Anthelminthic resistance, especially to macrocyclic lactones, is increasingly common in cattle and equine herds globally. Benzimidazoles are highly effective in most cattle herds (Suarez and Cristel, 2007, Demeler et al., 2010) and, although resistance is often reported in horses, resistance to this class is in the early stage in some countries, such as India, and monitoring is therefore necessary (Kumar et al., 2016).

The diagnosis of resistance is necessary, for which in vitro and in vivo methods can be used. In vivo methods are more expensive, require a larger number of animals and exert an influence on the animal's metabolism, whereas in vitro methods can identify resistance earlier using fewer animals and without interference from the animal (Coles et al., 2006).

The in vitro egg hatch test (EHT) is one way to monitor resistance to benzimidazoles (Coles et al., 2006). Although this is one of the best validated in vitro methods for the investigation of benzimidazoles, a major limitation is that the EHT must be performed within three hours after the collection of feces (Coles et al., 2006), as drug sensitivity is reduced after the onset of the embryonic development in the egg and the test becomes inaccurate (Le Jambre, 1976). In large countries, such as Australia, the United States and Brazil, ranches are located long distances from laboratories, which could be an obstacle to the routine use of this diagnostic test.

The eggs of parasites can remain viable and intact in well-stored fecal samples, impeding larval development within the egg. Anaerobiosis and temperature are two variables of fundamental importance to the adequate storage of fecal samples. Samples maintained under low O2 tension and at temperatures lower than 10ºC are viable for a longer period of time and can be used to determine the egg count per gram of feces. The influence of these variables on the development of helminth eggs is often considered to maintain the viability of samples for stool tests (Ciordia and Bizzell, 1963, Sengupta et al., 2016).

Anaerobiosis and refrigeration have been investigated in egg hatch tests, but with eggs of nematodes of sheep and only hatchability was assessed (Smith-Bujis and Borgsteede, 1986), with no investigation of the effect on sensitivity to benzimidazoles. Sensitivity to benzimidazoles has only been investigated qualitatively in the eggs of gastrointestinal nematodes of sheep and equines that were stored at room temperature and under anaerobiosis with sucrose (Whitlock et al., 1980). Sheep feces can be stored in an anaerobic medium for up to seven days (Hunt and Taylor, 1989). However, the storage of feces at room temperature can exert an influence on the result of the test due to the fermentation of the medium. Therefore, the storage of sheep feces under refrigeration for up to 12 days is indicated (Calvete et al., 2014). No previous studies have investigated the influence of temperature and anaerobiosis on the eggs of bovine and equine nematodes in the egg hatch test. Coles et al. (2006) indicate the storage of feces in bottles under anaerobiosis only for equines and small ruminants. Despite this indication, there is no validated protocol regarding storage time.

Therefore, the aim of the present study was to investigate the effect of temperature, anaerobiosis and feces conservation time on the hatchability and possible reduction in drug sensitivity due to the onset of embryonic development in eggs of gastrointestinal nematodes of bovines and equines.