Plasma Progesterone Concentration Depends On Sampling Site In Pigs

Animal Reproduction Science 86 (2005) 305–316

Plasma progesterone concentration depends on sampling site in pigs Juha V. Virolainena,b,∗ , Robert J. Loveb , Anssi Tasta , Olli A.T. Peltoniemia a

Department of Clinical Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, Pohjoinen Pikatie 800, 04920 Saarentaus, Finland b Department of Clinical Veterinary Sciences, Faculty of Veterinary Science, University of Sydney, Camden, NSW 2570, Australia

Received 11 March 2004; received in revised form 13 July 2004; accepted 20 July 2004

Abstract The objective of this study was to examine a possible difference in progesterone concentrations between the systemic venous blood and the caudal vena cava in early pregnant gilts. Nineteen crossbred pregnant gilts were offered three different regimens of feeding to examine influence of feeding on the secretion pattern of progesterone. The groups were high (H–H), low (L–L) and low–high (L–H) receiving 3.6, 1.8 and 1.8/3.6 kg/day, respectively. Catheters were placed in a jugular vein and the caudal vena cava (to sample ovarian secretion) on day 19 of pregnancy. Two consecutive samples taken at 30-min intervals were collected four times a day for 5 days (days 20–24). In addition, three gilts were simultaneously sampled from both catheters at 30-min intervals for 12 h on day 22. Progesterone concentration was significantly lower in the jugular vein compared with the caudal vena cava in all three feeding groups (P < 0.001). An indication of episodic pattern of progesterone production occurred in plasma collected from the caudal vena cava, but not from the jugular vein. Dietary intake did not cause a profound effect on plasma progesterone concentrations during days 20–24 of gestation. It seemed that ovarian progesterone was released into the vena cava in an episodic pattern and there were implications that these episodes were temporally associated with LH pulses. © 2004 Elsevier B.V. All rights reserved. Keywords: Luteinizing hormone; Mechanisms of hormone action; Ovary; Pregnancy; Progesterone

∗

Corresponding author. Tel.: +358 19 5295323; fax: +358 19 6851181. E-mail address: [email protected] (J.V. Virolainen).

0378-4320/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2004.07.004

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1. Introduction Progesterone is considered to orchestrate uterine secretions, which in turn are crucial for embryonic development and growth (Adams et al., 1981; Vallet et al., 1998). Low feed intake in the immediate postmating period is associated with elevated progesterone concentrations and with improved embryonic survival (Jindal et al., 1996). Progesterone levels may be subjected to seasonal variation (Wrathall et al., 1986), possibly due to seasonally suppressed secretions of luteinizing hormone (LH). These hormonal changes during the late summer and early autumn might result in loss of the whole litter, which is known as one of the seasonal infertility manifestations (Love, 1978). In contrast to findings by Jindal et al. (1996), high feeding rate immediately after mating has been reported to provide benefits for reproduction. Love et al. (1995) reported that liberal feeding improved pregnancy rate during the known seasonal infertility period. Although those benefits are considered to be mediated by LH and progesterone, it appears that progesterone concentrations in the peripheral blood are negatively influenced by a high feed intake, also at the time of seasonal infertility, even though increased LH secretion was detected at the same time (Virolainen et al., 2004). In spite of the decreased progesterone level, the pregnancy rate was significantly higher in gilts with abundant feeding as compared with the other regimen (Virolainen et al., 2004). Progesterone concentration in the circulating blood reflects the balance between synthesis and metabolic clearance of progesterone. A high food intake results in increased portal blood flow and metabolic clearance of progesterone and is, therefore, linked to lower progesterone concentration in plasma of sheep (Parr et al., 1993) and pigs (Prime and Symonds, 1993). However, it is possible that the production of progesterone increases due to a high rate feeding, but the rise is negated by an increased portal blood flow and metabolism. The benefits of increased progesterone production could be mediated locally to the uterus by countercurrent transfer of progesterone from venous blood from the ovary into arterial blood supplying the uterus as demonstrated in early pregnant sows (Stefanczyk-Krzymowska et al., 1998). Krzymowski et al. (1986) demonstrated a close anatomical association and specific structures between the venous, arterial and lymphatic vessels along the mesometrium, which might enable penetration of steroids into the uterine arterial blood. The described mechanism might ensure an increased progesterone concentration in blood flow to the uterus. High food intake might improve progesterone production, which in turn might have a local effect on the uterus, since a decrease in progesterone concentration of the peripheral blood has been reported (Virolainen et al., 2000, 2004). However, catheterization of the utero-ovarian vein requires an intensive surgical procedure that might affect ovarian and uterine function. Therefore, catheterization of the caudal vena cava via the lateral saphenous vein would be an appropriate method to monitor progesterone production. The objective of this study was to examine the difference in progesterone concentrations in the jugular and vena cava blood in early pregnant gilts. Furthermore, three feeding levels were used to determine if nutrient intake had an effect on ovarian progesterone production.

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2. Materials and methods 2.1. Animals, management and feeding Nineteen crossbred (Landrace and Large White) pregnant gilts (138 ± 15.7 kg, mean ± S.D.) from a commercial farm (Hillcrest Park piggery, Menangle, NSW, Australia) were used in this study. Gilts were transferred to light (12 h light and 12 h dark) and temperature (+23 ± 1 ◦ C) controlled climate rooms in two groups (8 and 11 gilts) with 6-week interval, 5–9 days after natural mating (day 0). Gilts were weighed after arrival and at the end of the trial. The gilts were housed in individual stalls and fed once a day at 9.00 a.m. with a commercial diet (Amitie Dry Sow diet; Vella Stock Feeds, Plumpton, NSW, Australia; 13 MJ/kg) of 2.5 kg/day from arrival until day 12 or 13 of pregnancy. The animals were then randomly assigned to three regimens, which were chosen based on earlier studies of authors (Peltoniemi et al., 1997a,b; Virolainen et al., 2004). From day 12 or 13, high feeding group (H–H) received 3.6 kg/day until the end of trial (day 24), low feeding group (L–L) received 1.8 kg/day and the low–high feeding group (L–H) had low rate (1.8 kg/day) until day 21 of pregnancy followed by a high rate feeding (3.6 kg/day) thereafter. Low level was about equal to maintenance requirements (metabolic body weight ∗ 0.458 MJ) and high level was 2.5 times maintenance per day. All pigs were tested for pregnancy by real time ultrasound. The sampling period, 20–24 days after mating, was chosen to minimize daily variations in progesterone concentration, since progesterone concentration appears to remain reasonably constant beyond day 20 (Tast et al., 2000). 2.2. Blood collection Gilts were fitted nonsurgically with an indwelling jugular vein cannulae via an ear vein on day 19 of pregnancy (Peacock, 1991). Gilts were fastened around the upper jaw with a rope snare and tied to fence while an i.v. catheter placement unit (14 G, 57 mm) was inserted into an ear vein and a vinyl tube (o.d. 1.5 mm × i.d. 1.0 mm) was passed through the catheter. The free tube was secured to the ear with adhesive tape wrapped around the ear and the ear was fixed to the neck by a collar of adhesive tape. The free end of tube was stored in a Velcro pouch attached to the neck for an easy sampling. The time taken for catheterization was approximately 5–10 min. In preparation for catheterization of the caudal vena cava (Benoit and Dailey, 1991), anesthesia was induced with an i.v. bolus injection of thiopentone sodium at a dosage of 1.25 g diluted in 25-ml sterile water. Anesthesia was maintained with halothane/oxygen mixture delivered by a facemask. Gilts were laid on the left side and lateral aspect of the right rear leg shaved approximately 20 cm dorsal to the hock and the area was disinfected with 70% EtOH. An incision was made through the skin approximately 3 cm dorsal to the hock and 2 cm lateral to the Achilles tendon. The subcutaneous fat (1–2 cm) was separated and the lateral saphenous vein (SV) isolated by blunt dissection. Tweezers were placed under the vessel and two loose ligatures were inserted approximately 1 cm apart. Slight traction was maintained on distal ligature, a small transversal incision made in the vein and a catheter (1.0 mm i.d., 1.5 mm o.d.) was inserted. Each catheter was marked previously at 4-cm intervals from 48 to 56 cm from the tip. Plasma samples were collected at

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the 48, 52 and 56 cm marks as the catheter was inserted. Approximately 2 ml of blood was withdrawn and discarded before collection of a 4-ml blood sample. A jugular sample was collected after the last (56 cm) sample from the vena cava. Sampling from all sites took approximately 3 min. Catheters were flushed after each sample with 3 ml of EDTA saline (0.9% K-EDTA, 0.9% NaCl). After samples were collected, catheters were pulled back to the 52 cm marked and secured with two ligatures around the vein. Before the catheter was secured, the distal ligature was used to tie off the vein. The wound was dressed with antibiotic powder (Terramycin Pinkeye Powder; Pfizer; Australia) and sutured (Coated Vicryl 0, 3.5 metric; Ethicon; Australia). The free end of catheter (1 m) was run dorsally along the caudal surface of rear leg lateral to the tail and ended on the back of the pig. Catheters were sutured to the skin and Velcro pouches were used to protect the free end of the catheter and they were fixed to the animal with adhesive tape wrapped around the abdomen. The legs were bandaged and catheters covered with adhesive tape. Catheterization took approximately 40–60 min and within 30 min after operation gilts walked back to their individual stall. Concurrent blood samples (4 ml) were collected from the jugular vein and vena cava starting on the day following the surgery. Approximately 2 ml of blood was withdrawn and discarded before collection of a 4-ml blood sample. Catheters were flushed with 3 ml of EDTA saline (0.9 % K-EDTA, 0.9% NaCl) after each sample collection. Two consecutive samples at 30-min intervals were collected four times a day for 5 days (day of pregnancy 20–24). Pre-prandial samples were taken at 8.00 a.m. (1 h before feeding) followed by post-prandial sampling (after meal) 2, 6 and 11 h later (at 8.00, 8.30, 11.00, 11.30, 15.00, 15.30, 20.00 and 20.30). Based on a great variation detected in concentrations of progesterone in plasma from the vena cava in the first group, three gilts (one of each feeding group) were additionally sampled from the both catheters at 30-min intervals for 12 h on day 22 in the second group in order to get information about that variation. Temporary difficulties with catheters in one of these animals (H–H) resulted an incomplete sample sets and therefore only two patterns (L–L and L–H) are presented here. Blood was collected in plastic tubes containing 100 ␮l of concentrated EDTA (250 mg/ml) and centrifuged within 1 h. Harvested plasma was stored at −20 ◦ C until assayed for progesterone or for LH. 2.3. Assays Blood samples were analyzed for progesterone using a direct commercial RIA (Spectria, Orion Diagnostica, Turku, Finland), which has been validated to measure progesterone in pig plasma (Peltoniemi et al., 1995). Fifty microliters of sample plasma and 500 ␮l of buffered 125 I label were added to the antibody-coated tubes. The sensitivity of the assay was ≤0.094 ng/ml. Intra-assay coefficients of variation (CV) for two different reference concentrations were 10.0 and 13.1%, while inter-assay CV were 12.8 and 7.9%. Four assays were performed (425 samples per assay). Plasma LH concentrations were determined using a previously validated direct homologous double antibody RIA (Niswender et al., 1970). The sensitivity of the assay was 0.14 ng/ml. The intra-assay CVs for three different reference concentrations were 11, 6 and 10%.

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2.4. Statistical analysis Three gilts were excluded when data were analyzed, because of technical problems with catheters. Eventually there were five, six and five gilts in the H–H, L–L and L–H groups, respectively. The values of progesterone concentration used in the statistical analysis were the original values of assay if they fell within the range of the assay (1.2–30.4 ng/ml). All values outside the range were diluted 50:50 and re-assayed (46 samples). If concentrations still exceeded the range, the highest value of 60 ng/ml was used (12 samples). All progesterone concentrations of the vena cava samples were compared to concentrations from the jugular vein samples. The mean of two consecutive samples (collected at 30-min interval) was calculated separately for both sampling sites in each gilt and the means were used as pre-prandial, 2-, 6- and 11-h post-prandial values. Pre-prandial concentrations were compared with post-prandial concentrations. In addition, the mean progesterone concentration for each day sampled was calculated and analyzed for both sampling sites separately. Analysis of variance for repeated measurements was used followed by pairwise comparisons with least significant difference (LSD) adjustment, when a significant difference was established. Day of pregnancy, time of bleeding and sample site were used as repeated values. Because data were not normally distributed, a log-transformation was used. Litter size and weight gain for the groups were compared using one way analysis of variance followed by Tukey’s pairwise comparisons when appropriate. Progesterone and LH patterns over 12 h were not statistically analyzed because of the low number of animals. However, the method for identification of LH pulses was based on the procedure described by (Virolainen et al., 2004). The method for identification of progesterone pulses was modified from the same procedure. Possible progesterone pulses were found from the profile after plotting an individual’s samples against time. A pulse was defined as at least two consecutive samples that exceeded the basal level by more than three standard deviations and in which the peak concentration was completed within two subsequent sampling intervals. The basal progesterone concentration in the vena cava was defined as the mean of three consecutive samples preceding every suspect progesterone pulse. If there were more than one possible pulse, the basal level was calculated using the basal value of progesterone pulse that gave the lowest progesterone concentrations.

3. Results The progesterone concentrations were higher (P < 0.001) in plasma from vena cava compared with concentrations measured from the jugular vein throughout the sampling period in all groups (Fig. 1) being 19 ± 11.5 ng/ml and 8 ± 2.6 ng/ml (mean ± S.D.), respectively. The variation (mean ± S.D.) between two consecutive 30-min progesterone samples was greater when measured in plasma from the caudal vena cava and compared with that from the jugular vein, being 8.7 ± 9.4 and 1.8 ± 0.9 ng/ml, respectively. Based on the significant difference between sampling sites, further analyses were performed separately for data from the jugular vein and the caudal vena cava. The mean progesterone concentration in plasma from both caudal vena and jugular vein decreased significantly (P < 0.001) as pregnancy progressed from day 20 to 24 in all groups (Fig. 2). Feeding level affected significantly

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Fig. 1. Means of progesterone concentrations in vena caval (VC) and in jugular (VJ) plasma for three feeding groups; high (H–H), low (L–L) and modified (L–H) per sampled days. Bleeding commenced on day 20 of pregnancy. Two consecutive samples were taken at 30-min interval four times per day; before feeding (pre-pr), 2, 6 and 11 h after feeding (2, 6 and 11, respectively).

(P < 0.05) concentration of progesterone in the jugular vein but not in the caudal vena cava. Progesterone concentration in jugular plasma from gilts in all groups decreased steadily after ingestion through a day. Post-prandial concentrations were significantly lower when compared to pre-prandial concentrations (P < 0.05, Figs. 1 and 3). Instead of decreasing tendency of progesterone, a great variation was observed in progesterone concentrations from the vena cava through a day. A significant interaction (P < 0.05) occurred between time and feeding group in the jugular samples but not in the vena cava samples. Progesterone

Fig. 2. Daily plasma progesterone concentrations (±S.E.M.) in the jugular vein (VJ) and the caudal vena cava (VC) for three feeding groups; high (H–H), low (L–L) and modified (L–H) (5, 6 and 5 gilts, respectively) on days 20, 21, 22, 23 and 24 of pregnancy. Plasma progesterone concentration decreased significantly in all groups and in both veins during the sampling period.

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Fig. 3. Means of plasma progesterone concentrations (±S.E.M.) depicted pre- and post-prandially for each feeding group; high (H–H), low (L–L) and modified (L–H) in both veins; the jugular vein (VJ) and the caudal vena cava (VC).

concentrations in the jugular vein in the L–L group were significantly lower compared to those in the H–H group, but there was only a tendency (P = 0.054) being lower when compared with the L–H group. No difference occurred in concentrations between the groups L–H and H–H. Individual patterns of progesterone and LH secretion were achieved by intensive bleeding at 30-min intervals for 12 h from two of three gilts (Fig. 4A and B). Characteristics of LH and progesterone for the both veins are shown in Table 1. An episodic pattern of progesterone production was evident in plasma samples collected from the vena cava but not from those collected from the jugular vein. The gilt from the L–L group seemed to have a basal progesterone concentration in the vena cava similar to the concentration in the jugular vein (Fig. 4A). Furthermore, there were three defined progesterone pulses and the amplitudes of those progesterone pulses (27.7 ng/ml) were threefold greater than the basal level (10.3 ng/ml) concentration. The average time interval between peaks of progesterone secretions was 5.0 h. In this gilt, LH pulse (three of five defined LH pulses) preceded every episodic release of progesterone seen in vena cava (Fig. 4A). The other gilt, from the group L–H, had higher progesterone concentrations in the vena cava than in the jugular vein Table 1 LH and progesterone characteristics determined from plasma samples (VC from caudal vena cava and VJ from jugular vein) collected for 12 h at 30-min intervals on day 22 of pregnancy Gilt/feeding

4/LLL

12/LHL

Hormone/site

Basal level (ng/ml)

Mean concentrations (ng/ml)

Number of pulses

Amplitude (ng/ml)

LH Progesterone/VC Progesterone/VJ

0.8 10.3 6.5

1.2 19.2 8.4

5 3 1

1.1 27.7 5.6

LH Progesterone/VC Progesterone/VJ

0.8 28.2 9.7

1.0 28.2 9.7

2 0 0

1.0

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Fig. 4. A and B. Plasma progesterone profiles from the caudal vena cava (P VC,
-
) and the jugular vein (P VJ, -) and luteinizing hormone (LH) profiles (-) during a 12 h bleeding window at 30-min intervals on day 22 of pregnancy for two gilts. Gilt with profile A had low level feeding since day 11 of pregnancy and gilt with profile B had high level feeding since day 20 of pregnancy. Animals were fed once a day at 9 a.m. after bleeding (shown with arrow). All of the feed offered was consumed within 10 min by the gilt a, but not until the late afternoon by the gilt b.

throughout the sampling period, but there were no pulses to fulfill the criteria described for a pulse (Fig. 4B). However, there were prolonged increments in progesterone with about 5-h interval, but those increments were not associated with two defined LH pulses (Fig. 4B). However, there was a further increment in progesterone concentration, when LH pulse occurred. One gilt aborted in L–H group a day after sampling period (on day 25). There was no indication of an acute infective cause as indicated by clinical signs although the gilt reduced feed consumption to half of offered on days 23–25. Circulating progesterone concentration did not decrease a day before the abortion. However, progesterone concentration detected in plasma from the vena cava was decreased nearly at the level of peripheral plasma concentration on days 23–25 (when measured eight times per day); whereas, concentrations in the vena cava were two times greater than concentrations in the jugular vein for first 2 days detected.

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The gilts in the H–H group gained significantly more weight (P < 0.05) than those in the L–L group. The means ±S.D. for each group (H–H, L–L and L–H) were 18.5 ± 3.5, 9.6 ± 5.6 and 11.5 ± 2.6 kg, respectively. The feeding regimen did not affect significantly total litter size or number of piglets born alive, 11.5 ± 1, 12.7 ± 3.3, 11.0 ± 2.6 and 11.5 ± 1, 11.75 ± 2.5, 11.0 ± 2.6 for groups H–H, L–L and L–H, respectively.

4. Discussion The results of present study showed a significant difference between progesterone concentrations measured in jugular blood and blood in the caudal vena cava. Vena cava sampling was more suitable for the study of ovarian production as samples obtained contained progesterone produced in both ovaries before its metabolism in the liver, since the ovarian and the uterine veins from both sides drain into the caudal vena cava. Higher progesterone concentration in the caudal vena cava might indicate local influence of progesterone in the uterus. Progesterone seemed to be produced in episodic manner, based on a great variation measured in progesterone concentrations from two consecutive samples from vena cava. Findings in late pregnant miniature pigs by Parvizi et al. (1976) and two individual patterns of progesterone from the caudal vena cava in the present study provided further indication for pulsatile secretion. In both of the gilts an increase of progesterone secretion was associated with the time of feeding. On average, the interval was 5.0 h for pulses (gilt 2) or prolonged increments of progesterone (gilt 12). Gilt 12 had additional two progesterone peaks, which did not fulfill the criteria for a pulse. However, the observed variation in concentration of progesterone between two samples taken randomly during a day is explained by episodic pattern of hormone secretion. No such episodes were detectable in jugular vein samples. Effects of episodic release of progesterone on the uterus or the uterine secretions remain unknown. Moreover, there was some indication of interaction between episodic secretion of LH and progesterone in early pregnant pigs as reported earlier in late pregnant miniature pigs (Parvizi et al., 1976), in sheep (Alecozay et al., 1988; McNeilly et al., 1992), in horses (Perkins et al., 1993) and during late luteal phase in monkeys (Ellinwood et al., 1984). However, not every LH pulse was followed by a progesterone pulse, which might indicate a threshold effect or a refractory period of the CL. There might also be some other factors effecting on progesterone secretion in early pregnancy. Further studies should be done to ensure a connection between progesterone pulses and LH pulses in early pregnant pigs. If there is a connection, it may be important in the seasonal disruption of pregnancy. Suppressed LH secretion in autumn might induce a reduction in progesterone concentration at the uterine level. In turn, decreased progesterone blood flow to the uterus might have direct or indirect effects on viability of embryos. A role of pulsatile secretion of progesterone in optimal secretion of the uterine proteins remains also unknown. The present study did not show a profound effect of the level of feeding on progesterone concentration. Eating affected progesterone concentration when compared within a day and this was reflected clearly in the pattern of progesterone in both sampling sites. However, there were inverse effects in the vena cava compared to those in the jugular

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vein. There was a significant decrease in progesterone concentration in the jugular vein when compared between pre- and post-prandial values; whereas, in the vena cava there was a slight post-prandial increase in progesterone production (except in the L–H group). The decrease in progesterone concentration in the jugular vein might be due to increased metabolism and increased portal blood flow (Prime and Symonds, 1993). However, an increment of progesterone in the caudal vena cava after ingestion might indicate a direct effect of metabolic mediator (such as insulin) on ovaries. The patterns of progesterone secretion of the intensively bled gilts supported those phenomena too. A mediator of feeding effect on progesterone secretion remains unclear. To reveal factors mediating this influence in early pregnant pigs, an intensive sampling especially from the caudal vena cava is needed in further studies. Earlier studies examined daily progesterone concentrations in peripheral blood for 3 weeks after mating and were able to demonstrate effects of feeding level on progesterone secretion in gilts (Jindal et al., 1996; Virolainen et al., 2004) and in sows (Virolainen et al., 2000; Zak et al., 1997). However, a period later in pregnancy was used in the present study to minimize variation in progesterone secretion seen during the 3 weeks after mating. It was thought that effects of feeding on progesterone would be easier to demonstrate at this later stage. However, the level of feeding did not affect progesterone concentrations profoundly. There was no indication of detrimental effects of high plane nutrition commencing a day 11, when compared litter size between the groups. This was consistent with findings by (Dyck and Strain, 1983) who reported no difference in embryonic survival when feeding level was increased on day 11. On the other hand, they reported that high feeding rate for 10 days after mating decreased embryonic survival, but thereafter increased pregnancy rate. Therefore, benefits of high feeding rate would be probably seen during the first 3 weeks of pregnancy. To reveal, if progesterone is involved in the mediating mechanism of early disruption of pregnancy, that period should be studied in detail. Effects of a high energy diet on progesterone production (measured from the vena cava) for the first 3 weeks of pregnancy still remain unclear. Furthermore, further studies are required to determine whether the pattern of progesterone secretion is pulsatile up to day 14 after mating, when the CL are not LH dependent but work autonomously (Anderson et al., 1967; Brinkley et al., 1964). To reveal a pattern of progesterone secretion, an intensive sampling such as used in the present study with those two individual is required. If progesterone is secreted in an episodic manner only after day 14 and there was a correlation between LH and progesterone pulses thereafter, this may provide further evidence for a critical role of LH in the mechanism of early disruption of pregnancy. In most studies, the role of progesterone as a mediator of the effects of post-mating feeding levels on uterine environment and embryonic viability has been examined by progesterone concentration in circulating plasma (Dyck et al., 1980; Jindal et al., 1996; Jindal et al., 1997; Mao and Foxcroft, 1998; Virolainen et al., 2000, 2004), which may not be informative enough according to the results of the present study. Therefore, it seems that to obtain more appropriate information about effects of feeding on progesterone and early pregnancy, it may be more valuable to follow changes of progesterone in plasma from the vena cava or the uterine veins with intensive sampling rather than in peripheral plasma with a single puncture. In conclusion, this study has shown that plasma progesterone concentrations are greater in the caudal vena cava draining the uterus and the ovaries than in the jugular vein. The

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shown difference is detectable when progesterone is measured concurrently from plasma representative of peripheral blood and blood containing uterine and ovarian hormones before their metabolism in the liver. This indicates that the uterus is supplied with a locally higher progesterone concentration compared to peripheral blood. Furthermore, the present study provided an indication for episodic pattern of progesterone secretion in early pregnant pigs. Further studies are needed to verify episodic nature of progesterone secretion and to determine a possible association between them and LH pulses. The significance of progesterone pulsatility has also to be determined.

Acknowledgments Hillcrest Park piggery at Menangle is acknowledged for providing gilts for the trial. I would like to acknowledge all the staff of Department of Veterinary Clinical Sciences at Camden who was involved in this trial. Kim Heasman is acknowledged for her assistance with the progesterone and the LH assays. Peter Thompson is acknowledged for his comments and assistance with statistical analysis.

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