Effect of Vegetable Oils Feed Additives on Endoparasites Associated with Dewormed Racing Horses

Endoparasites Associated with Dewormed Abstract: The effectiveness of commonly used parasiticides decreases due to the drug resistance developed by many organisms. Therefore, the application of feed additives possessing antiparasitic properties may be helpful in limiting the burden of parasites. The aim of this research was to evaluate parasitological coefﬁcients for endoparasites of regularly dewormed Arabian horses and Thoroughbreds, fed on a basal diet of oat and muesli with vegetable oils and/or without vitamin E. The observations revealed parasitic invasion in 25 of 27 examined horses. The most frequent in both breeds were Strongylidae—accounting for 86% of Thoroughbreds and 100% of Arabian horses. Strongyloididae were observed in 33% of individuals representing both breeds while botﬂies were found in one Arabian horse and one Thoroughbred. Mean EPG coefﬁcients for strongylids and strongyloidids were higher in Thoroughbreds (530 vs. 529 and 43 vs. 29, respectively), although the differences were not statistically signiﬁcant. The addition of pure linseed oil signiﬁcantly reduced the number of Strongylidae in fecal samples. The results indicate that properly selected plant additives obtained from arable crops may be helpful in limiting the numbers of drug-resistant strongylids.


Introduction
Internal horse parasites are a considerable issue permanently affecting the health of animals. Endoparasitic infestations are most often subclinical and frequently are unnoticed by veterinarians, breeders and horse owners [1]. Despite increasing numbers of antiparasitic drugs, in many cases deworming is performed incorrectly and thus it turns out to be ineffective [2]. The problem of parasitosis in horses comes mostly down to routine deworming, usually twice a year, without prior coproscopic examination. This procedure only temporarily reduces the number of adult parasites susceptible to the drug used [3]. The latter, combined with a recurrent application of the same and/or close pharmacological agents, with ivermectin and praziquantel being most commonly used [4][5][6] leads to partial or complete resistance developed by many parasites, especially gastro-intestinal, which cannot be eradicated with these drugs anymore [7][8][9][10][11][12]. The parasites that have become or have been gradually becoming resistant to currently used preparations are representatives Linseed n = 7 (3 oo, 4 xx) 3 linseed + vitamin E n = 6 (2 oo, 4 xx) 4 n = 7 (3 oo, 4 xx)

Samples Collection
Fresh stool samples were collected individually from each animal in the early morning hours on 22 June 2020. Each sample was collected into sterile containers (15 mL) immediately after defecation. Then, they were cooled down to the temperature of 4 • C for the time of transport (15 min) and then analyzed.

Parasitological Analysis
Qualitative parasitological analysis was carried out using the flotation method of 2 g of homogenized fecal samples in a supersaturated NaCl solution, prepared 24 h prior to the analysis. The quantitative analysis was performed with McMaster's method, under a light microscope (Delta Optical ProteOne) in McMaster chambers [34].
Parasitic larvae and eggs of botflies were identified based on egg chorion as well as oral hooks and body spines shape and number with the use of keys by Draber-Mońko [35] and Li et al. [36]. Families of Nematoda were determined based on the shape and measurements of the eggs.

Data Analysis
The obtained results were analyzed in Statistica software (v. 13.3, StatSoft Inc., Tulsa, OK, USA). The normality of the data distribution was checked using Shapiro-Wilk's test, which proved the non-normality distribution of the results. In consequence, the differences were determined with the nonparametric Mann-Whitney's U-test and Kruskal-Wallis test. Correlations were calculated using Spearman's correlation test.

Results
Stool examination revealed that among 27 horses dewormed in 25 February 2020 were confirmed to host eggs and larvae of members of at least one family of parasites in June 2020. The most prevalent endoparasites of examined horses were Strongylidae Baird, 1856 (ova)-accounting for 86% cases of parasitism in Thoroughbreds and 100% in Arabian horses (Figures 1 and 2a). In 33% of horses from both breed representatives of Strongyloididae Chitwood and McIntosh, 1934 (ova) were also observed ( Figure 2b). Eggs and larvae of botflies from Gasterophilus cf pecorum (Fabricius, 1794) (Diptera: Oestridae) occurred in one Arabian horse and one Thoroughbred (Figure 3a,b). Moreover, in one Arabian horse, the presence of alive Paramecium sp. specimen was noted (Figure 3c). 1856 (ova)-accounting for 86% cases of parasitism in Thoroughbreds and 100% in Arabian horses (Figures 1 and 2a). In 33% of horses from both breed representatives of Strongyloididae Chitwood and McIntosh, 1934 (ova) were also observed ( Figure 2b). Eggs and larvae of botflies from Gasterophilus cf pecorum (Fabricius, 1794) (Diptera: Oestridae) occurred in one Arabian horse and one Thoroughbred (Figure 3a,b). Moreover, in one Arabian horse, the presence of alive Paramecium sp. specimen was noted (Figure 3c).  1856 (ova)-accounting for 86% cases of parasitism in Thoroughbreds and 100% in Ara-bian horses (Figures 1 and 2a). In 33% of horses from both breed representatives of Strongyloididae Chitwood and McIntosh, 1934 (ova) were also observed ( Figure 2b). Eggs and larvae of botflies from Gasterophilus cf pecorum (Fabricius, 1794) (Diptera: Oestridae) occurred in one Arabian horse and one Thoroughbred (Figure 3a,b). Moreover, in one Arabian horse, the presence of alive Paramecium sp. specimen was noted (Figure 3c).  The McMaster stool analysis showed that the mean eggs per gram (EPG) coefficient for Strongylidae was 529 in Arabian horses and 530 in Thoroughbreds, whereas for Strongyloididae it was 29 vs. 43, respectively. The differences were not statistically significant. The minimal and maximal observed number of eggs in stool samples in Arabian horses were 200-1850 (Strongylidae) and  while in Thoroughbreds, the scopes were 50-1850 (Strongylidae) and 50-300 (Strongyloididae) (Figure 4).
Parasitic co-invasions were also observed. In 42% of Arabian horses Strongylidae occurred along with other parasites, while in Thoroughbreds the share of parasitic cooccurrence was 38% of all cases of parasitism ( Figure 4). In addition, when analyzing the infestation data on particular individuals, it was noticed that in the case of co-invasions of Strongylidae + botflies, horse no. 1 (group 2) was characterized by less than half the number of strongyles compared to Oestridae, while in horse no. 20 (group 3), Strongylidae were almost three times more numerous than botflies.   Parasitic co-invasions were also observed. In 42% of Arabian horses Strongylidae occurred along with other parasites, while in Thoroughbreds the share of parasitic co-occurrence was 38% of all cases of parasitism ( Figure 4). In addition, when analyzing the infestation data on particular individuals, it was noticed that in the case of co-invasions of   Parasitic co-invasions were also observed. In 42% of Arabian horses Strongylidae occurred along with other parasites, while in Thoroughbreds the share of parasitic co-occurrence was 38% of all cases of parasitism ( Figure 4). In addition, when analyzing the infestation data on particular individuals, it was noticed that in the case of co-invasions of Statistical analysis of the difference of eggs/larvae of parasites in stool samples of Arabian horses and Thoroughbreds was conducted using Mann-Whitney's U-test. In this case, it appeared that the differences are statistically insignificant for alfa 0.05. This is manifested by the value of the p coefficient (Strongylidae p = 0.492; Strongyloididae p = 0.456; Oestridae p = 0.709), supported by the box whiskers graphs presented in the Figure 5. It, therefore, can be concluded that the horses of different breeds are not more susceptible to parasites.
Effects on feed additives on prevalence values of parasites in four experimental groups are presented in Figure 6. The second group, fed with the addition of pure linseed oil, was characterized by the lowest prevalence of Strongylidae (71% vs. 100% in other groups), absence of Strongyloididae (vs. 14%, 50% and 71% in groups 1, 3 and 4, respectively) and the lowest share of horses infested with botflies (14%) compared to group 3 (17%). Groups 1 and 4, in turn, were free from Gasterophilus sp.
Statistical analysis of the difference of eggs/larvae of parasites in stool samples of Arabian horses and Thoroughbreds was conducted using Mann-Whitney's U-test. In this case, it appeared that the differences are statistically insignificant for alfa 0.05. This is manifested by the value of the p coefficient (Strongylidae p = 0.492; Strongyloididae p = 0.456; Oestridae p = 0.709), supported by the box whiskers graphs presented in the Figure 5. It, therefore, can be concluded that the horses of different breeds are not more susceptible to parasites. Effects on feed additives on prevalence values of parasites in four experimental groups are presented in Figure 6. The second group, fed with the addition of pure linseed oil, was characterized by the lowest prevalence of Strongylidae (71% vs. 100% in other groups), absence of Strongyloididae (vs. 14%, 50% and 71% in groups 1, 3 and 4, respectively) and the lowest share of horses infested with botflies (14%) compared to group 3 (17%). Groups 1 and 4, in turn, were free from Gasterophilus sp.  Effects on feed additives on prevalence values of parasites in four experimental groups are presented in Figure 6. The second group, fed with the addition of pure linseed oil, was characterized by the lowest prevalence of Strongylidae (71% vs. 100% in other groups), absence of Strongyloididae (vs. 14%, 50% and 71% in groups 1, 3 and 4, respectively) and the lowest share of horses infested with botflies (14%) compared to group 3 (17%). Groups 1 and 4, in turn, were free from Gasterophilus sp.  The impact of feed additives on the EPG coefficient for experimental groups was determined using Kruskal-Wallis analysis of ranks. This test was used due to the nonnormality distribution of the results, as well as the necessity of comparing more than two groups. Considering the number of eggs/larvae in 1 g of feces, it was apparent that the most justified option was to verify the difference of Strongylidae in between the groups. The Oeastridae were detected only in 2 of 4 groups, so the difference between the groups was evident. A similar tendency is visible in Strongyloididae. According to Kruskal-Wallis analysis, the difference between the groups of Strongylidae is statistically significant (p = 0.0143). More precisely-at least one group was found to differ from the others with a significance level of 0.05. Closer analysis revealed that only the second group is different from the 1 group (Table 2). Closer inspection of the p coefficient revealed that, although differences of group 1 and 2 are significant, all other relations are close to Agriculture 2021, 11, 525 7 of 13 being significant in terms of statistics. Apart from the clear relation of groups 1 vs. 2, the next statistical correspondence was visible for groups 2 vs. 4 where p = 0.054, which is close to being considered as statistically valid. Subsequently, the differences of groups 2 vs. 3 (p = 0.097) represented less significant differences compared to the previously mentioned groups but was still worthy of consideration. For a greater number of horses or tests performed in a longer duration, the differences of group 2 from the others could appear statistically important. Therefore focusing the investigation on this group seemed to be justified. Moreover, it should be noted that apart from group 2, the other of the p values were equal to 1, which are safe to be considered as unimportant from a statistical perspective.  The impact of feed additives on the EPG coefficient for experimental groups was determined using Kruskal-Wallis analysis of ranks. This test was used due to the non-normality distribution of the results, as well as the necessity of comparing more than two groups. Considering the number of eggs/larvae in 1g of feces, it was apparent that the most justified option was to verify the difference of Strongylidae in between the groups. The Oeastridae were detected only in 2 of 4 groups, so the difference between the groups was evident. A similar tendency is visible in Strongyloididae. According to Kruskal-Wallis analysis, the difference between the groups of Strongylidae is statistically significant (p = 0.0143). More precisely-at least one group was found to differ from the others with a significance level of 0.05. Closer analysis revealed that only the second group is different from the 1 group (Table 2). Closer inspection of the p coefficient revealed that, although differences of group 1 and 2 are significant, all other relations are close to being significant in terms of statistics. Apart from the clear relation of groups 1 vs. 2, the next statistical correspondence was visible for groups 2 vs. 4 where p = 0.054, which is close to being considered as statistically valid. Subsequently, the differences of groups 2 vs. 3 (p = 0.097) represented less significant differences compared to the previously mentioned groups but was still worthy of consideration. For a greater number of horses or tests performed in a longer duration, the differences of group 2 from the others could appear statistically important. Therefore focusing the investigation on this group seemed to be justified. Moreover, it should be noted that apart from group 2, the other of the p values were equal to 1, which are safe to be considered as unimportant from a statistical perspective.  Table 2. Multiple comparisons between the groups (group 1-supplemented with soybean oil, group 2-supplemented with linseed oil, group 3-supplemented with linseed oil and vitamin E, group 4-control group) (R stands for the value of an average rank; a bolded values indicates a statistically significant difference for α = 0.05). The test of Kruskal-Wallis is essentially ANOVA for ranks. The ranks are shown in Figure 8. In general, the greater the sum of ranks, the greater number of pieces in gram of feces for a given group. The test of Kruskal-Wallis is essentially ANOVA for ranks. The ranks are shown in Figure 8. In general, the greater the sum of ranks, the greater number of pieces in gram of feces for a given group. The correlation between the types of parasite found in the stool was verified with the aid of Spearman correlation of ranks. This was done in order to verify if the occurrence of a parasite can be a reason to suspect the occurrence of the other types. However, the analysis revealed no significant correlation, which means that the parasites must be considered individually. The occurrence of one type does not mean the other type is also present. The results of the correlation are shown in Table 3.  The correlation between the types of parasite found in the stool was verified with the aid of Spearman correlation of ranks. This was done in order to verify if the occurrence of a parasite can be a reason to suspect the occurrence of the other types. However, the analysis revealed no significant correlation, which means that the parasites must be considered individually. The occurrence of one type does not mean the other type is also present. The results of the correlation are shown in Table 3.

Discussion
The occurrence of endoparasites in animals treated with parasiticide containing ivermectin is accordant with the results of Kyvsgaard et al. [37], who observed the reappearance of strongyle eggs within 60 days post deworming.
The research indicated that the factor of breed does not significantly affect the number of gastrointestinal parasites. A comparison between horse breeds and their strongylid burden was included in the research by Kuzmina et al. [38], who revealed that Thoroughbreds, Ukrainian Saddlers and Russian Racers had much higher EPG values compared to non-breed horses. Still, the latter cannot be fully compared to our results, because we only analyzed two breeds of horses.
Mean EPG coefficients for Strongylidae, despite not being significantly different between Thoroughbreds and Arabian horses (530 vs. 529) in this study, were lower than the mean values in riding horses examined in Ukraine [38]. Below, the listed horses represented groups closest to our study, i.e., a similar sample size, dewormed 1-2 a year, with macrocyclic lactones, including: Ukrainian Saddlers (EPG = 796.9; n = 8 (Donetsk), EPG = 680.8; n = 13 (Kharkov) and EPG = 889.6; n = 12 (Voronkovsky)) and Russian Racers (EPG = 583.3; n = 9). As for the Thoroughbreds, in the cited research, this breed underwent one or no deworming procedures per year and thus its mean EPG was considerably higher than the present-1428.1. Our results were higher than observed by Forteau et al. [39], in which case horses grazing separately were characterized by a mean EPG of 400, while when mixed with cattle, it was 230. The difference was attributed to the dilution effect. Jenkins et al. [40] revealed that EPG values for wild horses of Sable Islands, Canada, range from 669 to 1105, depending on the season.
Repeatable observations of relatively high amounts of strongyle ova in feces of regularly dewormed animals may result, in addition to the aforementioned cessation of biological activity of ivermectin, from the development of the drug-resistance in representatives of Strongylidae. The latter, in turn, may be an outcome of incorrect use of the named anthelmintics [10,12]. This assumption is partly supported by already cited results by Kuzmina et al. [38], who revealed that a statistically significant difference in EPG values was present only between horses with rare to lack of anthelmintic treatments but not between horses dewormed 1-2 a year vs. 3-4 times or more with macrocyclic lactones.
In both breeds, the most numerous were found for Strongylidae, which are the most common and geographically widespread equine endoparasites [41,42]. Our data on prevalence correspond with those by Ramey and Nielsen [43], wherein 78% of examined horses were infested by strongylids. Also, studies by Eslami et al. [44], Bulgaru and Tudor [45] as well as Ola-Fadunsin et al. [46] indicated Strongylidae as the most abundant group; however, the values of prevalence were lower-28.3%, 27.92% and 33.33%, respectively, vs. 78% (Thoroughbreds) and 100% (Arabian horses) in the present research.
Shares of parasitic co-invasions observed in this study (42% in Arabian horses and 38% in Thoroughbreds) were slightly lower than in horses and donkeys studied by Wannas et al. [47] in Iraq-50% and 71.42%, respectively. Lower shares of mixed invasions in equids were recorded by Sultan et al. [48] in Ethiopia, wherein only donkeys (8.41%) were simultaneously infested with Oestridae + Ascarididae (1 case) and, as in this study, botflies + Strongylidae (25 cases). There is a possibility that upon decreasing efficiency of the most use, broad-spectrum parasiticides, shares of mixed infection in horses may increase.
An atypical observation were eggs along with the first and the second stage larvae of G. pecorum (Figure 3a,b) at the total absence of the third stadium, which typically completes development in the rectum after which larvae exit the host and pupation takes place in the external environment [35]. Immature larvae were observed only in the feces of individuals fed with the addition of linseed oil-pure and mixed with vitamin E. A possible explanation of this situation was providing these horses with hay infested with G. pecorum ova. Oviposition on vegetation, especially grasses, is a peculiarity of this botfly species. Eggs retain the ability to infect equidae for more than 250 days and usually hatch in the horse's mouth after being soaked for 3-5 min of chewing [35,49,50]. On the other hand, in the case of excessive dehydration of the egg shell, which may have occurred during the drying and storage of hay, the larvae either lost the ability to hatch or managed to do it too late, i.e., already in the gastric contents. There they could not continue the regular life cycle, i.e., parasitize on the soft palate/esophagus and/or stomach and complete development in the rectum as the third stage [35,51]. The inability of G. pecorum to hatch from dried eggs was stated by Liu et al. [49]. Moreover, unfavorable conditions for continuing the development in the stomach combined with the administration of feed rich in mucosa-protective linseed oil and loose consistency of faeces, in individuals with immature larvae detected, may have prevented parasites from proper attachment and, as a result, contributed to their premature elimination from the digestive system of horses, along with the feces. The loose consistency of the stool was only observed in the morningafter training. Intensive efforts and prevailing weather conditions (high temperature and air humidity) caused a greater demand for water while reducing the consumption of solid feed by horses, which directly influenced the dilution of the feces and caused faster passage of the intestinal content. During the fortnight preceding stool sampling, 60-90% relative humidity, as well as a temperature minimum range of 14-19 • C and a maximum range of 18-32 • C were observed (data were obtained from the Institute of Meteorology and Water Management in Wrocław, Poland). The presence of ova in the samples was, in turn, an outcome of swallowing and excretion by animals completely dried and so inactive eggs. This explanation was also supported by the fact that G. pecorum has been listed in Poland [35,52]. Furthermore, the presence of other representatives of Gasterophilus spp., the oviposition site of which is horse hair and coat, besides above mentioned taxonomic traits of eggs and larvae, was excluded by the daily care of animals, including brushing and washing, as well as the lack of access to pastures where the infestation with other botflies could have been the easiest. In the research by Zhang et al. [53], it was revealed that the third stage larvae of G. pecorum are naturally excreted from the host for about 7 months a year. Two peaks of the mentioned process were also observed: peak I-from early April to early May and peak II-from mid-August to early September, even though G. pecorum is an univoltine species, i.e., it produces only one generation a year. During the first peak, larvae were characterized by lower survival and pupation rates along with a longer eclosion period than the parasites derived from the second peak. That could have been attributed to both overdrying of the egg shells and a long period of waiting for the proper host under unfavorable conditions, as in the present case.
Still, our data on the prevalence of Oestridae are considerably lower than presented by Tolliver et al. [54]-USA, Sweeney [55]-Ireland and Lyons et al. [56]-USA, wherein dewormed horses were infested with Gasterophilus larvae at the levels of 94%, 90% and 20%, respectively. The low prevalence of botflies revealed in this study corresponds, in turn, with observations by Sallé et al. [57] on sports horses in northern France, indicating a significant decrease in the numbers of these parasites in the digestive system of horses occurred in recent years. The observed differences may result from distinct methods of research (analysis of dissected digestive tracts vs. coproscopy) and, as already mentioned, the different extent of applied hygienic treatments between slaughter and sports horses. Also, as indicated by literature data, a few other factors affect infestation by Gasterophilus spp. in horses, among which are geographical location and climate [49], as well as changeable weather conditions (temperature, rainfall, RH) in a given locality [53,58]. Huang et al. [59] revealed 100% prevalence in studied equids (Equus przewalskii, E. hemionus, E. ferus caballus; n = 11) in northern China. These results are convergent with Liu et al. [60], who examined 90 ivermectin-dewormed horses. The prevalence of Gasterophilus larvae was 100%. Identical, 100% prevalence of G. pecorum was recorded in Iran by Moshaverinia et al. [61] in dissected animals. It is noteworthy to mention that this was the first observation of the species in this country. However, one should remember that in the era of climate change and the extensive transport of horses between countries and continents, in the future, more Oestridae species are very likely to be introduced in areas where they did not exist before.
In horse no. 1 we could observe an antagonistic interaction between co-occurring strongyles and botflies. This mechanism has already been described in humans [62]; however, the above presented results of statistical analysis excluded a supposition of any correlation between the presence of a different type of parasites. Another explanation may be an influence of pure linseed oil on parasites. Horse no. 1 was assigned to group number 2 (pure linseed oil), whereas number 20 was assigned to the third group (linseed oil + vitamin E). This confirms already revealed statistical differences between groupsin particular animals-representing these groups. Possible antiparasitic effects of pure, and/or mixed with Chenopodium, linseed oil were mentioned by Morcos [17] and Lyons et al. [18]; however, these mentions come from the 1950s and 1960s, i.e., before the invention of the contemporary parasiticides. However, present results seem to confirm the sense of these, already historical, attempts to control helminths with the use of natural substances. As stated in the beginning, even macrocyclic lactones are being slowly resisted by many parasites, so any additional, non-chemical, antiparasitic agents may be crucial in breeding animals in the future. This makes feed additives properties worth further detailed research.
Our study revealed no impact of the addition of vitamin E+linseed oil mixture on parasites. It is inconsistent with the only available data on tocopherol addition and helminth burden in animals, i.e., the work by De Wolf et al. [63] who investigated its effect on an experimental Haemonchus contortus (Trichostrongylidae) infestation in lambs. Authors found a positive influence of the D-α-tocopherol on the parasitic burden, which was 49% lower in lambs from the experimental group (n = 10), compared to the control (n = 10). The discrepancies in observations may be due to few factors, i.e., different species and age of animals, different parasites studied and an experimental (five weeks from the start of supplementation with the third stage of the larvae) vs. natural infection with helminths. Still, contradictory results and a lack of additional data on the subject point to the need for more studies.
Finally, an interesting side observation was the alive representative of Parameciidae (Figure 3c) in one sample; we did not find a similar case in the available literature.

Conclusions
The main conclusion arising from the results of the present paper is that the addition of pure linseed oil significantly reduces the burden of Strongylidae in examined horses. The statistical analysis revealed that only linseed oil and soybean represent valid differences, but considering the p values of multiple comparisons, and the fact that these studies are preliminary, further consideration of linseed oil appears to be justified. Therefore, the substance is worth analyzing further, including through tests on other groups of farm animals. The study also indicates that there is no impact of tocopherol addition on the parasitic burden in horses, which is in contradiction to, however, limited, literature data and thus should be studied in more detail. Moreover, among tested horse breeds, none are more susceptible to the parasitic infestation, regardless of the parasite's taxon, and the presence of particular taxa does not affect one another.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available for privacy reasons.