Danila Marini

Do I turn left or right? Effects of sex, age, experience and exit route on maze test performance in sheep

Brain development and function are susceptible to perturbation by environmental factors. Sheep are increasingly being used as a neurodevelopmental model due to timing similarities with humans, but effects of age, experience and sex on cognition are not well characterised in this species. We therefore studied memory and reversal learning in sheep using a modified Y-maze at two ages: naive 18 weeks old (18N: 23 male, 17 female), experienced 40 week old sheep that had previously been tested at 18 weeks (40E: 22 male, 17 female), and naive 40 weeks old (40N: 4 male, 10 female). Younger naive animals (18N) required more trials and time to solve the first reversal task (task R1) than 40E (P=0.007 and P<0.001 respectively). Experience also improved outcomes, with 40N sheep requiring more time to solve tasks L (P=0.034) and R1 (P=0.002) than 40E. Increasing age (40N cf. 18N) decreased bleat frequency in tasks R1, M2 and R2 (each P<0.05). In 40N females, outcomes also differed by exit method in task R1, with those that exited via an indirect route taking less time to pass tasks R1 (P=0.009) and R2 (P=0.015) than those that used a direct route. Age plus experience improved learning outcomes, demonstrating knowledge retention for 22 weeks in this species, whilst age alone affected mostly behavioral responses. These results provide comparison data, and can be utilised to improve experimental design, for studies of neurodevelopment in the sheep.

Placental and fetal growth restriction, size at birth and neonatal growth alter cognitive function and behaviour in sheep in an age- and sex-specific manner.

Intrauterine growth restriction and slow neonatal growth in humans are each associated with poorer learning, memory and cognitive flexibility in childhood and adulthood. The relative contributions of pre- and post-natal growth to cognitive outcomes are unclear, however. We therefore compared performance in learning, memory and reversal tasks using a modified Y-maze at 18 and 40 weeks of age in offspring of placentally-restricted (PR: 10 M, 13 F) and control (23 M, 17 F) ovine pregnancies. We also investigated relationships between size at birth, neonatal growth rates and cognitive outcomes. PR had limited effects on cognitive outcomes, with PR males requiring more trials to solve the initial learning task than controls (P=0.037) but faster completion of reversal tasks in both sexes at 18 weeks of age. In males, neonatal growth rate correlated inversely with numbers of trials and total time required to solve memory tasks at 40 weeks of age. In females, bleat frequency in the first reversal task at 18 weeks of age correlated positively with birth weight (r=0.734, P<0.05) and neonatal growth rate (r=0.563, P<0.05). We conclude that PR induces limited effects on cognitive outcomes in sheep, with some evidence of impaired learning in males, but little effect on memory or cognitive flexibility in either sex. Rapid neonatal growth predicted improved memory task performance in males, suggesting that strategies to optimize neonatal growth may have long-term cognitive benefits but that these may be sex-specific.

Developing an Ethically Acceptable Virtual Fencing System for Sheep

Simple Summary Virtual fencing has the potential to be implemented in animal management systems where conventional fencing cannot be applied. Previous work has been conducted on cattle with virtual fencing technology that uses a collar that emits a warning audio when an animal approaches a GPS set boundary. If the animal continues walking towards the boundary an electric stimulus is applied. Less research has been conducted on sheep virtual fencing, so alternatives need to be explored to test the proof of concept on the application of virtual fencing with sheep. Using manually controlled training collars, 30 crossbred sheep were trained to respond to an audio cue in order to avoid receiving a low-level electrical stimulus. Abstract To ensure animal welfare isn’t compromised when using virtual fencing, animals must be able to associate a benign conditioned stimulus with an aversive stimulus. This study used an associative learning test to train 30, four-year-old, Merino x Suffolk ewes, to associate an audio cue with an electric stimulus. Collars manually controlled by a GPS hand-held unit were used to deliver the audio and electric stimuli cues. For the associative learning, when sheep approached an attractant at a distance of three m from the trough, an audio cue was applied for one s. If the sheep stopped or changed direction, the audio cue ceased immediately and no electrical stimulus was applied. If the sheep did not respond to the audio cue it was followed by a low-level electrical stimulus. Approaches to the attractant significantly decreased from day one to day two. It took a mean of three pairings of the audio cue and electrical stimulus for a change in behaviour to occur, after which sheep that approached the attractant had a 52% probability of avoiding the electrical stimulus and responding to the audio cue alone. Further research is required to determine whether sheep can be trained to associate an audio cue with a negative stimulus for use in group grazing situations.

Palatability and pharmacokinetics of flunixin when administered to sheep through feed

Applying analgesics to feed is a potentially easy method of providing pain-relief to sheep and lambs that undergo painful husbandry procedures. To be effective, the medicated feed needs to be readily accepted by sheep and its consumption needs to result in therapeutic concentrations of the drug. In the present experiment, pelleted feed was supplemented with flunixin (4.0 mg/kg live weight) and offered to eight sheep. To test the palatability of flunixin, the individually penned sheep were offered normal feed and feed supplemented with flunixin in separate troughs for two consecutive days. A trend for a day by feed-type (control versus flunixin supplemented) interaction suggested that sheep may have had an initial mild aversion to pellets supplemented with flunixin on the first day of exposure, however, by on the second day there was no difference in consumption of normal feed and feed supplemented with flunixin. To test pharmacokinetics, sheep were offered 800 g of flunixin supplemented feed for a 12 h period. Blood samples were taken over 48 h and plasma drug concentrations were determined using ultra-high-pressure liquid chromatography, negative electrospray ionisation and tandem mass spectrometry. The mean ± S.D. time required to reach maximum concentration was 6.00 ± 4.14 h and ranged from 1 to 12 h. Average maximum plasma concentration was 1.78 ± 0.48 µg/mL and ranged from 1.61 to 2.80 µg/mL. The average half-life of flunixin was 7.95 ± 0.77 h and there was a mean residence time of 13.62 ± 1.17 h. Free access to flunixin supplemented feed enabled all sheep to obtain inferred therapeutic concentrations of flunixin in plasma within 6 h of starting to consume the feed. Provision of an analgesic in feed may be an alternative practical method for providing pain relief to sheep.

Randomised trial of the bioavailability and efficacy of orally administered flunixin, carprofen and ketoprofen in a pain model in sheep

OBJECTIVE To determine the efficacy and bioavailability of non-steroidal anti-inflammatory drugs (NSAIDs) when administered orally to sheep. DESIGN Randomised experimental design with four treatment groups: three NSAID groups and one control group (n = 10/group). The study animals were 40 18-month-old Merino ewes with an average weight of 31.4 ± 0.5 kg. METHODS Treatment was given orally at 24 h intervals for 6 days at dose rates expected to achieve therapeutic levels in sheep: carprofen (8.0 mg/kg), ketoprofen (8.0 mg/kg) and flunixin (4.0 mg/kg). Oil of turpentine (0.1 mL) was injected into a forelimb of each sheep to induce inflammation and pain; responses (force plate pressure, skin temperature, limb circumference, haematology and plasma cortisol) were measured at 0, 3, 6, 9, 12, 24, 36, 48, 72 and 96 h post-injection. NSAID concentrations were determined by ultra-high-pressure liquid chromatography. RESULTS The NSAIDs were detectable in ovine plasma 2 h after oral administration, with average concentrations of 4.5-8.4 µg/mL for ketoprofen, 2.6-4.1 µg/mL for flunixin and 30-80 µg/mL for carprofen. NSAID concentrations dropped 24 h after administration. Pain response to an oil of turpentine injection was assessed using the measures applied but no effect of the NSAIDs was observed. Although this pain model has been previously validated, the responses observed in this study differed from those in the previous study. CONCLUSIONS AND CLINICAL RELEVANCE The three NSAIDs reached inferred therapeutic concentrations in blood at 2 h after oral administration. The oil of turpentine lameness model may need further validation.

A randomised field study evaluating the effectiveness of buccal meloxicam and topical local anaesthetic formulations administered singly or in combination at improving welfare of female Merino lambs undergoing surgical mulesing and hot knife tail docking

This study was a field-based behavioural assessment of the pain responses to surgical mulesing modulated by a buccal formulation of meloxicam (Buccalgesic) and a topical local anaesthetic wound dressing (Tri-Solfen). 20 lambs were randomly allocated to each of: 1) Placebo and sham handled (Sham); 2) Placebo and mulesing (Mules); 3) Buccalgesic and mulesing (Mules+B); 4) Tri-Solfen and mulesing (Mules+T); 5) Placebo, Tri-Solfen and mulesing (Mules+T+P); 6) Buccalgesic, Tri-Solfen and mulesing (Mules+T+B). Lamb behaviour was observed by scan sampling every 15 min for 6 h post mulesing then for 1.5 h daily over the subsequent 10 days. Wound score, wound sensitivity and body weight were recorded on day 4, 7 and 10. On the day of mulesing, abnormal behaviours were reduced for all groups that received the analgesic drugs compared to the Mules group (P < 0.05). Tri-Solfen reduced expression of abnormal behaviours in the first 4 h; Buccalgesic reduced expression of abnormal behaviours between 2 and 6 h; and combination treatment reduced expression of abnormal behaviours over the entire observation period. On the subsequent two days, the drug combination resulted in fewer abnormal postures than Tri-Solfen alone. The drug combination tended to result in lower pain sensitivity (965.3 g tolerated) than either Mules+T+P (828.8 g), or Mules+B (791.2 g) on day 7 (P < 0.05). Use of Tri-Solfen and Buccalgesic singly or in combination improved the welfare of lambs undergoing surgical mulesing. The residual effect of pain and discomfort caused by mulesing, were evident despite provision of analgesic drugs.

Controlling Within-Field Sheep Movement Using Virtual Fencing

Simple Summary Virtual fencing has the potential to increase the implementation of spatial grazing management and targeted grazing without the use of conventional fencing. Current virtual fencing that uses an algorithm that is patented through CSIRO involves a collar that emits a warning audio when an animal approaches a set boundary. If the animal continues walking towards the boundary, an electric stimulus is applied. Using manually operated collars to implement a similar virtual fence, a small group of sheep were restricted from accessing a section of a small paddock. Sheep were successfully kept out of the excluded zone of the paddock. By the third day, the sheep were able to avoid receiving an electrical stimulus by turning away from the boundary when the warning audio was applied. When the sheep were allowed full access to the paddock again, then they were quick to use the once restricted area. Abstract Virtual fencing has the potential to greatly improve livestock movement, grazing efficiency, and land management by farmers; however, relatively little work has been done to test the potential of virtual fencing with sheep. Commercial dog training equipment, comprising of a collar and GPS hand-held unit were used to implement a virtual fence in a commercial setting. Six, 5–6 year-old Merino wethers, which were naïve to virtual fencing were GPS tracked for their use of a paddock (80 × 20 m) throughout the experiment. The virtual fence was effective at preventing a small group of sheep from entering the exclusion zone. The probability of a sheep receiving an electrical stimulus following an audio cue was low (19%), and declined over the testing period. It took an average of eight interactions with the fence for an association to be made between the audio and stimulus cue, with all of the animals responding to the audio alone by the third day. Following the removal of the virtual fence, sheep were willing to cross the previous location of the virtual fence after 30 min of being in the paddock. This is an important aspect in the implementation of virtual fencing as a grazing management tool and further enforces that the sheep in this study were able to associate the audio with the virtual fence and not the physical location itself.

Effects of induced placental and fetal growth restriction, size at birth and early neonatal growth on behavioural and brain structural lateralization in sheep

ABSTRACT Poor perinatal growth in humans results in asymmetrical grey matter loss in fetuses and infants and increased functional and behavioural asymmetry, but specific contributions of pre- and postnatal growth are unclear. We therefore compared strength and direction of lateralization in obstacle avoidance and maze exit preference tasks in offspring of placentally restricted (PR: 10M, 13F) and control (CON: 23M, 17F) sheep pregnancies at 18 and 40 weeks of age, and examined gross brain structure of the prefrontal cortex at 52 weeks of age (PR: 14M, 18F; CON: 23M, 25F). PR did not affect lateralization direction, but 40-week-old PR females had greater lateralization strength than CON (P = .021). Behavioural lateralization measures were not correlated with perinatal growth. PR did not alter brain morphology. In males, cross-sectional areas of the prefrontal cortex and left hemisphere correlated positively with skull width at birth, and white matter area correlated positively with neonatal growth rate of the skull (all P < .05). These studies reinforce the need to include progeny of both sexes in future studies of neurodevelopmental programming, and suggest that restricting in utero growth has relatively mild effects on gross brain structural or behavioural lateralization in sheep.

A pen study evaluation of buccal meloxicam and topical anaesthetic at improving welfare of lambs undergoing surgical mulesing and hot knife tail docking

Mulesing is a painful husbandry procedure commonly used to reduce the risk of breech strike in sheep. This study assessed the behavioural (over 6 h), cortisol, haptoglobin and haematology responses to surgical mulesing plus tail hot knife docking (mulesing); modulated by a buccal meloxicam formulation (Buccalgesic), a topical local anaesthetic wound dressing (Tri-Solfen) or both agents. 24 lambs were allocated to each of: 1) Placebo and sham handled (Sham); 2) Placebo and mulesing (Mules); 3) Buccalgesic and mulesing (Mules + B); 4) Tri-Solfen and mulesing (Mules + T); 5) Placebo, Tri-Solfen and mulesing (Mules + T + P); 6) Buccalgesic, Tri-Solfen and mulesing (Mules + T + B). Compared with Mules, Mules + T had a lower cortisol response (72.5 ± 8.7 nmol/L v 122.9 ± 8.7 nmol/L) at 30 min, reduced statue standing at 2 h (3.9% v 11.4%) and increased lying (20.9-25.0% v 7.3-12.5%). Mules + B had reduced cortisol response at 6 h (48.1 ± 8.5 nmol/L), reduced Neutrophil:Lymphocyte ratio at 6 h (Mules + B: 1.25; Mules: 2.44), reduced statue standing at 2 h and 4-6 h (4.1-8.3%), and increased lying at 5 h (27.4%). Mules + B + T had lower cortisol concentrations at 30 mins (86.51 ± 8.71 nmol/L), TWCC not significantly different from Sham at 6 h (9.07 vs 8.09) and 24 h (9.05 vs 8.38). Mules + T + B had significantly lower TWCC than Mules at 12 h (9.56 vs 11.05) and 24 h (9.05 vs 10.42). Mules + T + B did not.