Heat stress (HS) exposure to animals not only reduces resistance to diseases but also lowers their growth and reproductive efficiency (1). In female animals, growing ovarian follicles are affected, ultimately impairing conception. In male animals, plasma hormones, spermatogenesis, and sperm quality are badly affected (1). Spermatogenesis is a highly complex mechanism involving mitosis and meiosis, and it requires coordination of germ cell differentiation (2). To achieve optimized animal production, managing heat stress is essential. The literature shows that environmental factors, including heat stress, have a deleterious effect on animal reproduction. For normal spermatogenesis, mammalian testes should be kept at 4–6 °C below internal body temperature (3). Managing heat stress is equally important for seasonal breeders, as they breed during a specific season. Ideal climatic conditions of air temperature and humidity have not yet been reported for Dezhou donkeys. From previous studies, it is evident that one spermatogenic cycle lasts for 10.5 days in donkeys and that 4.5 spermatogenic cycles complete the cycle of spermatogenesis in 47.2 days (3). Based on the acrosomal system, the seminiferous epithelium (SE) in donkeys can be divided into 12 stages, which are further classified into undifferentiated spermatogonia (A_und) and differentiated (A₁, A₂, A₃, B_1, and B₂) spermatogonia (4). Germ cells near the basal membrane of the seminiferous epithelium (spermatogonia) are more vulnerable to heat stress due to their intense cell division activity (5). In rats, humans, and bovines, younger spermatids and pachytene spermatocytes are the most susceptible to hyperthermia (6, 7). The duration of spermatogenesis in donkeys (Equus asinus) is 42.7 days, and the entire process of spermatogenesis takes 4.5 cycles (each cycle lasts for 10.5 days), which are further divided into 12 stages, including undifferentiated spermatogonia (Aund) and differentiated spermatogonia (A1, A2, A3, B1, and B2) (8).

In mammals, germ cells are strategically positioned within the seminiferous epithelium in a well-organized cyclic manner, forming specific groupings known as stages. The seminiferous epithelium cycle involves a series of events that occur between these stages. This process includes the disappearance of a particular cellular arrangement, followed by its reappearance in a designated region of the seminiferous epithelium. These cell groupings can be identified using two methods: (a) by evaluating the morphological traits of spermatids and the overall alignment of germ cells and (b) by monitoring the development of the acrosomal system (9). Regarding the impact of heat stress (HS) on reproductive function in mammals, spermatogenesis is particularly susceptible, often resulting in varying degrees of reduced fertility (10). Regional variations in the extent of ambient temperature increase are significant, and there are also notable differences among species and individuals in their sensitivity to heat stress (11). High temperatures can substantially impact various aspects of reproductive processes in mammals (10). These effects include disruptions in sperm and egg formation, impaired egg maturation, compromised early embryonic development, altered fetal and placental growth, and reduced lactation. These harmful consequences of heat stress arise either from the elevated levels of body temperature or from the physiological adjustments that heat-stressed animals make to maintain their body temperature. All germ cells are sensitive to heat stress, including Leydig and Sertoli cells (12), but the most vulnerable are pachytene spermatocytes, spermatids, and epididymal sperm (13–15). The effects of elevated testicular temperature depend on the magnitude and duration of heat exposure. While a short-term, mild increase in testicular temperature may cause only a temporary decline in sperm quality, continuous or intense heating is more likely to lead to infertility (16). Additionally, prolonged and severe thermal injury could halt spermatogenesis permanently.

Although a substantial body of literature documents the impacts of heat stress in female animals, studies on male animals—especially donkeys—remain limited. Therefore, the purpose of the present study was to evaluate the effects of heat stress during June, July, and August on testicular histoarchitecture, including alterations in seminiferous epithelium dimensions, Jonhson’s score, stages of spermatogenesis, and testes weight, as well as plasma concentrations of testosterone, luteinizing hormone (LH), inhibin A, and inhibin B. This investigation will contribute to understanding the effects of short-term heat stress on reproductive physiology and spermatogenesis in Dezhou donkeys.

Heat stress is a major challenge for livestock. It not only compromises animal welfare but also causes health and productivity losses. To the best of our knowledge, this is the first study to investigate the effects of short-term heat stress on donkeys. Hyperthermia has detrimental effects on testosterone and inhibits spermatogenesis in mine, horse, sheep, cows, and pigs (26–31). Although the effects of short-term heat stress are well documented in other mammalian species, studies in donkeys are lacking. The process of spermatogonial differentiation into sperm is highly complex, and various factors, such as hormones, growth factors, seasonality, and testicular temperature, regulate it (32, 33). Our results are consistent with previous studies showing that heat stress induces oxidative stress, leading to apoptosis and damaged germ cells. Elevated testicular temperature disrupts the normal thermoregulatory mechanism of the testes and induces oxidative stress in germ cells, ultimately reducing sperm function and resulting in male infertility (34, 35). Normal development of spermatogonia, spermatocytes, and spermatids is essential for achieving high fertility, optimal semen quality, and spermatogenesis (36). Heat stress leads to apoptosis and germ cell autophagy, which can ultimately damage sperm DNA and increase the production of reactive oxygen species (ROS) (37). In mice, exposure to heat stress reduced the ability of sperm to fuse with oocytes both in vivo and in vitro (38). The hot season damaged the seminiferous epithelium in rams, leading to abnormal and non-viable sperm during mid-summer (39). Early stages of spermatogenesis were affected by heat stress in rams (30). This evidence suggests that heat stress induces oxidative stress in the testes, leading to germ cell apoptosis and ultimately lowering donkeys’ semen quality. The lower number of germ cells in the heat-stressed group suggests apoptosis in the seminiferous epithelium.

Body weight, testicular weight, and the testes-to-body weight ratio showed no significant differences and a similar ascending and descending order in all animals, as shown in Figure 1. These results indicate that short-term heat stress has no effect on testicular morphology. Mariane et al. (40) concluded that stallions are seasonal breeders, but seasonality in reproduction depends on the latitude of origin; alterations in seasonality affect gonadal activity in stallions (40). Our findings align with those of Freitas et al. (40), who concluded that seasonality and higher altitudes affect variation in reproduction. It may be one of the reasons why the hot months did not affect testicular weight. Figure 2 depicts the normal progression of spermatogenesis and the normal development of germ cells in all seminiferous tubules, including spermatogonia, spermatocytes, and spermatids.

The acrosomal system is considered the most effective method for analyzing various morphological alterations in the seminiferous epithelium. The accuracy of this method depends on stages and species [for example, six stages in humans (41) and 14 stages in rats (42)]. In previous studies, a tubular system has been applied to equine and asinine subjects. In the study by Chiarini-Garcia et al. (4), only the acrosomal method was used to analyze pre- and post-meiotic stage frequencies (~27%; stages VIII–XI, and ~55%; stages I–VII). Comparisons with the tubular morphological method showed similar results (8, 43, 44). In the present study, for the first time, the acrosomal system was combined with Johnson’s score, seminiferous tubule (ST) diameter, and germ cell quantification to evaluate alterations in the seminiferous epithelium of donkeys exposed to short-term heat stress. Our findings support previous observations that, within the same mammalian family, pre- and post-meiotic stage frequencies can be phylogenetically determined (45, 46).

Examination of spermatogenesis allowed us to distinguish distinct groups of spermatogonia based on their appearance. Using high-resolution light microscopy, we observed variations in nuclear morphology, allowing the separation of the earliest spermatogonia into two distinct categories: undifferentiated type A (A_und) and differentiated type A spermatogonia. The morphological characteristics distinguishing these two groups closely resembled those observed in mice (22), rats (47), and golden hamsters (48). However, unlike observations in other species, the chromatin in donkeys appeared coarse in all animals examined in this study. This discrepancy may have resulted from the method of cell preservation, such as fixation, or other tissue-processing factors. Nonetheless, we observed subtle morphological features in neighboring cells, including spermatocytes, spermatids, and, especially, Sertoli cells.

In 1991, Johnson classified horse spermatogonia into five subtypes: A1, A2, A3, B1, and B2. However, Aund spermatogonia are similar to those found in rodents, as described by previous studies (48–50). As shown in Figure 2, we observed A_und spermatogonia in the seminiferous epithelium across all 12 stages of the seminiferous epithelial cycle, mirroring the kinetics reported in rodents, which have been extensively studied (51). As shown in Figure 2, the Aund spermatogonial population reached its lowest level during stage VIII of the seminiferous epithelial cycle. Subsequently, their numbers gradually increased, peaked at stage VI of the following cycle, and declined again at stage VIII. Swierstra et al. (44) observed the same trend in type A spermatogonial kinetics during the cell cycle in horses. A possible explanation for this kinetic pattern is that, during stages VII and VIII, A_und spermatogonia differentiate into A₁ spermatogonia, giving rise to a new wave of spermatogonial differentiation, with a consequent reduction in the number of Aund spermatogonia, as described previously in rodents (52, 53). Our combined morphologic and stereological analyses allowed us to distinguish three types of differentiated spermatogonia (A1, A2, and A3) and two types of B spermatogonia (B1 and B2) in Dezhou donkeys. These cell types displayed distinct morphology under high-resolution light microscopy, and the kinetics along the cycle, from A₁ to B2, showed that cell numbers increased progressively. Each spermatogonial type was observed in the stage where it originated via mitosis from the predecessor cell and in the next one (or two) stages when the spermatogonial type would rise by mitosis of the next spermatogonial type, as observed in mice (52) and golden hamsters (48). Therefore, it was also demonstrated in Dezhou donkeys that spermatogonial types overlap in their kinetics across the seminiferous epithelial cycle. In stage VIII, a further reduction was observed, mirroring the pattern observed in stallions by Swierstra et al. (44) for type A spermatogonial kinetics during the cell cycle. This trend may be explained by the differentiation of type A undifferentiated spermatogonia into type A1 spermatogonia during stages VII and VIII, initiating a new wave of spermatogonial differentiation and consequently reducing the number of type A undifferentiated spermatogonia, as previously documented in rodents (52).

Figure 2 shows a positive correlation between STD, EH, and LTD. It is widely accepted that there is a positive correlation between ST diameter, EH, testicular weight, and the process of spermatogenesis (18, 36). Similarly, a positive correlation was observed between ST/field and germ cell numbers (spermatogonia, spermatocytes, and spermatids). A positive correlation among these parameters indicates the normal progression of spermatogenesis and the development of germ cells within the testicular histoarchitecture. As previously reported, similar findings were observed in Yanghou ganders (18, 36). Despite differences between species, the principle remains the same for testicular histoarchitecture. As the experiment progressed, animals in Group A showed normal germ cell development and consistently exhibited slightly higher values at all stages compared to the experimental group. Based on testicular histoarchitecture and germ cell development, we can speculate that short-term heat stress reduces the number of germ cells through apoptosis, whereas normal temperature helps maintain normal testicular histoarchitecture.

Endocrinological changes after temporary heat stress in jacks are poorly understood. LH showed an ascending pattern in the control group (Group A), while there existed a descending pattern in the experimental group (Group B), as shown in Figure 4A. In July and August, plasma hormone concentrations in the control group were significantly higher than those in the experimental group. Similar reductions in plasma LH due to heat stress have been reported in sheep (54), cattle (55), and goats (56). Heat stress has been shown to reduce plasma LH in horses (57), lactating cows (58), and working horses (59). Our findings are consistent with previous observations reported by researchers. In most mammalian species, inhibin B is the predominant form regulating reproduction, except in rams, where inhibin A is more important (60). Inhibin A and inhibin B showed variations during the experimental period. Inhibin B is the predominant form of inhibin involved in male reproductive function compared to inhibin A (60). It is a major form of circulating inhibin in hamsters, miniature pigs, and rats (61, 62, 63). These findings, together with previously reported data in mares, indicate a sexually dimorphic pattern of inhibin isoform expression in horses, with inhibin B being more prevalent in stallions and inhibin A being more prevalent in mares (64). Our findings are in accordance with previous research. Inhibin B concentrations were four times higher than inhibin A throughout the experimental period. This study suggests that inhibin B exerts negative feedback on follicle-stimulating hormone (FSH). It can be speculated that FSH secretion is suppressed by inhibin B and that weak signaling in the heat-stressed group affected Sertoli cell function, resulting in reduced testicular weights. The consistently lower plasma testosterone concentrations observed in the experimental group compared to the control group can be attributed to heat stress-induced loss of Leydig cells and germ cells, leading to reduced testosterone (T) levels. Testosterone plays a vital role in the progression of spermatogenesis in the seminiferous tubules (65). The plasma testosterone concentration was significantly higher in the control group at the end of August. These findings highlight that even short-term heat stress reduces plasma testosterone concentrations in donkeys. Disrupted spermatogenesis and lower Jonson’s score are closely associated with the lower testosterone concentrations observed in the experimental group. Additional research is required to explore the molecular mechanisms underlying apoptosis after short-term heat stress, focusing on genes in germ, Sertoli, and Leydig cells and their effects on semen in donkeys.

The present study, conducted on donkeys, shows that temporary heat stress is a type of stress that affects testicular histoarchitecture, the seminiferous epithelium, and plasma concentrations of LH, inhibin A, inhibin B, and testosterone. Temporary heat stress affects Sertoli and Leydig cells without altering the stages of spermatogenesis. Inhibin B is the primary circulating form of inhibin in Dezhou donkeys. Maintaining an optimal temperature of 25 °C can help prevent these harmful effects during the summer months in commercial donkey farming, while also considering the economic factors of the enterprise. There are some limitations in our study. Daily temperature and humidity were not recorded. In addition, the experimental duration was limited due to funding constraints. These factors should be addressed in future research.