Efficacy of dual intracerebroventricular and intravitreal CLN5 gene therapy in sheep prompts the first clinical trial to treat CLN5 Batten disease.

Mutations in the CLN5 gene cause the fatal, pediatric, neurodegenerative disease CLN5 neuronal ceroid lipofuscinosis. Affected children suffer progressive neuronal loss, visual failure and premature death. Presently there is no treatment. This study evaluated dual intracerebroventricular (ICV) and intravitreal (IVT) administration of a self-complementary adeno-associated viral vector encoding ovine CLN5 (scAAV9/oCLN5) into CLN5 affected sheep (CLN5-/-) at various disease stages. CLN5 disease progression was slowed in pre-symptomatic sheep who received a moderate dose of scAAV9/oCLN5, whilst a higher ICV dose treatment in early and advanced symptomatic animals delayed or halted disease progression. Intracranial (brain) volume loss was attenuated in all treatment cohorts, and visual function was also sustained in both the early and advanced symptomatic treated sheep over the 24-month duration of the study. Robust CLN5 protein expression was detected throughout the brain and spinal cord, and improvements in central nervous system and retinal disease correlates were observed. These findings hold translational promise for extending and improving the quality of life in both pre-symptomatic and symptomatic CLN5 patients, and prompted the initiation of the first in-human Phase I/II clinical trial testing ICV/IVT administration of scAAV9 encoding human CLN5 (https://clinicaltrials.gov/; NCT05228145).

Long-term safety and dose escalation of intracerebroventricular CLN5 gene therapy in sheep supports clinical translation for CLN5 Batten disease.

CLN5 neuronal ceroid lipofuscinosis (NCL, Batten disease) is a rare, inherited fatal neurodegenerative disorder caused by mutations in the CLN5 gene. The disease is characterised by progressive neuronal loss, blindness, and premature death. There is no cure. This study evaluated the efficacy of intracerebroventricular (ICV) delivery of an adeno-associated viral vector encoding ovine CLN5 (scAAV9/oCLN5) in a naturally occurring sheep model of CLN5 disease. CLN5 affected (CLN5-/-) sheep received low, moderate, or high doses of scAAV9/oCLN5 at three disease stages. The treatment delayed disease progression, extended survival and slowed stereotypical brain atrophy in most animals. Of note, one high dose treated animal only developed mild disease symptomology and survived to 60.1 months of age, triple the natural life expectancy of an untreated CLN5-/- sheep. Eyesight was not preserved at any administration age or dosage. Histopathologic examination revealed that greater transduction efficiency was achieved through higher ICV doses, and this resulted in greater amelioration of disease pathology. Together with other pre-clinical data from CLN5-/- sheep, the safety and efficacy data from these investigational new drug (IND)-enabling studies supported the initiation of the first in-human CLN5 gene therapy clinical study using the ICV delivery route for the treatment of CLN5 NCL. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT05228145.

Reproductive performance of a cohort of Standardbred mares under a commercial breeding system.

BACKGROUND: Despite being a large commercial breeding industry, there is little published data on the reproductive success of Standardbred mares. OBJECTIVES: To quantify the reproductive performance of Standardbred mares under artificial breeding systems in a commercial setting and determine the incidence of early embryonic and other pre-partum losses. STUDY DESIGN: Retrospective cohort study. METHODS: Data from four commercial farms were collected across four breeding years, and all mares were bred via artificial insemination. A total of 3995 mares contributed 7229 mare years. First-cycle pregnancy rate (FCPR) and end of season pregnancy rate (SPR) were analysed in mixed-effects logistic regression models. Time-to-conception interval was analysed in a Cox regression model. RESULTS: The mean FCPR was 61.4% (confidence interval [CI] 60.3%-62.6%), the mean end of SPR was 84.7% (CI 83.8-85.5%), the mean live foal rate (FR) was 73.1% (CI 72.1%-74.2%). Mares located on-farm were more probable to be pregnant in terms of both FCPR (odds ratio [OR] 1.168, CI 1.018-1.340, p = 0.026) and SPR (OR 2.026, CI 1.673-2.454, p < 0.001), mares inseminated with thawed-frozen semen were less probable to be pregnant in terms of FCPR (OR 0.598, CI 0.457-0.783, p < 0.001) and SPR (OR 0.479, CI 0.354-0.647, p < 0.001) compared with insemination with fresh-extended semen. Older mares (14 years and older) were less probable to be pregnant in terms of FCPR (OR 0.795, CI 0.688-0.919, p = 0.002) and SPR (0.435, CI 0.352-0.538, p < 0.001) compared with young mares (3- to 8-year old). MAIN LIMITATIONS: Retrospective data relied on accurate record keeping of stud farms and no mare-treatment or ovulation induction records were available. Live FRs relied mostly on annual foaling returns so fetal/foal deaths may be underrepresented. CONCLUSION: This study provides substantial baseline data on reproductive performance for Standardbred mares managed under a commercial artificial breeding system.

Characterization of neuropathology in ovine CLN5 and CLN6 neuronal ceroid lipofuscinoses (Batten disease).

Sheep with naturally occurring CLN5 and CLN6 forms of neuronal ceroid lipofuscinoses (Batten disease) share the key clinical features of the human disease and represent an ideal model system in which the clinical efficacy of gene therapies is developed and test. However, it was first important to characterize the neuropathological changes that occur with disease progression in affected sheep. This study compared neurodegeneration, neuroinflammation, and lysosomal storage accumulation in CLN5 affected Borderdale, CLN6 affected South Hampshire, and Merino sheep brains from birth to end-stage disease at ≤24 months of age. Despite very different gene products, mutations, and subcellular localizations, the pathogenic cascade was remarkably similar for all three disease models. Glial activation was present at birth in affected sheep and preceded neuronal loss, with both spreading from the visual and parieto-occipital cortices most prominently associated with clinical symptoms to the entire cortical mantle by end-stage disease. In contrast, the subcortical regions were less involved, yet lysosomal storage followed a near-linear increase across the diseased sheep brain with age. Correlation of these neuropathological changes with published clinical data identified three potential therapeutic windows in affected sheep-presymptomatic (3 months), early symptomatic (6 months), and a later symptomatic disease stage (9 months of age)-beyond which the extensive depletion of neurons was likely to diminish any chance of therapeutic benefit. This comprehensive natural history of the neuropathological changes in ovine CLN5 and CLN6 disease will be integral in determining what impact treatment has at each of these disease stages.

Nitrogen Balance of Dairy Cows Divergent for Milk Urea Nitrogen Breeding Values Consuming Either Plantain or Perennial Ryegrass.

Inefficient nitrogen (N) use from pastoral dairy production systems has resulted in environmental degradation, as a result of excessive concentrations of urinary N excretion leaching into waterways and N2O emissions from urination events into the atmosphere. The objectives of this study were to measure and evaluate the total N balance of lactating dairy cows selected for milk urea N concentration breeding values (MUNBVs) consuming either a 100% perennial ryegrass (Lolium perenne L.) or 100% plantain (Plantago lanceolata L.) diet. Sixteen multiparous lactating Holstein-Friesian × Jersey cows divergent for MUNBV were housed in metabolism crates for 72 h, where intake and excretions were collected and measured. No effect of MUNBV was detected for total N excretion; however, different excretion characteristics were detected, per urination event. Low MUNBV cows had a 28% reduction in the concentration of urinary urea nitrogen (g/event) compared to high MUNBV cows when consuming a ryegrass diet. Cows consuming plantain regardless of their MUNBV value had a 62% and 48% reduction in urinary urea nitrogen (g/event) compared to high and low MUNBV cows consuming ryegrass, respectively. Cows consuming plantain also partitioned more N into faeces. These results suggest that breeding for low MUNBV cows on ryegrass diets and the use of a plantain diet will reduce urinary urea nitrogen loading rates and therefore estimated nitrate leaching values, thus reducing the environmental impact of pastoral dairy production systems.

Electroretinography data from ovine models of CLN5 and CLN6 neuronal ceroid lipofuscinoses.

This article presents datasets associated with the research article entitled "Intravitreal gene therapy protects against retinal dysfunction and degeneration in sheep with CLN5 Batten disease" (Murray et al., [1]). The neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of fatal inherited diseases caused by mutations in a number of CLN genes that lead to degenerative and fatal encephalopathies in children. Naturally-occuring sheep models of NCL exist. Affected sheep share the clinical and pathological features of the human disease, including retinal degeneration. Electroretinography (ERG) was employed to characterise the physiological changes in the degenerating retina of CLN5 and CLN6 forms of ovine NCL. ERGs were performed every two months from 3 to 17 months of age in 11 NCL affected (6 CLN5-/ - and 5 CLN6-/- ) sheep and 12 clinically normal heterozygous controls (6 CLN5+/ - and 6 CLN6 +/-) under three different adaptation conditions. A-wave and b-wave amplitudes were collected from each eye using the Eickemeyer Veterinary ERG system. These are the first longitudinal datasets assessing the progression of retinal degeneration in ovine NCL, aiding in characterisation of the disease process and providing insight into optimal therapeutic windows for subsequent studies.

Plantain (Plantago lanceolata) reduces the environmental impact of farmed red deer (Cervus elaphus).

The objective of this study was to evaluate the effect of plantain (Plantago lanceolata L.) on water dynamics and balance, as well as nitrogen (N) excretion by red deer (Cervus elaphus L.) as a potential forage tool to reduce negative environmental impacts. This experiment used a crossover design with red deer (n = 8) in metabolism crates to determine how fresh-cut herbage diets of either plantain or ryegrass (Lolium perenne L.) compared in terms of dry matter intake (DMI), diet digestibility, water dynamics, and N dynamics. Deer consuming plantain had greater water intake from herbage (P < 0.01) compared with ryegrass. Additionally, when fed plantain, deer had greater water excretion from urine (P < 0.01; 69.4%) and feces (P < 0.01; 29.4%) and, thus, total water excretion (P < 0.01; 61.7%) than when fed ryegrass. When consuming plantain, deer had greater DMI (P = 0.02; +11.2%) and fecal output (P < 0.01; +36.8%) and lower apparent dry matter digestibility (P = 0.03; -8.3%) compared with ryegrass. Plantain (15.9%) contained 30% less crude protein than ryegrass (22.8%) so that even with the greater DMI of plantain, plantain had lower (P < 0.01; -23%) N intake (g/d). Deer consuming plantain had lower urine N concentration (P < 0.01) than when consuming ryegrass. Additionally, deer consuming plantain had much less daily urine N (P < 0.01; -34.9%) excretions. Our results indicate deer fed plantain had greater DMI, ingested more water, and excreted more water than those consuming ryegrass, with lower urinary N (UN) concentration and lesser daily urine N excretion. Thus, we conclude that offering red deer plantain may reduce the environmental impact associated with UN output, such as nitrate leaching or N2O emissions to the atmosphere.

An Appraisal of Methods for Measuring Welfare of Grazing Ruminants.

Although disturbances in body function of animals can be measured to determine whether a state of stress may exist, there is growing interest in finding ways to assess their emotional status as an indicator of good or bad welfare status. Generally it is easier to determine poor states of well-being than positive ones. For grazing ruminants some indicators of well-being include absence of illness, good growth and productivity, and longevity. Motion detectors can provide automated remote monitoring of behavior and it is likely that there will be advances in the interpretation software to increase the utility of this technology for assessing well-being. Cortisol levels in body fluids, feces and pelage are prominent as a marker of poor animal welfare, but like many of the other objective measures that are used, are not wholly reliable at the individual animal level. These other measures include: plasma serotonin, heart rate variation, infra-red thermography, cytokines, salivary alpha amylase, and acute phase proteins. Use of automated facial expression recognition may supplement electrophysiological recording as means to quantify the pain experience of animals. Although the measures described in the literature do not necessarily provide the final answer for determination of welfare in grazing ruminants, they all have some merit and deserve further investigation.

Computed tomography provides enhanced techniques for longitudinal monitoring of progressive intracranial volume loss associated with regional neurodegeneration in ovine neuronal ceroid lipofuscinoses.

INTRODUCTION: The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are a group of fatal neurodegenerative lysosomal storage diseases of children caused by various mutations in a range of genes. Forms associated with mutations in two of these, CLN5 and CLN6, are being investigated in well-established sheep models. Brain atrophy leading to psychomotor degeneration is among the defining features, as is regional progressive ossification of the inner cranium. Ongoing viral-mediated gene therapy trials in these sheep are yielding encouraging results. In vivo assessment of brain atrophy is integral to the longitudinal monitoring of individual animals and provides robust data for translation to treatments for humans. METHODS: Computed tomography (CT)-based three-dimensional reconstruction of the intracranial volume (ICV) over time reflects the progression of cortical brain atrophy, verifying the use of ICV measurements as a surrogate measure for brain size in ovine NCL. RESULTS: ICVs of NCL-affected sheep increase for the first few months, but then decline progressively between 5 and 13 months in CLN5-/- sheep and 11-15 months in CLN6-/- sheep. Cerebral ventricular volumes are also increased in affected animals. To facilitate ICV measures, the radiodensities of ovine brain tissue and cerebrospinal fluid were identified. Ovine brain tissue exhibited a Hounsfield unit (HU) range of (24; 56) and cerebrospinal fluid a HU range of (-12; 23). CONCLUSIONS: Computed tomography scanning and reconstruction verify that brain atrophy ovine CLN5 NCL originates in the occipital lobes with subsequent propagation throughout the whole cortex and these regional differences are reflected in the ICV loss.

Longitudinal In Vivo Monitoring of the CNS Demonstrates the Efficacy of Gene Therapy in a Sheep Model of CLN5 Batten Disease.

Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are neurodegenerative lysosomal storage diseases predominantly affecting children. Single administration of brain-directed lentiviral or recombinant single-stranded adeno-associated virus 9 (ssAAV9) vectors expressing ovine CLN5 into six pre-clinically affected sheep with a naturally occurring CLN5 NCL resulted in long-term disease attenuation. Treatment efficacy was demonstrated by non-invasive longitudinal in vivo monitoring developed to align with assessments used in human medicine. The treated sheep retained neurological and cognitive function, and one ssAAV9-treated animal has been retained and is now 57 months old, almost triple the lifespan of untreated CLN5-affected sheep. The onset of visual deficits was much delayed. Computed tomography and MRI showed that brain structures and volumes remained stable. Because gene therapy in humans is more likely to begin after clinical diagnosis, self-complementary AAV9-CLN5 was injected into the brain ventricles of four 7-month-old affected sheep already showing early clinical signs in a second trial. This also halted disease progression beyond their natural lifespan. These findings demonstrate the efficacy of CLN5 gene therapy, using three different vector platforms, in a large animal model and, thus, the prognosis for human translation.

Molecular forms of C-type natriuretic peptide in cerebrospinal fluid and plasma reflect differential processing in brain and pituitary tissues.

C-type natriuretic peptide (CNP) is a paracrine growth factor widely expressed within tissues of the central nervous system. Consistent with this is the high concentration of CNP in cerebrospinal fluid (CSF), exceeding levels in the systemic circulation. CNP abundance is high in hypothalamus and especially enriched in pituitary tissue where - in contrast to hypothalamus - processing to CNP-22 is minimal. Recently we have shown that dexamethasone acutely raises CNP peptides throughout the brain as well as in CSF and plasma. Postulating that molecular forms of CNP would differ in central tissues compared to forms in pituitary and plasma, we have characterized the molecular forms of CNP in tissues (hypothalamus, anterior and posterior pituitary gland) and associated fluids (CSF and plasma) using size-exclusion high performance liquid chromatography (SE-HPLC) and radioimmunoassay in control (saline-treated) and dexamethasone-treated adult sheep. Three immunoreactive-CNP components were identified which were consistent with proCNP (1-103), CNP-53 and CNP-22, but the presence and proportions of these different fragments differed among tissues. Peaks consistent with CNP-53 were the dominant form in all tissues and fluids. Peaks consistent with proCNP, conspicuous in hypothalamic extracts, were negligible in CSF whereas proportions of low molecular weight immunoreactivity (IR) consistent with CNP-22 were similar in hypothalamus, posterior pituitary gland and CSF. In contrast, in both plasma and the anterior pituitary gland, proportions of higher molecular weight IR, consistent with CNP-53 and proCNP, predominated, and low molecular weight IR consistent with CNP-22 was very low. After dexamethasone, proCNP like material - but not other forms - was increased in all samples except CSF, consistent with increased synthesis and secretion. In conclusion, immunoreactive forms of CNP in central tissues differ from those identified in anterior pituitary tissue and plasma - suggesting that the anterior pituitary gland may contribute to systemic levels of CNP in some physiological settings.

Dexamethasone increases production of C-type natriuretic peptide in the sheep brain.

Although C-type natriuretic peptide (CNP) has high abundance in brain tissues and cerebrospinal fluid (CSF), the source and possible factors regulating its secretion within the central nervous system (CNS) are unknown. Here we report the dynamic effects of a single IV bolus of dexamethasone or saline solution on plasma, CSF, CNS and pituitary tissue content of CNP products in adult sheep, along with changes in CNP gene expression in selected tissues. Both CNP and NTproCNP (the amino-terminal product of proCNP) in plasma and CSF showed dose-responsive increases lasting 12-16 h after dexamethasone, whereas other natriuretic peptides were unaffected. CNS tissue concentrations of CNP and NTproCNP were increased by dexamethasone in all of the 12 regions examined. Abundance was highest in limbic tissues, pons and medulla oblongata. Relative to controls, CNP gene expression (NPPC) was upregulated by dexamethasone in 5 of 7 brain tissues examined. Patterns of responses differed in pituitary tissue. Whereas the abundance of CNP in both lobes of the pituitary gland greatly exceeded that of brain tissues, neither CNP nor NTproCNP concentration was affected by dexamethasone, despite an increase in NPPC expression. This is the first report of enhanced production and secretion of CNP in brain tissues in response to a corticosteroid. Activation of CNP secretion within CNS tissues by dexamethasone, not exhibited by other natriuretic peptides, suggests an important role for CNP in settings of acute stress. Differential findings in pituitary tissues likely relate to altered processing of proCNP storage and secretion.

Nutrient restriction in early ovine pregnancy stimulates C-type natriuretic peptide production.

C-type natriuretic peptide (CNP), a paracrine growth factor promoting vasodilation and angiogenesis, is upregulated in human and ovine pregnancy in response to vascular stress or nutrient restriction (NR) in late gestation. Postulating that maternal plasma CNP products are increased by modest NR (50% of metabolisable energy requirement) early in pregnancy, and further enhanced by litter size, we studied serial changes of maternal plasma CNP in pregnant ewes receiving a normal (NC, n=12) or restricted (NR, n=13) diet from Day 30 to Day 93 or 94 of gestation. Liveweight of NR ewes was 10kg less than that of NC ewes at slaughter. Plasma CNP products increased progressively after Day 40 and were higher in NR (P<0.05) ewes after Day 60; they were also enhanced by litter size (P<0.01) and were positively associated with increased placental efficiency. In contrast, whereas fetal and placental weight were reduced by NR, fetal plasma CNP products (Day 93/94) were not affected. We conclude that increases in CNP during rapid placental growth are further enhanced by both increasing nutrient demands and by reduced supply, presumably as part of an adaptive response benefitting placental-fetal exchange.

Sustained increases in plasma C-type natriuretic peptides fail to increase concentrations in cerebrospinal fluid: Evidence from pregnant sheep.

C-type natriuretic peptide (CNP) is a paracrine growth factor with high abundance in CNS tissues and cerebrospinal fluid (CSF). Consistent with findings of CNP transcripts in the cerebral microvasculature and hypothalamus, CNP increases the permeability of the blood-brain barrier and reduces food intake when administered intracerebroventricularly in rodents. Whether high concentrations of CNP in plasma can affect CSF levels is unknown. Accordingly we have studied changes (days 4, 87 and 116) in concurrent plasma and CSF concentrations of CNP peptides in pregnant sheep - a physiologically unique setting in which plasma CNP is elevated for prolonged periods. Preliminary studies in non pregnant sheep showed stable CNP levels in CSF during repetitive sampling. Compared with values in non pregnant controls, plasma concentrations of CNP peptides were markedly raised (30-fold) at days 87 and 116 in pregnant sheep, yet CSF levels in the two groups did not differ. CNP peptides in CSF decreased from day 4 to day 87 in pregnant sheep, possibly reflecting an adaptive response of the cerebral vasculature to increased hemodynamic load. We conclude that sustained high concentrations of CNP - far exceeding levels encountered in human pathophysiology - fail to affect CNP peptide levels in CSF.

Differential response of C-type natriuretic peptide to estrogen and dexamethasone in adult bone.

C-type natriuretic peptide (CNP) is crucial in promoting endochondral bone growth in mammals including humans but whether this paracrine hormone participates in maintaining bone integrity in the mature skeleton is unknown. Accordingly we studied changes in plasma and bone tissue CNP in anoestrus adult ewes receiving short term anabolic (estrogen) or catabolic (dexamethasone) treatment for 7days. CNP and the aminoterminal fragment of the CNP prohormone (NTproCNP) were measured in plasma and extracts of cancellous bone excised from vertebral, iliac, tibial and marrow tissues. Concentrations of CNP peptides were much higher in vertebral and iliac extracts than those of tibial or marrow. Both plasma CNP and NTproCNP increased rapidly after estrogen followed by a later rise in bone alkaline phosphatase. Vertebral and iliac (but not tibial or marrow) CNP peptide content were significantly increased by estrogen. Consistent with a skeletal source, plasma NTproCNP was significantly associated with vertebral tissue CNP. In contrast, bone tissue CNP peptide content was unaffected by dexamethasone despite suppression of plasma CNP peptides and bone alkaline phosphatase. We postulate that increases in trabecular bone CNP reflect new endosteal bone formation in these estrogen responsive tissues whereas reduced plasma CNP peptides after dexamethasone, without change in cancellous bone content, reflects reductions in cortical bone turnover.

Milk composition in the Weddell seal Leptonychotes weddellii: evidence for a functional role of milk carbohydrates in pinnipeds.

We propose that secretion of milk sugar has important consequences for the metabolic economies of lactating phocid seals and their pups. Milk was collected from 21 Weddell seals Leptonychotes weddellii in McMurdo Sound, Antarctica, and assayed by standard methods. Milk composition changed over the course of lactation, but at mid- to late lactation (16-40 d postpartum), Weddell seal milk composition was relatively constant at 33.8% +/- 0.82% water, 54.0% +/- 0.80% fat, 10.1% +/- 0.16% crude protein, 0.84% +/- 0.03% sugar, 0.75% +/- 0.02% ash, and 23.3 +/- 0.3 kJ g−1 whole milk (WM). At this stage, milk composition varied among individual seals in all assayed constituents except ash. The concentration of sugar in the aqueous phase of Weddell seal milk ( 24.9 +/- 0.6g sugar L−1 water) was ca. 44%-77% of levels found in terrestrial carnivores, indicating that the low sugar concentration of WM is primarily due to its high fat content, not alteration of the aqueous phase. In early lactation, fasting Weddell seals were estimated to devote 39 g d(-1) glucose to milk sugar synthesis, an amount similar to the estimated demand of the maternal brain. This additional glucose demand must be covered by gluconeogenesis in fasting animals and represents a considerable additional drain on maternal resources. However, provision of sugar to offspring at rates sufficient to meet neonatal substrate requirements appears to be essential for efficient fat and protein deposition and thus may be an important component of the phocid reproductive strategy of rapid growth and early weaning.

Effect of nutrition on plasma C-type natriuretic peptide forms in adult sheep: evidence for enhanced C-type natriuretic peptide degradation during caloric restriction.

Previous studies in lambs and children show that the plasma concentration of amino terminal pro-C-type natriuretic peptide (NTproCNP), a stable product of proCNP, is strongly correlated with skeletal growth and markers of bone formation. Consistent with these findings, CNP expression is sensitive to nutritional status and is reduced by caloric restriction (CR) in both the fetus and the postnatal lamb. However, the effect of nutritional status on CNP in the adult, once linear growth is complete, is unknown. Hypothesizing that reduced CNP synthesis during CR is contingent on the presence of active growth plates, we studied the effect of CR ( 25% of maintenance) or loading (CL, 200% of maintenance) on CNP forms and alkaline phosphatase (ALP) in adult ewes and compared the findings to responses in a control group (C) fed a maintenance diet of 10.6 MJ of metabolizable energy. Live body weight was reduced (17%) in the CR group and increased (10%) in the CL group after 16 days of intervention. Plasma CNP concentration and ALP both fell in CR sheep and were significantly lower than C (P < .05 for both), returning toward basal levels 1 week after refeeding. In contrast, plasma NTproCNP did not differ (CR vs C). There were no significant changes in CNP forms and ALP in CL sheep compared with C. Fall in plasma CNP but not in NTproCNP in CR adult sheep suggests that CNP degradation (not synthesis) is altered, and contrasts with previous findings in growing lambs where CR reduces both CNP forms.

C-type natriuretic peptide forms in pregnancy: maternal plasma profiles during ovine gestation correlate with placental and fetal maturation.

Circulating concentrations of C-type natriuretic peptide (CNP) and a related amino terminal fragment (NTproCNP) were measured at weekly intervals from preconception to 3 wk postpartum in ewes with twins (n = 8) and nonpregnant ewes (n = 8). In contrast to low and stable values in nonpregnant ewes (CNP, 0.75 +/- 0.08; NTproCNP, 22 +/- 2 pmol/liter), CNP forms increased abruptly at 40-50 d of gestation and rose to peak values (CNP, 31 +/- 5, NTproCNP, 270 +/- 16 pmol/liter) at about d 120. Approximately 7 d prepartum, the concentration of both CNP forms fell precipitously to preconception values immediately postpartum. In separate studies, circulating maternal CNP forms were positively related to fetal number at d 120. Consistent with a major contribution from the placenta to circulating levels, the concentrations of CNP forms were elevated in the placentome (cotyledon: CNP, 18 +/- 4, NTproCNP, 52 +/- 10 pmol/g; caruncle: CNP, 13 +/- 3, NTproCNP, 31 +/- 6 pmol/g) and much higher than those of intercaruncular uterine tissue (CNP, 0.19 +/- 0.05, NTproCNP, 0.98 +/- 0.2 pmol/g) in late-gestation ewes (P < 0.001, n = 4). These distinctive patterns of maternal plasma CNP forms, positive relation with fetal number, and greatly elevated protein concentrations in the placentome demonstrate the hormone's strong relation to placental and fetal maturation. The findings provide a firm basis for future studies of the functional role of CNP in fetal-maternal welfare.

Effect of sex steroids on plasma C-type natriuretic peptide forms: stimulation by oestradiol in lambs and adult sheep.

Although C-type natriuretic peptide (CNP) is crucial to post-natal endochondral growth, roles for the hormone in pubertal bone growth and the physiological effects of sex steroid substitution on CNP synthesis are not known. Using a plasma marker of CNP tissue synthesis (amino-terminal proCNP, NTproCNP), we have studied the effect of exogenous oestrogen (E(2)) or testosterone (T) on plasma CNP forms and bone alkaline phosphatase (bALP) in pre-pubertal lambs. Responses to E(2) in non-cycling adult ewes were also studied. In 15-week-old intact ewe lambs, E(2) promptly increased plasma NTproCNP and CNP (P<0.001) to peak on day 2, and bALP (P<0.001 peaking on day 7), whereas no significant stimulation in response to T was observed in male lambs. Linear bone growth and live weight were unaffected. In adult anoestrous ewes, basal concentrations of CNP forms and bALP were lower than in ewe lambs, in keeping with skeletal maturity, but adults responded similarly to E(2). Prompt and sustained increases in NTproCNP and CNP, and a later threefold rise in bALP (all P<0.001), were induced by E(2). Possible contributions to these increases in plasma CNP forms by reproductive tissues (a known site of E(2)-induced CNP expression) were excluded by showing similar E(2)-induced CNP responses in adult ewes after surgical removal of reproductive tissues. These results are the first to show that E(2) stimulates plasma CNP forms and bALP in lambs and adult sheep and raise the possibility that CNP also participates in bone formation in the mature skeleton.

Response of plasma CNP forms to acute anabolic and catabolic interventions in growing lambs.

Using a novel marker of C-type natriuretic peptide (CNP) synthesis [amino-terminal pro-CNP (NT-proCNP)], we have recently shown that plasma NT-proCNP is strongly correlated with skeletal growth and markers of bone formation and is reversibly reduced by glucocorticoids. The effects on CNP of other catabolic or anabolic factors, known to affect skeletal growth, are unknown. Accordingly, we have studied the response of plasma CNP forms to acute catabolic (caloric restriction) and anabolic [growth hormone (GH) stimulation] interventions in lambs and related the findings to circulating IGF-I levels, growth velocity, and markers of bone formation. Lambs fed a reduced caloric intake (25% of normal) for 6 days exhibited reduced live weight, plasma urea, and IGF-I (P < 0.001 for all) compared with control lambs. Basal levels of NT-proCNP (40.1 +/- 0.9 pmol/l) fell promptly to a nadir (28.1 +/- 0.8 pmol/l, P < 0.001) on day 6, returning rapidly to basal levels upon refeeding. Although plasma alkaline phosphatase (ALP) fell (P < 0.001), reductions in metacarpal growth velocity were not significant within the 12-day period of study. In contrast to caloric restriction, long-acting bovine recombinant GH (2.5 mg/kg on days 0 and 6), as expected, increased plasma IGF-I more than twofold above control for 12 days (P < 0.001). Growth velocity did not differ during the 30 days of observation, and, consistent with unchanged growth velocity, plasma NT-proCNP and ALP were also unaffected. In conclusion, CNP synthesis and markers of bone formation are acutely sensitive to catabolism but unaffected by doses of GH that fail to stimulate skeletal growth.