Parental bias in expression and interaction of genes in the equine placenta.

Most autosomal genes in the placenta show a biallelic expression pattern. However, some genes exhibit allele-specific transcription depending on the parental origin of the chromosomes on which the copy of the gene resides. Parentally expressed genes are involved in the reciprocal interaction between maternal and paternal genes, coordinating the allocation of resources between fetus and mother. One of the main challenges of studying parental-specific allelic expression (allele-specific expression [ASE]) in the placenta is the maternal cellular remnant at the fetomaternal interface. Horses (Equus caballus) have an epitheliochorial placenta in which both the endometrial epithelium and the epithelium of the chorionic villi are juxtaposed with minimal extension into the uterine mucosa, yet there is no information available on the allelic gene expression of equine chorioallantois (CA). In the current study, we present a dataset of 1,336 genes showing ASE in the equine CA (https://pouya-dini.github.io/equine-gene-db/) along with a workflow for analyzing ASE genes. We further identified 254 potentially imprinted genes among the parentally expressed genes in the equine CA and evaluated the expression pattern of these genes throughout gestation. Our gene ontology analysis implies that maternally expressed genes tend to decrease the length of gestation, while paternally expressed genes extend the length of gestation. This study provides fundamental information regarding parental gene expression during equine pregnancy, a species with a negligible amount of maternal cellular remnant in its placenta. This information will provide the basis for a better understanding of the role of parental gene expression in the placenta during gestation.

Transcriptomic analysis of equine placenta reveals key regulators and pathways involved in ascending placentitis†.

Improved understanding of the molecular mechanisms underlying ascending equine placentitis holds the potential for the development of new diagnostic tools and therapies to forestall placentitis-induced preterm labor. The current study characterized the equine placental transcriptome (chorioallantois [CA] and endometrium [EN]) during placentitis (placentitis group, n = 6) in comparison to gestationally-matched controls (control group, n = 6). Transcriptome analysis identified 2953 and 805 differentially expressed genes in CA and EN during placentitis, respectively. Upstream regulator analysis revealed the central role of toll-like receptors (TLRs) in triggering the inflammatory signaling, and consequent immune-cell chemotaxis. Placentitis was associated with the upregulation of matrix metalloproteinase (MMP1, MMP2, and MMP9) and apoptosis-related genes such as caspases (CASP3, CASP4, and CASP7) in CA. Also, placentitis was associated with downregulation of transcripts coding for proteins essential for placental steroidogenesis (SRD5A1 and AKR1C1), progestin signaling (PGRMC1 and PXR) angiogenesis (VEGFA, VEGFR2, and VEGFR3), and nutrient transport (GLUT12 and SLC1A4), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could explain placental insufficiency during placentitis. Placentitis was also associated with aberrant expression of several placenta-regulatory genes, such as PLAC8, PAPPA, LGALS1, ABCG2, GCM1, and TEPP, which could negatively affect placental functions. In conclusion, our findings revealed for the first time the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during equine placentitis, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways.

Paternally expressed retrotransposon Gag-like 1 gene, RTL1, is one of the crucial elements for placental angiogenesis in horses†.

RTL1 (retrotransposon Gag-like 1) is an essential gene in the development of the human and murine placenta. Several fetal and placental abnormalities such as intrauterine growth restriction (IUGR) and hydrops conditions have been associated with altered expression of this gene. However, the function of RTL1 has not been identified. RTL1 is located on a highly conserved region in eutherian mammals. Therefore, the genetic and molecular analysis in horses could hold important implications for other species, including humans. Here, we demonstrated that RTL1 is paternally expressed and is localized within the endothelial cells of the equine (Equus caballus) chorioallantois. We developed an equine placental microvasculature primary cell culture and demonstrated that RTL1 knockdown leads to loss of the sprouting ability of these endothelial cells. We further demonstrated an association between abnormal expression of RTL1 and development of hydrallantois. Our data suggest that RTL1 may be essential for placental angiogenesis, and its abnormal expression can lead to placental insufficiency. This placental insufficiency could be the reason for IUGR and hydrops conditions reported in other species, including humans.

Equine cervical remodeling during placentitis and the prepartum period: a transcriptomic approach.

Cervical remodeling is a critical component in both term and preterm labor in eutherian mammals. However, the molecular mechanisms underlying cervical remodeling remain poorly understood in the mare. The current study compared the transcriptome of the equine cervix (cervical mucosa (CM) and stroma (CS)) during placentitis (placentitis group, n = 5) and normal prepartum mares (prepartum group, n = 3) to normal pregnant mares (control group, n = 4). Transcriptome analysis identified differentially expressed genes (DEGs) during placentitis (5310 in CM and 907 in CS) and during the normal prepartum period (189 in CM and 78 in CS). Our study revealed that cervical remodeling during placentitis was dominated by inflammatory signaling as reflected by the overrepresented toll-like receptor signaling, interleukin signaling, T cell activation, and B cell activation pathways. These pathways were accompanied by upregulation of several proteases, including matrix metalloproteinases (MMP1, MMP2, and MMP9), cathepsins (CTSB, CTSC, and CTSD) and a disintegrin and metalloproteinase with thrombospondin type 1 motifs (ADAMTS1, ADAMTS4, and ADAMTS5), which are crucial for degradation of cervical collagens during remodeling. Cervical remodeling during placentitis was also associated with upregulation of water channel-related transcripts (AQP9 and RLN), angiogenesis-related transcripts (NOS3, ENG1, THBS1, and RAC2), and aggrecan (ACAN), a hydrophilic glucosaminoglycan, with subsequent cervical hydration. The normal prepartum cervix was associated with upregulation of ADAMTS1, ADAMTS4, NOS3 and THBS1, which might reflect an early stage of cervical remodeling taking place in preparation for labor. In conclusion, our findings revealed the possible key regulators and mechanisms underlying equine cervical remodeling during placentitis and the normal prepartum period.

Transcriptomic analysis of equine chorioallantois reveals immune networks and molecular mechanisms involved in nocardioform placentitis.

Nocardioform placentitis (NP) continues to result in episodic outbreaks of abortion and preterm birth in mares and remains a poorly understood disease. The objective of this study was to characterize the transcriptome of the chorioallantois (CA) of mares with NP. The CA were collected from mares with confirmed NP based upon histopathology, microbiological culture and PCR for Amycolatopsis spp. Samples were collected from the margin of the NP lesion (NPL, n = 4) and grossly normal region (NPN, n = 4). Additionally, CA samples were collected from normal postpartum mares (Control; CRL, n = 4). Transcriptome analysis identified 2892 differentially expressed genes (DEGs) in NPL vs. CRL and 2450 DEGs in NPL vs. NPN. Functional genomics analysis elucidated that inflammatory signaling, toll-like receptor signaling, inflammasome activation, chemotaxis, and apoptosis pathways are involved in NP. The increased leukocytic infiltration in NPL was associated with the upregulation of matrix metalloproteinase (MMP1, MMP3, and MMP8) and apoptosis-related genes, such as caspases (CASP3 and CASP7), which could explain placental separation associated with NP. Also, NP was associated with downregulation of several placenta-regulatory genes (ABCG2, GCM1, EPAS1, and NR3C1), angiogenesis-related genes (VEGFA, FLT1, KDR, and ANGPT2), and glucose transporter coding genes (GLUT1, GLUT10, and GLUT12), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could elucidate placental insufficiency accompanying NP. In conclusion, our findings revealed for the first time, the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during NP, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways.

Estrogens Regulate Placental Angiogenesis in Horses.

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p < 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17ß-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.

Elevated blood urea nitrogen alters the transcriptome of equine embryos.

High blood urea nitrogen (BUN) in cows and ewes has a negative effect on embryo development; however, no comparable studies have been published in mares. The aims of the present study were to evaluate the effects of high BUN on blastocoele fluid, systemic progesterone and Day 14 equine embryos. When a follicle with a mean (±s.e.m.) diameter of 25±3mm was detected, mares were administered urea (0.4g kg-1) with sweet feed and molasses (n=9) or sweet feed and molasses alone (control; n=10). Blood samples were collected every other day. Mares were subjected to AI and the day ovulation was detected was designated as Day 0. Embryos were collected on Day 14 (urea-treated, n=5 embryos; control, n=7 embryos). There was an increase in systemic BUN in the urea-treated group compared with control (P<0.05), with no difference in progesterone concentrations. There were no differences between the two groups in embryo recovery or embryo size. Urea concentrations in the blastocoele fluid tended to be higher in the urea-treated mares, with a strong correlation with plasma BUN. However, there was no difference in the osmolality or pH of the blastocoele fluid between the two groups. Differentially expressed genes in Day 14 embryos from urea-treated mares analysed by RNA sequencing were involved in neurological development, urea transport, vascular remodelling and adhesion. In conclusion, oral urea treatment in mares increased BUN and induced transcriptome changes in Day 14 equine embryos of genes important in normal embryo development.

Equine placentitis is associated with a downregulation in myometrial progestin signaling†.

The current study aimed to elucidate the mechanisms underlying myometrial activation during equine placentitis related to progestogens and the progesterone receptor signaling pathways. Placentitis was induced via intracervical inoculation with Streptococcus equi ssp zooepidemicus in mares at approximately 290 days of gestation (placentitis group; n = 6) with uninoculated gestationally matched mares as controls (n = 4). Mares in the placentitis and control groups were euthanized, and myometrial samples were collected from two regions: region 1-parallel to active placentitis lesion with placental separation in placentitis group (P1) or caudal pole of the placenta in control group (C1); and region 2-parallel to apparently normal placenta without separation in placentitis group (P2) or uterine body in control group (C2). In the current study, SRD5A1 and AKR1C23, which encode for the key P4 metabolizing enzymes, were downregulated in P1 in comparison to C1, C2, and P2, and this was associated with a decline (P < 0.05) in 5αDHP, allopregnanolone (3αDHP), and 20αDHP in P1 in comparison to C1. Further, myometrial expression of PR was downregulated (P < 0.05) in P1 in comparison to C1 and P2, and this was associated with activation of the inflammatory cascade as reflected by significant upregulation of IL-1ß and IL-8 in P1 in comparison to C1, C2, and P2, and supported by increased tissue leukocytes in P1 in comparison to C1. In conclusion, equine placentitis is associated with a localized withdrawal of progestins and a downregulation of the PR in the myometrium concomitant with upregulation of inflammatory cytokines and subsequent myometrial activation.

Extraction of RNA from formalin-fixed, paraffin-embedded equine placenta.

Archived formalin-fixed, paraffin-embedded (FFPE) samples represent a valuable resource for the determination of gene expression for physio/pathological conditions. In the present study, we validated a protocol for the extraction of RNA from FFPE samples collected from healthy and diseased equine placenta. The quality and quantity of the extracted RNA from the FFPE and matching RNAlater™-preserved samples and expression levels of common housekeeping genes and reference microRNAs were evaluated. Precision of the expression data was evaluated by comparing relative expression of CYP19A1 and HSD3B1 in FFPE and RNAlater™ samples. The median RNA concentration recovered from FFPE samples was 316.8 ng/mm3 of tissue (ranging between 61.6 and 917.4 ng/mm3 ), average RNA integrity number was 2.3 ± 0.9 (mean ± standard deviation), and 84% of samples had RNA fragments longer than 200 nucleotides (DV200 ). RNA concentrations and CT values for GAPDH, ACTB, miR-8908a and miR-369 in FFPE samples were significantly correlated (r = -0.8, -0.7, -0.4 and -0.4, respectively; p < 0.001). Expression pattern of normalized CYP19A1 and HSD3B1 in paired FFPE and RNAlater™ samples was significantly correlated (r = 0.97 for CYP19A1 and HSD3B1; p < 0.001). This study demonstrates that RNA can be extracted from FFPE equine placental tissue and used for downstream transcriptomic analysis. Similar RNA expression patterns were obtained using RNAlater™ and FFPE tissue samples.

Kinetics of the chromosome 14 microRNA cluster ortholog and its potential role during placental development in the pregnant mare.

BACKGROUND: The human chromosome 14 microRNA cluster (C14MC) is a conserved microRNA (miRNA) cluster across eutherian mammals, reported to play an important role in placental development. However, the expression kinetics and function of this cluster in the mammalian placenta are poorly understood. Here, we evaluated the expression kinetics of the equine C24MC, ortholog to the human C14MC, in the chorioallantoic membrane during the course of gestation. RESULTS: We demonstrated that C24MC-associated miRNAs presented a higher expression level during early stages of pregnancy, followed by a decline later in gestation. Evaluation of one member of C24MC (miR-409-3p) by in situ hybridization demonstrated that its cellular localization predominantly involved the chorion and allantoic epithelium and vascular endothelium. Additionally, expression of predicted target transcripts for C24MC-associated miRNAs was evaluated by RNA sequencing. Expression analysis of a subset of predicted mRNA targets showed a negative correlation with C24MC-associated miRNAs expression levels during gestation, suggesting the reciprocal control of these target transcripts by this miRNA cluster. Predicted functional analysis of these target mRNAs indicated enrichment of biological pathways related to embryonic development, endothelial cell migration and angiogenesis. Expression patterns of selected target mRNAs involved in angiogenesis were confirmed by RT-qPCR. CONCLUSION: This is the first report evaluating C24MC kinetics during pregnancy. The findings presented herein suggest that the C24MC may modulate angiogenic transcriptional profiles during placental development in the horse.

Inhibition of 5α-reductase alters pregnane metabolism in the late pregnant mare.

In the latter half of gestation in the mare, progesterone concentrations decline to near undetectable levels while other 5α-reduced pregnanes are elevated. Of these, 5α-dihydroprogesterone and allopregnanolone have been reported to have important roles in either pregnancy maintenance or fetal quiescence. During this time, the placenta is necessary for pregnane metabolism, with the enzyme 5α-reductase being required for the conversion of progesterone to 5α-dihydroprogesterone. The objectives of this study were to assess the effects of a 5α-reductase inhibitor, dutasteride on pregnane metabolism (pregnenolone, progesterone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3ß,20α-diol and allopregnanolone), to determine circulating dutasteride concentrations and to assess effects of dutasteride treatment on gestational parameters. Pregnant mares (n = 5) received dutasteride (0.01 mg/kg/day, IM) and control mares (n = 4) received vehicle alone from 300 to 320 days of gestation or until parturition. Concentrations of dutasteride, pregnenolone, progesterone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3ß,20α-diol, and allopregnanolone were evaluated via liquid chromatography-tandem mass spectrometry. Samples were analyzed as both days post treatment and as days prepartum. No significant treatment effects were detected in pregnenolone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3ß,20α-diol or allopregnanolone for either analysis; however, progesterone concentrations were increased (P < 0.05) sixfold in dutasteride-treated mares compared to control mares. Dutasteride concentrations increased in the treated mares, with a significant correlation (P < 0.05) between dutasteride concentrations and pregnenolone or progesterone concentrations. Gestational length and neonatal outcomes were not significantly altered in dutasteride-treated mares. Although 5α-reduced metabolites were unchanged, these data suggest an accumulation of precursor progesterone with inhibition of 5α-reductase, indicating the ability of dutasteride to alter progesterone metabolism.

Endocrine changes, fetal growth, and uterine artery hemodynamics after chronic estrogen suppression during the last trimester of equine pregnancy.

Equine pregnancy is characterized by very high circulating concentrations of estrogens. The physiological roles of estrogens during equine gestation are largely unknown, although some studies suggest a role in the regulation of uterine artery hemodynamics and a relationship between low circulating estrogen concentrations and late pregnancy loss. The objectives of this experiment were to evaluate the effects of estrogen suppression on uterine artery hemodynamics and on pregnancy outcome. Estrogen synthesis was suppressed using letrozole, a potent aromatase inhibitor. Twelve pregnant mares were randomly assigned to a control (n = 6) or treatment (n = 6; 500 mg letrozole orally every 4 days) group with treatment starting at 240 days of gestation and continuing until parturition. Weekly serum samples were analyzed to determine testosterone, dehydroepiandrosterone sulfate, estradiol, estrone sulfate, progestins, and prostaglandin F2α metabolite concentrations. Ultrasonographic examinations were performed biweekly and measurements included uterine artery hemodynamics (diameter, pulsatility, and resistance indices), fetal growth using the diameter of the fetal eye, and placental evaluation using the combined thickness of the uterus and placenta. At parturition, gestational length, foal weight, and neonatal viability were determined. Letrozole suppressed estrogen synthesis during gestation by approximately 90% compared to control values. This large reduction in circulating estrogens had no effect on uterine artery hemodynamics, normal placental development, maintenance of pregnancy, or neonatal viability. However, neonates from letrozole-treated mares had lower (P < 0.05) birth weights than controls, suggesting that estrogens may play a role in fetal growth that is not mediated through regulation of uterine blood flow.

Selection of developmentally competent immature equine oocytes with brilliant cresyl blue stain prior to in vitro maturation with equine growth hormone.

Immature oocytes synthesize a variety of proteins that include the enzyme glucose-6-phosphate dehydrogenase (G6PDH). Brilliant cresyl blue (BCB) is a vital blue dye that assesses intracellular activity of G6PDH, an indirect measure of oocyte maturation. The objective was to evaluate the BCB test as a criterion to assess developmental competence of equine oocytes and to determine if equine growth hormone (eGH) enhanced in vitro maturation (IVM) of equine oocyte. Cumulus-oocytes complexes (COCs) were recovered by aspirating follicles <30 mm in diameter from abattoir-derived ovaries and were evaluated morphologically. Thereafter, COCs were exposed to BCB (26 µM) for 90 min at 39°C and selected based on the colour of their cytoplasm (BCB positive/BCB+ or BCB negative/BCB-). The COCs were allocated as follows: (a) IVM medium; (b) eGH group; (c) BCB-/IVM; (d) BCB+/IVM; (e) BCB-/eGH; and (f) BCB+/eGH. Then, COCs were cultured in vitro for 30 h, at 39°C in a 5%CO2 humidified air atmosphere. Cumulus-free oocytes were incubated in 10 µg/ml of bis-benzamide for 20 min at 39°C and nuclear maturation was evaluated with epifluorescence microscopy. Of the 39 COCs selected morphologically and subjected to BCB staining, 18/39 (46.2%) were classified as BCB+ and 21/39 (53.8%) as BCB- (P > 0.05). Maturation was not affected significantly by BCB classification, but the maturation rate was higher for oocytes that had been exposed to exogenous eGH versus controls (16/28, 57.1% versus 8/26, 30.8%, P < 0.05). In the present study, the BCB test was not useful for predicting competent equine oocytes prior to IVM. However, eGH enhanced equine oocyte maturation in vitro.

The effects of selected sedatives on basal and stimulated serum cortisol concentrations in healthy dogs.

Background: Hormone assessment is typically recommended for awake, unsedated dogs. However, one of the most commonly asked questions from veterinary practitioners to the endocrinology laboratory is how sedation impacts cortisol concentrations and the adrenocorticotropic hormone (ACTH) stimulation test. Butorphanol, dexmedetomidine, and trazodone are common sedatives for dogs, but their impact on the hypothalamic-pituitary-adrenal axis (HPA) is unknown. The objective of this study was to evaluate the effects of butorphanol, dexmedetomidine, and trazodone on serum cortisol concentrations. Methods: Twelve healthy beagles were included in a prospective, randomized, four-period crossover design study with a 7-day washout. ACTH stimulation test results were determined after saline (0.5 mL IV), butorphanol (0.3 mg/kg IV), dexmedetomidine (4 µg/kg IV), and trazodone (3-5 mg/kg PO) administration. Results: Compared to saline, butorphanol increased basal (median 11.75 µg/dL (range 2.50-23.00) (324.13 nmol/L; range 68.97-634.48) vs 1.27 µg/dL (0.74-2.10) (35.03 nmol/L; 20.41-57.93); P < 0.0001) and post-ACTH cortisol concentrations (17.05 µg/dL (12.40-26.00) (470.34 nmol/L; 342.07-717.24) vs 13.75 µg/dL (10.00-18.90) (379.31 nmol/L; 275.96-521.38); P ≤ 0.0001). Dexmedetomidine and trazodone did not significantly affect basal (1.55 µg/dL (range 0.75-1.55) (42.76 nmol/L; 20.69-42.76); P = 0.33 and 0.79 µg/dL (range 0.69-1.89) (21.79 nmol/L; 19.03-52.14); P = 0.13, respectively, vs saline 1.27 (0.74-2.10) (35.03 nmol/L; 20.41-57.93)) or post-ACTH cortisol concentrations (14.35 µg/dL (range 10.70-18.00) (395.86 nmol/L; 295.17-496.55); (P = 0.98 and 12.90 µg/dL (range 8.94-17.40) (355.86 nmol/L; 246.62-480); P = 0.65), respectively, vs saline 13.75 µg/dL (10.00-18.60) (379.31 nmol/L; 275.86-513.10). Conclusion: Butorphanol administration should be avoided prior to ACTH stimulation testing in dogs. Further evaluation of dexmedetomidine and trazodone's effects on adrenocortical hormone testing in dogs suspected of HPA derangements is warranted to confirm they do not impact clinical diagnosis.

Letrozole administration effects on the P450aromatase expression and the reproductive parameters in male sheep (Ovis aries).

Letrozole comprises a non-steroid aromatase inhibitor that has been applied as a preventive for many uses, such as breast cancer prevention, treatment of hormonal dysfunction, and male infertility. Precisely in Northeast Brazil, ovine consist of the leading livestock produced, and their reproduction in captivity has been demonstrated difficult. Thus, we hypothesized whether the application of letrozole will improve male sheep reproduction. One group of 6 animals received a daily dosage of 0.5mg/Kg of letrozole for 60 days, while the other six animals were used as control. Samples were collected from control and treated animals after 30 and 60 days of the experiment. Blood samples were collected to measure the steroid hormone levels. Semen was collected from control and treated groups using an artificial vagina and cryopreserved for spermatozoa morphology and CASA analysis. The testicles were collected for histological analysis, gene expression, and immunohistochemistry of P450aromatase protein. Hormone levels demonstrate no differences in the estradiol/testosterone levels among the control and both treated groups. Immunohistochemistry analysis revealed the presence of P450aromatase protein in spermatogonia cells and Leydig cells in the control and treated groups in both periods analyzed. Moreover, there were no differences in the P450aromase gene expression in the control and treated group. Morphological analysis of the spermatozoa revealed that letrozole treatment did not affect mitochondrial activity or cause any deformities. In addition, motility parameters in the sperm from the treated group were not affected by letrozole treatment compared to the control group. Morphological analysis of the testis demonstrated that letrozole treatment increase the seminiferous tubule area but no signs of germ cell damage. Our results show that letrozole has a morphological effect on the testicles in the ovine model but no pathological or severe effect caused by hormone level imbalance. Overall, letrozole comprises a non-steroid aromatase inhibitor, which can be applied to ovine reproduction.

Binding of Equine Seminal Lactoferrin/Superoxide Dismutase (SOD-3) Complex Is Biased towards Dead Spermatozoa.

Sperm-neutrophil binding is an important facet of breeding and significantly impacts fertility. While a specific seminal plasma protein has been found to reduce this binding and improve fertility (CRISP-3), additional molecule(s) appear to promote binding between defective sperm and neutrophils. Recent work has suggested one of these proteins is lactoferrin (LF), an 80 kDa iron-binding protein found throughout the body, but the purity of the protein was not confirmed. It is unknown if LF binds to sperm selectively based on viability, and if receptors for LF are located on equine sperm. To evaluate this, we attempted to purify equine seminal LF from five stallions (n = 5), biotinylate LF, and evaluate potential binding site(s) on spermatozoa. LF was consistently associated with superoxide dismutase (SOD-3), and all attempts to separate the two proteins were unsuccessful. Flow cytometric and microscopic analyses were used to compare LF/SOD-3 binding to viable and nonviable spermatozoa. Additionally, various methods of biotinylation were assessed to optimize this methodology. Biotinylation of seminal plasma protein was an effective and efficient method to study seminal plasma protein properties, and the binding site for LF/SOD-3 was found to be broadly localized to the entire sperm cell surface as well as selective towards nonviable/defective sperm. Although we were not able to determine if the binding to equine spermatozoa was through LF or SOD-3, we can conclude that equine seminal LF is tightly bound to SOD-3 and this protein complex binds selectively to nonviable spermatozoa, possibly to mark them for elimination by neutrophil phagocytosis.

Effects of isoflurane anesthesia on cerebrovascular autoregulation in horses.

OBJECTIVE: To test a hypothesis predicting that isoflurane would interfere with cerebrovascular autoregulation in horses and to evaluate whether increased mean arterial blood pressure (MAP) would increase cerebral blood flow and intracranial pressure (ICP) during isoflurane anesthesia. ANIMALS: 6 healthy adult horses. PROCEDURES: Horses were anesthetized with isoflurane at a constant end-tidal concentration sufficient to maintain MAP at 60 mm Hg. The facial, carotid, and dorsal metatarsal arteries were catheterized for blood sample collection and pressure measurements. A sub-arachnoid transducer was used to measure ICP Fluorescent microspheres were injected through a left ventricular catheter during MAP conditions of 60 mm Hg, and blood samples were collected. This process was repeated with different-colored microspheres at the same isoflurane concentration during MAP conditions of 80 and 100 mm Hg achieved with IV administration of dobutamine. Central nervous system tissue samples were obtained after euthanasia to quantify fluorescence and calculate blood flow. RESULTS: Increased MAP did not increase ICP or blood flow in any of the brain tissues examined. However, values for blood flow were low for all tested brain regions except the pons and cerebellum. Spinal cord blood flow was significantly decreased at the highest MAP. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that healthy horses autoregulate blood flow in the CNS at moderate to deep planes of isoflurane anesthesia. Nonetheless, relatively low blood flows in the brain and spinal cord of anesthetized horses may increase risks for hypoperfusion and neurologic injury.

Use of Tubo-Ovarian Ligation Via Colpotomy as A Potential Method for Sterilization in Mares.

The goal of this study was to develop a safe, effective, and economical method for permanent sterilization of mares based upon tubo-ovarian ligation performed via colpotomy. In this study, we evaluated the application of a nylon cable tie (zip-tie) to the ovarian pedicle and oviduct of mares to induce ovarian ischemia and tubal ligation without removal of ovaries. Initially, efficiency of zip-ties on the ovarian pedicle was tested in vitro and in vivo. Based on the absence of leakage through the zip-tie ligated vessels in anatomic specimens, we confirmed the potential efficacy of the technique. Next, ligation of the ovarian pedicle via a standing colpotomy was conducted in five mares. Although the surgical procedure in these mares appeared to be quick and efficient, all five mares were noted to develop ovarian adhesions to surrounding abdominal viscera in either one or both ovaries postoperatively. Ovarian ischemia led to loss of ovarian activity based upon ultrasound examination, which was confirmed by a low plasma progesterone concentration in four of the five mares. During the postoperative period, four mares demonstrated clinical signs related to the ovarian adhesions and were euthanized. The postoperative complications associated with ovarian adhesions to abdominal viscera presented significant challenges, limiting the success of this study. While this technique resulted in ovarian ischemia and atrophy in four out of the five mares, we were unable to assess long-term effects on the health and reproduction of the mares due to the ovarian adhesions to the surrounding tissues and the potential for secondary complications. Although technically feasible, tubo-ovarian ligation via colpotomy does not appear to be a viable option for sterilization of mares using the described technique due to ovarian adhesions post procedure.

Kinetics of placenta-specific 8 (PLAC8) in equine placenta during pregnancy and placentitis.

Placenta-specific 8 (PLAC8) is one of the placenta-regulatory genes which is highly conserved among eutherian mammals. However, little is known about its expression in equine placenta (chorioallantois; CA and endometrium; EN) during normal and abnormal pregnancy. Therefore, the current study was designed to 1) elucidate the expression of PLAC8 in equine embryonic membranes during the preimplantation period, 2) characterize the expression profile of PLAC8 in equine CA (45d, 4mo, 6mo, 10 mo, 11 mo and postpartum) and EN (14d, 4mo, 6mo, 10 mo, and 11 mo) obtained from pregnant mares (n = 4/timepoint), as well as, d14 non-pregnant EN (n = 4), and 3) investigate the expression profile of PLAC8 in ascending placentitis (n = 5) and in nocardioform placentitis (n = 6) in comparison to normal CA. In the preimplantation period, PLAC8 mRNA was not abundant in the trophectoderm of d8 equine embryo and d14 conceptus, while it was abundant later in d 30, 31, 34, and 45 chorion. In normal pregnancy, PLAC8 mRNA expression in CA at 45 d gradually decline to reach nadir at 6mo before gradually increasing to its peak at 11mo and postpartum CA. The mRNA expression of PLAC8 was significantly upregulated in CA from mares with ascending and nocardioform placentitis compared to control mares. Immunohistochemistry revealed that PLAC8 is localized in equine chorionic epithelium and immune cells. Our results revealed that PLAC8 expression in equine chorion is dynamic during pregnancy and is regulated in an implantation-dependent manner. Moreover, PLAC8 is implicated in the immune response in CA during equine ascending placentitis and nocardioform placentitis.

Relationships between blood and follicular fluid urea nitrogen concentrations and between blood urea nitrogen and embryo survival in mares.

High blood urea nitrogen (BUN) concentration is linked to low fertility in cows and ewes; however, this relationship has not been reported in mares. The study characterized the relationship between BUN and follicular fluid urea nitrogen (FUN) during follicle growth (Experiment 1) and the impact of BUN from embryo donors on the pregnancy outcome of recipient mares (Experiment 2). In experiment one, follicular fluid and blood samples were collected from mares during diestrus with growing follicles and during estrus with pre-ovulatory follicles (n = 16 and 10 mares, respectively). In experiment two, BUN concentrations of embryo donors were related to pregnancy outcome after embryo transfer. In experiment one, there was a strong positive correlation between BUN and FUN (R = 0.83; P < 0.0001), with higher BUN in mares with growing follicles than with preovulatory follicles (P = 0.004) and higher FUN in growing follicles than in preovulatory follicles (P = 0.031). In experiment two, BUN was higher in donor mares that produced unsuccessful embryos compared to donor mares that produced embryos resulting in successful pregnancies at D14 (P < 0.03). Additionally, there was an effect of age (P = 0.01) and interaction between age and lactation (P = 0.009) in donor mares for embryo survival after embryo transfer. Donor mares with unsuccessful embryos were older than donor mares with successful embryos. Therefore, these experiments showed that BUN was related to follicular fluid environment as well as to the survival of Day 7-8 embryos after transfer to recipient mares.