Steroid precursors, steroids, neuroactive steroids, and neurosteroids concentrations in serum and saliva of healthy neonatal heifer Holstein calves

Abstract Background Persistence of high neurosteroid concentrations in blood is associated with neonatal encephalopathy and septicemia in foals. This has not been investigated in calves. Objectives To determine concentrations of steroid compounds in serum and saliva within the first 48 hours after birth in healthy neonatal calves, identify potential markers for disease, and investigate the association between serum steroid compounds concentrations in calves and their respective dams within 2 hours after birth. Animals Twelve healthy neonatal heifer Holstein calves and their dams. Methods Prospective study. Serum and saliva were collected from calves at 2, 6, 24, and 48 hours after birth. Steroid compounds were analyzed using liquid chromatography‐mass spectrometry. A nonlinear regression model was used to determine half‐lives of the neurosteroids. Serum concentrations of neurosteroids between the cows and calves were compared using the Wilcoxon signed rank test. Results Half‐lives (95% confidence intervals) of dehydroepiandrosterone (DHEA) and 17α,20α‐dihydroxyprogesterone in calf serum were 2.9 (2.1, 4.3), and 2.1 (1.3, 3.0) hours, respectively. Pregnanediol in saliva had a half‐life (95% confidence interval) of 24.5 (14.2, 66.5) hours. Serum DHEA (1718.7 ± 2313 vs 57.7 ± 44) and 17α,20α‐dihydroxyprogesterone (207.8 ± 198.2 vs 43.5 ± 33.5) concentrations respectively were higher (P < .05) in calves compared to cows. Conclusions and Clinical Importance Dehydroepiandrosterone, 17α,20α‐dihydroxyprogesterone, and pregnanediol could be potential markers of disease in neonatal heifer calves with unexplained failure to thrive or encephalopathy. However, because of the wide 95% confidence interval of the half‐life, pregnanediol in saliva might not be a potential marker.


| INTRODUCTION
Neurosteroids (NS) and neuroactive steroids (NAS) are steroid compounds produced in the brain and other tissues, respectively, both of which have effects in the nervous system. 1,2 These compounds are synthesized from cholesterol which is converted into pregnenolone and then into all other endogenous steroids. 3 These compounds modulate states of arousal, cognitive function, mood changes, stress response, central nervous disorders, seizures, and play an important role in brain development and neuroprotection. 1,[3][4][5][6][7][8] and NAS concentrations at birth have been investigated in infants and neonatal foals. [9][10][11] Neonatal maladjustment syndrome (NMS) is the most common neurologic disorder affecting foals within the first 72 hours of life, and is also known as neonatal encephalopathy (NE), hypoxic-ischemic encephalopathy, perinatal asphyxia, and dummy foal syndrome. [12][13][14][15][16] This syndrome encompasses foals suffering from perinatal hypoxia, and those suspected of having impaired transition from intra-to extrauterine life as the result of persistent increase of NS and NAS concentrations after birth. 11,17,18 This persistent increase in steroid compounds is presumed to cause alterations in behavior including lack of bonding with the mare and nursing, states of consciousness, and in some foals causing seizures. 19,20 Furthermore, IV infusion of a neurosteroid (allopregnanolone) into healthy neonatal foals produced alterations in behavior similar to those observed in NE. 21,22 A similar syndrome occurs in neonatal calves termed NE. 23,24 Other terminology includes dummy calf and weak calf syndrome (WCS) to describe neonatal calves with weakness and failure to thrive of undetermined etiology. 25 Other etiologies of WCS include infectious and nutritional causes. 26 A recent study of 200 neonatal calves classified 29% as NE and identified being male, dystocia, prolonged labor, and calf malpresentation as risk factors for the development of NE. 23 If the pathophysiology of WCS is similar to that of NMS in neonatal foals, the role of neurosteroid compounds in the development of NE in neonatal calves warrants investigation. 17,18 The majority of studies evaluating concentrations of NS have analyzed serum or plasma samples. 9,11,18 A potential alternative to blood include saliva and urine as in other species. 27 However, comparison of concentrations of several NS in serum and saliva have not been investigated in veterinary medicine, especially during times of large shifts in concentrations within the first 48 hours after birth. 28 There are mixed results in correlating serum and salivary cortisol concentrations in horses. 28 Neurosteroids and NAS concentrations have not been investigated in neonatal calves to determine if similar shifts in concentrations occur during the first 48 hours of life similar to other species. 11 The calf is an ideal species to investigate such a process as they have a large average birth weight allowing safe collection of multiple blood collections. Furthermore, they secrete large amounts of saliva making it easier to collect and monitor. Therefore, the objective of the study was to determine concentrations of a panel of NS and NAS in both, serum and saliva within the first 48 hours of life in healthy neonatal calves, and identify those with a rapid decline in concentrations during this time to serve as potential markers of neonatal disease. A secondary objective in this study was to investigate the association between serum steroid compounds concentrations in calves and their respective dams within 2 hours after birth.

| Sample collection and handling
After a physical and neurological examination at 2 hours after birth, blood from the jugular vein and saliva samples were collected at 2, 6, 24, and 48 hours after birth. For each calf and cow, venipuncture was performed on the left or right jugular vein. At each time point, a venous blood sample (3 mL) was collected with a 20-gauge needle and aliquoted into a glass vacuum blood tube with no anticoagulant (Monojet red top, Pulmolab, Northridge, California). The blood was allowed to clot at room temperature for 1 hour and blood samples were then centrifuged for 10 minutes at 1000g. Serum was aliquoted into 2 mL cryotubes (Thermo Fisher Scientific, West Sacramento, California) and immediately stored at −80 C until analysis.
At each time point, saliva was collected from each of the calves using a salivette and dual chambered centrifuge tubes (Salimetrics LLC, State College, Pennsylvania) and dual chambered centrifuge tubes to allow for easy extraction of the saliva. The calves were allowed to suckle on the absorbent swab for 2 minutes, and then the swabs were placed within 10 seconds on ice in the provided tube. The tubes were then centrifuged for 10 minutes at 1000g, and the saliva was collected and frozen at −80 C until further analysis.   cortexone, corticosterone, testosterone, 5α-dihydroprogesterone, 21-hydroxypregnanolone, 17α,20α-dihydroxyprogesterone, pregnenolone, and pregnanediol. However, only DHEA and 17α,20αdihydroxyprogesterone were considered reference compounds because the 95% CIs for the half-lives were determined. The serum half-lives (95% CI) for DHEA ( Figure 1A) and 17α,20αdihydroxyprogesterone ( Figure 1B) were 2.9 (2.1, 4.3) and 2.1 (1.3,   3.0) hours, respectively. The 95% CI for half-lives of the other 8 compounds were either very wide or undefined suggesting that the data points for these compounds did not unambiguously define the half-lives, and therefore were not appropriate targets.

| Serum and saliva neurosteroid concentrations
Although serum progesterone and cortisol were not found to be target compounds in this study, they have been used as reference compounds in other species. Therefore, their 1-phase decay curves were reported and depicted for comparison ( Figure 1C,D). The half-lives for progesterone and cortisol were 0.2 (undetermined, wide 95% CI) and 11 (undetermined, wide 95% CI) hours, respectively.

| Comparison of serum and saliva neurosteroid concentrations
Concentrations of cortisol and pregnanediol were compounds that were best fitted by 1-phase exponential decay in both serum and saliva. The rate constants for the nonlinear regression models were not different (0.06 for serum vs 0.21 for saliva; P = .29) between serum and saliva cortisol concentrations ( Figure 3). The rate constant for the nonlinear regression models were different (0.19 for serum vs 0.03 for saliva; P = .01) between serum and saliva for pregnanediol concentrations (Figure 4).

| Calf and cow NS at birth
Comparison of cow to calf serum data at within 2 hours after birth yielded 3 group categories of NS. Concentrations for the NS were either higher, lower, or not different in calves compared to cows (  1.3, 3.0) hours. C, Decay curve for progesterone. R 2 = 0.93. The halflife for progesterone was 0.2 hours. However, 95% confidence interval, and rate constant were very wide, and undetermined suggesting that the decay curve was ambiguous and the half-life estimate was not precise. D, Decay curve for cortisol. R 2 = 0.99. The half-life for cortisol was 11 hours. However, 95% confidence interval for the half-life and rate constant were wide, and undetermined suggesting that the decay curve was ambiguous and the half-life estimate was not precise rapid, and steady decline within the 48 hours of sampling. These NS were DHEA and 17α,20α-dihydroxyprogesterone in serum, and pregnanediol in saliva. Dehydroepiandrosterone and 17α,20αdihydroxyprogesterone had the most precipitous decrease by 24 hours after birth, reaching their lowest concentrations that remained throughout the next 24 hours (48 hours after birth).
Pregnanediol concentrations in saliva had a steady but less precipitous decline reaching low levels by 48 hours after birth. This rapid decline was because of the short half-life of these NS, 2.9 and 2.1 hours in serum for DHEA and 17α,20α-dihydroxyprogesterone, respectively; whereas 24.5 hours in saliva for pregnanediol. This rapid decline likely reflected a placental origin or termination of endogenous production. 11  To assess if similar steroid compounds and concentrations were present in neonatal calves to those of their cows, serum samples were collected from both groups 2 hours after birth of their respective calves. Our results showed a wide variation in these compounds concentrations (Table 2). Neurosteroids and NAS concentrations that were significantly higher in calves than in cows included DHEA, cortisol, cortexone, corticosterone, 20α-dihydroprogesterone, 17α,20α-dihydroxyprogesterone, cally regulated from before birth and have a key role in modulating early brain development, plasticity, and behavior during childhood. 33 Therefore, it is possible that high DHEA concentrations at birth might reflect the importance of this compound in early neurodevelopment during fetal life and modulation of behavior after birth in neonatal calves. Parturition in cattle is initiated via the fetal pituitary-adrenal axis. 34 During late gestation, ACTH from the fetal pituitary gland stimulates the fetal adrenals to produce increased concentrations of cortisol. 34 Fetal cortisol induces synthesis of placental 17α-hydroxylase and aromatase, increased production of estrogen, and decreased production of progesterone in preparation for parturition. 35,36 Therefore, the increased cortisol concentrations in these neonatal calves also reflected endogenous production. Compounds found in significantly lower concentrations in calves included androstenedione, allopregananolone, and progesterone. Progesterone is needed to maintain pregnancy and assumed to be the reason for being higher in cows than in neonatal calves. 37 Furthermore, through a series of enzymatic reactions allopregnanolone is derived from progesterone 2 ; potentially also explaining why allopregnanolone is also higher in cows than in neonatal calves. Additionally, allopregnanolone increases during pregnancy and peaks in late gestation in rats and mares. [38][39][40] The remaining steroid compounds studied here were not significantly different between calves and their cows and could have reflected a placental contribution. This was fur-   thrive, and signs such as lack of bonding with the mare, not nursing, unawareness of the environment, obtundation, abnormal sleep, and in some cases seizures. 17,18,41 Similarly, neonatal calves with NE display failure to thrive and inability to nurse. 23  Therefore, our study results cannot entirely exclude potential targets including progesterone that have been identified in foals.
In conclusion, DHEA and 17α,20α-dihydroxyprogesterone were found to rapidly decline within 24 hours of life and remained at low

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
The study was reviewed and approved by UCD-IACUC as described to collect blood and saliva samples from neonatal calves at 2, 6, 12, 24, and 48 hours of age. The study was non-invasive and not terminal. No animals were harmed.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.