973). The question therefore arises whether a single insemination with a large
number of motile spermatozoa (e.g. 360 X 10 6 ) will produce fertility results equivalent
Fertility of Frozen Ram Semen
425
to two inseminations (each with 180 X 106 motile cells) and hence economize on the
labour involved. Further investigation is needed to examine the merits of each procedure. It should, however, be taken into account that while a large number of motile
spermatozoa in the appropriate inseminate volume can be achieved by reconcentration
of the thawed semen, the number of cells in the unconcentrated inseminate is dependent mainly on the pre-freezing dilution ratio, which in turn can influence the cell
recovery upon thawing (Lightfoot and Salamon 1969).
Acknowledgments
The authors thank Professor T. J. Robinson for advice and constructive comments.
Acknowledgment is also made to Mr G. P. Walker and sons, 'Ledgworth', Yass,
N.S.W., for provision of sheep and facilities. The work has been aided by grants from
the Australian Research Grants Committee and the Australian Wool Research Trust
Fund.
References
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prior to freezing on survival of spermatozoa. Aust. J. BioI. Sci. 22, 1547.
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Aust. J. BioI. Sci. 26, 513.
Manuscript received 12 November 1973