Hot and humid environments may adversely affect stallion fertility [1]. The present rise in global temperature is alarming for the horse industry, especially the stud market. Moreover, in contrast to other domestic species, stallions are selected for breeding, primarily based on their track record and conformation rather than reproductive soundness and heritable traits, which results in a low fertility index and rates of conception per cycle [2]. Hence, high ambient temperature is not only a threat for breeders, but it also raises questions regarding the afflicted stallion’s capacity to generate healthy spermatozoa. Spermatogenesis is a temperature-sensitive process, occurring optimally at 2 °C–4 °C below body temperature [3]. Numerous environmental conditions cause heat stress, which increases scrotal temperature, thereby contributing to reduced fertility or complete sterility in stallions [4]. Stallion spermatozoa exposed to scrotal heat stress exhibit fewer disulfide linkages and higher susceptibility of DNA to denaturation [5]. Thermal damage effectively degenerates stallions’ testicles, and total sperm abnormalities increase with decreased sperm kinetics and plasma membrane integrity [6]. Recently, Griffin et al. [7] studied heat-induced subfertility and found that while DNA damage is consistently negatively correlated with sperm fertility, it is consistently positively correlated with DNA environmental temperature and humidity in stallions vulnerable to heat stress [7].

Various types of cells in the testicles respond differently when exposed to heat stress. They exhibit varied levels of susceptibility and reactions and both normal and abnormal changes in their physiological state [8]. Owing to rapid cell division and the lack of superoxide dismutase, the spermatogenic cells are highly susceptible to heat-stress damage [9]. Research indicates that certain stages of spermatocytes and mature sperm cells are particularly sensitive to temperature fluctuations. For instance, spermatocytes in the zygotene and pachytene stages and spermatozoa in the early-round stage have been identified as the most heat-sensitive cells in rats [10]. Heat damages DNA and weakens double strands, which lead to spermatogenesis-related heat stress-induced apoptosis. Spermatocyte integrity is severely impacted by heat, and this leads to DNA damage [11]. It is crucial for meiotic recombination that DNA repair occurs during spermatogenesis in developing germ cells. However, heat stress may cause unpairing of Y chromosomes during meiosis, which leads to spermatocyte death. This may also be caused by abnormal segregation of sex chromosomes due to heat stress [12]. Despite the influence of heat stress on these processes, spermatogonial stem cells (SSCs) tend to recover well on their own and rarely undergo apoptosis because of heat stress. A prior investigation showed that exposing SSCs to elevated temperatures alters their self-renewal behavior and changes the localization and folding of certain proteins, thus arresting the cell cycle [13].

Cryptorchidism impairs spermatogenesis and fertility by disrupting germ cell development, reduced sperm quality, and altered testicular microenvironment [14]. It poses significant challenges for both animals and their owners. One major factor for this is the lack of definitive evidence regarding the heritability of equine cryptorchidism. Consequently, there is no consistent policy across studbooks and breeding organizations regarding whether unilateral cryptorchids should be permitted or not for registration as breeding stallions [15]. Cryptorchidism impairs spermatogenesis and fertility through various processes. However, the specific impact of long-term heat stress associated with cryptorchidism on SSCs in stallions is not well understood. Therefore, this study is designed to investigate the effects of long-term heat stress on SSCs, associated with cryptorchidism in stallions. Knowing how cryptorchidism affects spermatogenesis and gaining a deeper understanding of the pathological changes that occur in cryptorchid testes may help in improving the knowledge regarding characteristics of germ cells in response to heat stress. We hypothesize that the undifferentiated SSCs are not significantly affected by long-term exposure to heat stress, while germ cells after differentiation phase may directly impacted by heat-stress conditions such as cryptorchidism in stallions.