Early Embryonic Loss Research Articles

Effect of post-mating administration of ketoprofen on serum progesterone concentration and fertility in Akkaraman ewes.

A major cause of early embryonic losses is inadequate secretion of progesterone (P4) hormone due to luteal insufficiency in farm animals. Post-mating applications that directly or indirectly increasing serum P4 concentrations have a positive effect on fertility. The aim of this study was to investigate the effect of post-mating administration of ketoprofen on serum P4 concentration and fertility in Akkaraman ewes synchronized with a short-term protocol during the breeding season. Oestrus monitoring ewes after synchronization were hand-mated and randomly assigned to two equal groups (Ketoprofen vs. Control). Ewes in the ketoprofen group (KPG) (n = 40) were administered with ketoprofen (Rifen, Richter pharma, Austria) intramuscularly (im) at a dose of 3mg/kg on days 9 and 10 after mating. In the control group (CG) ewes (n = 40) were administered with saline im on the same days. Blood samples were collected from ewes in both groups at four different time points of post-mating days (9, 12, 15 and 18 days). The results showed that there were no statistical differences between the KPG and CG groups on fertility parameters; pregnancy rates (85% vs. 72.5%), lambing rates (100% vs. 100%), single birth rates (55.9% vs. 55.2%), multiple birth rates (44.1% vs. 44.8%), litter sizes (1.56 vs. 1.55). In pregnant ewes, serum P4 concentrations on day 18 (4.35 ± 0.34 ng/mL) in the KPG group were higher than (3.27 ± 0.27 ng/mL) in CG group (P < 0.05). It was concluded that post-mating ketoprofen administration have no significant effect on fertility, but significantly increased the serum P4 concentration on day 18 in pregnant ewes.

Elavl1 is dispensable for appendicular skeletal development.

Elavl1/HuR is a RNA binding protein implicated in multiple developmental processes with pleiotropic roles in RNA life cycle. Loss of Elavl1 is incompatible with life with early embryonic loss of Elavl1 in epiblast cells being lethal with defects in placental branching and embryonic tissue growth. Postnatal global deletion of Elavl1/HuR results in lethality with atrophy in multiple tissues mainly due to loss of progenitor cells. However, roles of Elavl1 specifically during embryonic limb development is not well understood. Here we report that deletion of Elavl1 in limb bud mesenchyme in mouse did not reveal any abnormalities during embryonic development with normal development in pre- and postnatal limb skeleton. Analyses of skeletal patterning, morphogenesis and skeletal maturation including skeletal elements in stylopod, zeugopod and autopod during development did not reveal any significant differences between long bones from control and Elavl1 conditional knockout animals. Our study indicates differential dependency and susceptibility to loss of Elavl1 in different stem cell lineages with its functions being dispensable during limb development.

391 The impact of trace mineral source on systemic immunological status associated with early embryonic loss in lactating dairy cattle

Abstract Milk production has doubled from 1970 to 2009 but has been associated with a decline in fertility. Around 25% of failed pregnancies in dairy cattle are due to embryonic loss. Mineral bioavailability may affect embryonic retention, so we hypothesized that Zn, Cu, and Mn sources will influence systemic immunological status, which affects early embryonic retention. Holstein cows (n = 58) were randomly assigned to one of two daily oral drench trace mineral (Zn, Cu, and Mn) treatments 30 d before breeding (d -30): 1) Inorganic (ITM) or 2) Metal Methionine Hydroxy Analog Chelate (MMHAC). Before treatments began, cows were assigned to a semen type, beef vs. Holstein, equally, across treatments. All cows underwent a double Ovsynch protocol and were artificially inseminated on d 0. Humoral immunological status was assessed in serum samples taken on d 0 and d 7. Immune activity was quantified via a D2Dx systemic immunity score, which assesses samples for the presence of IgG, IgM, cytokines, and complement proteins. Ultrasonography was utilized for pregnancy confirmation on d 35, classifying cows as pregnant (P) or open. Progesterone analysis from d 21 plasma was used to categorize open cows as either non-pregnant (NP) or early embryonic loss (EEL). Statistical analyses in R Studio were completed, in two models based on pregnancy (P vs EEL and NP vs EEL), to determine the effects of mineral treatment, pregnancy status, semen type, and all interactions on humoral immune status. In the P vs EEL model, there were no effects (P &amp;gt; 0.30) of semen type, mineral treatment, pregnancy status, or any interactions on systemic immunity score on the day of breeding (d 0) or d 7. On d 0, cows provided MMHAC had a systemic immunity score of 0.073 ± 0.008 but were not different (P &amp;gt; 0.34) from ITM cows (0.063 ± 0.006). This trend continued on d 7 with systemic immunity scores of MMHAC cows not differing (P &amp;gt; 0.32) from ITM cows (0.055 ± 0.004 and 0.061 ± 0.003; respectively). There was a tendency (P = 0.10) for the three-way interaction of semen type x pregnancy x treatment to influence the immunity score change from d 0 to d 7. Pregnant cows on ITM treatment bred with beef semen had a positive change in immunity score (0.020 ± 0.0085). Whereas ITM cows bred with Holstein semen that underwent EEL had a negative change (-0.015 ± 0.0085); all other means were intermediate. In the NP vs EEL model, there were no effects (P &amp;gt; 0.14) of semen type, treatment, pregnancy status, or any interactions on systemic immunity score on d 0, d 7, or the change between d 0 and d 7. In summary, trace mineral bioavailability tended to affect systemic immunological status in P vs EEL cows but not in NP vs EEL cows, which may allow for the elucidation of immunological status differences during pregnancy establishment.

Molecular detection of Chlamydia abortus in endometrial biopsies of mares from western Canada.

Chlamydia abortus is a reported cause of infertility and endometritis in sheep, cattle, and pigs; however, the association between uterine disease and C. abortus is poorly understood in horses. Recently, a high prevalence of C. abortus in equine aborted chorioallantoises was reported in horses in western Canada. Based on this high prevalence, investigation into the effects of C. abortus on infertility and endometritis in western Canadian mares is prudent. We examined 98 formalin-fixed, paraffin-embedded endometrial biopsies from western Canada submitted between 2014 and 2022 using a Chlamydia-specific 16S rRNA PCR test; 40 samples tested positive for Chlamydia on PCR, and 28 were sequenced as C. abortus. The C. abortus-positive cases were primarily associated with a history of failure to conceive, early embryonic loss, or abortion. Our findings suggest that C. abortus may be a cause of conception failure and abortion in horses in western Canada.

Mouse developmental defects, but not paraganglioma tumorigenesis, upon conditional Complex II loss in early Sox10+ cells.

In humans, loss of heterozygosity for defective alleles of any of the four subunits of mitochondrial tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH, also Complex II of the electron transport chain) can lead to paraganglioma tumors in neuroendocrine cells. With the goal of developing mouse models of this rare disorder, we have developed various SDH conditional loss strategies. Based on recent lineage tracing studies, we hypothesized that conditional SDHC loss in early embryogenesis during migration of primordial neural crest cells that form the susceptible chromaffin cells of the adrenal medulla might induce paraganglioma. We triggered low levels of detectable SDHC loss in Sox10+ cells at E11.5 of mouse development. We report that, rather than developing adrenal medulla paraganglioma (pheochromocytoma), offspring survived with evidence of neural crest cell dysfunction. Phenotypes included mild lower extremity gait anomalies suggestive of neural tube closure defects and patches of unpigmented fur consistent with neural crest-derived melanocyte dysfunction. These defects were not observed in mice lacking Sdhc knockout. Our results add to existing data suggesting that, unlike humans, even early embryonic (Sox10-driven) SDHx loss is inadequate to trigger paraganglioma in mice of the genetic backgrounds that have been investigated. Instead, low levels of tricarboxylic acid cycle-deficient neural crest cells cause mild developmental defects in hind limb and melanocyte function. This new model may be of interest for studies of metabolism during early neural crest cell development.

Imputation accuracy and carrier frequency of deleterious recessive defects in Australian dairy cattle

Widespread genotyping has enabled the identification of putative recessive mutations that affect fertility through early embryonic fetal loss, or that compromise neonate or calf viability. The use of artificial insemination in the global dairy population can rapidly spread these harmful mutations, and testing for multiple mutations can become relatively expensive if not all tests are available on the same SNP panel. However, it is possible to provide heifer and cow predicted carrier status to farmers at no additional cost if the animals are genotyped with a standard SNP panel. Additionally, for defects where the causal mutation is unknown but a haplotype of markers has been associated with the defect, the carrier status can be predicted based on that haplotype. The aims of this study were 3-fold: (1) to determine the accuracy of imputation of putative causal mutations for recessive deleterious conditions in Australian dairy cattle, (2) to impute carrier status for known recessive deleterious conditions in all genotyped Australian Holstein, Jersey, and Red breed cows, and (3) to determine the changes in carrier frequencies across time for these recessive deleterious mutations. We used the F1 statistic, combining precision and recall, to assess the accuracy of carrier status prediction. We showed that known deleterious mutations can be accurately imputed in Australian Holstein and Jersey cattle that are not directly genotyped for the causal mutation, with F1 ranging between 0.88 and 0.99. For recessive deleterious conditions not included on the standard Australian SNP panel, carrier status could be predicted using a marker haplotype, with F1 ranging from 0.91 to 0.92. Most putative causals and haplotypes were either stable with a low carrier percentage or had a declining carrier percentage. However, several recessive mutations showed a relatively high or increasing percentage, highlighting the importance of detecting carriers to reduce the number of at-risk matings. Furthermore, the high carrier percentage of the recently identified bovine lymphocyte intestinal retention defect mutation emphasizes the importance of detection of novel mutations.

Microwave Dielectric Response of Bovine Milk as Pregnancy Detection Tool in Dairy Cows.

The most reliable methods for pregnancy diagnosis in dairy herds include rectal palpation, ultrasound examination, and evaluation of plasma progesterone concentrations. However, these methods are expensive, labor-intensive, and invasive. Thus, there is a need to develop a practical, non-invasive, cost-effective method that can be implemented on the farm to detect pregnancy. This study suggests employing microwave dielectric spectroscopy (MDS, 0.5-40 GHz) as a method to evaluate reproduction events in dairy cows. The approach involves the integration of MDS data with information on milk solids to detect pregnancy and identify early embryonic loss in dairy cows. To test the ability to predict pregnancy according to these measurements, milk samples were collected from (i) pregnant and non-pregnant randomly selected cows, (ii) weekly from selected cows (n = 12) before insemination until a positive pregnancy test, and (iii) daily from selected cows (n = 10) prior to insemination until a positive pregnancy test. The results indicated that the dielectric strength of Δε and the relaxation time, τ, exhibited reduced variability in the case of a positive pregnancy diagnosis. Using principal component analysis (PCA), a clear distinction between pregnancy and nonpregnancy status was observed, with improved differentiation upon a higher sampling frequency. Additionally, a neural network machine learning technique was employed to develop a prediction algorithm with an accuracy of 73%. These findings demonstrate that MDS can be used to detect changes in milk upon pregnancy. The developed machine learning provides a broad classification that could be further enhanced with additional data.

Effects of Mixed Pasture Legume Phytoestrogens on Superovulatory Response and Embryo Quality in Angus Cows.

Ovulation and artificial insemination rates have been observed to decrease in sheep and cows when exposed to dietary phytoestrogens at concentrations greater than 25 mg/kg DM. A grazing trial was undertaken to investigate the effects of coumestrol and other key phytoestrogens on the superovulatory response, embryo numbers and quality in beef cows grazing legume pastures. A 7-week controlled grazing trial was conducted with legume and ryegrass pasture treatments, with cows exposed to legumes at two timed treatments, 4 and 7 weeks. Twenty Angus cows were subjected to a conventional estrus synchronization and superovulation protocol. Embryos were recovered via conventional uterine body flushing 7 days post artificial insemination (AI). Numerous phytoestrogens were identified in both pasture and plasma samples, including coumestrol and formononetin. Concentrations of phytoestrogens in the pasture ranged from 0.001 to 47.5 mg/kg DM and 0 to 2.6 ng/mL in plasma. Approximately 50% of cows produced viable embryos 7 days post AI. A significant interaction between the effect of treatment groups on the embryo stage was observed (p < 0.05). The results suggest that concentrations of >25 mg/kg DM of phytoestrogens less than 20 days preceding AI may negatively affect oocyte developmental competence, reduce progesterone production and thus contribute to early embryonic loss.

MUC1 regulation in the left and right uterine horns and conceptus trophectoderm during the peri-implantation period of dromedary camel

Pregnancy maintenance in dromedary camels poses significant challenges, including early embryonic loss in the left uterine horn (LH) and unsuccessful pregnancy in the right uterine horn (RH), suggesting a potential asynchrony between conceptus signaling and uterine receptivity. The transition of the uterine epithelium from a pre-receptive to a receptive state requires a delicate balance of adhesion-promoting and anti-adhesion molecules. Mucin-1 (MUC1) acts as an anti-adhesive molecule on the uterine luminal (LE) and glandular (GE) epithelium. Downregulation of MUC1 is believed to be crucial for successful embryo attachment in various mammals. This study aimed to investigate the temporospatial expression of MUC1 in the LH and RH on Days 8, 10, and 12 pregnant dromedaries and their conceptuses. Quantitative real-time polymerase chain reaction (qrt-PCR), Western blot analysis, immunohistochemistry, and immunofluorescence techniques were employed to assess MUC1 expression at the mRNA and protein levels. The results demonstrated a reduction in MUC1 mRNA expression on Day 8, then increased on Day 10, followed by a decrease on Day 12 in LH. While the RH exhibited progressive increases, peaking on Day 12. However, MUC1 expression constantly exhibited higher levels in RH than in LH in all days. Two bands were detected at 150-kDa and 180-kDa, with the highest intensity observed on Day 10. Spatially, MUC1 was localized in the apical, cytoplasmic, and lumen of uterine glands only. MUC1 was barely detectable on Day 8 but gradually increased on Days 10 and 12 in both horns. Likewise, the RH exhibited higher MUC1 signals than the LH on Days 10 and 12. In the conceptuses, MUC1 mRNA increased on Day 8, peaked on Day 10, and declined on Day 12. Notably, MUC1 protein was detected in both the trophectoderm and endoderm, with high expression observed on Day 10 and reduced by Day 12. In conclusion, the decrease in MUC1 expression on Day 8 in the LH may be associated with maternal recognition of pregnancy (MRP), and the increase on Day 10 may related to embryo protection and movement, while the subsequent decrease on Day 12 could be linked to the embryo attachment and preparation for the implantation. Conversely, the increase of MUC1 in the RH implies a role in the anti-adhesion mechanism. These findings contribute to understanding MUC1's involvement in reproductive processes and provide insights into the complex mechanisms underlying successful pregnancy establishment and maintenance in dromedary camels.

From the laboratory to the field: how to mitigate pregnancy losses in embryo transfer programs?

Pregnancy losses negatively affect the cattle industry, impacting economic indices and consequently the entire production chain. Early embryonic failure has been an important challenge in the embryo industry because proper identification of embryo death at the beginning of gestation is difficult. This review aimed to provide a better understanding on reproductive failure and the relationship between early embryonic loss and different reproductive biotechniques. This review also considers insights and possible strategies for reducing early embryonic loss. The strategies addressed are as follows: i) great impact of rigorous embryo evaluation on reducing embryo losses; ii) selection of recipients at the time of transfer, taking into account health and nutritional status, and classification of the corpus luteum using ultrasound, either in area or vascularization; and iii) paternal effect as one of the factors that contribute to pregnancy losses, with a focus on embryo transfer.

Reproductive performance of a cohort of Standardbred mares under a commercial breeding system.

Despite being a large commercial breeding industry, there is little published data on the reproductive success of Standardbred mares. To quantify the reproductive performance of Standardbred mares under artificial breeding systems in a commercial setting and determine the incidence of early embryonic and other pre-partum losses. Retrospective cohort study. Data from four commercial farms were collected across four breeding years, and all mares were bred via artificial insemination. A total of 3995 mares contributed 7229 mare years. First-cycle pregnancy rate (FCPR) and end of season pregnancy rate (SPR) were analysed in mixed-effects logistic regression models. Time-to-conception interval was analysed in a Cox regression model. The mean FCPR was 61.4% (confidence interval [CI] 60.3%-62.6%), the mean end of SPR was 84.7% (CI 83.8-85.5%), the mean live foal rate (FR) was 73.1% (CI 72.1%-74.2%). Mares located on-farm were more probable to be pregnant in terms of both FCPR (odds ratio [OR] 1.168, CI 1.018-1.340, p = 0.026) and SPR (OR 2.026, CI 1.673-2.454, p < 0.001), mares inseminated with thawed-frozen semen were less probable to be pregnant in terms of FCPR (OR 0.598, CI 0.457-0.783, p < 0.001) and SPR (OR 0.479, CI 0.354-0.647, p < 0.001) compared with insemination with fresh-extended semen. Older mares (14 years and older) were less probable to be pregnant in terms of FCPR (OR 0.795, CI 0.688-0.919, p = 0.002) and SPR (0.435, CI 0.352-0.538, p < 0.001) compared with young mares (3- to 8-year old). Retrospective data relied on accurate record keeping of stud farms and no mare-treatment or ovulation induction records were available. Live FRs relied mostly on annual foaling returns so fetal/foal deaths may be underrepresented. This study provides substantial baseline data on reproductive performance for Standardbred mares managed under a commercial artificial breeding system.

Cathepsin L regulates oocyte meiosis and preimplantation embryo development.

Early embryonic loss, caused by reduced embryo developmental competence, is the major cause of subfertility in humans and animals. This embryo developmental competence is determined during oocyte maturation and the first embryo divisions. Therefore, it is essential to identify the underlying molecules regulating these critical developmental stages. Cathepsin L (CTSL), a lysosomal cysteine protease, is involved in regulating cell cycle progression, proliferation and invasion of different cell types. However, CTSL role in mammalian embryo development is unknown. Using bovine in vitro maturation and culture systems, we show that CTSL is a key regulator for embryo developmental competence. We employed a specific CTSL detection assay in live cells to show that CTSL activity correlates with meiotic progression and early embryo development. Inhibiting CTSL activity during oocyte maturation or early embryo development significantly impaired oocyte and embryo developmental competence as evidenced by lower cleavage, blastocyst and hatched blastocyst rates. Moreover, enhancing CTSL activity, using recombinant CTSL (rCTSL), during oocyte maturation or early embryo development significantly improved oocyte and embryo developmental competence. Importantly, rCTSL supplementation during oocyte maturation and early embryo development significantly improved the developmental competence of heat-shocked oocytes/embryos which are notoriously known for reduced quality. Altogether, these results provide novel evidence that CTSL plays a pivotal role in regulating oocyte meiosis and early embryonic development.

Pregnancy monitoring in mares: Ultrasonographic and endocrine approaches.

Methods to diagnose and monitor equine pregnancy continue to advance with improved instrumentation enabling the development of novel, non-invasive approaches to assess fetal well-being and viability using ultrasound and endocrine testing. From early embryonic loss to placentitis, that is typically encountered later in gestation, fetal viability and development as well as placental function can be evaluated using two fundamentally different, structural and functional, approaches. Ultrasound provides structural information on embryonic and fetal growth using such parameters as combined thickness of the uterus and placenta (CTUP), visual assessment of fetal fluids, activity, heart rate and multiple biometrics involving the fetal head and eyes, limbs and joints among many others, depending on the stage of gestation. Endocrine profiles that include progesterone and 5α-dihydroprogesterone, other metabolites, androgens and estrogens can be evaluated simultaneously using liquid chromatography-tandem mass spectrometry (LC-MS/MS) providing more functional information on fetal and placental competence and development. Endocrine information can be used in making clinical decisions including the need for progestin supplementation or when it can cease, and even estimating gestational stage in mares that cannot be easily palpated or scanned, as with mini-breeds or rancorous animals most notably. When used together, monitoring gestation by ultrasound and hormonal analysis provides unusual insight into feto-placental well-being and the progress of pregnancy, helping to identify problems needing therapeutic intervention.

Trophoblast PR-SET7 dysfunction induces viral mimicry response and necroptosis associated with recurrent miscarriage

Recurrent miscarriage (RM) is a distressing pregnancy complication. While the etiology of RM remains unclear, growing evidence has indicated the relevance of trophoblast impairment to the pathogenesis of RM. PR-SET7 is the sole enzyme catalyzing monomethylation of H4K20 (H4K20me1) and has been implicated in many pathophysiological processes. However, how PR-SET7 functions in trophoblasts and its relevance to RM remain unknown. Here, we found that trophoblast-specific loss of Pr-set7 in mice led to defective trophoblasts, resulting in early embryonic loss. Mechanistic analysis revealed that PR-SET7 deficiency in trophoblasts derepressed endogenous retroviruses (ERVs), leading to double-stranded RNA stress and subsequent viral mimicry, which drove overwhelming interferon response and necroptosis. Further examination discovered that H4K20me1 and H4K20me3 mediated the inhibition of cell-intrinsic expression of ERVs. Importantly, dysregulation of PR-SET7 expression and the corresponding aberrant epigenetic modifications were observed in the placentas of RM. Collectively, our results demonstrate that PR-SET7 acts as an epigenetic transcriptional modulator essential for repressing ERVs in trophoblasts, ensuring normal pregnancy and fetal survival, which sheds new light on potential epigenetic causes contributing to RM.

Global gene expression in equine endometrium around the time of maternal recognition of pregnancy

The conceptus-maternal communication during the preimplantation period, also known as maternal recognition of pregnancy (MRP), is crucial for the establishment and maintenance of pregnancy. This communication affects the endometrial transcriptome and proteome with subsequent reduction of endometrial pulsatile prostaglandin F2α secretion and the consequent extension of the corpus luteum's lifespan during early pregnancy in the mare. Also, this communication results in the preparation of the uterine environment for implantation. Any alteration of these processes will result in early embryonic death. Therefore, a better understanding of the normal endometrial transcriptomic changes around the time of MRP might be the key to understanding and preventing early embryonic loss. This study aimed to evaluate the endometrial transcriptome from Day 14 (Day 0 = ovulation) of pregnant and non-pregnant (i.e. diestrus) mares. RNA was isolated from pregnant (n=5) and non-pregnant (n=4) endometrial tissue samples. RNA-Seq was performed using Illumina NovaSeq6000 and differentially expressed genes (DEGs) were evaluated using DESeq.2 based upon an FDR<0.05. Our study identified 1193 DEGs (570 upregulated DEGs and 623 downregulated DEGs) in the pregnant endometrium as compared to the non-pregnant endometrium. In the current study, the pregnant endometrium was associated with downregulation (9.6-fold change) of mRNA forprostaglandin-endoperoxide synthase 2 (PTGS2, also known as cyclooxygenase-2 enzyme; COX2), which encodes for the rate-limiting enzyme in prostaglandin production. Gene ontology analysis of DEGs revealed that these genes were associated with relevant biological processes such as immune response (TNFAIP1, TNF, CXCR2, TNFSF15, IL15, TGFBR3, IL1B, TNFSF9, TLR8), regulation of trophoblast cell proliferation and migration (BOK, LIF, RBM46, TIMP1, CALR, GJA1), embryo growth and development (PTCH1, PTCH2R, DH10, GLI3, SHOX2, ZDBF2, EVX1, GREM2, KLF2, TWIST1), cell adhesion (COL28A1, EGFL6, ITGA3, FN1, NEDD9, LAMC2, RGMB, THBS4, RHOB, SELP, CHL1, CDHR1, MXRA8, FOLR3, NECTIN3), embryo implantation (MMP9 and LIF), placenta development (ADAM19, MAPK14, BIRC3), amino acids transportation (SLC1A1, SLC25A29, SLC36A2, SLC38A2, SLC7A5), steroid biosynthesis and metabolism (SRD5A1, HSD11B2, HSD17B7, CYP20A1), and progesterone receptors (PAQR5, and PGRMC1). Several of the DEGs identified in the current study are known to play a role in controlling endometrial receptivity to the conceptus in species other than the equine. These findings improve our understanding of the molecular changes taking place in equine endometrium around the time of MRP which might help us to understand and forestall early embryonic loss in the future.

Embryo transfer technique affects pregnancy rates in recipient mares

Embryo transfer in horses started in the 1970′s and has evolved since then in several technical and biological areas that may influence the overall outcome. The transfer method evolved from surgical to non-surgical (NSET), with pregnancy rates for NSET related closely to operator skills. Recently, high pregnancy rates have been reported by non-specialized operators using the cervical forceps method (Cuervo Arango et al., Veterinary Record. 2018,183;10,1-5). The aim of this retrospective study was to compare pregnancy and early embryonic loss rates between conventional and cervical forceps transfer techniques (Wilsher and Allen, Equine Vet. Educ. 2004. 16; 1, 39-44), operated by two veterinarians with different degrees of experience. During the last 12 breeding seasons in a commercial equine embryo transfer program in Chile, 345 embryos were transferred by two operators with low / moderate (n=86) or high experience (n=259), using a conventional manual technique and 228 embryos were transferred by the same two operators (n=81) and (n=147) respectively, using a bovine cervix forceps/Polansky speculum technique. Embryo flushes were performed at 8 or 9 days after ovulation on 102 donor mares aged 4 to 24 years old and transferred to recipient mares (n=387) aged 3 to 12 years old. Ovulation synchrony ranged from -1 to +4 days when comparing the recipient to the donor. No medications were used during embryo transfer and procedures were done at the samefacilities. Data were analyzed by a binary logistic regression model in which pregnancy and embryo loss were treated as dependent variables, and the experience of the operator and the ET technique as independent variables. Pregnancy rates at first diagnosis (12 to 14 days post ovulation) and embryo loss rate until 60 days of gestation were compared between and within operator groups. Overall pregnancy rates were higher (P<0.001) by the cervical forceps / speculum embryo transfer technique (195/228; 85%) than with the conventional technique (236/345; 65%) in both groups combined. Early embryonic loss rate did not differ (P>0.1) between the groups (6.0 vs. 4.6%, respectively). For the conventional technique, there was a significant operator effect (P=0.015) on the pregnancy rates at the first diagnosis (57 vs. 72%); no operator effect (P>0.1) was observed when using cervical forceps (84 vs. 86 %). These results agree with the report of Cuervo-Arango et al. (2018). We also confirmed that simple bovine cervical forceps work as well as the equine specific forceps reported by Wilsher and Allen (2004). In conclusion, the cervical forceps technique yielded higher pregnancy rates, with no effect of the operator's experience compared to the conventional technique in which the skill and experience of the operator is crucial and needs time and losses to achieve.

In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos.

In vitro production (IVP) of equine embryos is increasingly popular in clinical practice but suffers from higher incidences of early embryonic loss and monozygotic twin development than transfer of in vivo derived (IVD) embryos. Early embryo development is classically characterized by two cell fate decisions: (1) first, trophectoderm (TE) cells differentiate from inner cell mass (ICM); (2) second, the ICM segregates into epiblast (EPI) and primitive endoderm (PE). This study examined the influence of embryo type (IVD versus IVP), developmental stage or speed, and culture environment (in vitro versus in vivo) on the expression of the cell lineage markers, CDX-2 (TE), SOX-2 (EPI) and GATA-6 (PE). The numbers and distribution of cells expressing the three lineage markers were evaluated in day 7 IVD early blastocysts (n = 3) and blastocysts (n = 3), and in IVP embryos first identified as blastocysts after 7 (fast development, n = 5) or 9 (slow development, n = 9) days. Furthermore, day 7 IVP blastocysts were examined after additional culture for 2 days either in vitro (n = 5) or in vivo (after transfer into recipient mares, n = 3). In IVD early blastocysts, SOX-2 positive cells were encircled by GATA-6 positive cells in the ICM, with SOX-2 co-expression in some presumed PE cells. In IVD blastocysts, SOX-2 expression was exclusive to the compacted presumptive EPI, while GATA-6 and CDX-2 expression were consistent with PE and TE specification, respectively. In IVP blastocysts, SOX-2 and GATA-6 positive cells were intermingled and relatively dispersed, and co-expression of SOX-2 or GATA-6 was evident in some CDX-2 positive TE cells. IVP blastocysts had lower TE and total cell numbers than IVD blastocysts and displayed larger mean inter-EPI cell distances; these features were more pronounced in slower-developing IVP blastocysts. Transferring IVP blastocysts into recipient mares led to the compaction of SOX-2 positive cells into a presumptive EPI, whereas extended in vitro culture did not. In conclusion, IVP equine embryos have a poorly compacted ICM with intermingled EPI and PE cells; features accentuated in slowly developing embryos but remedied by transfer to a recipient mare.

Microdeletions on chromosomes 2 and 20 are associated with equine early pregnancy loss

Copy number variants (CNVs) are chromosomal microdeletions and microduplications greater than 1000 bp and can confer disease risk, if they impact gene dosage. Specific pathogenic CNVs have been described as associated with miscarriage and stillbirth in women but are unexplored in equine pregnancy. This study aimed to identify and characterize pathogenic CNVs associated with equine early embryonic loss (EEL) and early fetal losses collectively referred to as early pregnancy loss (EPL). Conceptuses were submitted to the laboratory by the attending veterinarians following clinical confirmation of pregnancy failure between gestation days 15 and 65. DNA was isolated from the allantochorion (ALC) (n=52) and fetus (n=13) of EPL pregnancies. As a control, DNA was isolated from allantochorion and fetuses of manually terminated clinically normal pregnancies (CNP n=20), and peripheral blood mononuclear cells (PBMCs) were isolated from adult horses (n=371). DNA was hybridized to 96 sample Axiom Equine 670K Arrays. DNAcopy was used to identify autosomal CNVs >1000 bp in length after excluding samples with <95% call rate, MAPD <0.35, log2 intensity between -0.2 and +0.2, and a significance of p>0.05. Two microdeletions were found in at least two EPL conceptuses that were absent in all control samples. A review of the literature confirmed both microdeletions were unreported to date (n=3040 individuals). In this study, PCR confirmed the deletions(del2:91608493-91610830 and del20:30652320-30656312). Bioinformatic analysis of del2:91608493-91610830 using ENSEMBL revealed that the 2.3 kb deletion region contained part of exon1 and intron1 of the mastermind like transcriptional coactivator 3 (MAML3) gene, a co-activator of Notch signaling known to play a key role in early development. This deletion in MAML3 was identified in the ALC of two EPLs lost at 42 and 64 days of gestation. Del20:30652320-30656312 was identified in seven EPLs, including both the fetal and ALC genome of two EPLs and only the fetal genome of an additional five EPLs. Bioinformatic analysis revealed the deletion in ECA20 encompassed a 4 kb region that included exon1 and partial intron1 of both transcripts for tubulin beta class 1 (TUBB), which is essential to neurogenesis. Five of the seven EPLs failed at day 35-40, and three of the fetuses had abnormalities of the central nervous system, including one failure of neural tube closure. Whilst causality can't be confirmed from these experiments, the rarity of microdeletions in viable horses, and the biological plausibility of the deleted gene impacting early development is supportive of a role for both microdeletions in lethality of the conceptus in the first two months of pregnancy. Further experiments are required to determine if the deletions are acquired through the germline.

Influence of Intrauterine Fluid Detection, Number of Transfers and Age of the Recipient on Pregnancy Rate and Early Embryonic Loss in a Commercial Embryo Transfer Program.

The selection of the recipient mare is one of the most important factors involved in the success of equine embryo transfer. The aim of this study was to determine whether the age of the recipient, the number of transfers and the detection of intrauterine fluid during the follicular phase or after ovulation can affect pregnancy rate at 14 and 45 days (PR 14 and PR 45) or early embryonic loss (EEL). A total of 1222 ETs were included in the study. Mares receiving the first embryo of the year had a higher PR 14 and 45 days compared to mares at the third transfer (78.8% and 70.1% vs. 65.6% and 54.1%, respectively). The detection of intrauterine fluid post ovulation negatively affected PR 14 (60.5% vs. 77.6%) and should therefore be considered an abnormal finding, probably being a sign of uterine inflammation or delayed uterine clearance. On the contrary, the age of the recipient mare and detection of fluid during follicular phase did not affect PR 14. Only the age of the recipient mare influenced the EEL, since mares aged 10-13 years had a higher EEL compared to mares aged 3-5 years (15.6% vs. 6.4%). Embryo size and grade affected PR 14 and 45.

Developmental Hurdles That Can Compromise Pregnancy during the First Month of Gestation in Cattle.

Several key developmental events are associated with early embryonic pregnancy losses in beef and dairy cows. These developmental problems are observed at a greater frequency in pregnancies generated from in-vitro-produced bovine embryos. This review describes critical problems that arise during oocyte maturation, fertilization, early embryonic development, compaction and blastulation, embryonic cell lineage specification, elongation, gastrulation, and placentation. Additionally, discussed are potential remediation strategies, but unfortunately, corrective actions are not available for several of the problems being discussed. Further research is needed to produce bovine embryos that have a greater likelihood of surviving to term.