Studies on early fetal loss in mares

Early pregnancy loss (EPL) is an important multifactorial condition that affects up to 20% of equine pregnancies, and mare age, parity, previous endometritis and twin pregnancy have all been implicated as causes of early pregnancy loss. The timing of the abortion strongly influences the outcome on the fertility. The regression of the endometrial cups, which form from trophoblastic cells by 35 days of gestation, is a self-regulated mechanism that is independent from fetal death, therefore mares that abort after the endometrial cup formation will present abnormalities in the ovulations, follicular growth and cyclicity due to the concurrent presence of equine chorionic gonadotropin (eCG). Early pregnancy loss has short- and long-term consequences to the horse industry, therefore, the development of novel diagnostic and management tools for early pregnancy loss are warranted. Blood markers and ultrasonographic parameters were assessed in mares undergoing experimentally induced early fetal loss. It was hypothesized that intrauterine infusion of cloprostenol results in earlier fetal compromise than systemic administration. Ovarian structures (number and sizes of follicles and corpora lutea area), fetal heartbeat, and fetal mobility of thirteen singleton pregnancies were assessed daily by transrectal ultrasonography until induction of pregnancy termination (60±2 days of gestation). Mares received 500g of cloprostenol intramuscularly every 12 hours (IM, n=7) or once transcervically (TC, n=6). After initial cloprostenol administration, ultrasonographic examinations were repeated at 6-hours-intervals until loss of fetal heartbeat. Plasma progesterone, estradiol-17β, and alpha-fetoprotein were assessed for five days before and after pregnancy loss. In addition, plasma PGFM concentrations were assessed immediately before cloprostenol administration (0 min), and then 15, 30, and 45 minutes, and 1, 2, 3, 4, 6, 12 hours after administration. Data were analyzed using the MIXED procedure with repeated measures in SAS. Significance was set at P<0.05. All mares lost their pregnancies within forty-eight hours after initial cloprostenol administration, with no difference in time to pregnancy loss. There were significant effects of time starting by 12 h post-induction of pregnancy termination but there was no time by group interaction for progesterone concentrations. Estradiol-17β and alpha-fetoprotein concentrations were not altered upon impending abortion. Concentrations of PGFM increased significantly by two hours after cloprostenol administration, but there were no differences between groups. No time effects or time by group interaction for fetal mobility and heartbeat was detected. Expectedly, the number and area of corpora lutea decreased significantly after cloprostenol administration with no significant differences between groups. In conclusion, intrauterine administration of cloprostenol was not different than repeated systemic administration to terminate the pregnancy. Both models for early fetal loss were equivalent for the endpoints assessed herein. Next, to explore the use of infusions of kerosene to enhance the regression of endometrial cups, thirteen mares had intrauterine kerosene infusions at 21- and 35-days post-abortion. Anecdotally, intrauterine infusion with kerosene has been proposed as a method to treat endometrial cup retention, however there are no controlled studies evaluating kerosene’s usefulness for enhancing endometrial cup regression following abortion. We hypothesized that intrauterine kerosene infusions would hasten regression of endometrial cups without detrimental effects on the endometrium and the mare’s general health. The objectives of this study were to assess the uterine response, systemic side effects and efficacy of intrauterine kerosene infusions to enhance regression of endometrial cups. Nine light-breed mares were enrolled in the study after an experimentally-induced abortion with cloprostenol (n=12) by 60 (602 days) days of gestation. Mares were randomly allocated an intrauterine infusion with 500mL of kerosene (Ker. n=6) or 500mL saline (Cont. n=6) on day 21 and 35 after the pregnancy termination. Uterine biopsies were collected at day 7, 21, 35 and 49, stained with H&E and graded according to the Kenney & Doig 1986 classification. Lymphocyte B CD20, lymphocyte T CD3 and macrophage IBA cell populations were characterized by immunohistochemistry in endometrial biopsies from a subset of mares (n=4/group). Physical examinations, complete blood cell counts, and serum biochemistry were performed before each infusion and then repeated for 2 days after each uterine infusion. Uterine lavage was performed 24 hours after each infusion. Serum samples were collected right before the abortion induction, then at 7, 21, 28, 35, 42, and 49 days after the pregnancy termination for assessment of serum eCG. Continuous data were analyzed with MIXED procedure with repeated measures in SAS, categorical data with LOGISTIC procedure in SAS. Significance was set at p<0.05. Kerosene infusion did not affect complete blood cell counts and serum chemistry parameters. There were no appreciable abnormalities on the physical examinations following kerosene infusions. Concentrations of eCG decreased over time (p<0.001), but there were no differences between groups or time by group interactions (p=0.7128). Histology samples from the uterus showed no signs of increased fibrosis or degeneration in the treatment group. In conclusion, while kerosene infusions did not appear to have detrimental effects on mare health, our findings suggest that the use of kerosene in the uterus does not enhance the regression of endometrial cups

Steroidogenic Enzyme and Steroid Receptor Expression in the Equine Accessory Sex Glands

The expression pattern and distribution of sex steroid receptors and steroidogenic enzymes during development of the equine accessory sex glands has not previously been described. We hypothesized that equine steroidogenic enzyme and sex steroid receptor expression is dependent on reproductive status. Accessory sex glands were harvested from mature stallions, pre-pubertal colts, geldings, and fetuses. Expression of mRNA for estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), androgen receptor (AR), 3β-Hydroxysteroid dehydrogenase/Δ5-4 isomerase (3βHSD), P450,17α hydroxylase, 17–20 lyase (CYP17), and aromatase (CYP19) were quantified by RT-PCR, and protein localization of AR, ER-α, ER-β, and 3βHSD were investigated by immunohistochemistry. Expression of AR, ESR2, CYP17, or CYP19 in the ampulla was not different across reproductive statuses (p \u3e 0.1), while expression of ESR1 was higher in the ampulla of geldings and fetuses than those of stallions or colts (p \u3c 0.05). AR, ESR1 and ESR2 expression were decreased in stallion vesicular glands compared to the fetus or gelding, while AR, ESR1, and CYP17 expression were decreased in the bulbourethral glands compared to other glands. ESR1 expression was increased in the prostate compared to the bulbourethral glands, and no differences were seen with CYP19 or 3β-HSD. In conclusion, sex steroid receptors are expressed in all equine male accessory sex glands in all stages of life, while the steroidogenic enzymes were weakly and variably expressed

Dynamic regulation of the transcriptome and proteome of the equine embryo during maternal recognition of pregnancy

During initial maternal recognition of pregnancy (MRP), the equine embryo displays a series of unique events characterized by rapid blastocyst expansion, secretion of a diverse array of molecules, and transuterine migration to interact with the uterine surface. Up to date, the intricate transcriptome and proteome changes of the embryo underlying these events have not been critically studied in horses. Thus, the objective of this study was to perform an integrative transcriptomic (including mRNA, miRNAs, and other small non-coding RNAs) and proteomic analysis of embryos collected from days 10 to 13 of gestation. The results revealed dynamic transcriptome profiles with a total of 1311 differentially expressed genes, including 18 microRNAs (miRNAs). Two main profiles for mRNAs and miRNAs were identified, one with higher expression in embryos ≤5 mm and the second with higher expression in embryos ≥7 mm. At the protein level, similar results were obtained, with 259 differentially abundant proteins between small and large embryos. Overall, the findings demonstrated fine-tuned transcriptomic and proteomic regulations in the developing embryo associated with embryo growth. The identification of specific regulation of mRNAs, proteins, and miRNAs on days 12 and 13 of gestation suggested these molecules as pivotal for embryo development and as involved in MRP, and in establishment of pregnancy in general. In addition, the results revealed new insights into prostaglandin synthesis by the equine embryo, miRNAs and genes potentially involved in modulation of the maternal immune response, regulation of endometrial receptivity and of late implantation in the mare

Uterine extracellular vesicles as multi-signal messengers during maternal recognition of pregnancy in the mare

In contrast to other domestic mammals, the embryo-derived signal(s) leading to maternal recognition of pregnancy (MRP) are still unknow in the mare. We hypothesize that these embryonic signals could be packed into uterine extracellular vesicles (uEVs), acting as multi-signal messengers between the conceptus and the maternal tract, and contributing to MRP. To unveil these signals, the RNA and protein cargos of uEVs isolated from uterine lavages collected from pregnant mares (P; day 10, 11, 12 and 13 after ovulation) and cyclic control mares (C; day 10 and 13 after ovulation) were analyzed. Our results showed a fine-tuned regulation of the uEV cargo (RNAs and proteins), by the day of pregnancy, the estrous cycle, and even the size of the embryo. A particular RNA pattern was identified with specific increase on P12 related to immune system and hormonal response. Besides, a set of proteins as well as RNAs was highly enriched in EVs on P12 and P13. Differential abundance of miRNAs was also identified in P13-derived uEVs. Their target genes were linked to down- or upregulated genes in the embryo and the endometrium, exposing their potential origin. Our study identified for first time specific molecules packed in uEVs, which were previously associated to MRP in the mare, and thus bringing added value to the current knowledge. Further integrative and functional analyses will help to confirm the role of these molecules in uEVs during MRP in the mare

Sexual differentiation and primordial germ cell distribution in the early horse fetus

It was the aim of this study to characterize the development of the gonads and genital ducts in the equine fetus around the time of sexual differentiation. This included the identification and localization of the primordial germ cell population. Equine fetuses between 45 and 60 days of gestation were evaluated using a combination of micro-computed tomography scanning, immunohistochemistry, and multiplex immunofluorescence. Fetal gonads increased in size 23-fold from 45 to 60 days of gestation, and an even greater increase was observed in the metanephros volume. Signs of mesonephros atrophy were detected during this time. Tubular structures of the fetal testes were present from day 50 onwards, whereas cell clusters dominated in the fetal ovary. The genital ducts were well-differentiated and presented a lumen in all samples. No sign of mesonephric or paramesonephric duct degeneration was detected. Expression of AMH was strong in the fetal testes but absent in ovaries. Irrespective of sex, primordial germ cells selectively expressed LIN28. Migration of primordial germ cells from the mesonephros to the gonad was detected at 45 days, but not at 60 days of development. Their number and distribution within the gonad were influenced (p < 0.05) by fetal sex. Most primordial germ cells (86.8 ± 3.2% in females and 84.6 ± 4.7% in males) were characterized as pluripotent according to co-localization with CD117. However, only a very small percentage of primordial germ cells were proliferating (7.5 ± 1.7% in females and 3.2 ± 1.2% in males) based on co-localization with Ki67. It can be concluded that gonadal sexual differentiation in the horse occurs asynchronously with regard to sex but already before 45 days of gestation.Fil: Scarlet, Dragos. University of Veterinary Medicine Vienna; Suiza. Universitat Zurich; SuizaFil: Handschuh, Stephan. University of Veterinary Medicine Vienna; SuizaFil: Reichart, Ursula. University of Veterinary Medicine Vienna; SuizaFil: Podico, Giorgia. University of Illinois. Urbana - Champaign; Estados UnidosFil: Ellerbrock, Robyn E.. University of Illinois. Urbana - Champaign; Estados UnidosFil: Demyda Peyrás, Sebastián. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Canisso, Igor F.. University of Illinois. Urbana - Champaign; Estados UnidosFil: Walter, Ingrid. University of Veterinary Medicine Vienna; SuizaFil: Aurich, Christine. University of Veterinary Medicine Vienna; Suiz

Intrauterine Blood Plasma Platelet-Therapy Mitigates Persistent Breeding-Induced Endometritis, Reduces Uterine Infections, and Improves Embryo Recovery in Mares

Microorganisms, including pathogenic or opportunistic bacteria and fungi, may gain access to the uterus during breeding, and infectious endometritis plays a major role in equine subfertility. This study aimed to assess the post-breeding inflammatory response, endometrial culture, and embryo recovery of mares susceptible to persistent breeding-induced endometritis (PBIE) treated with plasma-rich (PRP) or -poor (PPP) plasma. Mares (n = 12) susceptible to PBIE had three cycles randomly assigned to receive intrauterine infusions of lactate ringer solution (LRS, control), or autologous PRP or PPP pre- (−48 and −24 h) and post-breeding (6 and 24 h). Mares were bred with fresh semen from one stallion. Intrauterine fluid accumulation (IUF) and endometrial neutrophils were assessed every 24 h up to 96 h post-breeding. Uterine cytokines (Ilβ, IL6, CXCL8, and IL10) were evaluated before (0 h), 6, and 24 h post-breeding, and endometrial culture three and nine days after breed. Embryo flushing was performed 8 days post-ovulation. Data were analyzed with mixed model, Tukey’s post-hoc test, and multivariate regression. PRP treatment reduced endometrial neutrophils, post-breeding IUF, and pro-inflammatory cytokines when compared to control-assigned cycles, but not significantly different than PPP. Controls had a significantly higher percentage of positive bacterial cultures (33%) in comparison to PRP-assigned cycles (0%), whereas cycles treated with PPP were not significantly different from the other groups (25%). The PRP-assigned cycles had significantly greater embryo recovery rates (83%) than the control (33%), though not significantly different than PPP (60%). Plasma infusion reduced the duration and intensity of the post-breeding inflammatory response and improved embryo recovery in mares susceptible to PBIE. Platelets incrementally downregulate PBIE and appear to have a dose-dependent antimicrobial property

Retrograde Flushing Followed by Slicing Float-Up as an Approach to Optimize Epididymal Sperm Recovery for the Purpose of Cryopreservation in Equids

This study aimed to assess the parameters of epididymal sperm harvested by retrograde flushing (RF) followed by slicing float-up (SF). Epididymides from donkeys (n = 18) and horses (n = 28) were subjected to RF with a freezing extender and then SF technique. The retrieved sperm after RF and SF was evaluated for volume, concentration, and total sperm and then cryopreserved separately. Post-thaw total motility (TM) and progressive motility (PM) were evaluated with CASA. Sperm membrane integrity (SMI) and mitochondrial membrane potential (MMP) were assessed with flow cytometry. Sperm concentration was greater in donkeys than horses (684 &plusmn; 62.9 vs. 494 &plusmn; 50.9 million sperm/mL) (p = 0.02). The total sperm harvested was lower in SF (3.6 &plusmn; 0.7 billion) than RF (10.4 &plusmn; 1.5 billion) and in horses (4.6 &plusmn; 0.8 billion) than in donkeys (10.7 &plusmn; 1.8 billion) (p &lt; 0.05). RF followed by SF resulted in 57% and 31% more sperm per harvest in donkeys and horses. Results of TM and PM before freezing were not affected by technique or species (p &gt; 0.05). Post-thawing SMI and MMP did not vary with technique or species (p &gt; 0.05); TM and PM were not influenced by the technique or the species (p &gt; 0.05) but by their interaction (p = 0.005). In conclusion, using RF followed by SF enhances sperm recovery without affecting cryopreservation in equids

Luteal Tissue Area and Immunoreactive Concentration of Progesterone in Plasma of Bred and Non-bred Mares

Progesterone is pivotal to maintain pregnancy in the first trimester and low concentration ( .05). Multiple ovulations were associated with greater progesterone concentration and luteal tissue area (P = .0001). There was a moderate positive association between the number of ovulations and luteal tissue area (r = 0.54; P = .0001). The lack of change in the progesterone concentration and luteal tissue area between bred and non-bred mares suggests that horse seminal plasma does not affect luteal function in mares. As all mares had progesterone above 4 ng/mL after 5-days post-ovulation; it is possible that if mares with abnormal progesterone concentration were used, the results could have been different. In conclusion, pregnancy was not associated with greater progesterone concentration or luteal tissue area

Sexual Differentiation and Primordial Germ Cell Distribution in the Early Horse Fetus

It was the aim of this study to characterize the development of the gonads and genital ducts in the equine fetus around the time of sexual differentiation. This included the identification and localization of the primordial germ cell population. Equine fetuses between 45 and 60 days of gestation were evaluated using a combination of micro-computed tomography scanning, immunohistochemistry, and multiplex immunofluorescence. Fetal gonads increased in size 23-fold from 45 to 60 days of gestation, and an even greater increase was observed in the metanephros volume. Signs of mesonephros atrophy were detected during this time. Tubular structures of the fetal testes were present from day 50 onwards, whereas cell clusters dominated in the fetal ovary. The genital ducts were well-differentiated and presented a lumen in all samples. No sign of mesonephric or paramesonephric duct degeneration was detected. Expression of AMH was strong in the fetal testes but absent in ovaries. Irrespective of sex, primordial germ cells selectively expressed LIN28. Migration of primordial germ cells from the mesonephros to the gonad was detected at 45 days, but not at 60 days of development. Their number and distribution within the gonad were influenced (p < 0.05) by fetal sex. Most primordial germ cells (86.8 ± 3.2% in females and 84.6 ± 4.7% in males) were characterized as pluripotent according to co-localization with CD117. However, only a very small percentage of primordial germ cells were proliferating (7.5 ± 1.7% in females and 3.2 ± 1.2% in males) based on co-localization with Ki67. It can be concluded that gonadal sexual differentiation in the horse occurs asynchronously with regard to sex but already before 45 days of gestation

Spatiotemporal endometrial transcriptome analysis revealed the luminal epithelium as key player during initial maternal recognition of pregnancy in the mare

During the period of maternal recognition of pregnancy (MRP) in the mare, the embryo needs to signal its presence to the endometrium to prevent regression of the corpus luteum and prepare for establishment of pregnancy. This is achieved by mechanical stimuli and release of various signaling molecules by the equine embryo while migrating through the uterus. We hypothesized that embryo's signals induce changes in the endometrial gene expression in a highly cell type-specific manner. A spatiotemporal transcriptomics approach was applied combining laser capture microdissection and low-input-RNA sequencing of luminal and glandular epithelium (LE, GE), and stroma of biopsy samples collected from days 10-13 of pregnancy and the estrous cycle. Two comparisons were performed, samples derived from pregnancies with conceptuses ≥ 8 mm in diameter (comparison 1) and conceptuses ≤ 8 mm (comparison 2) versus samples from cyclic controls. The majority of gene expression changes was identified in LE and much lower numbers of differentially expressed genes (DEGs) in GE and stroma. While 1253 DEGs were found for LE in comparison 1, only 248 were found in comparison 2. Data mining mainly focused on DEGs in LE and revealed regulation of genes related to prostaglandin transport, metabolism, and signaling, as well as transcription factor families that could be involved in MRP. In comparison to other mammalian species, differences in regulation of genes involved in epithelial barrier formation and conceptus attachment and implantation reflected the unique features of equine reproduction at the time of MRP at the molecular level