Search Results (73)

The corpus luteum (CL) is a transient gland that can directly influence follicular dynamics and oocyte quality. The objective of this study was to evaluate the influence of the absence or presence of a small (≤3 mm), medium (4–8 mm), or large (>8 mm) CL in slaughterhouse ovaries on in vitro embryo production. Cumulus–oocyte complexes (COCs) were collected from each group of ovaries and matured in TCM-199 medium, plus hormones and fetal bovine serum. Fertilization was performed with fresh semen from a Katahdin ram of known fertility. Embryo development was carried out in commercial sequential media for 72 and 96 h, until the blastocyst stage. The number of follicles (2–6 mm in diameter) and COCs were influenced by the presence of CL, which was higher (p < 0.05) in the Large CL group (5.51 ± 0.33 and 3.62 ± 0.27) compared to the Without CL group (4.54 ± 0.19 and 2.62 ± 0.14, respectively), with no difference between the CL sizes. Likewise, the diameter and area of the COCs were higher in the Small CL group of ovaries compared to the Without CL group. In the Large CL group of ovaries, 9% more morulae (p < 0.05) were obtained compared to the Without CL group; in the Medium CL group, 13% more blastocysts were obtained compared to the Without CL group. However, in the hatching capacity and diameter of blastocysts, no statistical difference was evident (p > 0.05). In conclusion, the presence and size of the CL in the ovaries of slaughtered sheep influence the productive efficiency of embryos in vitro under the conditions in which the present study was carried out. Full article

A divergent selection for resilience was carried out in rabbits over 12 generations. The selection criterion was increased (the HO line) and decreased litter size variability at birth (the HE line). The HO line (more resilient) shows higher litter size than the HE line (less resilient). The HO line sows higher litter size and embryo development than the HE line. The aim of this work is to investigate the plasma organic acid profile in both lines at mating and early gestation in order to analyze the effect of selection by resilience in the ovulation rate and early gestation. A total of 19 and 18 nonlactating multiparous females from the HE and HO lines were used. The ovulation rate, normal embryos, and percentage of compacted morulae at 72 h post-coitum (hpc) were studied, and blood samples were obtained at mating and 72 hpc. The organic profile was determined by HPLC. Bayesian methodology was used for statistical analysis. The HE line had 1.5% fewer normal embryos and 12.3% fewer compacted morulae than the HO line. The ovulation rate was similar in both lines. α-ketoglutaric acid and cis-aconitic acid were higher in the HE line than in the HO line. Citric acid, lactic acid, and pyruvic acid were higher at mating than at early gestation. In conclusion, the lower efficiency in the utilization of energy sources in the HE line could explain the reduced embryo production observed. The organic profile varies depending on the reproductive state in the female. Full article

A time-lapse live embryo monitoring system provides a powerful approach to recording dynamic developmental events of cultured embryos in detail. By obtaining continuous short-interval images, blastocyst formation can be predicted and embryos can be selected. The objective of this study was to investigate the morphokinetic parameters of fishing cat–domestic cat interspecies somatic cell nuclear transfer (iSCNT) embryos from one-cell to blastocyst stages, and in particular, the cleavage patterns of the first division in iSCNT and IVF embryos, as these play a central role in euploidy. Domestic cat in vitro fertilization (IVF) embryos were set up as controls. The results show that morula and blastocyst development rates were significantly lower in the iSCNT embryos compared to their IVF counterparts. All earlier time points of embryonic development before the onset of blastulation in the iSCNT embryos were significantly delayed when compared with their IVF counterparts. In iSCNT, normal embryos (defined as those that developed to the blastocyst stage) took a longer time to reach the morula stage, and these morulas were more likely to undergo compaction, compared to their arrested embryo counterparts. Direct cleavage in the first division is a morphological aberration, and was seen with greater prevalence in iSCNT embryos than control IVF embryos; these aberrant embryos displayed a significantly lower blastocyst development rate than embryos that had undergone normal cleavage. In conclusion, the morphokinetic parameters of fishing cat–domestic cat iSCNT embryos at early stages could be used to predict their potential for development to the blastocyst stage. A time-lapse imaging system is potentially a powerful tool for selecting early embryos with developmental potential for transfer, and hence, for improving feline iSCNT efficiency. Full article

In eukaryotes, mRNAs with long poly(A) tails are translationally active, but deadenylation and uridylation of these tails generally cause mRNA degradation. However, the fate of uridylated mRNAs that are not degraded quickly remains obscure. Here, using tail-seq and microinjection of the 3′ region of mRNA, we report that some mRNAs in starfish are re-polyadenylated to be translationally active after deadenylation and uridylation. In oocytes, uridylated maternal cyclin B mRNAs are stable without decay, and they are polyadenylated to be translated after hormonal stimulation to resume meiosis, whereas they are deadenylated and re-uridylated at the blastula stage, followed by decay. Similarly, deadenylated and uridylated maternal ribosomal protein mRNAs, Rps29 and Rpl27a, were stable and inactive after hormonal stimulation, but they had been polyadenylated and active before hormonal stimulation. At the morula stage, uridylated maternal ribosomal protein mRNAs were re-polyadenylated, rendering them translationally active. These results indicate that uridylated mRNAs in starfish exist in a poised state, allowing them to be recycled or decayed. Full article

Retrotransposable elements are implicated in genome rearrangements and gene expression alterations that result in various human disorders. In the current study, we sought to investigate the potential effects of long interspersed elements-1 (LINE-1) overexpression on the integrity and methylation of DNA and on the expression of three major pluripotency factors (OCT4, SOX2, NANOG) during the preimplantation stages of human embryo development. Human MI oocytes were matured in vitro to MII and transfected through intracytoplasmic sperm injection (ICSI) either with an EGFP vector carrying a cloned active human LINE-1 retroelement or with the same EGFP vector without insert as control. The occurrence of retrotransposition events was screened by fluorescent microscopy. The in vitro preimplantation development as well as the methylation, pluripotency, and DNA double-strand breaks (DSBs) of the transfected embryos were examined. LINE-1 retrotransposons gave rise to new retrotransposition events in the transfected embryos. LINE-1 injected embryos were characterized by accelerated asymmetrical cell division, multiple cellular fragments, cleavage arrest, and degeneration. Early OCT4 expression remained unaltered, but cleavage arrest and a high fragmentation rate hindered the expression of SOX2/NANOG at the morula stage. Increased DNA DSBs were observed in cleavage-stage blastomeres, while no methylation changes were detected before the cleavage arrest. Our data provide evidence that LINE-1 retrotransposition in human preimplantation embryos may induce DNA DSBs, while at the same time, it appears to interfere with the expression patterns of pluripotency factors. The morphological, structural, and cleavage abnormalities of the transfected embryos show that aberrant retroelement expression may negatively affect human embryo development. Full article

The complement system is a pivotal component of innate immunity, extensively studied in vertebrates but also present in invertebrates. This study explores the existence of a terminal complement pathway in the tunicate Botryllus schlosseri, aiming to understand the evolutionary integration of innate and adaptive immunity. Through transcriptome analysis, we identified a novel transcript, BsITCCP, encoding a protein with both MACPF and LDLa domains—a structure resembling that of vertebrate C9 but with a simpler organization. Phylogenetic reconstruction positions BsITCCP between invertebrate perforins and vertebrate terminal complement proteins, suggesting an evolutionary link. Localization studies confirmed that bsitccp is transcribed in cytotoxic morula cells (MCs), which are also responsible for producing other complement components like BsC3, BsMBL, BsMASP, and BsBf. Functional assays demonstrated that bsitccp transcription is upregulated in response to nonself challenges and is dependent on BsC3 activity; inhibition of BsC3 led to a significant reduction in BsITCCP expression. Electron microscopy revealed that MCs form contact with perforated yeast cells, indicating a possible mechanism of cell lysis similar to the immunological synapse observed in vertebrates. These findings suggest that a C3-governed lytic complement pathway exists in B. schlosseri, challenging the assumption that a C5 ortholog is necessary for such a pathway. This work enhances our understanding of the evolution of the complement system and suggests that invertebrates possess a terminal complement complex capable of mediating cell lysis, regulated by C3. Future studies will focus on confirming the pore-forming ability of BsITCCP and its role in the immunological synapse. Full article

The use of C-type natriuretic peptide (CNP) in the interaction with the oocyte and in the temporary postponement of spontaneous meiosis resumption has already been well described. However, its action in pre-implantation developmental-stage embryos is yet to be understood. Thus, our study aimed to detect the presence of the canonical CNP receptor (natriuretic peptide receptor, NPR2) in germinal vesicle (GV)-, metaphase II (MII)-, presumptive zygote (PZ)-, morula (MO)-, and blastocyst (BL)-stage embryos and, later, to observe possible modulations on the embryos when co-cultured with CNP. In Experiment I, we detected and quantified NPR2 on the abovementioned embryo stages. Further, in Experiment II, we intended to test different concentrations (100, 200, or 400 nM of CNP) at different times of inclusion in the in vitro culture (IVC; inclusion from the beginning, i.e., day 1, or from day 5). In Experiment III, 400 nM of CNP was used on day 1 (D1) in the IVC, which was not demonstrated to be embryotoxic, and it showed potentially promising results in the blastocyst production rate when compared to the control. Thus, we analyzed the embryonic development rates of bovine embryos (D7) and hatching kinetics (D7, D8, and D9). Subsequently, morula and blastocyst were collected and evaluated for transcript abundance of their competence and quality (apoptosis, oxidative stress, proliferation, and differentiation) and lipid metabolism. Differences with probabilities less than p < 0.05, and/or fold change (FC) > 1.5, were considered significant. We demonstrate the presence of NPR2 until the blastocyst development stage, when there was a significant decrease in membrane receptors. There was no statistical difference in the production rate after co-culture with 400 nM CNP. However, when we evaluated the abundance of morula transcripts, there was an upregulated transcription in ADCY6 (p = 0.057) and downregulated transcripts in BMP15 (p = 0.013), ACAT1 (p = 0.040), and CASP3 (p = 0.082). In addition, there was a total of 12 transcriptions in morula that presented variation FC > 1.5. In blastocysts, the treatment with CNP induced upregulation in BID, CASP3, SOX2, and HSPA5 transcripts and downregulation in BDNF, NLRP5, ELOVL1, ELOVL4, IGFBP4, and FDX1 transcripts (FC > 1.5). Thus, our study identified and quantified the presence of NPR2 in bovine pre-implantation embryos. Furthermore, 400 nM of CNP in IVC, a concentration not previously described in the literature, modulated some transcripts related to embryonic metabolism, and this was not embryotoxic morphologically. Full article

Background: Receptor-interacting protein kinases (RIPKs) and mixed-lineage kinase domain-like protein (MLKL) are crucial in regulating innate immune responses and cell death signaling (necroptosis and apoptosis), and are potential candidates for genetic improvement in breeding programs. Knowledge about the RIPK family and MLKL in sea cucumber remains limited. Methods: We searched the genomes of sea cucumber Holothuria leucospilota for genes encoding RIPKs and MLKL, performed phylogenetic tree, motif and functional domain analyses, and examined tissue distribution and embryonic development patterns using qPCR. Results: RIPK5 (Hl-RIPK5), RIPK7 (Hl-RIPK7) and MLKL (Hl-MLKL) were identified in sea cucumber H. leucospilota. Hl-RIPK5 and Hl-RIPK7 were mainly expressed in coelomocytes, suggesting that they play a role in innate immunity, whereas Hl-MLKL exhibited relatively low expression across tissues. During embryonic development, Hl-MLKL was highly expressed from the 2-cell stage to the morula stage, while Hl-RIPK5 and Hl-RIPK7 were primarily expressed after the morula stage, indicating different roles in embryonic development. In primary coelomocytes, Hl-RIPK5 transcriptional activity was significantly depressed by LPS, poly(I:C), or pathogen Vibrio harveyi. Hl-RIPK7 expression levels were unchanged following the same challenges. Hl-MLKL mRNA levels were significantly decreased with poly(I:C) or V. harveyi, but did not change with LPS. Conclusions: These findings provide valuable insights into the evolutionary tree and characterization of RIPK and MLKL genes in sea cucumber, contributing to the broader understanding of the RIPK gene family and MLKL in ancient echinoderms. Full article

This study evaluates the utilization of in vitro embryo production (IVEP) technology for the conservation and breeding of the Auliekol cattle breed, a primary beef breed in Kazakhstan facing population decline due to the cessation of breeding programs and the incursion of transboundary diseases. We assessed the effect of consecutive ovum pick-up (OPU) procedures on oocyte yield and embryo production in Auliekol and Aberdeen Angus cows. A total of 2232 and 3659 oocytes were aspirated from Auliekol and Aberdeen Angus donors, respectively, with significantly higher yields and embryo production observed in Aberdeen Angus cows. The application of a meiotic block using Butyrolactone I (BLI) and subsequent in vitro fertilization (IVF) protocols was employed, with embryo development monitored up to the morula/blastocyst stage. Results indicated that Auliekol cows exhibited lower oocyte recovery, cleavage, and blastocyst rates compared to Aberdeen Angus cows, likely due to genetic characteristics. Despite the challenges, IVEP presents a valuable tool for the preservation and future propagation of the Auliekol breed, highlighting the need for further research to enhance reproductive outcomes and conservation strategies. Full article

The main objective of this study was to determine the influence of the recipient dairy cows’ breed, lactation number, estrus condition, the type, location and volume of the corpus luteum (CL) and the time of year that the embryo transfer (ET) was performed on the pregnancy rates of a large, fresh in vitro fertilization–embryo transfer program for dairy cows in a commercial herd in China. The recipients were from a herd of dairy cows in Ningxia, a province in northwest China, and we statistically analyzed the data of 495 cows from 2021 to 2023. Cumulus oocyte complexes (COCS) were isolated from follicular fluid obtained through ovum pick-up (OPU) and oocytes were incubated 20–22 h for in vitro maturation (IVM). Embryos were obtained after 10–12 h of in vitro fertilization (IVF) and six days of in vitro culture (IVC). Embryos at the morula or blastocyst stage were transferred to randomly chosen recipients (n = 495). The influence of recipients’ breed (Holstein or other), recipients’ lactation number (heifers or cows), estrus type (natural or synchronized), CL type (homogeneous, CL_hom or cavitary, CL_cav), CL side (left or right), volume of the CL and season of transfer (spring, autumn or winter) on pregnancy rates were determined. The pregnancy rates were analyzed by binomial logistic regression with IBM SPSS statistics software, version 26. Pregnancy rates after ET to Holstein cows and other breeds were 43.49% and 42.68%, respectively (p > 0.05). Regarding age, pregnancy rates were 45.56% for heifers and 30.77% for cows (p < 0.05). Pregnancy rates following ET during natural and synchronized estrus were 44.41% and 41.5%, respectively (p > 0.05). Pregnancy rates with a left- or right-side CL were 40.18% and 45.65%, respectively (p > 0.05). The pregnancy rates achieved with a CL_hom and CL_cav were 44.44% and 39.68%, respectively (p < 0.05). The rates obtained in spring, autumn and winter were 49.26%, 46.02% and 34.64%, respectively (p < 0.05). Moreover, it was found that pregnancy rates were higher in recipients with a CL volume measuring greater than 10 cm³ compared with those with a CL volume measuring less than 10 cm³ (p < 0.05). The comparisons showed that recipients’ breed, estrus type or side of the CL had no effect, but the recipients’ lactation number, ET season and the type and volume of the CL have significant effects on pregnancy rates during ET. Full article

Preimplantation genetic testing for aneuploidy (PGT-A) has become a useful approach for embryo selection following IVF and ICSI. However, the biopsy process associated with PGT-A is expensive, prone to errors in embryo ploidy determination, and potentially damaging, impacting competence and implantation potential. Therefore, a less invasive method of PGT-A would be desirable and more cost-effective. Noninvasive methods for PGT-A (ni-PGT-A) have been well-studied but present limitations in terms of cf-DNA origin and diagnostic accuracy. Minimally invasive pre-implantation genetic testing (mi-PGT-A) for frozen-thawed embryo transfer is a promising, less studied approach that utilizes a combination of spent culture media (SCM) and blastocoelic fluid (BF)-derived cell-free (CF)-DNA for genetic testing. This study aimed to optimize the effectiveness of mi-PGT-A for aneuploidy diagnosis by investigating the optimal temporal sequence for this protocol. SCM+BF was collected at either 48 or 72 h of culture after thawing day 3 preimplantation embryos. cf-DNA in the SCM+BF was amplified, analyzed by next-generation sequencing (NGS) and compared with results from the corresponding whole embryos (WEs) obtained from human embryos donated for research. Fifty-three (42 expanded blastocysts, 9 early blastocysts, and 2 morula) WE and SCM+BF samples were analyzed and compared. The overall concordance rate between SCM+BF and WE was 60%. Gender and ploidy concordance improved with extended culture time from 48 h (73% and 45%) to 72 h (100% and 64%), respectively. These results demonstrate that SCM+BF-derived cf-DNA can be successfully used for mi-PGT-A. Our findings indicate that longer embryo culture time prior to SCM+BF-derived cf-DNA analysis improves DNA detection rate and concordance with WEs and decreases the proportion of false positive results. Full article

Equine granulocytic anaplasmosis (EGA) is a tick-borne disease affecting horses worldwide, caused by Anaplasma phagocytophilum. The disease ranges from non-specific clinical signs to fatal outcomes. This paper aimed to analyze EGA cases reported in peer-reviewed journals, particularly on clinico-pathological findings, diagnosis, and therapeutic management. Overall, 189 clinical cases from 31 publications were included in the study. Extensive symptomatology for the EGA cases was reported, of which mostly was fever (90.30%), followed by limb edema (48.51%), anorexia (41.79%), depression (32.84%), icterus (22.39%), ataxia (17.91), tachycardia (16.42%), and lethargy (15.67%). Laboratory tests revealed thrombocytopenia (90.32%), anemia (75%), decreased hematocrit (70.59%), leukopenia (55.88%), lymphopenia (58.14%), and neutropenia (41.67%) as the most common hematological abnormalities. For a subset of tested animals, hyperbilirubinemia (20/29), hyperfibrinogenemia (13/15), and hyponatremia (10/10) were also reported. The diagnosis was established by microscopic identification of morulae (in 153 cases), and/or PCR (120 cases), isolation (1 case), or serology (56 cases). For treatment, oxytetracycline was used in the majority (52.24%) of EGA cases, but recovery without antibiotherapy (10.34%) was also noted. In conclusion, the variety of clinical and pathological findings and the challenging therapeutic approaches reported suggest that EGA should be included in the differential diagnosis when fever occurs. Full article

Domestic cat blastocysts cultured without the zona pellucida exhibit reduced implantation capacity. However, the protein expression profile has not been evaluated in these embryos. The objective of this study was to evaluate the protein expression profile of domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were generated: (1) domestic cat embryos generated by IVF and cultured in vitro (zona intact, (ZI)) and (2) domestic cat embryos cultured in vitro without the zona pellucida (zona-free (ZF group)). The cleavage, morula, and blastocyst rates were estimated at days 2, 5 and 7, respectively. Day 7 blastocysts and their culture media were subjected to liquid chromatography–tandem mass spectrometry (LC–MS/MS). The UniProt Felis catus database was used to identify the standard proteome. No significant differences were found in the cleavage, morula, or blastocyst rates between the ZI and ZF groups (p > 0.05). Proteomic analysis revealed 22 upregulated and 20 downregulated proteins in the ZF blastocysts. Furthermore, 14 proteins involved in embryo development and implantation were present exclusively in the culture medium of the ZI blastocysts. In conclusion, embryo culture without the zona pellucida did not affect in vitro development, but altered the protein expression profile and release of domestic cat blastocysts. Full article

Pre-implantation embryos release extracellular vesicles containing different molecules, including DNA. The presence of embryonic DNA in E-EVs released into the culture medium during in vitro embryo production could be useful for genetic diagnosis. However, the vesicles containing DNA might be derived from embryos suffering from apoptosis, i.e., embryos of bad quality. This work intended to confirm that embryos release DNA that is useful for genotyping by evaluating the effect of embryonic apoptosis on DNA content in E-EVs. Bovine embryos were produced by parthenogenesis and in vitro fertilization (IVF). On Day 5, morulae were transferred to individual cultures in an EV-depleted SOF medium. On Day 7, embryos were used to evaluate cellular apoptosis, and each culture medium was collected to evaluate E-EV concentration, characterization, and DNA quantification. While no effect of the origin of the embryo on the apoptotic rate was found, arrested morulae had a higher apoptotic rate. E-EVs containing DNA were identified in all samples, and the concentration of those vesicles was not affected by the origin or quality of the embryos. However, the concentration of DNA was higher in EVs released by the arrested parthenogenetic embryos. There was a correlation between the concentration of E-EVs, the concentration of DNA-positive E-EVs, and the concentration of DNA. There was no negative effect of apoptotic rate on DNA-positive E-EVs and DNA concentration; however, embryos of the best quality with a low apoptotic rate still released EVs containing DNA. This study confirms that the presence of DNA in E-EVs is independent of embryo quality. Therefore, E-EVs could be used in liquid biopsy for noninvasive genetic diagnosis. Full article

The study of pig preimplantation embryo development has several potential uses: from agriculture to the production of medically relevant genetically modified organisms and from rare breed conservation to acting as a physiologically relevant model for progressing human and other (e.g., endangered) species’ in vitro fertilisation technology. Despite this, barriers to the widespread adoption of pig embryo in vitro production include lipid-laden cells that are hard to visualise, slow adoption of contemporary technologies such as the use of time-lapse incubators or artificial intelligence, poor blastulation and high polyspermy rates. Here, we employ a commercially available time-lapse incubator to provide a comprehensive overview of the morphokinetics of pig preimplantation development for the first time. We tested the hypotheses that (a) there are differences in developmental timings between blastulating and non-blastulating embryos and (b) embryo developmental morphokinetic features can be used to predict the likelihood of blastulation. The abattoir-derived oocytes fertilised by commercial extended semen produced presumptive zygotes were split into two groups: cavitating/blastulating 144 h post gamete co-incubation and those that were not. The blastulating group reached the 2-cell and morula stages significantly earlier, and the time taken to reach the 2-cell stage was identified to be a predictive marker for blastocyst formation. Reverse cleavage was also associated with poor blastulation. These data demonstrate the potential of morphokinetic analysis in automating and upscaling pig in vitro production through effective embryo selection. Full article