Animal Reproduction Science 81 (2004) 295–312

Hormonal profiles and embryo survival of

sows subjected to induced stress during
days 13 and 14 of pregnancy
P. Razdan b,∗ , P. Tummaruk a , H. Kindahl b , H. Rodriguez-Martinez b ,
F. Hultén b , S. Einarsson b
aDepartment of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Sciences,
Chulalongkorn University, Bankok, Thailand
b Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine,

Centre for Reproductive Biology in Uppsala (CRU), Swedish University of Agricultural Sciences (SLU),
P.O. Box 7039, SE-750 07 Uppsala, Sweden
Received 16 April 2003; received in revised form 29 September 2003; accepted 29 September 2003

Abstract
Group housing of sows during the mating and gestation period has become the overall common
management practice in Sweden. Loose housing is probably less stressful for the animals because
it allows them more opportunities to behave naturally, but mixing unfamiliar sows does create a
stressful situation due to aggressive interactions, which can lead to food deprivation. The objective
of the present study was to investigate and compare the effects of stress in form of food deprivation
and ACTH administration at days 13 and 14 of pregnancy (day 1, first day of standing oestrus) in
sows. The hormonal secretion of the sows and foetal survival by day 30 of pregnancy was, therefore,
studied in 17 crossbred multiparous sows. The sows were randomly allocated into three different
groups: one control (C-) group; one food deprived (FD-) group, which was deprived of food from the
morning of day 13 of pregnancy until the evening meal on day 14; and a third group (A-), which was
given intravenous injections of synthetic ACTH (Synachten® Depot), at a dose of 0.01 mg/kg body
weight every sixth hour from 6 a.m. on day 13 until 6 a.m. on day 15 of pregnancy. All sows were
slaughtered at 30 ± 2 day of pregnancy and the genital tracts recovered. Total number of corpora
lutea (CL), total number of viable or nonviable embryos and foetal survival rates were determined.
Samples from the peripheral blood circulation were collected four times a day from day 12 until
slaughter, except during days 13–15 when blood was collected every second hour. The blood samples
were analysed for cortisol, progesterone, oestrone, prostaglandin F2␣ -metabolite, oestrone-sulphate,
insulin, free fatty acids and triglycerides. FD-sows had increased levels of cortisol, free fatty acids
and progesterone, as well as a lowered level of insulin in the peripheral blood plasma, while A-group

∗ Corresponding author. Tel.: +46-18-671342; fax: +46-18-673545.

E-mail address: pia.razdan@og.slu.se (P. Razdan).

0378-4320/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2003.09.005
296 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312

sows had increased levels of both cortisol and insulin compared with the C-group. Treatment with
ACTH seemed to cause a 2-day delay in the increase of oestrone, from day 19, as seen in the FD-
and C-group, to day 21 of pregnancy. At the time of slaughter, there were no significant differences
among groups in terms of total number of foetuses and foetal survival rate. The results of the present
study suggest a capacity of the sow to compensate for the influence of induced moderate stress at
the time of pregnancy when maternal recognition occurs.
© 2003 Elsevier B.V. All rights reserved.

Keywords: Stress; Maternal recognition of pregnancy; Cortisol; Progesterone; PGF2␣ ; Insulin; Free fatty acids

1. Introduction

The negative impact of stress on pigs and their reproductive performance is well es-
tablished, and therefore, an important factor to consider in animal husbandry. Due to new
laws based on animal welfare considerations, group housing of sows during the mating and
gestation period has become the overall common management practice in Sweden today.
Group housing of sows is considered to be less stressful for the animals because it allows
them more opportunities to behave naturally. To minimise stress, it is necessary to keep
groups of familiar sows intact, but this is often not possible in commercial pig production.
Mixing unfamiliar sows does create a stressful situation due to aggressive interactions that
can lead to food deprivation (Mendel et al., 1992; Brouns and Edwards, 1994; Tsuma et al.,
1996b). However, the periods in early pregnancy at which the sows and their foetuses are
most vulnerable to stress is yet to be evaluated.
The majority of embryonic death occurs before day 18 of pregnancy (Pope and First,
1985; van der Lende and Schoenmaker, 1990; Lambert et al., 1991). The extent of embryo
mortality may be affected by many different factors, both physiological such as breed
difference, and nonphysiological such as stress during early pregnancy. Induced stress in
the forms of food deprivation and/or ACTH-treatment has been shown to cause hormonal and
metabolic disturbances during the first 2 days after ovulation (Mburu et al., 1998; Mwanza
et al., 2000a,b; Razdan et al., 2001; Razdan et al., 2002). Stress also has negative effects on
the rates of oviductal sperm transport, of spermatozoa attached to the zona pellucida and
on embryo cleavage, in sows slaughtered up to 6 days after ovulation (Mburu et al., 1998).
Another possibly critical period during early pregnancy of the sow is day 13 (counting
first day of oestrus as day 1). This is the time when maternal recognition of pregnancy
occurs and the initiation of foetus oestrogen synthesis coincides with rapid trophoblastic
elongation (Geisert et al., 1982; Bazer et al., 1994). During the maternal recognition of
pregnancy, pig blastocysts signal their presence by synthesising and releasing oestrogens
and possibly other substances that interact with the maternal system to allow pregnancy
to continue. The hypothesis of the present study was that the period of pregnancy when
maternal recognition of pregnancy occurs would be more sensitive to endocrine changes
due to the finely orchestrated hormonal interaction between the sow and her foetuses which
is necessary for the maintenance of pregnancy. The purpose of the present study was,
therefore, to investigate whether repeated administration of ACTH and/or food deprivation
during days 13 and 14 of pregnancy (day 1, first day of standing oestrus), might alter
 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312 297

hormonal secretion of the sows, as well as negatively affect foetal survival by day 30 of
pregnancy.

2. Materials and methods

2.1. Animals

The research plan and all procedures involving the use of animals were reviewed and
approved by the Ethical Committee for Experimentation with Animals, Sweden. From
the day of arrival to the pro-oestrus of the second oestrus after weaning, the health sta-
tus (appetite and general condition) of the sows was carefully monitored. In three cases
of persistent clinical infections—one endometritis, one lameness and one with unknown
cause of illness—the sows were excluded from the study. The experiment was performed
with 17 crossbred (Swedish Landrace × Swedish Yorkshire) sows being in their second to
fourth parity. The sows were brought from one commercial farm on the day of weaning
and weighed between 180 and 230 kg. They were housed in individual pens on straw in
the vicinity of a boar, at the Department of Obstetrics and Gynaecology, SLU, Uppsala,
Sweden. Sows were fed 2.9 kg of a commercial feed, containing 15.5% crude protein and
12.4 MJ metabolisable energy (ME)/kg, divided into two meals at 7 a.m. and 3 p.m., which
is according to Swedish standards (Simonsson, 1994). Water was provided ad-libitum.
The sows were randomly divided into three groups: one control group (C, n = 6), one
group that was deprived of food (FD, n = 5) and one group treated with synthetic ACTH
(A, n = 6).
One of the sows in the A-group, no. 7138, returned to oestrus at day 28. She was not
excluded from the study since it was assumed that the abortion was an effect of treatment.
The parameters obtained from this sow, which significantly influence the results of the study,
will be presented separately. One of the food-deprived sows had to be excluded from the
blood sampling part of the study due to a failing jugular vein catheter.

2.2. Oestrous detection and ovulation

Heat detection was performed twice daily (morning and evening) by using the back-
pressure test in front of a boar. Standing oestrus was defined when sows responded with a
standing reflex to the back-pressure test. The first day of standing oestrus was defined as
day 1 of pregnancy.
The time of ovulation was determined through transrectal ultrasonography using an an-
nular array sector scanner (type Scanner 250, Pie Medical b.v Maastricht, the Netherlands)
with a 5 MHz multiple-scan angle transducer as described by Mburu et al. (1995). Each
sow was monitored during the first oestrus after weaning to predict the interval from onset
of standing oestrus to ovulation in the second oestrus. From about 20 h after the onset of
oestrus, ultrasonography was performed every fourth hour until ovulation had occurred.
Time of ovulation was set as the time when all large follicles had collapsed.
The sows were inseminated 18–8 h prior to ovulation in the second oestrus after weaning.
The animals were inseminated with 100 ml of fresh boar semen extended with Beltsville
298 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312

Thawing Solution (BTS, Pursel and Johnson, 1976) to a total of 10 × 109 spermatozoa,
pooled from two boars with proven fertility.

2.3. Treatment

To achieve easy and stress-free blood collection, a jugular vein catheter was inserted
under general anaesthesia (Rodriguez and Kunavongkrit, 1983) a few days before the second
oestrus after weaning was expected (days 15–18). A permanent Silastic tube (Still Werner,
i.d. 1.016 mm, o.d. 2.159 mm) was inserted into the right jugular vein, passed subcutaneously
to the back and exteriorised through the skin where it was connected to a cannula. The tubing
was flushed once a day with heparinised saline solution (25 IE/ml) until the blood collection
and treatment began.
The treatment was performed during days 13 and 14 of pregnancy, denoting the first
day of standing oestrus as day 1 of pregnancy. The A-group sows were given intravenous
injections of tetracosactid (Synachten® Depot), a synthetic ACTH, at a dose of 0.01 mg/kg
body weight. Each dose was diluted with saline solution to a total volume of 5 ml. Catheters
were flushed with 5 ml of saline solution after each injection. Injections were given ev-
ery sixth hour from 6 a.m. on day 13 until 6 a.m. on day 15 of pregnancy. FD-sows
were deprived of food from the morning of day 13 of pregnancy until the morning of
day 15 when feeding was resumed. C-group sows were fed and given intravenous in-
jections of 10 ml of saline solution according to the procedures applied on the A-group
sows.

2.4. Blood sampling

Blood collection in heparinised tubes (Teruma, Venoject, evacuated blood collection

tubes) started at day 11 of pregnancy and was performed four times a day (9 a.m., 11 a.m., 1
p.m. and 3 p.m.) until slaughter, except during days 13–15 where blood was collected every
second hour. The tubes were immediately centrifuged (1500 × g; 20 ◦ C; 10 min) and the
collected plasma was frozen and stored at −20 ◦ C. At days 13, 14 and 15, blood was also
collected at 10 a.m. and 6 p.m. in tubes without additive to obtain serum. Before storage,
and within a period of 60 min after sampling, the tubes were centrifuged twice at the same
speed and duration as above.

2.5. Slaughter and recovery of the genital tract and foetuses

The sows were slaughtered at 30 ± 2 day of pregnancy and the internal genital tracts were
immediately recovered and examined. The total number of corpora lutea (CL) was counted
to determine the ovulation rate. Embryo survival rate was determined by dividing the total
number of intact foetuses with the total number of CL. The number of allantoic sacs was
counted by palpation of the uterine horns. They were then separated from each other by a
pair of forceps and examined one-by-one, beginning with the sac situated most closely to
the ovary in the left uterine horn (L1). The examination of each foetal unit included weight
of total foetal unit, volume of allantoic fluid, weight of placenta, weight of allantochorion
and the weight and length of the foetus.
 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312 299

2.6. Hormone assays

2.6.1. Cortisol
Plasma cortisol was determined by solid-phase, radioimmunoassay (Diagnostic Products,
Los Angeles, CA, USA) according to the manufacturer’s instructions (10 ␮l, two replicates
per sample). Serial dilutions of porcine plasma with high concentrations of cortisol produced
displacement curves parallel to the standard curve. The intra-assay coefficient of variation
was between 4.6 and 8.8%. The inter-assay coefficient of variation was between 4.0 and
6.2%. The detection limit of the assay was 5.5 nmol/l.

2.6.2. Progesterone
The concentration of progesterone in the peripheral blood plasma was determined using
a solid-phase radioimmunoassay kit (Coat-A-Count® Progesterone, Diagnostic Products
Corporation, Los Angeles, CA, USA). The kit was used according to the manufacturer’s
instructions (100 ␮l, two replicates per sample). The intra-assay coefficient of variation was
between 6.1 and 19.8%. The inter-assay coefficients were up to 6.3%. The average detection
limit of the assay was 0.06 nmol/l.

2.6.3. Oestrone
The concentration of oestrone in peripheral blood plasma was determined using the
DSL-8700 (Diagnostic Systems Laboratories Inc., Webster, USA) oestrone radioimmunoas-
say kit, which allows quantitative measurement of oestrone in serum or plasma. The kit
was used according to the manufacturer’s instructions (50 ␮l, two replicates per sample).
The intra-assay coefficient of variation was between 5.7 and 1.2%. The inter-assay coeffi-
cient of variation was between 4.3 and 7.5%. The average detection limit of the assay was
4.44 pmol/l.

2.6.4. Prostaglandin F2␣ metabolite

The main initial blood plasma metabolite of prostaglandin F2␣, 15-keto-13,14-dihydro-
PGF2␣ (15-ketodihydro-PGF2␣ ), was analysed by radioimmunoassay as described previ-
ously by Kunavongkrit et al. (1983), the sample volume used was 100 ␮l and there were
two replicates per sample. The relative cross-reactions of the antibody were 16% with
15-keto-PGF2␣ , 4% with 13,14-dihydro-PGF2␣ and 0.4% with PGF2␣ . The intra-assay co-
efficient of variation ranged between 3.4 and 7.6% for different ranges of the standard curve
and the inter-assay coefficient of variation was approximately 14%. The practical limit of
sensitivity for the assay analysing 0.2 ml of plasma was 60 pmol/l.

2.6.5. Oestrone-sulphate
The concentration of oestrone-sulphate in peripheral blood plasma was determined using
a radioimmunoassay kit (DSL-5400, Diagnostic Systems Laboratories Inc., Webster, USA),
which allows quantitative measurement of oestrone-sulphate in serum or plasma. The kit was
used according to the manufacturer’s instructions (50 ␮l, two replicates per sample). The
intra-assay coefficient of variation was between 5.7 and 7.7%. The inter-assay coefficient
of variation was up to 7.7%. The average detection limit of the assay was 3.7 nmol/l. The
cross-reactivity of the oestrone-sulphate antiserum against the oestrone-sulphate, oestrone
300 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312

and oestrone glucuronide was 100, 4.9 and 3.4%, respectively, and the cross-reactivity
against other oestrogens was less than 1%.

2.6.6. Insulin
The concentration of insulin in peripheral blood plasma was determined using a solid-
phase 125 I radioimmunoassay kit (Coat-A-Count® Insulin, Diagnostic Products Corpo-
ration, Los Angeles, CA, USA) designed for the quantitative measurement of insulin in
serum. The kit was used according to the manufacturer’s instructions (200 ␮l, two repli-
cates per sample). Intra-assay coefficients of variation (%) were 7.08% at 12.1 ␮U/ml,
10.0% at 45.4 ␮U/ml and 4.19% at 128 ␮U/ml, for low, medium and high assay con-
trols, respectively. The minimal detection concentration (MDC) of the assay was
1.55 ␮U/ml.

2.7. Blood serum metabolite assays

2.7.1. Free fatty acids

Blood serum free fatty acids were analysed with an enzymatic colourimetric method
on Cobas FARA (Roche, Basel Switzerland) using reagents from (Wako NEFA C, Neuss,
Germany), the sample volume used was 50 ␮l and there were two replicates per sam-
ple. The intra-assay coefficients of variation (%) for two quality control samples were
3% at 0.4 mmol/l and 2% at 0.8 mmol/l and the corresponding inter-assay coefficients
were 5.7 and 6.4%, respectively. The minimal detection concentration of the assay was
0.05 mmol/l.

2.7.2. Triglycerides
Blood serum triglycerides were analysed with an enzymatic colourimetric method
(GP/PAP method) on Cobas FARA (Roche) using reagents (UNIMATE 5 TRIG) from the
same company, the sample volume used was 50 ␮l and there were two replicates per sam-
ple. The intra-assay coefficient of variation (%) was 1.2% at 1.4 mmol/l and the inter-assay
coefficient of variation was 4% at 1.4 mmol/l. The minimal detection concentration of the
assay was 0.01 mmol/l.

2.8. Statistical analysis

Statistical analyses were carried out using the mixed procedure in the SAS software
package (SAS Institute Inc., 1989). The statistical model used to compare differences in
ovulation rate, total number of foetuses, embryo survival rate and day of slaughter, in-
cluded the fixed effect of treatment (three groups); sow was set as a random factor to
account for repeated sampling within groups. To compare differences in the hormonal
baseline levels between groups, the hormonal observations were grouped into suitable
time periods depending on the length of the sampling period. Before and during time
of treatment, one period was equal to 1 day of pregnancy. For hormones analysed un-
til time of slaughter, the time periods after day 15 of pregnancy consisted of approx-
imately 2 days. The statistical evaluation of hormonal concentrations was based on
average values calculated for each sow and time period. The model applied included
 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312 301

the fixed effect of treatment (three groups), time period (three to seven periods), the
interaction between treatment and time period and the random effect of sow within
treatment.

3. Results

3.1. Macroscopical examination of the genital tract

There were no significant differences in number of corpora lutea, number of foetuses or

embryo survival rate between the groups (Table 1). In the A-group, sow no. 7138 returned
to oestrus at day 28. At the time of slaughter (day 30) she had no foetuses, but scars from
the placental attachment sites were observed. Calculations performed, omitting this sow,
did not change the outcome of the significance test.

3.2. Blood plasma levels of hormones

3.2.1. Cortisol
There was no significant difference in average cortisol levels prior to the treatment pe-
riod between any of the treatment groups (Fig. 1). During the first day of treatment (day
13) there was a significantly (P < 0.05) higher cortisol level in the A-group and the
FD-group compared with the C-group. Sows in the A-group also had a significantly higher
(P < 0.0001) level of cortisol on day 14 compared with the C-group and the FD-group,
while there were no differences between the FD-group and the C-group at day 14 and
onwards. At day 15, the C-group and the FD-group sows had normal daily variation
in the cortisol level while the cortisol level of the A-group sows was low and without
variation.

3.3. Progesterone

There was no difference between the groups in the average blood plasma level of proges-
terone before the treatment period (Fig. 2). The FD-group had a significantly (P < 0.01)
higher plasma level of progesterone from the onset of treatment at day 13 until the day

Table 1
Day of slaughter, number of corpora lutea (CL), total number of viable and nonviable foetuses, as well as em-
bryo survival rate (no. of viable foetuses/no. of CL) (mean ± S.D.) in control (C-), food-deprived (FD-) or
ACTH-stimulated (A-) sows on days 13 and 14 of pregnancy
C-group lsmeans FD-group lsmeans A-group
(lsmean±S.D.) (lsmean±S.D.) (lsmean±S.D.)
Day of slaughter 30.3 ± 0.4 30.8 ± 0.5 30.0 ± 0.5
Total no. of CL 19.0 ± 1.3 20.6 ± 1.4 18.5 ± 1.3
Total no. of viable foetuses 14.3 ± 2.0 15.2 ± 2.2 11.0 ± 2.0
Total no. of nonviable foetuses 0.7 ± 0.2 0.0 ± 0.0 0.5 ± 0.3
Embryo survival rate (%) 75.6 ± 11.2 75.7 ± 12.2 62.0 ± 11.2
302 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312

C-group FD-group A-group

450 Treatment period

400

350
Plasma cortisol nmol/l

300

250

200

150

100

50

0
Time -21 -19 -17 -15 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 75 81
Day 12 13 14 15 16

Fig. 1. Plasma levels of cortisol (lsmean ± S.D.) before, during and after treatment in control (C-), food-deprived
(FD-) and ACTH-stimulated (A-) sows. Time (h) 0, start of treatment.

C-group FD-group A-group

180

160
Plasma progesterone nmol/l

140

120

100

80

60

40

Treatment period
20

0
Time -43 -36 -21 -15 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 75 81
Day 11 12 13 14 15 16

Fig. 2. Plasma levels of progesterone (lsmean ± S.D.) before, during and after treatment in control (C-),
food-deprived (FD-) and ACTH-stimulated (A-) sows. Time (h) 0, start of treatment.
 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312 303

C-group FD-group A-group

400

350

300
Oestrone pmol/l

250

200

150

100

50

0
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Day of pregnancy

Fig. 3. Plasma levels of oestrone (lsmean ± S.D.) from day 16 of pregnancy in control (C-), food-deprived (FD-)
and ACTH-stimulated (A-) sows.

after treatment (day 15), compared with the C- and A-groups. There were no significant
differences in plasma progesterone levels between the C- and A-groups.

3.3.1. Oestrone and oestrone-sulphate

In all three groups, the plasma oestrone concentration was 20–40 pmol/l from day 13 to
about day 15 when it decreased to become significantly (P < 0.05) lower between days
16 and 18 (Fig. 3). In the C- and FD-groups, there was a significant (P < 0.001) increase
in the blood plasma level of oestrone starting at day 19 of pregnancy, while the oestrone
level in the A-group did not start to increase until day 21 of pregnancy. The oestrone level
in the A-group sows was significantly lower compared with the oestrone level of the C-
and FD-group sows between days 19 and 22 (P < 0.05). From day 23 and onwards, there
were no significant differences between the groups. The concentration of oestrone-sulphate
in the peripheral blood plasma was below the detection limit in all groups until about
day 22, and there were no significant differences between the groups in any time interval
(Fig. 4).

3.3.2. Prostaglandin F2␣ -metabolite

An overall significant (P < 0.001) increase in the blood plasma concentration of prosta-
glandin F2␣ (PGF2␣ )-metabolite at day 12 of pregnancy was observed (Fig. 5a), which is
consistent with the findings of Tsuma et al. (1996a). The level of PGF2␣ -metabolite did not
differ significantly between the groups until day 14 when it tended to decrease more rapidly
in the A-group compared with the C- and FD-group (P = 0.06 and P = 0.09, respectively)
304 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312

14
C-group FD-group A-group

12
Plasma oestrone-sulphate nmol/l

10

0
22 23 24 25 26 27 28 29
Day of pregnancy

Fig. 4. Plasma levels of oestrone-sulphate (lsmean ±S.D.) from day 22 of pregnancy in control (C-), food-deprived
(FD-) and ACTH-stimulated (A-) sows.
C-group FD-group A-group
2500
Treatment period
Fig. 5b
Plasma prostaglandin F2α-metabolite pmol/l

2000

1500

1000

500

0
1 2 34 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
(a) Day of pregnancy

Fig. 5. (a) Plasma levels of PGF2␣ -metabolite (lsmean ± S.D.) before, during and after time of treatment in
control (C-), food-deprived (FD-) and ACTH-stimulated (A-) sows. (b) Plasma levels of PGF2␣ -metabolite
(lsmean ± S.D.) on every sampling occasion during days 10–15 of pregnancy in control (C-), food-deprived
(FD-) and ACTH-stimulated (A-) sows. Time (h) 0, start of treatment. (c) Plasma level of PGF2␣ -metabolite
(lsmean ± S.D.) in sow 7138 before and during time of treatment and until she returned to oestrus at day 28.
 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312 305

C-group FD-group A-group

3000

2500

2000
Prostaglandin F2α pmol/l

1500

1000

500

Treatment period
0
9

36
28
12

16

20

32

40

44

48

52

56

60

64
1

24
5

Time
-6
-6

-2
-4
-4

-1

(b) Day 10 11 12 13 14 15

9000

7138
8000

7000
PGF2α-metabolite pmol/l

6000

5000

4000

3000
Treatment period

2000

1000

0
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
(c) Day of pregnancy

Fig. 5. (Continued ).
306 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312

60 treatment period

50
C-group FD-group A-group
Plasma insulin nmol/l

40

30

20

10

0
13a.m 13 p.m 14 a.m 14 p.m 15 a.m 15 p.m
Day of pregnancy

Fig. 6. Serum levels of insulin (lsmean ± S.D.) during the time of treatment and the day after in control (C-),
food-deprived (FD-) and ACTH-stimulated (A-) sows.

(Fig. 5b). At day 15, the concentration of PGF2␣ -metabolite was significantly (P < 0.05)
lower in the A-group compared with the C- and FD-group until 8 a.m. From day 16 and
onwards, there was no significant difference in the blood plasma level of PGF2␣ -metabolite
between the three groups. Sow no. 7138 was considered to be an outlier regarding this
parameter and was, therefore, excluded from the calculations. The PGF2␣ -metabolite values
for this sow increased dramatically starting at day 22, reached their maximum at day 23
(>5 nmol/l) and declined thereafter rapidly (Fig. 5c).

3.3.3. Insulin
During the treatment period, the blood plasma insulin values in the FD-group was signif-
icantly (P < 0.01) lower than the level of C-group sows, while the insulin concentration of
the A-group sows was significantly (P = 0.0001) higher than both the C- and FD-groups
(Fig. 6). At day 15, no significant difference between groups was observed.

3.3.4. Free fatty acids and triglycerides

In the FD-group, serum levels of free fatty acids increased to be significantly higher
(P < 0.0001) than the C- and A-groups from the afternoon at day 13 until the morning at
day 15 (day after treatment) (Fig. 7). There was no significant difference in the serum levels
of free fatty acids between the C- and A-groups. In addition, there was no difference in the
levels of triglycerides between the groups on the first day of treatment. During the second
day of treatment, however, a significant (P < 0.05) decrease of triglycerides in the A-group
sows was observed (Fig. 8). The day after the end of treatment (day 15), FD-group sows
had lower levels of triglycerides compared with the C-group sows (P = 0.08).
 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312 307

1,4
Treatment period
Plasma level of free fatty acids in nmol/l

1,2
C-group FD-group A-group
1

0,8

0,6

0,4

0,2

0
13 a.m 13 p.m 14 a.m 14 p.m 15 a.m 15 p.m
Day of pregnancy

Fig. 7. Serum levels of free fatty acids (lsmean ± S.D.) during the time of treatment and the day after treatment
in control (C-), food-deprived (FD-) and ACTH-stimulated (A-) sows.

1,2 C-group FD-group A-group

Treatment period
1
Plasma triglycerides nmol/l

0,8

0,6

0,4

0,2

0
13 a.m 13 p.m 14 a.m 14 p.m 15 a.m 15 p.m
Day of pregnancy

Fig. 8. Serum levels of triglycerides (lsmean ± S.D.) during the time of treatment and the day after treatment in
control (C-), food-deprived (FD-) and ACTH-stimulated (A-) sows.

4. Discussion

In this study, no significant differences were found between control and treatment groups
in ovulation and foetal survival rate, as determined by post mortem examination of the
genital tract and foetuses.
308 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312

During the time of treatment, sows in both the FD- and A-group had elevated levels of
cortisol, which confirms a physiological stress-situation. These findings are consistent with
previous findings by Mburu et al. (1998), Mwanza et al. (2000b) and Razdan et al. (2002).
Acute stress is known to be associated with increased levels of cortisol, but with the daily
variation kept intact as well as the negative endocrine feedback mechanism (Rosmond and
Björntorp, 2000). The endocrine negative feedback is probably the cause of the low level of
cortisol, without daily variation, which was observed in the A-group sows the day after the
end of treatment (day 15). In the FD-group, cortisol concentration was elevated only on the
first day of treatment, which suggests an adaptation to the situation. This is in line with the
behaviour of the sows. They were mostly very unsettled during the first day of treatment,
but had generally become calmer by the second day. This behavioural pattern is different
from the real-life regrouping situation, where disputes will progress for approximately 2
days before a new rank-order is established (Tsuma et al., 1996b). Therefore, it could be
argued that food deprivation together with repeated ACTH injections might mimic the live
situation better than when sows were treated with either ACTH or food deprivation, as done
in the present study. ACTH injections and food deprivation would together probably give
more pronounced effects on embryo development and survival.
Elevated progesterone levels were seen during the time of treatment in the FD-group. One
of several mechanisms that may contribute to the nutritionally induced changes in the
progesterone concentration in peripheral blood is the metabolic clearance rate of the hepatic
portal blood flow (Prime and Symonds, 1993). A catabolic metabolism will lower the
clearance rate, which results in elevated progesterone levels. High planes of nutrition have,
on the other hand, been shown to have a detrimental effect on embryo survival in connection
with reduced plasma progesterone concentration (Ashworth, 1991; Jindal et al., 1997). In
the present study, the elevated levels of progesterone during days 13 and 14 of pregnancy did
not have any significant effects on embryo survival. The explanation for this could be that
the critical window of time during which progesterone-mediated effects will be observed
might be limited to the first 3–4 days after ovulation (Foxcroft, 1997).
The plasma oestrone concentration was on average higher during days 13 and 14 than
during days 16–18 when it was under the detection limit in most of the sows, especially in
the A-group. The plasma oestrone concentration started to rise again at day 19 in the C-
and FD-group and a 2 days delay was noted in the A-group. This is in good agreement with
other studies where a biphasic synthesis and release of oestrogens from porcine blastocysts
is illustrated; uterine content of oestrogen increases during rapid conceptus elongation,
declines and remains low for 2 days to be followed by a second sustained increase after
day 14 (Geisert et al., 1982; Stone and Seamark, 1985). The level of oestrogen production
reflects the development of the foetuses and it might, therefore, be speculated that the
delayed second phase of oestrogen release seen in the A-group sows could be due to less
developed foetuses at this stage of the pregnancy, compared with the foetuses of the C- and
FD-group animals.
It is generally accepted that oestrogens produced by the foetuses are involved in the
protection of the CL from PGF2␣ produced by the endometrium and its luteolytic effects, and
that oestrogens thereby act as the messenger for maternal recognition of pregnancy (Bazer
et al., 1986). However, it has also been suggested that prostaglandin produced by the foetuses
is involved in the maintenance of the early pregnancy since inhibition of prostaglandin
 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312 309

secretion has resulted in pregnancy failure (Kraeling et al., 1985). In the present study,
there was a significant increase in the blood plasma level of PGF2␣ -metabolite at day 12 of
pregnancy that lasted throughout day 14. Previous reports indicate no persistent increase in
the peripheral blood plasma level of PGF2␣ -metabolite between days 12 and 25 of pregnancy,
which is proposed to be due to an inhibition of PGF secretion into the uterine venous drainage
or an inhibition of PGF production (Shille et al., 1979; Guthrie and Rexroad, 1981). The
latter suggestion is not supported by the findings of Zavy et al. (1980) who demonstrated that
total recoverable PGF was about 50 times greater in uterine flushings from pregnant gilts
at day 18 of pregnancy compared with non-pregnant gilts at day 18 of the oestrous cycle.
The results of the present study illustrate an increase in PGF2␣ -metabolite in synchrony
with the rapid increase in blastocyst production of oestrogens at day 13, supporting the
theory that the increase in oestrogen concentration would enhance the PGF synthesis by
the endometrium and possibly also the blastocysts. The oestrogens produced by blastocysts
probably also alter the direction of PGF secretion towards the uterine lumen (Bazer and
Thatcher, 1977), resulting in the rapid decrease of PGF2␣ -metabolite seen between days 13
and 15 in the peripheral blood plasma in this study. During days 14 and 15, the level of
PGF2␣ tended to be lower in the A-group sows compared with the C- and FD-groups but
after day 16, there were no differences between groups in the level of PGF2␣ -metabolite.
This effect of treatment is consistent with earlier findings where repeated injections with
synthetic ACTH have decreased the basal level of PGF2␣ -metabolite (Razdan et al., 2002).
The most likely explanation for this is that ACTH stimulates cortisol production, which in
turn inhibits release of arachidonic acid. Sow 7138 had a second increase in the level of
PGF2␣ -metabolite at days 23 and 24, and at day 28 she returned to oestrus. At autopsy,
scars from the implantation sites in the endometrium were present, supporting the theory
that foetal loss after day 12 of pregnancy resulted in a delayed return to oestrus (more than
23 days). The sows had, therefore, been exposed to the first phase of oestrogen produced
by the blastocysts, but not the second one (van der Meulen et al., 1991).
Measurement of plasma oestrone sulphate concentration reflects endometrial sulphation
of conceptus oestrogens (oestradiol and oestrone) released into the maternal circulation
(Roberts et al., 1993). In the present study, the concentration of oestrone-sulphate in the
peripheral blood plasma of the sows in all groups was below the detection limit until the
afternoon at day 21 or the morning of day 22, when it started to increase. This is not consistent
with earlier studies, where the concentration of oestrone-sulphate in the peripheral blood
plasma of pregnant females has been reported to reveal a similar biphasic pattern as the
oestrogens measured in the uterine flushing (Robertson and King, 1974). Madej et al. (1998)
also observed a significant increase in urinary oestrone-sulphate excretion on days 13 and
14 of pregnancy followed by a second one on days 19–20 of gestation. One explanation for
this could be that the antibody used in the radioimmunoassay is very specific and has a very
low cross-reactivity to other oestrogens.
The elevated levels of insulin seen in the A-group sows might be due to gluconeogenesis
and a glycogen breakdown promoted by high cortisol levels, which also leads to an insulin
resistance that diverts energy from storage to active muscle (Dimitriadis et al., 1997; Robert
et al., 2000). This will lead to a high glucose level in blood plasma and, secondly, a high
insulin level. The elevated level of free fatty acids and the decrease in insulin shows that the
FD-sows were in a catabolic state but compensated for the lack in energy intake, which is
310 P. Razdan et al. / Animal Reproduction Science 81 (2004) 295–312

in agreement with earlier findings (Mburu et al., 1998; Mwanza et al., 2000a; Razdan et al.,
2001).
The treatments induced hormonal changes similar to those observed in stressful situa-
tions. An indication of a disturbance in the foetal development around day 19 of pregnancy
in the A-group was apparent, but at the time of slaughter there were no significant differ-
ences between the groups in embryo survival rate. This could be due to large individual
variations or because the simulated stress created in the present trial was not severe enough
to significantly influence the survival of the embryos. It is also possible that the sows were
able to recover from the hormonal imbalance caused by the treatments.

5. Conclusions

Food deprivation during days 13 and 14 of pregnancy increased the levels of cortisol, free
fatty acids and progesterone and lowered the level of insulin in the blood plasma of sows.
Injections with synthetic ACTH during the same period of pregnancy increased the levels
of cortisol and insulin and caused a 2 days delay in the increase of oestrone, from day 19
to 21 of pregnancy. At the time of slaughter, day 30 of pregnancy, there were no significant
effects on embryo survival rate, suggesting a large individual variation or capacity of the
sow to compensate for moderate stress-like effects during this stage of pregnancy.

Acknowledgements

The authors wish to thank FORMAS and Swedish Meats for financial support, Lotta
Sundström for analysing the PGF2␣ -metabolite and Dr. Mats Forsberg for his help with
analysing the other blood parameters. Heléne Gille, Carola Jansson, Mari Wallbring and
Ulrika Mattson are gratefully acknowledged for their help with the experimental animals
and Kjell-Ove Eklund for computer assistance.

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