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Article

The Bidirectional Interaction Between Insulin and the Hypothalamus–Pituitary–Adrenal Axis in Normal Pregnant Mares

by
Katiuska Satué
1,*,
Deborah La Fauci
2,
Pietro Medica
2,
Maria Gemma Velasco-Martinez
1,
Cristina Cravana
2,
Giuseppe Bruschetta
2 and
Esterina Fazio
2
1
Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, CEU-Cardenal Herrera University, Tirant lo Blanc, 7, Alfara del Patriarca, 46115 Valencia, Spain
2
Department of Veterinary Sciences, Veterinary Physiology Unit, Polo Universitario Annunziata, Via Palatucci 13, 98168 Messina, Italy
*
Author to whom correspondence should be addressed.
Stresses 2025, 5(1), 4; https://doi.org/10.3390/stresses5010004
Submission received: 22 October 2024 / Revised: 12 December 2024 / Accepted: 2 January 2025 / Published: 7 January 2025
(This article belongs to the Collection Feature Papers in Human and Animal Stresses)
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Abstract

:
The aim of this study was to determine if the changes in plasma insulin, glucose (GLU), fructosamine (FRUCT), adrenocortical hormone (ACTH), and cortisol (CORT) concentrations in mares of different ages were substantial enough to indicate the need to also establish specific reference intervals for pregnant Spanish Purebred mares with a heterogeneous body conditional score (BCS). A total of 45 mares were used in the study, which were classified according to age into 24 <10 years (from 4 to 9 years) and 21 >10 years (from 10 to 18 years). According to the BCS, mares <10 and >10 years were distinguished into three groups as follows: underweight (BCS < 4–5; n = 8), moderate (BCS = 6–7; n = 8), and overweight (BCS = 8; n = 8) (BCS < 4–5 (n = 7), BCS = 6–7 (n = 7), and BCS = 8 (n = 7)), respectively. The main results of this study were that (I) circulating insulin, GLU, FRUCT, ACTH, and CORT concentrations were altered throughout the whole duration of pregnancy in mares; that (II) aging and BCS significantly affected insulin, ACTH, and CORT changes; and that (III) ACTH-CORT significantly correlated with insulin, FRUCT, and GLU. The results may have implications for health and disease and warrant future prospective investigations on the bidirectional interaction between insulin and the hypothalamus–pituitary–adrenal (HPA) axis in equine species, affecting the GLU and FRUCT profile through the entire physiological pregnancy.

1. Introduction

The metabolism of pregnant mares undergoes pivotal adaptations to respond and adapt to the growing fetal–placental unit requirements. The last trimester of pregnancy is considered the particular time in which the greatest substrate mobilization is observed in response to the increased fetal development. The mare’s circulating glucose (GLU) represents the principal source among different substrates [1,2], and its evolution is different in pregnant mares with different BCSs [3]. Specifically, an increase in GLU in mares with moderate BCSs, a decrease in overweight or fat mares, and an absence of variations in underweight or ∼thin mares were detected [3]. What is more, the evaluation of GLU and insulin dynamics during gestation in mares [4,5,6,7] and their dynamics according to cortisol curves and BCS [8] were also described, with the presence of insulin resistance (IR) and its relation to the weight, height, and clinical changes of the neonate being identified; nevertheless, no correlation was found between maternal GLU and insulin concentrations and foal weight and height [8].
Moreover, previous studies have shown that gestation in a mare is represented by an increase in HPA axis activity [9], in which, in the early stages, the adrenocortical hormone (ACTH) could be related to the secretion of corticotropin-releasing hormone (CRH), which is triggered when the developing placenta stimulates maternal ACTH [9,10]. However, other research refutes this, since CRH does not increase until delivery, possibly due to differences in placentation [11]. ACTH stimulates the biosynthesis of CORT [12]. In addition, the magnified synthesis of CORT in pregnancy is due to the increased placental and maternal ACTH, to the peripheral anti-glucocorticoid effect produced by the highest progesterone (P4) concentrations [13], and to the stimulation of the synthesis of placental estrogens. It is therefore feasible that the elevated free CORT contributes to IR during this period [5,14], while insulin normally provides an inhibitory tone to the HPA axis [15].
Limited studies have examined the effect of a whole pregnancy on the interaction between insulin and HPA function in mares; moreover, recently, the effects of ovulation and early pregnancy on ACTH and insulin concentrations have been described in healthy mares [13]. No significant effects of age, reproductive cycle, or pregnancy were detected on the CORT, GLU, or insulin concentrations; on the other hand, a significant effect of early pregnancy and ovulation on ACTH concentrations was recorded [16].
Some studies in humans and experimental animals have documented that GLU homeostasis depends on humoral and neuronal signals [17]. Humoral signals mediated by stress-induced hyperglycemia are directly dependent on adrenal activity [18,19]. The neuronal pathway involves CRH neurons in the paraventricular nucleus of the hypothalamus (PVN) which regulates HPA axis activity by stimulating the release of ACTH and adrenal corticosteroids [20]. The neuropeptide CRH orchestrates neuroendocrine stress signaling through two G protein-coupled receptors, CRH receptor subtype 1 (CRHR1) and CRH receptor subtype 2 (CRHR2), widely distributed in the hypothalamus [21]. The hypothalamus is an important metabolic center for the regulation of “gut–brain” activity and has been well characterized in GLU metabolism [17,18,22,23,24]. Despite this, hypothalamic CRH signaling and the type of receptor involved in regulating GLU metabolism remain unknown [25], particularly in mares.
On these bases, we hypothesized that mares’ age could be correlated to changes in maternal metabolism during the whole pregnancy, according to their BCS. The aim of this study was to determine if the changes in plasma insulin, GLU, FRUCT, ACTH, and CORT in mares of different ages were substantial enough to indicate the need to also establish specific reference intervals for pregnant Spanish Purebred mares with heterogeneous BCSs. The shift in the changes in these parameters and the related metabolic effects could be used as diagnostic tools for assessing the dismetabolic evidence involved during complete pregnancy in equine species. The results may have implications for health and disease and warrant future prospective investigations.

2. Results

2.1. Insulin

The mean insulin concentrations decreased from the 1st month until the 7th month of pregnancy, with a tendency to increase from the 8th to 11th month. Two significant peaks were found in the 1st and 2nd month, where the maximum concentrations were obtained both in mares aged < 10 and >10 years compared to others (p < 0.05). On the other hand, lower concentrations were observed from the 6th to 8th month compared to the 3rd, 4th, and 11th month (p < 0.05), with lower concentrations also in the 10th rather than the 11th month (p < 0.05). Compared to <10 year-old mares, those aged >10 showed lower insulin concentrations in both the 7th and 9th month of pregnancy (p < 0.05) (Figure 1A).
Related to the BCS, compared to the younger mares, overweight and moderate mares >10 years old showed higher insulin concentrations in the 1st, 9th, and 10th month (p < 0.05), and only overweight mares showed lower values in the 5th and 6th month (p < 0.05) (Figure 1B).

2.2. Glucose

The mean GLU concentration showed a tendency to constantly increase from the 1st month until the end of pregnancy, with higher values from the 8th to 10th month than the 1st and the 2nd month (p < 0.05). No significant differences were observed between mares of different ages (Figure 2A). Related to the BCS, compared to the younger mares, those that were older showed lower GLU concentrations (p < 0.05) independent of different BCSs (Figure 2B).

2.3. Fructosamine

The mean FRUCT concentrations showed a tendency to increase from the 1st to the 11th month, where the maximum concentrations were obtained in mares aged both < 10 and >10 years, with concentrations generally higher in the second half of pregnancy than the first (p < 0.05). No significant differences were observed between mares of different ages and with different BCSs (Figure 3A).

2.4. ACTH

The mean ACTH concentrations showed a variable trend, with two peaks recorded in the 6th and 9th month obtained in mares aged both < 10 and >10 years compared to the 1st, 10th, and 11th month of pregnancy (p < 0.05), in which the lowest values were observed. Compared to <10 year-old mares, those aged >10 showed lower ACTH concentrations in both the 2nd and 3rd month, but higher concentrations in the 6th, 8th, 10th, and 11th month of pregnancy (p < 0.05) (Figure 4A). Related to the BCS, compared to the older mares, overweight, moderate-weight, and underweight mares < 10 years old showed lower ACTH concentrations along the whole gestation (p < 0.05). Overweight mares >10 years old showed higher ACTH concentrations in the 1st, 3rd, 6th, and 8th month (p < 0.05) than those with other BCSs, and moderate-weight mares >10 years old showed higher ACTH concentrations in the 5th–7th months than underweight mares (p < 0.05) (Figure 4B).

2.5. Cortisol

The mean CORT concentrations remained at lower constant values during the first 4 months than others (p < 0.05) and increased progressively from the first half of pregnancy, achieving maximum mean values in the 8th and 10th month. A significant decrease was observed in the 11th month of pregnancy compared to the others (p < 0.05). Compared to <10 year-old mares, those aged >10 showed lower CORT concentrations in the 5th month but higher concentrations from the 6th to 8th month of pregnancy (p < 0.05) (Figure 5A). Related to the BCS, compared to the younger mares, overweight, moderate-weight, and underweight mares > 10 years old showed generally lower cortisol concentrations from the 2nd to 6th month and significantly lower levels in the 7th–8th month (p < 0.05). Mares >10 years old with moderate BCSs showed higher cortisol concentrations in the 5th–7th month (p < 0.05) than underweight mares (p < 0.05) (Figure 5B).
The correlations between the parameters evaluated in the study have been included in Table 1.

3. Discussion

The main results of this study were that (I) circulating insulin, GLU, FRUCT, ACTH, and CORT concentrations were altered throughout the whole duration of pregnancy in mares; that (II) aging and BCS significantly affected insulin, ACTH, and CORT changes; and that (III) ACTH-CORT significantly correlated with insulin, FRUCT, and GLU.

3.1. Insulin, Glucose, Fructosamine

Data obtained showed that significant changes in carbohydrate metabolism and pancreatic β-cell function occurred throughout the whole pregnancy in mares. The tendency for insulin to increase from the 8th to 11th month confirmed that pregnant mares, consuming high-starch feeds in the third period, increased insulin synthesis and glycemic responses to feeding than nonpregnant mares or matched pregnant mares consuming a fat and fiber-based diet [5].
On the other hand, the decreasing insulin trend recorded from the 1st month until the 7th month of pregnancy did not corroborate the results that showed that up to approximately 270 days of gestation, reduced pancreatic β-cell sensitivity to GLU resulted in hyperinsulinemia; what is more, it is well known that after this period, the fetus reaches approximately 45% of its final birth weight, consequently showing the highest GLU demand. In addition, the uterine GLU uptake removes 75% of that lost from the maternal circulating pool. Therefore, maternal GLU usage is reduced to a minimum to allow this transfer to the fetal development. Insulin concentrations and pancreatic-cell sensitivity to GLU are also reduced compared with that in earlier gestation [2,26].
The trend of circulating GLU during pregnancy, characterized by an increase in the first trimester of pregnancy, was in line to that obtained in Lipizzaner mares [27], in which the highest circulating GLU concentrations were observed in the last three months of gestation but were different to data obtained by others in which GLU did not show any changes along pregnancy [28,29,30]. The same trend described for the FRUCT further corroborates the considerations made for the GLU, even further supporting the vital role of carbohydrate metabolism, especially during the later stages of fetal growth.
However, the elevation of GLU observed in the mares with moderate BCSs was probably due to the progression of maternal IR cells, enabling the redirection of maternal nutrients to fetal development. Considering the BCS, a contradictory result is the finding of the lowest GLU concentrations in the overweight mares at the end of the gestation, since it is accepted that obesity conditions and higher circulating GLU concentrations occurred [31].
Considering only the effect of age, compared to mares < 10 years old, insulin concentrations were higher during the first 6 months but were even higher in months 9, 10, and 11 in mares > 10 years old, with no differences in GLU and FRUCT concentrations. The significant differences between mares of different ages showed that this variable represented an additional factor which may modify the insulin trend in pregnant mares, confirming other studies in which age and pregnancy are common factors shown to influence insulin secretion [26,32,33]. The significant increases in insulin concentrations observed in older mares than younger mares are in line with several studies that have identified an increase in insulin concentration in aged mares when compared to younger mares [33]; although no clear mechanism has been demonstrated, this effect could be attributed to being the compensation for a decrease in peripheral tissue insulin sensitivity [33] that was not present in our aged mares. Although both insulin secretion and sensitivity have been shown to be altered during pregnancy, with decreased peripheral tissue GLU uptake and the possibly to ensure an adequate supply of nutrients to deliver to the fetus [5], in our study, it is not surprising that this occurred during the last gestation period. Although no clear mechanism has been demonstrated, some factors such as reduced physical activity or reduced liver and kidney function in older mares could result in a decrease in insulin clearance, prolonging the half-life and duration of a higher circulating insulin concentration while maintaining GLU and FRUCT levels between both age groups [34,35]. On the other hand, insulin concentrations can also be affected by seasonal changes in healthy horses, with increased insulin concentrations noted during spring when compared to winter [36].
Jointly considering the effects of pregnancy, age, and BCS, the evolution of the GLU trend was different, reflecting an increase in carbohydrate metabolism in an anabolic or catabolic manner according to the different phases of pregnancy. By not evaluating the weight and size of the foals at the time of birth, it was not possible to verify whether the decrease in blood GLU in the BCS 8 was due to the increased needs of GLU by the growing fetus, as has been suggested in experimental animals [37]. It would have been interesting to evaluate to what extent the age and BCS of the dam could have influenced the foal weight at birth. Future studies are necessary to elucidate this mother–foal relationship and its effects on the health and viability of the foal.
As a product of the non-enzymatic reaction of GLU with total serum proteins, FRUCT reflects blood GLU levels accumulated over the past 2–3 weeks. Thus, FRUCT provides short-term monitoring of GLU when insulin and glucose are changing rapidly, as happens in gestation [38]. The dynamics experienced by the FRUCT were like those previously shown in pregnant mares by the same authors, ranging between 213.0 ± 13.67 in the 1st month and 277.69 ± 34.14 mg/mL in the last month of gestation [39], as also shown by Filipovic et al. [40] during the last 60 days of gestation.
The energy substrate requirements allow for rapid fetal growth during the last two periods of gestation [41,42]. Since the equine fetus has a limited capacity for gluconeogenesis, it depends largely on the transplacental supply of GLU [43] and this in turn depends on the maternal–fetal concentration gradient. The increase in this gradient is facilitated by the progressive development of IR [44]. This IR state, which implies a lower sensitivity to insulin in maternal adipose and muscle tissue, allows the preferential direction of GLU to the fetoplacental unit. IR promotes lipolysis and postprandial hyperglycemia, allowing a greater supply of nutrients to the fetus [5,26,45]. Muscle cells, adipose tissue, and liver, which become resistant to insulin, require the highest concentrations of insulin to stimulate GLU uptake [45]. Furthermore, the efficiency of pancreatic ß cells increases after 270 days of gestation; in this regard, Hoffman et al. [11] reported a significantly higher basal concentration of GLU and insulin upon the oral administration of GLU and its slow decrease during the oral administration of GLU in late pregnant mares compared to early lactation. This endocrine regulation is the sum of two processes, (a) the increased synthesis of pancreatic insulin and (b) the regulation of the function of pancreatic ß cells at the placental level [46]. The increase in FRUCT from the 6th month to the end of gestation indicates the activation of gluconeogenesis, possibly to accommodate the development of uterine content in the later stages of gestation, as occurs in women [38] and in experimental animals [47]; however, no age-related differences were obtained.

3.2. ACTH, Cortisol

The lowest ACTH values observed during the first 5 months, followed by a progressive increase from the second half of the pregnancy, achieving the maximum peak values in the 7th and 9th month, confirm the significant effect of pregnancy on ACTH concentrations [16].
A previous study carried out both in pregnant Spanish Purebred and Standardbred mares showed an increase in ACTH and cortisol in specific periods of gestation [9]. Specifically, the increase in ACTH was only observed in the early stages of pregnancy, until the 3rd month, while the increase in cortisol was only observed after 5 months of pregnancy [9]. This is partially consistent with our findings, as both hormones increased during the second half of pregnancy, with ACTH achieving the maximum peak values in the 6th and 9th month, followed by cortisol in the 8th and 10th. On the other hand, ACTH and cortisol concentrations showed the lowest values during the first half of pregnancy. This consensual trend observed both for ACTH and cortisol could be due to the expected activation of the HPA axis along the whole pregnancy, causing the secretion of ACTH and cortisol, which act on multiple organ systems to redirect energy resources to meet real or anticipated demand in the last or the first period of pregnancy, respectively.
The discrepancy observed in the results of other authors could, therefore, be due to the different timings considered in the studies mentioned above. Other authors have suggested that a possible explanation for the increase in ACTH concentration in early pregnancy could be the secretion of CRH, triggered when the developing placenta stimulates maternal ACTH secretion [9,10]. Further studies have, however, failed to confirm this hypothesis as no increase in CRH has been detected in pregnant mares, possibly due to differences in placentation [11].
The absence of a correlation between ACTH and CORT confirmed that it is frequently reported in horses, and CORT is less frequently measured in clinical practice given its larger variability. Moreover, the absence of a correlation between ACTH and CORT has previously been described in different conditions beyond pregnancy [16,29,48,49].
The lowest CORT values observed during the first 4 months, followed by a progressive increase from the second half of the pregnancy, achieving the maximum values in the 8th and 10th month, followed by a significant decrease in the 11th month, do not confirm previous results described in mares of the same breed, in which the peak mean value was reached in both the 4th and 5th month [50]; however, although our trend was different, the superimposed lowest values were recorded as being limited to the 11th month only, as previously observed by Satué et al. [47]. In addition, the CORT pattern was not entirely overlapping to that which occurred in mares of different breeds, such as Standardbred [51,52], Thoroughbred [53,54], and Arabian mares [55], during the same reproductive period. Indeed, Fazio et al. [54] showed a progressive decrease in CORT concentrations from the 3rd to the 6th month in Thoroughbred mares, in agreement with the data observed by Flisinska-Bojanowska et al. [51], who observed a decrease in CORT concentrations, with the lowest concentrations being observed from the 3rd to the 10th month. Moreover, in this study, the highest CORT concentrations were observed in both the 8th and 10th month of pregnancy in Spanish Pure mares, conforming partially with the peak reached in Thoroughbred mares, but only in the 8th month [54].
Compared with mares <10 years old, those >10 years old showed a lower ACTH concentration in the 2nd and 3rd month but a higher concentration in the 6th, 8th, 10th, and 11th month and a lower CORT concentration in the 5th month, but this was higher from the 6th to the 8th month. The significant differences between mares of different ages showed that this variable presented an additional factor which may modify the ACTH-CORT trend in pregnant mares. Moreover, mares aged >10 showed the lowest ACTH and CORT concentrations in the 2nd–3rd and 5th month, respectively, but the highest two hormone values from the 6th to 8th month of pregnancy. These differences confirmed the significant effect of age on previously observed ACTH changes [16] but are not in line with the findings of the same authors [16], who detected no significant effect of age or pregnancy on the cortisol changes. Therefore, these results support the idea that the rise in CORT concentration during pregnancy may lead to an increase in P4 release [56]. It is well known that during pregnancy, there is a competitive mechanism of inhibition between CORT and P4, and consequently, high circulating P4 concentrations could be responsible for the suppression of CORT [13]. However, some of the variations between mares could be related to circadian and circannual changes. It is known that HPA axis activity increases in late summer and autumn, leading to higher concentrations of ACTH and CORT [16,57]. Considering that the samples were collected over 11 months and were not in the same month of gestation, it is possible that these factors could have had an influence.
However, in Spanish Purebred mares, the correlations between P4 and CORT appear to indicate that a competitive mechanism does not exist between both mineralocorticoids in this breed. It is interesting to note that the highest CORT levels were observed in the 8th month of pregnancy in Thoroughbred pregnant mares; Fazio et al. [54] related this pattern with the increase in maternal plasma progestogen, supporting the hypothesis that this increase occurring in late gestation is the result of fetal adrenocortical activity. Fazio et al. [54] also related this increase in CORT with the existence of a negative correlation, specific to equines, between CORT and estrogen concentrations, which increase between the third and the eighth month in pregnant mares.
Pregnancy significantly alters the metabolism of substrates in the mother due to fetal demands [14,45]; therefore, the higher CORT levels maintained during the first half of pregnancy may be related with the period of intense lactation in Spanish Purebred broodmares. Like humans, the mare becomes insulin resistant during late pregnancy [5,14], as corroborated by the increase in GLU concentrations in this same period. Insulin resistance is associated with the anti-insulin effects of CORT [58], as well as the functional modification of pancreatic β cells [45]. The anti-insulin effects of this hormone inhibit the entry of glucose into cells, glycogen synthesis, glycolysis, and lipogenesis and activate glycogenolysis, proteolysis in muscle, and gluconeogenesis from amino acids. These actions aim to increase the plasma glucose concentrations, preserving it for consumption by organs like the brain, and to improve the maternal and placental transfer of glucose to meet fetal demands [38,59].
The results obtained confirmed that the activation of the HPA axis causes the secretion of glucocorticoids, which act on multiple organ systems to redirect energy resources to meet real or anticipated demand [60]. Activation of the hypothalamus–pituitary–adrenocortical (HPA) axis represents a primary hormonal response to homeostatic challenge, also according to age and BCS. The older mares showed generally higher ACTH and lower CORT concentrations than younger mares, confirming that control of glucocorticoid release is mediated by glucocorticoid feedback at varying levels of the HPA axis, serving to limit prolonged exposure to catabolic actions of glucocorticoids [61]. In addition, the highest ACTH and insulin concentrations observed in overweight and moderate older mares and the highest GLU and CORT concentrations observed in moderate older mares are certainly more difficult to explain, probably to be ascribed to the concept of resilience, which is better represented in older individuals that appear more IR than younger individuals.

4. Materials and Methods

4.1. Animals

All methods and procedures used in this study were in compliance with the guidelines of the Spanish law (RD 37/2014) that regulates the protection of animals used for scientific purposes. The Animal Ethics Committee for the Care and Use of Animals of the CEU-Cardenal Herrera University (Spain) concluded that the proposed study did not need ethical approval as it is a non- experimental clinical veterinary practice (CEEA 22/01). Forty-five mares were used in this study, and all were assigned a BCS on a scale from 1 to 9 (1 is poor, 5 is moderate, and 9 is extremely fat) by their owner and by an experienced person, based on the classification of Henneke et al. [62,63]. To analyze the changes in insulin–HPA axis parameters as a function of age, mares were divided into two groups, consisting of 24 subjects <10 years old, from 4 to 9 years, and of 21 subjects >10 years old, from 10 to 18 years. According to the BCS, mares <10 years and >10 years old were distinguished into three groups as follows: underweight (BCS < 4–5; n = 8), moderate (BCS = 6–7; n = 8), and overweight (BCS = 8; n = 8); BCS < 4–5 (n = 7), BCS = 6–7 (n = 7), and BCS = 8 (n = 7), respectively.
All animals were subjected to the same treatments for feeding and management. Feeding was administered in relation to BCS and gestation time. The daily diet of the mares consisted of the administration of concentrate and alfalfa. The concentrate was composed of a grain mix of oats, corn, barley, wheat, and flax, and fiber was provided by alfalfa hay and wheat straw: 10–12.5 kg of dry matter was administered daily, with a ratio of forage to concentrate of 80%/20% during the first 8 months of pregnancy. The amounts of concentrate and hay fed to mares by month of gestation in order to maintain BCS are included in Table 2. From the 9th month, they received a supplementation with a mineral vitamin supplement (Pavo Podo Lac pellets, Pavo Feed Excellence, Madrid, Spain) at a rate of 0.42 kg/100 kg of live weight per day. All mares had free access to a salt block. Water was provided ad libitum.

4.2. Evaluation of Reproductive Activity of Mares

The experiment began at the beginning of the breeding season (late February). From the beginning of the reproductive season, mares that showed estrus behavior were subjected to rectal palpation to evaluate the degree of follicular growth. In addition, an ultrasound evaluation of the ovary was performed using a transrectal probe (Sonosite 180 Plus) to determine the enlargement of the preovulatory follicle, to determine the presence of uterine edema, and to predict the ovulation time. Hence, when the follicular diameter reached ≥35 mm, mares were intramuscularly treated with 1500 IU of human chorionic gonadotropin—hCG (Chorulon®, Intervet, Salamanca, Spain)—to induce ovulation. Insemination was carried out 30 h after this hormonal treatment using cooled semen (Spanish State Stallion Deposit, Zaragoza, Spain). Once artificial insemination was performed, ovulation was verified by ultrasound examination at 48 h. At the time of pregnancy confirmation on day 16 post insemination, the first blood sample was drawn and was carried out monthly for 11 months. All mares were inseminated and were confirmed as pregnant within 40 days (late February, March, and early April). The duration of pregnancy was nearly the same and was regular in all animals studied. Mares foaled spontaneously, eutocic delivery, and all foals were healthy and viable. After foaling, all mares were in the lactation period and had foals on their sides. The stage of pregnancy by time of blood sampling was the same in all mares. The last blood samples were taken from 7 to 15 days before parturition.

4.3. Blood Samples

Blood collections were always performed by jugular venipuncture between 8:00 and 11:00 A.M., using 20 mL disposable syringes (Becton Dickinson Discardit II) and 18–20 G needles (Sterican, Braun Melsungen AG, Melsungen, Germany). A total of 20 mL was collected, and each blood sample was added to glass tubes with lithium heparin (Tapval). Samples were refrigerated at 4 °C for transport, and then were centrifuged at 3000 rpm× for 10 min (p Selecta Centrifuge); the plasma obtained was stored at −20 °C until analyses.

4.4. Determination of Insulin, Glucose, Fructosamine, ACTH and Cortisol Concentrations

Circulating insulin (mUI/mL) was analyzed with a commercial enzyme immunoassay for the determination of insulin in equine plasma (DE4747, Demeditec Co., Kiel, Germany) validated for equine species. The inter-assay coefficient of variation (CV) was 10.3% and the intra-assay CV was 6.6%. The minimal detectable concentration was 3 pg/mL. According to the manufacturer’s information, cross-reactivity of the antiserum is 100% with porcine insulin, 22% with human insulin, and <0.2% with porcine and human proinsulin and C-peptide.
Circulating GLU (mg/dL) concentrations were analyzed by spectrophotometry using the Spin 200E spectrophotometer (Spinreact, Barcelona, Spain) and reagents of the same commercial house. The glucose oxidase–peroxidase (GOD-POD) liquid method was used, the sensitivity was 1 mg/dL = 0.0039 absorbance, the detection limit was 0.3709 mg/dL, and the linearity limit was 500 mg/dL. Intra- and inter-assay CVs were 0.48–0.59 and 2.57–2.98, respectively.
Circulating FRUCT (µg/mL) concentrations were determined by spectrophotometry (Spin 200E, Spinreact, Tarragona, Spain), using reagents from the same commercial house (Spinreact®). The detection limit and linearity were 1.31 and 1000 mmol/L, respectively. The intra- and inter-assay CVs were 2.17% and 2.12%, respectively. The sensitivity of the technique is 1 mmol/L.
Circulating ACTH concentrations (pg/mL) were measured with a commercial radioimmunoassay RIA (ACTH, Phoenix Pharmaceutical Inc., Burlingame, CA, USA). The technique shows cross-reactivity with ACTH (100%), β-endorphin (1%), corticotrophin-releasing factor (CRF) (0%), alpha-melanocyte stimulating hormone (MSH) (0%), metenkephalin (0%), alpha-atrial natriuretic peptide (ANP) (0%), and brain natriuretic peptide (BNP32) (0%).
Circulating CORT concentrations were quantified in 100 µL of serum by a competitive immunoassay using C97 polyclonal antibodies (Endocrinology laboratory, Department of Physiology, Complutense University Madrid, Madrid, Spain). This laboratory procedure shows high specificity for cortisol; however, it cross-reacts with prednisolone (15.71%), prednisone (18.9%), cortisone (10.8%), corticosterone (6.4%), 11-deoxycortisol (40.31%), 21-deoxycortisol (5.31%), and dexamethasone < 0.1%). None of the mares in the present study were on steroidal anti-inflammatory therapy, primarily due to their physiological state. The sensitivity of the technique was 3 pg/100 µL. The intra-assay CVs were between 3.7% and 6.63% at low concentrations and between 3.92% and 9.93% at high concentrations. The percentage recovery of known amounts of the sample was 95%.

4.5. Statistical Analyses

Descriptive statistics (mean, standard deviation, SD, and maximum and minimum values) were obtained for each group of mares by age and BCS. The normality of data was verified by means of the Shapiro–Wilk test. Between-period differences in BCS groups were assessed using a Friedman test for repeated samples, followed by a Wilcoxon test. For the analysis within each period of the differences between BCS groups, a Mann–Whitney test was performed. The relationship between these concentrations was examined by linear regression analysis, and the correlation was expressed by Pearson’s correlation coefficient. The level of significance was p < 0.05.

5. Conclusions

Data obtained confirmed that both insulin and the HPA axis, together with GLU and FRUCT, are critical for energy storage, mobilization, and distribution in multiple organ systems and are needed to ensure energy availability along the whole pregnancy. Aging and BCS significantly affected insulin, ACTH, and CORT in pregnant mares. The advancement of age induces important changes in the analyzed parameters in pregnant mares characterized with an increase in insulin in the first 6 months and in months 9, 10, and 11; an increase in ACTH in the 6th, 8th, 10th, and 11th month; and an increase in CORT in months 6 to 8 without differences in GLU and FRUCT concentrations.
Therefore, the deep time gap between the historical and iconic works of the 1980s and the current updates confirm the importance of new data, ranges, and/or additional considerations regarding these issues. These results may have implications for health and disease and warrant future prospective investigations on the bidirectional interaction between insulin and the HPA axis in equine species, also affecting the GLU and FRUCT profile thorough the entire pregnancy.

Author Contributions

Conceptualization, K.S., E.F. and P.M.; formal analysis, C.C., D.L.F. and G.B.; investigation, K.S., G.B. and M.G.V.-M.; data curation, C.C., D.L.F. and M.G.V.-M.; writing—original draft preparation, K.S., P.M. and E.F.; writing—review and editing, E.F. and P.M.; visualization, P.M. and K.S.; supervision, E.F. and C.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data that support this study will be shared upon reasonable request to be corresponding author.

Acknowledgments

The authors wish to thank the technical staff of the Clinical Analysis Laboratory of the CEU-Cardenal Herrera University of Valencia and the Laboratory of Endocrinology of University Complutense of Madrid, Spain.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. (A). Circulating insulin concentrations (mean ± S.D.) in pregnant Spanish Purebred mares aged <10 and >10 years old along entire gestation. Asterisk indicates significant differences vs. <10 years (p < 0.05); letters indicate significant differences vs. other months (p < 0.05): a = vs. 4–10 months; b = vs. 6–8 months: c = vs. 11 months. (B). Mean circulating insulin concentrations in pregnant Spanish Purebred mares aged < 10 and >10 years old and with different BCSs along entire gestation. Letters indicate significant differences vs. <10 years (p < 0.05): A (BCS 6–7, 8); B (BCS 8).
Figure 1. (A). Circulating insulin concentrations (mean ± S.D.) in pregnant Spanish Purebred mares aged <10 and >10 years old along entire gestation. Asterisk indicates significant differences vs. <10 years (p < 0.05); letters indicate significant differences vs. other months (p < 0.05): a = vs. 4–10 months; b = vs. 6–8 months: c = vs. 11 months. (B). Mean circulating insulin concentrations in pregnant Spanish Purebred mares aged < 10 and >10 years old and with different BCSs along entire gestation. Letters indicate significant differences vs. <10 years (p < 0.05): A (BCS 6–7, 8); B (BCS 8).
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Figure 2. (A). Circulating glucose concentrations (mean ± S.D.) in pregnant Spanish Purebred mares aged <10 and >10 years old along entire gestation. Letters indicate significant differences vs. other months (p < 0.05): a = vs. 8–10 months. (B). Mean circulating glucose concentrations in pregnant Spanish Purebred mares aged <10 and >10 years old along entire gestation. Letters indicate significant differences vs. <10 years (p < 0.05): A (BCS 4–5); B (BCS 6–7); C (BCS 8).
Figure 2. (A). Circulating glucose concentrations (mean ± S.D.) in pregnant Spanish Purebred mares aged <10 and >10 years old along entire gestation. Letters indicate significant differences vs. other months (p < 0.05): a = vs. 8–10 months. (B). Mean circulating glucose concentrations in pregnant Spanish Purebred mares aged <10 and >10 years old along entire gestation. Letters indicate significant differences vs. <10 years (p < 0.05): A (BCS 4–5); B (BCS 6–7); C (BCS 8).
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Figure 3. (A). Circulating fructosamine concentrations (mean ± S.D.) in pregnant Spanish Purebred mares aged < 10 and >10 years old along entire gestation. Letters indicate significant differences vs. other months (p < 0.05): a = vs. 4–11 months; b = vs. 5–11 months; c = vs. 6–11 months; d = vs. 7–11 months; e = vs. 8–11 months. (B). Mean circulating fructosamine concentrations in pregnant Spanish Purebred mares aged < 10 and >10 years old with different BCSs along entire gestation.
Figure 3. (A). Circulating fructosamine concentrations (mean ± S.D.) in pregnant Spanish Purebred mares aged < 10 and >10 years old along entire gestation. Letters indicate significant differences vs. other months (p < 0.05): a = vs. 4–11 months; b = vs. 5–11 months; c = vs. 6–11 months; d = vs. 7–11 months; e = vs. 8–11 months. (B). Mean circulating fructosamine concentrations in pregnant Spanish Purebred mares aged < 10 and >10 years old with different BCSs along entire gestation.
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Figure 4. (A). Circulating ACTH concentrations (mean ± S.D.) in pregnant Spanish Purebred mares aged <10 and >10 years old along entire gestation. Asterisk indicates significant differences vs. <10 years (p < 0.05); letters indicate significant differences vs. other months (p < 0.05). a = vs. 2, 3, 6, 9 months; b = vs. 6, 10, 11 months; c = vs. 10, 11 months. (B). Circulating ACTH concentrations (mean ± S.D.) in pregnant Spanish Purebred mares aged < 10 and >10 years old with different BCSs along entire gestation. Letters indicate significant differences vs. <10 years (p < 0.05): A (BCS 8); B (BCS 6–7).
Figure 4. (A). Circulating ACTH concentrations (mean ± S.D.) in pregnant Spanish Purebred mares aged <10 and >10 years old along entire gestation. Asterisk indicates significant differences vs. <10 years (p < 0.05); letters indicate significant differences vs. other months (p < 0.05). a = vs. 2, 3, 6, 9 months; b = vs. 6, 10, 11 months; c = vs. 10, 11 months. (B). Circulating ACTH concentrations (mean ± S.D.) in pregnant Spanish Purebred mares aged < 10 and >10 years old with different BCSs along entire gestation. Letters indicate significant differences vs. <10 years (p < 0.05): A (BCS 8); B (BCS 6–7).
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Figure 5. (A). Circulating cortisol concentrations (mean ± S.D.) in pregnant Spanish. Purebred mares aged < 10 and >10 years old along entire gestation. Asterisk indicates significant differences vs. <10 years (p < 0.05); letters indicate significant differences vs. other months (p < 0.05): a = vs. 5–11 months; b = vs. 10–11 months; c = vs. 11 months. (B). Mean circulating cortisol concentrations in pregnant Spanish Purebred mares aged < 10 and >10 years old and different BCSs along entire gestation. Letters indicate significant differences vs. < 10 years (p < 0.05): A = (BCS 4–5, 6–7, 8); α = BCS 6–7 vs. BCS 4–5.
Figure 5. (A). Circulating cortisol concentrations (mean ± S.D.) in pregnant Spanish. Purebred mares aged < 10 and >10 years old along entire gestation. Asterisk indicates significant differences vs. <10 years (p < 0.05); letters indicate significant differences vs. other months (p < 0.05): a = vs. 5–11 months; b = vs. 10–11 months; c = vs. 11 months. (B). Mean circulating cortisol concentrations in pregnant Spanish Purebred mares aged < 10 and >10 years old and different BCSs along entire gestation. Letters indicate significant differences vs. < 10 years (p < 0.05): A = (BCS 4–5, 6–7, 8); α = BCS 6–7 vs. BCS 4–5.
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Table 1. Correlations among the parameters considered in the study.
Table 1. Correlations among the parameters considered in the study.
CORT (ng/dL)Insulin (mUI/mL)FRUCT (µg/mL)GLU (mg/dL)
ACTH (pg/mL)0.01−0.13−0.26−0.10
CORT (ng/dL) −0.010.360.13
Insulin (mUI/mL) 0.060.04
FRUCT (µg/mL) 0.39
Table 2. Amounts of concentrate and hay (kg) in each month of pregnancy considering the BCS. Calculation for 500 kg of BW.
Table 2. Amounts of concentrate and hay (kg) in each month of pregnancy considering the BCS. Calculation for 500 kg of BW.
Month of pregnancyKg BCS
4–5		BCS
6–7		BCS
8
1 to 8Concentrate 2.51.00
Hay 10.09.07.5
9Concentrate 2.751.10
Hay 11.09.98.25
10Concentrate 2.871.150.25
Hay 11.510.38.5
11Concentrate 3.02.40.5
Hay 12.010.88.5
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Satué, K.; La Fauci, D.; Medica, P.; Velasco-Martinez, M.G.; Cravana, C.; Bruschetta, G.; Fazio, E. The Bidirectional Interaction Between Insulin and the Hypothalamus–Pituitary–Adrenal Axis in Normal Pregnant Mares. Stresses 2025, 5, 4. https://doi.org/10.3390/stresses5010004

AMA Style

Satué K, La Fauci D, Medica P, Velasco-Martinez MG, Cravana C, Bruschetta G, Fazio E. The Bidirectional Interaction Between Insulin and the Hypothalamus–Pituitary–Adrenal Axis in Normal Pregnant Mares. Stresses. 2025; 5(1):4. https://doi.org/10.3390/stresses5010004

Chicago/Turabian Style

Satué, Katiuska, Deborah La Fauci, Pietro Medica, Maria Gemma Velasco-Martinez, Cristina Cravana, Giuseppe Bruschetta, and Esterina Fazio. 2025. "The Bidirectional Interaction Between Insulin and the Hypothalamus–Pituitary–Adrenal Axis in Normal Pregnant Mares" Stresses 5, no. 1: 4. https://doi.org/10.3390/stresses5010004

APA Style

Satué, K., La Fauci, D., Medica, P., Velasco-Martinez, M. G., Cravana, C., Bruschetta, G., & Fazio, E. (2025). The Bidirectional Interaction Between Insulin and the Hypothalamus–Pituitary–Adrenal Axis in Normal Pregnant Mares. Stresses, 5(1), 4. https://doi.org/10.3390/stresses5010004

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