Reference genes for gene expression profiling in mouse models of Listeria monocytogenes infection.

RT-qPCR dissects transcription-based processes but requires reference genes (RGs) for data normalization. This study prospected RGs for mouse macrophages (pMØ) and spleen infected with Listeria monocytogenes. The pMØ were infected in vitro with L. monocytogenes or vehicle for 4 h. Mice were injected with L. monocytogenes (or vehicle) and euthanized 24 h post-injection. The RGs came from a multispecies primer set, from the literature or designed here. The RG ranking relied on GeNorm, NormFinder, BestKeeper, Delta-CT and RefFinder. B2m-H3f3a-Ppia were the most stable RGs for pMØ, albeit RG indexes fine-tuned estimations of cytokine relative expression. Actß-Ubc-Ppia were the best RGs for spleen but modestly impacted the cytokine relative expression. Hence, mouse models of L. monocytogenes require context-specific RGs for RT-qPCR, thus reinforcing its paramount contribution to accurate gene expression profiling.

Gene Regulatory Networks: Improving Inferences with Transfer Learning.

Deep transfer learning improves the inference of gene regulatory networks in human cells, reveals disease-associated genes, and identifies network-based druggable targets in human heart disease.

Cloning by SCNT: Integrating Technical and Biology-Driven Advances.

Somatic cell nuclear transfer (SCNT) into enucleated oocytes initiates nuclear reprogramming of lineage-committed cells to totipotency. Pioneer SCNT work culminated with cloned amphibians from tadpoles, while technical and biology-driven advances led to cloned mammals from adult animals. Cloning technology has been addressing fundamental questions in biology, propagating desired genomes, and contributing to the generation of transgenic animals or patient-specific stem cells. Nonetheless, SCNT remains technically complex and cloning efficiency relatively low. Genome-wide technologies revealed barriers to nuclear reprogramming, such as persistent epigenetic marks of somatic origin and reprogramming resistant regions of the genome. To decipher the rare reprogramming events that are compatible with full-term cloned development, it will likely require technical advances for large-scale production of SCNT embryos alongside extensive profiling by single-cell multi-omics. Altogether, cloning by SCNT remains a versatile technology, while further advances should continuously refresh the excitement of its applications.

Evaluation of quality and gene expression of goat embryos produced in vivo and in vitro after cryopreservation.

In the present study, we aimed to identify morphological and molecular changes of in vivo and in vitro-produced goat embryos submitted to cryopreservation. In vivo embryos were recovered by transcervical technique from superovulated goats, whereas in vitro produced embryos were produced from ovaries collected at a slaughterhouse. Embryos were frozen by two-steps slow freezing method, which is defined as freezing to -32 °C followed by transfer to liquid nitrogen. Morphological evaluation of embryos was carried out by assessing blastocoel re-expansion rate and the total number of blastomeres. The expression profile of candidate genes related to thermal and oxidative stress, apoptosis, epigenetic, and implantation control was measured using RT-qPCR based SYBR Green system. In silico analyses were performed to identify conserved genes in goat species and protein-protein interaction networks were created. In vivo-produced embryos showed greater blastocoel re-expansion and more blastomere cells (P < 0.05). The expression level of CTP2 and HSP90 genes from in vitro cryopreserved embryos was higher than their in vivo counterparts. Unlikely, no significant difference was observed in the transcription level of SOD gene between groups. The high similarity of CPT2 and HSP90 proteins to their orthologs among mammals indicates that they share conserved functions. In summary, cryopreservation negatively affects the morphology and viability of goat embryos produced in vitro and changes the CPT2 and HSP90 gene expression likely in response to the in vitro production process.

Housekeeping genes for RT-qPCR in ovine preimplantation embryos.

Housekeeping genes (HKG) are paramount for accurate gene expression analysis during preimplantation development. Markedly, quantitative reverse transcription polymerase chain reaction (RT-qPCR) in ovine embryos currently lacks HKGs. Therefore, we tested 11 HKGs for RT-qPCR normalization during ovine parthenogenetic preimplantation development. Seven HKGs reached the qPCR efficiency threshold (97.20-105.96%), with correlation coefficients ranging from -0.922 to -0.998 and slopes from -3.22 to -3.59. GeNorm ranked glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-binding protein (TBP) as the best HKG pair, while H3 histone, family 3A (H3F3A) was the third HKG. Relative gene expression was measured for zinc finger protein X-linked (ZFX) and developmental pluripotency-associated 3 (DPPA3) transcripts during ovine parthenogenetic preimplantation development. ZFX did not show any transcript abundance fluctuation among oocytes, cleavage-stage embryos, and morulae. DPPA3 transcript abundance was also similar among all developmental stages, therefore suggesting that it may not display a maternal gene expression profile. In silico analysis of ovine DPPA3 mRNA and protein showed high conservation to bovine orthologues. However, DPPA3 orthologues differed in regulatory motifs. In conclusion, GAPDH, TBP and H3F3A are stable HKGs in ovine parthenogenetic embryos and allow accurate RT-qPCR-based gene expression analysis.

Reproductive efficiency of nellore (Bos indicus) cows subject to both FTAI and homeopathic supplementation / Eficiência reprodutiva em vacas nelore (bos indicus) submetidas à IATF e a suplementação homeopática

The aim of this work was to determine the effect of homeopathic supplementation on both ovarian dynamics and conception rate in Nellore cows subjected to fixed-time artificial insemination (FTAI). Cows (n = 150) were randomly distributed to the control (CG) and the homeopathy group (HG). The HG cows were supplemented with Pró-cio in the mineral salt for 60 days and both experimental groups were further subjected to FTAI. Cows were evaluated for ovarian dynamics (n = 16), progesterone (P4) concentration (n = 16), and conception rates (n = 150). Ovarian dynamics determined by ultrasonography and showed similar findings for CG and HG, respectively. Thus follicular diameter (8.7 ± 1.0 mm vs. 10.0 ± 0.8 mm), mean pre-ovulatory follicle volume (0.46 ± 0.15 mL vs. 0.61 ± 0.12 mL), and mean follicular growth (3.65 ± 1.41 mm vs. 4.60 ± 1.21 mm) did not differ between groups. Moreover, corpus luteum diameter was similar between groups (CG: 16.28 ± 0.7 mm vs. HG: 15.6 ± 0.8 mm; P > 0.05), although P4 levels did differ (CG: 2.55 ± 0.85 ng mL-1 vs. HG: 6.52 ± 1.19 ng mL-1; P < 0.05). The conception rate after FTAI was not affected by homeopathic supplementation (CG: 74.67 %, and did HG: 77.33 %; P > 0.05). In conclusion, the homeopathic supplementation Pró-cio increases P4 concentrations but does improve the reproductive efficiency of Nellore cows subject to FTAI.

O objetivo foi determinar o efeito da suplementação homeopática na dinâmica ovariana e taxa de concepção em vacas Nelore cows submetidas à inseminação artificial em tempo fixo (IATF). As vacas (n = 150) foram distribuídas aleatoriamente nos grupos controle (GC) e grupo homeopático (GH). As vacas do GH foram suplementadas com Pró-cio® no sal mineral mineral por 60 dias. Ambos os grupos foram submetidos à IATF. As vacas foram avaliadas quanto à dinâmica ovariana (n = 16), concentração de progesterona (P4; n = 16) e taxa de concepção (n = 150). A dinâmica ovarina foi determinada por ultra-sonografia e mostrou resultados semelhantes para o GC e o GH, respectivamente. Portanto, para diâmetro folicular (8,7 ± 1,0 mm vs. 10,0 ± 0,8 mm), volume médio do folículo pré-ovulatório (0,46 ± 0,15 mL vs. 0,61 ± 0,12 mL) e crescimento folicular médio (3,65 ± 1,41 mm vs. 4,60 ± 1,21 mm) não diferiram entre os grupos. Além disso, o diâmetro do corpo lúteo foi semelhante entre os grupos (CG: 16,28 ± 0,7 mm vs. HG: 15,6 ± 0,8 mm; P > 0.05), apesar dos níveis de P4 diferirem (CG: 2,55 ± 0,85 ng mL-1 vs. GH: 6,52 ± 1,19 ng mL-1; P < 0.05). A taxa de concepção após a IATF não foi afetada pela suplementação homeopática (GC:74.67 % vs. GH: 77.33 %; P > 0.05). Em conclusão, a suplementação homeopática com Pró-cio aumenta a concentração de P4 mas não melhora a eficiência reprodutiva de vacas Nelore cows submetidas à IATF.

Freezing of Stallion Semen: In Vitro Evaluation of Motility and Acrosin Activity in Sperm Cells Cryopreserved Using Different Semen Extenders.

The work described here aimed to verify the efficiency of different extenders for cryopreservation of equine semen using sperm motility and acrosin activity as spermatic parameters. The semen was fractioned into two equal parts and resuspended in an 11% lactose solution in a 1:1 proportion, where it remained for 20 minutes at room temperature. The semen was centrifuged at 600 g for 10 minutes, and after the second centrifugation, each pellet received the freezing extender (Merck or Zorlesco) and was loaded into 4 mL straws. Each straw was placed in liquid nitrogen vapor steam for 15 minutes and further immersion in liquid nitrogen at -196°C for long-term storage. After thawing, semen samples were initially evaluated for sperm motility, both total and progressive, and acrosin activity. Moreover, semen was incubated at 37°C and further assessed at 60 and 120 minutes in a thermoresistance test (TRT) for sperm motility and acrosin activity. Immediately after thawing, both progressive and total motility, and acrosin activity were lower (p < 0.05) in thawed semen than in fresh semen. During the TRT, total sperm motility and acrosin activity after 60 minutes were lower (p < 0.05) than those obtained after thawing. Similarly, total sperm motility and acrosin activity were lower (p < 0.05) after 120 minutes than at 60 minutes of the TRT. The analysis of motility and acrosin activity allowed the conclusion that both extenders have a similar capacity to preserve the integrity of sperm cells subject to freezing and thawing.

Conceptus loss in Santa Inês ewes carrying twin pregnancies by natural mating or embryo transfer.

Commercial application of reproductive biotechnologies such as multiple ovulation and embryo transfer depends on its overall efficiency. Sheep embryo transfer is gradually gaining wider adoption, but pregnancy rates after embryo transfer remain lower than those derived from natural mating for most breeds. The work was aimed to evaluate embryonic and fetal losses in Santa Inês ewes carrying twin pregnancies by natural mating or embryo transfer. Ewes were subjected to synchronized natural mating by ram effect or used as recipients for embryo transfer. Ewes diagnosed as carrying twin pregnancies at day 25 were used in the experiment (n = 42). Conceptus viability was monitored by ultrasonography on days 30, 35, 40, 45, 50, and 55 after conception. Conceptus loss was similar (P > 0.05) within natural mating 11/42 (26.19%) and embryo transfer 14/42 (33.34%). However, overall embryonic loss (80.0%) was greater (P < 0.05) than fetal loss (20.0%), with no difference within groups The results allow the conclusion that conceptus loss after embryo transfer is similar to natural mating and occurs predominantly during the embryonic stages.

Full-term potential of goat in vitro produced embryos after different cryopreservation methods.

Cryopreservation of preimplantation embryos represents a major challenge due to their shape and relatively large cells. Embryo source and cryopreservation method are key factors to cryotolerance efficiency and few reports have investigated more promising protocols for goat embryos. The study was aimed to compare different cryopreservation methods for goat in vitro produced (IVP) embryos. Goat blastocysts were subjected to conventional freezing (CF), Dimethyl sulfoxide vitrification (DMSO-V) and Dimethylformamide vitrification (DMF-V). Cryopreserved blastocysts were assessed for re-expansion, cell viability and in vivo development rates. Blastocyst re-expansion after cryopreservation was similar between groups, but cell viability was lower for DMF-V (32%) than CF (68%) and DMSO-V (60%). Pregnancy and delivery rates were similar for CF (60% and 50%) and DMSO-V (50% and 45%) and higher then DMF-V (20% and 15%), respectively. Finally, kidding rates were also indistinguishable for CF (40%) and DMSO-V (35%), but higher then DMF-V (12.5%). In conclusion, conventional freezing and vitrification using DMSO have similar efficiencies for cryopreservation of goat IVP embryos and cryoprotectant for vitrification affects its outcome.

Use of retinyl acetate, retinoic acid and insulin-like growth factor-I (IGF-I) to enhance goat embryo production.

Experiments were carried out to investigate the beneficial effects of retinyl acetate (RAc) and retinoic acid (RA) on goat oocyte maturation as well as the effects of insulin-like growth factor-I (IGF-I), RAc and RA during embryo culture under chemically defined conditions. In Experiment 1, in vitro maturation (IVM) was performed in a chemically defined basic maturation medium (bMM) supplemented with 0.3 µM RAc or 0.5 µM RA. Presumptive zygotes and embryos (2-4 cells) were cultured in droplets of potassium simplex optimised medium (KSOM); however, none of the embryos reached the blastocyst stage. In Experiment 2, oocytes were matured in bMM + RAc or bMM + RA. Presumptive zygotes and 2- to 4-cell embryos were placed in fresh KSOM droplets supplemented with RAc, RA, IGF-I, RAc+IGF-I or RA+IGF-I. In Experiment 1, addition of RAc and RA to bMM increased (P < 0.05) the proportion of 2- to 4-cell embryos reaching the morula stage as compared to the control. In Experiment 2, supplementation of embryo culture media with retinoids and IGF-I increased (P < 0.05) the proportion of 2- to 4-cell stage embryos developing to the morula and blastocyst stage. Our data demonstrate that goat embryo production in chemically defined media could be improved by exogenous RAc or RA and by the interaction between retinoids and IGF-I, and that goat embryos can be produced in vitro from oocytes following protocols similar to those currently used for cattle.

Analysis of actinomycin D treated cattle oocytes and their use for somatic cell nuclear transfer.

The present work aimed to evaluate the transcription and replication inhibitor, actinomycin D, for oocyte chemical enucleation. Cattle oocytes matured in vitro were treated with actinomycin D according to the following treatments: T1, control; T2=1.0 microg/ml for 16 h; T3=1.0 microg/ml for 14 h; T4=2.5 microg/ml for 14 h; T5=5.0 microg/ml for 14 h. The oocytes were denuded and activated during 24-26 h of maturation. Oocytes were fixed to determine the maturation status and for chromosome morphology evaluation. Furthermore, oocytes treated with actinomycin D were used for somatic cell nuclear transfer (SCNT). Parthenogenetic and SCNT embryos were fixed to evaluate the percentage of apoptotic nuclei by the TUNEL assay. The maturation (T1=90.4%; T2=82.3%; T3=79.1%; T4=83.4%; T5=74.7%), cleavage (T1=68.9%; T2=46.0%; T3=49.7%; T4=33.4%; T5=29.3%) and blastocyst rate at D8 (T1=41.1%; T2=1.8%; T3=1.3%; T4=0.9%; T5=0.0%) after actinomycin D treatment were significantly different. There was a significant chromosome uncoiling when treated with greater concentrations (2.5 and 5.0 microg/ml). After SCNT, the cleavage rate (61.3%) was similar to the actinomycin D-treated control group (61.3%) and less than the non-treated control (70.2%), although the blastocyst rate was greater in the SCNT group (11.8%) comparing with the treated control (3.6%) and less than the untreated control (38.0%). Treated parthenogenetic embryos had more apoptotic cells than the parthenogenetic controls (24.2% compared with 4.8%). However, the SCNT group using treated cytoplasts was similar from the SCNT control (9.3 compared with 13.0%). Actinomycin D treatment was efficient in blocking embryonic development. Moreover, it was possible to obtain reconstructed embryos that possess an apoptotic cell index indistinguishable from controls.