How Useful Are Existing Protocols in the Quick Assessment of the Welfare of Semi-Feral Horses? Pilot Study on Konik Polski Horses Living in the Forest Sanctuary.

Scientifically validated and standardised methods for the evaluation of the welfare of free-living horses are urgently needed by both the owners and managers of these populations and those responsible for implementing national welfare legislation. The aim of the study was to test the feasibility and usefulness of two welfare protocols that could be applied to semi-feral populations: a prototype of welfare assessment template (WAT) for Carneddau semi-feral ponies and the IFCE/INRAE Horse Welfare Protocol. Additionally, the body condition scale designed by Henneke (BCS-H) was employed. The study took place in July/August 2022 and April 2023 to evaluate the welfare of a pilot population of nineteen semi-feral Konik polski horses. The horses scored high or satisfactory under indicators across both protocols; only body condition scores were significantly lower in early spring (BCS-WAT: 1.11 ± 0.57; BCS-H: 3.84 ± 1.17) than in the summer (BCS-WAT: 1.58 ± 0.61; BCS-H: 5.63 ± 1.01). Our study confirmed the feasibility of utilising most of the WAT and IFCE/INRAE welfare indicators in semi-feral horses. Some adaptations, such as considering validation of scales, positive welfare indicators and animals' free-choice of conditions, have been suggested for future in-field application.

Human-controlled reproductive experience may contribute to incestuous behavior observed in reintroduced semi-feral stallions (Equuscaballus).

Equine reproductive behavior is affected by many factors, some remaining poorly understood. This study tested the hypothesis that a period of captivity during the juvenile period and human-controlled reproduction may potentially be involved in the disruption of the development of incestuous mating avoidance behavior in sanctuary-reintroduced male Konik polski horses. Between 1986 and 2000, cases of incestuous behavior in harem stallions born and reared until weaning in the sanctuary were studied. Eight males lived in the sanctuary's feral herd for the rest of their lives (the non-captive group; nC). They gained their own harem of mares without human intervention (no human-controlled reproductive activity, nHC). Another five stallions were removed as weanlings, reared in captivity and then reintroduced as adults (captive, C). Three of these C stallions were used as in-hand breeding stallions, one as a "teaser" (human-controlled reproductive activity, HC) and one was not used for reproduction in captivity (nHC). Reproductive records for 46 mares, daughters of all 13 harem stallions, were scrutinized and cases of incestuous breeding were recorded by interrogation of foal parentage records. C stallions failed to expel more daughters than nC stallions (33% vs. 18%, P = 0.045), and mated with significantly more of them (28% vs. 11%, P = 0.025). Interestingly, HC stallions expelled fewer (60%) and successfully mated with more (33%) daughters that nHC stallions (84% expelled, P = 0.013, and 10% successful mating with daughters, P = 0.010). All HC stallions bred incestuously at least once. We propose that human intervention during a critical period of development of social and reproductive behavior in young stallions, by enforced separation from their natal herd and in-hand breeding, may contribute to their later aberrant behavior and disruption of inbreeding avoidance mechanisms in these stallions. The previous occurrence of human-controlled breeding may be one of the factors promoting incestuous behavior of stallions in natural conditions. The uninterrupted presence of stallions in their harems and herd member recognition may also play important roles in inbreeding avoidance in horses.

Some Genetic and Environmental Effects on Equine Asthma in Polish Konik Horses.

Current knowledge of the genetic and environmental backgrounds of equine asthma seems to be insufficient, especially for primitive horse breeds. The main objectives of this study were to estimate the effects of sex, birth period, stud, parentage line and inbreeding on asthma morbidity in Polish Konik horses. Records of 274 horses (housed in two studs) were analysed. These animals were allocated to maternal and paternal lines. Individual inbreeding coefficients were extracted from the additive relationship matrix. Horses underwent diagnosis based on observation of the basic symptoms (high frequency of coughing and excessive nasal discharge). Subsequently, some horses (28 individuals) were clinically examined to confirm the earlier observations. Generally, no significant effects of parentage line on heaves morbidity were identified by the use of logistic regression, although the Pearson's chi-squared test had shown that individuals of some maternal and paternal lines had a predisposition to severe equine asthma. It was concluded that the individual inbreeding level is not associated with the incidence of EA, but a significant effect of the maternal inbreeding coefficient may be observed. It was also found that there is some variability in the incidence of this disease between studs.

Investigations of the maintenance system of the Konik Polski horse and its effects on fecal microbiota activity during the winter and summer seasons.

Gastrointestinal microbiota play a key role in the nutrients digestion and hence maintaining animal health and welfare. The diet offered to the animals in captivity may differ considerably from that on natural pastures. In a stabled maintenance system, horses have a limited choice of habitat and feed. Time spend for feeding is relevant for equine welfare because the reduction of the time devoted for foraging may be responsible for inducing gastric inflammation and ulceration. Therefore, in the present study, it was hypothesized that fecal bacterial fermentative processes differ between free-roaming and stabled Konik Polski Horses (KPHs) with respect to microbial enzymatic activity, and thus the fecal concentration of short-chain fatty acids (SCFA), which can be further utilized for assessing the feeding behavior and welfare in free-roaming versus stabled horses. The SCFA concentration and profile, as well as the extracellular and intracellular activities of selected bacterial enzymes, were characterized in horse feces collected during the winter and summer feeding seasons. The results showed higher enzymatic activity and SCFA production in the feces excreted by free-roaming versus stabled horses, especially during summer. An increase in pasture plant diversity may be beneficial for the gastrointestinal microbiota and hence for maintaining health and welfare.

The examination of biophysical parameters of the skin in Polish Konik horses.

This study aimed to assess the biophysical parameters of the skin in Polish Konik horses (Polish primitive horses). According to the authors, this is the first assessment performed on such a wide scale in this group of animals. The evaluation carried out is innovative both with regards to the breed of the animals and the wide scope of the physicochemical skin assessment. The study group comprised mares, stallions and geldings, and the evaluations concerned transepidermal water loss, corneometry, pH, skin temperature assessment and mexametry. These parameters were assessed in five skin regions: the lips, the right ear, the prosternum, the right side of the neck and the chest. The measurements were taken after spreading the hair apart, with the use of a Multiprobe Adapter System (MPA®) and dedicated probes (Courage + Khazaka electronic GmbH, Cologne, Germany). The measurements revealed statistically significant differences in the values of transepidermal water loss in the lips in mares compared with stallions (P = 0.023) and also in stallions compared with geldings (P = 0.009). Corneometry showed significantly higher results in the neck region in mares compared with stallions (P = 0.037) and the prosternum areas in mares and geldings compared with stallions (P = 0.037 and P = 0.018). Skin pH measurement on the right side of the neck rendered significantly higher values in stallions than in mares (P = 0.037). In geldings, the skin temperature was significantly higher than in stallions (P = 0.049). Once the appropriate physicochemical values for specific animal species and breeds are determined, non-invasive methods of skin examination in many diseases and also methods of evaluation of the efficacy and/or adverse effects of applied medications can be established.

Transcriptome Profiling of the Retained Fetal Membranes-An Insight in the Possible Pathogenesis of the Disease.

Retained fetal membranes (RFM) is one of the most common post-partum diseases of a complex etiology. Moreover, its pathogenesis is still not elucidated. Detailed transcriptomic analysis of physiological and retained placenta may bring profound insight in the pathogenesis of the disease. The aim of the study was to compare the transcriptome of the retained and physiologically released placenta as well as biological pathways and processes in order to determine the possible pathogenesis of the disease. Samples of the endometrium and the allantochorion were taken within 2 h after parturition from control mares (n = 3) and mares with RFM (n = 3). RNA sequencing was performed with the use of all samples and mRNA expression of chosen genes was validated with Real Time PCR. Analysis of RNA-seq identified 487 differentially expressed genes in the allantochorion and 261 in the endometrium of control and RFM mares (p < 0.0001). Within genes that may be important in the release of fetal membranes and were differentially expressed, our report pinpointed BGN, TIMP1, DRB, CD3E, C3, FCN3, CASP3, BCL2L1. Gene ontology analysis showed possible processes which were altered in RFM that are apoptosis, inflammatory-related processes, and extracellular matrix metabolism and might be involved in the pathogenesis of RFM. This is the first report on the transcriptome of RFM and physiologically released placenta in mares.

Expression profile of proinflammatory mediators in the placenta of mares during physiological detachment and retention of fetal membranes.

Physiological parturition is characterized by sterile, inflammatory-like processes. During parturition, the placenta expresses various proinflammatory mediators, such as chemokines and IL-17. Nevertheless, inflammatory processes present in the parturient mare are poorly characterized. The aim of this study was to investigate the expression of selected chemokines and IL-17 in the allantochorion and the endometrium of mares that retained fetal membranes (RFM) and expelled them physiologically. We hypothesized that the expression of these mediators may be altered in the placenta of mares with RFM and result in RFM occurrence. Differences in mRNA expression in the placenta of investigated groups of mares were detected for CCL2, CCL3, CCL4, CCL8, CXCL1, CXCL8, CXCL10, CX3CL1 and IL-17. There were no differences in mRNA expression of CCL5 and CXCL6. Gene ontology network analysis showed enrichment in genes related to leukocyte migration, cell chemotaxis and response to chemokine in tissues of RFM mares. Analysis of association network suggested denotations between CXCL6, CXCL8, CXCL1, CCL5, CCL4, CX3CL1 and CXCL10. Moreover, possible inhibition of CXCL10 by IL-17A and prostaglandin peroxide synthase 2 (PTGS2) by CXCL1 was detected. Our results suggest that, based on differences in chemokines and IL-17 expression, recruited subsets of leukocytes might differ between the analyzed groups of mares, which in turn may impair the separation of fetal membranes in the group of RFM mares. In addition, the results of the expression analysis suggest that macrophages might be one of the most abundant cells infiltrating the equine placenta during the expulsion of fetal membranes. Furthermore, we suspect that the synthesis of PTGS2 might be inhibited in mares with RFM.

Welfare of Free-Roaming Horses: 70 Years of Experience with Konik Polski Breeding in Poland.

To prevent abuse and to assure the welfare of domestic horses, attempts to assess welfare in a standardized way have been made. Welfare-assessment tools often refer to the physical and social environments of feral domestic horses as examples of welfare-friendly conditions for horses. However, free-roaming horses are often exposed to conditions or states that may be regarded as welfare threats or abuse. The aim of this review was to present cases of welfare compromises as well as natural ways to restore high standards of welfare to Konik polski horses (Koniks) living in semiferal conditions in a forest sanctuary over the course of 70 years. Welfare problems in Koniks related to feeding, locomotor, social, reproductive, and comfort behavior, as well as health issues concerning hoof trimming and parasitism in Koniks, are discussed. Periodic food scarcity or abundance, stressful events around weaning and gathering, the consequences of fights among stallions, exposure to sire aggression during dispersal, lameness during "self-trimming," exposure to insect harassment, high levels of parasitism, and specific landscape formations may endanger free-roaming horses. It has to be underlined that despite the excellent adaptability of horses to free-roaming conditions, one should be aware that welfare problems are to be expected in any semiferal population. Here, we present the management system applied for 70 years in free-roaming Konik polski horses that minimizes welfare threats. It allows close follow-up of individual horses, the strict monitoring of health and welfare on a daily basis, and if necessary, instant reactions from caretakers in cases of emergency. Moreover, it addresses the problem of starvation due to overgrazing and thus, the ethical controversy related to the eradication of surplus animals causing environmental damage.

Inter- and intrabreed diversity of the major histocompatibility complex (MHC) in primitive and draft horse breeds.

BACKGROUND: Polymorphism of major histocompatibility complex (MHC) genes ensures effective immune responses against a wide array of pathogens. However, artificial selection, as performed in the case of domestic animals, may influence MHC diversity. Here, we investigate and compare the MHC diversity of three populations of horses, for which different breeding policies were applied, to evaluate the impact of artificial selection and the environment on MHC polymorphism. METHODS: Samples of DNA were taken from 100 Polish draft horses, 38 stabled Konik Polski horses and 32 semiferal Konik Polski horses. MHC alleles and haplotype diversity within and between these populations of horses was estimated from 11 MHC microsatellite loci. RESULTS: MHC diversity measured based on allelic richness, observed heterozygosity, expected heterozygosity and polymorphism content was similar across the MHC microsatellite loci in all three populations. The highest expected heterozygosity was detected in semiferal primitive horses (He = 0.74), while the lowest was calculated for draft horses (He = 0.65). In total, 203 haplotypes were determined (111 in Polish draft horses, 43 in semiferal Konik Polski horses and 49 in stabled Konik Polski horses), and four haplotypes were shared between the two populations of Koniks. None of these haplotypes were present in any of the previously investigated horse breeds. Intra-MHC recombination events were detected in all three populations. However, the population of semiferal Konik horses showed the highest recombination frequency among the three horse populations. In addition, three recombination events were detected. CONCLUSIONS: These results showed that despite the different breeding policies, the MHC allele and haplotype diversity was similarly high in all three horse populations. Nevertheless, the proportion of new haplotypes in the offspring was the highest in semiferal Konik Polski horses, which indicates the influence of the environment on MHC diversity in horses. Thus, we speculate that the genetic makeup of the domestic horse MHC might be more strongly influenced by the environment than by artificial selection. Moreover, intra-MHC conversion, insertion, and deletion and intra-MHC recombination may be proposed as mechanisms underlying the generation of new MHC haplotypes in horses.

The oxytocin-prostaglandins pathways in the horse (Equus caballus) placenta during pregnancy, physiological parturition, and parturition with fetal membrane retention.

Despite their importance in mammalian reproduction, substances in the oxytocin-prostaglandins pathways have not been investigated in the horse placenta during most of pregnancy and parturition. Therefore, we quantified placental content of oxytocin (OXT), oxytocin receptor (OXTR), and prostaglandin E2 and F2 alpha during days 90-240 of pregnancy (PREG), physiological parturition (PHYS), and parturition with fetal membrane retention (FMR) in heavy draft horses (PREG = 13, PHYS = 11, FMR = 10). We also quantified OXTR and prostaglandin endoperoxide synthase-2 (PTGS2) mRNA expression and determined the immunolocalization of OXT, OXTR, and PTGS2. For relative quantification of OXT and OXTR, we used western blotting with densitometry. To quantify the prostaglandins, we used enzyme immunoassays. For relative quantification of OXTR and PTGS2, we used RT-qPCR. For immunolocalization of OXT, OXTR, and PTGS2, we used immunohistochemistry. We found that OXT was present in cells of the allantochorion and endometrium in all groups. PTGS2 expression in the allantochorion was 14.7-fold lower in FMR than in PHYS (p = 0.007). These results suggest that OXT is synthesized in the horse placenta. As PTGS2 synthesis is induced by inflammation, they also suggest that FMR in heavy draft horses may be associated with dysregulation of inflammatory processes.

Evidence for Increased Content of PGF2α, PGE2, and 6-keto-PGF1α in Endometrial Tissue Cultures From Heavy Draft Mares in Anestrus With Endometritis.

Endometritis is one of the most important causes of infertility in mares. Mares may suffer from endometritis outside the breeding season; however, pathological condition is often undiagnosed in anestrus. The aim of this study was to examine whether the secretion profiles of prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2), and a metabolite of prostacyclin I2 (6-keto-PGF1α) differ in endometrial tissue cultures of heavy draft mares in anestrus with endometritis compared to those without endometritis. The endometrial biopsies were collected from 51 heavy draft mares. Inclusion criteria for the control group were absence of endometritis confirmed by histology and no ovarian activity. Inclusion criteria for the experimental group were presence of endometritis showing polymorphonuclear cells and/or lymphocytes infiltration in endometrium and no ovarian activity. Retrospectively, 22 mares were enrolled in this study. The content of PGF2α (P < .05) and PGE2 (P < .001) in the culture medium was distinctly elevated in mares suffering from endometritis, compared to control mares. The relative mRNA abundance responsible for prostaglandins synthesis, that is, PGF2α synthase (PGFS; P < .01), PGE2 synthase (PGES; P < .01), and prostaglandin-endoperoxide synthase (PTGS2; P < .01), were also increased in endometrial tissue of mares with endometritis compared to control mares. The content of 6-keto-PGF1α (P < .0001) in endometrial tissue cultures from mares with endometritis was strikingly elevated compared to those without endometritis; however, plasma concentration of 6-keto-PGF1α was not significantly different between experimental and control groups. This leads to the conclusion that augmented endometrial secretion of PGF2α, PGE2, and 6-keto-PGF1α is associated with endometritis even in mares in anestrus.

Advanced age in mares affects endometrial secretion of arachidonic acid metabolites during equine subclinical endometritis.

Even if mares continue to breed up to an advanced age, in aging mares reproductive failure is quite common. Subclinical endometritis, which occurs more often in aging mares than in younger counterparts, may cause prolongation or shortening of the inter-estrus period or the corpus luteum lifespan. We hypothesized that during subclinical endometritis the secretion of selected arachidonic acid metabolites may differ in aging mares compared to younger females. To verify this thesis, ex vivo organ cultures of endometrium were established with subsequent measurements of concentrations of prostaglandin E2 (PGE2), 6-keto-PGF1α and both leukotrienes (LTs), LTB4 and LTC4 in the culture supernatants. The endometrial biopsies were obtained from 82 mares of known breeding history. This study revealed that the concentrations of the selected arachidonic acid metabolites, which act both as immunological mediators and endocrine modulators in the reproductive organs, depends on the mares' ages. Spontaneous endometrial secretion of PGE2, 6-keto-PGF1α and LTC4 was increased in mares aged 16-23 years that suffered from subclinical endometritis, compared with control counterparts. Moreover, secretion of these metabolites was higher in endometritis-positive mares aged 16-23 years than in younger females. We conclude that advanced age in mares further disturbs the immuno-endocrine balance in endometritis-positive mares.

Cells expressing CD4, CD8, MHCII and endoglin in the canine corpus luteum of pregnancy, and prepartum activation of the luteal TNFα system.

In the dog, knowledge about involvement of the immune system in controlling luteal function is restricted to observations showing a time-dependent invasion of immune cells into the corpus luteum (CL) of non-pregnant bitches. Therefore, this study investigated the presence of CD4-, CD8-, MHCII- and endoglin-expressing cells in CL collected throughout pregnancy from pre-implantation until prepartum luteolysis. Immunohistochemistry and semi-quantitative RT-PCR were applied. The time-dependent expression of CD4, CD8 and endoglin was more strongly related to formation of the CL, whereas MHCII was induced during luteolysis. Next, the luteal expression of TNFα and its receptors, TNFR1 and TNFR2, was analyzed in non-pregnant dogs between days 5-65 after ovulation and during pregnancy. Moreover, the effects of progesterone withdrawal were investigated in mid-pregnant dogs treated with an antigestagen aglepristone. The TNFα system was induced in the early CL of non-pregnant dogs. In pregnant dogs, expression of TNFα did not vary much, contrasting with increased expression of both receptors in the post-implantation period and significantly decreased expression at mid-gestation; prepartum luteolysis was characterized by increased TNFR2 expression. Apart from the downregulated expression of TNFR1, the changes observed following antigestagen treatment resembled those observed during normal prepartum luteolysis. A modulatory function of the TNFα system during formation of the canine CL is suggested, possibly related to the strong accompanying vascularization and luteal infiltration with activated macrophages. Contrasting with the slow luteal regression in non-pregnant dogs, in pregnant animals the upregulation of TNFR2 expression during prepartum luteolysis implies functional involvement of the TNFα system during that time.

Programmed necrosis - a new mechanism of steroidogenic luteal cell death and elimination during luteolysis in cows.

Programmed necrosis (necroptosis) is an alternative form of programmed cell death that is regulated by receptor-interacting protein kinase (RIPK) 1 and 3-dependent, but is a caspase (CASP)-independent pathway. In the present study, to determine if necroptosis participates in bovine structural luteolysis, we investigated RIPK1 and RIPK3 expression throughout the estrous cycle, during prostaglandin F2α (PGF)-induced luteolysis in the bovine corpus luteum (CL), and in cultured luteal steroidogenic cells (LSCs) after treatment with selected luteolytic factors. In addition, effects of a RIPK1 inhibitor (necrostatin-1, Nec-1; 50 µM) on cell viability, progesterone secretion, apoptosis related factors and RIPKs expression, were evaluated. Expression of RIPK1 and RIPK3 increased in the CL tissue during both spontaneous and PGF-induced luteolysis (P < 0.05). In cultured LSCs, tumor necrosis factor α (TNF; 2.3 nM) in combination with interferon γ (IFNG; 2.5 nM) up-regulated RIPK1 mRNA and protein expression (P < 0.05). TNF + IFNG also up-regulated RIPK3 mRNA expression (P < 0.05), but not RIPK3 protein. Although Nec-1 prevented TNF + IFNG-induced cell death (P < 0.05), it did not affect CASP3 and CASP8 expression. Nec-1 decreased both RIPK1 and RIPK3 protein expression (P < 0.05). These findings suggest that RIPKs-dependent necroptosis is a potent mechanism responsible for bovine structural luteolysis induced by pro-inflammatory cytokines.

Identifying diagnostic endocrine markers and changes in endometrial gene expressions during pyometra in cats.

Pyometra is a significant reproductive problem in cats. The aims of this study were to evaluate (i) the immunological profile of queens by studying plasma concentrations of metabolites of prostacyclin I2 (6-keto-PGF1α), leukotriene B4 (LTB4) and leukotriene C4 (LTC4); and (ii) the gene transcription profiles of Toll-like receptors (TLRs) 2 and 4 (TLR2/4), PGE2-synthase (PGES), PGF2α-synthase (PGFS) and prostaglandin-endoperoxide synthase 2 (PTGS2) in the feline endometrium throughout the estrous cycle, after medroxyprogesterone acetate (MPA) treatment and during pyometra. The concentration of plasma 6-keto-PGF1α in pyometra was increased in comparison to other groups studied (p<0.01). Endometrial mRNA coding for TLR2 was up-regulated in cats suffering from pyometra compared to other groups (p<0.001). Expression of mRNA for TLR4 was up-regulated in endometria originating from MPA-treated cats, pyometra and late diestrus cats, compared with tissues from cats during estrus and anestrus (p<0.05). As expected, endometrial mRNA for PTGS2 was up-regulated only in pyometra, compared with other groups (p<0.001). Similarly, endometrial mRNA for PGFS was up-regulated in pyometra, compared with endometria from anestrus, late diestrus and from MPA-treated cats (p<0.05), or from cats during estrus (p<0.01). Overall, these results indicate that plasma concentrations of LTB4 and LTC4 are not useful diagnostic markers since they were not increased in queens with pyometra, in contrast to 6-keto-PGF1α. In addition, treatment with MPA evoked neither endocrine nor molecular changes in endometria of cats.

Synthesis and reception of prostaglandins in corpora lutea of domestic cat and lynx.

Felids show different reproductive strategies related to the luteal phase. Domestic cats exhibit a seasonal polyoestrus and ovulation is followed by formation of corpora lutea (CL). Pregnant and non-pregnant cycles are reflected by diverging plasma progesterone (P4) profiles. Eurasian and Iberian lynxes show a seasonal monooestrus, in which physiologically persistent CL (perCL) support constantly elevated plasma P4 levels. Prostaglandins (PGs) represent key regulators of reproduction, and we aimed to characterise PG synthesis in feline CL to identify their contribution to the luteal lifespan. We assessed mRNA and protein expression of PG synthases (PTGS2/COX2, PTGES, PGFS/AKR1C3) and PG receptors (PTGER2, PTGER4, PTGFR), and intra-luteal levels of PGE2 and PGF2α Therefore, CL of pregnant (pre-implantation, post-implantation, regression stages) and non-pregnant (formation, development/maintenance, early regression, late regression stages) domestic cats, and prooestrous Eurasian (perCL, pre-mating) and metoestrous Iberian (perCL, freshCL, post-mating) lynxes were investigated. Expression of PTGS2/COX2, PTGES and PTGER4 was independent of the luteal stage in the investigated species. High levels of luteotrophic PGE2 in perCL might be associated with persistence of luteal function in lynxes. Signals for PGFS/AKR1C3 expression were weak in mid and late luteal stages of cats but were absent in lynxes, concomitant with low PGF2α levels in these species. Thus, regulation of CL regression by luteal PGF2α seems negligible. In contrast, expression of PTGFR was evident in nearly all investigated CL of cat and lynxes, implying that luteal regression, e.g. at the end of pregnancy, is triggered by extra-luteal PGF2α.

Type of Inflammation Differentially Affects Expression of Interleukin 1ß and 6, Tumor Necrosis Factor-α and Toll-Like Receptors in Subclinical Endometritis in Mares.

Mares that fail to conceive or lose their embryos, without showing typical signs of clinical endometritis, should be suspected of subclinical endometritis (SE). In this study, the question was addressed: does SE fully activate selected mechanisms of innate immunity in mares? For this aim, expression of mRNAs for Toll-like Receptor 2 and 4 (TLR 2/4), interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor α (TNF) was examined in control mares versus either mares suffering from chronic endometritis (ChE) or subacute suppurative endometritis (SSE). The concentrations of IL-1ß, IL-6 and TNF-α in supernatants from endometrial tissue cultures after 4 h incubation were measured using the enzyme immunoassay (EIA) method. Eighty-two warmblood mares, of known breeding history, were enrolled in this study. Based on histopathological assessment, mares were classified as suffering from ChE, SSE or as being healthy. In addition, immuno-localization of both TLR2 and TLR4 as well as TNF-α was investigated in the equine endometria. The mRNA expression of TLR2 (P < 0.01), IL-1ß (P < 0.0001), IL-6 (P < 0.0001) and TLR4 and TNF (P < 0.05) was up-regulated in endometria of mares suffering from SSE compared with unaffected mares. Concentrations of IL-6 and TNF-α were increased only in mares exhibiting SSE, compared with unaffected (P < 0.01 for both) and ChE mares (P < 0.05 for both). Immuno-localization of TNF-α and TLRs was confirmed, both in unaffected and SE-affected endometria, and was present in the luminal and glandular epithelia and stromal cells. The severity of inflammation impacts the immune response and fosters activation of innate immunity mechanisms, as observed in the endometria of mares. The intracellular localization of TLRs and TNF-α in the endometria indicates a key role of endometrial epithelial and stromal cells in the immune response and inflammation.

Comparison of the effect of lipopolysaccharide on tumor necrosis factor α (TNF-α) secretion and TNF and TNFR1 mRNA levels in feline endometrium throughout the estrous cycle during pyometra and after medroxyprogesterone acetate treatment.

Endotoxins released by Gram-negative bacteria are potent stimulators of tumor necrosis factor α (TNF-α) production. The objectives of this study were to evaluate plasma levels of TNF-α, TNF-α secretion, and mRNA levels of TNF and TNF-α receptor type 1 (TNFR1) following exposure to lipopolysaccharide (LPS). For this, we used cultured endometrial cells or organ cultures, throughout the estrous cycle, after hormone treatment with medroxyprogesterone acetate (MPA), and during pyometra. Plasma TNF-α concentrations were increased in animals at estrus (P < 0.05) compared to other groups. In the LPS-challenged endometrium, secretion of TNF-α by tissues collected during estrus increased (P < 0.001) compared to that of other groups. LPS, alone or combined with TNF-α, upregulated TNF gene expression in the feline endometrium at diestrus (P < 0.001 for both treatments), in queens treated short-term with MPA (P < 0.01 and P < 0.05, respectively) and in queens treated long-term with MPA (P < 0.01 and P < 0.001, respectively). During pyometra, TNF and TNFR1 mRNA were increased only after tissues were challenged with TNF-α and LPS (P < 0.001 and P < 0.01, respectively). When cultured endometrial cells were challenged with LPS, the concentration of TNF-α increased only in epithelial cells after 4 h and 12 h (P < 0.05 and P < 0.01, respectively). Since LPS did not affect stromal cells, but TNF-α increased its own transcript after 2 h (P < 0.01), 4 h (P < 0.05) and 12 h (P < 0.001), we assume that stromal cells are not directly involved in pathogen recognition, as was the case for epithelial cells.

Prostaglandin release by cultured endometrial tissues after challenge with lipopolysaccharide and tumor necrosis factor α, in relation to the estrous cycle, treatment with medroxyprogesterone acetate, and pyometra.

To better understand the pathogenesis of endometrial changes in cats associated with administration of progestagen contraceptives and with pyometra, we examined local variability of the prostaglandin synthesis system after challenge with either tumor necrosis factor α (TNF-α) or lipopolysaccharide (LPS) in organ cultures of endometrial tissues derived from cyclic cats, cats treated with medroxyprogesterone acetate (MPA), or cats with pyometra, as well as in cultured endometrial epithelial and stromal cells. In addition, spontaneous prostaglandin secretion was compared in endometria from different experimental groups. Data gathered in the present study show that the concentration of PGE2 in supernatants was increased only in endometrial organ cultures from cats with pyometra (P < 0.001) compared with other groups. This was also true for PGF2α in pyometra, compared with cats treated either short- or long-term with MPA and cats during late diestrus (P < 0.001), anestrus (P < 0.01), and estrus and middiestrus (P < 0.05). Treatment with LPS and TNF-α combined stimulated PGE2 secretion in all groups compared with the control (P < 0.001 for endometria of cats during anestrus or middiestrus, cats treated short-term with MPA, and those with pyometra; P < 0.01 for endometria of cats treated long-term with MPA; and P < 0.05 for the endometria of cats during estrus and late diestrus). The combined treatment with LPS and TNF-α increased PGF2α secretion in the endometria of cats treated short-term with MPA (P < 0.001), during anestrus and pyometra (P < 0.01 for both), and estrus and middiestrus (P < 0.05 for both), compared with the control. Spontaneous secretion of prostaglandins was several times greater in the endometria of queens with pyometra, compared with other groups, which may further regulate the local inflammatory response. Data gathered from endometrial cell culture and endometrial organ culture lead to the conclusion that disturbances in prostaglandin release contribute to pyometra in cats.

Altered secretion of selected arachidonic acid metabolites during subclinical endometritis relative to estrous cycle stage and grade of fibrosis in mares.

Mares that fail to become pregnant after repeated breeding, without showing typical signs of clinical endometritis, should be suspected of subclinical endometritis (SE). Contact with infectious agents results in altered synthesis and secretion of inflammatory mediators, including cytokines and arachidonic acid metabolites, and disturbs endometrial functional balance. To address the hypothesis that SE affects the immune endocrine status of the equine endometrium, spontaneous secretion of prostaglandin E(2) (PGE(2)), prostaglandin F(2α) (PGF(2α)), 6-keto-PGF(1α )(a metabolite of prostacyclin I(2)), leukotriene B(4) (LTB(4)), and leukotriene C(4) (LTC(4)) was examined. In addition, secretion of these factors was examined relative to the grade of inflammation, fibrosis, and estrous cycle stage. Eighty-two warmblood mares, of known breeding history, were enrolled in this study. On the basis of histopathologic assessment, mares were classified as suffering from first-grade SE, second-grade SE, or being healthy. The grade of fibrosis and the infiltration of endometrial tissue with polymorphonuclear leukocytes were examined by routine hematoxylin-eosin staining. In mares suffering from SE, the secretion profiles of PGE(2), 6-keto-PGF(1α), LTB(4), and LTC(4) were changed compared to mares that did not suffer from endometritis. The secretion of PGE(2) and 6-keto-PGF1α was increased, whereas that of LTB(4) and LTC(4) was decreased. Secretion of 6-keto-PGF(1α) was increased in first- and second-grade SE (P < 0.01). The concentration of PGI(2) metabolite was increased only in inflamed endometrium, independently of the inflammation grade, but was not affected by fibrosis. Prostaglandin E(2) secretion was increased in second-grade SE (P < 0.05). The secretion of LTB(4) decreased in both first- and second-grade SE (P < 0.05), whereas secretion of LTC(4) was decreased only in second-grade SE (P < 0.05). Fibrosis did not change the secretion profile of PGE(2), PGF(2α), and 6-keto-PGF(1α) during the course of SE. However, the secretion profile of LTB(4) was affected during the course of fibrosis. Evident divergences between PGE(2) and PGF(2α) profiles and in PGE2:PGF(2α) ratios in the control versus SE mares observed during the course of diestrus contribute to shortened or prolonged interestrous intervals observed clinically in SE mares.