Comparative stereological evaluation of the term allantochorion membrane in the mare pregnant with mule foals and equine foals.

Mules are derived from crossing horse mares with a donkey, in which the interest is due to gentleness and ability to work and equestrian sports. As the placenta is responsible for fetal development and maturation, knowing its typical microstructure allows us to understand how fetomaternal interactions occur in this interspecific pregnancy. Thus, the study performed a comparative stereological evaluation of volumetric composition and fetomaternal contact surface in the uterine body (UB), gravid uterine horn (GUH), and nongravid uterine horn (NGUH) of Mangalarga Paulista mare's term allantochorion membrane in mule and equine pregnancies. In equine gestation, the UB microcotyledon surface density was negatively correlated with the NGUH absolute area and the total volume of microvilli. In mule gestation, the base width and the number of microcotyledon were negatively correlated with the height and number of microcotyledons in the NGUH. Mule also showed a negative correlation between (1) the UB microcotyledon surface density and the GUH microcotyledons number per unit of membrane length, (2) the GUH total volume and the NGUH microcotyledon number. Such differences demonstrate a compensatory mechanism in conversion capacity among macrocompartments. A trend toward a greater total volume of allantoid vessels and total volume of allantoid mesoderm in UB microvilli was found in the equine and mule groups, respectively. There was a significant increase in the base width of microcotyledons in the NGUH of mules versus horses. These finds possibly influence the exchange capacity of each placental microregion and suggest a difference between mule versus horse term allantochorion membrane.

Low plasma progesterone concentration during the early luteal phase delays endometrial development and the beginning of placentation in mares.

While detrimental effects of reduced plasma progesterone concentration in the early luteal phase on conceptus development in horses have recently been demonstrated, there is no information on associated effects on the endometrium, allantochorion (AC), and chorionic girdle (CG) in this species. We hypothesised that reduced early postovulatory progesterone concentration in pregnant horses is detrimental to endometrial function and development of the embryonic membranes and is an underlying cause of delayed conceptus development. After insemination and ovulation, mares (n = 11) were assigned to treatment (TREAT) or control (CON) during two pregnancies. In TREAT pregnancies, mares received a PGF2α analogue for four consecutive days starting on the day of ovulation with the aim to reduce progesterone secretion. Mares were left untreated in CON pregnancies and thus served as their own controls. Endometrial biopsies for analysis of histomorphology, epidermal growth factor (EGF) and EGF receptor (EGFR) mRNA and protein expression in the endometrium, AC, and CG as well as abundance of regulatory T lymphocytes (Tregs) were collected on day 34 of pregnancy. Histomorphometric analysis revealed a higher luminal endometrium and a higher CG epithelium in CON compared to TREAT pregnancies. Abundance of mRNA for EGF and EGFR was large in the endometrium, AC and CG but did not differ between TREAT and CON pregnancies. The number of endometrial regulatory T lymphocytes was reduced in TREAT compared to CON pregnancies, adding further aspects to the potentially detrimental effects of reduced progesterone concentrations on equine pregnancy.

The transcriptome landscapes of allantochorion and vitelline-chorion in equine day 30 conceptus.

During equine early gestation, trophectoderm forms chorion tissue, which is composed of two parts that one is covering allantoin, called allantochorion (AC) and another is covering yolk sac, which here we call vitelline-chorion (VC). Given that little is known about the equine trophoblast-derived chorion differentiation at an early stage, we first compared the transcriptome of AC and VC of day 30 equine conceptus based on RNA-sequencing. As a result, we found that compared to VC, there are 484 DEGs, including 305 up- and 179 down-regulated genes in AC. GO and KEGG analysis indicated that up-regulated genes in AC are mainly cell proliferation and cell adhesion-related genes, participating in allantois expansion and allantochorionic-placenta formation; dominant genes in VC are extracellular exosome and other cell adhesion-related genes implicated in direct and indirect conceptus-maternal communication. Additionally, as for the progenitor chorion tissue of equine chorionic gonadotropin secreting endometrium cup-the chorionic girdle (CG), which locates at the junction of the dilating AC and regressing VC, we revealed its unique gene expression pattern and the gene regulation during its further differentiation in vitro. Collectively, this study sheds light on the molecular events regarding the trophoblast differentiation and function at an early stage of the equine preimplantation conceptus.

Evaluation of Biochemical Composition of Amniotic and Allantoic Fluids at Different Stages of Pregnancy in Queens.

Fetal fluid contents have functions in protecting fetuses and are essential for fetal development and maturation. However, little is known about the exact physiological functions of fetal fluids in fetal development, as well as the changing composition throughout the gestational period in cats. In this study, the biochemical composition of amniotic (AMN) and allantoic (ALL) fluids was investigated, as well as in the maternal serum of pregnant queens. Eighteen queens were included in this study and assigned to six different groups, D20, D25, D30, D40, D45 and D60, according to the gestational stage of fetal development. A total of 44 amniotic and 37 allantoic samples were collected. Fetal fluids contained lesser concentrations of alanine aminotransferase, albumin, cholesterol, triglycerides, creatine kinase, amylase, total protein and globulin than maternal serum. Other variables, such as aspartate aminotransferase, gamma-glutamyl transferase, bilirubin and alkaline phosphatase, were in different concentrations at specific stages of gestation when compared to maternal serum. There were no differences between fetal fluids and maternal serum for lactate dehydrogenase, urea, lipase or glucose concentrations. There were greater concentrations of creatinine in amniotic fluid than in allantoic fluid or maternal serum. Based on the results of this study, fetal fluids do not accumulate as a result of the simple filtration of maternal blood, but rather, the fetus produces many of these components as a consequence of organ development and maturation.

Evolution of 17-ß-estradiol, estrone and estrone-sulfate concentrations in late pregnancy of different breeds of mares using Liquid Chromatography and Mass Spectrometry.

This study describes 17-ß-estradiol (E2), estrone (E1) and estrone-sulfate (E1S) concentrations between 4 and 11 months in healthy equine pregnancies of two different breeds using Liquid Chromatography coupled to Mass-Spectrometry (LC-MS). In 2 stud-farms including 15 Spanish PureBred (SPB) and 11 Showjumping (SJ) types mares, combined thickness of the uterus and the placenta (CTUP) was measured and blood was sampled monthly between 4 and 11 months of gestation. Concentrations of E2, E1 and E1S were assayed with LC-MS in mares with normal CTUP. Effects of breed, day of pregnancy and mare's parity and age on estrogens concentrations were investigated. Peak of E2 was observed at 5 months (median: 46.4 pg/mL; maximum: 201.5 pg/mL). A strong correlation was observed between E1 and E1S (p < 0.0001, r = 0.85). Peak of E1 (median: 571.0 pg/mL; maximum: 1641.9 pg/mL) and E1S (median: 573.6 ng/mL; maximum: 997.6 ng/mL) concentrations was observed at the 5th month and then E1S decreased quicker than E1 until the end of pregnancy. Higher E2 and E1 concentrations were observed in SJ than in SPB mares between the 6th and the 8th months. No difference between breeds was observed for E1S monthly evolution. Estrogen peak values were all observed at 5 months. Unlike recent LC-MS studies, E1S values observed here were in the same range than those previously established using immuno-assays. After the 6th month, E1S decreased quicker than E1. Effect of breed only observed on non-sulfonated estrogens should be further confirmed. These findings confirm that sulfonation activity of the allantochorion may be limited after the 6th month.

Pregnancy and placental development in horses: an update.

Horses have been domesticated by man and historical information mostly associates horses with men. Nowadays, however, horse riding is essentially by women. Women are also very much involved in equine sciences, with a large contribution to the understanding of fetoplacental development. While highlighting the work of female scientists, this review describes the recent advances in equine fetoplacental studies, focusing on data obtained by new generation sequencing and progress on the understanding of the role of placental progesterone metabolites throughout gestation. A second emphasis is made on fetal programming, a currently very active field, where the importance of maternal nutrition, mare management or the use of embryo technologies has been shown to induce long term effects in the offspring that might affect progeny's performance. Finally, new perspectives for the study of equine pregnancy are drawn, that will rely on new methodologies applied to molecular explorations and imaging.

Morphological Characteristics of the Placenta and Umbilical Cord of Arabian Mares Foaling in the United Arab Emirates.

A total of 127 normal placentas from Arabian mares resident in the United Arab Emirates were examined. The mean linear dimensions of the placenta were, on average, 84% of those previously recorded for the placentas of the Thoroughbred. Significant differences in the size of the allantochorion between primigravid and multiparous mares were seen only in the linear dimensions of the body portion. The pregnant horn was more commonly on the right than left side of the uterus (P = .01; 74/127; 58%). Cord attachment was primarily at the base of the two placental horns (112/127; 88%), with the remainder showing anomalies from this position. The mean (±SEM) total length of the umbilical cords was 62.2 ± 1.2 cm, being composed of the allantoic portion (29.7 ± 0.9 cm) and amniotic portion (32.5 ± 0.6 cm), which averaged 53 ± 0.01% of the total length. The amniotic portion was usually, but not always, longer than the allantoic portion (79/127; 62%). Longer cords were associated with a greater proportion of allantoic length. An enlarged yolk sac remnant (YSR) was present in 16/127 (13%) placentas. Cords with YSRs displayed a significantly longer allantoic portion than those without (P = .02). The total cord length was not correlated with the weight or area of the allantochorion or amnion, any linear measurement of the allantochorion, gestation length, the month of foaling, parity of the dam, or birthweight or sex of the foal. The purpose for which the mare was bred (i.e., racing or showing) influenced the cord length, those of show mares being significantly longer.

The feto-maternal immune response to equine placentitis.

PROBLEM: Ascending placentitis is one of the leading causes of abortion in the horse. Minimal work has focused on its effect on fetal fluids or the antenatal immune response of the fetus. METHODOLOGY: Placentitis was induced via transcervical inoculation of Streptococcus equi ssp Zooepidemicus, and fluids/serum/tissues were collected 4-6 days later following euthanasia. Cytokine concentrations were detected using a multiplex immunoassay within fetal fluids (amniotic and allantoic) and serum (maternal and fetal) in inoculated and control mares. In addition, tissues from fetal (spleen, liver, lung, umbilicus, amnioallantois) and maternal (spleen, liver, lung, chorioallantois, endometrium) origin were analyzed in inoculated and control mares utilizing qPCR for expression of cytokines. RESULTS: No difference in cytokine concentrations in maternal or fetal serum was noted between inoculated and control mares. Concentrations of IL-1ß, IL-6, IL-10, and GRO were upregulated in the amniotic fluid following inoculation, with a trend toward higher IL-6 concentration in allantoic fluid. The amnioallantoic tissue separating the two fluids had higher expression of IL-1ß and IL-6 following inoculation, while chorioallantois and endometrium upregulated IL-1ß and IL-8 expression. IL-1ß was upregulated in the maternal spleen following inoculation. Fetal spleens were upregulated in expression of IL-1ß, GRO, and IL-6, while IL-6 was higher in fetal liver after inoculation than in controls. CONCLUSION: The maternal response to placentitis is primarily pro-inflammatory while the fetus appears to play a regulatory role in this inflammation. Additionally, amniotic fluid sampling may be more diagnostic of ascending placentitis than circulating cytokines.

Major histocompatibility complex class I in the horse (Equus caballus) placenta during pregnancy and parturition.

BACKGROUND: Major histocompatibility protein class I (MHC-I) is believed to be expressed in the horse allantochorion only in limited areas at limited times. However, its expression has only been investigated in early pregnancy with non-quantitative techniques that cannot reliably detect small amounts of protein. OBJECTIVE: To quantify the relative expression of MHC-I in the allantochorion and endometrium during days 90-240 of pregnancy (PREG), parturition with physiological delivery of fetal membranes (PHYS), and parturition with retention of these membranes (FMR). Also, to visualize protein expression and determine whether classical or non-classical MHC-I mRNA is expressed. ANIMALS: Heavy draft horses. SETTING: PREG horses (n = 12) were sampled postmortem at a slaughterhouse. PHYS (n = 6) and FMR (n = 5) horses were sampled at farms in the vicinity of Olsztyn, Poland. METHODS: For relative quantification of MHC-I, western blotting with densitometry was used. To visualize MHC-I, immunohistochemistry was used. For mRNA identification, RT-PCR was performed. RESULTS: Although the quantity of MHC-I was lower during PREG than parturition, it was present in the allantochorion and endometrium during PREG. During parturition, MHC-I expression was upregulated in the allantochorion (PHYS vs. PREG: 2.7-times higher, 95% confidence interval, 1.3- to 5.7-times higher; FMR vs. PREG: 3.2-times higher, 95% confidence interval, 1.5- to 6.7-times higher). At parturition, staining for MHC-I was detected in the microcotyledons. Classical and non-classical MHC-I were expressed in both tissues during PREG, PHYS, and FMR. CONCLUSION: MHC-I protein is present in the horse allantochorion and endometrium for at least the first two-thirds of pregnancy and at parturition.

Color-Doppler signals of blood flow in the corpus luteum and vascular perfusion index for ovarian and uterine arteries during expansion of the allantochorion in Bos taurus heifers.

Hemodynamics of the CL and each main uterine artery during expansion of the allantochorion from the ipsilateral side (CL side) to the contralateral side were studied in heifers (n = 8 nonbred, 9 pregnant). Progesterone concentration, vascular perfusion index for each uterine artery (by spectral ultrasonography), extent of blood flow in the CL (percentage of CL tissue with color-Doppler signals of blood flow), and intrauterine location of the expanding allantochorion (by gray-scale ultrasonic imaging) were determined daily from Days 14-60 (Day 0 = ovulation). In the pregnant group, but not in the nonbred group, the percentage of CL tissue with blood-flow signals increased (P < 0.003) from Days 16 (66.7 ± 4.2%) to 23 (79.4 ± 2.5) and then more slowly increased (P < 0.02) from Days 24 (76.7 ± 2.9%) to 50 (85.0 ± 2.0%). The volume of CL increased (P < 0.0001) progressively from Days 26 (6.1 ± 0.4 cm3) to 60 (7.3 ± 0.7 cm3). The vascular perfusion index in the ipsilateral uterine artery did not change in the nonbred group and progressively increased in the pregnant group beginning on Day 17 in approximate temporal association with the increase in luteal blood-flow signals. Functional increases in the CL during early pregnancy were attributed to the greater uterine arterial blood flow on the ipsilateral or CL side and the reported prominent anastomosis from a branch of the uterine artery to the ovarian artery that supplies the CL ovary. After an initial significant decrease in vascular perfusion index in each uterine artery in the pregnant group, the index began to increase on Day 17 in the ipsilateral artery and on Day 18 in the contralateral artery. The perfusion continued in the ipsilateral artery but discontinued in the contralateral artery on Day 21. Perfusion and diameter of the uterine artery were greater for the ipsilateral side until the vesicle entered the contralateral horn on Day 33 and increased for the contralateral horn between vesicle entry and filling of the horn on Day 44. During Days 35-60, the perfusion index (P < 0.04) and artery diameter (P < 0.001) were lower in the contralateral than in the ipsilateral artery. Results supported the hypotheses that (1) CL function increases during early pregnancy in temporal association with an increase in blood flow in the ipsilateral uterine artery and (2) blood flow in each of the ipsilateral and contralateral uterine arteries increases as the allantochorion expands in each uterine horn.

Comparison between allantochorion membrane and amniotic sac detection by per rectal palpation for pregnancy diagnosis on pregnancy loss, calving rates, and abnormalities in newborn calves.

The objectives of the present investigation were to evaluate the pregnancy diagnosis by detection of either the allantochorion membrane (FMS) or amniotic sac (ASP) by per rectum palpation (PRP) during late embryonic or early fetal period on pregnancy loss (PRL) at reexamination, calving rates, and abnormalities in newborn calves. A controlled randomized blind design with 800 lactating dairy pregnant cows diagnosed by transrectal ultrasonography (TRUS) between Days 35 and 57 of gestation from one dairy farm were included. The cows were randomly divided according to detection of allantochorion membrane (FMS group; n = 264), detection of amniotic sac (ASP group; n = 266), and TRUS (control [CON] group; n = 270). TRUS was considered as the criterion standard method of comparison. The entire PRP was performed by one experienced veterinarian. Then, all the cows were reexamined only by TRUS between 2 and 4 weeks later by two independent veterinarians to assess PRL. The calving rate one (number of cows calved divided by the number of cows initially pregnant) and calving rate two (number of cows calved divided by the number of cows pregnant at reexamination) for each group was calculated. All abortions and stillborns were necropsied, and calves alive were followed for 5 days. The overall initial PRL (between initial pregnant cows and reexamination) for FMS, ASP, and CON groups was 7.4% (19/258), 8.8% (23/262), and 9.2% (24/260), respectively (P = 0.75). The overall late PRL (between reexamination and calving) for FMS, ASP, and CON groups was 4.2% (9/213), 5.7% (12/209), and 4.2% (9/216), respectively (P = 0.71). The calving rate one for FMS, ASP, and TRUS groups was 79.1% (204/258), 75.2% (197/262), and 79.6% (207/260), respectively (P = 0.63). The calving rate two for the same groups was 85.4% (204/239), 82.4% (197/239), and 87.7% (207/236), respectively (P = 0.27). The number of fetuses aborted late, premature, and mature dead from FMS, ASP, and CON groups was 6, 4, and 5, respectively (P = 0.85), and no abnormalities at necropsy were detected. One stillborn male calf with atresia coli after 281 days of gestation from a cow examined by ASP at Day 51 was diagnosed. It was concluded that the use of either FMS or ASP for pregnancy diagnosis during late embryonic or early fetal period did not increase the PRL, affect calving rates, or produce calves with congenital abnormalities.

Effect of oleic acid supplementation on prostaglandin production in maternal endometrial and fetal allantochorion cells isolated from late gestation ewes.

INTRODUCTION: Elevated circulating non-esterified fatty acids including oleic acid (OA) are associated with many pregnancy related complications. Prostaglandins (PGs) play crucial roles during parturition. We investigated the effect of OA supplementation on PG production using an in vitro model of ovine placenta. METHODS: Maternal endometrium (ME) and fetal allantochorion (FC) were collected in late pregnancy (day 135). Confluent cells were cultured in serum-free medium supplemented with 0, 20 or 100 µM OA and challenged with control medium, oxytocin (OT, 250 nM), lipopolysaccharide (LPS, 0.1 µg/ml) or dexamethasone (DEX, 5 µM). Spent medium was harvested at 2 and 24 h after challenge for quantifying PGs. RESULTS: In ME cells OA increased PGE2 production moderately but attenuated PGF2α production leading to a doubling of the PGE2:PGF2α ratio (E:F) (P < 0.01). Without OA, both OT and LPS stimulated PG production for about 3-fold (P < 0.01) without changing the E:F ratio. In the ME cells challenged with OT, OA decreased both PGE2 and PGF2α production by up to 70% (P < 0.01) whereas in LPS treated cells OA increased the E:F ratio. In FC cells PGE2 production at 2 h was stimulated by 100 µM OA (P < 0.05). In these cells LPS caused a 3-fold increase in PGE2 (P < 0.01), an effect which was completely inhibited by DEX. DISCUSSION: OA supplementation favours basal PGE2 production in both ME and FC. In ME OA increased E:F ratios and antagonized the stimulatory effect of OT on PG production. This suggests that raised circulating OA may affect both the initiation and progression of parturition.

Expression of oxytocin receptors is greatly reduced in the placenta of heavy mares with retained fetal membranes due to secondary uterine atony.

REASONS FOR PERFORMING STUDY: Fetal membrane retention can be a life-threatening condition and its incidence exceeds 50% in heavy draught mares. Although fetal membrane retention is commonly treated with repeated injections of oxytocin, based on the suggestion that it is caused mainly by secondary atony of the uterus, this treatment sometimes fails. This led us to ask if expression of oxytocin receptors differs in mares that retain fetal membranes due to secondary uterine atony. OBJECTIVES: To determine whether expression of oxytocin receptors in equine placental tissues differs when heavy draught mares expel fetal membranes or retain them because of secondary uterine atony. STUDY DESIGN: Controlled study using archived tissues. METHODS: Placental biopsies (containing the endometrium and allantochorion) were taken from 8 heavy draught mares during parturition. Four mares expelled fetal membranes shortly after foaling (control mares) and 4 mares retained them (expulsion time was >3 h from delivery). The 4 mares that retained fetal membranes had secondary atony of the uterus. The amount of oxytocin receptors was estimated by measuring the intensity of western blot bands. The presence and location of oxytocin receptors were determined by immunocytochemistry. RESULTS: Oxytocin receptor expression was nearly 50 times less intense in mares with placenta retention due to secondary atony of the uterus and immunocytochemical staining was barely visible. In the control mares, oxytocin receptors were found in both epithelial and endothelial cells of the placenta and staining was most intense where the endometrium contacts the allantochorion. CONCLUSIONS: Inadequate expression of oxytocin receptors may be a cause of uterine atony leading to fetal membrane retention.