Comparing microbiotas of foals and their mares' milk in the first two weeks after birth.

BACKGROUND: The mare-foal relationship is essential for the well-being and growth of a foal. Mare's milk provides a foal with nutrients, protective immunity, and microbes. Within the first two weeks of life, there is a risk for a foal to suffer from diarrhea, particularly "foal heat diarrhea" which happens at about the time of a mare's estrus cycle but is more likely due to transitions in the microbiota in the foal's gastrointestinal (GI) tract. We hypothesized that this GI microbiota transition could be caused by changes in lysozyme and microbial populations in the mare's milk. To test this hypothesis, fifteen mare-foal pairs were followed in the first 15 days post-foaling. Every other day milk was collected from mares and rectal swabs were collected from foals. Lysozyme activity in the mare's milk was measured using a fluorescence assay. Microbial DNA was isolated from the milk and swabs and the V4 domain of 16 S rRNA genes were PCR amplified and sequenced using Illumina MiSeq technology. Microbial populations were analyzed using DADA2 and phyloseq within R. RESULTS: Mare's milk lysozyme activity peaked for samples at Day 1 and levels dropped to 72.5% of Day 1 activity by Day 15; however, microbial populations in the mare's milk did not vary significantly over the two weeks. Furthermore, levels of microbial diversity found in foal rectal swabs were initially similar to microbial diversity seen in mare's milk; however, over the first fifteen days, diversity increased for the foal rectal swab microbiota and swab microbial populations differed from milk microbes. A transition occurred shifting from microbes from the phylum Proteobacteria early in rectal swabs to those primarily from the phyla Firmicutes and Bacteroidota after the first few days post-foaling. These phyla contained several families and genera of microbes that promote utilization of milk components in healthy gut transition. Microbial abundance levels correlated more with days post-parturition than with lysozyme activity and mare's milk microbial populations. CONCLUSIONS: The findings suggest that much of the microbial populations responsible for the transition of the foal's gut comes from sources outside of mare's milk species and levels of lysozyme activity.

Development of a fluorometric Cuboni test for the semi-quantitative measurement of urinary estrogen levels and pregnancy detection in mares.

The aim of the present study was to develop a semi-quantitative urine pregnancy test for mares based on the Cuboni reaction and to verify the reliability of this test. The urine specimens were hydrolyzed by heating in the presence of hydrochloric acid. The resulting free estrogens were extracted from the urine matrix using toluene. Sulfuric acid was added to the toluene extract and the mixture was heated again. The lower layer in the test tube containing sulfuric acid was used for fluorescence measurements with excitation at 355 nm and measurement at 535 nm. The fluorometric Cuboni test revealed that the fluorescence counts in urine samples collected after the second trimester of gestation were significantly higher than those obtained from barren mares. The levels of estrogens, including equilin, estrone and estardiol-17ß exhibited a dose-dependent increase in fluorescence counts, whereas other steroids, such as progesterone, testosterone, and cortisol, did not affect fluorescence. Heat treatment of urine samples with hydrochloric acid significantly increased the fluorescence counts in those collected after the second trimester of gestation compared to non-pregnant samples, implying the presence of large amounts of conjugated estrogens in pregnant mare urine. Fluorescence counts in urine samples obtained during pregnancy showed a positive relationship with estrone concentrations as measured by enzyme immunoassay. The results of the present study showed that the fluorometric Cuboni test facilitates urine fluorescence counts depending on the urinary estrogen content and is capable of discriminating between pregnancy and non-pregnancy states beyond the second trimester of gestation in mares.

Potential role of camel, mare milk, and their products in inflammatory rheumatic diseases.

Milk and dairy products serve as a significant dietary component for people all over the world. Milk is a source of essential nutrients such as carbohydrates, protein, fats, and water that support newborns' growth, development, and physiological processes. Milk contains various essential biological compounds that contribute to overall health and well-being. These compounds are crucial in immune system regulation, bone health, and gut microbiota. Milk and dairy products are primarily from cows, buffalos, goats, and sheep. Recently, there has been a notable increase in camel and mare milk consumption and its associated products due to an increasing attraction to ethnic cuisines and a greater awareness of food biodiversity. Camel and mare milk possess diverse nutritional and therapeutic properties, displaying potential functional foods. Camel milk has been linked to various health advantages, encompassing antihypertensive, antidiabetic, antiallergic, anticarcinogenic, antioxidant, and immunomodulatory properties. Camel milk has exhibited notable efficacy in mitigating inflammation and oxidative stress, potentially offering therapeutic benefits for inflammatory disorders. Nevertheless, although extensively recorded, the potential health benefits of mare's milk have yet to be investigated, including its impact on inflammatory conditions. This article highlights the therapeutic potential of camel and mare milk and its derived products in treating inflammatory rheumatic disorders, specifically focusing on their anti-inflammatory and immune-regulatory capabilities. These alternative types of milk, which do not come from cows, offer potential avenues for investigating innovative strategies to regulate and reduce inflammatory conditions.

Follicular Fluid-Derived Extracellular Vesicles Influence on In Vitro Maturation of Equine Oocyte: Impact on Cumulus Cell Viability, Expansion and Transcriptome.

Cumulus cell (CC) expansion is pivotal for oocyte maturation, during which CCs release factors that initiate paracrine signaling within the follicular fluid (FF). The FF is abundant in extracellular vesicles (EVs) that facilitate intercellular communication. Although bovine and murine EVs can control cumulus expansion, these effects have not been observed in equines. This study aimed to assess the impact of FF-derived EVs (ffEVs) on equine CC expansion, viability, and transcriptome. Cumulus-oocyte complexes (COCs) that underwent in vitro maturation (IVM) in the presence (200 µg protein/mL) or absence (control) of ffEVs were assessed for cumulus expansion and viability. CCs were isolated after 12 h of IVM, followed by RNA extraction, cDNA library generation, and subsequent transcriptome analysis using next-generation sequencing. Confocal microscopy images illustrated the internalization of labeled ffEVs by CCs. Supplementation with ffEVs significantly enhanced cumulus expansion in both compacted (Cp, p < 0.0001) and expanded (Ex, p < 0.05) COCs, while viability increased in Cp groups (p < 0.01), but decreased in Ex groups (p < 0.05), compared to the controls. Although transcriptome analysis revealed a subtle effect on CC RNA profiles, differentially expressed genes encompassed processes (e.g., MAPK and Wnt signaling) potentially crucial for cumulus properties and, consequently, oocyte maturation.

Analysis of the Influence of Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus Strains on Changes in the Hexachlorobenzene Content in Fermented Mare Milk during Refrigerated Storage.

(1) Background: Hexachlorobenzene (HCB) is a persistent organic pollutant that is possibly carcinogenic to humans. It is still found in the environment, humans and animals, and in foods, including milk and dairy products; (2) Methods: The influence of the probiotic cultures Lacticaseibacillus rhamnosus LCR and Lactiplantibacillus plantarum subsp. plantarum LP on the possibility of effecting the biodegradation of HCB in dairy products fermented from mare milk was investigated, taking into account the product storage time (maximum 21 days). HCB content was determined using the GC/MS method; (3) Results: A strong negative Pearson correlation (p < 0.05) was found between HCB concentration and the refrigeration storage time of the fermented beverages. The highest HCB reduction was observed in milk fermented with both Lacticaseibacillus rhamnosus LCR and Lactiplantibacillus plantarum subsp. plantarum LP (78.77%), while the lowest was noted when only Lactiplantibacillus plantarum subsp. plantarum LP was used (73.79%); (4) Conclusions: This pilot study confirmed that probiotics commonly used to give products health-promoting properties can also contribute to reducing the content of undesirable substances, and the bacterial cultures used might provide an alternative method for reducing HCB residues in fermented drinks.

[Influence of lactoferrin and enzymatiс hydrolysates of cow's and mare's milk proteins on anaphylactic sensitivity and cytokine profile of rats].

The development of innovative ingredients of specialized formula for children with intolerance to cow's milk proteins requires accounting the influence of the protein component on the allergic sensitivity. The aim of the research was to study the effect of lactoferrin (LF) from cow colostrum, cow's milk protein hydrolysate (CMPH) and mare's milk protein hydrolysate (MMPH) on the severity of the systemic anaphylaxis reaction, the levels of specific IgG antibodies and cytokines in rats parenterally sensitized with ovalbumin (OVA). Material and methods. The experiment was carried out on 4 groups of 26 male Wistar rats, which were sensitized intraperitoneally with chicken egg OVA and a systemic anaphylaxis reaction was induced on the day 29 by intravenous administration of a challenge dose of the antigen (6 mg per kg body weight). LF, CMPH and MMPH were introduced into the diet in doses of 1.4-2 g/kg body weight per day (on an average 1.59±0.04, 1.53±0.05 and 1.48±0.05 g per kg body weight respectively). The content of IgG antibodies in the blood serum was determined by an indirect ELISA; the levels of cytokines IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, GM-CSF, IFN-γ and TNF-α were detected by multiplex immunoassay. Results. Dietary LF did not have a significant effect on the severity of active anaphylactic shock (AAS), concentrations of antibodies and cytokines in the blood of animals. As a result of CMPH consumption, there were no significant changes in AAS severity and IgG antibodies to OVA but significant increase in TNF-α level was observed as well as a significant decrease in IL-1α (p<0.05). In animals receiving MMPH, there were also no significant changes in the severity of AAS, but a 1.9-fold decrease in the level of IgG antibodies to OVA (p<0.001) was noticed along with a significant increase in IL-12(p70) (p<0.05) and IL-10 (p<0.10) level. Conclusion. Cosumption of LF by sensitized rats didn't significantly affect their anaphylactic sensitivity and cytokine profile, while CMPH intake induced some signs of pro-inflammatory processes. Consumption of MMPH was accompanied by the formation of an anti-inflammatory cytokine profile, which corresponds to a decrease in the intensity of the humoral immune response to the model allergen. Differences in the effects of two hydrolisates, which have similar degrees of hydrolysis, may be associated with the specific composition of glycopeptides formed during the enzymatic cleavage of milk protein produced by these two species of dairy animals.

The evolutionary journey of freshwater crabs of the genus Potamon (Decapoda: Brachyura: Potamidae).

AIM: Freshwater ecosystems host a rich biodiversity, including freshwater crabs. The family Potamidae is a diverse group of freshwater crabs with a Palearctic and Oriental distribution. Specifically, the genus Potamon is found in the Middle East, Southern Europe, and Northern Africa. Potamon species are considered true freshwater crabs due to their total independence from the marine environment, which makes them idea organisms to study historical freshwater connectivity dynamics. In this study we aim to elucidate the phylogenetic relationships of the genus Potamon and discuss the historical freshwater connectivity in the Mediterranean region. METHODS: Here we use up to eleven genetic markers to reconstruct a robust phylogenetic tree of the group and use Bayesian methods to time-calibrate the tree. We further use historical biogeography methods implemented in a Bayesian framework to assess the ancestral distribution ranges of the group. RESULTS: In this study we generate the most complete dataset for the group, covering practically the whole distribution range of the genus. We obtained a robust phylogenetic hypothesis and evaluated the times of divergence of the group. The result of the historical biogeography shows the evolution of historical distribution ranges of species of Potamon. MAIN CONCLUSIONS: The Mediterranean Sea is placed in a key intersection for the exchange of fauna and flora. Here we present evidence for the potential of freshwater fauna exchange, through the Mediterranean Sea after a period of desiccation and the loss of contact with the ocean. The origin of Potamon genus is found to be in Western Asia, probably Eastern Iran.

Classification of Bisgaard taxon 6 and taxon 10 as Exercitatus varius gen. nov., sp. nov.

Forty-one isolates of Bisgaard taxon 6 obtained from guinea pigs, pandas, pigs and muskrat and isolates of taxon 10 from horses and horse bites in humans were subjected phenotypic characterization. Production of acid from (-)-d-mannitol, (-)-d-sorbitol and (+)-d-glycogen separated taxon 10 (positive) from taxon 6 (negative), while from two to 11 phenotypic characteristics separated taxa 6 and 10 from the 32 genera of Pasteurellaceae reported so far. Forty-four strains were genetically characterized. Sequencing of 16S rRNA genes documented a monophyletic relationship at the species level and the highest 16S rRNA gene sequence similarity of 95.6 % to other species was found between strain CCUG 15568T and the type strain of Mannheimia glucosida (CCUG 38457T). Digital DNA-DNA hybridization (dDDH) values predicted from whole genomic sequences between CCUG 15568T and other characterized strains of taxa 6 and 10 were 69.3-99.9 %. The average nucleotide identity values were higher than 95 % for all strains. The highest dDDH value of 29 % outside the taxa 6 and 10 group was obtained with the genome of the type strain of [Actinobacillus] succinogenes, indicating a separate taxonomic status at species level to taxa 6 and 10. The phylogenetic comparison of concatenated conserved protein sequences showed the unique position of the taxa investigated in the current study which qualified for the status of a new genus since the highest identity was found with Basfia with 79 %, well below the upper threshold between genera of 85 %. Based upon the low genetic similarity to other genera of the family Pasteurellaceae and a unique phenotype, we suggest that Bisgaard taxa 6 and 10 should be classified as Exercitatus varius gen. nov., sp. nov. The G+C of the type strain of Exercitatus varius, 8.5T (=CCUG 15568T=DSM 115565T), is 46.2 mol%, calculated from the whole genome.

The relationship between Th1/Th2 cytokine polarization and milk composition in the postnatal period in Kyrgyz mares and foals.

Immune compatibility between mare and foal is one of the important topics of reproductive immunology. At this point, although there are many studies on antibodies, there are not many publications on the relationship between the cytokine levels of mare, foal and milk and the effects of milk composition on this relationship. Here we investigate the relationship between Th1/Th2 cytokine balance and milk composition in Kyrgyz mares and foals. Samples were taken soon after the foal was born and on days 5, 10 and 20 after birth. Th1 (IFN-γ, IL-2, TNF-α) and Th2 (IL-4, IL-5, IL-10) cytokine levels in blood samples were measured using a commercial ELISA kit. Dry matter, fat, protein and non-fat dry matter ratios were determined in the mare's milk and colostrum. We detected no Th1/Th2 polarization in the mare's milk on the day of the foal's birth (day 0) and day 10, but there was Th1 polarization on day 5 and Th2 cytokine polarization on day 20. There was no polarization in the blood sera of the mares on days 0, 5, and 10, and Th1 cytokine polarization was also detected on day 20. We detected no Th1/Th2 cytokine polarization in the blood sera of the foals on any of the days. Dry matter (19.66 ± 0.39%), protein (16.56 ± 0.18%), fat (2.13 ± 0.17), and non-fat dry matter (17.59 ± 0.44%) were higher in colostrum than the other days. When the correlations between cytokine levels in milk samples and milk composition were examined, there was a positive correlation between IL-5 level and protein ratio on day 10. We detected a positive correlation between IL-2 level and the fat rate on day 20. There was a positive correlation between the IL-2 level and the non-fat dry matter ratio on day 20. Further studies are now needed to determine the relationship between the changes in the composition of mare's milk in the postnatal period and Th1/Th2 cytokine polarization in mares and foals at the time of birth and in the postnatal period. Our finding that protein and fat ratios in mare's milk in the postnatal period are positively correlated with IL-2 levels should be considered in terms of foal and human nutrition.

The post-ovulatory rise in progesterone is lower and the persistence of oestrous behaviour longer during the first compared with the second cycle of the breeding season in mares.

Mares are seasonally polyoestrous breeders. Therefore, the first ovulation of the season, following winter anoestrus, is the only cycle in which mares ovulate without the presence of an old CL from the previous cycle. The objective of this study was to compare the length of oestrous behaviour, and plasma progesterone concentrations during the early post-ovulatory period between mares after the first and second ovulation of the breeding season. Overall, 38 mares and 167 oestrous periods were used in the study. From those, 11 mares were used during the first and subsequent oestrous period to measure and compare the post-ovulatory rise in progesterone concentration, whereas all the mares were used to compare the length of the post-ovulatory oestrous behaviour between the first and subsequent cycles of the breeding season. The persistence of the post-ovulatory oestrus was longer (p < .001) following the first ovulation of the year (median of 52 h) compared with the subsequent ovulations (median of 36 h for second and later ovulations groups; n = 38 mares). The progesterone concentration at any of the four 8 h-intervals analysed (28, 36, 76 and 84 h post-ovulation) was lower (p < .01) following the first versus the second ovulation of the year. By 36 h post-ovulation the progesterone concentration of mares at the second ovulation of the year had passed the threshold of 2 ng/ml (2.1 ± 0.33 ng/ml), whereas in the first cycle it was 1.2 ± 0.13 ng/ml. In conclusion, mares had lower progesterone concentrations in their peripheral circulation and longer persistence of oestrous behaviour following the first ovulation of the year compared with the second and subsequent ovulatory periods of the breeding season.

Amniotic Mesenchymal-Derived Extracellular Vesicles and Their Role in the Prevention of Persistent Post-Breeding Induced Endometritis.

Persistent post-breeding induced endometritis (PPBIE) is considered a major cause of subfertility in mares. It consists of persistent or delayed uterine inflammation in susceptible mares. There are many options for the treatment of PPBIE, but in this study, a novel approach aimed at preventing the onset of PPBIE was investigated. Stallion semen was supplemented with extracellular vesicles derived from amniotic mesenchymal stromal cells (AMSC-EVs) at the time of insemination to prevent or limit the development of PPBIE. Before use in mares, a dose-response curve was produced to evaluate the effect of AMSC-EVs on spermatozoa, and an optimal concentration of 400 × 106 EVs with 10 × 106 spermatozoa/mL was identified. At this concentration, sperm mobility parameters were not negatively affected. Sixteen susceptible mares were enrolled and inseminated with semen (n = 8; control group) or with semen supplemented with EVs (n = 8; EV group). The supplementation of AMSC-EVs to semen resulted in a reduction in polymorphonuclear neutrophil (PMN) infiltration as well as intrauterine fluid accumulation (IUF; p < 0.05). There was a significant reduction in intrauterine cytokine levels (p < 0.05) for TNF-α and IL-6 and an increase in anti-inflammatory IL-10 in mares in the EV group, suggesting successful modulation of the post-insemination inflammatory response. This procedure may be useful for mares susceptible to PPBIE.

Production of Mare Chorionic Girdle Organoids That Secrete Equine Chorionic Gonadotropin.

The equine chorionic girdle is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (day 0) and invade the endometrium to become endometrial cups. These specialized trophoblast cells transition from uninucleate to differentiated binucleate trophoblast cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG; formerly known as pregnant mare serum gonadotropin or PMSG). This eCG has LH-like activity in the horse but variable LH- and FSH-like activity in other species and has been utilized for these properties both in vivo and in vitro. To produce eCG commercially, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare due to repeated blood collections and the birth of an unwanted foal. Attempts to produce eCG in vitro using long-term culture of chorionic girdle explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and can remain genetically and phenotypically stable throughout long-term culture (i.e., months). Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin (hCG) and proliferate long-term (>1 year). The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. Here we show generation of chorionic girdle organoids for the first time and demonstrate in vitro production of eCG for up to 6 weeks in culture. Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for chorionic girdle development of early equine pregnancy.

Neonates and Periparturient Mares: Tips and Tricks for Diagnosis and Management.

Colic is a common presenting complaint in foals. Unfortunately, evaluation of foals with colic can be difficult due to the common clinical presentation of acute abdominal pain attributable to a wide variety of disorders and a broad range of etiologies. Similar to foals, colic is one of the most common diseases of pregnant and periparturient mares. Several conditions are unique or occur more commonly in broodmares. This article reviews the most commonly encountered types of colic events in neonatal foals and periparturient mares, how to correctly diagnose these conditions, as well as details concerning appropriate medical management and/or surgical intervention.

Maternal age affects equine day 8 embryo gene expression both in trophoblast and inner cell mass.

BACKGROUND: Breeding a mare until she is not fertile or even until her death is common in equine industry but the fertility decreases as the mare age increases. Embryo loss due to reduced embryo quality is partly accountable for this observation. Here, the effect of mare's age on blastocysts' gene expression was explored. Day 8 post-ovulation embryos were collected from multiparous young (YM, 6-year-old, N = 5) and older (OM, > 10-year-old, N = 6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure or inner cell mass (ICM) enriched trophoblast, obtained by embryo bisection, were RNA sequenced. Deconvolution algorithm was used to discriminate gene expression in the ICM from that in the trophoblast. Differential expression was analyzed with embryo sex and diameter as cofactors. Functional annotation and classification of differentially expressed genes and gene set enrichment analysis were also performed. RESULTS: Maternal aging did not affect embryo recovery rate, embryo diameter nor total RNA quantity. In both compartments, the expression of genes involved in mitochondria and protein metabolism were disturbed by maternal age, although more genes were affected in the ICM. Mitosis, signaling and adhesion pathways and embryo development were decreased in the ICM of embryos from old mares. In trophoblast, ion movement pathways were affected. CONCLUSIONS: This is the first study showing that maternal age affects gene expression in the equine blastocyst, demonstrating significant effects as early as 10 years of age. These perturbations may affect further embryo development and contribute to decreased fertility due to aging.

Relationship between estrus endometrial edema and progesterone production in pregnant mares two weeks after ovulation.

BACKGROUND: 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 years) in which a normal embryonic vesicle with a diameter ≥ 14 mm and a corpus luteum with a diameter ≥ 15 mm were found 14 days after ovulation, and group B (n = 22; age 9.4 ± 4 .0 years) in which 14 days after ovulation, a small (< 15 mm) corpus luteum and/or a small embryonic vesicle was observed (diameter < 14 mm). Mares from group A had a significantly higher progesterone concentrations at 14 days after ovulation compared with group B mares. The presence of IUF, the number of treatments against IUF, and the time from AI to the day when uterus was found free of fluid did not affect progesterone concentration measured 14 days after ovulation. In group B, a significant correlation was found between progesterone concentration measured 14 days after ovulation and endometrial edema evaluated during estrus. CONCLUSIONS: In some cases poor development of endometrial edema during estrus can be associated with lower progesterone production 14 days after ovulation. Nevertheless, scientific explanation for this finding cannot be given based on our study.

Evaluation of growth, viability, and structural integrity of equine endometrial organoids following cryopreservation.

Reproductive diseases in mares are a significant cause of subfertility and profound economic loss in the equine industry. Utilizing a 3D in vitro cell culture system that recapitulates the in vivo physiology will reduce time, cost, and welfare concerns associated with in vivo reproductive research in mares. If this 3D model is combined with effective cryopreservation, reproductive research on mares can occur year-round, which is not currently possible in this seasonal species. Endometrial organoids, 3D in vitro cell clusters that exhibit in vivo uterine physiology, have been established in mice, women, and mares. Here we report the first comprehensive assessment of cryopreservation of endometrial organoids in the domestic mare. Organoid growth rate was not affected by the type of freezing media. However, growth rate varied among non-cryopreserved controls, organoids cryopreserved at passage 0 (P0), and organoids cryopreserved at passage 3 (P3). Additionally, there was no difference in organoid viability among freezing media or freezing timepoint (passages). Furthermore, fresh and frozen-thawed organoids displayed positive immunohistochemical staining for ZO-1, which is a marker for intercellular tight junctions, and for periodic acid-Schiff staining as marker for organoid function through mucin production. Results demonstrate that equine endometrial organoids can be cryopreserved with 10% dimethyl sulfoxide with minimal detrimental effects while maintaining intercellular tight junctions (ZO-1) and secretory function. Availability of cryopreserved endometrial organoids may permit expanded research on uterine pathologies that negatively affect mare fertility and improve efficiency, reduce cost, and minimize animal welfare concerns associated with in vivo research in the domestic mare.

The effect of priming and duration of oestradiol benzoate treatment before progesterone administration on embryo development and survival in anestrous recipient mares.

The effect of three different hormonal protocols to prepare anestrous recipient mares on embryo survival was evaluated. The first group consisted of only progesterone administration (NE) 4 days before embryo transfer, while the recipients from the other two groups received a single administration of 2.5 mg of oestradiol benzoate (SE) 2 days earlier or 8 mg of oestradiol split in increasing doses for 5 consecutive days (LE) ending 3 days before progesterone treatment. The likelihood of recovering an embryo 2 days after transfer was 46.1% (6/13), 62.5% (5/8) and 85.7% (6/7) for recipient mares from the no oestrus, short and long oestrous groups respectively (p = .09). In conclusion, the presence and duration of oestradiol treatment before progesterone administration tended to influence the embryo survival in anestrous recipients 2 days after transfer. The surviving embryos recovered from the three different groups of recipients did not show any difference in size and morphology.

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.

Advances in the ultrasound diagnosis in equine reproductive medicine: New approaches.

Ultrasound technology has led to new lines of research in equine reproduction, and it has helped to greatly improve clinical diagnosis and reproductive outcomes in equine practice. This review aims to discuss the potential clinical uses and new approaches of ultrasonography in equine reproduction. Doppler modalities are usually used to evaluate the vascularization of the follicles, corpus luteum (CL), and the uterus in the mare for diagnostic purposes. Inclusion of Doppler ultrasound in artificial insemination and embryo transfer programs could improve the reproductive outcome of these techniques. Better selection of recipients based on CL functionality, early pregnancy diagnosis 7-8 days postovulation of the donor before flushing or diagnosis of mares with endometritis with pathological increases of blood flow are examples of clinical applications in the mare. In the stallion, colour Doppler ultrasound has improved the diagnostic potential of B-mode ultrasound, improving the differential diagnosis of pathologies such as testicular torsion (decrease or absence of blood flow in the cord) and orchitis (increased blood flow in the cord). The incorporation of pulsed Doppler ultrasound into the reproductive evaluation of the stallion has enabled early identification of stallions with testicular dysfunction, thus allowing administration of timely treatment and subsequent improvements of the fertility prognosis for these animals. In addition, this technique has been used in the monitoring of patients undergoing medical and surgical treatments, thus verifying their efficacy. Recently, computer-assisted pixel analysis using specific software has been performed in research work in order to semi-quantitatively evaluate the vascularization (colour and power Doppler) and echotexture of different organs. These softwares are now being developed for clinical purposes, as is the case with Ecotext, a computer program developed for the evaluation of testicular echotexture, providing information on testicular functionality.

Variables Affecting Veterinary Students' Ability to Accurately Interpret Ovulation in Live Mare Palpation.

In a veterinary medicine curriculum, students' hands-on practice is essential but is still considered one of the major deficiencies in veterinary schools in Europe. After theoretical and basic practical training, students, under the control of experienced veterinarians (supervisors), monitored the reproductive cycle of embryo recipients by transrectal palpation and ultrasound. To evaluate the skills of students, the question "Has she ovulated?" was posed when a dominant follicle ≥ 35 mm was recorded in the previous day's examination and a score of 1 or 0 was assigned in the case of a correct or incorrect answer (test palpation), respectively. Study 1 involved the retrospective evaluation of 3,509 test palpation records of 43 students (31 females, 12 males) and showed a statistically significant positive correlation between the number of test palpations performed and the proportion of correct answers. There was a statistically significant effect of the number of test palpations performed by each student, their gender, and the season on the correct answers. When performing > 50 test palpations, a statistical difference between gender was observed (p < .05). Study 2 involved the prospective evaluation of 687 records on 52 standardbred or thoroughbred recipient mares collected from nine right-handed female students. The different mares, breed, occurrence of ovulation on the left or right ovary, and the presence of one or more large follicle(s) per ovary had no effect on the correct answers (p > .05). Individual students' performances were statistically different (p < .05), ranging from 60% to 92%.