United Kingdom

Introduction

Herbal medicines have been used in human and animal medicine for centuries to treat parasitic diseases; few examples have been investigated for genuine anti-parasitic activity. In developing countries access to effective anthelmintic treatment for livestock is often limited by cost, availability and variable quality. Reports of resistance to benzimadazoles in ruminants in Ethiopia serve as a warning that anthelmintic resistance may also be an emerging problem [1,2]. In light of these issues there is increasing interest in plant remedies as alternatives to synthetic anthelmintics. This study used a participatory rural appraisal (PRA) to identify plants with potential anthelmintic activity in the Oromia region of Ethiopia; five plant extracts were shortlisted and tested for efficacy against cyathostomins using in vitro assays. Current attitudes to ethnoveterinary medicine were discussed.

Methods

Focus group discussions with 29 groups of donkey owners from the Oromia region of Ethiopia explored the use of plants to treat GI parasites in livestock. Current attitudes to herbal medicines were discussed and recorded using thematic analysis. Plants of interest were collected and identified at the National Herbarium, Addis Ababa, Ethiopia. Plants were shortlisted for in vitro tests based on four criteria; ranking in the PRA, supportive literature, no evidence of toxicity and availability. Hydro-alcoholic extraction of dried plant material from shortlisted species was performed. The efficacy of extracts was evaluated in the egg hatch assay (EHA) using cyathostomin eggs recovered from the faeces of donkeys at the Donkey Sanctuary, UK. Dose response curves were produced and ED-50 values calculated using probit analysis.

Results

The focus groups identified 21 plants used as anthelmintics in livestock. A general move away from traditional medicines in the younger generation was observed, although when asked if they would use plants in future many would consider this if they had been tested scientifically and were approved by professionals. The five plants shortlisted for in vitro analysis were Acacia nilotica, Cucumis prophetarum, Rumex abysinnicus, Vernonia amygdalinia and Withania somnifera. Three showed efficacy in the EHA; Acacia nilotica, Cucumis prophetarum and Rumex abysinnicus, with EC-50 values of 0.7, 1.1 and 1.3mg/ml respectively.

Conclusion

Three out of five of the plants identified in the PRA showed efficacy in vitro suggesting that plant remedies used by livestock owners in the Oromia region of Ethiopia may contain compounds with genuine anthelmintic activity. Evaluation of current attitudes suggests that plant remedies are not used unless there is no other option, but that they would be considered should scientific evidence of efficacy and safety be presented to them by animal health professionals [3]. It is therefore essential that a randomised controlled trial is used to verify whether in vitro anthelmintic activity can be translated in vivo and thus whether the plants identified in this study have potential as safe alternatives to synthetic anthelmintic drugs. This study has highlighted that local practices pertaining to the health of working equids are a rich source of information that may help to inform sustainable and effective treatment strategies in future.

In the developed world the control of equid gastrointestinal (GI) nematodes, in particular cyathostomins, is increasingly challenging due the threat of anthelmintic resistance. In developing countries such as Ethiopia despite high parasite burdens, access to genuine anthelmintic treatment is limited. In both situations there is a need for alternative treatment and management regimens for effective parasite control and consequently there has been increasing interest in the use of bioactive plant extracts (BPEs) [1]. This study identified candidate plants in the UK and Ethiopia and evaluated their in vitro efficacy against cyathostomin populations derived from donkeys. A participatory rural appraisal (PRA) approach was used to identify five ethnoveterinary medicines for use in donkeys in Ethiopia. In the UK three plants were identified following extensive review of literature citing efficacy against GI nematodes of other host species and two extracts were provided courtesy of Prof Jerzy Behnke, University of Nottingham. Hydro-alcoholic extraction of dried plant material was carried out for the eight extracts prior to reconstitution in both water and DMSO. Efficacy of each extract was evaluated by egg hatch assay (EHA) and larval migration assay (LMA) using eggs and larvae recovered from the faeces of donkeys at the Donkey Sanctuary, UK. Dose response curves were produced and ED-50 values were calculated using probit analysis. Of the five Ethiopian plant extracts tested, four showed efficacy in the EHA and/or LMA. The two most efficacious were Acacia nilotica and Rumex abyssinicus in the EHA with ED-50 values of 0.72mg/ml and 1.29mg/ml respectively. Of the five UK extracts four showed efficacy in the EHA and/or LMA. The two most efficacious were Carica papaya (papaya) in the LMA and Allium sativum (garlic) in the EHA with ED-50 values of 18.9µM and 0.65mg/ml respectively. The two BPEs most efficacious in vivo for Ethiopia and the UK are to be carried forward to in vivo trials. This study has demonstrated in vitro efficacy of nine plant extracts against cyathostomins. There is evidence in the literature that these plant extracts show efficacy both in vitro and in vivo against GI nematodes in other species. Therefore these results have identified potential alternatives to synthetic anthelmintics for the treatment of cyathostomins that require further investigation. [1] Githiori JB, Athanasiadou S, Thamsborg SM. Use of plants in novel approaches for control of gastrointestinal helminths in livestock with emphasis on small ruminants. Vet Para 2006;139, 308–320.

Anecdotal evidence from animal charities indicates that the number of overweight donkeys in the UK is increasing. Donkeys commonly have daily access to pasture therefore knowledge of grass intake is essential if feeding advice is to be relevant. The effects of herbage mass and length of grazing time on diet composition and dry matter intake (DMI) by mature donkeys were determined.

There were two measurement periods: period 1 during autumn when pasture was sparse (herbage mass 92 + 7g DM/m2) and period 2 during summer when pasture was more abundant (herbage mass 197 + 12g DM/m2). Twenty mature donkeys were selected and split into three grazing groups (8, 12 and 23 h daily grazing access). Barley straw was fed ad libitum and each donkey was given 150mg per day of an n-alkane marker Dotriacontane (C32) for the 12 d of each study period. Herbage mass significantly affected total DMI and diet composition. During summer DMI of donkeys in the 8 and 23h groups was significantly greater than during autumn (P<0.05). The proportion of grass in the diets of all donkeys was also greater in summer compared to autumn (P<0.001). Grazing time did not significantly influence total daily DMI during either season due to donkeys consuming more straw when grass intake was reduced. Restricting donkeys to 12h or less grazing per day in summer significantly (P<0.001) reduced their grass intake compared to that of donkeys with 23h access. When grazing sparse pastures (autumn) time allowed for grazing did not influence grass intake. The results show that time allowed for grazing per se was less important than the herbage mass available to the donkey in terms of grass DMI.

Measuring daily food intake of foraging animals is essential if accurate feeding rations are to be implemented. The alkane technique, which has recently been validated in equines, now provides the opportunity to measure intake at pasture. The aims of this study were to determine the effects of herbage mass and grazing time allowance on dry matter intakes in mature donkeys in the UK. The effect of grazing time allowance on diet composition was also measured. Two study periods took place; period 1 when pasture was sparse (herbage mass 133.1±10 g dry matter/m2) and period 2 when pasture was more abundant (herbage mass 284.5±17.2 g dry matter/m2). Eighteen mature donkeys, male and female, were selected for the study and split into three grazing groups. Groups 1 and 2 were restricted to 8 and 12 hours grazing time per day, respectively. Group 3 was allowed 23 hours grazing time daily. Access to a yarded area and shelter was available to all donkeys during grazing periods. Barley straw was fed ad libitum to all donkeys and was available at all times. Each donkey was administered with 150 mg per day of an n-alkane marker Dotriacontane (C32) in the form of a labelled wheat biscuit fed three times daily for the 12 days of each study period. During period 1 grazing time allowance had no significant effect on daily DMI although the donkeys with 23 hours access did consume more than donkeys with only 12 and 8 hours grazing access (2.61, 2.54 and 2.26 kg, respectively). The proportion of grass and straw comprising daily intake was affected by grazing time allowance (P<0.05). Grass comprised 18% of daily intake for the 8 and 12-hour groups and 11% in the diet of the 23-hour group, although this difference was not significant. During period 2 daily DMI remained unaffected by grazing time allowance. The proportions of grass and straw within the diet were significantly affected (P<0.001), grass comprised 25 and 29% of daily intake for the 8 and 12-hour groups but made up 41% of daily intake of the 23-hour group. These results show that grazing time allowance has little effect on overall DMI but when given the opportunity donkeys increase their grass intake.

Donkeys and horses differ substantially in their evolutionary history, physiology, behaviour and husbandry needs. Donkeys are often kept in climates that are colder and wetter than those they are adapted to and therefore may suffer impaired welfare unless sufficient protection from the elements is provided. We compared the shelter-seeking behaviours of donkeys and horses in relation to temperature, precipitation, wind speed and insect density. Our study collected 13,612 day-time data points (location of each animal, their activity such as feeding, resting, moving, etc., and insect-related behaviours) from 75 donkeys and 65 horses (unclipped and un-rugged) with free access to man-made and natural shelters between September 2015 and December 2016 in the South-West of the UK. Each animal was observed at least once a week, with an average of 65 observations per individual overall. Even though the UK climate is quite mild (1 to 33 degrees Celsius in our sample), the preliminary results showed clear differences in the shelter seeking behaviour between donkeys and horses. Overall donkeys were observed far more often inside their shelters than horses (χ2(1)=1,783.1, P<0.001). They particularly sought shelter when it was raining: there was a 54.4%-point increase (35 to 89.4%) in the proportions of donkeys sheltering in rainy conditions, in comparison to a 14.5%-point increase in horses (9.6 to 24.1%). Results of binary logistic regressions indicated that there was a significant association between species, precipitation and shelter-seeking behaviour (χ2(3)=2,750.5, P<0.001). Horses sought shelter more frequently when it got hotter, whereas donkeys sought shelter more often in colder weather (χ2(3)=2,667.3, P<0.001). The wind speed (range 0 to 8 m/s – calm to moderate breeze) had an effect on location choice, and this again differed significantly between donkeys and horses (χ2(3)=1,946.5, P<0.001). In a moderate breeze, donkeys tended to seek shelter whereas horses moved outside. The insect-related behaviours were closely related to temperature and wind speeds. The donkeys’ shelter-seeking behaviour strongly suggests that in temperate climates they should always have access to shelters that provide sufficient protection from the environment.

At present there are no published feeding guidelines specific to donkeys. Current recommendations are to feed 0.75 of horse energy needs on a body weight basis. However, it has been shown that donkeys have a greater digestive efficiency than horses and ponies and thus, feeding them as though they were small horses results in excess energy intake and, as a consequence they become obese. The formation of feeding guidelines begins with the estimation of the energy requirement for maintenance, as this is the value upon which nutrient requirements are related to. The aim of the study was to determine the maintenance digestible energy requirements of mature donkeys during each UK season. Twenty mature donkeys (10 male, 10 female) were selected for use in the study. The donkeys were fed a diet of hay and barley straw in quantities that were adjusted to maintain body weight. An equilibration period of minimum 16 days was followed by a five day total faecal collection; this was carried out for each season. Food and faecal samples were analysed for dry matter (DM), organic matter (OM), digestible energy (DE), crude protein (CP), neutral-detergent fibre (NDF) and acid-detergent fibre (ADF); nutrient and energy intakes were calculated. There was a significant seasonal effect on all intakes except NDF and ADF. DM and DE intakes showed significant increases in winter compared to summer, rising by 42 and 35%, respectively. Digestible crude protein (DCP) intakes increased during winter and spring but were significantly lower in autumn compared to all other seasons. A strong seasonal effect was exerted on all in vivo digestibilities although there was no effect of sex. OM, NDF and ADF intakes were affected by sex with male animals having higher intakes compared to female animals. Comparison of these results with NRC (1989) horse requirements showed a considerably reduced requirement by donkeys for energy and protein and thus it was concluded that feeding tables specific to horses are not suitable for calculating donkey requirements.

The female donkey is often known as a mare or jenny, the male as a donkey stallion or jack.

Reproductive behaviours

Sexual behaviour is often more exaggerated in the donkey and stallion-like behaviour may persist in the male donkey after castration. It is recommended that, unless being used for breeding, all male donkeys are castrated between 6 and 18 months of age.

Females will start cycling regularly between 10 and 22 months old with a wide variation in oestrus duration of 2–10 days. Seasonality of ovarian activity is variable and likely to be influenced by photoperiod, nutrition and temperature. Older females will tend to show longer interovulatory intervals. Signs of oestrus observed in females include mounting (females will mount each other with the oestrous female on the bottom), mouth clapping, winking (eversion of the clitoris), raising the tail, urinating, posturing (abducted hindlegs, arched tail) and backing up towards the jack. During dioestrus, females will show a lack of interest in the male and will move away or kick if male interest is persistent.

Puberty in the male donkey occurs between 16 and 20 months, with sexual maturity at around 3 years of age; however, males may show mounting behaviours from young foals. Male sexual behaviour differs from horses in that jacks are often slow to achieve erection (10–40 min) [1] and may mount a jenny several times before becoming fully erect. Several periods of sexual interaction are usual, separated by periods where the jack will withdraw away from the jenny. Donkey stallions are territorial and can become very aggressive, especially in the presence of competing males and females in season.

Reproductive anatomy

Donkey reproductive organs are proportionally larger than horse reproductive organs and ligation of the testicular artery is mandatory when castrating donkey stallions [2]. Even slim donkeys can have large amounts of scrotal fat so care should be taken post castration that fat does not prolapse from the surgical site. Castration via the inguinal approach is recommended for large or mature male donkeys over 4 years of age. Donkeys castrated after 18 months of age are more likely to retain stallion-like behaviours.

Testing for anti-Müllerian hormone (AMH) has welfare and practical advantages over the hCG stimulation test for diagnosing cryptorchid donkeys. It only requires a single blood sample and following castration, AMH concentrations are undetectable within approximately 2 days, making AMH a useful test if there is any doubt over the success of a recent castration [3].

The jenny’s reproductive organ anatomy is generally similar to that of the horse mare although due to size, rectal examination of miniature donkeys may be challenging. AI techniques are similar to those described in the horse although the anatomy of the cervix, coning towards the caudal end [4], may make manipulation of the cervix for uterine flushing or other techniques requiring access to the uterine cavity during dioestrus difficult.

Pregnancy

Owner education is essential as many donkey owners are often inexperienced, unprepared or unaware that their donkey is in foal. Gestation is longer and has greater variability than horses and ponies; ranging from 11 to 14.5 months. The incidence of twin foaling at full gestation is reportedly higher than in horses and ponies.

Pregnancy diagnosis can include transrectal ultrasound; optimal time for early diagnosis is 14 days post ovulation, transrectal palpation from day 40 [2] and oestrone sulfate testing from day 120.

Pregnant females should be vaccinated following recommended equine guidelines, and prior to foaling, parasite prevention should be put into place including appropriate pasture management during and after pregnancy. Body condition should be regularly assessed and feed adjusted appropriately. Pregnancy and lactation are risk factors for hyperlipaemia.

Electrolyte levels in the mammary secretions can be used to predict foaling. A sodium:potassium ratio of <1 is indicative of foaling occurring in the next 24–48 h [2]. Calcium levels are less reliable but can also be used.

Jennies have a higher tendency to exhibit foal heat than horses and ponies, with higher pregnancy rates [2].

Foal management

The incidence of failure of passive transfer ranges from 3 to 40% [5]. Risk factors are similar to those found in the horse and the IgG level considered normal in horse foals is used. There is a problem with recognition of prematurity or dysmaturity when compared with horses. In horses, the covering date is usually known and the gestation period is more defined. In donkeys, especially in miniatures, the variation in gestation length can make it very hard to define a premature donkey foal [4]. Despite their thick fluffy coat (appearance of warmth and hardiness compared to the horse foal), donkey foals are not very hardy and require warmth and suitable shelter [5].

References

1. S. Purdy. (2019) Small herd behaviour in domestic donkeys. Equine Veterinary Education 31, 199-202.
2. The Donkey Sanctuary. (2018) The Reproductive System. In: The Clinical Companion of the Donkey, 1st edition, Matador, Leicestershire, pp 73-86.
3. N. Matthews, T. Taylor, V. E. N. Blanchard South, A. E. Durham. (2017) Use of Anti-Müllerian hormone (AMH) for the diagnosis of cryptorchidism in donkeys. ECEIM Congress 2016 Abstracts. Journal of Veterinary International Medicine 31, 604-618.
4. N. Matthews, T. Taylor, T. Blanchard. (2003) An overview of reproduction in donkeys. International Animal Health News: A publication of Christian Veterinary Mission 18.
5. N. Aronoff. (2010) The donkey neonate. In: Veterinary Care of Donkeys, Editors: N. Matthews, T. Taylor, International Veterinary Information Service, Ithaca NY. Last updated: 29 March 2010.

Cyathostomins, the predominant parasitic nematodes of equids, have developed varying degrees of resistance to all three classes of anthelmintic licensed for use in horses. It is essential that the effectiveness of alternative methods of control for these pathogens are quantified, including incorporating climatic data and the commonly advocated practice of removal of faeces from pasture. Here, we obtained monthly faecal worm egg counts (FWEC, n=4,460 individual counts) from 803 donkeys based at The Donkey Sanctuary (Devon, UK). The dataset also included age, sex, field, FWEC history and previous anthelmintic administrations in each individual, as well as the pasture hygiene management method applied in the field where the donkey was grazed. FWEC were analysed alongside local climatic data using a generalised linear mixed model to assess associations between these variables and each observed monthly FWEC. The preferred model was identified using a model selection algorithm based on penalised likelihoods, and associated a 2.1% decrease in FWEC per day with air frost two calendar months ago (p<0.001) and a 38% lower FWEC in groups with twice weekly manual faecal removal compared to those with no faecal removal (p=0.004). Other weather effects, both alone and as interaction terms with the average FWEC of the field were included in the model, alongside individual FWEC history with anthelmintic administration as interaction terms and date as a single term. Our study identifies factors that may be useful as part of on-going predictive modelling based methods of improving targeted selective therapy.