Cryopreservation of epididymal sperm allows final preservation of the gene reserve from valuable sires in case of unexpected injury terminating the breeding career. This case report describes the birth of a healthy foal following insemination with frozen-thawed epididymal sperm. The testes and epididymides were removed under general anaesthesia and sent cooled to the laboratory overnight. The cauda epididymidis was dissected and 17.79 x 10 (9) sperm were harvested by a retrograde flush technique. A fertile mare was inseminated 1 year later with frozen-thawed epididymal sperm. Sperm were deposited into the tip of the uterine horn ipsilateral to the ovulation site. The mare did not become pregnant after the 1st cycle. In the 2nd breeding cycle, additional homologous seminal plasma was delivered into the uterus at the time of insemination and the mare was diagnosed as pregnant 14 days post ovulation. A healthy colt was born after 334 days of gestation. The method for preparing gonads for transportation to an appropriate laboratory is described for veterinarians and the different steps of semen collection and preservation are presented.

ZusammenfassungDie Tiefgefrierkonservierung von Nebenhodenspermien stellt eine gute Möglichkeit zur Erhaltung der Genreserve wertvoller Vatertiere dar, wenn ein plötzlicher Unfall eine herkömmliche Samengewinnung unmöglich macht. Im vorliegenden Fall wird von der Geburt eines gesunden Fohlens berichtet, das aus der Besamung mit Nebenhodenspermien entstand. Die Hoden und Nebenhoden wurden in Allgemeinanästhesie entfernt und gekühlt über Nacht in das Labor verbracht. Durch retrograde Spülung der Nebenhodenschwänze konnten 17,79 × 109 Spermien konserviert werden. Eine fruchtbare Stute wurde 1 Jahr später mit tiefgefrorenen Nebenhodenspermien tief intrauterin besamt. Im 1. Zyklus wurde die Stute nicht tragend. Im 2. Zyklus wurde homologes Seminalplasma zusammen mit den Nebenhodenspermien in den Uterus verbracht und 14 Tage nach der Ovulation konnte eine Trächtigkeit festgestellt werden. Nach einer Graviditätsdauer von 334 Tagen wurde ein gesundes Hengstfohlen geboren. Die Vorbereitung von Hoden und Nebenhoden nach einem Unfall oder Todesfall für die weitere Bearbeitung im Labor und die einzelnen Arbeitsschritte für die Aufbereitung von Nebenhodenspermien werden beschrieben.

Chromosomal abnormalities are notable causes of infertility in horses. Mares show various degrees of estrous behavior, and ultrasound examination often reveals an underdeveloped genital tract. This article reports investigations on fertility in a Haflinger sibship with a healthy, normally developed, fertile mare with at least three healthy offspring. Chromosomal analysis performed incidentally and blinded for this mare revealed 63,X/64,XX/65,XXX mosaicism. Two closely related mares were also mosaics (63,X/64,XX), and one of them was a carrier of a marker chromosome. Repeated examinations of the mare and seven relatives (four mares and three stallions) did not provide evidence for sub- or in-fertility. They had no developmental abnormalities or conspicuous body conditions. Peripheral blood samples were collected for analysis of the karyotype and molecular analyses. Chromosomes were Giemsa stained and 4',6-diamidino-2-phenylindole banded to identify numerical or structural aberrations of chromosomes and identification of sex chromosomes, respectively. Fluorescence in situ hybridization was performed with an equine Y-chromosome painting probe to identify and count the sex chromosomes, and polymerase chain reaction analysis was used to test for the presence of the SRY gene and investigating chimerism. The present article demonstrates the necessity of further studies analyzing chromosomal X0 mosaics to improve the predictive value of chromosomal aberrations on fertility.

The aim of the present study was to evaluate the effect of different freezing procedures on sperm motion, viability, the acrosome status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity on epididymal stallion sperm. Therefore, the sperm of 10 healthy stallions was harvested by retrograde flushing after testectomy, diluted with a semen extender containing defined milk proteins and a freezing extender containing egg yolk and glycerol and frozen according to 4 different protocols, using a programmable freezer and a floating rack performing a slow (processes 1 and 2) or a fast cooling rate (processes 3 and 4, respectively). Post-thaw total motility and slow sperm values were lower when using process 4 compared with processes 1 and 2 (P < .05) after 1 hour of incubation. Progressive motility was lower in process 4 compared with process 1 immediately after thawing and after 1 hour of incubation (P < .05). The amount of rapid sperm was lower when using process 4 compared with process 1 immediately after thawing (P < .05). After 1 hour of incubation, the amount of rapid sperm was lower when using process 4 compared with processes 1 and 2 (P < .05). Higher values for viable sperm were seen in processes 1 and 2 compared with process 4 (P < .05) after 1 hour of incubation. Immediately after thawing, more viable sperm with high MMP (hMMP) were observed when using process 3 compared with process 2 (P < .05). After 1 hour of incubation, a significantly higher amount of viable hMMP sperm were detected when using processes 1 and 2 compared with process 4 (P < .05). Process 2 yielded a lower percentage of sperm containing low calcium (lCa) than process 3 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of lCa sperm was observed using process 4 (P < .05). The subpopulation of viable/hMMP/lCa sperm was higher when using process 3 compared with process 2 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of this subpopulation was detected in process 4 (P < .05). The DNA integrity was similar in all groups. In conclusion, a slow cooling rate with a controlled rate freezer resulted in best sperm quality after thawing. Using a floating rack in nitrogen vapor as an alternative to a programmable freezer, equilibration in a cooled environment is advantageous.

Cryopreservation of epididymal sperm allows the saving of genetic material in case of unexpected death or emergency castration. The aim of the present study was the comparison of five different combinations of extenders commercially available for equine frozen semen processing for cryopreservation of epididymal sperm. Epididymal sperm were harvested from gonads of 10 healthy stallions after routine castration by retrograde flush technique. Then, samples were split and diluted with (1) INRA96 + INRA Freeze, (2) BotuSemen + BotuCRIO, (3) EquiPlus + Gent Freeze, (4) EquiPlus + EquiPlus Freeze, and (5) Gent + Gent Freeze. Extenders 1 and 2 showed higher values for total and progressive motility after thawing compared with extender 4 (P < .05). Extender 3 was in between 1 and 2 (P > .05), and extender 5 resulted in the lowest values (P < .05). The subpopulation of viable frozen-thawed sperm with high mitochondrial membrane potential and low intracellular calcium content was higher using extender 1 compared with extenders 3, 4, and 5 (P < .05) and higher in extender 2 compared with extenders 4 and 5 immediately after thawing (P < .05). After 1 hour of incubation, this subpopulation yielded the highest values in extender 2 (P < .05). Immediately after thawing, extender 1 yielded higher values for percentage of DFI and mean DFI than extenders 3, 4, and 5 (P < .05). Following 1 hour of incubation after thawing, sperm processed with extender 1 resulted in the highest values for percentage of DFI and mean DFI (P < .05). Using extender 2, mean DFI values were lower than those in extender 1 and higher than the extenders 3, 4, and 5 (P < .05). The study revealed that according to the examined sperm quality parameters, freezing extenders (extender 1, extender 2) using low concentrations of glycerol either combined with or without methylformamide were beneficial for cryopreservation of stallion epididymal sperm. For processing of stallion epididymal sperm, an extender containing milk proteins (extenders 1-4) for initial dilution after sperm harvesting is preferable to an extender including egg yolk (extender 5).

Background The CEA gene family is one of the most rapidly evolving gene families in the human genome. The founder gene of the family is thought to be an ancestor of the inhibitory immune checkpoint molecule CEACAM1. Comprehensive analyses of mammalian genomes showed that the CEA gene family is subject to tremendous gene family expansion and contraction events in different mammalian species. While in some species (e.g. rabbits) less than three CEACAM1 related genes exist, were in others (certain microbat species) up to 100 CEACAM1 paralogs identified. We have recently reported that the horse has also an extended CEA gene family. Since mechanisms of gene family expansion and diversification are not well understood we aimed to analyze the equine CEA gene family in detail. Results We found that the equine CEA gene family contains 17 functional CEACAM1 -related genes. Nine of them were secreted molecules and eight CEACAMs contain transmembrane and cytoplasmic domain exons, the latter being in the focus of the present report. Only one ( CEACAM41 ) gene has exons coding for activating signaling motifs all other CEACAM1 paralogs contain cytoplasmic exons similar to that of the inhibitory receptor CEACAM1. However, cloning of cDNAs showed that only one CEACAM1 paralog contain functional immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail. Three receptors have acquired a stop codon in the transmembrane domain and two have lost their inhibitory motifs due to alternative splicing events. In addition, alternative splicing eliminated the transmembrane exon sequence of the putative activating receptor, rendering it to a secreted molecule. Transfection of eukaryotic cells with FLAG-tagged alternatively spliced CEACAMs indicates that they can be expressed in vivo. Thus detection of CEACAM41 mRNA in activated PBMC suggests that CEACAM41 is secreted by lymphoid cells upon activation. Conclusions The results of our study demonstrate that alternative splicing after gene duplication is a potent mechanism to accelerate functional diversification of the equine CEA gene family members. This potent mechanism has created novel CEACAM receptors with unique signaling capacities and secreted CEACAMs which potentially enables equine lymphoid cells to control distantly located immune cells.

The aim of the present study was the investigation of stallion epididymal sperm frozen with seminal plasma within the same straw separated by an air bubble before freezing. We hypothesized that mixing both components after thawing improves the sperm quality compared with conventionally frozen-thawed epididymal sperm. Cryopreservation of epididymal sperm allows the preservation of spermatozoa in case of death or termination of the breeding carrier. However, these sperm lack contact with the seminal plasma, which plays an important role in the development of sperm functionality and the regulation of uterine inflammatory response after insemination. Motion characteristics, morphology, and viability were assessed in epididymal sperm mixed with seminal plasma after thawing (group SP) and compared with conventionally packed cryopreserved epididymal sperm that had no contact with seminal plasma (group FT). Total and progressive motility, curvilinear velocity (VCL), and average lateral head displacement were significantly higher in group SP (P < .05). During post-thaw incubation, VCL decreased in group FT but not in group SP (P < .05), and beat cross-frequency was significantly lower in group FT than in group SP after post-thaw incubation (P < .05). Straightness did not differ between the groups (P > .05). Although the percentage of tail abnormalities and isolated sperm heads increased significantly only in group SP after cryopreservation (P < .05), the proportion of morphologically abnormal and nonviable sperm was not affected by seminal plasma (P > .05). Mixing both components after thawing increased sperm motility and velocity but did not negatively affect morphology or viability. This packaging system is promising for routine artificial insemination of stallion epididymal sperm.

During semen processing for cryopreservation, most seminal plasma is usually removed, and components with protective effects on sperm may be missing after thawing and within the female reproductive tract. The present study evaluated the effect of postthaw addition of autologous seminal plasma on motion characteristics of stallion sperm with fair (n = 4) or poor (n = 3) freezability. Therefore, pure seminal plasma (group SP1), seminal plasma combined with fresh semen extender (group SP2), or seminal plasma mixed with freezing extender (group SP3) were used to fill 0.5 mL straws and frozen similar to stallion semen. Postthawing, semen samples (n = 42) were diluted either with semen extender (group FT) or with seminal plasma (n = 126) of groups SP1 to SP3 to 25 × 106 sperm/mL. In fair freezer stallions, total and progressive motilities were higher in group FT than in group SP1 (P < .05), but there was no difference in poor freezing stallions among groups (P > .05). However, comparing individual stallions, positive effects of seminal plasma on total or progressive motility were detected in two stallions. Curvilinear velocity increased in groups SP2 and SP3 in fair freezer stallions and in all groups with seminal plasma compared with group FT in poor freezer stallions (P < .05). Although straightness was higher in groups SP2 and SP3 compared with group FT in fair freezer stallions (P < .05), there was no difference among groups in stallions with poor freezability (P > .05). Average lateral head displacement did not change among groups of fair freezer stallions (P > .05) but was higher in groups SP2 and SP3 than in group FT in poor freezer stallions (P < .05). Beat cross frequency was higher in all groups diluted with seminal plasma postthawing in fair freezer stallions (P < .05), but only in group SP1 than in group FT in poor freezer stallions (P < .05). The addition of autologous seminal plasma to frozen-thawed semen can improve motion characteristics of stallions with fair and poor freezability. This is a valuable additional protocol for laboratories dealing with cryopreservation of stallion semen and for veterinarians working with fair or poor freezer stallions.

The Anti-Muellerian-Hormone (AMH) is a dimeric glykoprotein of the transforming growth factor superfamily and plays an important role in sexual differentiation during embryo development but also during follicle development. AMH can be used as a biomarker in artificial reproduction technologies, predicting success of superovulation. There is also a relation between AMH and fertility, and AMH correlates well with primordial follicular pool leading to a diagnostic value for the ovarian reserve. The present study investigated the effect of the ovulation inducing agent human choriongonadotropin (hCG) on the concentration of AMH in serum of cyclic mares. Serum concentrations of AMH were evaluated during oestrus immediately before (T-2) and 24 hours following administration of hCG (T-1), at ovulation (T0) and day 7 to 9 post ovulation (T+ 1). The concentration of AMH (ng/ml, median; minimum-maximum) did not differ between mares treated with hCG (T-2: 0.33; 0.16-2.00; T-1: 0.40; 0.17-2.06; T0: 0.42; 0.20-2.07; T+ 1: 0.38; 0.21-1.82) and controls (T-2: 0.48; 0.13-1.47; T-1: 0.59; 0.17-1.48; T0: 0.62; 0.14-1.70; T+ 1: 0.47; 0.17-1.17; p> 0.05). Among single measurements on different days of oestrous cycle, there was no difference in serum AMH (p> 0,05). Administration of hCG to induce ovulation had no influence on the pregnancy rate (control group: 80%; treated group 70%; p> 0.05) and significantly enhanced time of ovulation (control group: 4, 2 to 6; treated group: 2, 2 to 2 days; median, minimum to maximum; p< 0.05). Serum AMH concentration during oestrus and on the day of ovulation did not differ dependent from pregnancy success (pregnant: 0.37; 0.13-1.47; 0.45; 0.17-2.06; 0.61; 0.14-1.70; non-pregnant: 0.67; 0.33-2.00; 0.71; 0.32-2.00; 0.58; 0.34-2.07ng/ml at T-2, T-1 and T0, respectively; p> 0.05). However, in mid dioestrus AMH concentration had the tendency to be higher in non-pregnant mares (pregnant: 0.42; 0.17-1.17; non-pregnant: 0.63; 0.32-1.82ng/ml; at T+ 1; p= 0.08). Further studies are needed to evaluate AMH concentration on defined days during oestrous cycle and early pregnancy to evaluate the prognostic value of AMH concentration for mare fertility.

Persistent breeding-induced endometritis (PBIE) or delayed uterine clearance (DUC) are major causes of mare subfertility. Oxytocin and its receptor are thought to play significant roles in the pathogenesis of DUC but the specific roles of oxytocin receptor (OR) distribution and gene expression remain undefined. In this study both OR distribution and gene expression in the endometrium, myometrium and cervix during both luteal and non-luteal phases in non-pregnant mares (n = 27) of differing age (young: 2-9 years, n = 17; old: > 10 years, n = 10) and endometrial biopsy score were described using immunohistochemistry (IHC) and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively. Immunohistochemistry showed a similar pattern of OR distribution in uterus and cervix, with the exception of the glandular epithelium, absent in the cervix. Uterine ORs were localized in endometrial luminal and glandular epithelia, transmural vascular endothelium, sub-epithelial and peri-glandular stromal cells and myometrial smooth muscle cells. The OR labeling intensity was consistently greatest in the vascular endothelium. Real-time qPCR showed a higher OR gene expression in myometrium compared to cervix (P = 0.001) and endometrium (P = 0.009). There was no difference in OR gene expression between cervix and endometrium (P = 1.0). Oxytocin receptor gene expression was significantly higher during the non-luteal phase in both combined uterine tissues (endometrium and myometrium) and myometrium. Oxytocin receptor distribution and gene expression were not influenced by a mare's age or endometrial biopsy score. As endometrial biopsy score and mare age were not predictors of OR gene expression, deficient OR gene expression is unlikely to be associated with DUC. (C) 2018 Elsevier Inc. All rights reserved.

The present report describes a 4-year-old Trakehner mare which was referred to the clinic for a breeding soundness evaluation. Clinical, histological, and postmortem examination revealed an underdeveloped genital tract, the absence of a cervix uteri, and small inactive ovaries without male gonadal tissue. Blood lymphocyte analysis revealed an unusual mosaic karyotype consisting of 2 cell lines. For the majority of cells (70%), monosomy X (63,X) was observed. The remaining cells (30%) contained 64 chromosomes including one X chromosome and a small rudimentary Y chromosome consisting mostly of heterochromatin. The centromere was retained, but its full functionality was questionable. PCR analysis revealed that the entire male-specific region of Y (Yq14), including the SRY gene, was deleted. It remained unclear if the pseudoautosomal region (Yq15) and parts of the heterochromatic region (Yq13) were affected by this deletion. The phenotype of the mare with this disorder of sex development associated with sex chromosome abnormalities is genetically comparable to 63,X monosomy which fully explains the clinical findings.

Zusammenfassung Gegenstand und Ziel: Im peripartalen Zeitraum der Stute kann es eine besondere Herausforderung sein, Veränderungen im Allgemeinverhalten und der Vitalparameter richtig zu interpretieren. In der Studie wurde untersucht, ob sich bei gesunden Stuten unterschiedlichen Pferdetyps in der Spätträchtigkeit und in der 1. Woche post partum (p. p.) die Herz- (HF) und Atemfrequenz (AF) sowie die innere Körpertemperatur (IKT) von den in der Literatur angegebenen Referenzwerten für adulte Pferde unterscheiden. Material und Methoden: Im Rahmen der täglichen klinischen Untersuchung erfolgte eine Auswertung der HF, AF und IKT von Stuten, die zur Geburtsüberwachung stationär eingestellt waren. Unterschiede in Bezug auf die Pferdegröße wurden ausgewertet und bei Großpferden wurde der Einfluss einer Schwergeburt und von Nachgeburtsverhalten evaluiert. Ergebnisse: In den letzten Wochen ante partum (a. p.) stieg die HF deutlich an und war p. p. in allen Gruppen deutlich niedriger als a. p. (p < 0,05). Die AF stieg bei Groß- und Kleinpferden a. p. an und sank p. p. wieder deutlich ab (p < 0,05). Die IKT veränderte sich im peripartalen Zeitraum in allen Gruppen und war p. p. deutlich höher (p < 0,05). Bei Ponys waren HF, AF und IKT immer am höchsten (p < 0,05) und bei Großpferden wurde die niedrigste AF gemessen (p < 0,05). Bezüglich der HF ergab sich zwischen Pferden mit Eutokie und Dystokie kein Unterschied (p > 0,05). Am Tag 1 p. p. war die AF bei Stuten mit Dystokie allerdings signifikant höher (p < 0,05). Unterschiede bei der IKT an den Tagen a. p. und p. p. konnten nur bei Stuten mit ungestörtem Geburtsverlauf festgestellt werden (p < 0,05), doch blieben die Werte im Referenzbereich. Bei Stuten mit Nachgeburtsverhalten zeigte sich keine Änderung der HF (p > 0,05), die IKT war aber deutlich höher am Tag 1 p. p. (p < 0,05). Schlussfolgerung: Die Vitalparameter von Stuten in der Spätträchtigkeit lassen auf eine erhebliche körperliche Leistung über einen langen Zeitraum schließen. Die Interpretation der Vitalparameter im Zusammenhang mit dem Reproduktionsstatus der Stute ist wesentlich, um Störungen des Allgemeinbefindens richtig einzuschätzen und notwendige Behandlungen gezielt durchzuführen.

Objective: During the peripartal period, interpretation of basic clinical signs may be challenging. In the present study, heart rate (HR), respiratory rate (RR) and body temperature (BT) were evaluated in healthy mares of different breed types and compared to reference values for adult horses from the literature. Material and methods: During daily physical exams of periparturient mares, the HR, RR and BT were evaluated. Differences according to the horse's size were investigated and in large breeds, the influence of dystocia or retained placenta was analysed. Results: During the last weeks before parturition (a.p.), the HR significantly increased and was clearly lower after parturition (p.p.; p < 0.05). In larger horses, the RR increased a. p. and decreased p.p. (p < 0.05). The BT underwent changes in all groups during the periparturient period and was higher p.p. (p < 0.05). In general, values for HR, RR and BT were highest in ponies (p < 0.05) while the lowest RR was measured in large horses (p < 0.05). There was no difference in the HR between mares with eutocia or with dystocia (p > 0.05). By contrast, the RR was significantly higher in mares with dystocia on day 1 p.p. (p < 0.05). Differences in the BT a. p. and p. p. occurred only in mares with eutocia (p < 0.05) and remained within the normal values. Mares with retained placenta did not exhibit significant changes in the HR (p > 0.05), but the BT was higher on day 1 p.p. (p < 0.05). Conclusion: Increased HR, RR and BT in mares during late pregnancy suggest a distinct physical performance for a prolonged period of time. Interpretation of these parameters in relation to the mare's reproductive state is essential to diagnose potential disorders and to determine whether therapy is required.

Reducing seminal plasma (SP) is essential in stallion semen preservation; however, SP has an important role in sperm fertility as well as sperm protection and transportation in the female genital tract. In the present study, semen storage at high concentrations and low volumes and the effect of SP added immediately before insemination, volume, and deposition (corpus or uterine horn) of the insemination dose were evaluated. Semen processing protocols were investigated in experiment 1: dilution to a final volume of 20 mL (control); or centrifugation and dilution of the sperm pellet to a volume of 1.5 mL containing 500 × 106 sperm. Immediately before insemination, centrifuged samples were diluted with INRA96 extender containing 0%, 5%, 20%, or 80% SP to a final volume of 20 mL. In experiment 2, concentrated semen was not further processed before insemination. Pregnancy rates were lowest when adding 20% SP to the semen extender (100%, 89%, 67%, 30%, and 67% for control; 0%, 5%, 20%, and 80% SP, respectively; P < .05). Embryonic growth between days 12 and 14 was highest using 0% SP (292%, 515%, 290%, 343%, and 404% for control; 0%, 5%, 20%, and 80% SP, respectively; P < .05). Uterine fluid accumulation did not differ among groups (P > .05). The number of progressively motile sperm per dose was similar in pregnant (median, range: 347, 217–452) and nonpregnant cycles (314, 195–369; P > .05). Deep intrauterine insemination did not improve pregnancy outcome when using 500 × 106 sperm (P > .05). Stallion semen can be stored at 5°C for 24 hours highly concentrated with low volumes of extender, yielding normal pregnancy rates. Adding SP to ejaculated sperm immediately before insemination is not necessary after 24 hours of cooled storage.

To preserve epididymal sperm, only a limited number of sperm is available, and an optimum processing method that is applicable for the majority of stallions is therefore crucial for successful preservation. The aim of the present study was to evaluate the effect of four different extenders that are commercially available for chilled semen on the motion characteristics of stallion epididymal sperm. Sperm were harvested by retrograde flush from 20 epididymides after the routine castration of 10 stallions. Aliquots of sperm samples were diluted with each of the four extenders ([E1] skim milk-based, [E2] containing defined milk protein, [E3] containing egg yolk, and [E4] containing caseinate). Total motility (TMOT %) and progressive motility (PMOT %) assessed immediately after sperm harvesting and during prolonged storage were highest in extenders with selected milk proteins (E2: 54, 24–79 and 50, 19–78; E4: 57, 23–82 and 52, 14–80) compared to the skim milk-based extender (E1: 40, 1–66 and 37, 0–62) and the egg yolk-containing extender (E3: 21, 6–48 and 13, 2–40; median, min–max for TMOT and PMOT, stored for 48 hours at 4°C, respectively). Motility values were similar for extenders E2 and E4 during the entire storage period (P > .05), while extenders E1 and E3 yielded significantly lower values (P < .05). Using E1, motility increased during the storage period but did not reach values similar to those of E2 or E4. Curvilinear velocity (VCL), amplitude lateral head displacement (ALH), and beat-cross frequency (BCF) differed among all extenders after sperm harvesting (P < .05), but not after 24 (VCL, ALH) and 48 hours (VCL, ALH, BCF; P > .05). Based on the motility results, we recommend extenders containing defined milk protein to process epididymal sperm.

Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family that are secreted by trophoblast cells. PSGs may modulate immune, angiogenic and platelet responses during pregnancy. Until now, PSGs are only found in species that have a highly invasive (hemochorial) placentation including humans, mice and rats. Surprisingly, analyzing the CEACAM gene family of the horse, which has a non-invasive epitheliochorial placenta, with the exception of the transient endometrial cups, we identified equine CEACAM family members that seem to be related to PSGs of rodents and primates. We identified seven genes that encode secreted PSG-like CEACAMs Phylogenetic analyses indicate that they evolved independently from an equine CEACAM1-like ancestor rather than from a common PSG-like ancestor with rodents and primates. Significantly, expression of PSG-like genes (CEACAM44, CEACAM48, CEACAM49 and CEACAM55) was found in non-invasive as well as invasive trophoblast cells such as purified chorionic girdle cells and endometrial cup cells. Chorionic girdle cells are highly invasive trophoblast cells that invade the endometrium of the mare where they form endometrial cups and are in close contact with maternal immune cells. Therefore, the microenvironment of invasive equine trophoblast cells has striking similarities to the microenvironment of trophoblast cells in hemochorial placentas, suggesting that equine PSG-like CEACAMs and rodent and primate PSGs have undergone convergent evolution. This is supported by our finding that equine PSG-like CEACAM49 exhibits similar activity to certain rodent and human PSGs in a functional assay of platelet-fibrinogen binding. Our results have implications for understanding the evolution of PSGs and their functions in maternal-fetal interactions.

This study aimed to assess if Ecotext, a new software for evaluation of testicular echotexture, is a good method for diagnosis of stallions with testicular dysfunction (TD). Relationships between Ecotext parameters and sperm motility and production, testicular volume, and testicular blood flow were also studied. Ecotext provides a total of six echotexture parameters: Ecotext 1 (black pixels), 2 (white pixels) and 3 (grey pixels), and another 3 parameters related to hypoechogenic areas: Ecotext tubular density (ETD), Ecotext tubular diameter (ETd), and Ecotext tubular area (ETA). Stallions (n = 33) were assessed using proven diagnostic techniques (spermiogram, B-mode and Pulse Doppler ultrasound), and subsequent analysis with Ecotext. Animals were classified as "control stallions" (n:21, acceptable semen quality), and "stallions with TD" (n:12, poor semen quality (TM < 60%, PM < 45% and total nº of sperm with PM < 2000 × 106 spz), that were subdivided into "induced TD group" (immunized, anti-GnRH vaccine) and "acquired TD group". The acquired TD group showed differences in all Ecotext parameters in relation to controls (Ecotext 1:0.11 ± 0.17 vs 2.82 ± 2.52, Ecotext 2:1584.0 ± 575.8 vs 388 ± 368.2, Ecotext 3:134.2 ± 9.26; ETA: 2.14 ± 0.59 vs 5.40 ± 1.90; ETd: 65.66 ± 6.27 vs 86.93 ± 10.65 and ETD: 92.35 ± 11.24 vs 132.10 ± 16.35, p ≤ 0.001). Results suggest acquired TD stallions were suffering testicular degeneration with loss of architecture and function as all Ecotext parameters were altered in relation to controls. Induced TD horses only showed a reduction in ETD (116.2 ± 8.59 vs 132.10 ± 16.35, p ≤ 0.001), despite all sperm parameters being worse. These findings suggested immunized stallions probably only experience an acute loss of testicular functionality and parenchyma architecture is likely not affected since differences in Ecotext parameters with control stallions were not detected. ETD was the best parameter to identify animals with TD (AUC: 0.84, optimal cut-off value of 124.3 seminiferous tubules/cm2). Correlations were found between ETD and Doppler indices (PI: 0.60; RI: 0.47 p ≤ 0.001), total testicular volume (r: 0.48; p ≤ 0.05) and sperm motility (TM:0.51; and PM:0.54; p ≤ 0.001) and production (r:0.51; p ≤ 0.001). In summary, Ecotext could identify changes in testicular echotexture of stallions with TD. Results open the possibility for new research focused on establishing the relationship between Ecotext parameters and histomorphometry features in stallion testes.