DOI: 10.21451/1984-3143-AR2017-0035 Anim. Reprod., v.15, n.4, p.1214-1222, Oct./Dec. 2018 Morphological characteristics of mule conceptuses during early development Nathia Nathaly Rigoglio1, £, Gustavo de Sá Schiavo Matias1, Maria Angelica Miglino1, Andrea Maria Mess1, Julio Cesar Ferraz Jacob2, Lawrence Charles Smith3 1Department of Surgery, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Butanta, Sao Paulo, Brazil. 2Department of Reproduction and Animal Evaluation, Federal Rural University of Rio de Janeiro, Seropedica, Rio de Janeiro, Brazil. 3Department of Veterinary Biomedicine, Centre de recherche en reproduction et fertilité, Faculty of Veterinary Medicine, University of Montreal, QC J2S 2M2, Saint-Hyacinthe, QC, Canada. Abstract Hochholdinger and Hoecker, 2007; Chen, 2010a). Several investigations of the effects of heterosis have Hybrids between species are often infertile and been performed in the rabbit, pig and cattle (Long, extremely rare among mammals. Mules, i.e. crossing 1980; Blasco et al., 1993; Galvin et al., 1993; Newman between the horse and the donkey, on the other hand are et al., 1993). In addition, studies on hybrids allows for very common in agricultural and leisure practices due to investigations onthe specific role of genes in their enhanced post-natal physical characteristics that is developmental attributes (Goodwin, 2007). For instance, believed to occur for outbreeding or hybrid vigor. Since hybridization studies have revealed how epigenetic no reports are availableon the effects of hybrid vigor variation (Jirtle and Skinner, 2007; Simmons, 2008; during early development, this study focused on Skinner et al., 2010; Guerrero and Skinner, 2012; characterizing the intrauterine development of mule Groszmann et al., 2013) contributes to a better conceptuses during critical embryo-to-fetus transition understanding of the molecular mechanisms of complex period. Nine embryos and fetuses of early gestation, traits associated with hybrid vigor (Cubas et al., 1999; obtained after artificial insemination and transcervical Manning et al., 2006; Shindo et al., 2006; Ni et al., flushing, were evaluated by means of gross anatomy 2009; He et al., 2010). In domestic animal and plants, and histology and compared to data available for the hybrid vigor seems to minimize inbreeding and to equine. We found that some events, such as C-shape enhance breeding outcome (Gray, 1972; Anderson, turning, apearence of branchial archs, limb and tail 1988). buds, formation of primary and secondary brain Hybrids between true species are rare, vesicles, heart compartmentalization, and development especially among mammals. Nonetheless, the mixing of of somites, occurred slightly earlier in the mule. species has always fascinated humans and inspired Nonetheless, no major differences were observed in imaginations of creatures such as sphinx and centaurs in other developmental features, suggesting similarities mythology. However, due to natural barriers and between the mule and the horse development. In isolating mechanisms, interspecies mating is successful conclusion, these data suggest that the effect of hybrid only in a few closely related mammalian species vigor is present during intrauterine development in the (McGovern, 1976). Examples that are able to crossbreed mule, at least with regard to its maternal parent. are known for felids, ursids and bovides as well as several species of equides. Hybrids between the horse Keywords: embryology, development, mule. Equus caballus and the donkey Equus asinus are common, resulting in the mule as a hybrid of a female Introduction horse and a male donkey and the hinny as the reciprocal crossbreed (Allen and Short, 1997). They are usually Hybrids as crossbreeds of strains, varieties, or infertile, mainly because of chromosome species occur in both plants and animals. The effect of inconsistencies, i.e. the horse possesses 64, the donkey hybridization on an organism is variable; in some 62 and the hybrids 63 chromosomes that generally instances the offspring is similar to one parent or it may inhibit proper pairing during meiosis (Camillo et al., be intermediate between the parental traits. In addition, 2003). Besides this, mules are commonly used in hybrid offspring may have characteristics that are agriculture and other purposes. In particular, mules regarded to be superior or inferior in comparison to the often demonstrate hybrid vigour in physical traits of one or both parents. Offspring superiority is characteristics (Travis, 1990). They inherit a dense regarded as outbreeding enhancements, heterosis or musculature from their donkey farther and tend to be hybrid vigor. Briefly, hybrid vigor is a fundamental stronger for carrying more weight and have better biological phenomenon characterized by superior endurance than a horse but are associated with the performance of the hybrid offspring as heterozygote steadfast temperament and surefootedness of their between the two lines over its parents that are donkey mother. They are usually tall, relatively slow, representing the homozygote conditions. It may increase but without severe leg problems (Travis, 1990). The the biomass, stature, growth rate, fertility and/or fitness mules' performance in cognition tests is significantly in a population (Birchler et al., 2003, 2010; better than that of the parent species (Proops et al., _________________________________________ £Corresponding author: [email protected] Received: October 16, 2017 Copyright © The Author(s). Published by CBRA. Accepted: September 24, 2018 This is an Open Access article under the Creative Commons Attribution License (CC BY 4.0 license) Rigoglio et al. Mule growth during the embryo-to-fetus transition. 2009). Appart from a study based on ultrasonography each. The sections were diaphanized in xylene for 1h (Paolucci et al., 2012), little is known about the and embedded in paraffin (HistosecV-MERCK, Sao morphological effects of hybrid vigor during the critical Paulo, Brazil). The paraffin blocks were sectioned at phase of intrauterine development. Therefore, our 5µm in an automatic microtome (Leica, RM2165, objective here was to obtain accurate information of the Nussloch, Germany), mounted on histological slides, gross anatomy and histology of mule conceptuses and and incubated at 60°C. The sections were then compare these to developmental patterns observed in deparaffinized and stained with hematoxylin and eosin previous equines (Franciolli et al., 2011; Rodrigues et (H&E) . al., 2014). So far, no comparable data are available on donkey early ontogeny. Results Material and Methods Extra-embryonic membranes Animals At 17 days of gestation, the conceptus was oval in shape, loosely attached to the maternal system and All procedures using live animals were approved by an had a large yolk sac. At 33 days, the large yolk sac had animal ethical committee from Scholl of Veterinary an extended bilaminar omphalopleura, but also Medicine and Animal Science at University of Sao possessed blood vessels forming a trilaminar Paulo (CEUA/FMVZ-USP) (protocol n°2573/2012). omphalopleura. The chorioallantoic membrane was The mule conceptuses were collected in the Faculty of small, but well vascularized (Fig. 1A). At day 40, the Animal Science and Food Engineering, University of yolk sac was smaller with bi- and trilaminar São Paulo (FZEA-USP) and the Universidade Federal omphalopleura. The chorioallantoic membrane was Rural do Rio de Janeiro (UFRRJ - Seropédica). Using 1 relatively expanded (Fig. 1B). In the fetus of 51 days, to 2 liters of Ringer's lactate solution at 37°C (Camillo the yolk sac was even more regressed (Fig. 1C). At 63 et al., 2010), eight embryos and fetuses were collected days, a yolk sac lumen was absent and the tissue was by transcervical uterine flushing from mares at days 17 found only around the umbilical cord (Fig. 1D). The to 63 after insemination (Tab. 1). Samples were fixed in chorioallantoic membrane was continuously expanded 4% paraformaldehyde and crown-rump length (Fig. 1C,D). The amnion surrounded the fetus and measurements of the conceptuses were obtained after enlarged continuously during gestation (Fig. 1A-C). the dissection of the extra-embryonic membranes. The extra-embryonic membranes of mule conceptuses at 33, External features of the conceptuses 40, 51 and 63 days of gestation were analyzed macroscopically, as well as the external characteristics of Until day 33, the skin was translucent and then embryos at 17, 25 and 33 days of gestation and fetuses at became opaque (Fig. 2A-C). Optic vesicles were 45 and 63 days of gestation. Macroscopic appearance of apparent at day 25 (Fig. 2B) and became pigmented the brain, lung, kidney and fetal gonads was obtained from day 33 onwards (Fig. 2C-E). Forelimb and tail from the fetus at 63 days of gestation using a buds were present early in development whereas the stereomicroscope (Stemi DV4, Zeiss, USA). Results were hind limb buds were first observed only at day 25 (Fig. photographed with a Nikon Coolpix P510 digital camera. 2A and B). From day 33 onwards a forelimb and hind limb could be clearly identified, and the tail at day 45 Table 1. Crown-rump measurements according to the (Fig. 2B-D). Somites were present until day 25 (Fig. gestational age of embryos and fetuses. The crowm rump 2A-C). At the 45th day, external ears were present and of these embryos/fetuses increases as they develop. neck and nostrils could be observed (Fig. 2D). On day AGE (days) Crown-rump (cm) 63, lips, eyelids,