Effect Of Feeding Level On Progesterone Concentration In Early Pregnant Multiparous Sows

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 commercial 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 pregnancy. 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 significant 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: [email protected] (J.V. Virolainen).

0378-4320/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2005.01.012

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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 detrimental 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 injections in high-fed gilts (Ashworth, 1991; Jindal et al., 1997) implicates a role for progesterone 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 feeding 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 obscure. 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.

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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 commercial feed (Imetys-Pekoni, Suomen Rehu Ltd., Finland). Sows were imported to the Saari Unit, Department of Clinical Veterinary Sciences, University of Helsinki, Mantsala, Finland, 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 monitored 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 detection 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 animals 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 intravenous 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 heparin (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 analysis.

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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 intramuscularly 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 homologous 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 respective inter-assay CVs were 12% and 8% (eight assays performed). Serum cortisol concentrations were analysed by a solid-phase radioimmunoassay (Coat-A-count Cortisol, Diagnostic Products Corporation, Los Angeles, CA, USA), according to the manufacturer’s instructions. 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.

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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, although 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 concentration 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 concentration 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

Day of slaughter Crown-to-ramp length (cm) No. of CL Progesterone (ng/ml), al Progesterone (ng/ml), jug No. of viable foetuses Total no. of foetuses Embryo survival rate (%)

35.0 3.6 a 26.2 2.2 10.1 11 13 45

±S.D. 0 0.3 5.3 0.7 3.2 5.1 7 25

LLL

±S.D.

LHL

35.2 3.8 b 22.6 1.8 13.5 15.6 16.8 69

1.0 0.2 3.4 1.0 2.9 3.1 2.9 8

34.8 3.9 b 18.7 1.4 13.5 10.3 13 55

Different letters within a row indicate significant differences, P < 0.05.

±S.D. 1.5 0.4 2.5 1.0 3.3 2.9 1 10

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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 challenge. 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. Cortisol 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 significantly 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 concentrations. 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.

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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 pregnancy

n

Frequency

Amplitude

Basal level

LH concentration (mean ± S.D.)

HHH

14 21 14 21 14 21

6 6 5 5 4 4

2.17 ± 0.75 2.00 ± 0.89 2.40 ± 0.89 2.60 ± 0.89 2.50 ± 0.58 3.25 ± 0.50

1.19 ± 0.81 a 0.85 ± 0.43 b 1.08 ± 0.16 a 0.79 ± 0.20 b 1.18 ± 0.12 a 1.16 ± 0.27 b

0.73 ± 0.21 0.75 ± 0.26 0.94 ± 0.39 0.72 ± 0.28 0.69 ± 0.21 0.76 ± 0.24

0.90 ± 0.19 0.90 ± 0.27 1.13 ± 0.38 0.88 ± 0.25 0.91 ± 0.18 0.98 ± 0.23

LLL LHL

Sows diagnosed as pregnant by ultrasound on day 21 were included. Different letters indicate significant differences, 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 concentrations 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 physiological 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 concentration 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 indicate 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

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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 cortisol 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 duration. 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 discussed above, the dietary supplement in the form of straw ad libitum in all groups

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may have affected the observations on the feeding regimens. These sows were not socially stressed group and management was optimal. In addition, our study was not performed 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 concentrations 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 progesterone 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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