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RNA-Seq data from human semen suggests that the study of the sperm transcriptome requires the previous elimination from the ejaculates of somatic cells carrying a larger load of RNA. Semen purification is also carried to study the sperm transcriptome in other species including swine and it is often done by density gradient centrifugation to obtain viable spermatozoa from fresh ejaculates or artificial insemination doses, thereby limiting the throughput and remoteness of the samples that can be processed in one study. The aim of this work was to evaluate the impact of purification with density gradient centrifugation by BoviPure TM on porcine sperm. Four boar ejaculates were purified with BoviPure TM and their transcriptome sequenced by RNA-Seq was compared with the RNA-Seq profiles of their paired non-purified sample. Seven thousand five hundred and nineteen protein coding genes were identified. Correlation, cluster, and principal component analysis indicated high-although not complete-similarity between the purified and the paired non-purified ejaculates. 372 genes displayed differentially abundant RNA levels between treatments. Most of these genes had lower abundances after purification and were mostly related to translation, transcription and metabolic processes. We detected a significant change in the proportion of genes of epididymal origin within the differentially abundant genes (1.3%) when compared with the catalog of unaltered genes (0.2%). In contrast, the proportion of testis-specific genes was higher in the group of unaltered genes (4%) when compared to the list of differentially abundant genes (0%). No proportion differences were identified for prostate, white blood, lymph node, tonsil, duodenum, skeletal muscle, liver, and mammary gland. Altogether, these results suggest that the purification impacts on the RNA levels of a small number of genes which are most likely caused by the removal of epididymal epithelial cells but also premature germinal cells, immature or abnormal spermatozoa or seminal exosomes with a distinct load of RNAs., Competing Interests: SB was employed by the company Group Gepork S.A. The authors declare that this study received funding from MINECO, AGAUR, Generalitat de Catalunya and MICINN. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lian, Gòdia, Castello, Rodriguez-Gil, Balasch, Sanchez and Clop.)

Two of the most important problems in high-yielding dairy goat farms are early and accurate pregnancy diagnosis and the appropriate dry off of lactating does before the next kidding. The hypothesis posits that it could be possible to identify pregnant does through maximum progesterone milk levels at any time during the pregnancy, and that there is an optimal time to apply a lactation inhibitor to help dry off lactating does. Therefore, 114 Murciano-Granadina breed goats were used, from which 74 goats were inseminated at week 20 of lactation and samples of milk from pregnant and non-pregnant goats were taken at two-week intervals. The average maximum progesterone milk levels were higher outside the natural breeding season (40° latitude) than in the breeding season (11.6 ± 1.13 vs. 8.6 ± 1.02 ng/mL), although the levels from pregnant and non-pregnant goats were similar (10.85 ± 1.3 vs. 9.74 ± 1.6 ng/mL), except in the case of pregnancy with four foetuses (12.5 ± 1.3 ng/mL). Milk yield at mating does not affect fertility until a value of at least 3250 mL/day. Pregnancy started to affect milk yield up to the +7th week and was 59.9% lower in the +10th week after mating, so the use of lactation inhibitors could be more effective from this latter week. In conclusion, the results show that it is not possible to detect gestation in goats reliably through the maximum concentration of progesterone in milk at any time during lactation, except in the case of goats gestating four foetuses, that the milk yield of goats at mating does not affect fertility until a value of at least 3250 mL/day, and that from the 10th week post-mating, the application of lactation inhibitors would be optimal.

Context: Some vaginal lubricants and ultrasound gels are known to be detrimental to sperm function and therefore could negatively affect fertility., Aims: The aim of the current study was to develop a sperm motility index (SMI) to test the sperm toxicity of ultrasound gels and vaginal lubricants used in reproductive medicine., Settings and Design: Two ultrasound gels (Aquasonic ® and Kefus ® ) and five vaginal lubricants (Vaginesil™, Velastisa ® , K-Y Jelly ® , Control ® , and Durex ® ) were studied. Three different concentrations (1%, 5%, and 10%) of each lubricant were tested., Subjects and Methods: SMI was calculated dividing the percentage of progressively motile sperm in each tested gel by that in the control at 0.5, 1, 2, and 24 h of incubation at 5% of CO 2 and 37°C. SMI values <0.75 indicate sperm toxicity., Statistical Analysis Used: The main outcome measured was SMI for each concentration and time of incubation., Results: Only Durex ® did not show any deleterious effect on sperm quality. The rest of lubricants presented different degrees of toxicity. Vaginesil™ resulted in toxic for all concentrations and incubation periods (SMI < 0.12). Control ® and Velastisa ® presented toxicity at 10% after 2 h, while K-Y Jelly ® showed toxicity at 10% from 1 h of incubation. Regarding ultrasound gels, Aquasonic ® showed toxic effects after only 0.5 h (SMI = 0.70 ± 0.15), while Kefus ® showed slightly toxic effects after 2 h (SMI 0.69 ± 0.07)., Conclusions: SMI is an accurate tool to evaluate sperm toxicity. One of the main strengths of the article is the inclusion of representative semen samples and known products used worldwide. This study has a relevant clinical translation since it highlights the importance of evaluating the possible sperm toxicity of simple products used in reproductive medicine., Competing Interests: There are no conflicts of interest., (Copyright: © 2021 Journal of Human Reproductive Sciences.)

Circular RNAs (circRNAs) are emerging as a novel class of noncoding RNAs which potential role as gene regulators is quickly gaining interest. circRNAs have been studied in different tissues and cell types across several animal species. However, a thorough characterization of the circRNAome in ejaculated sperm remains unexplored. In this study, we profiled the sperm circRNA catalogue using 40 porcine ejaculates. A complex population of 1,598 circRNAs was shared in at least 30 of the 40 samples. Generally speaking, the predicted circRNAs presented low abundances and were tissue-specific. Around 80% of the circRNAs identified in the boar sperm were reported as novel. Results from abundance correlation between circRNAs and miRNAs together with the prediction of microRNA (miRNA) target sites in circRNAs suggested that circRNAs may act as miRNA sponges. Moreover, we found significant correlations between the abundance of 148 exonic circRNAs and sperm motility parameters. Two of these correlations, involving ssc&#95;circ&#95;1458 and ssc&#95;circ&#95;1321, were confirmed by RT-qPCR using 36 additional samples with extreme and opposite sperm motility values. Our study provides a thorough characterization of circRNAs in sperm and suggests that circRNAs hold potential as noninvasive biomarkers for sperm quality and male fertility.

Understanding the molecular basis of cell function and ultimate phenotypes is crucial for the development of biological markers. With this aim, several RNA-seq studies have been devoted to the characterization of the transcriptome of ejaculated spermatozoa in relation to sperm quality and fertility. Semen quality follows a seasonal pattern and decays in the summer months in several animal species. The aim of this study was to deeply profile the transcriptome of the boar sperm and to evaluate its seasonal changes. We sequenced the total and the short fractions of the sperm RNA from 10 Pietrain boars, 5 collected in summer and 5 five sampled in winter, and identified a complex and rich transcriptome with 4,436 coding genes of moderate to high abundance. Transcript fragmentation was high but less obvious in genes related to spermatogenesis, chromatin compaction and fertility. Short non-coding RNAs mostly included piwi-interacting RNAs, transfer RNAs and microRNAs. We also compared the transcriptome of the summer and the winter ejaculates and identified 34 coding genes and 7 microRNAs with a significantly distinct distribution. These genes were mostly related to oxidative stress, DNA damage and autophagy. This is the deepest characterization of the boar sperm transcriptome and the first study linking the transcriptome and the seasonal variability of semen quality in animals. The annotation described here can be used as a reference for the identification of markers of sperm quality in pigs.

Background: During cytoplasmic oocyte maturation, Ca(2+) currents are vital for regulating a broad range of physiological processes. Recent studies have demonstrated that DMSO and EG cause large transient increases in intracellular Ca(2+) in mouse oocytes. The CP used in vitrifying protocols also increases the intracellular calcium transient. The aim of this study is to evaluate the effects of vitrifying time (before and after IVM) and exposure to the vitrification solutions and ionomycin on oocyte quality and embryonic development., Methods: 221 GV-oocytes unsuitable for IVF-ICSI cycles were randomly distributed into one of the following three groups. G1 (control group): 41 GV-oocytes IVM until MII; G2: 43 oocytes vitrified at GV stage and IVM until MII stage; and G3: 53 GV-oocytes IVM until MII and then vitrified. In order to clarify the effect of vitrification solutions (VS) on human oocyte IVM through the intracellular Ca(2+) oscillation, the following two groups were also included. G4: 43 GV-oocytes exposed to VS and IVM until MII; and G5: 41 GV-oocytes exposed to ionomycin and IVM until MII. All GV-oocytes that reached MII-stage were parthenogenetically activated to assess oocyte viability. IVM was performed in IVF-medium (24-48 h). Chemical treatment (ionomycin) and osmotic treatment (vitrification solutions) were performed without liquid-N2 immersion. The following rates were evaluated: survival (SR), in-vitro maturation (IVMR), activation (AR), development to 2-cell (DRC), development to morula (DRCM) and development to blastocyst (DRB). Ratios between the different treatment groups were compared using contingency tables analysis (chi-square test)., Results: A high survival rate was obtained in G2 (95.5 %) and G4 (96.6 %). In-vitro maturation rate was significantly higher for G4 (86 %) and G2 (83.7 %) compared to G1 (63.4 %), G3 (56.6 %) and G5 (48.8 %). DRCM was significantly higher for G1 and G2 compared to G3 (G1: 15.8 %, G2: 20.7 % and G3: 0 %). DRB was only obtained for the oocytes vitrified before IVM (G2: 3.4 %). AR was also significantly higher for G2 and G4 compared to G5 (G2: 80.5 %, G4: 86.5 % and G5: 55 %). DRCM and DRB were only obtained in G2 and G4. DRCM was significantly higher for oocytes vitrified at GV stage (G2) and for oocytes exposed to the VS in G4 compared to the oocytes exposed to the ionomycin in G5 (G2: 20.7 %; G4: 37.5 % and G5: 0 %)., Conclusions: Vitrifying GV-oocytes improves their IVM. Further investigation could look to increase the oocyte pool and improve fertility preservation options.

The present study evaluated the effects of exposing liquid-stored boar semen to different red light LED regimens on sperm quality and reproductive performance. Of all of the tested photo-stimulation procedures, the best pattern consisted of 10 min light, 10 min rest and 10 min of further light (10-10-10 pattern). This pattern induced an intense and transient increase in the majority of motility parameters, without modifying sperm viability and acrosome integrity. While incubating non-photo-stimulated sperm at 37 °C for 90 min decreased all sperm quality parameters, this reduction was prevented when the previously-described light procedure was applied. This effect was concomitant with an increase in the percentage of sperm with high mitochondrial membrane potential. When sperm were subjected to 'in vitro' capacitation, photo-stimulation also increased the percentage of sperm with capacitation-like changes in membrane structure. On the other hand, treating commercial semen doses intended for artificial insemination with the 10-10-10 photo-stimulation pattern significantly increased farrowing rates and the number of both total and live-born piglets for parturition. Therefore, our results indicate that a precise photo-stimulation procedure is able to increase the fertilising ability of boar sperm via a mechanism that could be related to mitochondrial function.

The rearing of poultry for meat production (broilers) is an agricultural food industry with high relevance to the economy and development of some countries. Periodic episodes of extreme climatic conditions during the summer season can cause high mortality among birds, resulting in economic losses. In this context, ventilation systems within poultry houses play a critical role to ensure appropriate indoor climatic conditions. The objective of this study was to develop a multisensor system to evaluate the design of the ventilation system in broiler houses. A measurement system equipped with three types of sensors: air velocity, temperature and differential pressure was designed and built. The system consisted in a laptop, a data acquisition card, a multiplexor module and a set of 24 air temperature, 24 air velocity and two differential pressure sensors. The system was able to acquire up to a maximum of 128 signals simultaneously at 5 second intervals. The multisensor system was calibrated under laboratory conditions and it was then tested in field tests. Field tests were conducted in a commercial broiler farm under four different pressure and ventilation scenarios in two sections within the building. The calibration curves obtained under laboratory conditions showed similar regression coefficients among temperature, air velocity and pressure sensors and a high goodness fit (R(2) = 0.99) with the reference. Under field test conditions, the multisensor system showed a high number of input signals from different locations with minimum internal delay in acquiring signals. The variation among air velocity sensors was not significant. The developed multisensor system was able to integrate calibrated sensors of temperature, air velocity and differential pressure and operated successfully under different conditions in a mechanically-ventilated broiler farm. This system can be used to obtain quasi-instantaneous fields of the air velocity and temperature, as well as differential pressure maps to assess the design and functioning of ventilation system and as a verification and validation (V&V) system of Computational Fluid Dynamics (CFD) simulations in poultry farms.