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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.

Silver nanoparticles (AgNPs) are now widely used in many industry applications. Because there are no regulations on discharge limits, improper discharge of waste from these industries can lead to environmental contamination and damage to ecosystem organisms. In this study Aporrectodea caliginosa earthworms were exposed to 0 (control), 0.3, 3, 30, 300 mg/kg Ag as AgNPs and 0, 0.03, 0.3, 3, 10 mg/kg of Ag as AgNO3 in soil for 4 weeks and select biochemical and neurotoxicity studies were conducted weekly. The lipid peroxidation (measured using thiobarbituric acid reactive substances; TBARS) and activities of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase, glutathione S transferase, lipid peroxidation), and nerve conduction velocity (NCV) of the medial giant fibers (MGF) using a novel non-invasive electrophysiological technique were measured in earthworms weekly. The TBARS and antioxidant enzyme activities were elevated by both AgNO3 and AgNPs and this was most evident in earthworms at 4 weeks >3 > 2 > 1. In neurotoxicity studies, MGF NCV progressively decreased in A. caliginosa exposed to both AgNPs and AgNO3. The findings highlight oxidative stress and neurotoxic effects of Ag compounds on earthworms and the importance of government authorities to have legislations in place to prevent excessive soil contamination by AgNPs produced by the expanding nanotechnology industries.

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.

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.

International audience; Earthworms are important and useful soil organisms, but in agricultural soils, they are potentially exposed to a wide variety of pesticides. Insecticides represent the highest threat to earthworms and many are neurotoxic. There is a need for a reliable, relevant, simple biomarker to assess the sub-lethal effects of neurotoxic insecticides on earthworms under laboratory or field conditions. The Aporrectodea caliginosa earthworms were exposed to 0 (control), 0.5×, 1× (normal field application rate), and 5× concentrations of a carbamate (Pirimor®) and an organophosphate (Lorsban®) insecticides. The nerve conduction velocity (NCV) of the medial giant fibers of A. caliginosa earthworm was recorded on days 0, 1, 2, 3, 4, and 7 to quantify sub-lethal neurotoxic effects. Acetylcholinesterase (AChE) enzyme activity of A. caliginosa homogenates was measured at the conclusion of the experiment. Pirimor® but not Lorsban® induced a significant decrease in NCV on days 3, 4, and 7 at 1× and 5× doses. A significant dose-dependent decrease was observed on AChE activity to Pirimor® at the doses used but not Lorsban®. A clear relationship is observed between AChE activity and NCV in the case of Pirimor®. This study showed that NCV is a sensitive biomarker that correlates well with classical biomarker measurements such as AChE enzyme activity. This technique could be used to study the impact of insecticides on earthworms and also their recovery.

MPS IIIC is caused by mutations in the HGSNAT gene. HGSNAT deficiency affects lysosomal catabolism of heparan sulfate (HS), resulting in widespread CNS pathology in infants and children, leading to behavioural problems, cognitive decline and, finally, dementia and death before adulthood. Currently there are no existing treatments for MPS IIIC and the development of treatments is difficult as the deficient enzyme cannot cross the blood brain barrier or diffuse between cells. We have developed an adeno-associated virus (AAV) based gene therapy for MPS IIIC which is efficacious in a MPS IIIC murine model via injection into the brain parenchyma. Although effective in a small murine brain, the architecture and volume is considerably different from a human brain, and the failure of previous clinical trials for AAV vectors using intraparenchymal injection, means that large animal experiments are essential in optimising widespread distribution needed for clinical efficacy. Due to the inability of HGSNAT to cross-correct cells, intracerebral delivery of vector needs to target the maximum number of cells, and with the use of convection enhanced delivery (CED) this can be achieved. CED has the potential to treat diseases with global pathology such as MPS IIIC as it overcomes the issues with current delivery methods and can be administered to multiple sites. We compared different conditions to determine optimal vector spread of an AAV9-GFP vector in the sheep brain, which is 130mL in volume, at clinically applicable volumes and titres whilst keeping volume identical throughout. We compared 4 intraparenchymal sites, against intraventricular injection, and compared different flow rates at 2 single intraparenchymal sites. We found a rate of 1μL/min into the corpus striatum produced the greatest vector distribution, whilst intraventricular outcomes were comparatively poor. However, further studies are currently ongoing to improve this further with the aim of determining the optimal delivery system for gene therapy of MPS IIIC.

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.

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 (E2) or testosterone (T) on plasma CNP forms and bone alkaline phosphatase (bALP) in pre-pubertal lambs. Responses to E2 in non-cycling adult ewes were also studied. In 15-week-old intact ewe lambs, E2 promptly increased plasma NTproCNP and CNP (PP2. Prompt and sustained increases in NTproCNP and CNP, and a later threefold rise in bALP (all P2. Possible contributions to these increases in plasma CNP forms by reproductive tissues (a known site of E2-induced CNP expression) were excluded by showing similar E2-induced CNP responses in adult ewes after surgical removal of reproductive tissues. These results are the first to show that E2 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.

1. Continuous body temperature records from dairy cows for 46 days of summer and contemporary data for climate temperature humidity index (THI) were analysed. 2. A large component of the body temperature data was not explained by changes in THI. 3. Outside a normal range of values (above 72 for THI and 39.05 °C for body temperature), there was evidence of a causal relationship with a time delay of about 120 min. 4. Both variables had a prominent circadian component, but these were more likely to be due to common and/or independent causes than to any direct relationship.

The development of mouse hair follicles depends on the proliferation, differentiation and migration of epithelial matrix cells in the follicle bulb. In particular, induction of the proliferation of epithelial cells is thought to be signalled by the dermal papilla at the base of the bulb. Neonatal mouse skin is useful for studying changes in gene expression during development of the follicles, as the mitotic activity of skin cells changes shortly after birth. Using RNA differential display, a 248-bp message has been identified, which is expressed in the skin, specifically on day 2 and day 3 but not on day 4 after birth. Confirmation of expression of this gene by ribonuclease protection assay showed that strong expression is seen on day 2 and day 3, but weak expression is also shown on day 1, day 4 and day 5. In situ hybridization data revealed that it is mainly localized in the dermal papilla. Analysis of its nucleotide sequence showed 99% identity between nucleotide 2 and 232 of the mouse uncoupled S49 cell mRNA for stimulatory GTP-binding protein (G(S)) alpha subunit, suggesting it is a segment of G(S)alpha. As the G(S)alpha subunit is involved in transducing extracellular signals across the cell, the finding of its expression in the papilla suggests it may be a molecular signal to the induction of epithelial proliferation in the follicle bulb. Evidence of strong expression on day 2, at the time when the mitotic activity of epithelial matrix cells starts to increase, also suggests that the G(S)alpha is a potential candidate for involvement in the initiation of follicle growth.

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.

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.

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.

Three experiments were carried out to determine whether growth of suckling red deer calves is determined by the potential of their mothers to produce milk. In the first experiment red deer hinds (n = 10, calves 6 weeks old) were treated either with bovine somatotrophin (bST, 54 mg s.c. injected every 2 weeks for 8 weeks then 108 mg every 2 weeks for a further 8 weeks) or saline. There was no effect of bST treatment on calf or hind liveweight, calf liveweight gain or body condition score of hinds. The second experiment used red and red-wapiti crossbred deer calves (n = 8–11) suckled by red deer dams that had been treated with bST or had received excipient only for 12 weeks from when the calves were 5 weeks old. Calf liveweight was affected by genotype (wapiti-red crossbreds were heavier than their red counterparts) but there was no effect of bST treatment of the hinds on calf growth in either of the genotypes. Although bST treatment of the suckled hinds elevated their plasma insulin-like growth factor-1 concentration it had no effect on milk yield. A third experiment ruled out the possibility that bST ingested by calves in milk from treated hinds would have had any influence on growth of calves in the other experiments. From these results it is concluded that the inherent demand from suckling calves, rather than the ability of adequately nourished hinds to produce milk, determines growth rate of red deer calves from birth to weaning.

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.

Ecological health of 15 sites in two mining areas on the West Coast of the South Island, New Zealand, was assessed using a non-invasive electrophysiological technique. The conduction velocity (CV) changes in the medial giant fibres (MGF) of the terrestrial earthworm Aporrectodea caliginosa were measured on days 0, 1, 2, 4, 7, 14, and 21 following exposure to soil and/or sediment from six acid mine drainage (AMD) sites, and aquatic oligochaete Lumbriculus variegatus at 0, 3, 6, and 24 h following exposure to water from 14 AMD sites. The colour of the soil/sediments varied from red-brown to black with pH ranging from 4.46 to 7.37. The colour of AMD water samples varied from clear, black, brown to orange, and the pH ranged from 2.99 to 7.66. The CV decreased progressively in A. caliginosa exposed to most soil and sediment samples from the AMD sites (compared with controls exposed to soil from an organic farm) and this was most evident in measurements taken at 7 days. Based on the CV measurements taken on day 7, sites 321 were significantly (P0.05) the most toxic to earthworms. The CV of L. variegatus exposed to AMD water sampled from many sites also decreased progressively and this was significantly lower than the controls in the measurements taken at 24 h from sites 39711. It is proposed that MGF CV in A. caliginosa and L. variegatus worms can be used as a non-invasive, sensitive, biomarker to monitor the toxicity of AMD sites.

This study examined the effectiveness of leuprolide, a gonadotrophin-releasing hormone agonist, in suppressing rut-associated events in farmed male red deer. In mid-January (~6 weeks before the rut period in the southern hemisphere) adult red deer (Cervus elaphus scoticus) stags that had been allocated to three groups (n = 10 per group) received leuprolide, administered subcutaneously in a 90-day release formulation, at zero (control), low (22.5 mg) or high (45 mg) doses. Following treatment with leuprolide there was evidence of suppression of mean plasma luteinising hormone concentration that was significant (P < 0.05) at 9 weeks. Mean plasma testosterone concentration of all three groups rose following treatment, then declined prematurely in the low- and high-dose leuprolide-treated groups, so that it was significantly (P < 0.05) suppressed (0.66 ± 0.29 and 2.0 ± 0.88 ng mL–1, low and high dose respectively) in early April when the peak value (9.0 ± 1.94 ng mL–1) was recorded from control stags. A reduction in mean liveweight occurred in all three groups through February–April and this did not differ among treatments. However, a corresponding reduction in mean body condition score was greater in the control stags (P < 0.05). There was little effect of leuprolide treatment on aggressive behaviours, but it lowered roaring frequency in the latter period of the rut. The results indicate that this gonadotrophin-releasing hormone agonist has potential for application in the deer farming industry to suppress undesirable effects of the rut.

To determine its potential as a tool for studies of growth in suckling red deer calves, bovine somatotrophin (bST) was administered to lactating red deer hinds. The present study used twice-daily machine milking of bST-treated hinds (n = 10, 54 mg bST for 2 weeks then 108 mg for 1 week) and compared the milk yield with that of saline-treated controls (n = 9). Treatment with 54 mg bST tended to increase milk yield by ~16% and the 108-mg dose increased (P = 0.013) milk yield by ~32%. Both doses of bST increased (P < 0.05 and P < 0.001, respectively) plasma insulin-like growth factor-1 concentration but did not affect total solids or fat content of the milk, nor was there any effect on body condition score or liveweight of the hinds. This shows that milk production in red deer hinds is increased by administration of bST, which makes it a suitable experimental technique for investigating the lactational biology of red deer.

A preliminary study was conducted to examine whether the marked inhibition of ovulation in seasonally acyclic red deer could be overcome by use of kisspeptin to stimulate gonadotrophin release in these animals. After 14 days progesterone priming with intravaginal CIDR-G devices, non-lactating and lactating hinds (n = 8 in both cases) received 0.50 µmol murine kisspeptin-10 in saline solution delivered intravenously in 30 hours, either continuously or as 4-hourly boluses, and lactating hinds (n = 8) received saline solution intravenously as controls. Kisspeptin treatment caused an elevation (P < 0.05) of the mean plasma LH concentration at 2 h (0.36 + 0.237 versus 0.11 + 0.006 ng/ml, treated versus controls, respectively). Two weeks later there was a tendency (Chi-square = 9.286, 4 d.f., P = 0.054) for an increase in occurrence of medium and larger sized ovarian follicles in treated hinds. However, no ovulations resulted from the infusions and there was no effect on timing of the onset of ovulatory activity during the subsequent breeding season, as determined from circulating progesterone concentrations. It is concluded that kisspeptin stimulates the reproductive axis in seasonally acyclic red deer hinds, however the dose or mode of administration required to produce ovulations may be higher than what is effective for sheep in an equivalent reproductive state.