. Vetrepharm, Ontario, Canada , 10–14 h apart, commencing 48 h before sponge removal
and ending 30 h before insemination. Insemination was by laparoscopy at 46 h post pessary withdrawal. Ewes were slaughtered on day 5 post insemination and the numbers
of corpora lutea and large follicles on each ovary were recorded. Each uterine horn was flushed through the oviduct with approximately 20 ml PBS supplemented with 3 BSA.
The flushings were searched for embryos and the embryos were graded according to recognised procedures.
2.4. Statistical analysis Data from Experiment 1 for the incidence of cleavage and blastocyst formation were
Ž .
analysed using the GENMOD procedure of SAS 1996 to fit a generalised linear model with a logit link function. Information on the number of cells per blastocyst and on the
results from staining were assessed by least squares analysis of variance using the GLM Ž
. procedure of SAS 1996 . In all cases, the models employed had terms for replicate,
treatment and individual ram. Data from Experiment 2 on conception rate were analysed using the GENMOD procedure and foetal weights were analysed by ordinary least
squares. The incidence of fertilisation and oocyte recovery were analysed using the Wilcoxon rank test with the individual animal as the experimental unit. A significance
level of 0.05 was used for testing effects in the models.
3. Results
3.1. Experiment 1 Ž
. The proportions of oocytes, averaged over all replicates, fertilised cleaved and the
proportions undergoing development to the blastocyst stage are presented in Table 1. Overall, insemination with sperm frozen using the ‘‘fast’’ freeze protocol produced a
Ž significantly higher cleavage rate compared with the ‘‘slow’’ freeze 57 vs. 26,
. P - 0.001 . Insemination with ‘‘fast’’-frozen semen produced more than double the
Ž .
percentage of blastocysts from oocytes than the slow freeze P - 0.001 based on
oocytes in culture. However, when expressed as a percentage of cleaved oocytes, there was no effect of freezing rate on developmental competence.
Table 1 The effect of freezing rate on the incidence of fertilisation and blastocyst production from sheep oocytes
following maturation, fertilization and culture in vitro Ž
. Values, within columns, with different superscripts are significantly different P - 0.001 .
Ž . Freezing rate
No. of oocytes Cleavage percentage
Blastocyst yield Total
From cleaved
a a
Fast 899
57 28
49
b b
Slow 845
26 13
51
Table 2 Percentages of acrosome-intact, acrosome-damaged and acrosome-reacted ram spermatozoa following post-
Ž .
thaw analysis using the fluorescent probe PNA-FITC least squares meansS.E. No significant difference.
Treatment Acrosome-intact
Acrosome-damaged Acrosome-reacted
Fast frozen 45.74.14
39.72.34 14.73.39
Slow frozen 49.04.14
36.02.34 15.03.39
There were highly significant differences among rams in the fertilising ability of their Ž
. frozen-thawed semen P - 0.001 . Fertilisation rate ranged from 36 to 84 following
insemination with semen frozen by the fast freezing curve, and between 7 and 79 using semen frozen at a slow rate. For five of the six rams used in the experiment, there
was a clear difference in the rate of cleavage in favour of fast freezing; however, one ram had similar fertilisation rates following insemination with semen from either freeze
Ž
. 70 fast, 79 slow . This was reflected in a highly significant ram = freeze interaction
Ž .
Ž .
P - 0.001 . There was also a significant replicate = freeze interaction P - 0.001 that reflected variation in the magnitude of the advantage of ‘‘fast’’ freezing among
replicates rather than any reversal of ranking of the freezing treatments. Freezing rate had no significant effect on the number of cells per blastocyst. The least
Ž .
Ž .
squares means for cell number were 126 12.4 n s 256 and 141 13.0 n s 111 for the ‘‘fast’’ and ‘‘slow’’ treatments, respectively.
There was no significant difference between slow and fast freezing with respect to the viability of frozen-thawed ram spermatozoa as assessed by epifluorescent staining.
However, there was significant ram-to-ram variation in percentage live spermatozoa Ž
. post-thaw
P - 0.01 . Similarly, freezing regime had no significant effect on the percentage acrosome intact, damaged or reacted spermatozoa following post-thaw
Ž .
analysis Table 2 . There was no relationship between ram effects and the percentage viable sperm or between ram effects and IVF results.
3.2. Experiment 2 When the teaser rams were removed, 35 ewes remained unmarked, and 33 of these
were pregnant at slaughter. Method of insemination had a highly significant effect on Ž
. pregnancy rate P - 0.001 . There was a significant effect of freezing rate on pregnancy
Table 3 Effect of freezing rate and method of insemination on pregnancy in ewes
Freezing rate Site of insemination
No. of ewes Percentage of pregnant ewes
No. of foetuses Fast
Uterine 30
60 28
Slow Uterine
29 40
17 Fast
Cervical 30
13 4
Slow Cervical
30 –
Table 4 Ovulation rate, embryo recovery and fertilisation for superovulated ewes inseminated into the uterine lumen
using two frozen-thawed semen types Ž
. Values, within rows, with different superscripts are significantly different P - 0.001 .
Freezing rate Fast
Slow No. of ewes:
Inseminated 23
24 Ovulated
22 22
Yielded ovarembryos 18
21 Yielded embryos
18 14
Mean ovulation rate 11.4
7.5 No. of oocytes:
Ž .
Ž .
Recovered 95 36
118 73.6
a b
Ž .
Ž .
Fertilised 71 81.4
49 39.3
Ž .
rate P - 0.01 , with 22 of the pregnant animals having been inseminated with
Ž .
fast-frozen semen Table 3 . Rate of freezing had no significant effect on foetal weight and there was no individual ram effect on this trait.
The results from the evaluation of fertilisation rate in vivo using superovulated ewes are given in Table 4. The higher mean ovulation rate in the ‘‘fast’’ group is attributable
to two animals having in excess of 20 ovulations each. Freezing rate had a highly Ž
. significant P - 0.001 effect on the number of ewes that yielded fertilised eggs. In the
Ž .
fast freeze group, all ewes that ovulated and yielded oocytes on flushing 18r22 had some ova fertilised. Of the ewes on the slow freeze treatment that ovulated and yielded
Ž .
ova on recovery 21r22 , seven had all oocytes unfertilised. There was a significant Ž
freezing effect on the fertilisation rate of recovered ova 81.4 for fast freezing
. compared with 39.3 after slow freezing; P - 0.01 . Data in Table 5 summarise the
developmental stages of the embryos following recovery. There was no effect of freezing rate on the proportion of cleaved oocytes that proceeded to the morularblasto-
cyst stage. There was no significant difference between individual rams in terms of their fertilising ability.
Table 5 Stages of embryonic development following laparoscopic intrauterine insemination in superovulated ewes
MrB: morulaerblastocysts expressed over total oocytes recovered. Ž
. Values, within rows, with different superscripts are significantly different P - 0.001 .
Freezing No. of fertilized
Cell stage Percentage
Percentage
a
rate oocytes
attained MrB
MrB 2–8
8–16 Morula
Blastocyst cells
cells
a
Ž .
Ž .
Slow 49
2 13
34 29 34r118
69 34r49
b
Ž .
Ž .
Fast 71
10 5
51 5
59 56r95 79 56r71
a
Expressed as a percentage of fertilized oocytes.
4. Discussion
