Advanced periovulatory follicle progression in beef heifers: association with higher estrous-associated temperatures and estrous activity on intrafollicular levels of estradiol and metabolites

Abstract

Female cattle engaging in estrous activity exhibit varying levels of higher estrous-associated temperatures (H.E.A.T.) that are functionally impactful on fertility important components in the periovulatory follicle. To test this hypothesis, estrus was synchronized in virgin Angus heifers. Follicular fluid hormones and metabolites impacted by estrous activity (categorized as Estrous Active or Inactive), alone or in combination with H.E.A.T. were evaluated. Estrus was defined as the first time a heifer stood to be mounted; treatment was assigned alternately at first mount (comingled with others to remain Estrous Active, n = 13) or prevented from engaging in estrous active behaviors (Estrous Inactive, n = 12) by moving to an individual open rail pen, in sight of others and allowing nose-to-nose contact. Vaginal temperature was recorded using a Thermochron iButton. Periovulatory follicular fluid was aspirated ∼11.9 ± 0.1 h after first standing mount. Multiple linear regression was performed. Metabolite pathway enrichment analysis was conducted using Metaboanalyst 6.0. Estradiol levels (intrafollicular and serum) were negatively related to H.E.A.T. (P < 0.001). Intrafollicular estradiol was lower in Estrous Active heifers (P < 0.0001). Twelve follicular fluid metabolites were positively related to H.E.A.T. (P < 0.05). Pathways enriched with H.E.A.T.-related metabolites (FDR < 0.1) included “Pyrimidine metabolism.” Abundance of 25 other metabolites was lower in the periovulatory follicular fluid of Estrous Active versus Estrous Inactive heifers (P < 0.05). Pathways enriched with metabolites affected by estrous activity included “Arginine biosynthesis” and “Phenylalanine, tyrosine, and tryptophan biosynthesis.” Six out of 12 other metabolites interactively influenced by H.E.A.T. and estrous activity are known for their antioxidant properties. In summary, periovulatory follicle progression is more advanced in Estrous Active heifers and in those exhibiting higher levels of H.E.A.T. Intrafollicular metabolites in the more advanced periovulatory follicles are linked to thermoregulation, steroidogenesis, antioxidant defense, and energy metabolism. Results provide new insight into pivotal changes in the periovulatory follicle when estrogen dominance is coming to an end, the initial differentiative shift in granulosa cells is occurring, along with other preparatory changes that may be important for eventual ovulation and meiotic progression of the oocyte contained within.

Estrous activity, alone or in combination with higher estrous associated temperatures, advance periovulatory follicle progression. Results provide new insight into pivotal changes in the periovulatory follicle as estrogen dominance comes to an end and follicle cells begin to differentiate to eventually give rise to the corpus luteum.

Introduction

Follicular phase events leading up to and after the onset of estrus are critically important when striving for pregnancy in cattle. In this positive endocrine environment resulting from prostaglandin (PGF_2α)-induced luteolysis, increasing luteinizing hormone (LH) pulsatility drives preovulatory dominant follicle growth and further heightens estradiol production (Kojima 2003). After reaching a threshold level, estradiol induces estrous active behaviors and the massive gonadotropin releasing hormone (GnRH)-induced LH surge (Short et al. 1979; Stumpf et al. 1991) some 2 to 4 h after the onset of estrus (Dieleman et al. 1986; Stevenson et al. 1998). This short but pivotally transformative time signifies the end of estrogen dominance, sets the stage for the beginnings of changes toward corpus luteum formation, ovulation some 26 to 30 h thereafter (Saumande and Humblot 2005), and maturation of the oocyte resident within (Kruip et al. 1983) by inducing a myriad of intrafollicular biochemical, morphological, and endocrinological changes (Lipner 1988; Duffy et al. 2019).

While standing to be mounted is the most definitive sign of estrus, females engage in other estrous active behaviors. In their search for others, they may walk up to four times more than non-estrual females (Kiddy 1977). Forming sexually active groupings, estrual females may attempt to and mount others numerous times. They may also engage in head butting, chin resting, vulva sniffing, and other active behaviors (Kerbrat and Disenhaus 2004). This hormonally induced, heightened level of physical activity of a sexual nature has been positively related to cattle pregnancy outcomes (Yaniz et al. 2006; Madureira et al. 2015; Saini et al. 2023).

Body temperature of female cattle engaged in estrous active behaviors may become elevated by as much as 0.5 to 1.5 °C. When thermoneutral conditions predominated at the onset of a spring breeding season, these higher estrous-associated temperatures (H.E.A.T.) averaged 39.9 °C and persisted from 0.1 to 9.4 h in Angus heifers (Mills et al. 2024). In suckled Angus cows, H.E.A.T. averaged 40 °C and persisted from 0.4 to 24 h (Mills et al. 2024). Maximum H.E.A.T. often coincides with the LH peak (Higaki et al. 2019). In Fallon (1962), higher pregnancy outcomes were reported in estrual cows that had rectal temperatures ranging from 38.7 to 40.5 °C immediately before AI compared to those with rectal temperatures ranging from 37.1 to 38.6 °C (73.5% vs. 60.2%, respectively). Furthermore, in a fixed-timed AI protocol, a 1 °C increase in rectal temperature at the time of AI increased pregnancy odds 1.9 times in Bos indicus cows (Liles et al. 2022).

Because H.E.A.T. may persist for hours in cattle, direct effects on periovulatory follicle components are unavoidable and functionally impactful. Direct exposure to an acute elevation in temperature induces germinal vesicle breakdown sooner than oocytes matured under thermoneutral conditions (Hooper et al. 2015; Campen et al. 2018). Additional efforts of Klabnik et al. (2025) documented a shift in a targeted transcriptome where corresponding protein products are involved in or promote oocyte maturation, cumulus cell connectivity, and function (e.g. progesterone production) provide additional support for the heat-exposed cumulus oocyte complex to be more advanced. Lactating dairy cows experiencing an acute episode of heat stress-induced hyperthermia limited to a short time period after a pharmacologically induced LH surge had a 15-fold increase in abundance of intrafollicular kininogen, which is a precursor of kinin peptides that mediate inflammation, vasodilation and prostaglandin synthesis to set the stage for ovulation (Rispoli et al. 2019). In a different subset of cows, there were also transcripts upregulated in the cumulus enveloping the maturing oocyte and the periovulatory follicle granulosa cells (e.g. FYN proto-oncogene that promotes meiotic maturation; Calreticulin [CALR]) which potentiates ovulation (Klabnik et al. 2022).

Follicular fluid components in the periovulatory follicle, with origins from the mural granulosa, cumulus, other cell type secretions, and/or transudative from the circulation provide necessary substrates for follicle progression and oocyte competence (Rodgers and Irving-Rodgers 2010; Pan et al. 2024) and provide insight into the follicle’s physiological status. Different follicular fluid metabolome profiles have been related to fertility, follicle maturity, and oocyte metabolism (Read et al. 2021, 2022). Specific to changes occurring within the follicle during the 11 h after the onset of estrus, Hessock et al. (2023) noticed a rise in the abundance of 17 metabolites in the periovulatory follicular fluid, suggesting an increase in bioavailability of amino acids and metabolites that may be used by the cumulus surrounding the oocyte and the mural granulosa cells for energy production and shuttling to the oocyte to support meiotic maturation. Overlapping with this same time period, Pollock et al. (2023) identified several periovulatory follicular fluid metabolites involved in energy metabolism, thermoregulation, and oxidative stress management that were positively related to H.E.A.T. Interestingly, Li et al. (2025) demonstrated that heat stress, which is often coincident with elevated body temperature significantly alters follicular fluid metabolite abundance in dairy cows, particularly affecting pathways like steroid hormones biosynthesis.

Estrus induced or not, elevated body temperature is likely a by-product of increased metabolism and or muscle strain due to increased activity (humans, Gleeson 1998). While the impact of exercise-related changes in circulating metabolite abundance is undisputed in different species including cattle, horses and humans (Green and Fraser 1988; Rasanen et al. 1996; Mikami et al. 2000; Gleeson 2008; Luo et al. 2022), as is the occurrence of H.E.A.T. (Piccione et al. 2003; Sakatani et al. 2016), the literature remains scant pertaining to studies evaluating the impact of estrous activity in females exhibiting varying levels of H.E.A.T. on the periovulatory follicle fluid milieu. Pollock et al. (2023) while insightful about potential impact of H.E.A.T. on certain intrafollicular metabolites when periovulatory follicles were aspirated 14.9 ± 3.3 h after onset of estrus, level of estrous activity in the synchronized group of nonlactating Jersey cows utilized in that study was not recorded, nor did authors evaluate the relationship of H.E.A.T. on intrafollicular steroids. To address this critical knowledge gap and make progress toward testing our hypothesis related to functional significance of estrous activity and H.E.A.T, intrafollicular hormones and metabolites were compared in Angus beef heifers exhibiting varying levels of H.E.A.T. that were allowed to comingle with others in sexually active groups (Estrous Active) or prevented from engaging in estrous active behaviors (Estrous Inactive) by individual penning after 1^st observed standing mount.

Materials and methods

Institutional animal care and use approval at the University of Tennessee, Knoxville was obtained before study onset.

Animals, synchronization protocol, and study design

Virgin Angus heifers (n = 38) were maintained on a mixed grass pasture and had unrestricted access to hay and water. To synchronize estrus (Figure 1), PGF_2α was administered (25 mg i.m.; Lutalyse, Zoetis, Parsippany, NJ, USA). Eleven days later, GnRH was administered (100 µg i.m. Factrel, Zoetis) and a controlled internal drug release (CIDR) device, containing 1.38 g progesterone (Eazi-Breed CIDR, Zoetis) was inserted into the vagina. After seven days, the CIDR was removed, and PGF_2α was administered. Heifers were visually observed continuously by a team of experienced individuals for behavioral signs of estrus starting ∼24 h after PGF_2α was administered. Estrus was defined as the time when a heifer first stood to be mounted by another (n = 31 out of 38 total heifers displayed estrus; 81.6%). When estrual heifers first stood to be mounted by another, they were alternately assigned to one of two treatments per Figure 1. Treatments were 1) estrual heifers continued to be comingled with others to allow for sexually active groupings to increase engagement in estrous active behaviors (Estrous Active), or 2) estrual heifers were prevented-restricted from further engagement in estrous active behaviors by moving to an individual open 6-rail pen (12’ 2” wide × 16’ 3” long) in sight of others and allowing nose-to-nose contact with others (Estrous Inactive).

Figure 1.

Study Design. Schematic depicting protocol for synchronization of estrus (estrus was defined as the time when a heifer first stood to be mounted by another), study design (treatment allocation categorized as comingled for Estrous Active or individually penned for Estrous Inactive), blood sampling, ovarian ultrasound evaluations, follicular fluid aspiration, and timing related to when vaginal temperature was recorded every 5 min after prostaglandin F_2⍺ (PGF_2⍺) administration in beef heifers. Representative higher estrous associated temperature (H.E.A.T.) curve was derived after averaging each animal’s complete H.E.A.T. curve from the 11 h prior to maximum H.E.A.T. and up to 11 h after maximum H.E.A.T. GnRH: gonadotropin releasing hormone; CIDR: control internal drug release; BS: blood sample; US: ovarian ultrasound; SAGs: sexually active groups.

Ovarian ultrasound and periovulatory follicular fluid aspiration

The largest diameter of follicles > 5 mm was recorded utilizing an IBEX EVO® II ultrasound and an eL7 linear probe (E.I. Medical Imaging, Loveland, CO, USA) at CIDR removal and PGF_2α administration, at 1st mount, and 11.9 ± 0.1 h later when the follicular fluid was aspirated from the periovulatory follicle (Figure 1). Neat follicular fluid samples without macroscopic blood contamination were successfully collected from 26 out of the 31 estrual heifers by ultrasound-guided transvaginal aspiration using an IBEX EVO® and an eC9 OPU probe (Hessock et al. 2023; Pollock et al. 2023). Follicular fluid was centrifuged to eliminate cells and debris (2,000 x g for 10 min at 4 °C) then divided into different aliquots before being snap-frozen in liquid nitrogen and storage at −80 °C.

Serum and follicular fluid hormone assays

Blood samples were obtained from the coccygeal vein/artery at CIDR removal, when a heifer first stood to be mounted by another, and at follicle aspiration (Figure 1). After centrifugation, serum was stored at −80 °C until hormone assays were conducted. Serum estradiol (E₂) concentrations were determined by radioimmunoassay (Kirby et al. 1997); intra-assay coefficient of variation (CV) was 3.2%; assay sensitivity was 1.0 pg/mL. Follicular fluid was diluted 1:2500 before use of the DetectX® Serum 17β-Estradiol ELISA Kit (Arbor Assays, Ann Arbor, MI, USA); (Garcia-Guerra et al. 2018; Rispoli et al. 2019). Sensitivity of assay was 2.2 pg/mL. The intra- and inter-assay CVs for follicular fluid E₂ were 2.1% and 3.9%. Serum and intrafollicular fluid progesterone concentrations were measured using the Progesterone Double Antibody RIA kit from MP Biomedicals (Solon, OH, USA) previously validated by Pohler et al. (2016). Follicular fluid was diluted 1:20. Assay sensitivity was 0.1 ng/mL. Intra- and inter-assay CVs for serum progesterone were 4.3% and 7.4%, respectively. Intrafollicular fluid progesterone intra-assay CV was 3.5%.

Vaginal and ambient temperature assessment

Vaginal temperature was recorded using a Thermochron iButton 1922 L data logger (Embedded Data Systems, Lawrenceburg, KY, USA) affixed to a blank CIDR (Pollock et al. 2023; Mills et al. 2024) at 5-min intervals (0.065 °C resolution) starting 12 h after iButton insertion at PGF_2α and continued through iButton removal at follicle aspiration (Figure 1). Ambient temperature and humidity were recorded hourly using the HOBO U23 Pro v2 data logger (Onset Computer Corporation, Bourne, MA, USA) from CIDR removal/PGF_2α administration until the removal of the last iButton (Figure 2). Temperature humidity index (THI) was calculated per Abbott et al. (2018).

Figure 2.

Ambient conditions. Hourly ambient temperature, relative humidity, and temperature humidity index (THI) from PGF_2⍺ administration to follicle aspiration. The red diamonds denote the time when each of the estrual heifers first stood to be mounted by another. THI was calculated per Abbott et al. (2018).

Baseline and higher estrous associated temperature

A baseline temperature was calculated for each heifer by averaging vaginal temperatures starting 12 h after PGF_2α administration and continuing up to the next 11 h. The start of the H.E.A.T. in estrual heifers was defined as when vaginal temperature went above baseline by 0.1 °C and continued to increase thereafter; H.E.A.T. end was defined as the time when vaginal temperature returned to baseline or iButton was removed. Different aspects specific to the H.E.A.T. in Estrous Active and Estrous Inactive heifers are provided in Tables 1 and 2. To account for the magnitude and persistence of estrous related temperatures, H.E.A.T. was estimated and defined by calculating the total area under the H.E.A.T. curve (AUC start to end, Table 1) per Pruessner et al. (2003) using the trapezoid formula with baseline temperature for individual heifers as the lower limit.

Table 1.

Descriptive data related to higher estrous associated temperatures (H.E.A.T.) in beef heifers (average ± SD) that were comingled (Estrous Active) or restricted from engaging in estrous active behaviors (Estrous Inactive) after first mount, ambient conditions during study, and baseline vaginal temperature duration.

H.E.A.T. Variables	Definition	Estrous Active1	Estrous Inactive2
Number of Heifers		13	12
Vaginal Temp, °C
1st Mount VTp3	VTp at first observed mount	39.1 ± 0.5	39.4 ± 0.5
Baseline VTp	Baseline Vaginal Temperature	38.5 ± 0.2	38.5 ± 0.2
*Baseline VTp Change	1st Mount VTp minus Baseline	0.6 ± 0.4	0.9 ± 0.4
Max H.E.A.T. VTp	Maximum H.E.A.T.	39.8 ± 0.5	39.9 ± 0.5
Baseline change	Maximum H.E.A.T. minus Baseline	1.2 ± 0.5	1.4 ± 0.3
Average VTp	1st Mount to H.E.A.T. End	39.2 ± 0.4	39.0 ± 0.3
Average VTp	H.E.A.T. Start to H.E.A.T. End	39.1 ± 0.3	39.0 ± 0.3
Duration, h
*H.E.A.T. start to max	H.E.A.T. Start to Maximum H.E.A.T.	5.6 ± 3.6	9.1 ± 4.4
Max to H.E.A.T. end4	Maximum H.E.A.T. to H.E.A.T. End	7.5 ± 3.9	8.7 ± 3.2
1st Mount to max	1st Mount to Maximum H.E.A.T.	1.5 ± 4.3	0.1 ± 2.8
*H.E.A.T. length	H.E.A.T. Start to H.E.A.T. End	13.1 ± 3.9	17.8 ± 3.6
Time above 39.5 °C	Time VTp Above 39.5 °C	4.6 ± 2.6	3.3 ± 3.1
Time above 40 °C	Time VTp Above 40 °C	2.5 ± 2.9	1.2 ± 1.8
Area under the curve
AUC start to max	H.E.A.T. Start to Max H.E.A.T.	43.9 ± 37.0	57.4 ± 27.0
AUC max to end	Max H.E.A.T. to H.E.A.T. End	69.1 ± 42.9	70.8 ± 35.1
AUC start to end	H.E.A.T. Start to H.E.A.T. End	113.0 ± 59.5	128.1 ± 38.0
AUC 1st mount to end	1st Mount to H.E.A.T. End	90.9 ± 59.6	71.6 ± 37.4
AUC ≥ 39.5 °C	Above 39.5 °C	78.7 ± 53.3	54.2 ± 51.2
Ambient conditions
THI5 12 h before Max	Average THI 12 h before Max	65.8 ± 0.2	65.5 ± 4.4
THI CIDRout to Asp	Average THI CIDRout to Aspiration	62.5 ± 0.8	62.3 ± 0.8
Other
Baseline duration	PGF2α to H.E.A.T. Start	52.3 ± 13.9	48.1 ± 15

¹Estrous Active: Estrual heifers comingled with others to allow for sexually active groupings.

²Estrous Inactive: Estrual heifers prevented from engaging in estrous active behaviors by individual penning.

³VTp: Vaginal temperature.

⁴H.E.A.T. End: When vaginal temperature returned to baseline or when follicle was aspirated.

⁵THI: Temperature humidity index.

^*Baseline VTp Change at 1st Mount, H.E.A.T. Start to Max, H.E.A.T. Length significantly differs (P < 0.05) between Estrous Active versus Estrous Inactive heifers.

Table 2.

Higher estrous associated temperatures (H.E.A.T.), Max, Length, and time above 39.5 °C and 40 °C in estrual heifers where follicular fluid was successfully aspirated.

ID	Baseline VTp1, °C	Trt2	1st Mount VTp, °C	THI3 at 1st Mount	Max VTp, °C	H.E.A.T. Length, h	≥ 39.5°C, h	≥ 40°C, h
18	38.6	EA4	39.0	73.1	40.7	14.3	9.3	6.5
14	38.6	EA	40.0	68.0	40.4	18.6	7.4	3.0
15	38.8	EA	39.7	63.4	40.3	12.3	5.8	0.5
29	38.5	EA	39.0	70.4	40.0	17.4	3.1	0.2
31	38.4	EA	38.8	73.1	39.8	14.3	5.9	…
26	38.8	EA	39.4	57.0	39.8	10.3	3.8	…
16	38.2	EA	39.4	68.1	39.7	14.9	2.0	…
8	38.5	EA	39.1	69.8	39.6	13.3	2.2	…
22	38.7	EA	39.5	55.4	39.6	16.6	2.3	…
5	38.6	EA	38.8	59.7	39.3	13.2	…	…
20	38.3	EA	38.4	59.5	39.1	8.0	…	…
3	38.7	EA	38.7	57.8	39.1	3.9	…	…
9	38.3	EA	38.8	59.6	39.0	13.2	…	…
30	38.7	EI5	40.3	59.2	40.8	20.3	9.4	4.8
10	38.6	EI	39.2	64.2	40.2	17.1	6.6	1.2
23	38.7	EI	39.7	57.7	40.2	17.0	4.9	0.3
17	38.5	EI	40.2	64.2	40.1	16.4	4.0	0.6
4	38.6	EI	39.9	68.0	40.0	15.3	2.3	0.1
21	38.6	EI	39.7	59.6	40.0	13.7	4.3	0.1
1	38.4	EI	39.4	68.0	39.7	14.6	1.2	…
27	38.3	EI	39.1	53.0	39.5	18.8	0.1	…
11	38.4	EI	38.9	57.8	39.5	15.9	0.1	…
13	38.4	EI	38.8	57.8	39.5	27.5	0.1	…
25	38.4	EI	38.9	73.1	39.3	18.6	…	…
2	38.2	EI	39.0	59.6	39.3	18.4	…	…

¹VTp: Vaginal Temperature.

²Trt: Treatment.

³THI: Temperature humidity index.

⁴Estrous Active: Estrual heifers comingled with others to allow for sexually active groupings.

⁵Estrous Inactive: Estrual heifers restricted from engaging in estrous active behaviors by individual penning.

Metabolome profiling of the periovulatory follicular fluid of estrual heifers

Ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) of periovulatory follicular fluid samples was performed by The University of Tennessee Knoxville Biological and Small Molecule Mass Spectrometry Core Facility (RRID: SCR_021368) as previously described by Horn et al. (2022) and Pollock et al. (2023). One follicular fluid sample out of the 26 total submitted for metabolomic analysis was omitted because E2/P4 ratio was low (0.09).

Heifer and other descriptive data and statistical analyses

Descriptive data highlighting the equivalence of heifers used in this study before (e.g. at CIDRout) and at 1st mount when treatments were alternatively assigned (comingled-Estrous Active, n = 13 or individually penned-Estrous Inactive, n = 12) are provided in Table 3 (i.e. animal, hormonal, follicle size, and proestrus length data). In other words, no significant differences existed between heifers at treatment assignment for any of the variables recorded, P ≥ 0.22.

Table 3.

Descriptive data highlighting the equivalence^* for heifers used in this study before (e.g. at CIDRout) and at 1st mount when treatments were alternatively assigned (comingled-Estrous Active or individually penned-Estrous Inactive).

Variables	Assigned treatment estrous active	Assigned treatment estrous inactive
Number of Heifers	13	12
Age1, d	458.5 ± 45.3	449.2 ± 37.6
Weight, kg	355.5 ± 44.6	376.0 ± 44.2
1st Mount to follicle aspiration, h	12.0 ± 0.3	11.8 ± 0.5
Size & Growth of aspirated follicle, mm
CIDRout Ov12	10.6 ± 1.7	9.7 ± 2.0
1st Mount Ov1	13.3 ± 1.5	14.3 ± 3.3
Serum E2, pg/mL3
CIDRout E2	1.9 ± 1.0	2.7 ± 1.7
1st Mount E2	9.1 ± 1.6	9.4 ± 0.9
Serum P4, ng/mL4
CIDRout P4	8.4 ± 4.2	7.9 ± 5.0
1st Mount P4	0.4 ± 0.2	0.5 ± 0.3
Other
Proestrus length (h from PGF2α5 to 1st Mount)	56.3 ± 14.4	57.1 ± 16.5

¹Age: Age at CIDR insertion.

²Ov1: Preovulatory follicle size.

³E₂: 17 β-Estradiol.

⁴P₄: Progesterone.

⁵PGF_2α: ProstaglandinF_2α.

^*No significant differences existed between heifers when they were alternately assigned to treatment for any of the variables recorded, P ≥ 0.22.

Statistical analyses were conducted using R Studio (R version 4.3.1 [2023-06-16 ucrt]). Graphical assessment of linearity was performed using partial residual plots (Larsen and McCleary 1972; Moya-Larano and Corcobado 2008; Schneider et al. 2010). Data normality was checked using Shapiro–Wilk test and log-transformed when warranted. Outliers were identified using the interquartile range method. Collinearity was assessed using Variance Inflation Factor (VIF), a VIF ≥ 5 was used to identify collinear variables. To avoid issues related to collinearity in linear regression models, separate models were derived for each collinear variable. To examine the relationship between H.E.A.T. (independent variable), assess the impact of Estrous Activity (independent variable and categorized as Estrous Active versus Inactive) and consider the possibility of an interaction among them (H.E.A.T. × Estrous Activity) on intrafollicular and circulating steroids and intrafollicular metabolite abundance (dependent variables), multiple linear regression models were utilized using a backward stepwise approach, specifically taking low Akaike Information (AIC) and R² values into consideration, to derive best-fit models. Other covariates used to derive best-fit models were included in the model only if deemed significant in an initial simple linear regression model (P < 0.1). When intrafollicular and steroid hormones were used as the dependent variable, covariates included age, weight, baseline vaginal temperature and its duration, follicle size at aspiration and the change from first mount to aspiration, THI and hours from first mount to aspiration. When metabolite abundance was used as the dependent variable, covariates included age, weight, baseline vaginal temperature and duration, proestrus length (h from PGF_2α to 1st Mount), and serum steroids concentrations. Serum progesterone at CIDR removal and aspiration along with their change were considered, as well as estradiol concentrations at CIDR removal, 1st mount and aspiration, and their respective changes (CIDR removal to first mount, first mount to aspiration, and CIDR removal to aspiration). Follicle size at aspiration and its change from first mount and CIDR removal to aspiration were also considered. Additionally, intrafollicular estradiol, progesterone, their ratio, and THI were included as covariates. These were covariates that could have affected response variables but were not of direct interest to this study. The final models included H.E.A.T., Estrous Activity (categorized as Estrous Active or Inactive), H.E.A.T. x Estrous Activity interaction, and any significant covariates (P ≤ 0.05). Related to the backwards stepwise approach, H.E.A.T., Estrous Activity, and the respective interaction, which were a priori, remained in the model until all remaining other covariates were significant. Afterwards, H.E.A.T. or Estrous Activity when not significant, but starting with the interaction, were removed from the model in a stepwise manner until only significant variables remained.

Results

Relatedness of H.E.A.T. and the impact of estrous activity on follicle size, intrafollicular, and serum hormones

Intrafollicular and serum levels of estradiol ∼11.9 ± 0.1 h after a heifer first stood to be mounted by another were negatively associated with H.E.A.T. (Table 4; Figure 3). Per each unit increase in the total area under the H.E.A.T. curve, intrafollicular estradiol decreased by 1.4009 ng/mL (P = 0.0003); whereas serum estradiol decreased by 0.0366 pg/mL (P = 0.0058). Interestingly, Estrous Active heifers had lower intrafollicular estradiol levels compared to Estrous Inactive heifers (233 versus 388 ± 19.8 ng/mL; P = 0.0001; Table 4; Figure 3). Neither the total area under the H.E.A.T. curve nor Estrous Activity affected follicle size at aspiration, change in follicle size from 1st mount to aspiration, intrafollicular progesterone, intrafollicular estradiol/progesterone ratio, and serum progesterone levels at aspiration (Table 4).

Table 4.

Relatedness of higher estrous associated temperatures (H.E.A.T.) and the impact of Estrous Activity (categorized as Estrous Active or Inactive): periovulatory follicle size, intrafollicular, and serum hormones.

Dependent variables	Independent variables	P-value	Slope or LS Means ± SEM for Estrous Active1 and Estrous Inactive2 heifers, respectively
Follicle Size, mm
At aspiration3	H.E.A.T.	0.4437	0.0065
	Estrous Activity	0.9372	14.0 and 14.1 ± 0.6
	H.E.A.T. x Estrous Activity	0.1536	—
Change 1st Mt-aspiration	H.E.A.T.	0.6316	−0.0041
	Estrous Activity	0.9567	0.61 and 0.66 ± 0.6
	H.E.A.T. x Estrous Activity	0.8382	—
Intrafollicular hormones
Estradiol (E2)	H.E.A.T.	0.0003	−1.4009
	Estrous Activity	0.0001	233 versus 388 ± 19.8
	H.E.A.T. x Estrous Activity	0.2063	—
Progesterone (P4)	H.E.A.T.	0.4766	−0.2562
	Estrous Activity	0.2354	142 versus 183 ± 23.6
	H.E.A.T. x Estrous Activity	0.6769	—
E2/P4 Ratio	H.E.A.T.	0.6231	−0.0037
	Estrous Activity	0.1396	1.91 and 3.01 ± 0.5
	H.E.A.T. x Estrous Activity	0.3583	—
Serum Hormones
E2 at Aspiration	H.E.A.T.	0.0058	−0.0366
	Estrous Activity	0.2489	7.0 and 8.31 ± 0.8
	H.E.A.T. x Estrous Activity	0.2298	—
P4 at Aspiration	H.E.A.T.	0.1149	−0.0013
	Estrous Activity	0.3420	0.29 and 0.22 ± 0.05
	H.E.A.T. x Estrous Activity	0.3279	—

¹Estrous Active: Estrual heifers comingled with others to allow for sexually active groupings.

²Estrous Inactive: Estrual heifers prevented from engaging in estrous active behaviors after 1st mount by individual penning.

³At aspiration: Follicle size at aspiration at 11.9 ± 0.1 h after 1st mount.

Figure 3.

Partial regression plots of the negative relationship of H.E.A.T. with follicular fluid (A) and serum (B) levels of estradiol (n = 25) and the impact of estrous activity on follicular fluid (C) and serum (D) levels of estradiol (n = 13 Estrous Active and n = 12 Estrous Inactive).

Follicular fluid metabolites in estrual heifers

A total of 106 metabolites were identified in follicular fluid collected 11.9 ± 0.1 h after estrual heifers were first observed to stand to be mounted by another (Table S1). Periovulatory follicular fluid metabolites included amino acids, amino acid precursors and derivatives, bile acids and bile salts, carbohydrates and carbohydrate conjugates, lipids and lipid-like molecules, nucleosides, nucleotides and analogues, and vitamins. Several of the identified metabolites are known to be involved with amino acid metabolism, glycolysis and gluconeogenesis, the pentose phosphate pathway, and the TCA cycle.

Follicular fluid metabolites relating to H.E.A.T

The total area under the H.E.A.T. curve was associated with the differential abundance of 12 of the 106 metabolites (11.3%). Eleven metabolites had a positive relationship with the total area under the H.E.A.T. curve (in order of significance: phosphorylethanolamine, orotate, thymine, uridine, indole, glutathione disulfide, cytidine, pantothenate, uracil, ribose phosphate and 2-hydroxy-2-methylsuccinate), and one metabolite, cystine, had a negative relationship (P ≤ 0.05; Figure 4). One KEGG pathway was enriched with follicular fluid metabolites related to the total area under the H.E.A.T. curve: “Pyrimidine metabolism” (FDR = 0.0001; Table 5).

Figure 4.

Metabolites Associated with H.E.A.T. Partial regression plots for the final model for each metabolite related to H.E.A.T. Plots depict the relationship between H.E.A.T. and metabolite abundance while adjusting for additional variables included in the final model (n = 25).

Table 5.

KEGG pathway significantly enriched with follicular fluid metabolites that differed in abundance based on higher estrous associated temperatures (H.E.A.T.).

Pathway ID	Pathway name	FDR1	Match Status2	Differentially abundant metabolites in pathway
bta00240	Pyrimidine metabolism	0.00013268	5/38	C00299 (Uridine)
				C00475 (Cytidine)
				C00178 (Thymine)
				C00295 (Orotate)
				C00106 (Uracil)

¹FDR: False discovery rate.

²Match Status: Number of significant metabolites in pathway/total number of metabolites in pathway.

Follicular fluid metabolites impacted by estrous activity (estrous active versus estrous inactive)

Abundance of 25 follicular fluid metabolites was impacted by Estrous Activity (P ≤ 0.05; Table 6). All metabolites were lower in the periovulatory follicular fluid of Estrous Active heifers. Two KEGG pathways were enriched with follicular fluid metabolites associated with Estrous Activity: “Arginine biosynthesis” and “Phenylalanine, tyrosine, and tryptophan biosynthesis” (FDR ≤ 0.1; Table 7).

Table 6.

Abundance¹ of periovulatory follicular fluid metabolites impacted by estrous activity (least square means ± SEM).

Metabolites	Estrous Active2	Estrous Inactive3	P-value
Glutamine	637,946 ± 75,211	1,082,893 ± 78,282	0.0004
Arginine	36,760 ± 2,451	49,602 ± 2,552	0.0015
Carnitine	4,699 ± 277	6,047 ± 288	0.0027
Glycine	267,062 ± 19,476	352,871 ± 21,173	0.0071
3-Methylphenylacetic acid	218,799 ± 39,710	388,973 ± 41,333	0.0071
D-Glucarate	4,467 ± 748	7,587 ± 779	0.0086
Citrate/isocitrate	2,900,684 ± 925,989	6,950,502 ± 1,010,033	0.0086
1-Methylhistidine	4,138 ± 402	5,787 ± 418	0.0095
Xylose	119,807 ± 10,114	159,632 ± 10,114	0.0115
Phenylalanine	1,255,916 ± 136,414	1,799,980 ± 148,297	0.0134
Asparagine	67,343 ± 7,287	96,014 ± 7,616	0.0139
Allantoin	705,347 ± 92,051	1,024,920 ± 95,813	0.0251
Tyrosine	510,270 ± 48,221	679,655 ± 52,422	0.0269
Aminocaproic acid	3,348,058 ± 355,889	4,579,830 ± 386,892	0.0290
Methionine	157,095 ± 12,624	200,753 ± 13,724	0.0292
Leucine/Isoleucine	3,340,217 ± 355,202	4,567,608 ± 386,146	0.0293
N-Acetylglutamine	3,256 ± 474	4,768 ± 493	0.0378
2-Dehydro-D-gluconate	86,601 ± 14,530	132,689 ± 15,124	0.0389
Lysine	13,332 ± 1,196	17,075 ± 1,196	0.0399
Allantoate	15,273 ± 2,514	23,090 ± 2,617	0.0425
D-Gluconate	278,895 ± 41,411	406,928 ± 43,104	0.0436
Ascorbate	23,190 ± 3,336	33,755 ± 3,479	0.0437
N-Acetylornithine	41,054 ± 6,745	61,358 ± 7,020	0.0489
Uric acid	1,390,877 ± 201,532	1,996,209 ± 209,768	0.0494
Cholesterol sulfate	12.3 ± 0.155	12.8 ± 0.161	0.0506

¹Abundance: Peak area.

²Estrous Active: Estrual heifers comingled with others to allow for sexually active groupings.

³Estrous Inactive: Estrual heifers prevented from engaging in estrous active behaviors after 1^st mount by individual penning.

Table 7.

KEGG pathways significantly enriched with follicular fluid metabolites impacted by estrous activity.

Pathway ID	Pathway name	FDR1	Match Status2	Differentially abundant metabolites in pathway
bta00220	Arginine biosynthesis	0.030916	3/14	C00062 (L-Arginine)
				C00437 (N-Acetylornithine)
				C00064 (L-Glutamine)
bta00400	Phenylalanine, tyrosine and tryptophan biosynthesis	0.030916	2/4	C00079 (L-Phenylalanine)
				C00082 (L-Tyrosine)

¹FDR: False discovery rate.

²Match Status: Number of significant metabolites in pathway/total number of metabolites in pathway.

Follicular fluid metabolites interactively influenced by H.E.A.T. and estrous activity (estrous active versus estrous inactive)

Relatedness of the total area under the H.E.A.T. curve with 12 follicular fluid metabolites differed depending on Estrous Activity (H.E.A.T. × Estrous Activity, P ≤ 0.05, Table 8; Figure 5). Interestingly, the total area under the H.E.A.T. curve was positively related to the abundance of six metabolites in the follicular fluid of Estrous Inactive but not in Estrous Active heifers (taurodeoxycholate, taurine, 2-oxo-4-methylthiobutanoate, homoserine/threonine, citrulline and tryptophan; Table 8). In contrast, the total area under the H.E.A.T. curve was negatively related to the abundance of four other metabolites (aconitate, deoxycytidine, serine, citraconate) in Estrous Inactive but not in Estrous Active heifers (Table 8). Only one metabolite interactively influenced by the total area under the H.E.A.T. curve and Estrous Activity (i.e. CMP) was positively related to the total area under the H.E.A.T. curve in Estrous Active heifers (Table 8) but was not in Estrous Inactive heifers.

Table 8.

Periovulatory follicular fluid metabolites interactively influenced by higher estrous associated temperatures (H.E.A.T.) and estrous activity.

Metabolites	Interaction H.E.A.T. × Estrous activity P-value1	Estrous Active H.E.A.T. P-value2	Estrous Active H.E.A.T. Slope3	Estrous Inactive H.E.A.T. P-value4	Estrous Inactive H.E.A.T. Slope5
H.E.A.T. (+) Associated with metabolite abundance in estrous inactive heifers
Taurodeoxycholate	0.0004	0.0711	−1640.3	0.0011	6,147.0
Taurine	0.0038	0.6025	183.6	0.0005	2,350.4
2-Oxo-4-methylthiobutanoate	0.0069	0.9224	16.5	0.0016	946.9
Homoserine/Threonine	0.0245	0.8842	−73.1	0.0119	2,228.4
Citrulline	0.0318	0.9051	−72.2	0.0151	2,606.2
Tryptophan	0.0437	0.2969	1106.0	0.0044	5,296.0
Glycodeoxycholate	0.0433	0.1256	−2669.0	0.1249	5,042.0
H.E.A.T. (−) Associated with metabolite abundance in estrous inactive heifers
Aconitate	0.0119	0.7460	607.2	0.0099	−13,561.0
Deoxycytidine	0.0143	0.2713	28.4	0.0236	−158.4
Serine	0.0184	0.7131	48.4	0.0161	−822.4
Citraconate	0.0222	0.7336	135.2	0.0197	−2,556.0
H.E.A.T. (+) Associated with metabolite abundance in estrous active heifers
CMP	0.0092	0.0017	22.7	0.2505	−12.1

¹Interaction H.E.A.T. × Estrous activity P-values in order of significance per each category.

² Estrous Active H.E.A.T. P-value for periovulatory follicular fluid metabolites in comingled heifers to allow for sexually active groupings.

³ Estrous Active slope: Metabolite slope for comingled heifers.

⁴ Estrous Inactive H.E.A.T. P-value for follicular fluid metabolites in heifers prevented from engaging in estrous active behaviors after 1st mount by individual penning.

⁵ Estrous Inactive Metabolite slope for restricted heifers.

Figure 5.

Metabolites that were interactively influenced by H.E.A.T. and Estrous Activity categorized as Estrous Active (EA) and Estrous Inactive (EI). Predictions were generated from the final models while controlling for additional variables. Interaction (Int) P-values and slopes for EA and EI provided per each metabolite (n = 25).

Discussion

This is the first study we are aware of to highlight an impact of estrous activity (active or not) and H.E.A.T. on the periovulatory follicular fluid milieu sufficient to impact follicle progression. To this end, follicle progression was more advanced in estrous active females and was also positively related with H.E.A.T. Concentration of steroids in our study are within the range of what is expected for dominant follicles transitioning into the differentiative period after an LH surge (Fortune and Hansel 1985). Decreases in intrafollicular and circulating levels of estradiol are expected within 6 h after the LH surge and continue thereafter (Murdoch and Dunn 1982; Dieleman et al. 1986). In the current study, where the periovulatory follicle was aspirated ∼12 h after the onset of estrus or ∼8 h after the LH surge may have occurred (Dieleman et al. 1986; Stevenson et al. 1998), higher levels of H.E.A.T. were negatively related to intrafollicular and serum levels of estradiol. Intrafollicular estradiol was lower in the periovulatory follicle of estrous active heifers. Differences in follicle progression are most likely attributable to the physiological differences that go along with H.E.A.T. and continuing to be estrous active because the negative relationship of H.E.A.T. with intrafollicular and circulating levels of estradiol was observed when analysis of estrous active and estrous inactive heifers was run separately. Estrous inactive heifers were maintained separately in open six rail pens where nose-to-nose contact with others was always possible. Although serum concentrations of estradiol were not statistically different, levels decreased from 9.1 to 7.1 and 9.4 to 8.1 pg/mL, respectively, in estrous active and inactive heifers (from first mount to follicle aspiration; ∼11.9 ± 0.1 h). Estrous active heifers were likely more stressed as some estrual heifers may be mounted up to 90+ times depending on the number in a sexually active group (Kenny and Tarrant 1988; Stevenson et al. 1996). Mindful of this, it is unsurprising that cortisol-related increases during the peak of estrus, which is around the same time as the peak levels of estradiol and estrous activity occur (Lyimo et al. 2000). Whether the level of H.E.A.T. or estrous activity results in subtle differences in the timing of the LH surge remains unclear but possible. In support of this notion, intrafollicular ascorbic acid levels in our study were lower in the more advanced periovulatory follicle of estrous active heifers. Ovarian ascorbic acid depletion has been used as a measure of LH activity (Reichert 1966). In rats, Guarnaccia et al. (2000) showed that LH administration depleted ascorbic acid levels in the preovulatory follicles.

During the design of this study, the possibility for increased sexual activity in females approaching estrus onset to increase vaginal temperature was acknowledged. However, treatment allocation at the onset of estrous activity (i.e. visually observed to be mounted at least once) was prioritized to ensure that follicular aspiration was only performed in heifers exhibiting estrus. Treatment allocation at this pivotal time while necessary contributed to the overall parameters of H.E.A.T. not to differ very much between Estrous Active and Estrous Inactive heifers. For instance, average vaginal temperatures of Estrous Active and Inactive heifers were 39.1 and 39.4 + 0.5 C at 1st Mount, an increase of 0.6 to 0.9 + 0.4 C above a baseline vaginal temperature of 38.5 + 0.2 C. Also, maximum H.E.A.T. occurred soon after 1st mount in Estrous Active and Estrous Inactive heifers. Even so, it is notable that none of the follicular fluid metabolites related to H.E.A.T. (n = 12) or impacted by Estrous Activity (n = 25) overlapped nor did the metabolites that were interactively influenced by H.E.A.T. and Estrous Activity (n = 12).

Interestingly, the abundance of several intrafollicular metabolites implicated in steroidogenesis (directly or indirectly) differed in the periovulatory follicular fluid of Estrous Active versus Estrous Inactive heifers (e.g. glutamine, cholesterol sulfate, arginine, ascorbic acid). Based on the findings of others described below, potential consequences or involvement of these metabolites in the periovulatory follicle’s progression toward the end of estrogen dominance cannot be ruled out. For instance, glutamine levels were lower in the periovulatory follicle of estrous active heifers having lower levels of estradiol. Higher levels of intrafollicular estradiol have been coincident with higher glutamine levels in follicles in women undergoing in vitro fertilization (IVF) treatment (Wang et al. 2022). Additional effort in that study showed that adding glutamine to KGN cells, a human granulosa tumor cell line, increased estradiol, and estradiol concentration was reduced in the glutamine-deprived treatment group (Wang et al. 2022). Cholesterol sulfate levels, which were higher in the periovulatory follicular fluid of estrous inactive heifers, have been inversely related to rate of pregnenolone synthesis in adrenal cells (Xu and Lambeth 1989). Tsutsumi et al. (2008) showed that cholesterol sulfate induced inhibition of progesterone in human granulosa-like tumor cells through downregulation of StAR and P450scc mRNA. Others have shown that when arginine was added to bovine granulosa cells in vitro, hormone-induced estradiol production was suppressed to some extent (Feng et al. 2018). In cultured human luteal cells, L-arginine inhibited aromatase activity (Johnson et al. 1999). Finally, physiologically relevant ascorbic acid levels lowered aromatase activity in mutant rats without impairing responsiveness to gonadotropins (Tsuji et al. 1989). In 1990, Luck et al. showed that supplementation of bovine cultured granulosa cells with ascorbic acid promoted higher levels of estradiol in response to higher levels of testosterone. Although it remains unclear if any of these metabolites (glutamine, cholesterol sulfate, arginine, ascorbic acid) directly or indirectly affected estradiol production in the periovulatory follicle of estrual females, differential follicular fluid abundance in estrous inactive and estrous active heifers provides additional support that the periovulatory follicle is more advanced in females exhibiting estrous activity.

Several of the periovulatory follicular fluid metabolites that were positively related to H.E.A.T. (uridine, uracil, orotate, cytidine, thymine) were significantly enriched in the pyrimidine metabolism pathway, including the biosynthesis of pyrimidines (ribose 5-phosphate and glutamine; Tozzi et al. (2006); Walter and Herr (2022)). Pyrimidine biosynthesis is not only critical for multiple cellular processes but is involved in the promotion of tumor formation (He et al. 2022). This finding alone is unsurprising given that the cellular components of the periovulatory follicle ∼ 12 h after onset of estrus are in the initial phase of transformative changes to not only end estrogen dominance but undergo other important changes to “set the stage” for eventual corpus luteum formation. However, when this finding is coupled with the new finding highlighting a positive relationship of intrafollicular phosphoethanolamine with H.E.A.T. these outcomes are not only insightful but may be functionally relevant. In support of this notion, transformative events that are involved in corpus luteum formation have been likened to tumor development (Smith et al. 1994). In luteal cells, phospholipids, including phosphoethanolamine, have been shown to play a role in gonadotropin-receptor binding and receptor activity (Azhar et al. 1976). In other cell types, accumulation of phosphoethanolamine has been related to tumor growth under nutrient (i.e. glutamine) restriction (Osawa et al. 2019). Interestingly, glutamine levels were lower in periovulatory follicles in estrous active heifers. Although speculative in terms of a presumptive role of either of these two metabolites in the initial transition of the mural granulosa toward becoming luteal cells, estrous activity is functionally impactful on periovulatory follicle components with effects persisting well after estrus ceases. Higher progesterone concentrations 7 d after insemination have been reported in lactating dairy cows exhibiting higher intensity of estrous expression (induced and spontaneous); Madureira et al. (2021). Saini et al. (2023) also reported a positive correlation between intensity of estrous response using a scoring system described by Layek et al. (2011) and luteal phase progesterone concentrations 12 d after AI.

Although the impacts of H.E.A.T. and estrous activity on the cumulus oocyte complex were not examined in our study, intrafollicular changes described herein could be impactful. Glutamine addition to in vitro maturation medium was stimulatory to nuclear maturation of bovine oocytes (Bilodeau-Goeseels 2006) as was the addition of ascorbic acid to denuded porcine oocytes (Tao et al. 2004). Several of the metabolites impacted by estrous inactivity have been related to exercise physiology and thermoregulation (e.g. allantoin, arginine, glycine, methionine, asparagine, tyrosine, leucine, uric acid). This is unsurprising since heightened levels of physical activity in estrual females can be likened to an intense form of exercise when planned by synchronization and where mounting activity is one of the major goal-oriented changes in physical activity. Level of H.E.A.T. varies among individual animals (Mills et al. 2024). Higher serum levels of allantoin in athletes have been related to the ability to perform daily intensive physical training (Mikami et al. 2000). Arginine, glycine, and methionine have been implicated in mechanisms important for heat loss e.g. increased skin temperature, evaporative water loss through respiration, and activation of neurological pathways involved in body temperature control in rats, rabbits, and cows; (Glyn and Lipton 1980; Wanner et al. 2015; Izquierdo et al. 2024). Systemic asparagine was reported to promote thermogenesis and enhance glycolysis and lipogenesis through the asparagine-mTORC1 axis in brown adipose tissue (Xu et al. 2021), whereas supplementation resulted in a longer time to exhaustion in rats (Marquezi et al. 2003). In addition, an acute dose of tyrosine consumed 1 h pre-exercise was associated with an increase in exercise capacity during a constant load cycling trial in warm environment (30 °C) in humans (Tumilty et al. 2011). Although higher levels of uric acid have been reported in horses and humans after exercise (Green and Fraser 1988; Rasanen et al. 1996), levels were lower in estrous active heifers. Cytidine monophosphate (CMP), a product of phosphatidylcholine biosynthesis, was positively associated with H.E.A.T. in estrous active heifers. Rats treated with CMP/uridine monophosphate (UMP) were able to endure longer periods of exercise (Gella et al. 2008).

Five intrafollicular pyrimidines were positively related to H.E.A.T. (i.e. uridine, uracil, orotate, cytidine, thymine) and have been shown by others to have direct impacts on body temperature. Three of the five intrafollicular metabolites (uridine, uracil, and cytidine) in our study were also positively related to H.E.A.T. in nonlactating Jersey cows exhibiting estrus (Pollock et al. 2023). Notably, administration of uridine induces hyperthermia in humans, rabbits, and mice (Cradock et al. 1986; Peters et al. 1987a, 1987b). Furthermore, orotate supplementation alleviated heat-induced increases in the body temperature of chickens exposed to elevated ambient temperatures (Shimamoto et al. 2020). While the functional significance of these metabolites in the periovulatory follicle remains unclear, their roles in exercise physiology and thermoregulation suggest that the differences in abundance observed in the current study may reflect physiological adaptations associated with the expression of H.E.A.T alone or in combination with estrous activity.

Because body temperature is elevated to varying degrees in estrual heifers, it is unsurprising that H.E.A.T. was related to certain antioxidants within the periovulatory follicle (cystine and glutathione disulfide). Ovarian production of reactive oxygen species (ROS) is an indispensable preovulatory signaling event triggered by LH (Shkolnik et al. 2011). Glutathione is the principal antioxidant. Cystine, which was negatively related to H.E.A.T., is a precursor of glutathione. Glutathione’s reduced form after scavenging ROS can be oxidized to glutathione disulfide, which was positively related to H.E.A.T. (Wrotek et al. 2020). Collectively, these findings suggest that H.E.A.T. may be promoting increased production and use of glutathione to manage intrafollicular ROS.

Several other periovulatory intrafollicular antioxidants were either positively or negatively related with H.E.A.T., but only in inactive estrous heifers (e.g. taurine, taurodeoxycholate, 2-oxo-4-methylthiobutanoate and homoserine/threonine). Disparate effects of H.E.A.T. based on estrous activity is unclear because intrafollicular levels of taurine and taurodeoxycholate were both positively related to H.E.A.T. in estrous active Jersey cows (Pollock et al. 2023). Taurine is an antioxidant that plays a crucial role in the attenuation of apoptosis (Ripps and Shen 2012). Others have shown that 2-oxo-4-methythiobutanoate improves antioxidant capacity (Tang et al. 2011), enhances immune function, and can alleviate the stress response in animals caused by heat (Zhang et al. 2015). Specific to the antioxidants negatively related to H.E.A.T. in restricted heifers (citraconate and serine), both have roles related to oxidative balance. Pollock et al. (2023) also observed a negative association with H.E.A.T. and periovulatory follicular fluid levels of serine. Relevant to the follicle, oxidative stress during oocyte maturation is mediated by using serine for glutathione synthesis (Turathum et al. 2021). Serine uptake within the mouse oocyte peaks during the latter stages of metaphase I and then rapidly decreases once the oocyte reaches metaphase II (Zhang et al. 2020).

In summary, periovulatory follicle progression is more advanced in estrous active heifers and in those exhibiting higher levels of H.E.A.T. Intrafollicular metabolites in the more advanced periovulatory follicles are linked to thermoregulation, steroidogenesis, antioxidant defense, and energy metabolism. Collectively, these findings provide new insight into pivotal changes in the periovulatory follicle when estrogen dominance is coming to an end and the initial differentiative shift in granulosa cells is occurring, along with other preparatory changes that may be important for eventual ovulation and meiotic progression on the oocyte contained within.

Glossary

Abbreviations

AIC

Akaike information criterion

AUC

area under the curve

BS

blood sample

CIDR

controlled internal drug release

CL

corpus luteum

COC

cumulus-oocyte complex

E₂

estradiol

P₄

progesterone

AI

artificial insemination

GnRH

gonadotropin-releasing hormone

H.E.A.T.

higher estrous associated temperatures

LH

luteinizing hormone

PGF_2α

prostaglandin F_2α

THI

temperature humidity index

US

ovarian ultrasound

VTp

vaginal temperature

ELISA

enzyme-linked immunoassay

Author contributions

Pablo Fioravanti (Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing—original draft, Writing—review & editing), Megan D. Mills (Data curation, Investigation, Methodology, Writing—review & editing), Abigayle B. Pollock (Investigation, Methodology, Writing—review & editing), Ian E. Batey (Investigation, Methodology, Visualization, Writing—review & editing), Kennedy S. Ozburn (Formal analysis, Writing—review & editing), Michael A. O’Neil (Formal analysis, Writing—review & editing), Rebecca Payton (Data curation, Investigation, Methodology, Writing—review & editing), Emma A. Hessock (Investigation, Methodology, Visualization, Writing—review & editing), F. N. Schrick (Conceptualization, Data curation, Investigation, Methodology, Supervision, Writing—review & editing), Shawn R. Campagna (Formal analysis, Methodology, Writing—review & editing), Qudus Sarumi (Formal analysis, Methodology, Writing—review & editing), Sarah Moorey (Formal analysis, Investigation, Methodology, Supervision, Writing—original draft, Writing—review & editing), and J. Lannett Edwards (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing—original draft, Writing—review & editing)

Supplementary data

Supplementary data is available at Journal of Animal Science online.

Conflict of interest statement. The authors declare no real or perceived conflicts of interest.

Data availability

The data underlying this article may be shared on reasonable request to the corresponding author.