Animal Reproduction Science 90 (2005) 117–126

Effect of feeding level on progesterone concentration

in early pregnant multiparous sows
J.V. Virolainen a,∗ , O.A.T. Peltoniemi a , C. Munsterhjelm a ,
A. Tast a , S. Einarsson b
a University of Helsinki, Faculty of Veterinary Medicine, Department of Clinical Veterinary Sciences,
Pohjoinen Pikatie 800, 04920 Saarentaus, Finland
b Swedish University of Agricultural Sciences, Centre for Reproductive Biology,

Department of Obstetrics and Gynaecology, Uppsala, Sweden

Received 7 July 2004; received in revised form 3 January 2005; accepted 31 January 2005
Available online 14 March 2005

Abstract

The effect of three feeding regimens on progesterone level was tested during early pregnancy in
multiparous sows. A total of eighteen sows in their eighth parity (8.1 ± 2.8, mean ± S.D.) were used.
During lactation the sows were fed to appetite and after weaning they received 4 kg (52 MJ) a com-
mercial feed per day. Following ovulation, sows were allocated to one of three treatment groups and
fed 2 kg/day (low feeding, LLL) or 4 kg/day (high feeding, HHH) throughout the trial or 2 kg/day for
11 days, 4 kg/day for 10 days, and 2 kg/day for the remaining days of the study (modified feeding,
LHL). Blood for progesterone and cortisol analyses was collected daily throughout the study, and
for luteinizing hormone (LH) assay for 12 h at 15 min intervals on days 14 and 21 of pregnancy.
An adrenocorticotropic hormone (ACTH) challenge test was performed on all sows day 28 of preg-
nancy. Dietary treatment did not significantly affect hormonal parameters. However, progesterone
concentration tended to be lower (P = 0.08) in the HHH group than in the LLL group. In the LHL
group venous progesterone concentration seemed to fluctuate. No effects of feeding were observed
on progesterone concentration in allantoic fluid on day 35 of pregnancy. Venous cortisol level was
significantly higher (P < 0.05) during proestrus and oestrus in all groups and there was no signifi-
cant difference between groups in response to ACTH challenge. The mean amplitude of LH pulses
decreased significantly (P < 0.01) from days 14 to 21 of pregnancy in all groups. In addition, an in-

∗ Corresponding author. Tel.: +358 19 5295323; fax: +358 19 6851181.

E-mail address: juha.v.virolainen@helsinki.fi (J.V. Virolainen).

0378-4320/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2005.01.012
118 J.V. Virolainen et al. / Animal Reproduction Science 90 (2005) 117–126

teraction was found between feeding level and baseline LH concentration and also between feeding
level and mean LH concentration. Embryonic recovery was highest in the LLL (69%), lowest in
the HHH (45%) and moderate in the LHL (55%) group. Neither high feeding nor modified feeding
provided any benefits for reproductive performance in multiparous sows. A low feeding regimen
thus appears optimal for multiparous sows in early pregnancy at least with the management regime
described.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Progesterone; LH; Early pregnancy; Feeding; Sows

1. Introduction

Feeding is considered an important factor in post-weaning reproductive performance.

Fertility is associated with successful feeding during lactation before mating as well as
during early pregnancy. The advantage of flush feeding (Ashworth, 1991; Beltranena et
al., 1991) is well known, and this regimen is widely used in practice. However, some
contradictions for level of feeding exist immediately after ovulation. Restricted feeding
during early pregnancy has been associated with higher progesterone concentrations and
improved embryonic survival (Dyck and Strain, 1983; Jindal et al., 1996, 1997). A detri-
mental effect for high level feeding, has been indicated by impaired embryonic survival,
to occur within the first 3 days (Jindal et al., 1996) or the first 10 days of pregnancy
(Dyck and Strain, 1983). Improved embryonic survival with exogenous progesterone in-
jections in high-fed gilts (Ashworth, 1991; Jindal et al., 1997) implicates a role for pro-
gesterone as a mediator of these nutritional effects. However, it has been argued that
a higher feeding level may have beneficial effects on pregnancy performance, at least
in group-housed animals, despite lower progesterone levels in plasma (Virolainen et al.,
2004b). This is because an increase in feeding beyond the recommended restricted feed-
ing schedule (1.5 × maintenance) appears to reduce behavioural problems of group-housed
sows at feeding (Brouns and Edwards, 1994) and to improve reproductive performance
during seasonal infertility (Love et al., 1995; Virolainen et al., 2004b). The mechanism
mediating the effects of high feeding during the seasonal infertility period remains ob-
scure.
Apart from the well-known interaction between progesterone levels and feed restriction,
restricted food affects secretion of other hormones involved in reproductive performance. In
pigs restricted feeding impairs luteinizing hormone (LH) secretion (Cosgrove et al., 1993;
Prunier et al., 1993). Moreover, food deprivation during early pregnancy (on days 10 and
11 of pregnancy) has been reported to result in elevated plasma cortisol concentration in
primiparous sows (Tsuma et al., 1996). Turner et al. (1999) demonstrated that prolonged
elevation of plasma cortisol levels, considered a marker of stress, prohibits the LH surge in
gilts. Three different feeding regimens were therefore applied here to investigate the effect
of feeding on progesterone, LH and cortisol secretion and embryonic survival in multiparous
sows in early pregnancy.
 J.V. Virolainen et al. / Animal Reproduction Science 90 (2005) 117–126 119

2. Materials and methods

2.1. Animals, management and feeding

Eighteen multiparous sows (Finnish Yorkshire × Finnish Landrace) from a Finnish sow
pool (Emomylly Oy, Huittinen, Finland) were used. Sows were on average in their eighth
(range 2nd–11th) parity and weighed 252 ± 32 kg (mean ± S.D.). During the preceding
lactation, their litter size was standardized and they were fed to appetite with a commer-
cial feed (Imetys-Pekoni, Suomen Rehu Ltd., Finland). Sows were imported to the Saari
Unit, Department of Clinical Veterinary Sciences, University of Helsinki, Mantsala, Fin-
land, in five replicas on the day after weaning between November and May (two sows
rejected). They were housed in paired pens on straw with free access to water and received
4 kg/day of a commercial ration (Tiineys-Pekoni, Suomen Rehu Ltd., Finland; 13 MJ DE;
7.4 lysine/kg; 14.5% crude protein) as flush-feeding. The growing follicles were mon-
itored by transrectal real-time ultrasound scanning (Pie-Medical® , 5 MHz sector probe)
three times a day until ovulation. The day of ovulation was considered to be day 0. Oestrus
was determined twice a day by fence-line contact with a mature boar. At the first de-
tection of standing oestrus, sows were inseminated, and this procedure was repeated at
12 h intervals while ever the sows were in standing oestrus. After insemination, the ani-
mals were randomly allocated to one of three feeding regimens: group LLL: 26 MJ/day
until day 35; group HHH: 52 MJ/day until day 35; group LHL: 26 MJ/day until day 11,
52 MJ/day until day 21 and 26 MJ/day until day 35. The feeding level was increased
in one increment but decreased by 0.5 kg/day until the lowest level was reached (in 3
days).
Sows were pregnancy-tested by a transcutaneous ultrasound examination commencing
on day 19 of pregnancy, and sows were determined to be pregnant when embryonic sacs
were present in the uterus. On day 35 ± 0.9 day (mean ± S.D.) of pregnancy, all sows were
slaughtered at a local abattoir, and the embryos, allantoic fluid and ovaries were examined
immediately by a method described elsewhere (Virolainen et al., 2004b). A 10 ml sample
of allantoic fluid was taken from each allantoic sac closest to the ovary with a sterile needle
and syringe. The fluid was transferred into a plastic tube and chilled on ice and then stored
at (−20 ◦ C) until progesterone analysis.

2.2. Blood collection

The sows were bled once a day for progesterone and cortisol analyses (via vena saphena
medialis or vena coccygea) until day 13, when they were non-surgically fitted with an intra-
venous indwelling jugular catheter (Virolainen et al., 2004a). The first 2 ml of each sample
was discarded. Catheters were flushed with 3 ml of NaCl-solution containing 50 IU/ml hep-
arin (Heparin, Leo Pharma, Sweden) when the sample was taken. Blood samples for LH
assay were collected from each sow commencing at 7 am at 15 min interval for 12 h on
days 14 and 21 of pregnancy. All blood samples were collected into glass tubes with no
activators and centrifuged within 2 h, and the serum was stored at −20 ◦ C until analy-
sis.
120 J.V. Virolainen et al. / Animal Reproduction Science 90 (2005) 117–126

2.3. ACTH challenge

An adrenocorticotropic hormone (ACTH) challenge test was performed on day 28 of

pregnancy. A pre-test blood sample was taken at 1 pm, and sows were then treated intra-
muscularly with 0.50 mg of ACTH (500 IU) (Synacthen® , 0.25 mg/ml, Novartis, Sweden).
Sows were bled for 3 h at 30 min intervals to observe response to ACTH as indicated by
plasma cortisol levels.

2.4. Assays

Blood samples were analysed for progesterone using a direct commercial RIA (Spectria,
Orion Diagnostica, Turku, Finland), which has been validated to measure progesterone in
pig plasma (Peltoniemi, 1994). The sensitivity of the assay was 0.09 ng/ml. The intra- and
inter-assay coefficients of variation (CVs) for three reference concentrations (0.82, 4.8 and
8.4 ng/ml) were less than <6%.
Serum LH concentrations were determined using a previously validated direct homol-
ogous double antibody RIA (Niswender et al., 1970), with modifications reported by
(Peacock, 1991). The sensitivity of the assay was 0.14 ng/ml. The intra-assay CVs for
two different reference concentrations (1 and 2 ng/ml) were 14% and 11%, and the respec-
tive inter-assay CVs were 12% and 8% (eight assays performed). Serum cortisol concentra-
tions were analysed by a solid-phase radioimmunoassay (Coat-A-count Cortisol, Diagnostic
Products Corporation, Los Angeles, CA, USA), according to the manufacturer’s instruc-
tions. The intra-assay CVs for the three reference concentrations were all less than 5%,
while the inter-assay CVs were less than 6.5% (four assays performed). The sensitivity of
the assay was 2 ng/ml.

2.5. Statistical analysis

To compare hormonal baseline levels of progesterone and cortisol, the daily hormonal
observations were divided into five and nine 4-day time periods, respectively. The first

Fig. 1. Profile of progesterone concentration (mean ± S.D.) in the high (HHH), low (LLL) and modified (LHL)
feeding groups throughout the trial. Arrow shows when feed intake (day 11) increased in the LHL group from 2
to 4 kg/day.
 J.V. Virolainen et al. / Animal Reproduction Science 90 (2005) 117–126 121

period contained the mean of hormonal values for days before ovulation. Mean values
were calculated for each sow and time period. Repeated measurements followed by the
least significant difference test were used to analyse data (Gill and Hafs, 1971). Days −2
to 15 (n = 5) and −2 to 32 (n = 9) were treated as subplots for progesterone and cortisol,
respectively, and dietary treatments (HHH, LLL and LHL) as the main plot. As some data
were not normally distributed, logarithmic transformations were performed before analysis.
The method for identification of LH pulses has been described previously (Virolainen et
al., 2004a). In the final analysis, sows not pregnant on the second day of intensive sampling
were excluded (two sows in group LHL). LH pulse characteristics were analysed using
analysis of variance with repeated measurements (software: SPSS).

3. Results

3.1. Progesterone concentrations and embryonic survival

Progesterone concentrations did not differ significantly between treatment groups, al-
though a tendency (P = 0.08) was present for progesterone concentration to be lower in
HHH sows (Fig. 1). After ovulation, no significant difference was observed in progesterone
concentrations between groups, but LLL and LHL sows tended to reach a higher level
of progesterone (25 ng/ml) by day 11 than HHH sows (17 ng/ml). On day 12, LHL sows
responded to the increase in feed intake (on day 11) by reducing their progesterone concen-
tration to below 20 ng/ml, which was similar to the level maintained by group HHH sows.
Progesterone concentration in allantoic fluid was unaffected by feeding level (Table 1). A
negative correlation (P < 0.01) was observed between crown-to-rump length and concentra-
tion of progesterone in allantoic fluid and also between day of sacrifice and concentration
of progesterone in allantoic fluid. Embryonic recovery tended to be (P = 0.09) highest in
group LLL (69%), lowest in group HHH (45%) and moderate in group LHL (55%). The
length of viable embryos at day 35 was significantly shorter (P < 0.05) in the HHH group
than in the other groups. In groups LLL and LHL, the length of embryos did not differ. One

Table 1
Day of slaughter, crown-to-rump length, number of corpora lutea (CL), concentration of progesterone in allantoic
fluid (al) and in plasma of the jugular vein (jug), number of foetuses and embryo survival rate (means ± S.D.) in
the high (HHH), low (LLL) and modified (LHL) feeding groups on day of slaughter
Treatment HHH ±S.D. LLL ±S.D. LHL ±S.D.
Day of slaughter 35.0 0 35.2 1.0 34.8 1.5
Crown-to-ramp length (cm) 3.6 a 0.3 3.8 b 0.2 3.9 b 0.4
No. of CL 26.2 5.3 22.6 3.4 18.7 2.5
Progesterone (ng/ml), al 2.2 0.7 1.8 1.0 1.4 1.0
Progesterone (ng/ml), jug 10.1 3.2 13.5 2.9 13.5 3.3
No. of viable foetuses 11 5.1 15.6 3.1 10.3 2.9
Total no. of foetuses 13 7 16.8 2.9 13 1
Embryo survival rate (%) 45 25 69 8 55 10
Different letters within a row indicate significant differences, P < 0.05.
122 J.V. Virolainen et al. / Animal Reproduction Science 90 (2005) 117–126

sow in each group aborted before the end of the trial; two aborted after ACTH challenge
and one after 3 weeks of pregnancy.

3.2. Cortisol concentrations and ACTH challenge

Cortisol concentration was significantly higher (P < 0.05) during oestrus than afterwards.
The mean (±S.D.) of cortisol concentration during oestrus and after ovulation (days 1–32)
for the groups HHH, LLL and LHL were 43.3 ± 11.8 and 29.2 ± 16.7, 61.7 ± 31.9 and
23.0 ± 14.1, and 66.6 ± 36.2 and 31.3 ± 24.0 ng/ml, respectively. In addition, the mean
cortisol level of the first days after ovulation (days 1–4) was significantly higher than the
means after day 24 of pregnancy. Feeding level did not affect cortisol concentration in this
experiment.
The response to ACTH, as indicated by cortisol concentration, is depicted in Fig. 2.
No significant difference was found between feeding groups in response to ACTH chal-
lenge. All sows had the same baseline level of cortisol before ACTH treatment and all
responded to the challenge, reaching a maximum concentration within 90 min. Corti-
sol concentration did not fall back to baseline levels within the sampling time period
(3 h).

3.3. LH data

Means of LH characteristics are shown in Table 2. None of these measures were signifi-
cantly affected by feeding level. However, a significant interaction did occur between level
of feeding and baseline LH, and also between level of feeding and mean LH concentration.
While baseline LH remained constant in the HHH group, it decreased in the LLL group and
increased in the LHL group. The same observations were made with mean LH concentra-
tions. A significant difference was present (P < 0.01) when amplitude of LH pulses on days
14 and 21 of pregnancy were compared. Amplitude decreased significantly in all groups
with progression of pregnancy.

Fig. 2. Mean profiles of cortisol (mean ± S.E.) response to adrenocorticotropic hormone (ACTH) challenge for
3 h (0–180 min) after treatment on day 28 of pregnancy in the high (HHH), low (LLL) and modified (LHL) feeding
groups.
 J.V. Virolainen et al. / Animal Reproduction Science 90 (2005) 117–126 123

Table 2
Luteinizing hormone (LH) characteristics (mean ± S.D.) determined from plasma samples collected for 12 h at
15 min intervals on days 14 and 21 of pregnancy
Feeding group Day of n Frequency Amplitude Basal level LH concentration
pregnancy (mean ± S.D.)
HHH 14 6 2.17 ± 0.75 1.19 ± 0.81 a 0.73 ± 0.21 0.90 ± 0.19
21 6 2.00 ± 0.89 0.85 ± 0.43 b 0.75 ± 0.26 0.90 ± 0.27
LLL 14 5 2.40 ± 0.89 1.08 ± 0.16 a 0.94 ± 0.39 1.13 ± 0.38
21 5 2.60 ± 0.89 0.79 ± 0.20 b 0.72 ± 0.28 0.88 ± 0.25
LHL 14 4 2.50 ± 0.58 1.18 ± 0.12 a 0.69 ± 0.21 0.91 ± 0.18
21 4 3.25 ± 0.50 1.16 ± 0.27 b 0.76 ± 0.24 0.98 ± 0.23
Sows diagnosed as pregnant by ultrasound on day 21 were included. Different letters indicate significant differ-
ences, P < 0.05.

4. Discussion

Our findings suggest that changes in feed intake during early pregnancy result in rapid
changes in circulating levels of progesterone in multiparous sows. A clear decrease was
observed in progesterone concentration in the LHL group immediately after the increase
in daily feed consumption (Fig. 1), but a significant difference was not found when con-
centrations of progesterone were compared between feeding groups. However, there was a
tendency (P = 0.08) for the HHH group to have a lower progesterone concentrations than
in the LLL group during the first 15 days of pregnancy. The decrease in progesterone level
in LHL sows is more likely associated with a change in feeding level than with a physio-
logical hormonal decrease, which is seen later in LLL sows. Increased blood flow in the
portal vein and an elevated rate of clearance of progesterone due to higher feed level, as
demonstrated by Miller et al. (1999) and by Prime and Symonds (1993), may result in the
lower progesterone concentrations observed in higher-fed sows. Progesterone concentra-
tion in allantoic fluid was not affected significantly by feeding level, as measured on day
35 of pregnancy. This finding contrasts with that of Razdan et al. (2004), who reported a
higher progesterone concentration in allantoic fluid of food-deprived sows on day 30 of
pregnancy but in the present study, none of the sow groups was completely deprived of
food. A correlation (P < 0.05) was found between progesterone levels in allantoic fluid and
the number of corpora lutea in the ovaries. While a significant difference was not present,
the numbers of corpora lutea between groups tended to differ. This observation might in-
dicate that progesterone in allantoic fluid is more dependent on number of corpora lutea
than on diet, or alternatively, that date of sampling was too late to reveal any residual effect
of feeding level. A difference in progesterone concentration in allantoic fluid might only
be noticeable during the first 3 weeks, as in peripheral blood. Further studies are needed
to determine the effects of feeding on progesterone concentrations in allantoic fluid during
earlier stages of pregnancy.
The positive effects of higher feeding level reported in gilts (Virolainen et al., 2004b)
were not observed in multiparous sows. However, the survival rate of embryos did not differ
significantly between groups, despite the survival rate tending to be lowest in group HHH,
at 45%, as compared with 55% and 69% for groups LHL and LLL, respectively. These
findings are in accord with earlier reports of high mortality of embryos at high feeding
124 J.V. Virolainen et al. / Animal Reproduction Science 90 (2005) 117–126

levels (Ashworth, 1991; Jindal et al., 1996; Virolainen et al., 2004b). Despite the fact that
the embryo survival rate in groups HHH and LHL was somewhat lower, the total number
of foetuses remained that of an average-sized litter, being 13 ± 7 and 13 ± 1 (mean ± S.D.),
respectively.
LHL or HHH regimens did not provide the benefits for pregnancy rate reported in gilts
(Virolainen et al., 2004b). Our finding is supported by the field study of Love et al. (1995),
who reported that gilts and primiparous sows seemed to derive benefits from a high feed rate.
In contrast to gilts, a high feed rate seemed to have a detrimental effect on farrowing rate in
multiparous sows. However, litter size was unaffected by feeding rate in early pregnancy.
This might indicate that mortality of embryos is greater in early pregnancy with a higher
feeding rate, and also greater in late pregnancy with a lower feeding rate.
Cortisol concentrations were not affected by feeding level. However, in all groups cor-
tisol concentration was significantly higher during oestrus before ovulation (day 0). The
mean of first days of pregnancy (days 1–4) differed significantly from means after day 24
of pregnancy. Elevated plasma cortisol before ovulation might be caused by transportation
of sows or by their regrouping. Transport stress (Dalin et al., 1993b) and regrouping (Dalin
et al., 1993a) are known to increase mean plasma cortisol levels in pigs for a short du-
ration. Alternatively, elevated cortisol might be caused by oestrus per se, since increased
cortisol levels have been reported during oestrus in pigs (Ash and Heap, 1975; Dalin et al.,
1988).
The ACTH challenge test was performed with 0.50 mg of ACTH (500 IU) (Synacthen® ,
0.25 mg/ml, Novartis, Sweden). We originally intended to use only 50 IU, but there were
two interpretations of correspondence of 1 mg of Synacthen® to IU. According to previous
protocols in the Faculty (Prof. T. Soveri, personal communication), the interpretation of
100 IU/1 mg was used. When two sows aborted 1 day after ACTH treatment, the dose was
reconsidered. The international reference preparation of tetracoactide has an activity of
1 IU/␮g of peptide (1 mg of Synacthen® therefore corresponds to 1000 IU) (Stoning et al.,
1984). However, the high dose of Synaethen® was continued to the end of the trial, and
no other abortions were associated with ACTH treatment. The ACTH challenge was not
influenced by feeding level. We hypothesized that sows receiving a low level of feeding
might be more stressed that the other sows. However, the LLL group had the lowest response
to the ACTH challenge, while the LHL group had the highest. This might be explained by
the straw bedding that the sows used to supplement their diet, decreasing stress produced
by the low feeding rate.
Mean of LH amplitude decreased significantly in all groups from days 14 to 21 of
pregnancy. The role of LH is thought to be crucial for maintaining early pregnancy in pigs.
A decreasing LH amplitude might indicate a lesser role of LH in pigs during early pregnancy,
as suggested by Peltoniemi et al. (1995). These authors also observed that pregnancy failed
in all gilts treated with gonadotrophin releasing hormone (GnRH) agonist before day 29
of pregnancy, whereas 50% of the gilts treated on day 29 of pregnancy maintained their
pregnancies.
In previous studies with gilts, mean LH concentration (Virolainen et al., 2004b) and
mean LH frequency (Peltoniemi et al., 1997) were affected by feeding. Our study failed,
however, to reveal any effect of feeding on LH secretion in multiparous sows. As dis-
cussed above, the dietary supplement in the form of straw ad libitum in all groups
 J.V. Virolainen et al. / Animal Reproduction Science 90 (2005) 117–126 125

may have affected the observations on the feeding regimens. These sows were not so-
cially stressed group and management was optimal. In addition, our study was not per-
formed during a known period of infertility. However, an interaction was found between
baseline and mean levels of LH and feeding. Further studies are needed to examine,
whether this interaction has an impact on reproductive performance during a period of
infertility.
Our results demonstrate that restricted and liberal feeding have an effect on peripheral
progesterone concentrations beyond day 4. The impact of changes in progesterone con-
centrations on embryonic survival in multiparous sows remains, however, open. The rapid
reduction in progesterone in response to an increase in feed intake implies an increase in
hepatic blood flow and metabolic clearance. Although peripheral progesterone did show
a decrease, progesterone concentration might be greater if measured close to the uterus
(Virolainen et al., 2005). A difference in progesterone concentration between different sites
might explain the benefits observed in gilts with a high feeding level and lower peripheral
plasma progesterone. Whether metabolized peripheral progesterone differs from local pro-
gesterone in the uterus during the early days of pregnancy, and whether any difference in
metabolism of progesterone exists between gilts and multiparous sows remain question for
future studies.
Neither high feeding nor modified feeding provided any advantages for reproductive
performance in multiparous sows. This might indicate that in gilts and multiparous sows two
different feeding strategies should be used in early pregnancy. However, the experimental
animals received optimal handling and a good environment. If the risk factors involved in
seasonal infertility and social pressure are increased, the benefits of higher feeding level
seen in gilts might be evident also in multiparous sows.

Acknowledgements

Suomen Rehu Ltd. is acknowledged for providing food for experimental sows during the
study. Laboratory assistant Marja-Liisa Tasanko is acknowledged for her assistance with
the blood samples and Kim Heasman and Jonna Jantunen for their assistance with the LH
assays. The authors wish to thank also all colleagues and stuff at the Saari Unit, who were
involved in this project.

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