Assessment of Connective Tissue in the Equine Uterus and Cervix: Review of Clinical Impact and Staining Options.

Uterine diseases stand as the primary cause of infertility in mares; however, the diagnostic process often relies on obtaining endometrial biopsies and their hematoxylin-eosin staining. This review seeks to present the variability of uterine changes and their impact on fertility and underscore the utility of special stains, such as Masson trichrome, picrosirius red, elastica van Gieson, or periodic acid-Schiff, in enhancing diagnostic breadth. Connective tissue evaluation in the cervix is discussed, as it is subjected to cyclic changes and the impact on overall fertility. Vascular changes, particularly prevalent in multiparous mares, play a crucial role in adapting to physiological and pathological alterations, affecting early gestation and impeding placental development. Given that uterine vascular pathologies often involve fibrotic changes, connective tissue stains emerge as a valuable tool in this context. Moreover, equine endometriosis, predominantly associated with endometrial fibrosis, further highlights the relevance of special stains, suggesting their underutilization in the diagnostic process. Recognizing the subjective nature of diagnosing uterine pathologies and the need for additional diagnostic tools, we advocate for using dedicated stains in the histopathological evaluation of uterine samples. In conclusion, we encourage scientists and diagnosticians to embrace additional tools that enhance pathology visualization, enabling more reliable diagnoses concerning expected fertility.

The Role of NF-κB in Endometrial Diseases in Humans and Animals: A Review.

The expression of genes of various proinflammatory chemokines and cytokines is controlled, among others, by the signaling pathway of the nuclear factor kappaB (NF-κB) superfamily of proteins, providing an impact on immune system functioning. The present review addresses the influence and role of the NF-κB pathway in the development and progression of most vital endometrial diseases in human and animal species. Immune modulation by NF-κB in endometritis, endometrosis, endometriosis, and carcinoma results in changes in cell migration, proliferation, and inflammation intensity in both the stroma and epithelium. In endometrial cells, the NF-κB signaling pathway may be activated by multiple stimuli, such as bacterial parts, cytokines, or hormones binding to specific receptors. The dysregulation of the immune system in response to NF-κB involves aberrant production of chemokines and cytokines, which plays a role in endometritis, endometriosis, endometrosis, and endometrial carcinoma. However, estrogen and progesterone influence on the reproductive tract always plays a major role in its regulation. Thus, sex hormones cannot be overlooked in endometrial disease physiopathology. While immune system dysregulation seems to be NF-κB-dependent, the hormone-independent and hormone-dependent regulation of NF-κB signaling in the endometrium should be considered in future studies. Future goals in this research should be a step up into clinical trials with compounds affecting NF-κB as treatment for endometrial diseases.

The Postural and Body Surface Temperature Response of Leisure Horses to Lunging with Selected Lunging Aids.

Incorporating lunging into a horse's daily routine aims to enhance fitness, physical condition, and specific skills or exercises when using lunging aids (LAs). To assess the effectiveness of lunging, non-contact technologies like geometric morphometrics and infrared thermography can be employed. This study seeks to evaluate lunging efficiency based on the horse's posture and surface temperature when lunging with different head and neck positions. The study aims to determine if changes in a horse's posture correspond to increased metabolic activity, as indicated by body surface temperature. Thirteen horses included in the study were lunged with chambon (CH), rubber band (RB), and triangle side reins (TRs) as well as with a freely moving head (FMH). Images were taken in visible light and infrared. Principal Component Analysis (PCA) was used to analyze horse posture changes and a Pixel-Counting Protocol (PCP) was used to quantify surface temperature patterns. The horses' posture exhibited contrasting changes, reflected by a changing centroid shape (p < 0.0001) but not size (p > 0.05) when lunged with RB and TRs, but not CH. Different (p < 0.0001) surface temperature patterns were observed during lunging. FMH lunging resulted in lower temperatures over a larger surface, CH induced moderate temperatures on a smaller area, RB caused moderate to high temperatures across a broader surface, and TRs led to higher temperatures over a smaller region. The studied lunging cases returned different (p < 0.0001) surface temperature patterns. Lunging with FMH returned lower temperatures over a larger surface, CH moderate temperatures on a smaller area, RB moderate to high temperatures across a broader surface, and TRs higher temperatures over a smaller region. The proposed methods can be applied to evaluate the efficiency of lunging in horses.

Application of the Two-Dimensional Entropy Measures in the Infrared Thermography-Based Detection of Rider: Horse Bodyweight Ratio in Horseback Riding.

As obesity is a serious problem in the human population, overloading of the horse's thoracolumbar region often affects sport and school horses. The advances in using infrared thermography (IRT) to assess the horse's back overload will shortly integrate the IRT-based rider-horse fit into everyday equine practice. This study aimed to evaluate the applicability of entropy measures to select the most informative measures and color components, and the accuracy of rider:horse bodyweight ratio detection. Twelve horses were ridden by each of the six riders assigned to the light, moderate, and heavy groups. Thermal images were taken pre- and post-exercise. For each thermal image, two-dimensional sample (SampEn), fuzzy (FuzzEn), permutation (PermEn), dispersion (DispEn), and distribution (DistEn) entropies were measured in the withers and the thoracic spine areas. Among 40 returned measures, 30 entropy measures were exercise-dependent, whereas 8 entropy measures were bodyweight ratio-dependent. Moreover, three entropy measures demonstrated similarities to entropy-related gray level co-occurrence matrix (GLCM) texture features, confirming the higher irregularity and complexity of thermal image texture when horses worked under heavy riders. An application of DispEn to red color components enables identification of the light and heavy rider groups with higher accuracy than the previously used entropy-related GLCM texture features.

Molecular Mechanism of Equine Endometrosis: The NF-κB-Dependent Pathway Underlies the Ovarian Steroid Receptors' Dysfunction.

Endometrosis is a frequently occurring disease decreasing mares' fertility. Thus, it is an important disease of the endometrium associated with epithelial and stromal cell alterations, endometrium gland degeneration and periglandular fibrosis. Multiple degenerative changes are found in uterine mucosa, the endometrium. However, their pathogenesis is not well known. It is thought that nuclear factor-κB (NF-κB), a cell metabolism regulator, and its activation pathways take part in it. The transcription of the profibrotic pathway genes of the NF-κB in fibrotic endometria differed between the follicular (FLP) and mid-luteal (MLP) phases of the estrous cycle, as well as with fibrosis progression. This study aimed to investigate the transcription of genes of estrogen (ESR1, ESR2) and progesterone receptors (PGR) in equine endometria to find relationships between the endocrine environment, NF-κB-pathway, and fibrosis. Endometrial samples (n = 100), collected in FLP or MLP, were classified histologically, and examined using quantitative PCR. The phase of the cycle was determined through the evaluation of ovarian structures and hormone levels (estradiol, progesterone) in serum. The transcription of ESR1, ESR2, and PGR decreased with the severity of endometrial fibrosis and degeneration of the endometrium. Moreover, differences in the transcription of ESR1, ESR2, and PGR were noted between FLP and MLP in the specific categories and histopathological type of equine endometrosis. In FLP and MLP, specific moderate and strong correlations between ESR1, ESR2, PGR and genes of the NF-κB pathway were evidenced. The transcription of endometrial steroid receptors can be subjected to dysregulation with the degree of equine endometrosis, especially in both destructive types of endometrosis, and mediated by the canonical NF-κB pathway depending on the estrous cycle phase.

Comparison of Donkey, Pony, and Horse Dorsal Profiles and Head Shapes Using Geometric Morphometrics.

Since donkey breeding has increased due to their variety of uses, welfare evaluation has become more important. This study aimed to compare donkey, pony, and horse dorsal profiles and head shapes using geometric morphometrics (GM). Photographs of 14 donkeys, 14 ponies, and 14 horses were analyzed using GM, including the sliding semilandmarks method. The variations in the first three principal components (PCs) were PC1: 57.16%, PC2: 16.05%, and PC3: 8.31% for the dorsal profiles and PC1: 44.77%, PC2: 13.46%, and PC3: 7.66% for the head shapes. Both the dorsal profiles and head shapes differed between donkeys and horses (p < 0.0001) but not between donkeys and ponies (p > 0.05). Moreover, both the dorsal profiles and head shapes differed in size between ponies and horses (p < 0.0001) but not in shape (p > 0.05). Higher Mahalanobis and Procrustes distances were noted between donkeys and horses as well between donkeys and ponies than between ponies and horses. The use of geometric morphometrics revealed the differences in the dorsal profiles and head shapes between the studied equids. These differences should be taken into account when adapting welfare scales and methods from horses to donkeys.

The NF-κB-signalling pathway in mare's endometrium infiltrated with the inflammatory cells.

Endometritis is an important issue decreasing mares' fertility. In the case of endometritis, both inflammatory cells infiltration and proinflammatory molecules production are regulated by various cellular and gene regulatory mechanisms, including the nuclear factor-κB (NF-κB)-dependent pathway. NF-κB-signalling pathway has been recently studied in the equine endometrium in the context of endometrosis. Thus, this study aimed to determine gene transcription of NF-κB subunits (RelA; NF-κB1; NF-κB2), proinflammatory molecules (MCP-1; IL-6) and hyaluronan synthases (HAS 1; HAS 2; HAS 3) in endometritis and compare them with the intensity and type of inflammatory cell infiltration. Endometrial samples, collected post-mortem from cyclic mares in oestrus or dioestrus, were classified histologically and examined using quantitative PCR. Transcription NF-κB subunits genes did not differ with either inflammatory intensity or type of inflammatory cell infiltration. Transcription of MCP-1 and IL-6 genes increased with the severity of inflammation, with the involvement of HAS 3 and HAS 2 genes, as opposed to HAS 1 genes. These proinflammatory molecules and hyaluronan synthases in the equine inflamed endometrium do not seem to be regulated by the NF-κB pathway. Hence, separate signalling pathways for the development and progression of equine endometritis and endometrosis may be suggested.

Selection of Image Texture Analysis and Color Model in the Advanced Image Processing of Thermal Images of Horses following Exercise.

As the detection of horse state after exercise is constantly developing, a link between blood biomarkers and infrared thermography (IRT) was investigated using advanced image texture analysis. The aim of the study was to determine which combinations of RGB (red-green-blue), YUI (brightness-UV-components), YIQ (brightness-IQ-components), and HSB (hue-saturation-brightness) color models, components, and texture features are related to the blood biomarkers of exercise effect. Twelve Polish warmblood horses underwent standardized exercise tests for six consecutive days. Both thermal images and blood samples were collected before and after each test. All 144 obtained IRT images were analyzed independently for 12 color components in four color models using eight texture-feature approaches, including 88 features. The similarity between blood biomarker levels and texture features was determined using linear regression models. In the horses' thoracolumbar region, 12 texture features (nine in RGB, one in YIQ, and two in HSB) were related to blood biomarkers. Variance, sum of squares, and sum of variance in the RGB were highly repeatable between image processing protocols. The combination of two approaches of image texture (histogram statistics and gray-level co-occurrence matrix) and two color models (RGB, YIQ), should be considered in the application of digital image processing of equine IRT.

The Effect of Rider:Horse Bodyweight Ratio on the Superficial Body Temperature of Horse's Thoracolumbar Region Evaluated by Advanced Thermal Image Processing.

Appropriate matching of rider-horse sizes is becoming an increasingly important issue of riding horses' care, as the human population becomes heavier. Recently, infrared thermography (IRT) was considered to be effective in differing the effect of 10.6% and 21.3% of the rider:horse bodyweight ratio, but not 10.1% and 15.3%. As IRT images contain many pixels reflecting the complexity of the body's surface, the pixel relations were assessed by image texture analysis using histogram statistics (HS), gray-level run-length matrix (GLRLM), and gray level co-occurrence matrix (GLCM) approaches. The study aimed to determine differences in texture features of thermal images under the impact of 10-12%, >12 ≤15%, >15 <18% rider:horse bodyweight ratios, respectively. Twelve horses were ridden by each of six riders assigned to light (L), moderate (M), and heavy (H) groups. Thermal images were taken pre- and post-standard exercise and underwent conventional and texture analysis. Texture analysis required image decomposition into red, green, and blue components. Among 372 returned features, 95 HS features, 48 GLRLM features, and 96 GLCH features differed dependent on exercise; whereas 29 HS features, 16 GLRLM features, and 30 GLCH features differed dependent on bodyweight ratio. Contrary to conventional thermal features, the texture heterogeneity measures, InvDefMom, SumEntrp, Entropy, DifVarnc, and DifEntrp, expressed consistent measurable differences when the red component was considered.

Characteristics of the Donkey's Dorsal Profile in Relation to Its Functional Body Condition Assessment.

As the breeding of donkeys has increased due to different types of use, welfare evaluation importance increases. This equid's welfare state has been described using body condition indicators and the geometric morphometrics method. However, the dorsal profile has not yet been assessed in donkeys. In this study, the body condition score (BCS), fatty neck score (FNS), dental condition score (DCS), sex, and breed were used as criteria of dorsal profile deformations. Photographs of 40 donkeys were analyzed using geometric morphometrics. Within the entire set of dorsal profiles, the variance of the first three principal components (PCs) was PC1 = 37.41%, PC2 = 23.43%, and PC3 = 13.34%. The dorsal profiles displayed deformation as an effect of FNS and BCS on size (FNS p = 0.012; BCS p = 0.024) and shape (FNS p < 0.0001; BCS p < 0.0001), rather than as an effect of DCS (p < 0.0001), sex (p = 0.0264), and breed (p < 0.0001) only on shape. The highest distances among the categories (Mahalanobis distances: MD ≥ 13.26; Procrustes distances: PD ≥ 0.044) were noted for FNS. The lowest distances were noted between jennets and males (MD = 4.58; PD = 0.012) and between BCS 1 and BCS 2 (MD = 4.70; PD = 0.018). Donkeys' body condition affects their dorsal profile and both FNS and BCS measurements should be considered when a donkey's dorsal profile is investigated.

Equine Endometrosis Pathological Features: Are They Dependent on NF-κB Signaling Pathway?

Endometrosis is an important mares' disease which considerably decreases their fertility. As classic endometrial classification methods might be insufficient for tissue pathological evaluation, further categorization into active/inactive and destructive/non-destructive types was developed by Hoffmann and others. This study aimed to compare NF-κB pathway genes transcription among histopathological types of endometrosis, following Hoffmann and co-authors' classification. Endometrial samples, collected postmortem from cyclic mares (n = 100) in estrus or diestrus, were classified histologically and used for gene transcription assessment. Gene transcription of NF-κB subunits (RelA, NF-κB1, NF-κB2), pro-inflammatory molecules (MCP-1, IL-6), and hyaluronan synthases (HAS 1, HAS 2, HAS 3) was compared among endometrosis types (active, non-active, destructive, non-destructive). Most individual mRNA samples showed high expression of RelA, NF-κB1, and MCP-1 gene transcripts and the destructive type of endometrosis, simultaneously. The expression of RelA and NF-κB1 genes was higher in active destructive group than in the other groups only in the follicular phase, as well as being higher in the inactive destructive group than in the others, only in the mid-luteal phase. The increase in gene transcription of the NF-κB canonical activation pathway in destructive endometrosis may suggest the highest changes in extracellular matrix deposition. Moreover, the estrous cycle phase might influence fibrosis pathogenesis.

Association between the Area of the Highest Flank Temperature and Concentrations of Reproductive Hormones during Pregnancy in Polish Konik Horses-A Preliminary Study.

Determination of the pregnancy status is one of the most important factors for effective pregnancy management. Knowledge of the stage of pregnancy is important to interpret many of the reproductive hormones' concentrations, including progesterone (P4), estrone sulfate (E1S), 17-ß estradiol (E2), and relaxin (REL). However, it is limited in wildlife or captive equids that cannot be handled. Reproductive hormones affect regional blood flow, the proliferation of tissues, and local metabolism intensity. Therefore, this preliminary study aimed to assess changes in thermal features of the abdomen lateral surface and concentrations of reproductive hormones in Polish native pregnant mares. The study was carried out on 14 non-pregnant and 26 pregnant Polish Konik mares during eleven months of pregnancy. Infrared thermography was conducted to image the lateral surface of mares' abdomen (Px1) and flank area (Px2); P4, E1S, E2, and REL concentrations in serum were also determined. The evidence of the association between the area with the highest temperatures (Area of Tmax) and serum concentrations of P4 (the slope = 1.373; p = 0.9245) and REL (the slope = 1.342; p = 0.4324) were noted dependent across months of pregnancy. Measures of superficial body temperatures were found to change monthly, similarly to ambient temperatures, with no evidence of coincidence with changes in reproductive hormone concentrations. Individual thermal characteristics of the lateral surface of the abdomen differed between pregnant and non-pregnant mares in other periods. Differences in maximal and average temperature and Area of Tmax were observed from the sixth month of pregnancy, and those in minimal temperature were observed from the eighth month.

A novel approach to thermographic images analysis of equine thoracolumbar region: the effect of effort and rider's body weight on structural image complexity.

BACKGROUND: The horses' backs are particularly exposed to overload and injuries due to direct contact with the saddle and the influence of e.g. the rider's body weight. The maximal load for a horse's back during riding has been suggested not to exceed 20% of the horses' body weight. The common prevalence of back problems in riding horses prompted the popularization of thermography of the thoracolumbar region. However, the analysis methods of thermographic images used so far do not distinguish loaded horses with body weight varying between 10 and 20%. RESULTS: The superficial body temperature (SBT) of the thoracolumbar region of the horse's back was imaged using a non-contact thermographic camera before and after riding under riders with LBW (low body weight, 10%) and HBW (high body weight, 15%). Images were analyzed using six methods: five recent SBT analyses and the novel approach based on Gray Level Co-Occurrence Matrix (GLCM) and Gray Level Run Length Matrix (GLRLM). Temperatures of the horse's thoracolumbar region were higher (p < 0.0001) after then before the training, and did not differ depending on the rider's body weight (p > 0.05), regardless of used SBT analysis method. Effort-dependent differences (p < 0.05) were noted for six features of GLCM and GLRLM analysis. The values of selected GLCM and GLRLM features also differed (p < 0.05) between the LBW and HBW groups. CONCLUSION: The GLCM and GLRLM analyses allowed the differentiation of horses subjected to a load of 10 and 15% of their body weights while horseback riding in contrast to the previously used SBT analysis methods. Both types of analyzing methods allow to differentiation thermal images obtained before and after riding. The textural analysis, including selected features of GLCM or GLRLM, seems to be promising tools in considering the quantitative assessment of thermographic images of horses' thoracolumbar region.

Advances in Thermal Image Analysis for the Detection of Pregnancy in Horses Using Infrared Thermography.

Infrared thermography (IRT) was applied as a potentially useful tool in the detection of pregnancy in equids, especially native or wildlife. IRT measures heat emission from the body surface, which increases with the progression of pregnancy as blood flow and metabolic activity in the uterine and fetal tissues increase. Conventional IRT imaging is promising; however, with specific limitations considered, this study aimed to develop novel digital processing methods for thermal images of pregnant mares to detect pregnancy earlier with higher accuracy. In the current study, 40 mares were divided into non-pregnant and pregnant groups and imaged using IRT. Thermal images were transformed into four color models (RGB, YUV, YIQ, HSB) and 10 color components were separated. From each color component, features of image texture were obtained using Histogram Statistics and Grey-Level Run-Length Matrix algorithms. The most informative color/feature combinations were selected for further investigation, and the accuracy of pregnancy detection was calculated. The image texture features in the RGB and YIQ color models reflecting increased heterogeneity of image texture seem to be applicable as potential indicators of pregnancy. Their application in IRT-based pregnancy detection in mares allows for earlier recognition of pregnant mares with higher accuracy than the conventional IRT imaging technique.

The Effect of Lunging with Three Aids on the Thermographically Determined Temperatures of the Distal Portion of Horse Limbs.

The regulation of head and neck position during physical exercise may be obtained using lunging aids (LAs): Chambon (CH), rubber band (RB), or triangle side reins (TR). The use of LAs would shift the center of horse mass caudally, thereby change the limb load. The aim is to evaluate the pattern of superficial temperatures (PST) of hoof wall (HW), the coronary band (CB), and the dorsal aspect of the fetlock joint (FJ) obtained before and after lunging with LAs and freely moving head (FMH). Sixteen horses were imaged using an infrared camera. The temperatures of HW, CB, and FJ were measured, and differences between images obtained before and after lunging were evaluated. The correlations between HW, CB, and FJ were then described using respective coefficients (r, rho). Before lunging PST was similar (P < .05) in FMH, CH, RB, and TR, with the strong positive correlations for forelimbs (HW/CB:rho = 0.9022; HW/FJ:rho = 0.7064; CB/FJ:rho = 0.8010) and hindlimbs (HW/CB:rho = 0.7287; HW/FJ:r = 0.7831; CB/FJ:r = 0.7233). After lunging, PST and correlations changed (P > .05) depending on LAs application. No correlations for FMH and CH, and positive correlations for RB and TR were noted: HW/CB for forelimbs (RB:r = 0.6813; TR:r = 0.8396), CB/FJ for hindlimbs (RB:r = 0.5621; TR:rho = 0.4579). The PST of the distal portion of limbs and relations between measured temperatures change depending on used LAs.

The Pattern of Superficial Body Temperatures in Leisure Horses Lunged with Commonly Used Lunging Aids.

BACKGROUND: The natural head and neck position (HNP) of horses differs from the position in horse riding when bit is used. The special lunging aids (LAs) are applied in order to modify HNP. Different types of LAs have the potential to affect the work of horse muscles and the superficial thermographic patterns (STPs). The effects of thre LAs on STPs of neck, chest, back, and hindquarters were investigated. METHODS: Sixteen leisure horses were lunged with freely moving head (FMH), rubber band (RB), chambon (CH), and triangle side reins (TRs). The thermographic images (n = 896) were analyzed before/after lunging for mean temperatures (Tmean) and minimum-maximum difference (Tdiff). RESULTS: Superficial Tmean increased (p < 0.001) in cranial part of neck, back, thoracic area, and limbs after lunging regardless of LAs application or its type. In comparison to other LAs: With RB, Tmean was higher in regions of interest (ROIs) 2,7 and lower in ROIs 3-4 (p < 0.05); with CH, Tmean was higher in ROIs 2-4 and 7 (p < 0.01); and with TRs, Tmean was higher in ROIs 2-4,7,9-11 (p < 0.01). In ROIs 2-4 and 7, Tdiff was lower with LAs than with FMH (p < 0.01) and in ROIs 9-10 with TRs. CONCLUSIONS: The choice of LAs should be dictated by the expected effect; however, all LAs increase the quality of the leisure horse lunging. LA use is more desirable than lunging with FMH.

An application of temperature mapping of horse's back for leisure horse-rider-matching.

Leisure riding is a popular way of using horses however, unlike sport or racing horses, those are mostly not associated with one rider with high skills. Constant overload of equine musculoskeletal system causes pathologies, which are affecting horse mobility and decreases the horse-rider communication. The aim was to propose the new scoring system of thermograph analysis as an aspect of differences in heat distributions on horseback before and after leisure ridings. The study was conducted on sixteen Polish warmblood horses, scanned with a non-contact thermographic camera. Heat pattern of the thoracolumbar area was evaluated on thermograms taken before and after exercise. The criteria with point values for horse-rider-matching were created: heat points on the dorsal midline of saddle-back contact area and degree of muscle unit overload. The results of thermograph analysis were compared with the results of a questionnaire on horse-rider communication during riding in order to estimate the relevance of matching. The maximum score was obtained in 38.3% and 39.8% of combinations based on the thermograph analysis and questionnaire, respectively. Results of both scoring systems were strongly positive correlated (r = .937), demonstrating high sensitivity (61.72%) and specificity (90.23%) of the matching. The horse-rider matching may improve horse comfort during leisure type of work.