Seasonal breeders closely link their reproductive efficiency to environmental cues that mainly include photoperiod (day length), temperature, and nutritional status (15). The pineal gland produces melatonin as a response to dark conditions, which controls the seasonal pattern of GnRH secretion (16). From winter months’ short-day periods, melatonin secretion extends over time until it suppresses GnRH release, which reduces LH and FSH secretion and causes reproductive inactivity (17). The reduction of melatonin during long-day periods results in the reactivation of GnRH pulsatile action and restores reproductive capacity (18).

Relational dynamics of the HPG axis are most prominent in mares together with sheep, goats, and donkeys because their breeding patterns match photoperiod modifications of melatonin release (16). Donkeys share the reproductive pattern of horses by being long-day breeders, and their breeding season occurs during spring and summer when day length expands (11). The natural birth cycle results in foal births when environmental conditions offer the best resources.

The hypothalamic–pituitary–adrenal (HPA) axis that controls stress responses creates a feedback mechanism with the HPG axis. Between chronic stress and HPA axis activation arises the production of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) that stimulate cortisol production from adrenal glands (19). The release of GnRH diminishes when cortisol levels increase, which subsequently decreases LH and FSH production, thus leading to reproductive system suppression.

The release of GnRH decreases when cortisol levels increase, which subsequently reduces LH and FSH production, thereby leading to suppression of the reproductive system (20). The reproductive system of female seasonal breeders shows adverse effects from chronic stress because this results in anovulation together with irregular estrous cycles and reduced estrogen production, which frequently causes ovarian dysfunction and persistent follicles or ovarian cysts (21). The prolonged exposure to stress in male individuals decreases testosterone levels along with spermatogenesis and causes sperm quality to decline while diminishing sexual desire, so fertility remains impaired in breeding periods (22).

The neurochemical agents serotonin (5-HT), along with dopamine and norepinephrine (NE), function as vital elements for controlling HPG axis responses under stress conditions (23). GnRH release receives stimulation from serotonin, although the changing sensitivity of serotonin receptors during seasonal periods may contribute to reproductive suppression caused by environmental stressors like nutritional deficiencies, changes in social standing, and climate patterns (24).

The HPG axis operates under significant control from energy balance regulation. Reproductive function regulation occurs through the AMPK-mTOR signaling pathway that detects energy levels by influencing GnRH neurons (19). AMPK activation stops GnRH secretion to cause reproductive dormancy when the body faces nutritional hardships or negative energy conditions (such as winter months). The HPG axis receives a signal from adequate energy storage to activate mTOR signaling, which then triggers GnRH release and increases reproductive capability (25).

The reproductive hormone regulation of donkeys that experience seasonality depends on their body condition changes and how much they eat between seasons (26). Studies performed on mares and sheep proved that minimal body fat, together with low leptin levels, restrict normal GnRH signal pulsing which causes breeding season delays. The HPG axis becomes fully active once the nutritional condition improves, thus breeding occurs at the most appropriate time for the environment (27). The molecular pathway Hypothalamic–Pituitary-Gonadal (HPG) Axis Dysregulation is shown in Figure 1.

There is a different gonadotropin secretion pattern in donkeys. Jennies exhibit two FSH peaks during one estrous cycle and a long-lasting LH surge that frequently continues after ovulation (28). This differs from the single peak of FSH and closely timed LH surge in mares and ewes. These hormonal patterns can provide distinct follicular and luteal sustainability processes in donkeys (29).

An impressive molecular difference is seen in the ligand specificity of the FSH receptor (FSHR). Cloned FSHRs in donkeys can to bind FSH and LH/chorionic gonadotropin (CG) in a ligand promiscuous manner, which has not been observed in horses or sheep (30, 31). This is due to differences in amino acids of the extracellular domain of the receptor (~96% homology with equine FSHR). The physiological significance of such receptor flexibility on follicular development has yet to be understood (32).

Equine chorionic gonadotropin (eCG) is LH-like, as well as FSH-like, in non-equines (33). Nevertheless, in donkeys, LH and CG are largely LH-active with little FSH-like activity in in vitro assays. This also emphasizes species-specific hormone-receptor interactions that may have effects on ovulatory regulation and folliculogenesis (29).

The mechanism through the HPG axis functions in seasonal breeders complicates the management of reproduction and breeding operations. The following potential strategies can be used to improve reproductive efficiency in seasonal breeders (34):

The reproductive system of donkeys, alongside other seasonal breeders, controls the HPG axis through interactions between photoperiod signals, metabolic indicators, and stress responses (35). The reproductive efficiency of animals becomes compromised when the stress axis becomes nonfunctioning due to chronic stress combined with poor nutritional status, along with unsuitable environmental elements (36). Understanding seasonal regulatory processes better will allow scientists to create specific measures that enhance the breeding performance of seasonal species. The summary of the molecular pathway is shown in Table 1.

The reproductive patterns of seasonal breeders are controlled through environmental signals that primarily include changes in day length (photoperiod) (40). The adaptation brings about offspring births in optimal times, which usually matches the spring season when environmental factors create favorable conditions for survival. The main regulator for this process functions through melatonin, which the pineal gland produces because of daily darkness. Through its neuroendocrine role, melatonin carries photoperiodic data, which subsequently affects reproductive hormone release (41).

Horses, together with donkeys, show suppressed melatonin production when daylight stretches out, which activates their gonads (42). Short-day breeders like sheep and goats activate reproduction during periods when their nightly melatonin hormone production reaches higher levels. Knowledge of the melatonin signaling pathway stands vital for determining the seasonal mechanisms thatdonkey reproductive effectiveness (43).

The SCN acts as a circadian oscillator that manages melatonin production by controlling the expression of BMAL1 and CLOCK together with Period (Per1, Per2) and Cryptochrome (Cry1, Cry2). All these genes create a transcription-translation feedback loop that controls the length of melatonin production based on photoperiod (55).

Long-day conditions cause changes in the phase relationships of SCN neurons, which affect clock gene expression patterns and decrease melatonin production levels. The expression patterns of clock genes under short-day conditions extend melatonin production, that results in reproductive inhibition in donkeys long-day breeders (56).

Kisspeptin, originating from the Kiss1 gene, operates as a strong activator of GnRH release. The arcuate nucleus of the hypothalamus experiences decreased Kiss1 expression because of melatonin effects, which results in reproductive inactivity (57).

The reduction of melatonin levels in donkeys with long-day breeding patterns stimulates Kiss1 gene expression to trigger the activation of GnRH along with gonadotropins that initiate reproductive functions. Sheep display seasonal anestrus by having melatonin suppress Kiss1 expression, which prevents the release of GnRH (58).

The mammalian ortholog of gonadotropin-inhibitory hormone (GnIH) is RFamide-related peptide-3 (RFRP-3), which controls the activity of the HPG axis by suppression. The secretion of RFRP-3 shows both melatonin-regulated patterns and species-specific responses toward GnRH release (59). Sheep experience seasonal anestrus because RFRP-3 reduces GnRH secretion in their system. Short-day conditions stimulate RFRP-3 to boost GnRH secretion in the brains of hamsters, that helps the reproductive system to function normally (60). Researchers have not confirmed the function of RFRP-3 in donkeys, even though its relationship to Kisspeptin and thyroid hormone regulation might help understand seasonal reproductive patterns (61).

In sheep and horses, the melatonin TSH thyroid kisspeptin GnRH cascade regulates photoperiodic reproduction tightly (62). Donkeys might not be fully involved in this axis. Their reproduction physiology seems to be less responsive to a change of daylight, which suggests that they depend on other stimuli like dietary conditions, temperature, or socialization (63). This hypothesis has not been molecularly confirmed because there is a lack of neuroendocrine mapping.

The examination of the melatonin signaling pathway enabled researchers to create methods that control reproductive patterns in donkeys along with other seasonal breeders (64).

The reproductive efficiency of seasonal breeders, including donkeys heavily depends on the functioning of their melatonin signaling pathway. The neuroendocrine transducer function of melatonin depends on its ability to process photoperiodic cues with the SCN while working with PT and thyroid hormones alongside Kisspeptin and RFamide-related peptides (65). Knowledge advancement regarding these mechanisms enables developers to create successful breeding management strategies that improve reproductive success for donkeys and other seasonal breeders (66). The summary of the molecular pathway is shown in Table 2.

The ovary produces sex hormones through a coordinated process involving two different cell types as well as two different hormones (99). Luteinizing hormone (LH) activates cholesterol conversion to androgens in the theca cells so that these hormones move on to granulosa cells. The hormone FSH in granulosa cells turns on aromatase activity that transforms androgens into estradiol (100). The restrictive hormone control system completes the proper functioning of estrous cycles and ovulation while sustaining pregnancy in seasonal reproduction cycles. Hormone production becomes impaired through disruptions in this pathway, which occurs from environmental stressors or EDC exposure, thus causing irregular reproductive cycles along with infertility (101).

EDCs interrupt ovarian steroidogenesis either by blocking essential enzymes, duplicating natural hormones, or obstructing various receptors (97). Studies show that the chemical substances bisphenol A (BPA), phthalates, and Polychlorinated Biphenyls (PCBs) block aromatase activity, which decreases estradiol production. Pesticides together with dioxins disrupt the steroidogenic acute regulatory (StAR) protein required for cholesterol transport into mitochondria (102). These environmental toxins interfere with vital molecular pathways to change the regulation of the estrous cycle as well as the reproductive efficiency of species that align with seasonal mating patterns (103).

The reproduction of seasonal breeders such as donkeys strongly depends on environmental clues, including photoperiod and nutrition, because disruptions in steroidogenesis cause major reproductive effects (104). Endocrine-disrupting compounds affecting sex hormone equilibrium control the duration of the estrous cycle, delay ovulation, and decrease fertility potential (105). Juvenile animals become unable to sustain pregnancy because the corpus luteum function fails to maintain normal progesterone levels, and when estrogen production becomes disrupted, it affects follicle maturation. The dependence of these species on hormonal changes for seasonal reproduction makes exposure to EDCs a possible cause of declining fertility and reproductive problems (106).

The essential hormonal process of steroidogenesis controls reproductive efficiency, but environmental pollutants create major difficulties for seasonal breeders to maintain their reproductive functions (107). The impact of environmental pollutants on hormone biosynthesis steps results in reproductive breakdowns, which creates permanent effects on fertility (108). Research must be conducted to determine how much seasonal breeders encounter environmental pollutants while developing new approaches to lessen the reproductive health damage. The identification of environmental polluting factors will enable better decision-making regarding protection plans for species suffering from pollution exposure effects (109) (Figure 5).

The summary of the molecular pathway is shown in Table 5.

The metabolism of cells produces ROS byproducts mainly in mitochondria, which serve as crucial signaling agents during folliculogenesis and ovulation and corpus luteum development (114). High levels of ROS exceed the capacity of antioxidants to control them, which results in damage to lipids, proteins, and DNA, leading to gonadal cell death through apoptosis (115). The reproductive patterns of seasonal breeders respond directly to photoperiodic changes and metabolic status, thus making this research important for their breeding cycles (116). The level of oxidative stress tends to increase throughout the non-breeding cycle to maintain reproductive dormancy, yet specific ROS regulation helps execute important reproductive processes, including follicular rupture together with sperm capacitation during the breeding period (117).

Gonadal functionality depends on programmed cell death known as apoptosis since this process regulates the death of follicles while also controlling sperm formation (118). Seasonal breeders primarily depend on the intrinsic apoptotic pathway, which originates from mitochondrial dysfunction combined with oxidative damage to their cells (119). Excessive ROS activates cytochrome c release from mitochondria to activate caspases, which in turn causes the death of follicular cells and germ cells (120). The existence of a balance between pro-apoptotic proteins BAX and BAK and anti-apoptotic protein BCL-2 decides whether cells will survive. The reproductive efficiency of reproductive systems is impacted by season-dependent modifications of gonadotropin levels and melatonin signaling that control oxidative stress mechanisms and apoptosis rates in gonadal tissues (121).

The reproductive efficiency of seasonal breeders such as donkeys is directly affected by oxidative stress and apoptosis because they harm gametes and their reproductive organs’ functionality (122). Controlled oxidative signaling supports ovulation together with sperm maturation during the breeding season. Excessive oxidative damage during times outside the breeding period quickens the process of follicular atresia while causing sperm viability to decrease (123). Reproductive success suffers from environmental stressors such as heat exposure, poor nutrition, and toxic environmental substances, which increase the rate of oxidative damage in animals (124).

The improvement of reproductive performance in seasonal breeders depends on implementing methods that reduce oxidative stress damage (125). The combination of antioxidant supplements, including vitamins C and E, and selenium, and melatonin, leads to better gonadal function and fertility results (126). The reproductive potential can benefit from nutritional measures that activate endogenous antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase (127). Proper control of environmental stressors together with appropriate nutritional provision during breeding seasons will help reduce oxidative damage, which in turn leads to improved reproductive outcomes across donkeys and other seasonal breeders (128).

The regulation of reproductive efficiency in seasonal breeders depends heavily on oxidative stress together with apoptosis mechanisms (129). The body needs regulated ROS production to maintain normal reproductive functions, yet too much oxidative damage triggers problems with gamete quality along with hormonal imbalancing and infertility (130). Laboratory research on oxidative stress mechanisms interacting with seasonal reproductive signals will enable scientists to develop better treatments for enhancing donkey breeding performance alongside other seasonal breeders (131) (Figure 6).

The summary of the molecular pathway is shown in Table 6.

The duration of daylight throughout seasons strongly controls the production of melatonin and therefore controls the anterior pituitary’s release of prolactin (135). The hypothalamus, along with the pars tuberalis of the pituitary, contains receptors that allow melatonin to trigger seasonal endocrine responses (136). The control mechanism for both gonadotropins as well as prolactin functions through the shared regulation of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin by melatonin (137).

The mechanisms that regulate seasonal prolactin changes differ from gonadotropin patterns because prolactin relies on direct neuroendocrine regulation, but gonadotropins follow feedback-based control (138). Current research does not provide enough evidence to prove that winter prolactin reduction happens only through increased dopamine inhibition (139). At this time, pituitary becomes more responsive to dopamine, which could be a factor in the decrease of prolactin, secretion throughout seasonal cycles. The intricate relationship between melatonin, prolactin and gonadotropins demonstrates how the human body readjusts reproductive and metabolic systems because of seasonal variations (132).

The regulatory patterns of pituitary hormone secretion might be influenced by seasonal shifts observed in folliculostellate cells, which belong to the category of pituitary support cells (145). Research indicates that breeding_season triggers an increase in cell-adherens junctions between these cells while prolactin and gonadotropin interactions simultaneously evolve (146). The GnRH hormone canstimulate prolactin release, but its effect on this process depends on the season, the species, and the reproductive condition of the animal (147).

Many mammals use prolactin as their main seasonal regulatory agent while photoperiodic cues processed through melatonin pathways control its secretions (148). Prolactin plays an essential part in seasonal physiological adaptations since it controls molt, metabolism and energy balance beyond reproduction (149). The complex dynamic system of prolactin together with gonadotropins and hypothalamic regulatory components shows that it plays an essential role in reproductive adaptation to environmental changes while managing energy, and reproductive resource distribution annually (150) (Figure 7).

The summary of the molecular pathway is shown in Table 7.

Thus, the prolactin pathway serves as a vital neuroendocrine mechanism in seasonal breeders, regulating reproductive timing and physiological adjustments in response to the photoperiod (133). The regulation of prolactin secretion in response to melatonin affects both the reproductive and metabolic functions (132). The pituitary paracrine interactions and dopamine modulation indicate the association between prolactin and gonadotropin output (151). Thus, it indicates the dual role of prolactin in regulating internal hormonal rhythms and external seasonal changes.

The molecular pathways coordinate in response to photoperiod and nutritional availability, ensuring optimal reproduction during long days (151). The summary of these molecular pathways is shown in Table 8. It is well established that the reproductive activity in donkeys is regulated by interconnected hormonal, signaling, and metabolic pathways that respond to the environmental and seasonal variability (16). Thus, disruptions in the HPG axis, melatonin signaling, KNDy neurons, and prolactin pathway result in decreased production of GnRH and gonadocorticoids (10). Malnutrition also affects the AMPK-mTOR pathway, resulting in declined reproductive performance, and oxidative stress leads to gonadal cell damage. Understanding these mechanisms broadens our knowledge of seasonal fertility and opens the horizon for improving reproductive efficiency through modulation of molecular pathways using hormonal, nutritional, and management strategies in seasonal breeders, particularly in donkeys.

Thus, donkeys have seasonally regulated reproductive patterns which are mainly regulated by photoperiod (152). Reproductive traits of males (e.g., testicular size, semen quality, hormonal changes (e.g., testosterone)) differ in breeding and non-breeding seasons (153). The results of the immunohistochemical studies of the epididymis indicate the presence of higher epithelial activity and sperm in the spring, whereas higher markers of oxidative stress and autophagy are observed in off-seasons (154). These results deny the previous hypotheses of low seasonality and support the importance of time-adjusted breeding plans. In addition, Dezhou donkeys immunized against inhibin exhibited elevated levels of FSH, LH, testosterone, and activin A, especially out of breeding season (155).

Donkeys and horses are very different in their reproductive behavior, the duration of a cycle, and anatomical characteristics (27). The estrous cycle and the gestation period of Jennies are longer, and they also respond to factors other than photoperiod (12). Jacks have bigger reproductive organs, and they take more time to ejaculate (152). Behavioral peculiarities of the donkey reproduction, like territoriality and non-harem mating patterns, as well as reduced spermatogenic efficiency, also distinguish it among other equids (27). These characteristics require specific assisted reproductive technology (ART) regimens. It has been found that duration of the follicular phase, not luteolysis, is the main factor determining variability in interovulatory interval (IOI) (156). Longer IOIs are associated with longer estrus, slower follicle development, and larger follicles at ovulation. Such findings are important to schedule insemination and forecast fertility periods (27).

Thc sperm of donkeys has some cryobiological difficulties. The traditional freezing techniques produce uneven fertility outcomes, particularly in jennies (157). Nevertheless, the latest developments in sperm vitrification show positive results. Straws with outer covers, using 0.25 mL straws, showed similar or better motility and in vivo fertility than standard frozen semen (158). Remarkably, vitrified semen caused a less severe and short-term uterine inflammatory reaction (159). Moreover, Phospholipase C Zeta (PLCzeta) localization in donkey sperm showed that it was competent in oocyte activation, particularly during intracytoplasmic sperm injection (ICSI) into horse oocytes (160). These interspecies ICSI outcomes confirm the utilization of donkey sperm in the creation of mules and imply the expansion of ARTs (161).

Endometritis is a significant limitation to fertility in donkeys. The use of equine-based histopathological grading of donkey uteri has been effective, and cytological and biopsy-based assessment would improve the level of diagnosis (162). These aids enable a superior categorization of uterine health and an even more accurate treatment regimen. Donkeys are prone to metabolic problems that affect reproduction. Metabolic disorders like insulin dysregulation, hyperlipemia, and Pituitary Pars Intermedia Dysfunction (PPID) are usually compounded by obesity (29). These conditions either directly or through systemic effects lead to impaired reproductive performance (e.g., laminitis, organ dysfunction). Most unfortunately, the majority of hormonal reference ranges and treatment protocols are based on horses, in spite of pharmacokinetic differences (163).

Breed size also plays a great role in reproductive performance. Big-bodied breeds such as the Dezhou donkey are more fertile and produce more milk than smaller breeds (e.g., Cullen donkeys) (164). Surveys conducted in Northern China indicate that formalized farm activities, especially with national/provincial institutions, are associated with improved ART adoption and reproductive success (153). The survey shows that about 73 percent of the surveyed farms are using artificial insemination, indicating the rising use of ART. Such results support the importance of breed selection, genetic advancement, and farm standardization in optimizing fertility. Donkeys are increasingly being used in protecting endangered equids. ARTs have advanced in horses, but adaptation to donkeys and wild equids is continuing (12). Donkeys can be used as fertility models as well as surrogates in conservation programs, particularly due to their reproductive strength and availability (165). Nevertheless, the molecular variability of gamete behavior and endocrine response requires specific studies. The donkey ARTs should be aligned with the principles of conservation biology in order to save genetic diversity in Equus (159).

Although there has been an improvement, there are still considerable gaps in our knowledge of donkey reproductive physiology and molecular control. The principal areas in need of focus are:

To resolve these problems, interdisciplinary cooperation, the use of sophisticated molecular technologies, and the dedication to the species-specific research framework will be needed.