Metabolomic signature of spermatozoa established during holding time is responsible for differences in boar sperm freezability†.

Holding at room temperature is the first step in most boar semen cryopreservation protocols. It is well accepted that a holding time (HT) of 24 h increases sperm cryotolerance. However, the effect of HT on ejaculates with different freezability is not entirely clear. The aim of this study was to understand how HT influences spermatic and seminal plasma metabolite profiles of boar ejaculates and how these possible changes affect freezability. A total of 27 ejaculates were collected and extended to 1:1 (v: v) with BTS and split into two aliquots. The first aliquot was cryopreserved without HT (0 h), and the second was held at 17°C for 24 h before cryopreservation. Spermatozoa and seminal plasma were collected by centrifugation at two times, before HT (0 h) and after HT (24 h), and subsequently frozen until metabolite extraction and UPLC-MS analysis. After thawing, the semen samples were evaluated for kinetics, membrane integrity, mitochondrial potential, membrane lipid peroxidation, and fluidity. The ejaculates were then allocated into two phenotypes (good ejaculate freezers [GEF] and poor ejaculate freezers [PEF]) based on the percent reduction in sperm quality (%RSQ) as determined by the difference in total motility and membrane integrity between raw and post-thaw samples cryopreserved after 24 h of HT. The metabolic profile of the seminal plasma did not seem to influence ejaculate freezability, but that of the spermatozoa were markedly different between GEF and PEF. We identified a number of metabolic markers in the sperm cells (including inosine, hypoxanthine, creatine, ADP, niacinamide, spermine, and 2-methylbutyrylcarnitine) that were directly related to the improvement of ejaculate freezability during HT; these were components of metabolic pathways associated with energy production. Furthermore, PEF showed an upregulation in the arginine and proline as well as the glutathione metabolism pathways. These findings help to better understand the effect of HT on boar sperm freezability and propose prospective metabolic markers that may predict freezability; this has implications in both basic and applied sciences.

An Efficient Method to Prepare Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing.

Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes.

Spermatozoa and seminal plasma small extracellular vesicles miRNAs as biomarkers of boar semen cryotolerance.

Freeze boar semen is still the biggest challenge for the swine industry due to the high cold shock sensitivity of boar sperm cells and the variance of post-thaw results among individuals and ejaculates from the same boar. To solve this problem, we investigate if miRNAs present in sperm cells and small extracellular vesicles (EVs) from seminal plasma of raw boar ejaculates can predict high-quality ejaculates after underwent the freeze-thaw process. For this, we obtained miRNAs samples of sperm cells and EVs from raw seminal plasma from 27 ejaculates before the cryopreservation process. Two groups with different freezability considering the analysis post-thaw of structure and sperm functionality were formed: High freezability (HF; n = 04) and low freezability (LF; n = 04). That done, we investigated the miRNAs profile of sperm cells and EVs from seminal plasma in both groups. Three miRNAs were differently abundant in LF ejaculates, being the ssc-miR-503 found in higher levels in sperm cells (P < 0.10). The ssc-miR-130a and ssc-miR-9 most abundant in EVs from seminal plasma (P < 0.10), in LF ejaculates. Through enrichment analysis, it was possible to verify that these miRNAs could be performing modifications in the development of male germ cells and in the production of energy to spermatozoa to maintain their viability and functionality. Therefore, we can demonstrate that ssc-miR-503, ssc-miR-130a, and ssc-miR-9 are related to low sperm cryotolerance in boars semen. So those miRNAs can be used as a biomarker to predict their low ability to tolerate the cryopreservation process.

Effects of different equilibration times at 5 °C on boar sperm cryotolerance.

Equilibration time (ET) is the period during which sperm cells are in contact with cooling/freezing media components at a temperature of 5 °C, providing a proper osmotic balance between the intra- and extra-cellular milieu. The present study aimed to determine the ET (0, 2, and 4 h) that results in greater post-thaw sperm quality and functions. Based on the post-thaw sperm membrane integrity and motility ratios, 20 ejaculates collected from five boars were classified as having good (GFE, n = 5) or poor (PFE, n = 15) freezing capacity. Ratios of post-thaw sperm with intact plasma membrane and acrosome were similar between ET (0 h: 37.02 % ± 2.85 %; 2 h: 34.59 % ± 7.12 %; 4 h: 37.87 % ± 4.44 %) in GFE samples. In PFE, ratios of sperm with intact plasma membrane and acrosome at post-thaw were greater (P < 0.05) after an ET of 2 h than after an ET of 0 h (2 h: 26.16 % ± 1.54 % and 0 h: 16.74 % ± 1.59 %). Also, ratios of post-thaw sperm with relatively lesser membrane lipids disorder were greater (P < 0.05) after an ET of 2 h than for other ET in both GFE (2 h: 21.97 % ± 4.24 % and 0 h: 16.63 % ± 2.38 %) and PFE (2 h: 16.65 % ± 1.40 % and 0 h: 13.23 % ± 1.25 %) samples. In conclusion, an ET of 2 h results in greater sperm cryotolerance in both GFE and PFE samples, which suggests that modifying the freezing protocol lead to an increase post-thaw sperm function and survival.

Long-Term Cytotoxicity, pH and Dissolution Rate of AH Plus and MTA Fillapex.

The aim of the present study was to verify the long-term cytotoxic effects of the MTA Fillapex and to compare them with AH Plus. Dissolution rate and pH were also evaluated. Human osteoblast cells were incubated with elutes of fresh specimens from AH Plus and MTA Fillapex, and with elutes of the same specimens for 4 successive weeks. Elute's pH was evaluated at each time point. A multiparametric cell viability assay was performed. For dissolution rate, ISO methodology was used. The results were analyzed by one-way analysis of variance, complemented with the Tukey post-test (p<0.05). No significant difference was found among the materials when fresh mixed (p>0.05). After 1 week, AH Plus became non-cytotoxic on all three evaluated parameters. Conversely, MTA Fillapex remained severely and mildly cytotoxic over the entire experimental period (p<0.05). The dissolution rate of AH Plus was significantly lower than MTA Fillapex at all time points (p>0.05). The pH of AH Plus was significantly lower than MTA Fillapex at the second and third week (p<0.05). In the other tested time points no statistical difference was observed. In conclusion, MTA Fillapex remained cytotoxic after 4 weeks and its cytotoxicity may be related to the high dissolution rate of this material.

Long-Term Cytotoxicity, pH and Dissolution Rate of AH Plus and MTA Fillapex

Abstract The aim of the present study was to verify the long-term cytotoxic effects of the MTA Fillapex and to compare them with AH Plus. Dissolution rate and pH were also evaluated. Human osteoblast cells were incubated with elutes of fresh specimens from AH Plus and MTA Fillapex, and with elutes of the same specimens for 4 successive weeks. Elute's pH was evaluated at each time point. A multiparametric cell viability assay was performed. For dissolution rate, ISO methodology was used. The results were analyzed by one-way analysis of variance, complemented with the Tukey post-test (p<0.05). No significant difference was found among the materials when fresh mixed (p>0.05). After 1 week, AH Plus became non-cytotoxic on all three evaluated parameters. Conversely, MTA Fillapex remained severely and mildly cytotoxic over the entire experimental period (p<0.05). The dissolution rate of AH Plus was significantly lower than MTA Fillapex at all time points (p>0.05). The pH of AH Plus was significantly lower than MTA Fillapex at the second and third week (p<0.05). In the other tested time points no statistical difference was observed. In conclusion, MTA Fillapex remained cytotoxic after 4 weeks and its cytotoxicity may be related to the high dissolution rate of this material.

Resumo O objetivo do presente estudo foi verificar os efeitos citotóxicos de longo prazo da MTA Fillapex e comparar com os do AH Plus. Solubilidade e pH também foram avaliados. Osteoblastos humanos foram incubados com elutos de amostras frescas de AH Plus e MTA Fillapex, e com elutos dos mesmos espécimes pelas 4 semanas seguintes. O pH foi avaliado a cada semana. Um ensaio multiparamétrico de viabilidade celular foi realizado. Para solubilidade foi utilizada metodologia ISO. Os resultados foram analisados por ANOVA, complementada com o pós-teste de Tukey (p<0,05). Nenhuma diferença significativa foi encontrada entre os materiais frescos quando avaliados em relação a citotoxicidade (p>0,05). Depois de uma semana, o AH Plus tornou-se não-citotóxico em todos os três parâmetros avaliados. Por outro lado, MTA Fillapex permaneceu citotóxico durante todo o período experimental (p<0,05). A solubilidade do AH Plus foi significativamente menor do MTA Fillapex para todos os períodos avaliados (p>0,05). O pH da AH Plus foi significativamente menor do que o MTA Fillapex na segunda e na terceira semana (p<0,05). Nos outros períodos testados não houve diferença estatística. Em conclusão, o MTA Fillapex permaneceu citotóxico após 4 semanas e a sua citotoxicidade pode estar relacionada à elevada solubilidade deste material.