Although the basis of fertility in cattle is multifaceted, events occurring at or around estrus are essential for reproductive success. Estrus signifies the major endocrine transition in which the preovulatory follicle prepares for ovulation. To this end, the estradiol-induced gonadotropin-releasing hormone (GnRH)—luteinizing hormone (LH) surge (Stumpf et al., 1991) sets the stage for ovulation 24 h to 30 h thereafter (Malhi et al., 2005; Saumande and Humblot, 2005; Ginther et al., 2013). The mural granulosa undergoes luteinization (i.e., the beginnings of corpus luteum formation) and the oocyte contained within the preovulatory follicle undergoes meiotic resumption and progression to metaphase II (Smith et al., 1994; Duggavathi and Murphy, 2009).

Increasing estradiol levels during the follicular phase of the estrous cycle underlie much of the behavioral changes leading up to and during estrus (Lyimo et al., 2000). Female cattle approaching estrus become restless, may walk up to four times more than those not exhibiting estrus, and interact with others forming sexually active groups (Kiddy, 1977; Diskin and Sreenan, 2000; Sveberg et al., 2013). Other secondary signs of estrus activity include, but are not limited to, chin-resting, head butting, and vulva sniffing (Kerbrat and Disenhaus, 2004). The most definitive sign of estrus, however, is the willingness of a female to stand to be mounted by another cow. Interestingly, several studies report a positive relationship between the level of activity in an estrual female and the probability of pregnancy outcome (López-Gatius et al., 2005; Madureira et al., 2015; Madureira et al., 2019; Tippenhauer et al., 2023).

Increased body temperature is another hallmark feature of estrus-active females, even under thermoneutral environmental conditions (Piccione et al., 2003; Sakatani et al., 2016). Higher estrus-associated temperatures (HEAT) may range from 0.3°C to 1.3°C or greater above the baseline (Redden et al., 1993; Kyle et al., 1998; Piccione et al., 2003; Fisher et al., 2008; Suthar et al., 2011; Randi et al., 2018). An estrus-related increase in body temperature is short in duration (5 h to 18 h; Redden et al., 1993; Kyle et al., 1998; Sakatani et al., 2016); it typically occurs around the LH surge and persists for several hours thereafter (Fisher et al., 2008; Miura et al., 2017). In addition to being an informative indicator of estrus (Redden et al., 1993; Sakatani et al., 2016), varying levels of HEAT may be functionally relevant.

In support of HEAT’s functional impact on fertility important events specific to the cumulus–oocyte complex, an acute, but short episode of exposure at 41°C for 4 h to 6 h in vitro, induces germinal vesicle breakdown earlier than in thermoneutral controls (Edwards et al., 2005; Hooper et al., 2015) without compromising embryo development after fertilization (Rowinski et al., 2021). In terms of impacts on other preovulatory follicle components, two transcripts (i.e., calreticulin and serpin family F member 2) in granulosa cells have been shown by others to potentiate ovulation (Tsafriri et al., 1989; Tokuhiro et al., 2015). These transcripts were upregulated by varying degrees of hyperthermia occurring after a pharmacologically induced LH surge (Klabnik et al., 2022). Calreticulin can enhance the binding of bradykinin to its receptor (reviewed in Bedard et al., 2005). Interestingly, higher bradykinin levels were noted in the preovulatory follicular fluid of the same cows exhibiting varying levels of hyperthermia, from which the granulosa samples (Klabnik et al., 2022) were collected (Rispoli et al., 2019). Bradykinin has been shown to potentiate follicular rupture in other species (Yoshimura et al., 1988; Hellberg et al., 1991).

Follicular fluid provides the necessary substrates for follicle growth and progression, oocyte developmental competence, and subsequent embryo viability (Leroy et al., 2011; Sirard, 2011). The differences in follicular fluid metabolome profiles have recently been associated with fertility, follicle maturity, or follicle progression after the onset of estrus (Read et al., 2021; Read et al., 2022; Hessock et al., 2023). Although changes in milk, serum, and urine metabolome profiles have been noted in cattle experiencing elevated body temperature resulting from chronic heat stress (Liao et al., 2018; Yue et al., 2020), it remains unclear whether an acute episode of HEAT is associated with changes in the follicular fluid metabolome of the preovulatory follicle. Thus, the objective of this study was to examine the relationship between HEAT and the preovulatory follicular fluid metabolome.

The novel findings described in this study highlight the relationship of varying levels of HEAT with the abundance of multiple metabolites in the preovulatory follicle fluid (19.8% of 86 total) ~ 14 h after the onset of estrus. Interestingly, the majority of affected metabolites were positively related to HEAT (11/17; 65%). This finding is especially interesting because intra-follicular changes in the preovulatory follicle in the final hours leading up to the LH surge are important for promoting oocyte competence for embryo development (Atkins et al., 2013; Jinks et al., 2013) and impacting cumulus–oocyte complex metabolism (Read et al., 2021; Moorey et al., 2022; Read et al., 2022) in addition to affecting subsequent luteal function (Perry et al., 2005) and pregnancy outcome (Lamb et al., 2001; Perry et al., 2005; Atkins et al., 2013).

Regardless of species, sex, or reproductive status, heightened levels of activity for as few as 45 min to 1 hour increase body temperature (Murray and Yeates, 1967; Vajrabukka and Thwaites, 1984; Gleeson, 1998). Estrus-induced or not, resultant increases in body temperature are likely a by-product of increased metabolic activity, increased blood flow, and or muscle strain due to elevated activity (Gleeson, 1998). Higher estrus-associated temperatures in our study fell within a range reported by others (Lewis and Newman, 1984; Redden et al., 1993; Kyle et al., 1998; Fisher et al., 2008), with one cow exceeding 40°C. Although the THI during the final week of our study approached the upper limit of thermoneutral conditions (77 THI for non-lactating Holsteins; Ouellet et al., 2021), the varying levels of HEAT likely reflect those attributable to changes in estrus activity. Increases in vaginal temperature were most notable after a cow first stood to be mounted, which is not surprising when using non-lactating Jersey cows, as they are more thermotolerant (Seath and Miller, 1947; Harris et al., 1960; Muller and Botha, 1993; Lim et al., 2021). In addition, the calculated baseline body temperature for each individual cow fell within the range of normal body temperatures for cattle not experiencing heat stress (Gaalaas, 1945). It is also important to note that estrus onset occurred in the majority of cows when the ambient conditions were thermoneutral.

Although instances of chronic elevations in body temperature because of disease or heat stress are detrimental to reproductive success, short-term acute increases in temperature may in fact be beneficial. At the cellular level, Hoshino (2018) demonstrated that increased oocyte intracellular temperature during maturation may be an indicator of oocyte quality and developmental competence. Fallon (1962) reported rectal temperatures ranging from 98.8°F to 105°F in dairy cows undergoing artificial insemination (AI) the morning (a.m.) after estrus was first observed the preceding afternoon (p.m.). In this otherwise synchronized set of females, cows with rectal temperatures ranging from 101.7°F to 105.0°F had higher fertility (73.5%) than those with lower rectal temperatures (98.8°F to 101.6°F). Fallon speculated that cows with a lower rectal temperature may have been out of estrus or were too close to ovulation when AI was performed. However, because the pregnancy outcomes (60.2%) in the cows with a lower rectal temperature were similar to those in other reported cow groupings observed in estrus and bred at different times, it is also plausible that HEAT was functionally relevant and impactful. The positive benefits of elevated body temperature are not limited to dairy cattle. Recently, our laboratory reported that a higher rectal temperature at AI in beef cattle subjected to a fixed-time AI protocol was related to a higher likelihood of pregnancy (Liles et al., 2022).

Nonetheless, for HEAT-related changes to be related to estrus activity, Laitano et al. (2010) reported that whole-body heat stress-induced oxidative stress in humans. When hyperthermia was combined with moderate-intensity exercise, however, a parallel increase in antioxidant defense compensated for hyperthermia-induced oxidative stress. Laitano et al. (2010) further speculated that elevated body temperature in combination with exercise likely enhances antioxidant defense by avoiding redox imbalance, thereby preventing cellular damage. Antioxidants are essential components within mammals, as they maintain oxidative homeostasis within the body (Mulla et al., 2018). Glutathione, an important antioxidant, is a major component of animal cells (Wu et al., 2004) and has properties that have been deemed essential to successful reproduction through maintaining reactive oxygen species levels (ROS; De Matos et al., 1997; Edwards et al., 2001; Zuelke et al., 2003).

Pertaining to our study, HEAT was positively related to different glutathione precursors (cysteine and glycine). Interestingly, cysteine alleviates the negative consequences of prolonged exposure of bovine oocytes to elevated temperature through the stimulation of glutathione production (De Matos et al., 1996; Luberda, 2005; Nabenishi et al., 2012). Glutathione levels fluctuate throughout the estrous cycle, with the highest concentrations at or around meiotic maturation (Sutovsky and Schatten, 1997; Brad et al., 2003; Zuelke et al., 2003). Not only is glutathione involved in events related to meiotic maturation and subsequent development, but its levels have also been linked to thermal responses. When a glutathione inhibitor was administered, the thermotolerance of various cell types decreased (Mitchell et al., 1983; Russo et al., 1984; Harris et al., 1991). Given that cold stress depletes glutathione levels and the inhibition of glutathione results in decreased thermotolerance, metabolite abundance changes associated with HEATs are more likely the result of a combination of activity-induced elevations in temperature.

Serine was the only metabolite that had a negative association with vaginal temperature at the onset of estrus when expressed as a change from the baseline. This metabolite is a non-essential amino acid that serves as a precursor for numerous molecules, including glutathione, taurine, and cysteine (Kalhan and Hanson, 2012). Turathum et al. (2021) suggested that oxidative stress is managed during oocyte maturation through the utilization of serine for glutathione synthesis within the developing oocyte. In mice, serine uptake within the oocyte reaches its maximum during the latter stages of metaphase I and then rapidly decreases once the oocyte reaches metaphase II (Zhang et al., 2020). Given its role in mediating ROS accumulation in the maturing oocyte and that elevated temperatures are known to promote ROS production, in retrospect, it is not surprising that HEAT may be negatively related to serine abundance in the follicular fluid of the preovulatory follicle.

The increased bioavailability of other metabolites highlights other potential HEAT-related consequences on intrafollicular metabolism important to support oocyte maturation and maintain oxidative balance. The amino acid taurine was positively associated with the vaginal temperature at estrus onset when expressed as a change from the baseline. Taurine is present in many mammalian tissues and is involved in numerous cellular functions. Specifically, it is a cytoprotective molecule with antioxidant properties that is also thought to be involved in the thermoregulatory process (Schaffer and Kim, 2018; Page et al., 2019). Toward this end, when humans engaged in intense exercise, the oral administration of taurine resulted in increased exercise endurance and temperature regulation in hotter environments (Page et al., 2019). While the impacts of taurine supplementation before the LH surge and during oocyte maturation are not clear, it is interesting to note that taurine positively impacts bovine embryo development under thermoneutral conditions (Dumoulin et al., 1992; Takahashi and Kanagawa, 1998).

The generation of heat is a natural and normal byproduct of cellular function and processes (Holtzclaw, 2001). In our study, during times of elevated body temperature, three pyrimidines (i.e., cytidine, uridine, and uracil) that have been implicated to play a role in body temperature increases (Cradock et al., 1986; Peters et al., 1987b) were detected in preovulatory follicular fluid. One of these metabolites (uracil) was HEAT-related. Notably, the administration of uracil’s precursor, uridine, has been shown to induce hyperthermia in rabbits, mice, and humans (Cradock et al., 1986; Peters et al., 1987b). Because uridine’s effect on body temperature is delayed, it has been postulated that uridine’s catabolites may be responsible for the induction of hyperthermia (Peters et al., 1987a; Peters et al., 1987b). The administration of uracil to rabbits increased their body temperature by 0.3°C (Peters et al., 1987b). The uracil levels in the follicular fluid collected ~ 14 h after estrus onset were negatively related to the vaginal temperature at first mount when expressed as change from the baseline, maximum vaginal temperature change from the baseline (VT _Max), and AUC₂, representing a combination of temperature increase and duration. These findings, while still unclear, are the first that we are aware of that associate pyrimidine metabolites with HEAT.

In instances of HEAT, it is not surprising for thermoregulatory and other processes to be impacted as the body strives for homeostasis during bouts of hyperthermia (reviewed by Godyń et al., 2019). The change in vaginal temperature at VT _Max was also positively related to follicular fluid uric acid levels. Although the significance of these findings in estrus-active females remains unclear, it is interesting to note that increases in exercise intensity in both horses and humans increase plasma uric acid levels (Green and Fraser, 1988; Räsänen et al., 1996). In humans, uric acid may in certain instances act as an antioxidant through its reduction of exercise-induced oxidative stress (Waring et al., 2003).

Regardless of related or contributive factors, the consequences of elevated body temperature are largely systemic in nature (Finch, 1986; Hahn, 1999). Whether HEAT-related differences in metabolite abundance in the follicular fluid of the preovulatory follicle ~14 h after estrus onset are reflective of changes occurring in the circulation or altered function of cells comprising the preovulatory follicle (i.e., granulosa and or cumulus–oocyte complex) remains unclear. Toward having a potential impact or a putative role, granulosa cells, which are contributors to follicular fluid components, have been shown to increase metabolism and amino acid production approximately 6 h after the LH surge (Gilbert et al., 2011).

To our knowledge, this is the first study to investigate the relationship between estrus-induced changes in body temperature and preovulatory follicular fluid metabolites. The significant outcomes related to temperature changes at or around the time of estrus onset are suggestive of a functional relevance of HEAT in driving the preparatory mechanisms involved in thermoregulation, energy metabolism, and oxidative stress management, and, in turn, possibly impacting oocyte competence and other preovulatory follicle components.

Although outside the scope of this study, it is interesting to note that the best-fit models derived to assess the impact of different aspects of HEAT on follicular fluid metabolomes included, in some instances, covariates related to estradiol and follicle size (see Supplementary Tables 4 – 6 ). We are only aware of one other study examining follicular fluid metabolomics in estrual cattle (Hessock et al., 2023), but its authors did not relate their findings to hormone levels or follicle size. In non-estrual cows, metabolites in preovulatory follicular fluid have been reported (Bender et al., 2010; Read et al., 2021; Read et al., 2022). Although estradiol concentration and follicle diameter have been found to be related to a number of follicular fluid metabolites (Read et al., 2021; Read et al., 2022), these findings do not overlap with those in this study. This is not entirely unexpected due to the dynamic changes occurring in the follicle around estrus and the inherent impact of those changes on systemic hormones.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The animal study was approved by Institutional Animal Care and Use Committee at the University of Tennessee, Knoxville. The study was conducted in accordance with the local legislation and institutional requirements.

Conception and design of study: JE, SM, and FS. Investigation/methodology: AP, EH, JE, SM, JK, RP, FS, and SC. Data and statistical analyses: AP, SM, JE, and RP. Writing—original draft, review, and editing: AP, JE, and SM. Review and editing: RP, EH, JK, FS, and SC. All authors contributed to the article and approved the submitted version.