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The complex anatomy of the ovine cervix limits the success of trans-cervical artificial insemination in sheep. However, there is a degree of natural relaxation of cervix at oestrus that is accompanied by an increase in the water content. As hyaluronan (HA) has a high affinity for water molecules, in this study, we tested the hypothesis that the HA content of the cervix, the proportion of different size fragments of HA and expression of its receptor CD44 vary with the stage of the oestrous cycle. Oestrous was synchronized in 25 Welsh mountain ewes, and their cervices were collected either during luteal phase (n=8) or pre-LH (n=8) or post-LH (n=9) surge stage of the oestrous cycle. The pre-LH surge group had the highest HA content (2.96 ng/mg of cervical tissue), which was significantly (P≤0.05) higher than that observed for the post-LH surge (2.04 ng/mg) group. The luteal phase group had a mean HA content intermediate between the pre- and post-LH surge groups, and was significantly different from either. The frequency of cervical samples containing both sizes of HA fragments (small and large) was significantly higher (P≤0.05) in the pre-LH surge group compared with the luteal and the post-LH surge groups, whereas that in post-LH surge group was significantly (P≤0.05) higher than that in the luteal group. The number of cervical samples that contained only small HA fragments was significantly (P≤0.05) higher in the luteal group compared with both the pre- and post-LH surge groups, whereas the number of samples containing only large HA fragments was significantly (P≤0.05) higher in the post-LH surge group compared with the luteal or pre-LH surge groups. Overall mean expression of CD44 in the vaginal and mid regions was significantly (P≤0.001) higher than that in the uterine region, with no difference between the vaginal and mid regions of the cervix. Pattern of CD44 expression depended on the stage of the oestrous cycle. At the luteal stage, CD44 expression did not vary among epithelial, sub-epithelial, circular and longitudinal smooth muscle layers, whereas at the pre- and post-LH surge stages, the expression in the epithelial layer was significantly (P≤0.001) higher than that in the other three layers. In general, CD44 expression in the transverse smooth muscle layer was significantly (P≤0.05) lower than the expression in all the other layers at all the stages of the oestrous cycle. The results indicated that the HA varied with the steroid status. Higher HA values at a time when cervical relaxation is naturally higher may indicate its involvement in remodelling of the cervix at oestrus.

Early mammalian embryo development in vitro can be enhanced by co-culture with oviductal cells and by the addition of insulin-like growth factors (IGFs). This study examined the expression patterns of the oviductal IGF system in cattle in relation to the number of days after oestrus and the presence or absence of embryos. Oviducts were collected from: (i) 66 nulliparous heifers on day 3, day 6 or day 16 after insemination and from (ii) ten non-pregnant, lactating cows on day 0 or day 1 of the oestrous cycle. Oviducts were coiled, frozen whole and sectioned for in situ hybridization. Expression patterns of mRNAs encoding IGF-I, IGF-II, type 1 IGF receptor (IGF-1R), and the IFG binding proteins (IGFBP)-1, -3 and -5 were determined from autoradiographs. Separate measurements were made for the mucosa and muscle layers of the infundibulum, ampulla and isthmus. None of the parameters measured differed between heifers with or without the presence of an embryo. mRNAs encoding IGF-I and IGF-1R were present in the mucosa and muscle of all three oviductal regions, and the highest value of IGF-I mRNA was measured in heifers on day 3. IGF-II mRNA was expressed predominantly in the muscle wall. IGFBP-1 mRNA was not detectable, whereas mRNAs encoding IGFBP-3 and -5 were expressed in both the muscle and mucosa. IGFBP-3 expression was higher in cows on day 0 and day 1 of the oestrous cycle than in heifers on day 3, day 6 and day 16 after insemination. A peak of IGFBP-5 expression was reached on day 6. Locally or systemically produced IGFs, regulated by IGFBPs, may act directly on the embryo or indirectly via modulation of oviductal secretions and muscular activity to influence the success of early embryo development.

This study examined the expression patterns of oxytocin and steroid receptors in the bovine endometrium during the oestrous cycle and early pregnancy to elucidate their respective roles in the regulation of luteolysis and the maternal recognition of pregnancy. In Expt 1, uterine biopsies were collected from four cows throughout three oestrous cycles each, to provide daily samples. In Expt 2, uterine tissue was collected on days 12, 14, 16 and 18 of the oestrous cycle (n = 20) or early pregnancy (n = 16). Oxytocin receptor, oestrogen receptor alpha and progesterone receptor mRNAs were localized by in situ hybridization, and localization of oestrogen receptor and progesterone receptor was confirmed by immunocytochemistry. All three receptors showed time- and cell-specific expression patterns. Oestrogen receptor alpha increased in all regions at oestrus but high concentrations were also found in the luminal epithelium during the mid-luteal phase and in the deep glands throughout the oestrous cycle. Progesterone receptor expression was higher in the stroma than it was in the types of epithelial cell, and increased expression was observed at oestrus and during the early luteal phase. The cyclical upregulation of oxytocin receptors in the luminal epithelium on about day 16 was not related to preceding changes in the endometrial expression of either oestradiol alpha or progesterone receptors. During early pregnancy, oxytocin receptor expression was suppressed. Oestrogen receptor a concentrations increased in the non-pregnant cows and decreased in the pregnant cows between days 16 and 18, but these changes followed rather than preceded the upregulation of oxytocin receptors in the non-pregnant cows. It is concluded that the initial upregulation of oxytocin receptors in the luminal epithelium, which triggers luteolysis, is not associated directly with changes in expression of oestrogen receptor alpha.

Oestradiol treatment can increase uterine oxytocin receptor expression in vivo. The actions of oestrogen are usually mediated via its receptor, but it also has direct non-genomic effects in some cells. This study investigated the effect of oestradiol and the role of the oestradiol receptor in regulating endometrial oxytocin receptor expression in the bovine uterus. Explant cultures (in triplicate) from late luteal phase non-pregnant endometrium received the following treatments: control (serum-free medium), oestradiol (0. 1 and 0.01 micromol l(-1)), oestradiol (0.1 micromol l(-1)) with the oestradiol receptor antagonist ICI 182780 (0.5 micromol l(-1)), and ICI 182780 (0.5 micromol l(-1)) alone. Explants were collected 12, 24 and 48 h after the start of culture. Oxytocin receptor mRNA expression in the explants was measured by in situ hybridization and oxytocin protein concentrations were measured by autoradiography with the iodinated oxytocin receptor antagonist d(CH(2))(5) [Tyr (Me)(2) Thr(4) Tyr NH(2)(9)]-vasotocin ((125)I-labelled oxytocin receptor antagonist). Oxytocin receptor mRNA and protein expression were initially low but spontaneous upregulation occurred in the luminal epithelium between 24 and 48 h (P < 0.01). Oestradiol increased oxytocin receptor mRNA upregulation in the first 24 h (P < 0.05) but the effect on 125 I-labelled oxytocin receptor antagonist binding was not significant. ICI 182780 inhibited the oestrogenic effect but had no significant effect on oxytocin receptor mRNA expression when given alone. In conclusion, the results showed that oestradiol exerts its effect via the oestradiol receptor. Oestradiol facilitates oxytocin receptor gene transcription by increasing it more rapidly, but spontaneous upregulation of endometrial oxytocin receptor still occurs in the absence of oestradiol.

Both the production of cytokines and the distribution of immune cells within the uterus change during early pregnancy. Evidence obtained mainly from mice indicates that these changes are important for implantation and in preventing a maternal immune response to the conceptus. The ruminant embryo also produces interferon tau at this time, the signal for the maternal recognition of pregnancy. The relationship between these events in cows was studied using uteri from three groups of animals on day 16 after observed oestrus: (i) cyclic controls, (ii) pregnant and (iii) inseminated but with no embryo present. Embryo size and the antiviral activity in uterine flushings (indicative of the interferon tau concentration) were measured. Sections of intact uterus were frozen for the localization and quantitation of CD4(+) (T lymphocytes), CD14(+) (macrophages) and CD21(+) (B lymphocytes) uterine cells by immunohistochemistry. The expression of interleukin (IL)-1alpha, IL-2, IL-6 and IL-10 mRNAs in uterine extracts was measured by RT-PCR. Neither embryo size, interferon tau concentration nor pregnancy status influenced the distribution of CD4(+), CD14(+) or CD21(+) cells in the day 16 uterus. Endometrial IL-1alpha mRNA was detected in most cows across the groups, whereas IL-2 mRNA was only present in the non-pregnant uterus. IL-6 and IL-10 mRNAs were not detectable in any uteri. In conclusion, IL-2 mRNA expression is detectable in the non-pregnant but not the pregnant uterus on day 16 and interferon t is unlikely to play a role in the redistribution of immune cells in the uterus during early bovine pregnancy.

The aim of the present study was to investigate the effects of administering a high plane diet during early to mid-gestation on the uterine and placental insulin-like growth factor (IGF) system and on systemic IGF-I concentrations in pregnant adolescent ewes with restricted placental growth. Embryos recovered from superovulated ewes inseminated by a single sire were transferred in singleton to the uterus of adolescent recipients. After transfer ewes were offered a high (H) or moderate (M) amount of a complete diet calculated to promote rapid or normal maternal growth rates, respectively. Five ewes from each group were switched from either M to H or H to M diets at day 52 of gestation. Maternal and fetal blood samples and placental tissues were collected from all animals at day 104. Ewes on the high plane diet from mid-gestation (HH, MH groups) had restricted placental mass (P < 0.01) and tended to have smaller fetuses. This was associated with increased maternal plasma IGF-I concentrations (P < 0.001). The pattern of expression of components of the IGF system in the uterus and placenta was studied by in situ hybridization. IGF-I mRNA concentrations were below the limit of detection. IGF-II mRNA expression was high in the fetal mesoderm and present in maternal stroma, but was not influenced by nutritional treatment. In contrast, IGF binding protein 1 (IGFBP-1) mRNA expression was higher (P < 0.05) and IGFBP-3 mRNA expression was lower (P < 0.05) in the endometrial glands of ewes in HH and MH groups. In the fetal trophoblast, IGFBP-3 mRNA expression was higher in the MH group. Type 1 IGF receptor expression was increased (P < 0.01) in the luminal epithelium of the HM group and IGFBP-2 mRNA expression was highest in the placentome capsule of ewes in the HH group. Together, these results indicate that reprogramming of the uterine and placental IGF axis by maternal nutrition could contribute to placental growth retardation in growing adolescent sheep.

The oviduct is the site of fertilization, and factors present in the oviductal fluid appear to be crucial to the future success of conceptus development. The spatial and temporal localization of mRNA encoding components of the insulin-like growth factor (IGF) system (IGF-I, IGF-II, the type 1 IGF receptor, and IGF-binding proteins -2, -3 and -4) in the ovine oviduct were examined in tissue samples taken during the early and late stages of follicular development, and the early, mid-, and late luteal phases using in situ hybridization. Expression of mRNA encoding IGF-I showed a cyclical pattern, increasing sharply in the mucosa and muscularis during the late follicular phase (P < or = 0.001), then declining. In the muscularis, mRNA encoding IGF-II exhibited no temporal changes, but concentrations in the mucosa increased from the late follicular stage to the early luteal phase. mRNA encoding the type 1 IGF receptor was present throughout the oviduct. Concentrations increased during the follicular phase to peak in the early luteal phase in both the mucosa and muscularis (P < 0.001). IGFBP-2 gene transcripts were undetectable at all time points examined. mRNAs encoding IGFBP-3 and IGFBP-4 were localized primarily in the stromal region. IGFBP-3 expression peaked in the late follicular stage of the cycle P < or = 0.001). The concentration of mRNA encoding IGFBP-4 increased in the follicular phase and was maintained at a significantly higher concentration during the early and mid-luteal stages (P < 0.001). The co-ordinate maximum expression of mRNA for both IGF-I and IGF-II, the type 1 IGF receptor and IGFBP-3 during the period when the gametes and embryo are in transit suggests a role for IGF-I and possibly IGF-II peptides in providing an oviductal environment propitious to conception and early embryonic growth and metabolism.

Oxytocin infusions were initiated on day 10 of the oestrous cycle in ewes, and luteal regression was induced by injection of 100 micrograms cloprostenol on day 12. Blood samples were collected at frequent intervals via an indwelling jugular vein cannula to measure concentrations of progesterone and luteinizing hormone (LH) during the luteal and follicular phases in saline (n = 6) and oxytocin (n = 5) infused animals. The oxytocin infusion maintained peripheral plasma concentrations of 53 +/- 3.2 pg oxytocin ml-1 (mean +/- SEM) compared with values of about 1 pg ml-1 during oestrus in control ewes. Oxytocin infusion had no effect on luteal phase progesterone concentrations, the timing of luteolysis, basal luteinizing hormone (LH) secretion, LH pulse frequency, or the timing or height of the LH surge. Treated ewes came into oestrus significantly earlier than controls (P < 0.05) but ovulated normally. Uterine samples collected 96 h after cloprostenol injection (approximately day 2 of the cycle) showed that oxytocin receptor concentrations were significantly higher in the endometrium in ewes that had been given a 5 day oxytocin infusion than in control animals (556 and 262 fmol mg-1 protein, respectively: geometric means from ANOVA, P < 0.001), whereas myometrial receptor concentrations were not affected (113 and 162 fmol mg-1 protein, respectively). We conclude that the previously reported delay in luteal development caused by oxytocin infusion (Wathes et al., 1991) is not due to the inhibition or delay of ovulation, but must instead occur via a direct influence on the developing corpus luteum.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentrations of oxytocin were measured in corpora lutea obtained from heifers throughout the oestrous cycle and first 30 days of pregnancy. Values were low during the first 3 days of the cycle (less than 250 ng/g tissue), increasing to 1312 ng/g by Day 4. Values then further increased up to a maximum of 2344 ng/g on Day 12. Concentrations were similar in cyclic and pregnant animals throughout the midluteal phase and were maintained at approximately 1500 ng/g until the 18th (cyclic cows) or 19th (pregnant cows) day after oestrus, when they were again low. Values subsequently remained less than 250 ng/g in pregnant cattle. Concentrations of oxytocin in jugular venous plasma of cyclic (n = 5) and pregnant (n = 4) cows were measured in samples collected every 15 min for 8 h on Days 14, 16, 18 and 19 after oestrus. There were no significant differences in mean concentrations (range: 2.5-4.7 pg/ml) or in the number, frequency or area under the curve of episodes between either cyclic and pregnant animals, or between days. Mean basal concentrations were higher on Day 16 than on Day 14 (P less than 0.05), values on Days 18 and 19 being intermediate. These findings suggest that the corpus luteum contains a finite amount of releasable oxytocin, which is exhausted by Day 18-19 after oestrus, whether or not pregnancy occurs, and that there is no further accumulation of oxytocin in the animal during early pregnancy. The contribution of luteal oxytocin to jugular venous concentrations appears to be less than in sheep, in which values in the jugular vein closely parallel those within the corpus luteum.

Two experiments were designed to investigate release patterns of oxytocin into plasma during oestrus and the early luteal phase. In Exp. 1, blood samples were collected from 5 ewes every 30 min for 10 h during 6 days around oestrus and the early luteal phase. During oestrus concentrations of oxytocin were generally low (1.27 +/- 0.54 pg/ml; mean +/- s.d.) but with occasional pulses up to 6 pg/ml. By Day 5 mean basal concentrations had risen to 4.5 +/- 2.1 pg/ml with a fluctuating release pattern. In Exp. 2, a method was developed for continuous blood sampling from conscious, unrestrained ewes. On the predicted day of oestrus following an untreated oestrous cycle, 8-ml blood samples were collected every minute for two 35-min periods (8 ewes: 16 sampling periods). For 6 ewes a ram was introduced to the pen for part of this time, and resulting behaviour was recorded. Additional blood samples were assayed for LH and progesterone to determine the stage of the cycle. Overall mean oxytocin concentrations ranged from 1.5 +/- 0.53 to 6.8 +/- 5.25 pg/ml in different animals. Ewes which were both in oestrus and exposed to the ram showed a pulsatile oxytocin release pattern consisting of low baseline concentrations with short-duration pulses superimposed (duration 1-4 min; amplitude 2.5-31.7 pg/ml; frequency 3.18/h). Coitus was not temporally associated with pulsatile release. However, the importance of the presence of the ram was indicated by total separation of 2 oestrous ewes from the ram until after experimentation. In these animals only 1 pulse of oxytocin was detected in 2.7 h of sampling. It is concluded that, although mean oxytocin concentrations at oestrus were low, short duration pulses were released into the plasma at this time. This effect may be dependent on the presence of a ram.

The ultrastructure of cow placentomes, collected between 37 and 260 days of gestation, was examined. The microvillar junction and binucleate cell granules were selectively stained by phosphotungstic acid. Fetal binucleate cells interrupted the microvillar junction and penetrated as far as the basement membrane of the uterine epithelium. The uterine epithelium included not only binucleate cells which contained the distinctive granules but also non-granulated binucleate cells with pyknotic nuclei at the microvillar junction. Binucleate cells with pyknotic nuclei were also seen within chorion cells. It is suggested that the normal function of a mature chorionic binucleate cell at all the stages of bovine pregnancy is migration into the uterine epithelium to release its granules and subsequent condensaton to a cell remnant which is phagocytosed by the chorionic epithelium.

Corpora lutea and follicles were taken from the ovaries of 12 ewes at intervals from the start of luteolysis until 3 days after ovulation. RIA analysis of the tissue oxytocin content showed that luteal oxytocin concentrations declined during luteolysis to reach basal values at about the time of the next ovulation. Oxytocin was first measurable in the walls of 3 out of 6 preovulatory follicles during the LH surge, with a small increase in concentration to 26.1 +/- 6.6 pg/mg before ovulation, and a further increase in the young corpus luteum to concentrations exceeding 1 ng/mg 2-3 days later. After the LH surge, oxytocin was also found in the follicular fluid at a concentration of 3.4 +/- 0.3 ng/ml. Using immunocytochemical techniques, oxytocin and neurophysin were first detected in the follicle wall immediately before ovulation, and were localized in the granulosa cells. After ovulation the stained cells initially formed strands which appeared to break down to clusters and then to individual cells as the corpus luteum matured. The immunocytochemical picture also suggested that neurophysin immunoreactivity increased within a few hours of ovulation but that processing to oxytocin may be delayed. Measurements of circulating oxytocin concentrations revealed a pulsatile release pattern throughout the follicular phase with the height of the pulses decreasing from 25 +/- 5 pg/ml during luteolysis to a minimum of 11 +/- 2 pg/ml during the LH surge.

A group of 14 ewes was actively immunized against oxytocin coupled to carrier protein, and comparisons of the reproductive status of these animals were made against ewes immunized against carrier protein only (N = 5) and untreated controls (N = 6). The last two groups were indistinguishable and were therefore combined as a single control group for analysis of the results. Oestrous cycle lengths were significantly extended in oxytocin-immunized ewes (P less than 0.005) with 42% of cycles lasting greater than 18 days. Cloprostenol treatment in the mid-luteal phase resulted in apparently normal luteal regression and re-ovulation, but luteal phase FSH concentrations and follicular phase LH concentrations were elevated in the immunized ewes, although surge levels of both hormones were unaffected. Measurements of free oxytocin concentrations in the blood suggested that these were significantly raised in treated animals. Progesterone concentrations in peripheral plasma were not altered by treatment. Mating resulted in a conception rate of 91% in control ewes compared with only 28% in oxytocin-immunized animals (P less than 0.01). There was no evidence of any conceptus material in the uteri of non-pregnant immunized ewes 25 days after service. Some had re-ovulated, whereas the ovaries of others contained mature corpora lutea which had been maintained. Ovarian histology appeared normal. We conclude that active immunization against oxytocin influences gonadotrophin secretion and reduces fertility. The site(s) of action for both of these effects needs to be determined.

Anoestrous Romney Marsh ewes with (+P) and without (-P) progesterone pretreatment were induced to ovulate by multiple low-dose injection of GnRH followed by a bolus injection of GnRH. Luteal function was assessed by twice daily measurement of plasma progesterone. Animals were slaughtered on Days 3 or 5 after the end of GnRH treatment and CL and endometrium were recovered. In all Day-5 ewes, blood samples were collected at 30-min intervals for 8 h on Days 3 and 5 for measurement of PGFM and oxytocin. At slaughter 92% of the Group +P ewes had ovulated compared with 54% of the Group -P ewes. The ovaries of some of the Group -P ewes only contained luteinized cysts either alone or in association with CL. In the ewes that ovulated, progesterone profiles were normal in all Group +P ewes, whereas Group -P ewes had 'normal' or 'abnormal' profiles in which plasma progesterone was declining prematurely. All of the CL from ewes with abnormal progesterone profiles were associated with follicular cysts, and were significantly smaller and with a lower progesterone content on Day 5. PGFM levels decreased (P less than 0.05) between Days 3 and 5 in ewes in Groups +P and -P with 'normal' CL but increased (P less than 0.01) in Group -P ewes with 'abnormal' CL. Oxytocin levels were lower in Group -P ewes with 'abnormal' CL on Day 5, than in 'normal' ewes in Groups -P (P less than 0.01) or +P (P less than 0.05). In 3/5 Day-5 ewes with 'abnormal' CL there was a clear association between a major peak of oxytocin and a rise in PGFM during the frequent sampling period on Day 3 or Day 5, and endometrial oxytocin binding sites were present at slaughter. This suggests that the premature regression of 'abnormal' CL occurs via the normal luteolytic mechanism. Although ewes in Groups +P and -P with 'normal' CL had similar progesterone profiles, plasma oxytocin was significantly higher (P less than 0.05) in the Group -P ewes and oxytocin binding sites were present only in this group, suggesting that progesterone pretreatment can influence the production of both oxytocin and its receptor.

Bovine ovaries were obtained from the abattoir and corpora lutea were classified as: (1) early luteal phase (approximately Days 1-4); (2) mid-luteal phase (Days 5-10); (3) late luteal phase (Days 11-17); (4) regressing (Days 18-20) and (5) pregnant (Days 90-230). In addition, preovulatory follicles and whole ovaries without luteal tissue were collected. Concentrations of oxytocin, vasopressin, bovine neurophysin I and progesterone were measured in each corpus luteum by radioimmunoassay. Progesterone and neurophysin I levels increased from Stage 1 to Stage 2, plateaued during Stage 3 and declined by Stage 4. Oxytocin and vasopressin concentrations increased from Stage 1 to Stage 2 but declined during Stage 3 and were low (oxytocin) or undetectable (vasopressin) in follicles, whole ovaries and pregnancy corpora lutea. Therefore the concentrations of both peptide hormones were maximal during the first half of the cycle and declined before those of progesterone. The high concentration of oxytocin within the corpus luteum coupled with the presence of bovine neurophysin I suggests that oxytocin is synthesized locally.

Oestrus was synchronized in 8 cyclic heifers by progesterone treatment (PRID), after which the animals were monitored for one control cycle to measure the inter-oestrous interval. Osmotic minipumps containing saline (controls, N = 3) or oxytocin (N = 5) were implanted subcutaneously on Day 10 of the second cycle, and removed 12 days later. Jugular venous blood samples were collected daily for measurement of progesterone, and every 2 days for oxytocin. In addition, blood samples were taken every 10 min from 1 h before to 3 h after minipump insertion for measurement of plasma 15-keto-13,14-dihydroprostaglandin-F-2 alpha (PGFM) and every 30 min over the same period for measurement of progesterone and oxytocin. The lengths of the first untreated cycle in both groups of heifers were 20.2 +/- 0.56 (mean +/- s.e.m.) days compared with 25.4 +/- 0.81 days after oxytocin treatment (P less than 0.001). Oxytocin plasma concentrations in treated animals rose from less than 10 pg/ml to 70-500 pg/ml by 2 h after the start of oxytocin infusion and remained elevated until treatment was withdrawn. There was no increase in PGFM concentrations immediately after minipump insertion. Plasma progesterone concentrations were similar in treated and control animals but remained at mid-luteal levels for an average of 5 days longer in treated heifers. It is concluded that continuous administration of oxytocin can extend the luteal life-span in cattle.

Gilts were treated on Day 112 of gestation with saline or a prostaglandin (PG) F-2 alpha analogue. In control gilts there was a rise in the relaxin concentration from 48 h before the onset of delivery, peaking between 12 and 28 h pre partum followed by a steep fall. The relaxin concentrations at each 1-h time interval were analysed in relation to the farrowing interval for each gilt using correlation analysis. There was a significant (at least P less than 0.05) positive correlation between the relaxin concentration and the farrowing interval at every time period from 14 to 2 h before delivery. In contrast there was little relationship between concentrations of progesterone, oestrone and oestradiol-17 beta and farrowing intervals. The gilts treated with PGF-2 alpha analogues had steroid profiles indistinguishable from those in controls but differing relaxin secretion patterns. Relaxin concentrations peaked at 1-2 h after PGF-2 alpha injection and this was followed by a second smaller increase closer to the time of delivery in 7 out of 12 gilts. The 'two-peak' gilts had significantly higher relaxin concentrations at farrowing and took significantly longer to farrow than did the 'one-peak' gilts (P less than 0.005). These results suggest that high relaxin concentrations during the last 14 h before the onset of parturition are associated with increased farrowing times, but are not associated with any increase in neonatal mortality.