ANIMAL

REPRODUCTION
SCIENCE
ELSEVIER Animal ReproductionScience 39 (1995) 215-222

Superovulatory response of Chios sheep to PMSG

during spring and autumn
F. Samartzi a, C. Boscos a'*, E. Vainas b, P. Tsakalof
aDepartment of Clinics of Small and Large Animals, Veterinary Faculty, Aristolean University of Thessaloniki,
GR 540 06 Thessaloniki, Greece
bN.A.R.F., hzstitute of'Reproduction and AI, GR 570 08 Ionia, Greece
Accepted 5 January 1995

Abstract

To study the superovulatory response of Chios sheep to pregnant mares' serum gonadotrophin
(PMSG), two experiments were carried out; one in spring and one in autumn. Four doses of PMSG
( 1500 IU, Group 1; 1000 IU, Group 2; 750 IU, Group 3; 500 IU, Group 4; controls, Group 5) were
tested on 46 ewes. Oestrus was synchronised by means of MAP intravaginal sponges and PMSG was
injected i.m. at the time of sponge withdrawal. When in oestrus, ewes were naturally mated. On Day
7 after sponge removal, mid-ventral laparotomy was performed and the uterine horns and/or oviducts
were flushed with 20-40 ml Dulbecco's phosphate-buffered saline supplemented with 15% foetal
bovine serum (FBS). The embryos were examined under a dissecting microscope and were evaluated
according to morphological criteria.
The interval from sponge removal to the onset of oestrus was significantly (P<0.001) shorter in
autumn than in spring in all groups. No significant differences regarding superovulatory response,
collection and fertilisation rate or numbers of ova and embryos collected were found between spring
and autumn. The clinical signs of oestrus started earlier (P < 0.001 ) in all PMSG treated animals than
in the controls, both in spring and in autumn. The highest ovulation rate was recorded in Group 2
(5.9+: i.0), followed by Groups 1 (5.0+:0.9), 3 (3.9+:0.5), 4 (26+:0.4) and 5 (1.3+:0.1). The
increase observed in total ovarian response (corpora lutea + large anovulated follicles) parallelled the
increase of PMSG dose ( 10.7 +: 1.6, 7.7 +: 0.9, 4.5 + 0.6, 3.4 +: 0.5 and 1.8 +: 0.2 for Groups 1, 2, 3, 4
and 5, respectively). The highest mean number of ova was collected from Group 3 (3.4 + 0.5),
followed by Groups 2 (2.6+0.4), 4 (2.2+0.3), 1 (1.6+:0.5) and 5 (1.1 +:0.1 ). The higher doses
of PMSG ( 1500 and 1000 IU) significantly increased the mean number of anovulated follicles and
significantly decreased recovery rate. Mean number of high viability embryos collected per ewe
treated (0.9 +: 0.6, 1.5 + 0.4, 2.2 +: 0.5, 1.5 +: 0.4, 0.9 + 0. ! for Groups 1, 2, 3, 4 and 5, respectively)
was not improved by PMSG dose.
It is concluded that Chios sheep can be superovulated in autumn and in spring wi:th similar resuIt~.
Clinical signs of oestrus are initiated earlier in autumn than in spring. PMSG trea~.ment shortens the

* Corresponding author at: 28-Octovriou 19, 56728 Thessaloniki,Greece.

0378-4320/95/$09.50 © 1995Elsevier Science B.V. All rights reserved

SSD103 78 -4 320 ( 95 ) 01380-6
216 F. Samartzi et al. / Animal Reproduction Science 39 (1995) 215-222

interval from sponge removal to the onset of oesrrus. Although PMSG does not seem to be the most
suitable hormone for the induction of superovulation in Chios sheep, a dose of 750-1000 IU PMSG
gives satisfactory results; higher doses are associated with side effects in a significant number of
animals (many anovulated follicles, low recovery rate).

Keywords: Sheep; Superovulation; PMSG; Seasonal dynamics

1. Introduction

Superovulation is a prerequisite for the collection of a larger than normal number of

embryos and for the realisation of commercially applicable embryo transfer programmes.
Although much work has been done in this field, the wide variation in the superovulatory
response is still the weakest link in the chain of events required for successful embryo
transfer programmes (Seidel and Elsden, 1989); less than 40% of the causes for variability
in superovulatory response has been explained to date (Hahn, 1992). However, sheep
breed, dose of hormone and season have been recognised as some major causes of variation
in the superovulatory results (Gherardi and Lindsay, 1980; Mutiga and Baker, 1982b;
Bondurant, 1986; Torres et al., 1987).
Limited information concerning the superovulatory response of Chios sheep is currently
available. The Chios breed is a highl~ productive indigenous dai~ breed ;'hth high repro-
ductive rate (three births in 24 months), early !naturity (females are first fired at 8 months
of age), high incidence of twin and triple births (fecundity rate 1.75) and practically no
anoestrous period (high proportion of animals exhibit oestrus and have ovulations through-
out the year), which is bred in many regions of Greece (Katsaounis, 1980; Vosniakou,
1983). As a result of the increasing importanc; of embryo transfer and related techniques
in the livestock industry, a method for the sup~:rovulation of Chios ewes was investigated.
Pregnant mares" serum gonadotrophin (PMSG ! was chosen because its d,~sage is conven-
ient and, theret'ore, it can be easily applied in open sheep flocks. The main purposes of this
work were to study the superovulatory response of Chios sheep to PMSG,, to determine a
superovulatory treatment protocol lbr this breed and to investigate the possibility of using
Chios sheep as embryo donors both in spring (anoestrous period for sheep in Greece) and
in autumn (oestrous period for sheep in Greece).

2. Materials and methods

The experiments were conducted at the Institute of Reproduction and AI, Ionia, Thes-
saloniki, during 1990. Forty-six non-pregnant, non-lactating Chios ewes, 2--4 years old, of
42-50 kg iiveweight, were randomly allocated to ten experimental groups ( 1500 IU, Groups
IS and IA; 1000 IU, Groups 2S and 2A; 750 IU, Groups 3S and 3A; 500 IU, Groups 4S
and 4A; controls, Groups 5S and 5A; S representing spring and A, autumn). Fifteen of the
control ewes ( 12 from Group 5S and three from Group 5A), and 11 ewes from the other
groups (two from 4S, two from 3S, five from 4A and two from 3A) were used t,wiee (Table
I).
 F. Samartzi et al. / Animal RepnMuction Science 39 ¢1995) 215-222 217

Table 1
Experimental design (numbers of ewes used during the spring and the autunm experiments

PMSG dose Spring Autumn

(IU)
Group I st 2nd Tolal G~oup I st 2rid Total
embryo embryo embryo embryo
collection collection collection collection

1500 IS 4 2 6 IA 3 2 5
1000 2S 4 3 7 2A 4 3 7
750 3S 2 4 6 3A 4 3 7
500 4S 2 4 6 4A 5 - 5
0 (control) 5S 15 3 18 5A 3 2 5

Total 27 16 43 i9 I0 29

Oestrus was synchronised by means of MAP intravaginal sponges (Repromap, Upjohn;

Kalamazoo, MI) for 14 days and PMSG (lntergonan, Intervet International B V, Nether-
lands) was injected i.m. at the time of sponge withdrawal. Oestrus was detected twice daily
using vasectomised rams and each ewe was naturally mated to two to three different rams
of known fertility during oestrus. On Day 7 after sponge removal (Days 4--6 of the oestrous
cycle, onset of oestrus designated Day 0), animals were sedated with 0.05 mg kg - ' xylazine
(Rompun, Bayer; Leverkusen, Germany) and locally anaesthesised by infiltration of the
incision area with 12-15 ml 2% xylocaine; mid-ventral iaparotomy was performed and the
uterine horns and/or oviducts were flushed with 20--40 ml of medium, according to the
modified technique of Tervit and Havik (1976). The flushing medium consisted of Dul-
becco's phosphate-buffered saline (PBS) supplemented with 1 g I - ~ glucose, 0.036 g I - '
sodium pyruvate, antibiotics and 15% heat inactivated PBS. The embryos were examined
under a dissecting microscope and evaluated according to morphological criteria (Betteridge
and Moore, 1977; Lenh-Jensen, 1986). Embryos of excellent or good morphology were
characterised as 'high viability embryos' and embryos that were retarded or degenerated as
'low viability embryos'.

2.1. Statistical analysis

Data were analysed by analysis of variance (ANOVA). Original data were transtbrmed
to square roots; this enabled the transformed variables to approximate more closely to a
normal distribution and hence to stabilise the between-group variance. Multiple comparisons
were calculated using least significant difference (LSD), with significance being taken at
the level of P < 0.05.

3. Results

All ewes except one in Group 5 exhibited clinical signs of oestrus. The interval between
sponge removal and the onset of oestrus was significantly (P < 0.001 ) shorter during the
autumn experiment than during the spring experiment (Table 2).
218 F. &zmartzi et at./Animal Reprodz~ction Science 39 (1995) 215-222

Table 2
Interval from sponge withdrawal to the onset of oestrus (mean :!: SEM) during spring and autumn in Chios ewes superovulated
with PMSG

Spring Autumn

Group PMSG dose Ewes Interval to Group PMSG dose Ewes Interval to
(IU) (no.) oestrus onset (IU) (no.) oestrus onset
(days) (days)

IS 1500 6 1.94-0.1a IA 1500 5 1.2+0.1c

2S I000 7 2.04-0.1a 2A !000 7 1.4+0.2c
3S 750 6 1.9 "t-O.1a 3A 750 7 !.5 + O.lc
4S 500 6 2. I + 0. I a 4A 500 5 ! .4 4. 0.2c
~;S 0 (control) 18 2.7 4.0.1b 5A 0 (control) 5 2.1 4.0.1d

"'"'¢'dMeans followed by different letters are significantly different ( LSD test, P < 0.05).

Table 3
Superovulatory response and quality of embryos collected in five groups of Chios ewes superovulated with PMSG ( mean 4- SEM)
(a) Ovarian response

Group PMSG dose Ewes Corpora lutea per Largea anovulated Total ovarian responseb
(IU) (no.) ewe treated follicles per ewe per ewe treated
(ovulation rate) treated

I 1500 !! 5,0:i: 0.9a 5.7 4.1.3a 10.7 4-1.6a

2 1000 14 5.9 4-0.8a 1.8 4-0.4b 7.7 4-0.9a
3 750 13 3.9 4-0.5b 0.6 4- 0.2c 4.5 4-0.6b
4 500 II 2.6 4-0.4c 0.7 4-0.3c 3.4 ±0.5b
5 0 (control) 23 1.3 ±O.ld 0.5 4-0.2c 1.8 ±0.2c

(b) Numbers of ova collected and embryos recovered per ovum

Group PMSG dose Ewes Collection rate~ per Ova collected per Fertilization rated per ewe
( IU ) (no.) ewe treated ewe treated treated
(%) (%)

I 1500 II 30,9~ 10.Sa 1.6 ±0.5a 64.8± 14.8

2 1000 14 52.4 ±8.6a 2.6 ±0.4b 68.2:t: I!.1
3 750 13 86.3 4. 4.8b 3.4 ±0.5c 72.4 ± 1 !.4
4 500 !i 85.8 ± 5.5b 2.2 4.0.3b 59.1 ± 13.2
5 0 (control) 23 85.7 4. 7.0b I. ! 4-O.I a 90.5 + 5.6

(c) Quality of embryos collected

Group PMSG dose Ewes Embryos collected Proportion of high High viability embryos
( IU ) ( no. ) per ewe treated viability embryos per ewe treated
per ewe treated
(%)

I 1500 !! 0.9 4-0.5a ! 00.0 0.9 4-0.5

2 1000 14 !.8 4-0.3be 83.3 4.9.7 1.5 4.0.4
3 750 13 2.4 4. 0.5c 86.9 4. 8.3 2,2 4.0.5
4 500 !I !.6 ±0,4b 97.7 ± 2.3 1.5:1:0.4
5 0 (control) 23 1.04.0.1d 90.54.6.6 0.94.0.1

"Diameter over 0.5 cm.

~Corpora lutea plus large anovulated follicles.
~Number of ova collected per corpus luteum.
dNumber of embryos collected per ovum.
a,b,c,dMeans in the same column followed by different letters differ significantly (LSD test, P < 0.05).
 F. Samartzi et al. / Animal Reproduction Science 39 (1995) 215-222 219

With respect to superovulatory response, collection and fertilisation rate or numbers of

ova and embryos collected, neither significant differences between spring and autumn nor
significant interactions for season by PMSG dose were detected. Therefore, the data from
the two experiments were pooled. The overall results of this experiment are presented in
Table 3.
The hormone treatment significantly (P < 0.001 ) shortened the interval between sponge
withdrawal and onset of clinical oestrus in all experimental groups in comparison with the
controls. PMSG dose did not influence the time of the onset of oestrus (Table 2).
PMSG dose had a significant positive effect on ovulation rate (P < 0.001 ), number of
anovulated follicles (P <0.001), total ovarian response (P <0.001) and number of ova
collected (P < 0.001 ) per ewe treated (see Table 3).
In contrast, PMS.G dose had a significant (P < 0.001 ) negative effect on the collection
rate (Table 3). The mean collection rates in Groups 1 and 2 were significantly lower than
those in Groups 3, 4 and 5. The effect of hormone treatment on fertilisation rate was not
clear. While there were neither significant differences between each experimental group
and the controls nor among the four experimental groups (Table 3), the difference in
fertilisation rate between all the PMSG-treated animals regardless of the dose (66.6 5: 6.0%)
and the controls (90.5 + 5.6%) was statistically significant (P < 0.05).
The effect of PMSG dose on the number of embryos collected approached significance
(P = 0.0874). The LSD (P < 0.05) test revealed that ewes from Groups 2, 3 and 4 yielded
a significantly greater mean number of embryos than the controls. Ewes treated with 750
and 1000 IU PMSG yielded a significantly higher mean number of embryos than those
treated with 1500, 500 or 0 IU PMSG.
PMSG dose had no significant effect on either the percentage or the mean number of
high viability embryos collected per ewe in these experiments.

4. Discussion

The results of these experiments demonstrated that Chios sheep can be superovulated
with PMSG in spring (anoestrous period for sheep in Greece) as well as in autumn (oestrous
period for sheep in Greece). The time that elapsed from sponge withdrawal to the onset of
oestrus was significantly shorter in autumn than in spring, in all groups, but no significant
differences were found regarding all the other parameters studied. In contrast, seasonal
differences in superovulatory response are observed i.n sheep of most Euro~an, American
aad Australian breeds (Gherardi and Lindsay, 1980; Tones et al., 1987). Since the mech-
a~fism controlling the number of mature, oestrogen active follicles and therefore ovulation
rl.te
~.
is active during seasonal anoestrous (Webb et al., 1992), seasonal differences in
ovulauon rate (Hendy and Bowman, 1974) and superovulatory response observed in sheep
are associated with seasonal differences in the sensitivity of the ovary to exogenous gonad-
otrophins (Gherardi and Lindsay, 1980). Evidently, seasonal differences in the sensitivity
of the ovary in Chios sheep are minimal.
The response of Chios sheep to 1 5 ~ !!l PMSG was generally lower than that reported
for other sheep breeds, e.g. Romney (Whyman and Moore, 1980), Border Leices-
ter × Scottish Blackface (Scudamore et al., 1992), Booroo!a × Romn~,%Finnish Landrace,
220 F. Samartzi et el./Anintal Reproduction Science 39 (1995) 215-222

Timhadite, D'Mann and Galway (Quirke et al., 1987). In contrast, the response of ewes to
1000 and 750 IU PMSG was favourably comparable to that of other breedsmMerino
(Walker et al., 1989; Jabbour and Evans, 1991 ), Finnish Landrace, Timhadite, D'Mann,
Galway and Booroola × Romney (Quirke et al., 1987)wto the same doses. Finally, the
response of Chios ewes to 500 IU, although generally higher than that of other breedsm
Galway (Quirke et al., 1979), Timhadite, D'Mann and Booroola × Romney (Quirke et al.,
1987)--to similar doses of PMSG, cannot be considered as superovulation since ovulation
rate was 2.6 + 0.4 and only two out of 11 animals had more than three corpora lutea on their
ovaries.
Ovulation rate increased as PMSG dose increased only up to 1000 IU. This result confirms
those of several workers who showed that above a certain dose of hormone--PMSG (Mutiga
and Baker, 1982b), follicle-stimulating hormone (FSH) (Smith, 1984) or HAP (Crosby
et al., 1980)--the superovulatory response of ewes reaches a plateau or even decreases. It
is possible that either the amount of luteinising hormone (LH) is not sufficient for the
ovulation of all the follicles that have developed (Whyman and Moore, 1980) or that some
of the follicles are not capable of responding to ovulatory stimulus perhaps owing to
abnormal maturation after PMSG injection (Moor et al., 1985; Hafez, 1987).
Collection rate in Groups I and 2 was very low. The fact that the collection rate in Groups
3, 4 and 5 was high proves that the collection technique was applied satisfactorily. Moreover,
no case of premature luteai regression, frequently associated with loss of embryos (Bon-
durant, 1986; Torres et al., 1987), was observed. Therefore, the low collection rate in
Groaps I and 2 might be attributed to:
(a) loss of ova into the peritoneal cavity (Mutiga and Baker, 1982a);
(b) accelerated egg transport through the reproductive tract (Hafez, 1987; Hawk, 1988);
(c) alteration of the composition of oviductal or/and uterine secretions due to the altered
hormone pattern of superovulated animals (Wilmut et el., 1985) resulting in either a uterine
environment hostile to the vulnerable early embryo (Betteridge, 1977) or entrapment of
embryos in the oviduct (sticking to the uterine wall), the latter explanation is supported by
the fact that in three animals from Groups I and 2 (in which part of the embryos were
recovered) pregnancy was established in spite of the flushing and normal lambs were born;
(d) quick disintegration of unfertilised ova and/or degenerated embryos, even in the
short interval between ovulation and embryo collection because of abnormal maturation of
oocytes after PMSG induction of superovulation (Moor et al., 1985; Foote and Ellington,
1988).
The fertilisation rate during these experiments was similar to that in experimen.ts in ~,hich
natural mating had been used (Mutiga and Baker, 1982b; Walker et al., 1989), was higher
than that usually obtaiaed with artificial insemination (Whyman and Moore, 1980; Boland
et ai., 1983) but was lower than that obtained with intrauterine insemination (Walker et al.,
1989).
The mean number of high viability embryos collected from Chios sheep was lower than
the mean number in other sheep breeds after induction of superovulation with PMSG
(Walker et al., 1989; Scudamore et al., 1992) or FSH (Smith, 1984; Cognie et al., 1986).
The number of high viability embryos appears not to have improved with PMSG treatment,
mainly because of the extensive loss of ova/embryos from the ewes treated with 1500 or
1000 IU PMSG.
 F. Samartzi et al. / Animal Reproduction Science 39 (1995) 215-222 22 !

It is concluded that Chios sheep can be superovulated in autumn and in spring with similar
results. Clinical signs of oestrus are initiated earlier in autumn than in spring. P M S G
treatment shortens the interval from sponge removal to the onset o f oestrus (regardless of
the dose u s e d ) . T a k i n g into account the ovulation rate, the number of high viability embryos
collected and the incidence of side effects, a dose of 7 5 0 - 1 0 0 0 IU P M S G appears to be the
most appropriate for superovulation in Chios sheep, although P M S G does not seem to be
the most suitable hormone for the induction of superovulation of this breed. Higher doses
are associated with side effects ( m a n y anovulated follicles, low recovery rate) in a signif-
icant n u m b e r of animals.

Acknowledgements

The authors are indebted to Dr. A. Bourla and Dr. Th. Kouskoura for their assistance
during surgeries.

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