Anal fin morphology and gonadal histopathology in mosquitofish (Gambusia holbrooki) exposed to treated municipal sewage effluent.

Feral mosquitofish living in undiluted treated municipal sewage in Queensland, Australia, were examined for morphologic abnormalities indicative of reproductive dysfunction. Male and female mosquitofish were captured at two sites receiving undiluted treated sewage and compared with those captured at a reference site. Several morphologic end points were examined, including length of the fourth and sixth anal fin rays and gonadal histology. Both male and female mosquitofish at one site exhibited minor elongation of the fourth anal fin ray consistent with exposure to androgenic stimulation, although the spatial extent of the effect was limited and not significant at other sites further downstream. No incidences of intersex were found. These findings suggest that the level of treatment of domestic sewage at the two sewage treatment plants assessed is adequate to prevent reproductive abnormalities in exposed mosquitofish populations.

Neuronal ceroid-lipofuscinosis in Borderdale sheep.

AIM: To describe the gross and histological lesions of a neurological disease in Borderdale sheep characterised clinically by blindness and circling, as a basis to its classification. METHODS: Formalin-fixed tissues were processed into paraffin wax and epoxy resin for light and electron microscopy of variously stained sections. RESULTS: Lesions were those of a lysosomal storage disease with severe neurodegeneration of the cerebral cortex. The staining reactions, autofluorescence and ultrastructure of storage material allowed the diagnosis of neuronal ceroid-lipofuscinosis associated with the accumulation of subunit-c of mitochondrial ATP synthase. CONCLUSIONS: The severity of neurodegeneration and minor differences in the ultrastructure of storage material implied that this was a different disease from other forms of ovine ceroid-lipofuscinosis that accumulate subunit-c of mitochondrial ATP synthase. An autosomal recessive mode of inheritance is considered probable. Although of only minor economic importance, this disease may be important to research into the group of ceroid-lipofuscinoses as a whole.

Changes in GABAergic neuron distribution in situ and in neuron cultures in ovine (OCL6) Batten disease.

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited human and animal diseases characterized by progressive brain atrophy. A form in sheep is syntenic to the human CLN6 disease. Cell type specific neurodegeneration in these sheep was indicated by the distribution of GABAergic interneurons in coronal sections of normal and CLN6 affected sheep brains. A reduction of parvalbumin immunoreactive neurons in NCL cerebral cortex was the most striking feature. This was most pronounced in parietal cortex where very few positive cells remained. Calretinin immunoreactive somata in infragranular layers of the neocortex were also reduced while the number of calbindin positive cells was similar in affected and normal brains. There were fewer GAD immunoreactive neurons in the deeper layers of all NCL cortical areas examined. The parietal lobe was relatively more affected than frontal or temporal lobes while the cerebellum and the basal ganglia showed no signs of selective neuron loss. Since horizontally extending basket cells are mainly labelled by parvalbumin, the loss of these interneurons in the neocortex may render pyramidal neurons more excitable and compromise their co-ordinated output. In vitro, cultures of control and affected neurons from 60 to 70-day-old fetal brain hemispheres were examined for the presence of GABAergic and glutamatergic neurons. Different neurons developed distinct immunoreactivity to glutamate or GABA but the overall distribution was similar in normal and affected cultures. This culture system may provide a useful model to compare GABAergic cell function of normal and NCL affected neurons.

In vitro culture of neurons from sheep with Batten disease.

Specific storage of mitochondrial ATP synthase subunit c occurs in most forms of Batten disease, including the ovine form, but its relationship to the characteristic neurodegeneration is not clear. Storage occurs in most cell types but only neurons are functionally affected. Neurons were cultured from control and affected sheep. Ewes were superovulated and inseminated, and embryos were collected, frozen, stored, and later transplanted into surrogate dams for gestation at times to suit experimental demands. The optimal fetal age for cultures was investigated, from 50 to 125 days. There were no differences between control and affected embryos in this period of rapid growth. At 50 days brains consist of smooth-surfaced hemispheres and cerebellum with no obvious demarcation between gray and white matter. At 90 days they are like miniature adult brains. From 200 to 600 million viable cells were recovered from each fetus, regardless of age. DMEM/F12 with B27 was the most practical medium tested. Cell viability was not as good in medium containing serum. Treatment of surfaces with polylysine aided neuron adhesion. No developmental or viability differences were observed between normal and affected neuron cultures. At plating out cells were rounded. A day later single process outgrowths began. After 4 days these were over 200 microm and by Day 6 had created a network. Most neurons were bipolar. Neurons from 50 to 90-day old fetuses persisted in culture for over 100 days.

Disease-specific pathology in neurons cultured from sheep affected with ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCL, Batten disease) are a group of inherited neurodegenerative storage diseases in children. Mutations in different genes underlie different forms. Subunit c of mitochondrial ATP synthase is specifically stored in autofluorescent bodies in most of them, including a form in sheep. Mature bodies are lysosomal but the initial site of storage is not known, nor is it known how this leads to the characteristic neurodegeneration. Neurons were cultured in serum-free medium from control and affected sheep fetuses at 90 days gestation. They showed positive microtubule-associated protein staining, developed neurites, and had typical neuron morphology. Time-dependent accumulation of subunit c and of fluorescent storage bodies was observed in affected cells by immunocytochemistry and confocal microscopy. A small number of autofluorescent bodies were apparent after 4 days in culture. After 10 days these bodies were more numerous, more intensely autofluorescent, and often larger in size. By 14 and 21 days many neurons were packed with autofluorescent material. These bodies were not seen in control cultures. Immunocytochemistry revealed subunit c-positive storage material only in affected neurons and not in affected glial cells. Confocal microscope analysis, using organelle-specific dyes, demonstrated colocalization of autofluorescent bodies with lysosomes, not with mitochondria. Survival rates of the affected cells were unaffected by the storage body accumulation over a 3-month period. These cultures can now be used to study the mechanism of subunit c accumulation and of neurodegeneration and to test therapeutic possibilities.

Wool production in transgenic sheep: results from first-generation adults and second-generation lambs.

In sheep transgenic for a sheep insulin-like growth factor 1 (IGF-1) cDNA driven by a mouse keratin promoter, we assessed wool production and properties in 51 adults of the first generation (G1) and in 56 lambs of the second generation (G2). Transgenic G1 sheep had an increased rate of wool production during spring and summer of year 2 compared with nontransgenic half-sibs, with a maximum increase of 17% in December, but during the winter nadir rates were similar. At second- and third-year shearing, however, fleece weights were not significantly different. There was a trend for transgenic animals to have coarser wool of lower staple strength. A controlled feeding trial revealed no significant differences in feed intake. The transgene was expressed not only in skin but also in a wide range of other tissues. Circulating IGF-1 concentrations were not significantly different between transgenic and nontransgenic animals, suggesting that local mechanisms were more important than systemic mechanisms for wool production, but were significantly higher in males than in females. In the G2 sheep, transgenic fleece weight did not differ significantly from nontransgenic either as lambs or at the end of the lamb year. Although the transgene was inherited in Mendelian fashion and was widely expressed, the production advantage seen in animals of the first generation did not persist in the second generation.

Heritable testicular hypoplasia in Nguni (Bos indicus) bulls: vascular characteristics and testosterone production.

The biased unilateral occurrence of heritable gonadal hypoplasia was investigated by examining the gross- and microanatomy of the testicular artery and vein, testicular blood flow and testicular testosterone secretion in normal Nguni bulls and in Nguni bulls showing unilateral left, unilateral right and bilateral hypoplasia of the testis. A high incidence of branching of the testicular artery was found ipsilateral to hypoplastic testes. The branching occurs a short distance from the dorsal aorta: one branch proceeds to the testis, the other to the ipsilateral kidney. The association between arterial branching to the kidney and ipsilateral hypoplasia of the testis held for both unilaterally left and unilaterally right hypoplastic bulls. Variations in the anatomy of the testicular vein occurred in both normal and hypoplastic bulls but there was no specific association between the variations and ipsilateral hypoplasia. The lumen diameter of the testicular artery or branch correlated with testis mass. Wall thickness of the artery ipsilateral to hypoplastic testes was not different from that in normal bulls, discounting hyperplasia of the endothelium. Total blood flow to the testis correlated with testis mass. The secretion rate of testosterone from hypoplastic testes was lower than that of normal testes but there was no difference when compared on a unit mass basis.

Effect of surgical restriction of growth of the testicular artery on testis size and histology in bulls.

The growth of the testicular artery was restricted on one side in young bulls and subsequent testicular development was monitored. After the animals had been killed, the testes were studied histologically and compared with testes from hypoplastic bulls. The growth rate of testes from the experimental side was significantly lower than that of testes from the sham-operated side over a period of 578 days. At death, the experimental testes had a mean (+/- SD) mass of 76 (+/- 41) g compared with 220 (+/- 31) g for the control testes. The sham-operated testes accounted for 0.071 (+/- 0.008)% of live body mass compared with 0.025 (+/- 0.014)% for the experimental testes. The seminiferous tubules in the sham-operated testes had a mean diameter (+/- SD) of 211 (+/- 25) microns, whereas those of the artery-restricted testes and hypoplastic testes were significantly smaller (152 (+/- 37) and 145 (+/- 45) microns, respectively). In the artery-restricted and hypoplastic testes, the interstitial tissue accounted for a significantly greater proportion of the testicular tissue than in the sham-operated testes and spermatogenesis was either totally absent or present in only a small proportion of tubules. It is suggested that the artery-restricted testes could be used as a model for testicular hypoplasia.

Remote controlled sampling of cattle and buffalo blood.

1. Blood samples were obtained from cattle and buffaloes over extended periods of time by remote controlled methods at 4-hourly intervals. 2. Initial handling of the animals and deliberate exposure to stress at the end of the experimental times resulted in increased plasma concentrations of a number of variables. 3. Between initial handling and applied stress, the concentration of all variables investigated showed little change and no evidence of rhythms. 4. The results obtained reflect true plasma composition of free ranging cattle and buffaloes at rest.

Plasma constituents in Nguni cows over forty-eight hours.

Plasma constituents were investigated at ninety minute intervals over a forty-eight hour period on four unrestrained Nguni cows (in small cubicles) in order to study the presence or absence of rhythms. No circadian, ultradian or diurnal rhythm was found for any of the parameters investigated. Basal values over a forty-eight hour period were obtained for certain plasma constituents. These values are of use for studies relating to the effects of stress in cattle.