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Grazing behaviour of Miranda donkeys in a natural mountain pasture and parasitic level changes
Livestock Science 186 (2016) 16–21
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Grazing behaviour of Miranda donkeys in a natural mountain pasture and parasitic level changes M. Couto a,b, A.S. Santos b,c,n, J. Laborda d, M. Nóvoa d, L.M. Ferreira e, L.M. Madeira de Carvalho a a
CIISA, FMV, Lisboa, Portugal Escola Universitária Vasco da Gama, Coimbra, Portugal c CITAB-UTAD, Vila Real, Portugal d Associação para o Estudo e Proteção do Gado Asinino, 5225–011 Atenor Miranda do Douro, Portugal e CECAV-Departamento de Zootecnia, Universidade de Trás-os-Montes e Alto Douro, PO Box 1013, 5001-801 Vila Real, Portugal. b
art ic l e i nf o
a b s t r a c t
Article history: Received 15 January 2015 Received in revised form 2 January 2016 Accepted 8 January 2016
The Miranda donkey (Equus asinus) has an important role in the maintenance of rural landscape and traditions in inland of Portugal. Breeding and keeping of these animals rely on mountain pasture areas, known for their characteristic botanical diversity. However, their grazing behaviour in these areas has not been described and this knowledge is important to set the best management practices that promote an efficient use of these particular feed resources that could be related with parasitic level of these animals. In this way, this study aimed to assess feeding behaviour, diet selection, and relate the total phenolic and tannin content of ingested plants with the parasite load in a group of Miranda donkeys under free ranging system in natural mountain pastures. For this purpose, eight adult non-pregnant Jennies from the Miranda donkey breed were managed under continuous grazing on a 1.6 ha natural mountain pasture from late May to late July. Body condition score (BCS) and grazing behaviour of the Jennies was assessed in two different occasions. Additionally, the level of gastrointestinal nematode infections of the herd was assessed throughout the study. Results showed that animals spent 75.6% of total observation time in search/prehension activities corresponding to an average of 16 h/day. Jennies showed a preference for herbaceous species, although they were able to incorporate in their diet up to 30% of shrubs, suggesting that they can be used as biological tool for controlling and reducing shrub encroachment in these mountain areas. Parasite level increased from 0% positive samples in June to a maximum of 25% in July. The increase of EPG may be related with the observed decrease in the shrub consumption throughout the observation period. Having in account the presence of phenolic and condensed tannins in shrubs, their potential as natural anti-parasitic compounds and the possibility of taking part in donkey diet, these preliminary results can be of the utmost importance regarding an integrated approach of Miranda donkey parasite control. & 2016 Elsevier B.V. All rights reserved.
Keywords: Donkeys Diet selection Gastrointestinal parasites Tannins Mountain pastures
1. Introduction The Miranda donkey is one of the two native Portuguese donkey breeds. These animals are an integrating part of the local communities of the Trás-os-Montes region in the inland north of Portugal, they play an important role in maintaining rural communities and sustainable farming practices, and are a part of the local genetic biodiversity, being a key player in rural development of these territories nowadays. The Miranda donkey is well-
n Corresponding author at: Escola Universitária Vasco da Gama, Coimbra, Portugal. E-mail address: anasofi[email protected] (A.S. Santos).
http://dx.doi.org/10.1016/j.livsci.2016.01.005 1871-1413/& 2016 Elsevier B.V. All rights reserved.
conformed and characterized by a “rustic” appearance. The main coat is dark brown with lighter shades on the sidewall and bottom surface of the trunk, white muzzle and around the eyes, with hirsutism accented in broadsides, face and edges of ears and extremities, and abundant manes. Their average withers height is 1.35 m (Quaresma et al., 2005). Depending the season and the feed availability, nutritional requirements of these animals will vary along the year and changes in body condition score (BCS) may occur if proper supplementation is not given (McDonalds et al., 2002). These animals are still mostly used as working animals, both in farming work and more recently in equestrian tourism. They are traditionally bred under extensive systems often together
M. Couto et al. / Livestock Science 186 (2016) 16–21
with cattle, where pasture supply is the basis of their feeding regimes. Grazing areas mostly used by the Miranda donkey are the traditional mountain pastures, commonly known as “lameiros”. “Lameiros” are semi-permanent natural mountain pastures characterized by a high local botanical diversity including a considerable amount of shrub areas. These semi-permanent natural mountain pastures have been mostly abandoned, due to exodus of rural population. However, recently a new concern has arose to maintain these areas as a part of an integrated, selfsufficient production system since they have an incalculable environmental and landscape value and their conservation is an important objective to the maintenance of high levels of biodiversity in mountain areas. The establishment and development of livestock systems in less favoured areas can provide an environmental and economic benefit, contributing to the improvement of living conditions of rural population (Ferreira et al., 2013, Garcia et al., 2013). Both the Miranda Donkey and the lameiros play a role in its agroecosystem. Parasitic infections, especially by gastrointestinal parasites, particularly nematodes, are common in donkeys and are considered one of the major animal health and welfare problems, having an influence on their body condition and daily work routine (Yoseph et al., 2005). Infections occur by the ingestion of freeliving third stage larvae (also called L3), often when grazing in pastures. There is an increasing evidence of nematode resistance to conventional anthelmintic products in equids and therefore, natural alternative and/or complementary antiparasitic treatments are being pursued (Cernea et al., 2010; Payne et al., 2013). Recent evidence (Villalba et al., 2006) suggests that animals can self-medicate, and if so medicinal plants on diet could provide an important tool for parasite control. The use of plant secondary metabolites may be an effective way to treat animals, as much as phenolic compounds such as condensed tannins can provide beneficial medicinal effects to herbivores as they act against infectious conditions, such as parasitism (Juhnke et al., 2012). The “Lameiros” complex and biodiverse botanical composition, may allow animals to select specific plants that may have an influence in what concerns their natural resistance to gastrointestinal parasites, namely on the donkeys parasite resistance. However, information concerning grazing behaviour of donkeys on these types of pastures is very scarce and knowledge on how animals interact and select this specific botanical diversity could present interesting results to both natural parasite control in donkeys, as well as to the management of these type of biodiverse mountain pastures. In this way, the objective of this study was to observe the feeding behaviour, assess diet selection and relate the total phenolic and tannin content of ingested plants with the parasite load in a group of Miranda donkeys under free ranging system in these natural pastures, during the summer grazing season.
2. Material and methods 2.1. Experimental site, animals and study design The study was carried out in 2013 on a single plot of 1.6 ha pasture located at 651 m above sea level in the northern inland of Portugal, at Atenor, Miranda do Douro (41°24′48.12′′N; 6°29′15.38′ ′O). The climate characteristic of the region has high Mediterranean influence. Mean annual rainfall was 554.7 mm, and rainfall was persistent until the end of May, leading to a high pasture availability in the beginning of June. Mean monthly temperature recorded at the site during the study months was 15.3 °C in June and 23.4 °C in July. The plot used in this study is characterized by vegetation dominated by a mosaic of pasture grass sub-divided by shrub. At the beginning of the study, the lameiro plot was
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dominated by pasture with an average height of 50 cm, composed of several herbaceous species (grasses: Bromus sp., Dactylis glomerata, Lolium sp., Briza sp., Calamagrotis sp., Avena sp., Cynosurus sp., Agrostis sp., legumes: Lathyrus sp., Trifolium sp., Ononis sp., Anthylis sp., Vicia sp. and other non identified genera of herbaceous plants), and shrubs (Quercus sp. dominated: Quercus robur; Q. ilex; Q. pyrenaica, Q. faginea, and also Narrow-leafed ash: Fraxinus angustifolia, hawthorn: Crataegus monogyna, ulmus: Ulmus L., and gorse: Cytisus spp.). A total of 8 adult non-pregnant jennies from the Miranda donkey breed were managed under continuous grazing from late May to late July. Jennies’ BCS was assessed on a scale of one to five (one ¼poor, five¼obese) according to Smith and Wood (2008), at the beginning and end of the study. 2.2. Grazing behaviour and diet selection The study was distributed in two observational periods, period 1 carried out in June (1–4 June) and period 2 July (16–19 July) of 2013. These two close observation periods where chosen in order to allow the collection of data in two different situations: high and good quality pasture availability (period 1) and low quality and amount of pasture availability (period 2). Since climatic conditions in this area are characterize by a rapid temperature elevation together with an inexistence of rainfall in the summer months (from June to August), this leads to rapid changes in vegetation type and availability, and very often, pastures availability goes from high, to very low within a month time span. Each observational period (11 days) comprised a seven-day for adaptation of animals to the diet and experimental conditions; followed by four days of sample collection: faeces and diet components (herbaceous and shrub), and feeding behaviour observation: time spent grazing, by each animal, was assessed by recording the grazing activity every 15 mins during the light period of the day (from sunrise to sunset) on two consecutive days in two observation periods (2–3 June and 17–18 July). In the same periods, diet selected by each animal was estimated using the n-alkane markers (Dove and Mayes, 2006). For that, individual faecal samples were collected twice a day for four days in each observation period (i.e. 1–4 June and 16–19 July). Simultaneously, samples of the main herbaceous components found in the plot and shrub vegetation were collected. Since the plant species within the herbaceous vegetation and within the shrub vegetation presented resemblances in their alkane profile, only two diet components were considered: i.e. herbaceous and shrub. This procedure allowed us to discriminate these two distinct feed resources that was the main objective of this study. Diet composition was estimated for each animal using a nonnegative least-squares procedure in the “EatWhat?” software (Dove and Moore, 1995) which minimizes the discrepancies between the observed concentrations of each n-alkane (C25–C33) in the faeces and the estimated proportions of plant components in the diet. Alkane faecal concentrations of animals were not adjusted as previous results (Ferreira et al., 2007) showed that faecal recovery of these markers in equids is unaffected by carbon-chain length. 2.3. Parasitological evaluation All animals had been previously dewormed in January of 2013, by subcutaneous administration of ivermectin 2% (1 ml per 50 kg body weight). During the whole study periodic analysis of faecal samples was performed to control the parasitic load in the group between June and July, namely on the 2nd June, 2nd July, and on the 22nd July. Spot samples of donkey faeces were individually collected directly from the rectum to assess gastrointestinal
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nematode eggs excretion. The number of eggs per gram of faeces (EPG) or faecal egg count (FEC) was estimated using McMaster technique (Thienpont and Vanparijs, 1986; Madeira de Carvalho, 2001). Gastrointestinal nematode genera present in faeces were identified on the basis of infective third-stage larvae obtained by faecal cultures described by Roberts, O´Sullivan (1950), adapted by Madeira de Carvalho (2001) and Madeira de Carvalho et al. (2008), namely for the collection and identification of Cyathostomum sensu latum subpopulations.
3. Chemical analysis All samples (faeces and vegetation) were frozen ( 20 °C), dried (55 °C for 48 h) and then passed through a 1 mm sieve prior to analytical procedures. Chemical compositions of main vegetation components samples (herbaceous and shrub) were analysed following the AOAC procedures (Association of Official Analytical Chemists, 1990) for ash and CP (N Kjeldahl x 6.25); NDF, ADF and ADL were analysed by the methods of Robertson and Van Soest (1981). The total phenol (TP) and total tannin (TT) contents of the shrub were determined using the Folin- Ciocalteu assay in combination with polyvinyl-polypyrrolidone, using tannic acid (Merck, Damstadt, Germany) as the reference standard (Makkar, 1993). Alkane content of samples was analysed in duplicate according to the methods of Mayes et al. (1986), as modified by Oliván and Osoro (1999). The first stage involved a saponification of 0.5 g of faeces or 1.5 g of vegetation components for 14 h in 7 ml of 1 M KOH ethanolic. Afterwards, a hot extraction was performed with n-heptane at 65 °C (Oliván and Osoro, 1999). After the extraction, the sample was passed through a silica-gel (bed volume 5 ml), to separate the alkanes from pigments, sterols and alcohols. After the recovery proceeded with n-heptane, it was then re-dissolved in 500 μl of heptane for chromatographic analysis. Quantification of the alkanes was carried out by gas chromatography, using Perkin Helmer Clarus 580, equipped with flame ionisation detector (FID), an auto-sampler and a temperature-programmable, and by oncolumn injection on a 50-m column SGE of 0.220 mm internal
diameter. Helium was used as carrier gas at a constant flow of 2 ml min 1. Gradients of temperature were used for the injector (280 °C) and the column (170 °C for 4 min; 30 °C min 1 to 215 °C, 1 min at 215 °C, 6 °C min 1 to 300 °C, 4 min at 300 °C). The detector oven was maintained at 340 °C. Identification was performed by comparison of retention times of the samples components with the previously injected standard. Alkane concentrations were quantified relative to known amounts of the internal standards C22 and C34, added at the beginning of the extraction process. The use of two internal standards enabled the evaluation of the effectiveness of the extraction process and the correction of the peak areas for any discrimination detected during the solventextraction step.
4. Statistical analyses Comparison of diet composition (herbaceous vs shrub) in the two experimental periods was performed using one-way ANOVA (GenStat, 2011). Comparison of data (BCS, parasitic burden and tannins) in the two experimental periods was performed using non-parametric ANOVA through Kruskal–Wallis test with P o0.05 and association between parasitological data and ingested herbaceous and shrubs were analysed with non-parametric correlation Spearman test (GraphPad InStat, v.3.0.10., 2009).
5. Results 5.1. Chemical composition of grass and shrub vegetation Chemical composition of herbaceous and shrub vegetation collected is presented in Table 1. Cell walls components (NDF) represented the highest fraction in the two diet components, averaging 666.2 and 606.9 g/kg DM for herbaceous and shrub, respectively. In both periods, shrub showed high levels of ADF and ADL averaging 442.6 and 172.7 g/kg DM, respectively. TP and TT contents in shrub plants increased from the first to the second
Table 1 Chemical composition (g/kg DM), total phenol (TP) and total tannin (TT) (g tannic acid equivalents/kg DM), n-alkane concentration (mg/kg DM) of diet components consumed by the animals in the experiment and % of incorporation in the diet of vegetation types (herbaceous and shrub). June
Organic matter CP NDF ADF ADL TP TT C25 C26 C27 C28 C29 C30 C31 C32 C33 Total Total odd-chain (%) % in the diet
July
Effects
Herbaceous
Shrub
Herbaceous
Shrub
Period (P)
VC
PxVC
919.2 89.9 625.5 368.1 59.7 – – 14.1 2.3 31.3 7.3 196.4 9.0 224.7 8.5 57.8 551.5 95.1 65.8a
930.6 89.7 613.9 455.6 172.4 13.5 11.9 19.3 2.6 41.9 7.4 261.6 7.5 128.0 5.5 49.1 522.9 95.6 34.2b
905.8 77.0 706.9 427.5 72.0 – – 8.0 1.8 21.4 6.9 133.0 6.6 184.0 5.3 55.8 422.8 95.1 98.0a
924.3 85.3 599.9 429.5 172.9 14.6 13.1 10.1 2.1 91.0 8.7 158.2 5.5 106.8 6.6 66.8 455.9 95.0 2.0b
0.113 0.179 0.149 0.071 0.271 0.705 0.667 0.035 0.266 0.008 0.715 0.034 0.018 0.355 0.586 0.588 0.112 0.815 –
0.022 0.515 0.017 o 0.001 o 0.001 – – 0.331 0.644 o 0.001 0.512 0.292 0.129 0.021 0.656 0.970 0.912 0.968 o 0.001
0.556 0.494 0.048 o 0.001 0.311 – – 0.616 0.917 o 0.001 0.552 0.531 0.968 0.829 0.365 0.585 0.742 0.732 –
VC vegetation component; CP crude protein; NDF Neutral detergent fibre; ADF acid detergent fibre; ADL acid detergent lignin; TP total phenolics; TT total tannins. a b
Values within a row in the same period (June or July) with different superscripts differ significantly at Po 0.05. Values within a row in the same period (June or July) with different superscripts differ significantly at P o 0.05.
M. Couto et al. / Livestock Science 186 (2016) 16–21
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Table 2 Body condition score (BCS) of Jennies in the beginning (BCS1) and at the end (BCS2) of the study.
Fig. 1. Diurnal variation in search/prehension activities observed in June.
experimental periods, with values of 13.5 and 11.9 g tannic acid equivalents/kg DM, and of 14.6 and 13.1 g tannic acid equivalents/ kg DM, in periods 1 and 2 respectively.
Animal
BCS1
BCS2
A B C D E F G H Average Effect
3.0 3.5 3.5 3.0 3.0 3.0 3.5 3.0 3.2 0.424
3.0 3.5 3.0 3.0 3.0 2.5 3.5 3.0 3.1
5.2. Grazing behaviour Results showed no differences in the feeding behaviour patterns between the two experimental periods (Figs. 1 and 2). Animals spent 75.6% of total observation time in search/prehension activities corresponding to an average of 16 h/day. It seemed that, independently of the period (i.e. June or July), search/prehension activities were uniform across the day without clear resting moments. 5.3. Diet composition The alkane profile of the plant species is presented in Table 1. Concentrations of C22 and C34 are not shown as they are used as internal standards in GC analyses. C29 in herbaceous vegetation, and C31 in the shrubs where the dominant alkanes. Percentages of the main vegetation components selected by animals are also shown in Table 1. Significant differences were observed in the selection of herbaceous and shrub species in both periods (P o0.001), with the herbaceous vegetation representing the major diet component in both periods. A marked increase (P o0.001) of grass uptake was found in the second period, increasing from 65.8 to 98.0% of total ingestion. Animals showed an incorporation of about 30% of shrubs (Quercus spp.) in their diet in June, whereas this incorporation decreased (Po0.05) to 2% in July (Table 1).
Fig. 3. Variation of EPG, positive donkeys for EPG and percentage of grass and shrub intake in June and July.
5.4. Body condition score and gastrointestinal parasites The BCS data (Table 2) show that the Jennies presented similar mean values of 3.2 and 3.1, for the beginning and end of the study, respectively. Concerning parasitological tests, parasite level increased from 0% positive samples in June (average 0 EPG) to a maximum of 25%
Fig. 4. Variation of total L3/culture, positive donkeys for L3 and percentage of grass and shrub intake in June and July.
in July (average 88 EPG) (Fig. 3). Faecal cultures were positive for 12.5, 62.5 and 25% of the animals, with average total counts of 0.25, 154 and 3.63 L3, respectively in June, beginning and mid-July
Fig. 2. Diurnal variation in search/prehension activities observed in July.
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(Fig. 4). The EPG increase was not significant (P ¼0.3), as well as, the increase followed by a decrease of the total number of L3 (P ¼0.052). All positive cultures (33% of total) showed the presence of cyathostomines in the faecal culture, with predominance of Cyathostomum sensu latum morphotype A (83.33% of the total L3). The correlation between EPG/total L3 counts and percentage of ingested grass and brush was not significant (P 40.05), although some associations were negative (r ¼ 0.5 and r ¼ 0.2, respectively), mainly in July, when the increase of grass and TT concentration was followed by a decrease of L3 in faecal cultures.
6. Discussion 6.1. Chemical composition of grass and shrub vegetation Differences in chemical composition between diet components on each experimental period are consistent with the advancing growing season of plants in both herbaceous and shrub vegetation, with a gradual decrease of CP and increase of NDF. The CP values for mountain pastures in Portugal in a previous work (Moreira et al., 2001) conducted in the same type of natural pasture varied between 8 and 20%. TP and TT contents were low compared to the results obtained by Estiarte et al. (2007) for shrubs of the same type. These authors found average condensed tannin concentrations of 2.8% in Quercus ilex and Q. cerrioides. 6.2. Grazing behaviour and diet composition The amount of time animals spent in search/prehension activities in our study was similar to that observed by Fleurance et al. (2001) and Edouard et al. (2009) in equines grazing on different pasture conditions. Results showed that search/prehension activities were uniform across the day without clear resting moments in either observation periods, suggesting that the weather conditions (in this case heat) did not influenced their grazing behaviour. In fact, Osoro et al. (2012) refers that equids grazing behaviour is less affected by weather conditions when compared to ruminant species. Previous studies (Ferreira et al., 2013) showed that mares concentrated their diurnal grazing time at the beginning and the end of the days, which was not observed in our study. However, the experiment site had areas of pasture and shrub with shadow, which probably allowed grazing in times of greater heat. It was observed, throughout the whole study but with a higher incidence on the second observational period, the consumption of bark and woody plants which, according to Aganda and Tsopito (1998) may be associated with low feed availability. Other noticeable aspects of the observed grazing behaviour were that when herbage was very short and difficult to pull, Jennies would dug out the grass roots of with their forefeet, especially in the final phase of the second observation period. Differences observed in diet composition from June to July were not corroborated by the observational data. In fact, animals seemed to spend more time in searching/prehension activities in shrub areas in July while its incorporation in the diet was higher in June. This can be explained by the fact that although animals have occupied these shrub areas, they probably were in fact selecting the herbaceous material that grows mixed/under the shrubs. In fact, the Jennies impact on the plot under study was well observed after comparing the plot in the beginning and at the end of the study, with a clear decrease in vegetation availability. This ability of equines for grazing and their selective habits can be used as a fire control tool, allowing for a reduction in carbon emission with beneficial environmental and economic level.
6.3. Animal performance BCS is a useful indicator of donkey nutritional status and welfare. Although a certain level of subjectivity affects this method, the use of a single scale, practice and a high level of repeatability, will allow the standardization of the method (Smith and Wood, 2008). The average values for BCS measurements in our observation period show that the Jennies presented a three-point classification, which is considered optimal and apparently the study did not interfere with it. In fact, donkeys seem to be able to maintain their body condition in circumstances where other species do not (Muller et al., 2001). This can be due to lower of nutritional requirements, searching for high quality bites, greater tolerance to undesirable foods, increased consumption and passage of fibrous food (Muller et al., 2001). Intestinal parasitism is a common problem found in donkeys (Madeira de Carvalho et al., 2007). Animal nutrition can affect the development of parasitism, by improving the ability of the host to overcome the parasitism (natural resistance to parasitic disease), by increasing the ability of the host to manage the negative effects of parasitism (resilience), or by limiting the development of parasite population through the intake of anti-parasitic compounds (Coop and Kyriazakis, 2001). The use of selected bioactive plants, plants containing secondary metabolites that are considered for their beneficial effects on health rather than for their direct nutritional value, or exploring the possible ability to selfmedication of bioactive pastures, may have a positive influence on the control of animal parasitic disease (Hoste et al., 2006; Villalba 2009). In the present study, animals showed a decrease in the shrub consumption throughout the observation periods and this could be associated with the increase of EPG. In fact, the largest consumption of shrubs is in agreement with the lowest level of parasite infection. Juhnke et al. (2012) point out that animals “learned” about the anti-parasitic effects of condensed tannins and increased their preference for a tannin-rich feed, in a study conducted with lambs. On the other hand, the initial high intake of TT and the following increase of grass intake in July, could be associated with a resilient status, meaning that while EPG was increasing, animals did not show clinical signs. Although not significantly, it could have influenced too the low L3 yields in culture at the end of July and this could be explained by the negative correlations found for these factors. These findings suggest that this ancestral animal husbandry technique, allowing donkeys to select tannin and protein rich feeds, can enable a process of selfmedication in animals with a low parasite level. Parasite biodiversity in this study was similar to that observed by Sousa et al. (2012) and these results are consistent since 2007. Cyathostomins control is rather difficult due to their short prepatent period and resistance to anti-parasitic drugs, but in this study the increase of EPG was not relevant (only equids with EPG 200–500 should be dewormed) and L3 survival in laboratory conditions was low (only three L3/culture at the end of the trial). Cyathostomins prepatent period (Cpp) can be as short as 1.5–2 months (Madeira de Carvalho, 2001). According to previous parasite control data of this donkey population that occurred in January 2013, we can assume that Cpp was delayed around 6 months while the animals were in these natural mountain pastures, probably due to the high intake of TT. Therefore, taking into account the presence of phenolic and condensed tannins in shrubs, their potential as natural anti-parasitic compounds and the possibility of being part of the normal donkey diet, these preliminary results can be of the outmost importance. Taking into account results obtained in this preliminary study, using donkeys as a biological tool for maintaining these characteristic mountain pastures may have several benefits both the pastures as well as the animals. The next step would be to study
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rotational and rational grazing methods in order to achieve a maximization of the pasture throughout the year. This can allow keeping of livestock in free grazing, with high levels of animal welfare, decrease production cost with decreased the use of deworming and contribute to lower environmental impacts, as well as contributing to the maintenance of mountain pasture areas and controlling shrub encroachment.
7. Conclusions Results obtained in this study indicate that Miranda donkey breed Jennies, although showing a preference for herbaceous species, are able to incorporate in their diet up to 30% of shrubs. These results should be further explored, has these animals can be used as a biological tool for controlling and reducing shrub encroachment in mountain areas, prevents pasture degeneration and allowing for a reduction in carbon emissions. This ability can be seen has an alternative usage for this endangered donkey breed. The apparent 6 month delay of Cyathostomins prepatent period in our study eventually related to the high TT intake in June, should be, in our opinion further studied. This may highlight the need for a regular use of these natural pasture systems, aiming the integration of ancient agro-ecosystems with a natural and sustainable parasite control in this Miranda donkey population.
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García, R.R., Rodrigues, M.A.M., Osoro, K., 2013. Foraging behaviour of domestic herbivore species grazing on heathlands associated with improved pasture areas. Livest. Sci. 155, 373–383. Fleurance, G., Duncan, P., Mallevaud, B., 2001. Daily intake and the selection of feeding sites by horses in heterogeneous wet grasslands. Anim. Res. 50, 149–156. Garcia, R.R., Fraser, M.D., Celaya, R., Ferreira, L.M.M., Garcia, U., Osoro, K., 2013. Grazing land management and biodiversity in the Atlantic European heathlands:a review. Agroforest Syst. 87, 19–43. Hoste, H., Jackson, F., Athanasiadou, S., Thamsborg, S.M., Hoskin, S.O., 2006. The effects of tannin-rich plants on parasitic nematodes in ruminants. Trends Parasitol. 22, 253–261. Juhnke, J., Miller, J., Hall, J.O., Provenza, F.D., Villalba, J.J., 2012. Preference for condensed tannins by sheep in response to challenge infection with Haemonchus contortus. Vet. Parasitol. 188, 104–114. Madeira de Carvalho, L.M., 2001. Epidemiologia e controlo da estrongilidose em diferentes sistemas de produção equina em Portugal (Thesis Ph.D.). Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal. Madeira de Carvalho, L.M., Cernea, M.S., Martins, S., Sousa, S., Gersão, S., Cernea, L. C., 2008. Comparative study of cyathostomin horse infection in Portugal and Romania based in L3 subpopulations of Cyathostomum sensu latum. Rev. Sci. Parasitol. 9, 48–56. Makkar, H.P.S., Blummel, M., Borowy, N.K., Becker, K., 1993. Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. J. Sci. Food Agric. 61, 161–165. Mayes, R.W., Lamb, C.S., Colgrove, P.M., 1986. The use of dosed and herbage n-alkanes as markers for the determination of herbage intake. J. Agric. Sci. 107, 161–170. McDonald, P.R.A., Edwards, R., Morgan, C.A., Greenhalgh, J.F.D., 2002. Animal Nutrition, 6th ed. Prentice Hall, London, UK. Moreira, N., Aguiar, C., Pires, J.M., 2001. Lameiros e Outros Prados e Pastagens de Elevado Valor florístico-Pastagens de Montanha. Direcção Geral de Desenvolvimento Rural, Lisboa, Portugal. Oliván, M., Osoro, K., 1999. Effect of temperature on alkane extraction from faeces and herbage. J. Agric. Sci. 132, 305–311. Quaresma, M., Nóvoa, M., Monteiro, A., Almeida, J.M., Portas, M., 2005. A raça Asinina de Miranda. Rev. Port. De. Cienc. Vet. 100, 227–231. Roberts, F.H.S., O´Sullivan, P.J., 1950. Methods for egg counts and larval cultures for strongyles infesting the gastro-intestinal tract of cattle. Aust. J. Agr. Res. 1, 99–102. Robertson, J.B., Van Soest, P.J., 1981. The detergent system of analysis and its application to human foods. In: James, W.P.T., Theander, O. (Eds.), The Analysis of Dietary Fiber. Marcell Dekker, New York, USA, pp. 123–158. Sousa, S., Nóvoa, M., Paz Silva, A., Madeira de Carvalho, L., M., 2012. Parasite control in Miranda Donkeys as a way of keeping animal welfare, in: Saastamoinen. In: Fradinho, M., Santos, M.J., Miraglia, N., A.S. (Eds.), Forages and Grazing in Horse Nutrition. Wageningen Academic Publishers, Holanda, pp. 425–432. Smith, D., Wood, S., 2008. Donkey nutrition. In: Svendsen, E.D., Soulsby, L., Duncan, J., Emeritus, Hadroll, D. (Eds.), The Professional Handbook of the Donkey. Whittet Books, London, UK, pp. 10–27. Thienpont, D., Vanparijs, Rochette, F., 1986. Diagnóstico de las Helminthiasis por Médio del Examen Coprológico, 2nd ed. Janssen Research Foundation, Beerse, Belgium. Yoseph, S., Smith, D.G., Mengistu, A., Teklu, F., Firew, T., Betere, Y., 2005. Seasonal variation in the parasite burden and body condition of working donkeys in East Shewa and West Shewa regions of Ethiopia. Trop. Anim. Health Prod. 37, 35–45.
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Grazing behaviour of Miranda donkeys in a natural mountain pasture and parasitic level changes M. Couto a,b, A.S. Santos b,c,n, J. Laborda d, M. Nóvoa d, L.M. Ferreira e, L.M. Madeira de Carvalho a a
CIISA, FMV, Lisboa, Portugal Escola Universitária Vasco da Gama, Coimbra, Portugal c CITAB-UTAD, Vila Real, Portugal d Associação para o Estudo e Proteção do Gado Asinino, 5225–011 Atenor Miranda do Douro, Portugal e CECAV-Departamento de Zootecnia, Universidade de Trás-os-Montes e Alto Douro, PO Box 1013, 5001-801 Vila Real, Portugal. b
art ic l e i nf o
a b s t r a c t
Article history: Received 15 January 2015 Received in revised form 2 January 2016 Accepted 8 January 2016
The Miranda donkey (Equus asinus) has an important role in the maintenance of rural landscape and traditions in inland of Portugal. Breeding and keeping of these animals rely on mountain pasture areas, known for their characteristic botanical diversity. However, their grazing behaviour in these areas has not been described and this knowledge is important to set the best management practices that promote an efficient use of these particular feed resources that could be related with parasitic level of these animals. In this way, this study aimed to assess feeding behaviour, diet selection, and relate the total phenolic and tannin content of ingested plants with the parasite load in a group of Miranda donkeys under free ranging system in natural mountain pastures. For this purpose, eight adult non-pregnant Jennies from the Miranda donkey breed were managed under continuous grazing on a 1.6 ha natural mountain pasture from late May to late July. Body condition score (BCS) and grazing behaviour of the Jennies was assessed in two different occasions. Additionally, the level of gastrointestinal nematode infections of the herd was assessed throughout the study. Results showed that animals spent 75.6% of total observation time in search/prehension activities corresponding to an average of 16 h/day. Jennies showed a preference for herbaceous species, although they were able to incorporate in their diet up to 30% of shrubs, suggesting that they can be used as biological tool for controlling and reducing shrub encroachment in these mountain areas. Parasite level increased from 0% positive samples in June to a maximum of 25% in July. The increase of EPG may be related with the observed decrease in the shrub consumption throughout the observation period. Having in account the presence of phenolic and condensed tannins in shrubs, their potential as natural anti-parasitic compounds and the possibility of taking part in donkey diet, these preliminary results can be of the utmost importance regarding an integrated approach of Miranda donkey parasite control. & 2016 Elsevier B.V. All rights reserved.
Keywords: Donkeys Diet selection Gastrointestinal parasites Tannins Mountain pastures
1. Introduction The Miranda donkey is one of the two native Portuguese donkey breeds. These animals are an integrating part of the local communities of the Trás-os-Montes region in the inland north of Portugal, they play an important role in maintaining rural communities and sustainable farming practices, and are a part of the local genetic biodiversity, being a key player in rural development of these territories nowadays. The Miranda donkey is well-
n Corresponding author at: Escola Universitária Vasco da Gama, Coimbra, Portugal. E-mail address: anasofi[email protected] (A.S. Santos).
http://dx.doi.org/10.1016/j.livsci.2016.01.005 1871-1413/& 2016 Elsevier B.V. All rights reserved.
conformed and characterized by a “rustic” appearance. The main coat is dark brown with lighter shades on the sidewall and bottom surface of the trunk, white muzzle and around the eyes, with hirsutism accented in broadsides, face and edges of ears and extremities, and abundant manes. Their average withers height is 1.35 m (Quaresma et al., 2005). Depending the season and the feed availability, nutritional requirements of these animals will vary along the year and changes in body condition score (BCS) may occur if proper supplementation is not given (McDonalds et al., 2002). These animals are still mostly used as working animals, both in farming work and more recently in equestrian tourism. They are traditionally bred under extensive systems often together
M. Couto et al. / Livestock Science 186 (2016) 16–21
with cattle, where pasture supply is the basis of their feeding regimes. Grazing areas mostly used by the Miranda donkey are the traditional mountain pastures, commonly known as “lameiros”. “Lameiros” are semi-permanent natural mountain pastures characterized by a high local botanical diversity including a considerable amount of shrub areas. These semi-permanent natural mountain pastures have been mostly abandoned, due to exodus of rural population. However, recently a new concern has arose to maintain these areas as a part of an integrated, selfsufficient production system since they have an incalculable environmental and landscape value and their conservation is an important objective to the maintenance of high levels of biodiversity in mountain areas. The establishment and development of livestock systems in less favoured areas can provide an environmental and economic benefit, contributing to the improvement of living conditions of rural population (Ferreira et al., 2013, Garcia et al., 2013). Both the Miranda Donkey and the lameiros play a role in its agroecosystem. Parasitic infections, especially by gastrointestinal parasites, particularly nematodes, are common in donkeys and are considered one of the major animal health and welfare problems, having an influence on their body condition and daily work routine (Yoseph et al., 2005). Infections occur by the ingestion of freeliving third stage larvae (also called L3), often when grazing in pastures. There is an increasing evidence of nematode resistance to conventional anthelmintic products in equids and therefore, natural alternative and/or complementary antiparasitic treatments are being pursued (Cernea et al., 2010; Payne et al., 2013). Recent evidence (Villalba et al., 2006) suggests that animals can self-medicate, and if so medicinal plants on diet could provide an important tool for parasite control. The use of plant secondary metabolites may be an effective way to treat animals, as much as phenolic compounds such as condensed tannins can provide beneficial medicinal effects to herbivores as they act against infectious conditions, such as parasitism (Juhnke et al., 2012). The “Lameiros” complex and biodiverse botanical composition, may allow animals to select specific plants that may have an influence in what concerns their natural resistance to gastrointestinal parasites, namely on the donkeys parasite resistance. However, information concerning grazing behaviour of donkeys on these types of pastures is very scarce and knowledge on how animals interact and select this specific botanical diversity could present interesting results to both natural parasite control in donkeys, as well as to the management of these type of biodiverse mountain pastures. In this way, the objective of this study was to observe the feeding behaviour, assess diet selection and relate the total phenolic and tannin content of ingested plants with the parasite load in a group of Miranda donkeys under free ranging system in these natural pastures, during the summer grazing season.
2. Material and methods 2.1. Experimental site, animals and study design The study was carried out in 2013 on a single plot of 1.6 ha pasture located at 651 m above sea level in the northern inland of Portugal, at Atenor, Miranda do Douro (41°24′48.12′′N; 6°29′15.38′ ′O). The climate characteristic of the region has high Mediterranean influence. Mean annual rainfall was 554.7 mm, and rainfall was persistent until the end of May, leading to a high pasture availability in the beginning of June. Mean monthly temperature recorded at the site during the study months was 15.3 °C in June and 23.4 °C in July. The plot used in this study is characterized by vegetation dominated by a mosaic of pasture grass sub-divided by shrub. At the beginning of the study, the lameiro plot was
17
dominated by pasture with an average height of 50 cm, composed of several herbaceous species (grasses: Bromus sp., Dactylis glomerata, Lolium sp., Briza sp., Calamagrotis sp., Avena sp., Cynosurus sp., Agrostis sp., legumes: Lathyrus sp., Trifolium sp., Ononis sp., Anthylis sp., Vicia sp. and other non identified genera of herbaceous plants), and shrubs (Quercus sp. dominated: Quercus robur; Q. ilex; Q. pyrenaica, Q. faginea, and also Narrow-leafed ash: Fraxinus angustifolia, hawthorn: Crataegus monogyna, ulmus: Ulmus L., and gorse: Cytisus spp.). A total of 8 adult non-pregnant jennies from the Miranda donkey breed were managed under continuous grazing from late May to late July. Jennies’ BCS was assessed on a scale of one to five (one ¼poor, five¼obese) according to Smith and Wood (2008), at the beginning and end of the study. 2.2. Grazing behaviour and diet selection The study was distributed in two observational periods, period 1 carried out in June (1–4 June) and period 2 July (16–19 July) of 2013. These two close observation periods where chosen in order to allow the collection of data in two different situations: high and good quality pasture availability (period 1) and low quality and amount of pasture availability (period 2). Since climatic conditions in this area are characterize by a rapid temperature elevation together with an inexistence of rainfall in the summer months (from June to August), this leads to rapid changes in vegetation type and availability, and very often, pastures availability goes from high, to very low within a month time span. Each observational period (11 days) comprised a seven-day for adaptation of animals to the diet and experimental conditions; followed by four days of sample collection: faeces and diet components (herbaceous and shrub), and feeding behaviour observation: time spent grazing, by each animal, was assessed by recording the grazing activity every 15 mins during the light period of the day (from sunrise to sunset) on two consecutive days in two observation periods (2–3 June and 17–18 July). In the same periods, diet selected by each animal was estimated using the n-alkane markers (Dove and Mayes, 2006). For that, individual faecal samples were collected twice a day for four days in each observation period (i.e. 1–4 June and 16–19 July). Simultaneously, samples of the main herbaceous components found in the plot and shrub vegetation were collected. Since the plant species within the herbaceous vegetation and within the shrub vegetation presented resemblances in their alkane profile, only two diet components were considered: i.e. herbaceous and shrub. This procedure allowed us to discriminate these two distinct feed resources that was the main objective of this study. Diet composition was estimated for each animal using a nonnegative least-squares procedure in the “EatWhat?” software (Dove and Moore, 1995) which minimizes the discrepancies between the observed concentrations of each n-alkane (C25–C33) in the faeces and the estimated proportions of plant components in the diet. Alkane faecal concentrations of animals were not adjusted as previous results (Ferreira et al., 2007) showed that faecal recovery of these markers in equids is unaffected by carbon-chain length. 2.3. Parasitological evaluation All animals had been previously dewormed in January of 2013, by subcutaneous administration of ivermectin 2% (1 ml per 50 kg body weight). During the whole study periodic analysis of faecal samples was performed to control the parasitic load in the group between June and July, namely on the 2nd June, 2nd July, and on the 22nd July. Spot samples of donkey faeces were individually collected directly from the rectum to assess gastrointestinal
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nematode eggs excretion. The number of eggs per gram of faeces (EPG) or faecal egg count (FEC) was estimated using McMaster technique (Thienpont and Vanparijs, 1986; Madeira de Carvalho, 2001). Gastrointestinal nematode genera present in faeces were identified on the basis of infective third-stage larvae obtained by faecal cultures described by Roberts, O´Sullivan (1950), adapted by Madeira de Carvalho (2001) and Madeira de Carvalho et al. (2008), namely for the collection and identification of Cyathostomum sensu latum subpopulations.
3. Chemical analysis All samples (faeces and vegetation) were frozen ( 20 °C), dried (55 °C for 48 h) and then passed through a 1 mm sieve prior to analytical procedures. Chemical compositions of main vegetation components samples (herbaceous and shrub) were analysed following the AOAC procedures (Association of Official Analytical Chemists, 1990) for ash and CP (N Kjeldahl x 6.25); NDF, ADF and ADL were analysed by the methods of Robertson and Van Soest (1981). The total phenol (TP) and total tannin (TT) contents of the shrub were determined using the Folin- Ciocalteu assay in combination with polyvinyl-polypyrrolidone, using tannic acid (Merck, Damstadt, Germany) as the reference standard (Makkar, 1993). Alkane content of samples was analysed in duplicate according to the methods of Mayes et al. (1986), as modified by Oliván and Osoro (1999). The first stage involved a saponification of 0.5 g of faeces or 1.5 g of vegetation components for 14 h in 7 ml of 1 M KOH ethanolic. Afterwards, a hot extraction was performed with n-heptane at 65 °C (Oliván and Osoro, 1999). After the extraction, the sample was passed through a silica-gel (bed volume 5 ml), to separate the alkanes from pigments, sterols and alcohols. After the recovery proceeded with n-heptane, it was then re-dissolved in 500 μl of heptane for chromatographic analysis. Quantification of the alkanes was carried out by gas chromatography, using Perkin Helmer Clarus 580, equipped with flame ionisation detector (FID), an auto-sampler and a temperature-programmable, and by oncolumn injection on a 50-m column SGE of 0.220 mm internal
diameter. Helium was used as carrier gas at a constant flow of 2 ml min 1. Gradients of temperature were used for the injector (280 °C) and the column (170 °C for 4 min; 30 °C min 1 to 215 °C, 1 min at 215 °C, 6 °C min 1 to 300 °C, 4 min at 300 °C). The detector oven was maintained at 340 °C. Identification was performed by comparison of retention times of the samples components with the previously injected standard. Alkane concentrations were quantified relative to known amounts of the internal standards C22 and C34, added at the beginning of the extraction process. The use of two internal standards enabled the evaluation of the effectiveness of the extraction process and the correction of the peak areas for any discrimination detected during the solventextraction step.
4. Statistical analyses Comparison of diet composition (herbaceous vs shrub) in the two experimental periods was performed using one-way ANOVA (GenStat, 2011). Comparison of data (BCS, parasitic burden and tannins) in the two experimental periods was performed using non-parametric ANOVA through Kruskal–Wallis test with P o0.05 and association between parasitological data and ingested herbaceous and shrubs were analysed with non-parametric correlation Spearman test (GraphPad InStat, v.3.0.10., 2009).
5. Results 5.1. Chemical composition of grass and shrub vegetation Chemical composition of herbaceous and shrub vegetation collected is presented in Table 1. Cell walls components (NDF) represented the highest fraction in the two diet components, averaging 666.2 and 606.9 g/kg DM for herbaceous and shrub, respectively. In both periods, shrub showed high levels of ADF and ADL averaging 442.6 and 172.7 g/kg DM, respectively. TP and TT contents in shrub plants increased from the first to the second
Table 1 Chemical composition (g/kg DM), total phenol (TP) and total tannin (TT) (g tannic acid equivalents/kg DM), n-alkane concentration (mg/kg DM) of diet components consumed by the animals in the experiment and % of incorporation in the diet of vegetation types (herbaceous and shrub). June
Organic matter CP NDF ADF ADL TP TT C25 C26 C27 C28 C29 C30 C31 C32 C33 Total Total odd-chain (%) % in the diet
July
Effects
Herbaceous
Shrub
Herbaceous
Shrub
Period (P)
VC
PxVC
919.2 89.9 625.5 368.1 59.7 – – 14.1 2.3 31.3 7.3 196.4 9.0 224.7 8.5 57.8 551.5 95.1 65.8a
930.6 89.7 613.9 455.6 172.4 13.5 11.9 19.3 2.6 41.9 7.4 261.6 7.5 128.0 5.5 49.1 522.9 95.6 34.2b
905.8 77.0 706.9 427.5 72.0 – – 8.0 1.8 21.4 6.9 133.0 6.6 184.0 5.3 55.8 422.8 95.1 98.0a
924.3 85.3 599.9 429.5 172.9 14.6 13.1 10.1 2.1 91.0 8.7 158.2 5.5 106.8 6.6 66.8 455.9 95.0 2.0b
0.113 0.179 0.149 0.071 0.271 0.705 0.667 0.035 0.266 0.008 0.715 0.034 0.018 0.355 0.586 0.588 0.112 0.815 –
0.022 0.515 0.017 o 0.001 o 0.001 – – 0.331 0.644 o 0.001 0.512 0.292 0.129 0.021 0.656 0.970 0.912 0.968 o 0.001
0.556 0.494 0.048 o 0.001 0.311 – – 0.616 0.917 o 0.001 0.552 0.531 0.968 0.829 0.365 0.585 0.742 0.732 –
VC vegetation component; CP crude protein; NDF Neutral detergent fibre; ADF acid detergent fibre; ADL acid detergent lignin; TP total phenolics; TT total tannins. a b
Values within a row in the same period (June or July) with different superscripts differ significantly at Po 0.05. Values within a row in the same period (June or July) with different superscripts differ significantly at P o 0.05.
M. Couto et al. / Livestock Science 186 (2016) 16–21
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Table 2 Body condition score (BCS) of Jennies in the beginning (BCS1) and at the end (BCS2) of the study.
Fig. 1. Diurnal variation in search/prehension activities observed in June.
experimental periods, with values of 13.5 and 11.9 g tannic acid equivalents/kg DM, and of 14.6 and 13.1 g tannic acid equivalents/ kg DM, in periods 1 and 2 respectively.
Animal
BCS1
BCS2
A B C D E F G H Average Effect
3.0 3.5 3.5 3.0 3.0 3.0 3.5 3.0 3.2 0.424
3.0 3.5 3.0 3.0 3.0 2.5 3.5 3.0 3.1
5.2. Grazing behaviour Results showed no differences in the feeding behaviour patterns between the two experimental periods (Figs. 1 and 2). Animals spent 75.6% of total observation time in search/prehension activities corresponding to an average of 16 h/day. It seemed that, independently of the period (i.e. June or July), search/prehension activities were uniform across the day without clear resting moments. 5.3. Diet composition The alkane profile of the plant species is presented in Table 1. Concentrations of C22 and C34 are not shown as they are used as internal standards in GC analyses. C29 in herbaceous vegetation, and C31 in the shrubs where the dominant alkanes. Percentages of the main vegetation components selected by animals are also shown in Table 1. Significant differences were observed in the selection of herbaceous and shrub species in both periods (P o0.001), with the herbaceous vegetation representing the major diet component in both periods. A marked increase (P o0.001) of grass uptake was found in the second period, increasing from 65.8 to 98.0% of total ingestion. Animals showed an incorporation of about 30% of shrubs (Quercus spp.) in their diet in June, whereas this incorporation decreased (Po0.05) to 2% in July (Table 1).
Fig. 3. Variation of EPG, positive donkeys for EPG and percentage of grass and shrub intake in June and July.
5.4. Body condition score and gastrointestinal parasites The BCS data (Table 2) show that the Jennies presented similar mean values of 3.2 and 3.1, for the beginning and end of the study, respectively. Concerning parasitological tests, parasite level increased from 0% positive samples in June (average 0 EPG) to a maximum of 25%
Fig. 4. Variation of total L3/culture, positive donkeys for L3 and percentage of grass and shrub intake in June and July.
in July (average 88 EPG) (Fig. 3). Faecal cultures were positive for 12.5, 62.5 and 25% of the animals, with average total counts of 0.25, 154 and 3.63 L3, respectively in June, beginning and mid-July
Fig. 2. Diurnal variation in search/prehension activities observed in July.
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(Fig. 4). The EPG increase was not significant (P ¼0.3), as well as, the increase followed by a decrease of the total number of L3 (P ¼0.052). All positive cultures (33% of total) showed the presence of cyathostomines in the faecal culture, with predominance of Cyathostomum sensu latum morphotype A (83.33% of the total L3). The correlation between EPG/total L3 counts and percentage of ingested grass and brush was not significant (P 40.05), although some associations were negative (r ¼ 0.5 and r ¼ 0.2, respectively), mainly in July, when the increase of grass and TT concentration was followed by a decrease of L3 in faecal cultures.
6. Discussion 6.1. Chemical composition of grass and shrub vegetation Differences in chemical composition between diet components on each experimental period are consistent with the advancing growing season of plants in both herbaceous and shrub vegetation, with a gradual decrease of CP and increase of NDF. The CP values for mountain pastures in Portugal in a previous work (Moreira et al., 2001) conducted in the same type of natural pasture varied between 8 and 20%. TP and TT contents were low compared to the results obtained by Estiarte et al. (2007) for shrubs of the same type. These authors found average condensed tannin concentrations of 2.8% in Quercus ilex and Q. cerrioides. 6.2. Grazing behaviour and diet composition The amount of time animals spent in search/prehension activities in our study was similar to that observed by Fleurance et al. (2001) and Edouard et al. (2009) in equines grazing on different pasture conditions. Results showed that search/prehension activities were uniform across the day without clear resting moments in either observation periods, suggesting that the weather conditions (in this case heat) did not influenced their grazing behaviour. In fact, Osoro et al. (2012) refers that equids grazing behaviour is less affected by weather conditions when compared to ruminant species. Previous studies (Ferreira et al., 2013) showed that mares concentrated their diurnal grazing time at the beginning and the end of the days, which was not observed in our study. However, the experiment site had areas of pasture and shrub with shadow, which probably allowed grazing in times of greater heat. It was observed, throughout the whole study but with a higher incidence on the second observational period, the consumption of bark and woody plants which, according to Aganda and Tsopito (1998) may be associated with low feed availability. Other noticeable aspects of the observed grazing behaviour were that when herbage was very short and difficult to pull, Jennies would dug out the grass roots of with their forefeet, especially in the final phase of the second observation period. Differences observed in diet composition from June to July were not corroborated by the observational data. In fact, animals seemed to spend more time in searching/prehension activities in shrub areas in July while its incorporation in the diet was higher in June. This can be explained by the fact that although animals have occupied these shrub areas, they probably were in fact selecting the herbaceous material that grows mixed/under the shrubs. In fact, the Jennies impact on the plot under study was well observed after comparing the plot in the beginning and at the end of the study, with a clear decrease in vegetation availability. This ability of equines for grazing and their selective habits can be used as a fire control tool, allowing for a reduction in carbon emission with beneficial environmental and economic level.
6.3. Animal performance BCS is a useful indicator of donkey nutritional status and welfare. Although a certain level of subjectivity affects this method, the use of a single scale, practice and a high level of repeatability, will allow the standardization of the method (Smith and Wood, 2008). The average values for BCS measurements in our observation period show that the Jennies presented a three-point classification, which is considered optimal and apparently the study did not interfere with it. In fact, donkeys seem to be able to maintain their body condition in circumstances where other species do not (Muller et al., 2001). This can be due to lower of nutritional requirements, searching for high quality bites, greater tolerance to undesirable foods, increased consumption and passage of fibrous food (Muller et al., 2001). Intestinal parasitism is a common problem found in donkeys (Madeira de Carvalho et al., 2007). Animal nutrition can affect the development of parasitism, by improving the ability of the host to overcome the parasitism (natural resistance to parasitic disease), by increasing the ability of the host to manage the negative effects of parasitism (resilience), or by limiting the development of parasite population through the intake of anti-parasitic compounds (Coop and Kyriazakis, 2001). The use of selected bioactive plants, plants containing secondary metabolites that are considered for their beneficial effects on health rather than for their direct nutritional value, or exploring the possible ability to selfmedication of bioactive pastures, may have a positive influence on the control of animal parasitic disease (Hoste et al., 2006; Villalba 2009). In the present study, animals showed a decrease in the shrub consumption throughout the observation periods and this could be associated with the increase of EPG. In fact, the largest consumption of shrubs is in agreement with the lowest level of parasite infection. Juhnke et al. (2012) point out that animals “learned” about the anti-parasitic effects of condensed tannins and increased their preference for a tannin-rich feed, in a study conducted with lambs. On the other hand, the initial high intake of TT and the following increase of grass intake in July, could be associated with a resilient status, meaning that while EPG was increasing, animals did not show clinical signs. Although not significantly, it could have influenced too the low L3 yields in culture at the end of July and this could be explained by the negative correlations found for these factors. These findings suggest that this ancestral animal husbandry technique, allowing donkeys to select tannin and protein rich feeds, can enable a process of selfmedication in animals with a low parasite level. Parasite biodiversity in this study was similar to that observed by Sousa et al. (2012) and these results are consistent since 2007. Cyathostomins control is rather difficult due to their short prepatent period and resistance to anti-parasitic drugs, but in this study the increase of EPG was not relevant (only equids with EPG 200–500 should be dewormed) and L3 survival in laboratory conditions was low (only three L3/culture at the end of the trial). Cyathostomins prepatent period (Cpp) can be as short as 1.5–2 months (Madeira de Carvalho, 2001). According to previous parasite control data of this donkey population that occurred in January 2013, we can assume that Cpp was delayed around 6 months while the animals were in these natural mountain pastures, probably due to the high intake of TT. Therefore, taking into account the presence of phenolic and condensed tannins in shrubs, their potential as natural anti-parasitic compounds and the possibility of being part of the normal donkey diet, these preliminary results can be of the outmost importance. Taking into account results obtained in this preliminary study, using donkeys as a biological tool for maintaining these characteristic mountain pastures may have several benefits both the pastures as well as the animals. The next step would be to study
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rotational and rational grazing methods in order to achieve a maximization of the pasture throughout the year. This can allow keeping of livestock in free grazing, with high levels of animal welfare, decrease production cost with decreased the use of deworming and contribute to lower environmental impacts, as well as contributing to the maintenance of mountain pasture areas and controlling shrub encroachment.
7. Conclusions Results obtained in this study indicate that Miranda donkey breed Jennies, although showing a preference for herbaceous species, are able to incorporate in their diet up to 30% of shrubs. These results should be further explored, has these animals can be used as a biological tool for controlling and reducing shrub encroachment in mountain areas, prevents pasture degeneration and allowing for a reduction in carbon emissions. This ability can be seen has an alternative usage for this endangered donkey breed. The apparent 6 month delay of Cyathostomins prepatent period in our study eventually related to the high TT intake in June, should be, in our opinion further studied. This may highlight the need for a regular use of these natural pasture systems, aiming the integration of ancient agro-ecosystems with a natural and sustainable parasite control in this Miranda donkey population.
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