Open Veterinary Journal, (2025), Vol. 15(11): 6514-6526

Review Article

10.5455/OVJ.2025.v15.i12.36

Seasonal effects on follicular dynamics and hemodynamics, ovarian hormone levels, nitric oxide levels, and lactate dehydrogenase levels in native mares

Amal M. Aboelmaaty¹*, Abdalla E. A. Elgharieb², Hazem Ahmed El-Debaky¹ and Jamal M. H. Alkhadrawy^2,3

¹Animal Reproduction and AI Department, Veterinary Research Institute, National Research Center, Giza, Egypt

²Theriogenology Department, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt

³National Center of Animal Health, Ministry of Agriculture, Livestock and Marine Resources, Tripoli, Libya

*Corresponding Author: Amal M. Aboelmaaty. Animal Reproduction and AI Department, Veterinary Research Institute, National Research Center, Giza, Egypt. Email: am.aly [at] nrc.sci.eg; amalaboelmaaty1 [at] yahoo.com

Submitted: 22/10/2024 Revised: 20/10/2025 Accepted: 11/11/2025 Published: 31/12/2025

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Abstract

Background: Summer heat stress affects the regularity of the estrous cycle in different animal species.

Aim: This study aimed to investigate seasonal effects on follicular dynamics, hemodynamics, and associated biomarkers across the estrous cycle in native mares.

Methods: Doppler ultrasound examinations and blood samples were collected along three sequential estrous cycles during the cold (November to March) and the hot months of the year (May to August). The diameter, antrum diameter, and color area of the dominant follicle (DF) were determined. The diameters, uterine horns, and corpus luteum (CL) diameters, areas, and color areas were estimated. In blood samples, estradiol (E2), progesterone (P4), cholesterol, lactate dehydrogenase (LDH), and nitric oxide (NO) concentrations were determined.

Results: Season (Cold vs. Hot) significantly (p < 0.01) reduced the total number of follicles in the hot months compared with the cold months. Season × Days of estrous (20 days, -5 to 14) impacted (p < 0.05) total number of follicles. Estrous cycle days (p < 0.001) and Season × Days of estrous influenced (p < 0.001) DF diameter/cm, color area/pixel, and P4. Season (p < 0.001), Days of the estrous cycle (p < 0.0001), and Season × Days of estrous influenced (p < 0.001) the area/pixel, the antrum area/pixel, the circumference/pixel, the color area %, the granulosa color area, the granulosa color area %, the circulatory % of the DFs, and also CL diameter, uterine horn diameter/cm, uterine horn area/pixel, total cholesterol, NO, LDH, and E2. Either season (p < 0.001) or days of the estrous cycle (p < 0.001) influenced the DF antrum diameter/cm and uterine horn color area/pixel. Estrous days of the hot months increased the area, the antrum area, the circumference of the DFs, the area of the uterine horns, total cholesterol, NO, and LDH.

Conclusion: In mares, the season of the year did not disturb the ovarian macro-environmental dynamics, DF growth, and recruitment but disrupted blood flow mediators, enzymes, and biochemicals that may associate disturbances in the intrafollicular mediators and influence the oocyte quality. It is recommended that mares be breeded under suitable environmental conditions during the cold months when good quality green food is available.

Keywords: Mares, Season, Ovarian hormones, Dominant follicle, Luteal dynamics.

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Introduction

Under our subtropical conditions and in most of the southern Mediterranean countries, estrous regularity was recorded throughout the year in native and Arabian mares. For several decades before the introduction of ultrasound in equine reproduction, rectal palpation was not sufficient to detect estrous, but estrous detection depended on the observation and recording of behavioral signs of estrous. These previous reports confirmed the regularity of estrous behavioral signs throughout the year, which showed different lengths with little or no significant seasonal variations in Egypt (El-Ghannam and El-Sawaf, 1967). In horses bred in Pakistan, both Arabian and thoroughbred mares showed a higher frequency of estrous and conception during cold months (October to March) than during hot months [June to August (Warriach et al., 2014)]. After complete anestrum formation and the absence of winter ovulation in seasonal breeding mares, the mares expressed a transition period before starting their first ovulation in the breeding season. In seasonal breeding mares, irregular estrus cycle lengths are usually associated with a high incidence of dominant follicle (DF) growth and regression (Rizzo et al., 2020). The growth and regression of the DFs during these transition periods, this ovarian follicular resurgence was associated with not only irregular estrous cycle lengths but also irregularities in the estrus behaviors and irregularity in the resumption of secretion of gonadotrophins and ovarian steroids (Ginther, 1992). In Sweden, the month of mating did not influence the rate of first-cycle conception or second-cycle conception during the breeding or ovulatory season (Hemberg et al., 2004). In countries where mares exhibit seasonal breeding patterns and enter deep anestrus during winter months Donadeu and Ginther, (2002), the significant variation in photoperiod, ambient temperature, and other climatic conditions between different seasons of the year play significant roles in seasonality in breeding mares where the breeding season there commences in moths with long photoperiods in spring and summer (Coelho et al., 2023). In contrast to horses in subtropical countries with insignificant differences in the photoperiod between seasons or months of the year, the natural breeding season of horses in the Northern Hemisphere begins in April and ends in September, whereas, in the Southern Hemisphere, it begins in October and ends in March (Vilhanová et al., 2021). In many temperate zones, mares show anestrum during winter and start the breeding season during early spring with a transitional estrous (Cuervo-Arango and Clark, 2010). In New Zealand, thoroughbred mares begin breeding on the first of September (Hanlon and Firth, 2012). In seasonal breeding mares, the length of the transition period ranged from 60 and 80 days between the winter anestrous and the summer ovulatory breeding seasons was characterized by growth and regression of ovarian DFs with neither ovulation nor corpus luteum (CL) development associated with expressing erratic estrous behavior (Nagy et al., 2000; Donadeu and Watson, 2007). Increasing both the artificial light interval and the room temperature was based on protocols used to bring mares in estrus during their deep winter anestrus (Sharp and Ginther, 1975; Palmer et al., 1982; Sharp, 2011; Sharp et al., 2011; O’Brien et al., 2020). The exposure of seasonal breeding mares to artificial light during their deep anestrous period to induce ovulation requires a duration of 60–70 days (Guillaume et al., 2000). From another perspective, the use of artificial light programs did not show any efficacy in inducing fertile ovulation during winter anestrus, and the use of hormones to hasten the first ovulation of the year had been less than consistently successful under natural photoperiods (McCue et al., 2007; Raz et al., 2010, 2011). In seasonal breeding mares, antral follicles grow in the ovary in a wave-like pattern depending on the stage of the estrous cycle, season, pregnancy, age, breed, and individuality (Raz et al., 2011). During the transition from the anestrous season to the breeding season, the vascular wall of the growing and DF for the first ovulation in the breeding season was low on the day before the first ovulation in comparison with the number of vessels present in a fertile ovulating DF in the middle of the breeding season (Uliani et al., 2011).

In seasonal breeding mares maintained under natural daylight in the Northern Hemisphere, the first spontaneous ovulation was recorded from the middle of March to early May (McCue et al., 2007). In seasonally breeding mares, the unsuccessful breeding of mares in different months of the year depends on the length of the photoperiod (Yoon, 2012). A decrease in pregnancy rates following breeding mares in the first ovulation of the season was recorded compared with subsequent ovulations (Cuervo-Arango and Clark, 2010).

In seasonal breeding, the season of breeding starts when the day length is more than 16 hours (Yoon, 2012). After entering a state of ovarian inactivity or seasonal deep anestrus, sequential ovulations and estrus regularity continue from the first ovulation in the breeding season from spring to October or November (Sharp, 2011; Sharp et al., 2011). In the south Mediterranean and Middle Eastern countries where such a significant difference in the photoperiod is missing, breeding mares commenced during the winter and early spring and stopped during the summer, as indicated by higher foaling rates during the colder months, and this management of the breeding season aimed to overcome the hot seasonal temperature during the summer and early winter seasons (Abo El-Maaty and Gabr, 2010).

Other reports recorded seasonality in mare breeding, where mares expressed seasonal polyestrous species, and their estrous displayed from 20 to 22 days during their breeding season (Nagy et al., 2000). Yoon (2012) collected data from different cold countries and reported that the estrous cycle in mares was 21 days with 5–7 days of estrus and 14–15 days of a diestrus period. Moreover, he stated that only the estrous phase depended on the season and became shorter during the summer due to the faster follicular development, but the diestrus was stable throughout the different seasons of the year. Estrus behavior was observed in mares over an average of 5–7 days, with extreme variations ranging from 3 to 12 days (Yoon, 2012). The variation in the length of the estrous and luteal phases was attributed to individuality, season, stallion, and proximity of other horses (Raz and Aharonson-Raz, 2012). The longer length of the luteal development (52 hours) post-ovulatory estrus in mares was noticed after the first ovulation in the breeding season than that noticed (36 hours) in subsequent ovulations (Guillaume et al., 2000). This delay in the development of the CL in the first ovulation of the breeding season was accompanied by the presence of low progesterone concentrations and longer persistence of post-ovulation estrous behavior in mares compared with the next one and following ovulatory periods during the breeding season (Newcombe Et al., 2023).

In subtropical areas, Ali et al. (2014) reported that regular ovulations in Arab mares were independent of the photoperiodic period and that mares can reproduce efficiently all over the year with no district or season influencing their fertility. The season of the year with a favorable temperature-humidity index (THI) increased pregnancy maintenance, but higher to severe THI increased early embryonic loss by 25% (Yu et al., 2022). Lactate dehydrogenase (LDH) is one of the important enzymes present in muscle, liver, and hemopoietic cells and its levels increase with the increase of stress, damage to body cells, and also tumor development (Augoff et al., 2015). Therefore, the objectives of this study were to distinguish the effects of cold and hot months on DF dynamics and hemodynamics, CL development, and the associated ovarian hormones, nitric oxide (NO), LDH, and total cholesterol.

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Material and Methods

Animals, housing, and feeding

Normally, cycling and clinically healthy mares were included in the current study. Mares were kept in an open yard during the day and indoors at night with artificial light on the research farm belonging to the Department of Theriogenology (Faculty of Veterinary Medicine, Cairo University). Native mares (N=10) of 7–18 years and body weight from 400 to 450 kg were regularly dewormed and received medications for treating blood and internal parasites. The study was conducted from autumn to summer (November 2022–August 2023). Spontaneous ovulating estrous cycles were studied during the cold months (November 2022–February 2023) and the hot months from May to August.

Ultrasonographic (US) and Doppler examinations

The current study used a Doppler ultrasound scanner equipped with a real-time B-mode 12 MHz linear-array rectal transducer (Sonovet R3, Madison, Samsung, South Korea) to examine the DF and the CL in addition to the uterine dynamics and hemodynamics. The diameters of the DFs, developing corpora lutea, and uterine horns were determined using the electronic calipers of the ultrasound each other day during the study period. Once the DF achieved a diameter of 30 mm combined with the presence of uterine estrous edema and estrous behavior, daily reproductive US, and Doppler examinations were performed.

As soon as the DFs reach their maximum diameters and disappear or another smaller follicle is observed on the following examination, this day is designated as Day 0 or the day of ovulation (Abo El-Maaty and Abdelnaby, 2017). The electronic calipers of the ultrasound scanner were used to measure the diameter of the CL, starting from its first appearance until after completing luteolysis and the approach of the next ovulation (Abdelnaby and Abo El-Maaty, 2017b). The color Doppler mode of Doppler ultrasonography was used to assess the vasculature within the CL immediately after its first detection after ovulation.

The pulsed-wave Spectral Doppler mode of ultrasound was used to assess the diameters, blood flow velocities, and blood flow indices of the ovarian arteries, as previously recorded (Bollwein et al., 2004). During the spontaneous ovulation of all animals, the diameters of the horns were determined by the ultrasound grayscale mode, and their vasculature was assessed by the color Doppler mode from Day 5 before ovulation until Day 14 post-ovulation (Abdelnaby et al., 2016). The grayscale mode of ultrasound was used to determine the diameters of the DFs and the diameters of their antral cavities (Abdelnaby and Abo El-Maaty, 2017a). The color Doppler mode of the Doppler ultrasound scanner was used to estimate the vascularization area in the DFs (Abo El-Maaty and Abdelnaby, 2017). The granulosa area was estimated by subtracting the area (estimated by image analysis software) of the DF antrum from the area of the whole DF. To estimate the percentage of vascularization in the DFs, the color area/pixel (determined by image analysis software) is divided by the total area of the DF in percentage/pixel. The ovarian artery pulsatility (PI) and resistance (RI) indices were recorded (Bollwein et al., 2004).

The recorded images and video clips on the memory of the ultrasound scanner were exported using a portable removable disk for later analysis using image analysis software (Photoshop CS6).

Blood sampling

Blood samples were collected from the jugular vein of each mare and placed into blank vacuum tubes for hormone assays and blood biochemical measurements. All samples were immediately placed on ice in a specialized container bag and sent to the laboratory for centrifugation (10 minutes at 3,000 rpm) within 2 hours after collection. Then, sera were harvested and stored at 20°C until the hormone and biochemical assays at the end of the study.

Hormone assaying

The hormones progesterone (P4) and estradiol (E2) were assayed using a commercial ELISA kit (DRG Instruments GmbH, Germany). For progesterone, the range of the assay was 0.0–40 ng/ml, the sensitivity was 0.045 ng/ml, and the intra- and inter-assay variability was 6.81% and 7.25%, respectively. For estradiol, the range of the assay was 9.7–200 pg/ml, the sensitivity was 9.714 pg/ml, and the intra- and inter-assay variability were 6.86% and 5.59%, respectively. Total cholesterol was assayed using a colorimetric kit (MG, Science and Technology Center “STC”, Egypt). LDH (NS 283001) was assayed using a colorimetric kit (Salucea, Dutch technology in life science, Haansberg19,4874 NJ Etten Leur, The Netherlands). Total proteins, albumin, total cholesterol, and NO were assayed using colorimetric diagnostic kits (BioDiagnostics, Egypt).

Statistical analysis

Data are expressed as Mean ± SD. A simple one-way analysis of variance (ANOVA) was used to study the effect of days during spontaneous ovulation (Estrous Days -5 to 14) for the sequential analysis of data within either the cold or the hot seasons using SPSS version 21. Duncan’s multiple range test was used to distinguish between significant means at p < 0.05. An Independent sample t-test was performed to determine the effect of the season of the day of ovulation (Day 0) on the diameter, antrum diameter, area, antrum area, color area, granulosa area, color area %, and granulosa area %. Repeated measure Two-way ANOVA using Univariate Generalized Linear Mode was performed to study the effect of the two breeding seasons (cold and hot seasons), Days (21) of the estrous cycle (Estrous, from Day -5 to Day 14), and their interaction (2 × 21) on the studied parameters.

Ethical approval

The protocol was approved by the Animal Care and Ethical Use Committee of the Faculty of Veterinary Medicine at Cairo University before this study began (Approval ID: Vet- CU—03162023708 and Vet-CU—01122022586).

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Results

Effects of seasonal temperature on follicular dynamics

The breeding season (p < 0.001) and Season × Estrous (p=0.003) significantly impacted the number of total follicles but the days of the estrous cycle decreased (p < 0.0001; Fig. 1a) the number of total follicles during the hot months only especially on the day of ovulation (Day 0, Table 1). During the cold breeding season, the number of total follicles reached maximum values on Day -5 (6.60 ± 2.15) and Day 8 (7.00 ± 0.21) but reached the lowest number on Day -4 (2.00 ± 0.22), Day 10 (3.17 ± 0.96), and Day -1 (3.50 ± 2.12; Fig. 1a). During the hot breeding season, the total number of follicles did not vary greatly throughout the days of the estrous cycle. The comparison between the cold and hot breeding seasons showed an increased (p=0.0001) number in the total number of follicles during the estrous cycle of the cold breeding season (Table 1).

Fig. 1. Showing the mean number of total follicles (A), DF color area % (B), DF diameter/cm (C), DF antrum diameter/cm (D), DF area/pixel (E), and DF antrum area/pixel (F) during cold and hot months with standard deviation bars.

Table 1. Effect of breeding season on ovarian DF dynamics, uterine function, and hormonal parameters on the day of ovulation (Day 0).

The breeding season did not affect the mean diameter of the DF/cm throughout the estrous cycle (Fig. 1c) or the ovulating one on Day 0 (Table 1). The DF reached the maximum diameter on the days of ovulation in either the cold or hot breeding seasons, indicating the significant effect of the estrous days (p < 0.0001) and the season × Estrous (p < 0.0001) on its diameter.

Season, Estrous days, and estrous days during the cold months significantly (p < 0.0001; Fig. 1d) influenced the DF antrum diameter/cm. DFs that ovulated during the hot months had lower (p < 0.05) antrum diameters (Table 1).

Season, Estrous, and Season ×Estrous influenced (p < 0.01) DFs areas/pixel (Fig. 1e) and DFs antrum areas/pixel (Fig. 1f). Both the DF and antrum areas increased throughout the estrous cycles during the hot months. The DF and antrum areas increased insignificantly on the day of ovulation in the hot months, with a significant effect of estrous days during the hot and cold months on either the DF or antrum area (Table 1). The DF circumference/pixel was higher (p < 0.0001) during the estrous cycle of the hot months compared with the cold ones, except on Day 13 (Fig. 2c) and the Day of ovulation (Table 1). Estrous days (p < 0.0001), Season ×Estrous days (p=0.005; Fig. 2c), and Days of estrous during the hot months impacted the DF circumference.

Fig. 2. Showing the mean DF color area/pixel (A), DF granulosa color area/pixel (B), DF circumference/pixel (C), DF circulatory % (D), CL diameter/cm (Cl/cm; E), and the Cl area/ pixel (F) during cold and hot months with standard deviation bars.

The circulatory percentage of the DF did not differ on the day of ovulation (Table 1). Circulatory percentages of the DF increased during the estrous cycle days of the cold months on Days -4, 7–10, and 12–14 (Fig. 2d). Season (p=0.01), Estrous days (p < 0.001), Season ×Estrous days (p < 0.0001; Fig. 2d), and Days of estrous during both hot and cold months impacted (p < 0.0001) the DF circulatory %.

Effects of seasonal temperature on follicular hemodynamics

Generally, season tended (p > 0.05) to influence the DF color area percentage, and hot season tended (p > 0.05) to decrease it on the day of ovulation (Table 1). Both cold months’ estrous days (p=0.004), hot months’ estrous days (p=0.3), and season estrous days (p=0.018) and the interaction of Season × Estrous (p < 0.0001; Fig. 1b) affected the DF color area % (Table 1).

The DF color area/pixel did not vary between the cold and hot seasons (Fig. 2a) and on Day 0 with a slight non-significant decrease during the ovulation of the hot months (Table 1). Estrous days (p < 0.0001; Fig. 2a), Season ×Estrous days (p < 0.0001; Fig. 2a), days of estrous during the cold (p < 0.0001), and days of estrous during the hot months (p < 0.01) impacted the DF color area (Table 1). The color area/pixel of the DF granulosa cells decreased (p=0.02) on the day of ovulation in the hot months (Table 1) with a significant effect of the season (p < 0.012) and estrous days (p=0.016).

Season × Estrous days (p < 0.001; Fig. 2b), estrous days during the hot (p < 0.0001), and estrous days during the cold months (p < 0.0001) on its area. A significant decrease in the DF granulosa color area can be observed from Days 1 to 6 and Days 3 and 13 of the hot months.

Effect of seasonal temperature on uterine horn dynamics

The uterine horn diameter/cm (Ut H, p < 0.0001 Fig. 4a), area/pixel (p < 0.0001; Fig. 4b), and color area (p=0.005; Fig. 4c) were influenced by the season and the estrous cycle days. Season ×Estrous days significantly (p < 0.0001) affected Ut H diameter (p=.007) and Ut H area (p < 0.0001). On the day of ovulation, the Ut H area/pixel increased (p=0.001) during the cold month, with a nonsignificant decrease in the Ut H and color area. Days of estrous in the hot and cold months influenced (p < 0.0001) the Ut H diameter, area, and color area (Table 1).

Fig. 4. Showing the mean Ut H/cm (A), Ut H area/pixel (B), Ut H color area/pixel (C), total cholesterol mg/dl (D), NO (μmol/l; E), and LDH U/l (F) during cold and hot months with standard deviation bars.

Effects of seasonal temperature on CL dynamics

Compared with the cold months, the developing CL/cm showed a lower diameter during the first three days after ovulation in the hot months but obtained higher diameters from Days 7 to 10 and reached low diameters on Days 13, 14, -5, and -4. Season (p < 0.0001), Estrous days (p < 0.0001), Season × Estrous days (p < 0.001; Fig. 2e), and Days of estrous during both hot and cold months impacted (p < 0.0001) CL diameter/cm (Fig. 2e). The CL area/pixel was affected by Estrous days (p=0.008) and tended to be influenced by the interaction of Season × Estrous days (p > 0.05; Fig. 2f). Lower CL areas were observed during the hot months of the year on Days -4, -2 to 4, and 13–14 of the estrous cycle, which increased on Day -3, Days 6, 7, 9, and 10 compared with those of the cold months.

Effect of seasonal temperature on ovarian artery blood flow

The season had a significant seasonal effect on the blood flow indices of the ovarian artery. The seasonal effects on the PI index (PI, p=0.006) and RI index (RI, p=0.023) are presented in Figure 3A and B). Days of estrous influenced the ovarian artery PI (p=0.001) and RI (p=0.005) but Season × Estrous days influenced (p=0.003) only the RI. Hot months were characterized by low PIs on days -5 to -2 and Days 6–12 with a significant increase on Days 0, 1, and 4 compared with the cold months. The RI of the ovarian artery during the hot months increased on days 1, 0, and 4 compared with the cold months. Days of estrous during cold and hot months significantly impacted both PI and RI (p < 0.05).

Fig. 3. Figure showing the mean ovarian artery PI index (A), RI index (B), estradiol (E2 pg/ml, C), progesterone (P4 ng/ml, D) during cold and hot months, with standard deviation bars.

Effects of seasonal temperature on ovarian hormone levels

Estradiol levels increased (E2 pg/ml; p=0.002) on the day of ovulation during the hot months. The days of the estrous cycle affected the concentrations of E2 and progesterone (P4; p < 0.0001) during either hot or cold months (Table 2). During the hot months, E2 increased from Days 3 to 6 to Day 9, and the Days from 11 to 14 except for Days 2, 8, and 10 compared with the hot months (Fig. 3c). In contrast to the estrous cycles of the hot months, P4 increased during the estrous cycles of the cold months, except on Days 5, 4, 8, 10, and 13 (Fig. 3d). Season × Estrous days significantly (p < 0.0001) affected RI, E2, and P4. On the day of ovulation, total cholesterol (Fig. 4d; p < 0.0001), NO (Fig. 4e; p < 0.0001), and LDH (Fig. 4f, p < 0.0001) increased in the hot months than cold months with significant (p < 0.0001) effect of the days of the estrous cycle during the hot and cold months on their concentrations (Table 2). Season, Estrous days, and Season × Estrous days significantly (p < 0.0001) influenced NO, LDH, and total cholesterol (Fig. 4). Higher NO and total cholesterol levels are observed throughout estrous cycles during hot months. However, higher LDH levels were observed during the estrous days of the hot months, except on days 2, 3, 10, and 12.

Table 2. Mean ± SD circulating blood parameters on the day of ovulation (Day 0) in cold and hot months and the effect of estrous days on blood parameters within seasons.

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Discussion

The breeding season is one of the most important factors that must be considered to prevent economic losses caused by the high expenses of maintaining brood mares throughout the year with low fertility and conception rates (Katila et al., 2010). In mares, hot summer temperatures affect the duration of estrus (Vilhanova et al., 2021). Short estrous cycles or estrus periods were associated with lower conception rates than normal estrus cycles or periods (Solodova and Kalinova, 2022). In mares, hot summer temperatures affect the duration of estrus (Vilhanova et al., 2021). A similar pattern of decreased estrus duration with the progression of the breeding season was observed in Arab purebred mares maintained in Northern Tunisia (Najjar et al., 2019).

Although ovarian activity in mares in the northern hemisphere begins at the end of January every year with a DF diameter not exceeding 21.0 mm (Donadeu and Ginther, 2003), in the south Mediterranean countries, mare breeding can be performed throughout the year. However, the actual breeding time commences from October to May every year, with the foaling season occurring during the same months (Abo El-Maaty and Gabr, 2010), so that the birth of young foals and lactation occurs during the season most favorable for raising offspring. Because of the long estrous phase, the follicle diameter was used to predict the nearest time of ovulation in mares (Koskinen et al., 1989). The ovulating follicles and corpora lutea are not completely spherical. The preovulatory follicle changes its shape from a spherical to an ovoid, conical, or pear shape and becomes more irregular as ovulation approaches, with a progressive change in the shape to oblong within the last two hours before ovulation (Townson and Ginther, 1989). On-site, the follicle diameter is determined using ultrasound electronic calipers, but it is not suitable to compare the DFs between hot and cold seasons as measuring the circumferences or areas using image analysis software. This difference in the analysis of the diameter or area explains the difference between seasons observed in the area but lost in measuring the diameters. Because the ovulating follicles and the CL are not completely spherical but ovoid or pear shape, the circulatory percentage is one of the image analysis parameters that determine the shape of the DF and showed significant differences between hot and cold seasons and different days of the estrous cycle and their interaction. The decrease in the circulatory volume as the follicle approaches ovulation confirms the irregular shape that was used previously to predict ovulation (measured here indicates that they are not always special. Therefore, when both diameter and area were compared in the cold and hot months of the year, seasonal significant differences appeared in the DF area and antrum area than in the DF diameters and antrum diameter. This study recorded seasonal differences in the total number of follicles, DF area, and antrum area with no seasonal effect on the DF diameter and antrum diameter. However, the difference between seasons observed in the area and antrum area of the DFs but lost by measuring the diameter and antrum diameters throughout the estrous cycle by selecting and ovulating follicles of larger area and antrum area. Using the diameter to assess the seasonal differences in the DF diameter showed increased DF diameter during the cold months, reaching a maximum during February and a minimum during August. In horses exhibiting seasonal breeding, the transition from anestrous to the reproductive cycle affects follicle development and reproductive efficiency, which increases the follicle diameter at the start of the breeding season and compared to the end of the breeding season (Alvarez et al., 2023; de Lima et al., 2020; Newcombe and Cuervo-Arango, 2013). In agreement with the absence of any difference in the DF diameter between the hot and cold seasons, neither ambient temperature, photoperiod, nor rainfall influenced the diameter of the DF (Rua et al., 2019). In addition, the diameter at which the preovulatory follicle could be used as a reference to breed the mare could not be determined because of the low repeatability, even for the same mare (Lefrançois and Bruyas, 2016).

In this study, season showed no significant effect on the color signal in the DF, but by estimating the percentage of the color area by dividing the color area by the total follicle area, seasonal differences became obvious and tended to increase on the day of ovulation occurred during the cold months. Moreover, the determination of follicle thickness by determining the granulosa area and calculating the percentage of color signal revealed that preovulatory follicles during the cold months showed significantly higher granulosa area and percentage of color signal on the day of ovulation. Preovulatory follicles showed a progressive increase in their wall thickness compared with the early to mid-estrous phases (Pierson and Ginther, 1985) and a decrease in the color signal as it approached ovulation (Gastal et al., 2006).

The current study indicated seasonal changes in the size (diameter) of the developing corpora lutea after ovulation, but the CL area did not differ in the cold or hot months. This seasonal difference in the diameter of the CL between seasons could be referred to as the pear shape of the CL, and we used to measure the longest diameter during ultrasound (Kimura et al., 2005). In agreement with the decrease in the CL diameter that occurred 1–7 days after ovulation in the hot months of the year, the corpora lutea that developed after ovulation in the late summer were smaller and had less vascularization than those that formed in the early or mid-reproductive season (Panzani et al., 2016; Ishak et al., 2017; Ishak et al., 2019). The developed corpora lutea in autumn produces insufficient progesterone to maintain pregnancy (Duncan, 2021). Therefore, exogenous progesterone may be recommended to help autumn-bred mares avoid pregnancy loss (Panzani et al., 2017).

Estrous in mares is associated with uterine edema (Grabowska and Kozdrowski, 2022). The presence of estrous edema increases the diameters of the uterine body and horns. So, the uterine edema was high and significantly increased the uterine diameter during the hot months. Moreover, a significant seasonal effect on the uterine horn area is expressed by increased uterine horn area and color area throughout the cycle in the hot months, which is in agreement with the decrease in the endometrial edema scores recorded in late autumn (Spencer et al., 2022).

The significant increase in total cholesterol along the estrous cycle during the hot months compared with the cold months could be referred to as the difference in the breed and age of the mares (Asadi et al., 2006). In addition to the significant difference between seasons in the concentrations of NO reported in this study and its significant increase along the estrous cycles of the hot season, fluctuations and peaks in the NO concentrations were connected to ovulation and CL development (Abdelnaby and Abo El-Maaty, 2017a,b). NO plays a significant role in the regulation of gonadotropin secretion, steroidogenesis, follicular development, ovulation, luteal development and regression, pregnancy, parturition/labor, and oviductal, cervical, and vaginal function (Rosselli et al., 1998; Dixit and Parvizi, 2001). NO not only controls blood flow but is also one of the free radicals that play a significant role during stress, and its increase during hot months could be referred to as combating oxidative stress due to increased ambient temperature and the absence of good-quality green fodder. NO increased in mares that experienced early embryonic death and uterine infections (Kotp et al., 2015; Tsuzuki et al., 2016).

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Conclusion

The hot summer season increased the antrum diameter, area, antrum area, color area, and color area by %. On the day of ovulation, DFs in the hot season were nearly similar in diameter with a nonsignificant increase in the area, antrum area, and circumference. The ovulating DFs of the hot months had lower antrum diameter, color area, color area %, granulosa area, and granulosa color area % than those of the cold months. The uterine horns of the hot months had larger diameter, area, and color area than those of the cold months. Estradiol, total cholesterol, NO, LDH, total proteins, and albumins increased during the ovulations in the hot months compared to the cold months.

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Acknowledgments

All authors have approved the submitted manuscript for publication.

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