Incorporation of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men

This study investigated the incorporation of cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA) and trans-10,cis-12-CLA (t10,c12 CLA) into plasma and peripheral blood mononuclear cell (PBMC) lipids when consumed as supplements highly enriched in these isomers. Healthy men (n = 49, age 31 +/- 8 years) consumed one, two, and four capsules containing similar to600 mg of either c9,t11 CIA or t10,c12 CLA per capsule for sequential 8 week periods followed by a 6 week washout before consuming the alternative isomer. Both isomers were incorporated in a dosedependent manner into plasma phosphatidylcholine (PC) (c9,t11 CLA r = 0.779, t10,c12 CLA r = 0.738; P < 0.0001) and cholesteryl ester (CE) (c9,t11 CLA r = 0.706, t10,c12 CLA r = 0.788; P < 0.0001). Only t10,c12 CLA was enriched in plasma nonesterified fatty acids. Both c9,t11 CIA and t10,c12 CLA were incorporated linearly into PBMC total lipids (r = 0.285 and r = 0.273, respectively; P < 0.0005). The highest concentrations of c9,t11 CLA and t10,c12 CLA in PBMC lipids were 3- to 4-fold lower than those in plasma PC and CE. These data suggest that the level of intake is a major determinant of plasma and PBMC CLA content, although PBMCs appear to incorporate both CLA isomers less readily.

Incorporation of cis-9, trans-11 conjugated linoleic acid and vaccenic acid (trans-11 18 : 1) into plasma and leucocyte lipids in healthy men consuming dairy products naturally enriched in these fatty acids

The present study investigated whether consuming dairy products naturally enriched in cis-9, trans-11 (c9,t11) conjugated linoleic acid (CLA) by modification of cattle feed increases the concentration of this isomer in plasma and cellular lipids in healthy men. The study had a double-blind cross-over design. Subjects aged 34-60 years consumed dairy products available from food retailers for 1 week and then either control (0.17 g c9,t11 CLA/d; 0.31 g trans-vaccenic acid (tVA)/d) or CLA-enriched (1.43 g c9,t11 CLA/d; 4.71 g tVA/d) dairy products for 6 weeks. After 7 weeks washout, this was repeated with the alternate products. c9,t11 CLA concentration in plasma lipids was lower after consuming the control products, which may reflect the two-fold greater c9,t11 CLA content of the commercial products. Consuming the CLA-enriched dairy products increased the c9,t11 CLA concentration in plasma phosphatidylcholine (PC) (38 %; P=0.035), triacylglycerol (TAG) (22 %; P < 0.0001) and cholesteryl esters (205 %; P < 0.0001), and in peripheral blood mononuclear cells (PBMC) (238 %; P < 0.0001), while tVA concentration was greater in plasma PC (65 %; P=0.035), TAG (98 %; P=0.001) and PBMC (84 %; P=0.004). Overall, the present study shows that consumption of naturally enriched dairy products in amounts similar to habitual intakes of these foods increased the c9,t11 CLA content of plasma and cellular lipids.

Feeding rumen-inert fats differing in their degree of saturation decreases intake and increases plasma concentrations of gut peptides in lactating dairy cows

Our objective was to determine the effect of feeding rumen-inert fats differing in their degree of saturation on dry matter intake (DMI), milk production, and plasma concentrations of insulin, glucagon-like peptide 1 (7-36) amide (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin (CCK) in lactating dairy cows. Four midlactation, primiparous Holstein cows were used in a 4 x 4 Latin square experiment with 2-wk periods. Cows were fed a control mixed ration ad libitum, and treatments were the dietary addition (3.5% of ration dry matter) of 3 rumen-inert fats as sources of mostly saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), or polyunsaturated fatty acids (PUFA). Daily DMI, milk yield, and composition were measured on the last 4 d of each period. Jugular vein blood was collected every 30 min over a 7-h period on d 12 and 14 of each period for analysis of plasma concentrations of hormones, glucose, and nonesterified fatty acids. Feeding fat decreased DMI, and the decrease tended to be greater for MUFA and PUFA compared with SFA. Plasma concentration of GLP-1 increased when fat was fed and was greater for MUFA and PUFA. Feeding fat increased plasma glucose-dependent insulinotropic polypeptide and CCK concentrations and decreased plasma insulin concentration. Plasma CCK concentration was greater for MUFA and PUFA than for SFA and was greater for MUFA than PUFA. Decreases in DMI in cows fed fat were associated with increased plasma concentrations of GLP-1 and CCK and a decreased insulin concentration. The role of these peptides in regulating DMI in cattle fed fat requires further investigation.

Plasma concentrations of gut peptides in dairy cattle increase after calving

Effects of transition from late gestation to early lactation on plasma concentrations of glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1-(7-36) amide (GLP-1), and cholecystokinin (CCK) have not been reported in cattle. The objective of the present study was to measure plasma concentrations of GLP-1, GIP, CCK, insulin, glucose, and nonesterified fatty acids in blood plasma obtained from the coccygeal vein of 32 Holstein cows at an average of 11 d before, and 5, 12, and 19 d after calving. Feed dry matter intake (DMI) averaged 14.4, 17.7, and 19.9 kg/d on d 5, 12, and 19 of lactation, respectively, as milk yield increased (30.6, 36.6, and 39.7 kg/d, respectively). Plasma concentrations of insulin and glucose were lower postpartum than prepartum, but did not differ among samples collected after calving. In contrast, plasma concentration of gut peptides increased linearly after calving, perhaps as a consequence of increased feed intake and nutrient absorption; however, the increases in plasma concentrations of GIP and GLP-1 as lactation progressed were not associated with increased DMI per se, and likely reflect the endocrine and metabolic adaptations of lactogenesis. In contrast, increased concentration of CCK was related both to increasing days in milk and DMI. By 19 d postpartum, concentrations of GLP-1, GIP, and CCK increased by 2.3-, 1.8-, and 2.8-fold, respectively, compared with values at 11 d before calving. Although these peptides have direct and indirect effects that reduce appetite and DMI in other species (including increased insulin secretion), these may be glucose- or insulin-dependent functions, and insulin and glucose concentrations were reduced in early lactation.

Abomasal infusion of casein, starch and soybean oil differentially affect plasma concentrations of gut peptides and feed intake in lactating dairy cows

The effects of specific nutrients on secretion and plasma concentrations of gut peptides (glucagon-like peptide-1((7-36)) amide (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin-8 (CCK)) differ across species, but are not reported for cattle. Our objective was to determine acute (hours) and chronic (1 week) effects of increased abomasal supply of protein, carbohydrate, or fat to the small intestine on dry matter intake (DMI) and plasma concentrations of GLP-1, GIP, CCK, and insulin. Four mid-lactation Holstein cows were used in a 4 x 4 Latin square design experiment. Treatments were 7-day abomasal infusions of water, soybean oil (500 g/d), corn starch (1100 g/d), or casein (800 g/d). Jugular vein plasma was obtained over 7 h at the end of the first and last day of infusions. Oil infusion decreased DMI on day 7, but total metabolizable energy (ME) supply (diet plus infusate) did not differ from water infusion. Casein and starch infusion had no effect on feed DMI; thus, ME supply increased. Decreased DMI on day 7 of oil infusion was accompanied by increased plasma GLP-1 concentration, but decreased plasma CCK concentration. Increased plasma GIP concentration was associated with increased ME supply on day 7 of casein and starch infusion. Casein infusion tended to increase plasma CCK concentration on both days of sampling, and increased plasma GLP-1 and insulin concentration on day 1 of infusion. The present data indicate a sustained elevation of plasma concentration of GLP-1, but not CCK, may contribute to the reduced DMI observed in dairy cows provided supplemental fat. (C) 2008 Elsevier Inc. All rights reserved.

Metabolic profiles and progesterone cycles in first lactation dairy cows

This study investigated the ovarian function, metabolic profiles and fertility in first lactation Holstein-Friesian dairy cows (mean 305 day milk yield: 7417 +/- 191 kg, n = 37). Reproductive profiles obtained from milk progesterone analysis were categorized into normal (n = 17) and four abnormal profiles (delayed ovulation, DOV1, n = 9; DOV2, n = 2; persistent corpus luteum, PCL1, n = 6; PCL2, n = 4; 1: immediately post-calving, 2: subsequent cycles). Fifty-five percent of cows had abnormal profiles with half of these being categorized as DOV1. Fertility of DOV1 and DOV2 cows was reduced whereas PCL1 and PCL2 cows had similar reproductive competence to normal profile cows. DOV1 animals had higher milk energy values, lower energy balances, lower dry matter intakes (DMI) and greater body weight and body condition score (BCS) losses post-calving than normal profile animals. DOV1 animals also had lower insulin-like growth factor-I (IGF-I) and higher betahydroxybutyrate (BHB) concentrations and tended to have the lower insulin and glucose concentrations in the pre-service period than normal profile cows. All PCL animals had vulval discharges postpartum. Despite this, the DMI, body weight and BCS changes, IGF-I concentrations and fertility of PCL1 animals was similar to normal profile cows. In conclusion, the high prevalence of delayed ovulation post-calving (DOV1) in primiparous high yielding cows lasted long enough (71 +/- 8.3 days) to have a detrimental impact on fertility and was associated with significant physiological changes. This study did not establish any detrimental effects of PCL profiles on fertility or production parameters. (C) 2002 Elsevier Science Inc. All rights reserved.

First lactation ovarian function in dairy heifers in relation to prepubertal metabolic profiles

The aim of this study was to determine whether any differences in the GH-IGF-I axis in juvenile calves were predictive of fertility problems as adult cows. Endogenous metabolic hormone profiles before and after feeding and the response to a GH-releasing factor (GRF) challenge were measured in prepubertal (6 month) dairy calves. These metabolic parameters were subsequently related to physical characteristics at puberty and to ovarian function during the first lactation. Milk progesterone analysis was used to categorize the animals into those with normal progesterone profiles following calving (n = 17) and those that developed delayed ovulation (DOV1, n = 9) or persistent corpus luteum (PCL1, n = 6) profiles. There were associations between prepubertal GH parameters, glucose and non-esterified fatty acid (NEFA) concentrations and the body condition score at which the animals attained puberty. The calves which subsequently developed DOV1 profiles as cows tended to have a higher GH pulse amplitude during fasting than normal profile animals, they did not show the anticipated decrease in circulating glucose concentrations following a post-prandial rise in insulin and they also had the lowest IGF-I concentrations. The calves that later developed PCL1 had a significantly larger GH pulse amplitude and pulse area than normal profile animals in the fed period and had the highest IGF-I concentrations. There were no differences in prepubertal insulin or NEFA concentrations or in the GH response to a GRF challenge between the different progesterone profile categories. Plasma IGF-I concentrations in prepubertal animals were positively correlated with their post-calving concentrations, whereas glucose concentrations had a negative correlation between these time-periods. These results suggested that the different juvenile endocrine profiles of the DOV1 cows may predispose them to a higher rate of tissue mobilization during lactation and a consequent reduction in fertility, while altered GH and IGF-I levels in PCL1 cows may later contribute to the maintenance of the persistent corpus luteum. Therefore metabolic differences in prepubertal calves were later reflected by altered reproductive function during the first lactation.

Relationships between the plasma concentrations of insulin-like growth factor-I in dairy cows and their fertility and milk yield

The relationships between insulin-like growth factor-I (IGF-I) and the fertility and milk yield of Holstein-Friesian dairy cows were investigated. The concentration of IGF-I in blood was measured weekly from one week before to 12 weeks after calving in 177 multiparous cows and at four times during this period in 142 primiparous cows; the concentration of IGF-I in milk was measured in 50 of the multiparous cows. The plasma concentrations of IGF-I were higher in the primiparous than in the multiparous animals. in the primiparous cows, high concentrations of IGF-I before calving were associated with longer calving to conception intervals. Conversely, in the multiparous cows low concentrations of IGF-I before and after calving were associated with a failure to conceive, despite repeated services. Multiparous cows with IGF-I concentrations of greater than 25 ng/ml in the week after calving were 11 times more likely to conceive to first service than those with lower concentrations. Concentrations of IGF-I greater than 50 ng/ml at first service increased the likelihood of conception five-fold. Cows with higher peak milk yields had lower plasma concentrations of IGF-I and took longer to return to ovarian cyclicity. The negative relationship between milk yield and return to cyclicity was stronger in the multiparous cows (P<0(.)002) than in the primiparous cows (P<0(.)04). The concentrations of IGF-I in milk followed a different pattern and were not associated with the changes in plasma IGF-I or fertility.

Effect of plasma insulin and branched-chain amino acids on skeletal muscle protein synthesis in fasted lambs

The increase in fractional rate of protein synthesis (K-s) in the skeletal muscle of growing rats during the transition from fasted to fed state has been explained by the synergistic action of a rise in plasma insulin and branched-chain amino acids (BCAA). Since growing lambs Also exhibit an increase in K-s with level of feed intake, the objective of the present study was to determine if this synergistic relationship between insulin and BCAA also occurs in ruminant animals. Six 30 kg fasted (72 h) lambs (8 months of age) received each of four treatments, which were based on continuous infusion into the jugular vein for 6 h of: (1) saline (155 mmol NaCl/l); (2) a mixture of BCAA (0.778 mumol leucine, 0.640 mumol isoleucine and 0.693 mumol valine/min.kg); (3) 18.7 mumol glucose/min.kg (to induce endogenous insulin secretion): (4) co-infusion of BCAA and glucose. Within each period all animals received the same isotope of phenylalanine, (Phe) as follows: (1) L-[1-C-13]Phe; (2) L-phenyl-[ring H-2(5)]-alanine; (3) L-[N-15]Phe; (4) L-[ring 2,6-H-3]Phe. Blood was sampled serially during infusions to measure plasma concentrations of insulin, glucose and amino acids, and plasma free Phe isotopic activity; biopsies were taken 6 h after the beginning of infusions to determine K-s in in. longissimus dorsi and vastus muscle. Compared with control (saline-infused) lambs, K-s was increased by an average of 40% at the end of glucose infusion, but this effect was not statistically significant in either of the muscles sampled. BCAA infusion, alone or in combination with glucose, also had no significant effect on K-s compared with control sheep. K-s was approximately 60% greater for vastus muscle than for m. longissimus dorsi (P<0.01), regardless of treatment. It is concluded that there are signals other than insulin and BCAA that are responsible for the feed-induced increase in K-s in muscle of growing ruminant animals.

Limit-feeding a high-energy diet to meet energy requirements in the dry period alters plasma metabolite concentrations but does not affect intake or milk production in early lactation

Limit-feeding dry cows a high-energy diet may enable adequate energy intake to be sustained as parturition approaches, thus reducing the extent of negative energy balance after parturition. Our objective was to evaluate the effect of dry period feeding strategy on plasma concentrations of hormones and metabolites that reflect energy status. Multiparous Holstein cows (n = 18) were dried off 45 d before expected parturition, paired by expected calving date, parity, and previous lactation milk yield, and randomly assigned to 1 of 2 dry-period diets formulated to meet nutrient requirements at ad libitum or limited intakes. All cows were fed the same diet for ad libitum intake after parturition. Prepartum dry matter intake (DMI) for limit-fed cows was 9.4 kg/d vs. 13.7 kg/d for cows fed ad libitum. During the dry period, limit-fed cows consumed enough feed to meet calculated energy requirements, and ad libitum-fed cows were in positive calculated net energy for lactation (NEL) balance (0.02 vs. 6.37 Mcal/d, respectively). After parturition, milk yield, milk protein concentration, DMI, body condition score, and body weight were not affected by the prepartum treatments. Cows limit fed during the dry period had a less-negative calculated energy balance during wk 1 postpartum. Milk fat concentration and yield were greater for the ad libitum treatment during wk 1 but were lower in wk 2 and 3 postpartum. Plasma insulin and glucose concentrations decreased after calving. Plasma insulin concentration was greater in ad libitum-fed cows on d -2 relative to calving, but did not differ by dietary treatment at other times. Plasma glucose concentrations were lower before and after parturition for cows limit-fed during the dry period. Plasma nonesterified fatty acid concentrations peaked after parturition on d 1 and 4 for the limit-fed and ad libitum treatments, respectively, and were greater for limit-fed cows on d -18, -9, -5, and -2. Plasma tumor necrosis factor-alpha concentrations did not differ by treatment in either the pre- or postpartum period, but tended to decrease after parturition. Apart from a reduction in body energy loss in the first week after calving, limit feeding a higher NEL diet during the dry period had little effect on intake and milk production during the first month of lactation.

Follicle-stimulating hormone isoforms and plasma concentrations of estradiol and inhibin A in dairy cows with ovulatory and non-ovulatory follicles during the first postpartum follicle wave

Following parturition, all cows display a wave of ovarian follicular growth, but a large proportion fail to generate a preovulatory rise in estradiol, and hence fail to ovulate. Follicle-stimulating hormone (FSH) exists as multiple isoforms in the circulation depending on the type and extent of glycosylation, and this has pronounced effects on its biological properties. This study examined differences in plasma FSH, estradiol, and inhibin A concentrations, and the distribution of FSH isoforms in cows with ovulatory or atretic dominant follicles during the first postpartum follicle wave. Plasma FSH isoform distribution was examined in both groups during the period of final development of the dominant follicle by liquid phase isoelectric focusing. Cows with an ovulatory follicle had higher circulating estradiol and inhibin A concentrations, and lower plasma FSH concentrations. The distribution of FSH isoforms displayed a marked shift toward the less acidic isoforms in cows with ovulatory follicles. A higher proportion of the FSH isoforms had a pl>5.0 in cows with ovulatory follicles compared to those with atretic follicles. In addition, cows with ovulatory follicles had greater dry matter intake, superior energy balance, elevated circulating concentrations of insulin and insulin-like growth factor-I, and lower plasma nonesterified fatty acids. The shift in FSH isoforms toward a greater abundance of the less acidic isoforms appears to be a key component in determining the capability for producing a preovulatory rise in estradiol, and this shift in FSH isoforms was associated with more favorable bioenergetic and metabolic status. (C) 2008 Elsevier Inc. All rights reserved.

Amniotic fluid and umbilical cord plasma corticotropin-releasing factor (CRF), CRF-binding protein, adrenocorticotropin, and cortisol concentrations in intraamniotic infection and inflammation at term

Context: Pregnant tissues express corticotropin-releasing factor (CRF), a peptide modulating fetal and placental ACTH and cortisol secretion. These actions are modulated by the locally expressed CRF-binding protein (CRF-BP). Objective: The objective of the study was to determine whether CRF, CRF-BP, ACTH, and cortisol concentrations change in amniotic fluid and umbilical cord plasma in the presence of intraamniotic infection/inflammation (IAI) in women with spontaneous labor at term. Design: This was a cross-sectional study. Setting: The study was conducted at a tertiary referral center for obstetric care. Patients: Patients included women in active labor at term with (n = 39) and without (controls; n = 78) IAI. Main Outcome Measures: Amniotic fluid and umbilical cord plasma concentrations of CRF, CRF-BP, ACTH, and cortisol measured by RIA and immunoradiometric assays were measured. Results: In patients with IAI, amniotic fluid CRF (0.97 +/- 0.18 ng/ml) and CRF-BP (33.06 +/- 5.54 nmol/liter) concentrations were significantly (P < 0.001) higher than in controls (CRF: 0.32 +/- 0.04 ng/ml; CRF-BP: 14.69 +/- 2.79 ml). The umbilical cord plasma CRF and CRF-BP concentrations were significantly (P < 0.001 for all) higher in women with IAI than in controls (CRF: 2.96 +/- 0.35 ng/ml vs. 0.38 +/- 0.18 ng/ml; CRF-BP: 152.12 +/- 5.94 nmol/liter vs. 106.9 +/- 5.97 nmol/liter). In contrast, amniotic fluid and umbilical cord plasma ACTH and cortisol concentrations did not differ between groups. Conclusions: Amniotic fluid and umbilical cord plasma CRF and CRF-BP concentrations are increased in women with spontaneous labor at term and IAI. CRF-BP may modulate CRF actions on ACTH and cortisol secretion, playing a pivotal role in limiting the inflammatory process and thus avoiding an overactivation of the fetal/placental hypothalamus-pituitary-adrenal axis at birth.

Bone morphogenetic protein-6 enhances gonadotrophin-dependent progesterone and inhibin secretion and expression of rnRNA transcripts encoding gonadotrophin receptors and inhibin/activin subunits in chicken granulosa cells

The aims were to examine ovarian expression of bone morphogenetic protein (BMP) ligands/receptor mRNAs in the chicken and to test the hypothesis that theca-derived BMP(s) modulates granulosa cell function in a paracrine manner. RT-PCR revealed expression of multiple BMPs in granulosa and theca cells from prehierarchical and preovulatory follicles with greater expression in theca cells; both cell types expressed BMP receptors-1A, -1B and -II consistent with tissue responsiveness. Preovulatory granulosa cells F1, F2 and F3/4) were cultured with BMP-6 (expressed by theca but not granulosa) in the presence/absence of LH, FSH or 8-Br-cAMP. RMP-6 increased 'basal' and gonadotrophin-induced inhibin-A and progesterone secretion by each cell type but did not enhance the effect of 8-Br-cAMP. This indicates that the observed synergism between BMP-6 and gonadotrophin might involve BMP-induced up-regulation of gonadotrophin receptors. In support of this, BMP-6 alone increased LH-receptor (LHR) mRNA in F1 cells and FSH-receptor (FSHR) mRNA in F1, F2 and F3/4 cells. RMP-6 also enhanced LH/FSH-induced LHR transcript amount in each cell type but did not raise FSHR transcript amounts above those induced by BMP-6 alone. To further explore BMP6 action on inhibin-A secretion, we quantified inhibin/activin subunits (alpha, beta(A), beta(B)) mRNAs. Consistent with its effect on inhibin-A secretion, BMP-6 enhanced 'basal' expression of alpha- and beta(A)-Subunit mRNA in F1, F2 and F3/4 cells, and beta(B)-subunit mRNA in F3/4 cells. BMP-6 markedly enhanced FSH/LH-induced expression of alpha-subunit in all follicles and FSH-induced beta(A)-subunit in F2 and F3/4 follicles but not in F1 follicles. Neither BMP-6 alone, nor FSH/LH alone, affected 'basal' OB mRNA abundance. However, co-treatment with gonadotrophin and BMP-6 greatly increased beta(B)-subunit expression, the response being lowest in F1 follicles and greatest in F3/4 follicles. Collectively, these results support the hypothesis that intra-ovarian OMPs of thecal origin have a paracrine role in modulating granulosa cell function in the chicken in a preovulatory stage-dependent manner.

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.

Variation in pituitary expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and type-I and type-II activin receptors during the chicken ovulatory cycle

Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor beta (TGF beta) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24-25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n = 8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnP,H-R), LH beta subunit, FSH beta subunit and GAPDH. Levels of mRNA for inhibin/activin beta A and beta B subunits, inhibin alpha subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (< 2 amol/reaction). Significant changes in expression (P < 0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r=0.77; P < 0.001) and betaglycan (r=0.45; P < 0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P < 0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r=0.33; P=0.06) and ActRIIA (r=0.34; P=0.06) mRNA levels. Expression of mRNAs encoding LH beta and FSH beta subunit were both lowest (P < 0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.