Yawning is a stereotyped behavioral pattern that is characterized by deep inspiration followed by short expiration across all vertebrate species (Barbizet, 1958; Argiolas and Melis, 1998; Collins and Eguibar, 2010). In mammals, including rats, yawning frequency is very low, approximately 1 yawn/h (Baenninger, 1997).

We selectively inbred for more than 85 generations two sublines of Sprague–Dawley (SD) rats: a high-yawning (HY) subline, with a mean of 20 yawns/h, which is one order of magnitude higher than the low-yawning (LY) subline, with a mean of 2 yawns/h (Urbá-Holmgren et al., 1990; Eguibar et al., 2015). We used as a control group and outbred SD rats that have a mean of 1 yawn/h. HY rats allow us to analyze the environmental influences on yawning behavior. Yawning frequency has a circadian rhythm with a peak before dusk (Anías et al., 1984). The yawning circadian rhythm is not an endogenous mechanism because it is not free running, and it can be synchronized by a restricted feeding period, with food availability being a stronger zeitgeber than a light–dark cycle (Holmgren et al., 1991). These sublines also differ in their responses to stress, as seen in the open-field arena, where HY rats are more active than LY rats, indicating that HY rats are less emotionally reactive than the latter (Moyaho et al., 1995).

The physiological role of yawning has not been clearly established until now, and there are several hypotheses about the role of this innate motor pattern in respiratory and circulatory roles. Several studies have demonstrated that yawning can change the respiratory rhythm, increasing heart frequency and blood oxygenation, as well as vasodilatation, but these variables are not triggering yawning. In conclusion, there is not a clear physiological association between respiration or circulation and yawning (Guggisberg et al., 2010). Another hypothesis about the physiological role of yawning is the role of drowsiness as an inducer of yawning and a concomitant increase in arousal levels as a method of global activation of brain activity from brain stem to cortical areas, but there is no convincing evidence of such an association (Guggisberg et al., 2010). A third hypothesis is the role of yawning in cooling down the brain temperature, the so-called thermoregulatory hypothesis (Gallup and Gallup, 2008). Based on the high yawning frequency of HY male rats, we can demonstrate that when a yawn happens, there is a decrease in the cornea and ear concha temperatures, two hairless facial structures, using thermographic analysis. In fact, 10 s after a yawn happens, there is a reduction in both facial areas that correlates with the reduction in the cortical temperature measured with implanted thermocouple probes in non-HY rats that then returns to basal levels within a short time period (Shoup-Knox et al., 2010; Eguibar et al., 2017b). In another study, we demonstrated in budgerigars that the beak temperature decreased when a yawn happened (Gallup et al., 2017). These experimental data indirectly support the thermoregulatory role of yawning, but it is necessary to obtain more empirical data to support the hypothesis. Finally, the blood gas (CO₂/O₂) hypothesis was rejected because breathing neither pure oxygen nor high CO₂ had a significant effect on yawning frequency, although both increased the breathing rate (Provine et al., 1987).

On the other hand, HY male rats allow us to show that this innate behavioral pattern is strongly correlated with spontaneous penile erections in a very short time window of only 3 min; in fact, 50% of yawns and penile erections happen together (Holmgren et al., 1985). Yawning frequency also correlated with the number of penile erections after systemic administration of D₂-like dopaminergic agonists such as apomorphine in low doses, bromocriptine, or (−)-quinpirole (Urbá-Holmgren et al., 1993; Eguibar et al., 2003); this behavioral correlation is present even in a myelin mutant rat with progressive demyelination (Eguibar et al., 2012).

Yawning behavior is regulated by several neurotransmitters, including cholinergic, muscarinic, or D₂-like dopaminergic agonists; it is inhibited by opioids and GABAergic mechanisms (for review, see Argiolas and Melis, 1998; Collins and Eguibar, 2010); and it is increased by the central administration of adrenocorticotrophic, α-melanocyte-stimulating hormone (MSH), oxytocin, and prolactin peptides and inhibited by bombesin (Argiolas and Melis, 1998; Díaz-Romero et al., 2002; Collins and Eguibar, 2010). Among all of these neurotransmitters and neuromodulators, the dopaminergic system, through D₂-like receptors, is the most potent inducer of yawning and penile erections, acting in the paraventricular nucleus (PVN) of the hypothalamus (Sanna et al., 2012), and the motor output is regulated by the striatum (Dourish and Cooper, 1990).

On the other hand, yawning is part of a behavioral syndrome induced by exposure to a stressor, with a strict temporal organization and an initial increase in alertness, grooming, yawning, and finally somnolence or even sleep (Delius, 1988). Therefore, grooming and yawning are two behaviors that reduce stress responses and have adaptive properties (Fentress, 1988). In the case of HY rats, they groomed more when exposed to a novel environment (Eguibar and Moyaho, 1997) or after wetting the fur (Moyaho et al., 1995), indicating that they had different coping strategies to confront a stressor. This approach is supported when HY rats are exposed to an open-field arena because they ambulate more and have a lower number of fecal boluses; therefore, they are less emotionally reactive (Moyaho et al., 1995). This finding is corroborated by a preliminary study in which HY rats explore more the open arms in the elevated-plus maze, supporting that they are less anxious (Eguibar et al., 2017a).

It is well established that maternal care plays a substantial role in physiological and behavioral stress responses in adulthood (Caldji et al., 2000). In this context, HY dams spent less time in the nest, retrieved their pups faster, and showed a longer latency to licking and mouthing the pups than LY dams. The HY dams also had atypical retrieving, and they built nests of less quality, supporting that they are motivated to take care of their pups, but the “fine tuning” of maternal care is different (Ugarte et al., 2011). In a previous study, we analyzed yawning frequencies in male and female rats after cross-fostering, but all subjects were raised in individual cages to increase their stress responses. Our results showed that sex ratio and littermate size influenced yawning frequency in adulthood (Moyaho et al., 2009), but no further analysis was performed until now.

Based on these data, the aim of this study was to first analyze the role of maternal care in spontaneous yawning and penile erections in male rats raised in conditions of in- and cross-fostering between the sublines or with SD dams. Second, we analyzed the maternal behavior during the initial phase of the lactation period in conditions of in- or cross- fostering between the sublines or with SD dams. Third, in another group of rats, fostered by different types of dams, we determined yawning, penile erection, grooming, and scratching frequencies after systemic administration of a specific D₂-like receptor agonist (−)-quinpirole, and finally in another group of rats, we measured the relative expression of D₂ receptors in the striatum using Western blot analysis.

Our results showed that the higher number of spontaneous and (−)-quinpirole-induced yawning bouts and penile erections in HY male rats is independent of the type of dam that raises them, supporting that the yawning frequency is specific to each subline. Therefore, genetic backgrounds are the main component due to strong inbreeding, and nurturing conditions during the lactation period did not play a significant role in determining yawning frequencies (Figure 2). A similar trend was obtained with LY and SD male rats, which had lower yawning frequencies independent of the type of dam that raised them, supporting that the genetic background is the main variable that determines yawning frequency.

In the case of the spontaneous number of penile erections, we clearly demonstrated that HY dams were able to significantly increase the number of penile erections in LY male rats. It is relevant that male rats subjected to in-fostering generally had almost double the number of penile erections with respect to their nonfostered counterparts (Figure 3), but these differences did not reach significant levels due to, at least in part, the higher variability and lower incidence of noninduced penile erections. It has been demonstrated that the amount of prepubescent self–anogenital grooming is greater in male rats than in their female counterparts, and this grooming component is androgen-dependent because castration reduced it in male rats and androgenization in female rats increased it (Moore, 1986a). The amount of anogenital grooming is also context dependent because living in isolation conditions significantly increased genital grooming (Moore, 1986b). In our conditions, all groups of rats lived in groups, with 3–4 Ss in acrylic cages, and tests were performed in transparent glass cylinders. Therefore, considering that all the conditions are equal, it was outstanding and unexpected that HY dams could increase the number of penile erections in the LY male rats (Figure 3C), supporting a strong maternal component in the expression of penile erections out of a sexual context.

It has been demonstrated that cross-fostering is a postnatal environmental manipulation that is capable of inducing behavioral and physiological changes in mice and rats (Bartolomucci et al., 2004). In fact, mother–pup interaction in rats occurs within the nest and consists of approaching the litter, licking and grooming the pups, and nursing them in different positions (Fleming et al., 1999). To control the effects of manipulation due to the fostering of the pups, we also used an in-fostering group of rats as an additional control of pup manipulation, but we obtained an increase in yawning frequencies and in penile erections in in-fostered male rats, supporting that dams are able to discern that they are different pups and change in some way the strategy in which they care for them, which is not reflected in the percentage of time spent in the different behavioral measurements. However, these changes are due to neonatal handling, as already demonstrated in the Roman lines (Fernández-Teruel et al., 1997). It is important to mention that Roman lines share various behavioral characteristics with our yawning sublines (Melis et al., 2019).

Our results also showed that arched-back and blanket-nursing postures differed between both sublines, as well as the time spent in self-grooming and outside the nest or even the time that HY dams approach the litter, supporting a different organization of maternal care in the HY subline with respect to the LY and SD dams, as previously demonstrated (Ugarte et al., 2011). Importantly, the general trend is maintained within each group of dams instead of the characteristic organization of maternal care in the different fostered groups evaluated in these experiments as HY dams that had a different organization of maternal care (Ugarte et al., 2011). These results clearly support that the genetic background between the sublines is quite stable because, as already mentioned, HY and LY rats were maintained by a strict inbreeding process for more than 85 generations (~35 years), which implies that the presence of different pups with different genetic pools is not able to change maternal care. A similar trend has been obtained with the naturally occurring variations in maternal licking/grooming of pups that were significantly correlated when the mother nursed in the arched-back position and persisted in the first and even second litters, supporting a possible epigenetic mechanism (Caldji et al., 2000). In future experiments, we could analyze the relationship of the amount of anogenital grooming displayed by the mother and the frequency of spontaneous and drug-induced penile erections in both sublines in the first and second generations to evaluate persistent effects due to maternal care.

In this context, it is relevant that Fleming et al. (1999) showed that there is an interaction between the newborn and the mother that is capable of altering the basic mechanisms of behavioral expression in both, including in neuroanatomical, neurochemical, and behavioral aspects (Fleming et al., 1999). If we consider the degree of interaction of the dams with their offspring and the ways that individuals respond later in life and then changing the response of hypothalamic–hypophysis–adrenal gland axis regulation and affiliative and social behaviors (Fleming et al., 1999), in which yawning and grooming play a central role (Collins and Eguibar, 2010; Melis et al., 2019). So, nurture had a strong influence on behavior, but it is not the case with yawning and penile erections in our experimental conditions. Because yawning is an emphatic behavior that is also related to communication, it is highly remarkable that maternal care did not influence the spontaneous expression of yawning, and in the case of HY male rats, they were noted as the rats that yawned more with respect to LY and SD rats independent of the subline or SD dams that raised them. Another possibility is that social interaction between the dams and their littermates can reverse the effects due to the quality and quantity of maternal care.

A similar trend was obtained with spontaneous penile erections, with HY rats showing more of this spinal reflex without any sexual context, neither motivational nor somesthetic. In future experiments, we will analyze the penile erections induced by an inaccessible estrous female, the so-called noncontact penile erections, or in the case of the retraction of the preputial skin as a mechanism to increase the frequency of penile erections in both sublines (Sachs et al., 1988; Sachs, 2000). As we already demonstrated in sexually experienced male HY rats, they showed a different copulatory pattern because they had more sexual bouts, with longer interintromission intervals that delayed ejaculation (Eguibar et al., 2016). It is quite relevant that HY dams are able to significantly increase the number of penile erections in the LY male rats, and this effect is probable due to an increase in the genital grooming given by HY dams because the number of penile erections in adulthood is strongly dependent on the genital grooming displayed in the first week of life (Moore, 1986a, b). This is the first report that shows an increase in the number of penile erections after a cross-fostering technique, which implies that early-life experiences have an important impact on the central and/or peripheral mechanisms that participate in penile erections. Another possible explanation is that maternal care is capable of changing oxytocinergic and dopaminergic neurotransmission and perhaps nitric oxide levels in key integrative areas in the hypothalamus, such as in the PVN of the hypothalamus, which are capable of increasing penile erections (Argiolas, 1994; Giuliano and Rampin, 2000). In fact, the local administration of oxytocin, dopaminergic agonist, or even nitric oxide increased the number of penile erections, supporting a central role of the PVN (Argiolas, 1994).

In a previous study, we showed that HY male rats have a higher percentage of noncopulators with respect to SD rats (Portillo et al., 2010). In Roman high-avoidance and in the novel exploration higher responder sublines, all of them showed deficits in sexual behavior that are proposed to be due to changes in the dopaminergic tone (Melis et al., 2019). Another group of rats selected on the basis of susceptibility to cholinergic agonists was the sensible and resistant Flinders sublines, FSL and FSR, respectively. These two groups of rats also differed in their sexual performance because both had a marked decrease in the ejaculatory frequencies and reached exhaustion sooner with respect to SD controls (Ferreira-Nuño et al., 2002). Importantly, the FSL rats had higher levels of anxiety and depressive-like behavior that were like those obtained in the LY subline of rats (Overstreet, 1993; Overstreet et al., 2005; Eguibar et al., 2017b). When cross-fostered with SD dams, FSL rats did not show a change in depressive-like symptoms in the forced swing test, and in adulthood, an SD liter showed clearly deteriorated depressive-like behavior when raised by FSL dams (Malkesman et al., 2008). Similar results were obtained when cross-fostering was used between stress-vulnerable and stress-resilient rats (Uchida et al., 2010).

These results are relevant, considering that HY male rats being less emotionally reactive (Moyaho et al., 1995) and having an increased frequency of grooming bouts when exposed to a novel environment (Eguibar and Moyaho, 1997), supporting this hypothesis. A recent experiment showed that HY rats explore and enter more the open arms of an elevated plus maze, supporting that they are resilient to stress manipulations (Eguibar et al., unpublished results). Cross-fostering between Lewis rats, a group of rats with higher anxiety rates, and Fischer 344 rats, which are resilient to stress, is relevant. When cross-fostering was performed, dams induced a nonsignificant increase in the offspring’s time spent in the open arms in the elevated plus-maze, but they were able to induce an increase in the offspring’s exploration of the center in an open-field arena, supporting that maternal care was able to change the anxiety responses (McCarty, 2017; Siviy et al., 2017).

Another relevant aspect is that maternal care can impact spontaneous penile erections, but this is not the case when we determined D₂-like dopaminergic agonist-induced yawning and penile erections, in which the HY phenotype had higher frequencies independent of the dams that raised them. It was unexpected that LY dams were capable of increasing dopamine D₂ receptor expression in the striatum in SD male rats, supporting that, during neurodevelopment, maternal care and the presence of their siblings and nest environment are capable of changing dopaminergic function independent of the genetic background of the male rats, but not in the (−)-quinpirole-induced yawning and penile erections. We use a cumulative dosing because it allows us to quickly evaluate behavioral responses to this dopaminergic agonist in a valuable group of rats as they are the in- and cross-fostered which require shorter time to obtain a dose–response curve with similar distribution to that obtained with other dose schemes. Previously this type of approach has been used in rats under food restriction that decreases or with high-fat diet that enhances sensitivity to the actions of quinpirole (Baladi and France, 2009). It is also the case with streptozotocin-treated rats that had a reduced response to quinpirole-induced yawning (Sevak et al., 2007).

Additionally, (−)-quinpirole decreased grooming bouts and abolished scratching; in the case of grooming, this effect was mainly due to the stimulation of D₁ receptors (Berridge and Aldridge, 2000a, b; Eguibar et al., 2003). Our results showed a clear inhibitory effect in all groups of rats, with the HY rats being more sensitive than the LY and SD rats, supporting the participation of D₂-like dopamine receptors in the modulation of this behavior. Dopamine had a modulatory role in the striatum because it is a key component for the serial order of the sequence of grooming components, including the transitions in the face components through the lateral parts of the body (Aldridge and Berridge, 1998). It is relevant that in mice with a knockdown mutation in the dopamine transporter gene, extracellular dopamine levels in the striatum increase by 170% with respect to wild-type counterparts, and they show an increased grooming stereotypy that emulates obsessive–compulsive disorder (Berridge et al., 2005). In animal models of obsessive–compulsive symptoms, there were excessive dopamine levels, and it is relevant that subchronic treatment of (−)-quinpirole produced enhanced checking behavior in the open-field arena (Szechtman et al., 1998). Our results of decreased grooming support a role for dopamine D₂ receptors in this behavior, which can be a display of obsessive–compulsive disorder, as previously reported (Robbins et al., 2019), but further experimental data are necessary to support the role of each type of dopamine D₂-like receptor. HY male rats had higher D₁ binding in the striatum with respect to LY rats, which supports their higher grooming bout frequency (Eguibar and Moyaho, 1997; Díaz-Romero et al., 2005).

Scratching behavior depends directly on a spinal command and can be modulated by higher brain centers that are capable of exciting or inhibiting the activity of the scratch generator (Gelfand et al., 1988). On the other hand, central administration of gastric releasing hormones and related peptides, such as bombesin, induced scratching, and this spinal reflex can be inhibited by the administration of D₁ antagonists, such as SCH 23390 (Van Wimersma Greidanus and Maigret, 1991), supporting again a role of dopamine neurotransmission in scratching. In HY rats, intracerebroventricular administration of bombesin significantly reduced yawning frequency and increased scratching independent of grooming bouts (Díaz-Romero et al., 2002).

Our results showed that yawning is a stereotyped and genetically determined behavior with little influence of initial stages of development, including maternal care and nesting conditions and sibling interactions during the lactation period. In fact, the behavioral responses after the administration of dopaminergic D₂-like agonist (−)-quinpirole clearly showed higher responses in the HY male rats, supporting a strong role of the genetic background.

In future experiments, we will analyze the specific role of dopaminergic neurotransmission in the PVN of the hypothalamus or in the striatum by applying dopaminergic agonists directly to each area. The increase in spontaneous penile erections in LY male rats raised by HY dams and the increase in D₂ receptor expression levels induced by LY dams In SD male rats require further experimental data, but these results support a specific role of maternal care in the expression of yawning and penile erections changing some biochemical characteristics in the central nervous system taking into account due to the advantage of high spontaneous expression of yawning and penile erections of HY subline with respect to SD and even Wistar rats.

All datasets generated for this study are included in the article and are available by request.

The animal study was reviewed and approved by Benemérita Universidad Autónoma de Puebla Animal Care and Use Committee No. EGC SAL-G-2019.

Tasks of individual authors: JE and CC: conceptualization. ÁD-N, ÁC, CC, AU, AT, and JE: data curation and investigation. ÁD-N, ÁC, HC-F, and JE: formal analysis. CC and JE: funding acquisitions and resources. ÁD-N, CC, ÁC, and JE: methodology. ÁD-N, CC, AT, and JE: writing.

The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.