Urine and fecal excretion patterns of dairy cows divergent for milk urea nitrogen breeding values consuming either a plantain or ryegrass diet.

Environmental degradation has been attributed to inefficient nitrogen utilization from pastoral dairy production systems. This degradation has especially been associated with the urine patch, which has been identified as a key component of nitrate leaching to waterways. However, a lack of information exists regarding the pattern of urination events and individual urination characteristics across the day, which would help inform strategic management decisions. The aim of this study was therefore to evaluate and report the patterns and characteristics of fecal and urination events throughout the day for cows divergent for milk urea nitrogen breeding values (MUNBV) on either a plantain [Plantago lanceolata L. (PL)] or ryegrass [Lolium perenne L. (RG)] diet as ways to reduce environmental impact. Sixteen multiparous lactating Holstein Friesian × Jersey cows divergent for MUNBV were housed in metabolism crates for 72 h, with all excretion events captured and analyzed. Cows selected as low for MUNBV consistently had a 65.2-kg lower urinary urea nitrogen (UUN) load (kg/ha) than high MUNBV cows for all hours of the day when consuming RG. The association between lower urinary urea loading rates and less N leaching implies a reduced environmental impact from low MUNBV cows consuming RG. When cows consumed PL, regardless of MUNBV, they had on average a 137.5-kg (UUN/ha) lower loading rate compared with high MUNBV cows on RG and a 72.2-kg (UUN/ha) lower loading rate compared with low MUNBV cows consuming RG across the day. Cows on PL also exhibited a different diel pattern of UUN load compared with cows consuming RG. Differences in the diel pattern of N excreted in feces were also detected based on MUNBV and by diet, with low MUNBV cows excreting on average 3.06 g more N in feces per event for the majority of the day compared with high MUNBV cows when consuming RG. Lower UUN loading rates and more N excreted in feces indicate a potentially lower environmental impact from low MUNBV cows when consuming RG compared with high MUNBV cows. The use of the PL diet also resulted in lower UUN loading rates and greater levels of N excreted in feces compared with RG, therefore also indicating its ability to reduce environmental impact compared with RG.

Expression of regulatory neuropeptides in the hypothalamus of red deer (Cervus elaphus) reveals anomalous relationships in the seasonal control of appetite and reproduction.

Red deer are seasonal with respect to reproduction and food intake, so we tested the hypothesis that their brains would show seasonal changes in numbers of cells containing hypothalamic neuropeptides that regulate these functions. We examined the brains of male and female deer in non-breeding and breeding seasons to quantify the production of kisspeptin, gonadotropin inhibitory hormone (GnIH), neuropeptide Y (NPY) and γ-melanocyte stimulating hormone (γ-MSH - an index of pro-opiomelanocortin production), using immunohistochemistry. These neuropeptides are likely to be involved in the regulation of reproductive function and appetite. During the annual breeding season there were more cells producing kisspeptin in the arcuate nucleus of the hypothalamus than during the non-breeding season in males and females whereas there was no seasonal difference in the expression of GnIH. There were more cells producing the appetite stimulating peptide, NPY, in the arcuate/median eminence regions of the hypothalamus of females during the non-breeding season whereas the levels of an appetite suppressing peptide, γ-MSH, were highest in the breeding season. Male deer brains exhibited the converse, with NPY cell numbers highest in the breeding season and γ-MSH levels highest in the non-breeding season. These results support a role for kisspeptin as an important stimulatory regulator of seasonal breeding in deer, as in other species, but suggest a lack of involvement of GnIH in the seasonality of reproduction in deer. In the case of appetite regulation, the pattern exhibited by females for NPY and γ-MSH was as expected for the breeding and non-breeding seasons, based on previous studies of these peptides in sheep and the seasonal cycle of appetite reported for various species of deer. An inverse result in male deer most probably reflects the response of appetite regulating cells to negative energy balance during the mating season. Differences between the sexes in the seasonal changes in appetite regulating peptide cells of the hypothalamus present an interesting model for future studies.

Comparison of analytical and predictive methods for water, protein, fat, sugar, and gross energy in marine mammal milk.

Mammalian milks may differ greatly in composition from cow milk, and these differences may affect the performance of analytical methods. High-fat, high-protein milks with a preponderance of oligosaccharides, such as those produced by many marine mammals, present a particular challenge. We compared the performance of several methods against reference procedures using Weddell seal (Leptonychotes weddellii) milk of highly varied composition (by reference methods: 27-63% water, 24-62% fat, 8-12% crude protein, 0.5-1.8% sugar). A microdrying step preparatory to carbon-hydrogen-nitrogen (CHN) gas analysis slightly underestimated water content and had a higher repeatability relative standard deviation (RSDr) than did reference oven drying at 100°C. Compared with a reference macro-Kjeldahl protein procedure, the CHN (or Dumas) combustion method had a somewhat higher RSDr (1.56 vs. 0.60%) but correlation between methods was high (0.992), means were not different (CHN: 17.2±0.46% dry matter basis; Kjeldahl 17.3±0.49% dry matter basis), there were no significant proportional or constant errors, and predictive performance was high. A carbon stoichiometric procedure based on CHN analysis failed to adequately predict fat (reference: Röse-Gottlieb method) or total sugar (reference: phenol-sulfuric acid method). Gross energy content, calculated from energetic factors and results from reference methods for fat, protein, and total sugar, accurately predicted gross energy as measured by bomb calorimetry. We conclude that the CHN (Dumas) combustion method and calculation of gross energy are acceptable analytical approaches for marine mammal milk, but fat and sugar require separate analysis by appropriate analytic methods and cannot be adequately estimated by carbon stoichiometry. Some other alternative methods-low-temperature drying for water determination; Bradford, Lowry, and biuret methods for protein; the Folch and the Bligh and Dyer methods for fat; and enzymatic and reducing sugar methods for total sugar-appear likely to produce substantial error in marine mammal milks. It is important that alternative analytical methods be properly validated against a reference method before being used, especially for mammalian milks that differ greatly from cow milk in analyte characteristics and concentrations.

Caloric restriction, but not caloric loading, affects circulating fetal and maternal C-type natriuretic peptide concentrations in late ovine gestation.

The factors regulating the greatly elevated concentrations of maternal plasma C-type natriuretic peptide (CNP) forms in ruminant pregnancy are largely unknown, but nutrient status is likely to be important. Previous work has shown that increases in maternal plasma CNP, sourced from the placenta, occur in response to caloric restriction in late gestation. Whether oversupply of nutrients also regulates CNP secretion in pregnancy has not been studied. Hypothesising that CNP in fetal and maternal tissues will be responsive to both deficiency and excess, we studied changes in CNP and a cosecreted fragment, namely N-terminal pro-CNP (NTproCNP), during short-term periods of caloric restriction (CR) and loading (CL). Twin-bearing ewes received CR (fasted Days 121-124), CL (Days 110-124) or control maintenance diets. During CR, fetal plasma CNP forms, insulin-like growth factor (IGF)-1 and liveweight all fell, and maternal plasma NTproCNP increased. During CL, fetal IGF-1 increased, whereas CNP forms and liveweight were unchanged, as were maternal concentrations of CNP forms. The high abundance of CNP peptides in placental tissues was unaffected by these short-term changes in nutrient supply. We conclude that CNP in the fetal-maternal unit is acutely responsive to undernutrition, but is unaffected by oversupply in late gestation.

Grazing dairy cows with low milk urea nitrogen breeding values excrete less urinary urea nitrogen.

There is an increasing pressure on temperate pastoral dairy production systems to reduce environmental impacts, coming from the inefficient use of N by cows in the form of excessive urinary N excretion and subsequent N leaching to the waterways and NO2 emissions to the atmosphere, these impacts have spurred research into various mitigation strategies, which have so far overlooked animal-based solutions. The objectives of this study were first, to investigate the relationship between MUN breeding values (MUNBV) and urinary urea N (UUN) concentrations and total excretion in grazing dairy cows; and secondly, to evaluate such a potential relationship in the context of different sward compositions and stage of lactation. Forty-eight multiparous, lactating Holstein-Friesian dairy cows genetically divergent for MUNBV were strip-grazed on either a ryegrass-white clover (24 cows) or ryegrass, white clover and plantain sward (24 cows), during both early and late lactation. Cows were fitted with Lincoln University PEETER sensors to evaluate urination behaviour by measuring frequency and volume of urination, as well as daily urine excretion. Urine and faeces were sampled for urea N content. Milk yield and composition were measured for individual cows in both periods. There was a positive relationship between MUNBV and MUN (R2 = 0.67, P ≤ 0.05), with MUN decreasing 1.61 ± 0.19 mg/dL per unit decrease in MUNBV across both sward types and stages of lactation. Urinary urea N concentration decreased 0.67 ± 0.27 g/L (R2 = 0.46, P ≤ 0.05) per unit decrease of MUNBV, with no effect on urine volume or frequency (number of urination events per day), which resulted in a 165.3 g/d difference in UUN excretion between the animal with the highest and the lowest MUNBV. At the same milk yield, percentage of protein in milk increased by 0.09 ± 0.03 (R2 = 0.61, P ≤ 0.05,) per unit decrease in MUNBV. Our results suggest that breeding and selecting for dairy cows with low MUNBV can reduce urinary urea N deposition onto pasture and consequently the negative environmental impact of pastoral dairy production systems in temperate grasslands. Moreover, reducing MUNBV of dairy cows can potentially increase farm profitability due to greater partitioning of N to milk in the form of protein.

Role of the thyroid gland in seasonal reproduction. III. Thyroidectomy blocks seasonal suppression of gonadotropin-releasing hormone secretion in sheep.

Previous studies demonstrated that the thyroid gland is required for the changes in LH secretion that lead to the end of the breeding season of female sheep, a reproductive transition generated within the ewe by an endogenous annual rhythm. The present study tested the hypothesis that this role of the thyroid is mediated via the GnRH neurosecretory system. The pulsatile secretion of GnRH into hypophyseal portal blood and LH into peripheral blood, as well as the neuroanatomical distribution of GnRH neurons and their light microscopic morphology, were compared among anestrous ewes and thyroidectomized (THX) ewes that failed to make the transition to the anestrous season. The study was conducted under photoperiodic conditions in which the transition to anestrus was generated by the endogenous rhythm. Each animal was ovariectomized and treated sc with a constant release implant of estradiol to normalize gonadal steroid status among thyroid-intact and THX ewes. High frequency pulses of GnRH and LH were evident in THX ewes that failed to make the transition to anestrus, whereas pulsatile secretion of GnRH and LH was generally not observed in thyroid-intact controls that had entered anestrus. This marked effect of thyroidectomy on GnRH secretion was not associated with widespread changes in the total number, distribution, or light microscopic morphology of GnRH neurons in the hypothalamus and preoptic area. Our finding that pulsatile secretion of GnRH is elevated in THX ewes that fail to make the transition to anestrus supports the hypothesis that the thyroid gland is required for the endogenously generated switch in function of the GnRH neurosecretory system that leads to the end of the breeding season of the ewe.

Effects of pinealectomy on the secretion of luteinizing hormone, testosterone and prolactin in rams exposed to various lighting régimes.

Two experiments were carried out to study the effects of controlled lighting régimes on plasma levels of LH were low in all groups of rams, which made the detection of significant effects of any treatment very unlikely. Pinealectomy reduced the effects of changes in the daily photoperiod on the patterns of secretion of testosterone and prolactin. These findings establish the pineal gland as an organ which influences the endocrine responses of rams to photoperiodic stimuli and it is concluded that the pineal gland is probably important as a mediator of seasonal reproductive changes in these animals.

Absence of circadian patterns of secretion of melatonin or cortisol in Weddell seals under continuous natural daylight.

Blood samples were obtained from adult female Weddell seals in McMurdo Sound, Antarctica, utilizing four different seals every 2 h for 24 h on two occasions in November 1987. Plasma levels of melatonin were mostly near zero, whereas the values for cortisol were high (2101 +/- 54 and 1908 +/- 108 (S.E.M.) nmol/l, n = 52). For both hormones there was no evidence of a diurnal pattern of secretion. Juvenile seals sampled in December 1985 also had no evidence of a diurnal secretion pattern of melatonin, but blood levels of melatonin were higher than in adults. The results indicate that secretion of melatonin in adult Weddell seals ceases during the Antarctic summer.

FSH secretion in pinealectomized rams subjected to artificial photoperiods.

Eight mature Romney rams which had been pinealectomized and another eight sham-operated rams were allocated equally to one of two contrasting lighting regimes. They were housed indoors with the temperature held constant, offered a constant diet and plasma samples were collected each week for 9 months. Changes in plasma FSH in pinealectomized and sham-operated rams were similar when the rams were subjected to normal seasonal changes in photoperiod, with lower FSH levels recorded under increasing daily photoperiods than during the phase of decreasing (stimulatory) photoperiods. In contrast a reversed pattern of changes in lighting advanced the increase of plasma levels of FSH in the case of sham-operated rams, because the period of decreased daily photoperiods occurred earlier in this situation. In this case, however, pinealectomized rams did not behave like the control animals, but had raised plasma FSH levels at the same time as rams on the normal lighting regime. These results demonstrate that the pineal-mediated effects of photoperiod on reproduction in sheep directly involve FSH secretion and that seasonal cyclic changes in FSH secretion persist even after pinealectomy.

An enzyme linked immunosorbent assay (ELISA) for plasma medroxyprogesterone acetate (MPA).

A single extraction fixed antigen enzyme-linked immunosorbent assay (ELISA) that can be completed in less than 24 h is described for the measurement of medroxyprogesterone acetate (MPA) in plasma. MPA is covalently coupled to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to a standard 96-well microtitre plate where it competes with MPA in the extracted plasma sample for goat anti-MPA. Antibody binding to the solid phase is determined via binding of a horse-radish peroxidase second antibody which reacts colorimetrically with its substrate. The reaction is stopped by addition of 1.25 M H2SO4 and absorbance read at 492 nm. All steps except for sample addition and extraction can be performed on an automatic ELISA processing machine. The assay is sensitive, specific and precise, with intra- and inter-assay coefficients of variation of less than 10 and 15%, respectively. Assay sensitivity is 0.08 ng/ml. The assay follows established methodology for other assays in this laboratory which assists standardization, cost structure and sample throughput and thus is a useful alternative to radioimmunoassays for the determination of MPA in plasma.

Glycine betaine excretion is not directly linked to plasma glucose concentrations in hyperglycaemia.

Diabetes mellitus subjects, type 1 and type 2, have increased glycine betaine excretion compared to normal subjects that correlated with plasma glucose and HbA(1C) concentrations. The current study was undertaken to determine whether elevated glucose concentration directly increases glycine betaine excretion in an animal model. Non-pregnant female Coopworth sheep received an intravenous glucose load (12.5,25 and 50% w/v; rate 200 ml/h) for 6 h followed by a 12 h physiological saline washout (0.9% w/v). Plasma and urine samples were analyzed for glycine betaine and glucose. Urine volumes and osmolality were also measured. Using the non-parametric Kruskal Wallis analysis of variance test we found no difference in glycine betaine excretion between glucose loaded and saline infused control animals (P=0.861). However, a significant negative correlation (r=-0.28, P<0.001) was observed between urine osmolality and glycine betaine excretion independent of treatment. We conclude that acute elevations of plasma glucose concentrations did not result in increased glycine betaine excretion and is therefore unlikely to be directly responsible for elevated glycine betaine excretion observed in diabetes mellitus subjects.

Regional distribution of estradiol receptors in growing antlers.

This study of estrogen receptors (ER) was carried out to confirm their presence and to determine their localisation in antler bones. Partially grown antlers were amputated from red deer (Cervus elaphus) stags, the skin removed, and samples taken of periosteum, cartilaginous tissue including perichondrium, and bone. Capacity and binding of free ER in the samples were calculated by Scatchard analysis of data obtained from a radioreceptor assay which utilised [3H]estradiol as tracer. High affinity ER (ka 1.3-3.4 x 10(10)/M) were detected in all tissues sampled with the exception of bone. Receptor capacity ranged from 12-74 fmol/mg protein, ranking the tissues for capacity in the following descending order: periosteum, cartilage, calcified cartilage. These results demonstrate the presence of ER in growing antlers and indicate regional localization of the receptors within these structures. The absence of ER in bone tissue within the antler suggests that the effect of estradiol on stimulation of mineralization in this tissue is indirect and must occur via its binding to the non-calcified tissues of antlers, e.g., periosteum, perichondrium, and cartilage.

Thyroid hormones are required for the expression of seasonal changes in red deer (Cervus elaphus) stags.

Experiment 1, from 6 July 1990 (winter) to 8 November 1990 (spring), examined the effect of an early elevation of plasma thyroid hormone concentrations on the expression of seasonal changes in 20-month-old stags. Four stags were injected subcutaneously with 1 mg thyroxine (T4) daily, and four with vehicle only. Plasma T4 concentration was elevated by injection of T4; the mean concentration 24 h after injection was 192 +/- 31 nmol L-1 compared with 58 +/- 5 nmol L-1 in controls on 21 September. However, live weight, testicular diameter, plasma testosterone concentration and moulting underwent similar changes in both groups of animals. Experiment 2 tested the hypothesis that thyroid hormones are required for the expression of changes that occur during spring and early summer in red deer stags. Thyroidectomized (THX) stags (n = 4) of mixed age (> 22 months old) were given a replacement dosage of T4 or vehicle only (controls) during spring (from 7 September). T4-treated THX stags underwent testicular regression, antler regeneration and spring moult during spring and early summer (October-December), whereas control THX stags did not display these changes. During spring and summer, T4-treated stags gained more live weight (P < 0.05) than control stags. These results show that, in stags, thyroid hormones are required for the expression of seasonal changes and that these changes are not caused by the seasonal elevation of plasma concentrations of thyroid hormones.

Anti-fertility effects of oral medroxyprogesterone acetate in rabbits.

Studies on the anti-fertility effects of medroxyprogesterone acetate (MPA) were conducted in rabbits. The bioavailability of MPA and plasma concentrations of progesterone and luteinizing hormone (LH) after mating were monitored following a single meal containing MPA (1000 mg) in entire does (n = 4); the response to gonadotrophin-releasing hormone (GnRH; 250 ng) was also observed in MPA-treated, ovariectomized does (n = 6). The reproductive tracts of rabbits mated following MPA treatment were examined 28-30 h after mating. Another group of rabbits (n = 4) received a single dose of MPA on Days 1, 10 or 19 after mating or daily for five days from Day 24. After dosage with 1000 mg MPA, plasma concentrations of MPA were detectable for eight days. However, following multiple dosing (10 mg, 5 days) MPA was detectable in the plasma for two days. MPA reduced the rate of ovulation and suppressed the increase in plasma concentrations of progesterone and LH observed after mating for four days, but had no effect on the response to GnRH. When administered late in gestation, MPA caused the death of fetuses. These results demonstrate an inhibitory effect of MPA on ovulation, probably at the hypothalamic level, and impairment of gestation or parturition.

Immunocytochemical localization of oestrogen receptors in perichondrium of antlers in red deer (Cervus elaphus).

Remodelling of cancellous bone of antlers to compact bone can be stimulated by administration of oestrogens and previous work has demonstrated the presence of specific oestrogen binding in this tissue. In this study the presence of oestrogen receptors (ER) in antler tissue from red deer males was examined by immunocytochemistry using a monoclonal mouse anti-human ER serum. Strong positive staining was detected in the tip regions of immature antlers and was confined primarily to the cells forming a fibrous layer of the perichondrium. This finding indicates that the effects of oestrogens on remodelling of cancellous bone of the antler may be mediated indirectly by the surrounding connective tissue layers.

Involvement of serotoninergic pathways in the control of luteinizing hormone secretion in red deer hinds.

Two experiments were conducted to determine whether serotoninergic pathways, which are implicated in the neuroendocrine regulation of luteininzing hormone (LH) secretion in domestic animals, have a similar action in red deer hinds. In the non-breeding season (August), ovariectomized (n = 5) and ovariectomized-thyroidectomized (n = 5) hinds received a vehicle solution followed 4 h later by either serotonin (66 microg kg(-1) i.v.) every 10 min for a further 4 h or the serotonin antagonist, cyproheptadine (3 mg kg(-1) i.v.) as a single injection. This procedure was repeated in the breeding season (June). In the non-breeding season serotonin was without effect, but cyproheptadine reduced LH pulse frequency and amplitude in ovariectomized-thyroidectomized hinds (P<0.01). During the breeding season, serotonin reduced LH pulse amplitude in ovariectomized hinds (P<0.05) and cyproheptadine reduced LH pulse frequency in both ovariectomized and ovariectomized-thyroidectomized hinds (P<0.05 and P<0.01, respectively). On each occasion, cyproheptadine increased (P<0.01) plasma prolactin concentration, whereas serotonin had no effect. These results indicate a stimulatory role for serotoninergic neurons on the hypothalamic GnRH pulse generator mechanism of red deer hinds during the breeding season. In a second experiment, the LH response to GnRH (5 microg i.v.) was examined in ovariectomized hinds (n = 5) following administration of a serotonin infusion (6.6 microg kg(-1) min(-1) i.v. for 15 min), cyproheptadine (3 mg kg(-1) i.v. as a single dose) or vehicle, in the breeding season (July) after induction of halothane anaesthesia and in the non-breeding season (December) without anaesthesia. Halothane anaesthesia eliminated endogenous pulses of LH. In comparison with the vehicle-treated controls, the response of plasma LH to exogenous GnRH was not altered by serotonin or cyproheptadine in either season, which shows that serotonin has no effect on LH release at the pituitary gland level in these animals. It was concluded that in the regulation of LH release in red deer hinds, serotoninergic pathways have a stimulatory role operating at the hypothalamic level.

Artificial induction of red gut in sheep.

Eight out of 10 lambs died within six to 11 hours after occlusion of the cranial mesenteric blood vessels by a snare placed around both the artery and vein. Death resulted from shock and was accompanied by post mortem findings identical to those seen in red gut. It is suggested that the cause of death in the naturally occurring disease is from arrested mesenteric blood flow following intestinal torsion.

Major lymph nodes of the head of the fallow deer (Dama dama) and lymphatic drainage of antlers.

Heads from 10 fallow deer bucks were examined to provide a description of the major lymph nodes in this region. The distribution and size of these nodes were similar to those of sheep and goats. To determine whether there was drainage of lymph from the skin of antlers, and to follow the route of this drainage, a solution containing Evans blue was injected intradermally into the antlers of 2 bucks whilst the animals were anaesthetised. Dye appeared in tracheal lymph ducts 14 to 30 min after injection. The spread of blue colouration in lymphatic ducts and nodes, seen at post-mortem examination, indicated that lymph flowed from antlers laterally into the ipsilateral parotid lymph nodes and from these via medial and lateral retropharyngeal lymph nodes to the tracheal ducts.

Targeting gene expression to the wool follicle in transgenic sheep.

To establish the feasibility of overexpressing foreign genes in the wool follicle, transgenic sheep were produced by pronuclear microinjection of a DNA construct consisting of a mouse ultrahigh-sulfur keratin promoter linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Four of 31 lambs born were transgenic. The overall efficiency of transgenesis was 1.1% of zygotes injected and transferred. Two transgenic rams were mated to nontransgenic ewes, and both transmitted the gene to their offspring in Mendelian fashion. CAT expression was found in the skin of one G0 ram and in 9 out of 26 transgenic G1 progeny. Two G1 lambs were sacrificed to study tissue specificity. Both had high levels of expression in skin but One had high expression in spleen and kidney with lower levels of expression in lung; the other had low expression in spleen, lung, and muscle. In situ hybridization demonstrated that transgene expression in the skin was confined to the keratogenous zone of the wool follicle cortex. Expression of CAT activity in skin was correlated with diet-induced or seasonal changes in the rate of wool growth. This keratin promoter appears useful for overexpressing factors in the wool follicle that might influence wool production or properties.

The trophoblast binucleate cell is the source of maternal circulating C-type natriuretic peptide during ovine pregnancy.

Maternal plasma concentrations of C-type natriuretic peptide (CNP) and a co-secreted bioinactive amino-terminal fragment (NTproCNP) are elevated during ovine pregnancy. Although the uteroplacental unit has been implicated as a likely source of CNP, the relative contributions of specific uterine and placental tissues, and identity of the cellular site/s of production remain unknown. Therefore, we measured CNP and NTproCNP in intercaruncular uterine tissue and maternal (caruncle) and fetal (cotyledon) placental tissues throughout gestation. Concentrations of CNP forms in placental tissues greatly exceeded those in intercaruncular uterine tissue throughout pregnancy (P < 0.05). Mean caruncular concentrations (CNP 32 ± 4, NTproCNP 56 ± 6 pmol g(-1)) peaked at day 60 whereas in the cotyledon there was a progressive increase in CNP forms to peak values (CNP 66 ± 6, NTproCNP 134 ± 9 pmol g(-1)) at day 100-135 followed by a sharp decline just prior to term (day 143). At term CNP gene expression was 6-fold greater in placental tissue compared with intercaruncular uterine tissue. Changes in maternal plasma concentration of CNP forms closely followed those in cotyledonary tissue whereas fetal plasma levels fell progressively throughout gestation. Immunohistochemistry revealed staining in binucleate cells (BNC) and around placental blood vessels. CNP's localization to the BNC suggests a novel endocrine role during pregnancy, in addition to its paracrine actions within the placental vasculature. The function of CNP in maternal circulation remains to be determined, but as proposed for other BNC products, may involve manipulation of maternal physiology and placental function to favour fetal growth.