Hormonal Profiles And Embryo Survival Of Sows Subjected To Induced Stress During Days 13 And 14 Of Pregnancy

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 a

Department 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: [email protected] (P. Razdan).

0378-4320/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2003.09.005

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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 FDand 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 established, 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

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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 status (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 backpressure 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 annular 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

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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 every 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 injections 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.

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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 radioimmunoassay 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 coefficient 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-dihydroPGF2␣ (15-ketodihydro-PGF2␣ ), was analysed by radioimmunoassay as described previously 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 coefficient 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

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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 solidphase 125 I radioimmunoassay kit (Coat-A-Count® Insulin, Diagnostic Products Corporation, 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 replicates 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 controls, 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 sample. 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 sample. 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, included 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 until time of slaughter, the time periods after day 15 of pregnancy consisted of approximately 2 days. The statistical evaluation of hormonal concentrations was based on average values calculated for each sow and time period. The model applied included

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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 period 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 progesterone 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 embryo 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

Day of slaughter Total no. of CL Total no. of viable foetuses Total no. of nonviable foetuses Embryo survival rate (%)

C-group lsmeans (lsmean±S.D.)

FD-group lsmeans (lsmean±S.D.)

A-group (lsmean±S.D.)

30.3 ± 0.4 19.0 ± 1.3 14.3 ± 2.0 0.7 ± 0.2 75.6 ± 11.2

30.8 ± 0.5 20.6 ± 1.4 15.2 ± 2.2 0.0 ± 0.0 75.7 ± 12.2

30.0 ± 0.5 18.5 ± 1.3 11.0 ± 2.0 0.5 ± 0.3 62.0 ± 11.2

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450

FD-group

A-group

Treatment period

Plasma cortisol nmol/l

400 350 300 250 200 150 100 50 0 Time -21 -19 -17 -15 0 12 Day

4

8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 75 81 15 14 16 13

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

Plasma progesterone nmol/l

160 140 120 100 80 60 40 Treatment period

20 0 Time -43 -36 -21 -15 0 Day 11 12

4

8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 75 81 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.

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C-group

FD-group

303

A-group

400 350

Oestrone pmol/l

300 250 200 150 100 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 Cand 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 prostaglandin 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)

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Plasma oestrone-sulphate nmol/l

14

C-group

FD-group

A-group

12 10 8 6 4 2 0 22

23

24

25 26 Day of pregnancy

27

28

29

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

Plasma prostaglandin F2α-metabolite pmol/l

Fig. 5b

2000

1500

1000

500

0 1

(a)

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

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.

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C-group

FD-group

305

A-group

3000

Prostaglandin F2α pmol/l

2500

2000

1500

1000

500

Treatment period

10

11

12

14

64

56

60

52

48

44

40

36

28

13

32

24

20

16

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12

4

0

1 -2 1 -1 7

-4

Time Day

-4 5

(b)

-6 9 -6 5

0 15

9000 7138

8000

PGF2α-metabolite pmol/l

7000 6000 5000 4000 3000

Treatment period

2000 1000 0

(c)

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 Day of pregnancy

Fig. 5. (Continued ).

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60

50 Plasma insulin nmol/l

C-group

FD-group

A-group

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 significantly (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).

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Plasma level of free fatty acids in nmol/l

1,4 Treatment period

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 p.m

14 a.m

15 p.m

15 a.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. C-group

1,2

A-group

Treatment period

1

Plasma triglycerides nmol/l

FD-group

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.

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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 Cand 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

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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

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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 situations. 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 differences 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.

References Ashworth, C.J., 1991. Effects of pre-mating nutritional status and post-mating progesterone supplementation on embryo survival and conceptus growth in gilts. Anim. Reprod. Sci. 26, 311–321. Bazer, F.W., Ott, T.L., Spencer, T.E., 1994. Pregnancy recognition in ruminants, pigs and horses: signals from the trophoblast. Theriogenology 41, 79–94. Bazer, F.W., Thatcher, W.W., 1977. Theory of maternal recognition of pregnancy in swine based on estrogen controlled endocrine versus exocrine secretion of prostaglandin F2␣ by the uterine endometrium. Prostaglandins 14, 397–401. Bazer, F.W., Vallet, J.L., Roberts, R.M., Sharp, D.C., Thatcher, W.W., 1986. Role of conceptus secretory products in establishment of pregnancy. J. Reprod. Fertil. 76, 841–850. Brouns, F., Edwards, S.A., 1994. Social rank and feeding behaviour of group-housed sows fed competitively or ad libitum. Appl. Anim. Behav. Sci. 39, 225–235. Dimitriadis, G., Leighton, B., Parry-Billings, M., Sasson, S., Young, M., Krause, U., Bevan, S., Piva, T., Wegener, G., Newsholme, E.A., 1997. Effect of glucocorticoid excess on the sensitivity of glucose transport and metabolism to insulin in rat skeletal muscle. Biochem. J. 321, 707–712.

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

311

Foxcroft, G.R., 1997. Mechanisms mediating nutritional effects on embryonic survival in pigs. J. Reprod. Fertil. Suppl. 52, 47–61. Geisert, R.D., Renegar, R.H., Thatcher, W.W., Roberts, R.M., Bazer, F.W., 1982. Establishment of pregnancy in the pig: interrelationships between pre-implantation development of the pig blastocyst and uterine endometrial secretions. Biol. Reprod. 27, 925–939. Guthrie, H.D., Rexroad Jr., C.E., 1981. Endometrial prostaglandin F release in vitro and plasma 1314-dihydro-15-keto-prostaglandin F2␣ in pigs with luteolysis blocked by pregnancy, estradiol benzoate or human chorionic gonadotropin. J. Anim. Sci. 52, 330–337. Jindal, R., Cosgrove, R.J., Foxcroft, G.R., 1997. Progesterone mediates nutritionally induced effects on embryonic survival in gilts. J. Anim. Sci. 75, 1063–1063. Kraeling, R.R., Rampacek, G.B., Fiorello, N.A., 1985. Inhibition of pregnancy with indomethacin in mature gilts and prepuberal gilts induced to ovulate. Biol. Reprod. 32, 105–110. Kunavongkrit, A., Kindahl, H., Madej, A., 1983. Clinical and endocrinological studies in primiparous zero-weaned sows. Hormonal patterns of normal cycling sows after zero-weaning. Zbl. Vet. Med. A 30, 616–624. Lambert, E., Williams, D.H., Lynch, P.B., Hanarahan, T.J., McGeady, T.A., Austin, F.H., Boland, M.P., Roche, J.F., 1991. The extent and timing of prenatal loss in gilts. Theriogenology 36, 655–665. Madej, A., Romanowicz, K., Einarsson, S., Forsberg, M., Barcikowski, B., 1998. Urinary excretion of oestrone sulphate and cortisol in early pregnant gilts treated with glucocorticoids. Acta Vet. Scand. 39, 61–70. Mburu, J.N., Einarsson, S., Dalin, A.-M., Rodriguez-Martinez, H., 1995. Ovulation as determined by transrectal ultrasonography in multiparous sows: relationship with oestrous symptoms and hormonal profiles. J. Vet. Med. A 42, 285–292. Mburu, J.N., Einarsson, S., Kindahl, H., Madej, A., Rodriguez-Martinez, H., 1998. Effect of post-ovulatory food deprivation on oviductal sperm concentration, embryo development and hormonal profiles in the pig. Anim. Reprod. Sci. 52, 221–234. Mendel, M., Zanella, A.J., Broom, D.M., 1992. Physiological and reproductive correlates of behavioural strategies in female domestic pigs. Anim. Behav. 44, 1107–1121. Mwanza, A.M., Englund, P., Kindahl, H., Lundeheim, N., Einarsson, S., 2000a. Effects of postovulatory food deprivation on the hormonal profiles, activity of the oviduct and ova transport in sows. Anim. Reprod. Sci. 59, 185–199. Mwanza, A.M., Madej, A., Kindahl, H., Lundeheim, N., Einarsson, S., 2000b. Postovulatory effect of repeated intravenous administration of ACTH on the contractile activity of the oviduct, ova transport and endocrine status of recently ovulated and unrestrained sows. Theriogenology 54, 1305–1316. Pope, W.F., First, N.L., 1985. Factors affecting the survival of pig embryos. Theriogenology 23, 91–105. Prime, G.R., Symonds, H.W., 1993. The influence of plane of nutrition on portal blood flow. J. Agric. Sci. 121, 389–397. Pursel, V.G., Johnson, L.A., 1976. Frozen boar spermatozoa: methods of thawing. J. Anim. Sci. 42, 927–932. Razdan, P., Mwanza, A.M., Kindahl, H., Hultén, F., Einarsson, S., 2001. Impact of postovulatory food deprivation on the ova transport, hormonal profiles and metabolic changes in sows. Acta Vet. Scand. 42, 15–25. Razdan, P., Mwanza, A.M., Kindahl, H., Hultén, F., Einarsson, S., 2002. Effects of repeated ACTH-stimulation on early embryonic development and hormonal profiles in sows. Anim. Reprod. Sci. 70, 127–137. Robert, M., Sapolsky, L., Romero, M., Munck, A.U., 2000. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative action. Endocr. Rev. 21, 55–89. Roberts, R.M., Xie, S., Trout, W.E., 1993. Embryo–uterine interactions in pigs during week 2 of pregnancy. J. Reprod. Fertil. Suppl. 48, 171–186. Robertson, G.L., King, R.H., 1974. Plasma concentration of progesterone, estrone, estradiol-17␤ and estrone sulfate in the pig at implantation, during pregnancy and at parturition. J. Reprod. Fertil. 40, 133–141. Rodriguez, H., Kunavongkrit, A., 1983. Chronic venous catheterisation for frequent blood sampling in unrestrained pigs. Acta Vet. Scand. 24, 318–320. Rosmond, R., Björntorp, P., 2000. Låg kortisolproduktion vid kronisk stress. Läkartidningen 47, 4120–4124. Shille, V.M., Karlbom, I., Einarsson, S., Larsson, K., Kindahl, H., Edqvist, L.E., 1979. Concentration of progesterone and 15-keto-13,14-dihydroprostaglandin F2␣ in the peripheral plasma during the estrous cycle and early pregnancy in gilts. Zbl. Vet. Med. A 26, 169–181. Simonsson, A., 1994. Näringsrekommendationer och fodermedelstabeller till svin. Swedish University of Agricultural Sciences, SLU Info. Rapporter, Husdjur, p. 75.

312

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

Statistical Analysis Systems Institute Inc., 1989. SAS/STAT User’s Guide, Version 6, fourth ed., vol. 2. Cary, NC, SAS Institute Inc. Stone, B.A., Seamark, R.F., 1985. Steroid hormones in uterine washings and in plasma of gilts between days 9 and 15 after oestrus and between days 9 and 15 after coitus. J. Reprod. Fertil. 75, 209–221. Tsuma, V.T., Einarsson, S., Madej, A., Kindahl, H., Lundeheim, N., 1996a. Effect of food deprivation during early pregnancy on endocrine changes in primiparous sows. Anim. Reprod. Sci. 41, 267–278. Tsuma, V.T., Einarsson, S., Madej, A., Kindahl, H., Lundeheim, N., Rojkittikhun, T., 1996b. Endocrine changes during group housing of primiparous sows in early pregnancy. Acta Vet. Scand. 37, 481–490. van der Lende, T., Schoenmaker, G.J.W., 1990. Embryo mortality and prolificacy in the pig. Livestock Prod. Sci. 26, 217–229. van der Meulen, J., Elsaesser, F., Oudenaarden, C.P.J., Helmond, F.A., 1991. Effect of intra-uterine oestradiol-17␤ administration on inter-oestrous interval in the pig. Anim. Reprod. Sci. 24, 305–313. Zavy, M.T., Bazer, F.W., Thatcher, W.W., Wilcox, C.J., 1980. A study of prostaglandin F2␣ as the luteolysin in swine: V. Comparision of prostaglandin F, progestins, estrone and estradiol in uterine flushings from pregnant and nonpregnant gilts. Prostaglandins 20, 837–851.