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Topic:Hormones

Hormones in horses are chemical messengers produced by various glands and tissues, regulating numerous physiological processes essential for maintaining homeostasis. These hormones influence a wide range of functions, including growth, metabolism, reproduction, and stress responses. Key hormones in equine physiology include cortisol, estrogen, testosterone, and insulin, among others. The levels and effects of these hormones can vary based on factors such as age, sex, and environmental conditions, impacting overall health and performance. This page compiles peer-reviewed research studies and scholarly articles that explore the production, regulation, and physiological roles of hormones in equine biology.
Endocrine changes during the peripartal period related to colostrogenesis in mammalian species.
Journal of animal science    May 9, 2023   Volume 101 doi: 10.1093/jas/skad146
Bigler NA, Gross JJ, Baumrucker CR, Bruckmaier RM.This review discusses endocrine and functional changes during the transition from late gestation to lactation that are related to the production of colostrum in different mammalian species. Species covered in this article include ungulate species (cattle, sheep, goats, pigs, horses), rodents (rat, mouse), rabbits, and carnivores (cats, dogs), as well as humans. An immediate availability of high quality colostrum for the newborn after birth is crucial in species where a transfer of immunoglobulins (Ig) does not or only partially occur via the placenta during pregnancy. Declining activity of ges...
Evaluation of the effect of glucocorticoid treatment on adrenocortical functions by monitoring endogenous hydrocortisone in horses.
The Journal of veterinary medical science    May 8, 2023   Volume 85, Issue 6 647-652 doi: 10.1292/jvms.23-0011
Kikuchi M, Nagata SI, Ishige T, Minamijima Y, Hirota KI, Tozaki T, Kakoi H, Kizaki K.Glucocorticoid preparations have anti-inflammatory effects, and are commonly used in the equine clinical setting; however, such treatments can cause a number of side effects. Adrenal insufficiency is an adverse effect induced by the suppression of adrenal function following drug administration. This study aimed to investigate the influence of two glucocorticoid preparations, dexamethasone and hydrocortisone, on adrenocortical function in horses. The usual doses of dexamethasone and hydrocortisone preparations in equine practice were administered intramuscularly to six horses, and peripheral bl...
Detection of gonadotropin-releasing hormone and its analogues in equine and canine urine by high-resolution data-independent acquisition.
Drug testing and analysis    May 3, 2023   Volume 15, Issue 8 896-908 doi: 10.1002/dta.3486
Timms M, Botteon A, Manos C, Griffin J, Levina V, Steel R.Gonadotropin-releasing hormone (GnRH) and its synthetic analogues are considered banned substances by the racing industry. GnRH is used as a pharmaceutical to regulate the female oestrous cycle, but the hormone is also thought to increase the production of testosterone in male animals. Using liquid chromatography in conjunction with high-resolution mass spectrometry (LC-HRMS) and data-independent acquisition (DIA), a method is presented for the detection of intact and truncated peptides of GnRH and its analogues down to the low picogram level in equine urine. The study of the catabolism of GnR...
Validation of an ELISA kit to measure allopregnanolone in human and equine hair. Peric T, Ellero L, Comin A, Pividori I, Prandi A.In humans, allopregnanolone plays important roles in a number of different neurodegenerative disorders, and it has been proposed for use in some therapies. Horses are commonly used as animal models for human neurodegenerative diseases, mental and behavioral disorders, and neuropsychiatric disorders, and there is interest in using hair as a biological sample to study hormones in these conditions. We validated the use of a commercial ELISA kit (DetectX allopregnanolone kit; Arbor Assays), which was designed for serum, plasma, feces, urine, and tissue samples, to assess allopregnanolone in hair s...
Interplay Among the Daily Rhythm of the Thyroid Hormones, Uncoupling Protein 1 and the Clock Gene Per2 in Horses.
Journal of equine veterinary science    April 21, 2023   Volume 126 104496 doi: 10.1016/j.jevs.2023.104496
During husbandry, domestic animals are exposed to many factors that can influence their circadian physiology organization leading to an increase in animals' discomfort. Thermal homeostasis is at the basis of animal wellness, the aim of the present study was to investigate the daily fluctuation of serum concentrations of 3,5,3'-triiodothyronine (T3) and thyroxine (T4) in association with the daily fluctuation of uncoupling protein 1 (UCP1) and clock gene Per2 in healthy horses housed in individual box, to improve the knowledge on this matter. Seven clinically healthy female Italian Saddle horse...
Exercise-related changes in the anabolic index (testosterone to cortisol ratio) and serum amyloid A concentration in endurance and racehorses at different fitness levels.
Frontiers in veterinary science    April 17, 2023   Volume 10 1148990 doi: 10.3389/fvets.2023.1148990
Grzędzicka J, Dąbrowska I, Malin K, Witkowska-Piłaszewicz O.Increased training loads in both human and equine athletes are generally implemented to improve their physical performance. These loads are tolerated only within appropriate training periodization with regard to recovery time. Otherwise, training overload causes failure in the systemic adaptation, which at first leads to overreaching, and progressively to overtraining syndrome (OTS). Exercise endocrinology, and anabolic/catabolic balance as an indicator of athlete performance status and OTS has continued to attract attention. In human medicine, changes in testosterone and cortisol levels, as w...
Sustained-release deslorelin acetate implants disrupt oestrous cyclicity in the mare.
Australian veterinary journal    April 16, 2023   Volume 101, Issue 7 275-278 doi: 10.1111/avj.13244
Joonè CJ, Cavalieri J.There is a need for a safe, effective and practical method of oestrus suppression in the mare. The aim of this study was to monitor ovarian activity in mares exposed to either 9.4 or 28.2 mg deslorelin acetate, a GnRH agonist, in the form of a sustained-release implant. Following oestrus synchronisation, mares were randomly assigned to one of three groups (n = 4 per group) and administered either one (Des1 group; 9.4 mg) or three (Des3 group; 28.2 mg) implants of deslorelin acetate (Suprelorin-12, Virbac Australia) or one blank implant (Control group; Virbac Australia). Mares underwe...
Stress response as a contributing factor in horses with laminitis.
Journal of veterinary science    April 4, 2023   Volume 24, Issue 2 e33 doi: 10.4142/jvs.22039
Moss A, Leise B, Hackett E.Laminitis is a complex and debilitating disease of horses. Numerous predisposing factors contribute to laminitis development, however the exact pathogenesis remains undetermined. Serum T4, cortisol, and histamine are components of the innate stress response and could play a causative or contributory role. Stress hormone concentrations in laminitis are largely unknown. Objective: To evaluate parameters associated with stress response in horses with laminitis, and compare these to healthy horses and horses with gastrointestinal (GI) disease. Methods: Thirty-eight adult horses presenting for non-...
Contributions to Mare Reproduction Research by the Ginther Team.
Journal of equine veterinary science    March 28, 2023   Volume 126 104295 doi: 10.1016/j.jevs.2023.104295
Ginther OJ.Examples of research discoveries and first reports on mare reproduction by the O.J. Ginther team are (1) determined daily circulating concentrations of four hormones during the estrous cycle, (2) showed that mares can be induced to ovulate and superovulate by hormone treatment during both ovulatory and anovulatory seasons, (3) demonstrated that prostaglandin F2α was the luteolysin in mares, (4) described the mare's elaborate hormonal and biochemical mechanism for selecting the ovulatory follicle from a pool of like follicles, (5) developed the method for diagnosing fetal sex by Day 60 using l...
Dual use of breeding stallions is possible without affecting the sperm quality.
Reproduction in domestic animals = Zuchthygiene    March 16, 2023   Volume 58, Issue 6 691-698 doi: 10.1111/rda.14337
Hensel B, Jakop U, Schmicke M, Schröter F, Jung M, Schulze M.Artificial insemination (AI) is commonly used in the equine industry to enhance the genetic value in breeding programs and to effectively utilize ejaculates. Many stallions are used as breeding stallions as well as in high-level sports competitions to improve their market value. The goal of the present study was to investigate whether this dual use of stallions influences the animals´ stress levels and/or the quality of their ejaculates. For this purpose, 18 stallions were grouped into two categories: breeding stallions with (BSC = breeding stallion competition), and breeding stallions with...
The Effect of Treatment with Oestradiol Benzoate on Oestrus Expression and Endometrial Oedema in Anovulatory and Cyclic Mares.
Animals : an open access journal from MDPI    March 5, 2023   Volume 13, Issue 5 doi: 10.3390/ani13050938
Silva ESM, Newcombe JR, Cuervo-Arango J.Oestrogens treatment is often used to induce oestrus behaviour in anoestrous mares to aid in the collection of stallion semen and as recipient mares to receive embryos when combined with progesterone. However, there are no studies to describe the effect of dose and individual mare on the intensity and duration of the response, in both anoestrous and cyclic mares. In Experiment 1, 13 anoestrous mares were treated with one of five doses of oestradiol benzoate (OB) (1, 1.5, 2, 3 and 4 mg) per mare in five consecutive treatment periods (n = 65), to determine the response in terms of endometrial oe...
Dynamics of extracellular vesicle-coupled microRNAs in equine follicular fluid associated with follicle selection and ovulation.
Molecular human reproduction    March 1, 2023   Volume 29, Issue 4 doi: 10.1093/molehr/gaad009
Gebremedhn S, Gad A, Ishak GM, Menjivar NG, Gastal MO, Feugang JM, Prochazka R, Tesfaye D, Gastal EL.Innumerable similarities in reproductive cyclicity and hormonal alterations highlight the considerable utility of the mare to study aspects of follicular dynamics and reproductive function in view of the largely constricted, human research subjects. The bi-directional communication between the growing oocyte and the surrounding somatic cells embodies the hallmark of mammalian follicular development, partially mediated by extracellular vesicles (EVs) encapsulated with microRNAs (miRNAs) and present in the follicular fluid (FF). Here, we aimed to decipher the dynamics of the miRNAs in EVs from e...
Seasonality does not influence cortisol or testosterone production, or seminal quality of stallions located at low latitudes.
Animal reproduction science    February 23, 2023   Volume 250 107202 doi: 10.1016/j.anireprosci.2023.107202
Freitas ML, Viana JHM, Dode MAN, Maggiotto SR, Pivato I, Braga TRC, Lim AIPG, de Oliveira RA.The effects of seasonality on the reproduction of stallions vary based on the latitude. Although previous studies have shown the influence of seasonality in raw semen quality in south-eastern Brazil, data regarding the influence of seasonality in cooled and frozen stored semen in Brazil is limited. Therefore, in this study, we have analysed if seasonality influences the hormone production (i.e., cortisol and testosterone), spermatogenesis, and quality of fresh, cooled, and frozen semen of stallions in central Brazil, and established the season most suitable for semen cryopreservation in a lati...
Monorchidism in a Phenotypic Mare With a 64,XY, SRY-Positive Karyotype.
Journal of equine veterinary science    February 1, 2023   Volume 126 104232 doi: 10.1016/j.jevs.2023.104232
Middlebrooks B, McCue P, Nelson B, May E, Divine C, Barton C, Conley A.Disorders of sexual development (DSD) are associated with atypical chromosomal, gonadal, or phenotypic sex. It is likely that the number of cases of DSD are underestimated in the equine population. Monorchidism in the horse is very rare. This case report describes the clinical assessment of a phenotypic mare with stallion-like behavior which led to the diagnosis of a DSD. A 4-year-old Quarter Horse mare presented in good body condition, with normal external genitalia for a mare, and normal mammary glands with two bilaterally symmetric teats. No uterus, cervix, or gonads were detected on transr...
Cortisol Variations to Estimate the Physiological Stress Response in Horses at a Traditional Equestrian Event.
Animals : an open access journal from MDPI    January 24, 2023   Volume 13, Issue 3 396 doi: 10.3390/ani13030396
Olvera-Maneu S, Carbajal A, Serres-Corral P, López-Béjar M.In many countries, horses remain involved in traditional equestrian events such as those celebrated in Menorca (Balearic Islands, Spain) every year since at least the 14th century. The present study aimed to evaluate the variations in salivary cortisol concentrations to estimate the physiological stress response in horses at the Menorca patronal festivals. Two different editions (years 2016 and 2018) of the festivals in honor of the Virgin of Grace in Maó (Menorca, Spain) were studied. Nineteen and seventeen Pure Breed Menorca stallions were included in the study, respectively. The stallions ...
Computer-assisted assessment of ovarian echotexture parameters in mares following changes after ovulation determined by ultrasonography.
Polish journal of veterinary sciences    January 18, 2023   Volume 25, Issue 4 525-533 doi: 10.24425/pjvs.2022.142040
Akkus T, Korkmaz Ö, Emre B, Zonturlu AK, Yaprakci Ö.This study was carried out to determine the time-dependent changes in the ultrasonographic image of the ovary with computer-assisted analysis programs at certain intervals after ovulation and to determine whether computer-assisted analysis programs and ovulation programs can be managed in cases where the ovulation time is unknown. The study included 40 purebred Arab mares. The study was subdivided into 4 different time periods of 6 (Group 1), 12 (Group 2), 18 (Group 3) and 24 (Group 4) hours following ovulation. In addition, after ovulation and ultrasonographic examination, natural inseminatio...
The hypothalamic-pituitary-adrenal axis response to ovine corticotropin-releasing-hormone stimulation tests in healthy and hospitalized foals.
Journal of veterinary internal medicine    January 18, 2023   Volume 37, Issue 1 292-301 doi: 10.1111/jvim.16604
Johnson K, Peterson J, Kopper J, Dembek K.The hypothalamic-pituitary-adrenocortical axis (HPAA) response to sepsis can be impaired in critical illness. Corticotropin-releasing hormone (CRH) stimulation test might assess HPAA function in foals. Objective: To evaluate plasma cortisol, ACTH, arginine vasopressin (AVP), and endogenous CRH (eCRH) response to different doses of ovine CRH (oCRH). Methods: Healthy (n = 14) and hospitalized (n = 15) foals <7 days of age. Methods: In this prospective randomized study, oCRH (0.1, 0.3, and 1 μg/kg) was administered intravenously and blood samples were collected before, 15, 30, 60, and ...
Physicochemical Characterization of a Recombinant eCG and Comparative Studies with PMSG Commercial Preparations.
The protein journal    January 18, 2023   Volume 42, Issue 1 24-36 doi: 10.1007/s10930-023-10092-x
Rodríguez MC, Mussio PE, Villarraza J, Tardivo MB, Antuña S, Fontana D, Ceaglio N, Prieto C.Equine chorionic gonadotropin (eCG) is a glycoprotein hormone widely used in timed artificial ovulation (TAI) and superovulation protocols to improve the reproductive performance in livestock. Until recently, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG). Here, a bioactive recombinant eCG (reCG) produced in suspension CHO-K1 cells was purified employing different chromatographic methods (hydrophobic interaction chromatography and reverse-phase (RP)-HPLC) and compared with a RP-HPLC-purifi...
Short Communication: Identification of equine corticotropin-like intermediate lobe peptide (CLIP) binding to an adrenocortipcotrophic hormone (ACTH) assay capture antibody.
Domestic animal endocrinology    January 16, 2023   Volume 83 106785 doi: 10.1016/j.domaniend.2023.106785
Knowles EJ, Hyde C, Harris PA, Elliott J, Menzies-Gow NJ.A chemiluminescent immunoassay is commonly employed to measure adrenocorticotrophic hormone (ACTH) concentrations to assist pituitary pars intermedia dysfunction diagnosis. In a previous study, seasonally-dependent assay cross-reactivity to endogenous equine corticotropin-like intermediate lobe peptide (CLIP, ACTH 18-39) was suspected. The present study aimed to demonstrate binding of endogenous equine CLIP to the capture antibody of the ACTH chemiluminescent immunoassay. Liquid chromatography - mass spectrometry (LCMS) methods were optimised to identify selected ions from synthetic human ACTH...
Association between insulin dysregulation and adrenocorticotropic hormone in aged horses and ponies with no clinical signs of pituitary pars intermedia dysfunction.
Equine veterinary journal    January 15, 2023   doi: 10.1111/evj.13925
Li FI, Spence RJ, de Laat MA, Harris PA, Sonntag J, Menzies-Gow NJ, Durham AE, Bailey SR, Sillence MN.High concentrations of adrenocorticotropic hormone (ACTH) are used as an indicator of pituitary pars intermedia dysfunction (PPID), but other factors that may influence ACTH need to be understood, if diagnostic reference ranges for ACTH are to be used with confidence. Insulin dysregulation (ID) could be one such factor, as insulin affects pituitary hormones in other species. Objective: To test the hypothesis that a relationship exists between high insulin and high ACTH in aged (>15-year-old) animals with no clinical signs of PPID. Methods: A cohort study. Methods: Thirteen horses and eleven p...
Behavioral and hormonal assessment of stress in foals (Equus caballus) throughout the weaning process.
PloS one    January 6, 2023   Volume 18, Issue 1 e0280078 doi: 10.1371/journal.pone.0280078
Delank K, Reese S, Erhard M, Wöhr AC.This study had the aim to demonstrate the midterm effects (three weeks) of weaning on foals' welfare. For this purpose, foals' behavioral changes and fecal levels metabolites of cortisol were evaluated. The observations took place at the state stud farm of Baden-Wuerttemberg in Germany. Ten foals (six colts and four fillies) were observed from one day before weaning up until three weeks after weaning. Weaning was divided into three blocks, the first in September, the second in October, and the last in November. The behavioral observation was done during an eight-hour period between 7:00 a.m. a...
Follicle Selection in Mares as a Model for Illustrating the Many Hormonal and Biochemical Interactions That Drive a Single Physiological Mechanism.
Journal of equine veterinary science    December 22, 2022   Volume 121 104196 doi: 10.1016/j.jevs.2022.104196
Ginther OJ.The mechanism for selection of the future dominant or ovulatory follicle in mares involves a relatively abrupt separation in growth rates between the future dominant follicle and several subordinate follicles and is termed diameter deviation. The event is used to illustrate that a coordinated complex of many follicular, hormonal, and biochemical factors interact and interbalance during a single physiological mechanism. For example, a positive effect of follicle stimulating hormone (FSH) on development of all follicles during the growing phase can later involve a positive effect of luteinizing ...
The effect of dexamethasone and flunixin-meglumine on ovulation, endometrial oedema, and inter-ovulatory interval length in the mare.
Theriogenology    November 28, 2022   Volume 197 57-61 doi: 10.1016/j.theriogenology.2022.11.042
Martínez-Boví R, Plaza-Dávila M, Cuervo-Arango J.The use of flunixin-meglumine (a potent non-steroidal anti-inflammatory drug) during the critical period of intrafollicular prostaglandin production before ovulation (24 and 36 h after hCG treatment) results in a high rate of ovulatory failure and formation of haemorrhagic anovulatory follicles (HAF) in the mare. Dexamethasone is commonly used to prevent persistent mating-induced endometritis in susceptible mares, but the effect on ovulation blockage within the pre-ovulatory critical window of intrafollicular prostaglandins production following hCG administration has not been determined. Six ...
Relationship between estrus endometrial edema and progesterone production in pregnant mares two weeks after ovulation.
BMC veterinary research    November 21, 2022   Volume 18, Issue 1 414 doi: 10.1186/s12917-022-03512-0
Grabowska A, Kozdrowski R.Progesterone plays a crucial role in the maintenance of pregnancy from conception to about 100-120 days of gestation when placenta becomes the main source of gestagens. The aim of the study was to test progesterone concentration 14 days after ovulation in pregnant mares and relate it to peak estral endometrial edema and the presence of intrauterine fluid (IUF) after artificial insemination (AI), the number of treatments against IUF, and the time from AI to the day when the uterus was found free of fluid. Results: Mares were divided into two groups: group A (n = 13; age 10.8 ± 4.5 y...
Low plasma progesterone concentration during the early luteal phase delays endometrial development and the beginning of placentation in mares.
Animal reproduction science    November 8, 2022   Volume 247 107149 doi: 10.1016/j.anireprosci.2022.107149
Okada CTC, Kaps M, Reichart U, Walter I, Gautier C, Aurich J, Aurich C.While detrimental effects of reduced plasma progesterone concentration in the early luteal phase on conceptus development in horses have recently been demonstrated, there is no information on associated effects on the endometrium, allantochorion (AC), and chorionic girdle (CG) in this species. We hypothesised that reduced early postovulatory progesterone concentration in pregnant horses is detrimental to endometrial function and development of the embryonic membranes and is an underlying cause of delayed conceptus development. After insemination and ovulation, mares (n = 11) were assigned to...
Postnatal Dynamics of Circulating Steroid Hormones in Mule and Equine Neonates.
Veterinary sciences    October 28, 2022   Volume 9, Issue 11 598 doi: 10.3390/vetsci9110598
Boakari YL, Legacki E, Alonso MA, Dos Santos ACF, Nichi M, Conley AJ, Fernandes CB.It is necessary to study hormonal patterns from mules to recognize alterations and neonatal maladaptation. Our objective was to evaluate concentrations of hormones in mule (n = 6) and equine foals (n = 6). Blood was collected at T0, 1, 6 and 12 h after birth. Hormone concentrations were evaluated using liquid chromatography tandem mass spectrometry. Effects of time, group and interactions and regression analysis were evaluated (p < 0.05). There was a cubic and quadratic decline in mule and equine foals, respectively, for 3β,20α-dihydroxy-DHP. Mule foals were born with lower circulating 3Î...
Energy endocrine physiology, pathophysiology, and nutrition of the foal.
Journal of the American Veterinary Medical Association    October 26, 2022   Volume 260, Issue S3 S83-S93 doi: 10.2460/javma.22.08.0348
Kinsella HM, Hostnik LD, Toribio RE.Most homeostatic systems in the equine neonate should be functional during the transition from intra- to extrauterine life to ensure survival during this critical period. Endocrine maturation in the equine fetus occurs at different stages, with a majority taking place a few days prior to parturition and continuing after birth. Cortisol and thyroid hormones are good examples of endocrine and tissue interdependency. Cortisol promotes skeletal, respiratory, cardiovascular, thyroid gland, adrenomedullary, and pancreatic differentiation. Thyroid hormones are essential for cardiovascular, respirator...
Identification of Candidate Genes for Twinning Births in Dezhou Donkeys by Detecting Signatures of Selection in Genomic Data.
Genes    October 19, 2022   Volume 13, Issue 10 1902 doi: 10.3390/genes13101902
Xie T, Zhang S, Shen W, Zhang G, Guo R, Zhang W, Cao Y, Pan Q, Liu F, Sun Y, Liu S.Twinning trait in donkeys is an important manifestation of high fecundity, but few reports are available elucidating its genetic mechanism. To explore the genetic mechanism underlying the twin colt trait in Dezhou donkeys, DNA from 21 female Dezhou donkeys that had birthed single or twin colts were collected for whole-genome resequencing. FST, θπ and Tajima’s D were used to detect the selective sweeps between single and twin colt fecundity in the Dezhou donkey groups. Another set of 20 female Dezhou donkeys with single or multiple follicles during estrus were selected to compare concentrat...
Serum progesterone and oxytocinase, and endometrial and luteal gene expression in pregnant, nonpregnant, oxytocin, carbetocin and meclofenamic acid treated mares.
Theriogenology    October 18, 2022   Volume 198 47-60 doi: 10.1016/j.theriogenology.2022.10.019
Diel de Amorim M, Bramer SA, Rajamanickam GD, Klein C, Card C.Our objectives were to examine changes in endometrial and luteal gene expression during estrus, diestrus, pregnancy and treatments to induce luteolysis and putatively induce luteostasis. Groups were: Diestrus (DIEST), Estrus (ESTR), Pregnant (PREG), Oxytocin (OXY), Carbetocin (CARB), and Meclofenamic acid (MFA). Blood was obtained from day (D)12 to D15 for measurement of oxytocinase, also referred to as leucyl-cysteinyl aminopeptidase (LNPEP) and progesterone. Luteal biopsies were obtained on D12 and D15 and an endometrial biopsy on D15. Real-time RT-PCR was performed for the following genes: ...
Prospective Case Series of Clinical Signs and Adrenocorticotrophin (ACTH) Concentrations in Seven Horses Transitioning to Pituitary Pars Intermedia Dysfunction (PPID).
Veterinary sciences    October 17, 2022   Volume 9, Issue 10 572 doi: 10.3390/vetsci9100572
Kirkwood NC, Hughes KJ, Stewart AJ.Poor recognition of subtle clinical abnormalities and equivocal ACTH concentrations make early diagnosis of PPID difficult. Progressive clinical findings and corresponding ACTH concentrations in horses transitioning to PPID over time have not been documented. Seven horses with ACTH concentrations equivocal for PPID (utilizing locally derived, seasonally adjusted diagnostic-cut off values (DCOV)) and no clinical signs of PPID were selected. Sequential measurement of basal and thyrotropin-releasing hormone (TRH)-stimulated ACTH concentrations and recording of clinical findings occurred from Octo...