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Summary Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique mainly used to overcome severe infertility problems associated with the male factor, but in cattle its efficiency is far from optimal. Artificial activation treatments combining ionomycin (Io) with 6-dimethylaminopurine after piezo-ICSI or anisomycin after conventional ICSI have recently increased the blastocyst rate obtained. Compounds to capacitate bovine spermatozoa, such as heparin and methyl-β-cyclodextrin and compounds to destabilize sperm membranes such as NaOH, lysolecithin and Triton X-100, have been assessed, although they have failed to substantially improve post-ICSI embryonic development. Disulfide bond reducing agents, such as dithiothreitol (DTT), dithiobutylamine and reduced glutathione, have been assessed to decondense the hypercondensed head of bovine spermatozoa, the two latter being more efficient than DTT and less harmful. Although piezo-directed ICSI without external activation has generated high fertilization rates and modest rates of early embryo development, other studies have required exogenous activation to improve the results. This manuscript thoroughly reviews the different strategies used in bovine ICSI to improve its efficiency and proposes some alternative approaches, such as the use of extracellular vesicles (EVs) as ‘biological methods of oocyte activation’ or the incorporation of EVs in the in vitro maturation and/or culture medium as antioxidant defence agents to improve the competence of the ooplasm, as well as a preincubation of the spermatozoa in estrous oviductal fluid to induce physiological capacitation and acrosome reaction before ICSI, and the use of hyaluronate in the sperm immobilization medium.

Summary Over the last decades, extracellular vesicles (EVs) have been found to be implicated in a complex universal mechanism of communication between different cell types. EVs are nanostructures of lipid nature that have an exosomal or ectosomal biogenesis, responsible for the intercellular transport of proteins, lipids, carbohydrates, nucleic acids, ions, among other molecules. The content of EVs can vary due to various factors such as hormonal stimuli, non-physiological conditions, metabolic state, etc. Once EVs reach their target cell, they can modulate processes such as gene expression, metabolism, response to external factors, and can even be associated with the delivery of molecules involved in epigenetic inheritance processes in germ cells. In mammalian reproduction, EVs have been shown to play an important role, either in vivo or in vitro, modulating a variety of processes in sperm, oocytes and embryos, and in their respective environments. Moreover, EVs represent a biodegradable, harmless and specific vehicle, which makes them attractive allies to consider when improving assisted reproductive technologies (ARTs). Therefore, the present review aims to describe the content of the main EVs involved in mammalian reproduction and how they can vary due to different factors, as well as to detail how EVs modulate, directly or indirectly, different molecular processes in gametes and embryos. In addition, we will highlight the mechanisms that remain to be elucidated. We will also propose new perspectives according to the characteristics of each particular EV to improve the different ARTs.

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Sperm sexing is a technology that can generate great economic benefits in the animal production sector. Techniques such as sex-sorting promise over 90% accuracy in sperm sexing. However, for the correct standardization of the technique, some laboratory methodologies are required. The present manuscript describes in detail a standardized equine sperm sex-sorting protocol using an absolute qPCR-based methodology. Furthermore, the results of absolute qPCR were implemented and validated by generating equine/bovine heterologous embryos by intracytoplasmic sperm injection (ICSI) of presumably sexed equine spermatozoa into bovine oocytes using a piezoelectric system (Piezo-ICSI). Our results indicated that equine sex-sorting spermatozoa had a 97% and 94% certainty for X and Y sperm, respectively, while presumptive female and male equine/bovine hybrid embryos, generated by Piezo-ICSI, had an accuracy of 92% with respect to the desired sex. Therefore, it is concluded that the presented methodology is a reliable, cost-effective, and relatively simple option for standardizing sex-sorting of equine spermatozoa. This is supported by the results of the correct sexing of Piezo-ICSI heterologous embryos generated with the sexed spermatozoa, validating the correct sexing and viability of these gametes.

This study aimed to analyze the effects on the lipidome of juvenile Oncorhynchus mykiss muscle fed 90% Brassica napus “rapeseed” oil and different amounts of Durvillaea antarctica “Cochayuyo” meal (1.5, 3 and 6%) as a replacement for cellulose. The analysis allowed for the identification of 329 lipids, mainly represented by phospholipids and fatty esters. The inclusion of Brassica napus oil significantly increased the levels of C18:2 species and fatty esters of hydroxylated fatty acids, which could play a bioactive role in human health. One of the most abundant lipids in all fillets was Phosphatidylcholine 33:6, which, according to the literature, could be considered a biomarker for the identification of Oncorhynchus mykiss. In all experimental diets, the species Phosphatidylethanolamine 15:1-18:24 showed four-fold higher levels than the control; increments of n-3- and n-6-rich phospholipids were also observed. Diets containing Durvillaea antarctica meal did not generate more significant variation in fish muscle phospholipids relative to the muscle of the rapeseed-oil-only group. These lipid species consist of medium- and long-chain fatty acids with different degrees of unsaturation. Still, it appears that the rapeseed oil masks the lipid contribution of the meal, possibly due to the low levels of total lipids in the macroalgae.

The FASN enzyme plays a key role in fatty acids synthesis as the main long-chain fatty acid synthesizer. A non-synonymous SNP (single nucleotide polymorphism) (g.17925A>G) located in the thioesterase domain of this enzyme and an effect in fat deposition has been observed, but has not been evaluated in this breed and, moreover, the reason whereby this occurs remains unclear. The objective of this study was to evaluate the effect of this SNP on the activity of FASN enzyme in subcutaneous and intramuscular adipose tissue from Holstein Friesian steers. To achieve this, 196 animals were sampled in a local abattoir, genotyped for the FASN g.17924A>G SNP and characterized for fatty acid profile. Then a sub sample of 20 animals per genotype were selected to extract the total protein from subcutaneous and intramuscular adipose tissue to estimate the FASN enzyme activity. The FASN activity for each genotyped animal was assessed indirectly by measuring the decrease in the absorbance of NADPH at 340 nm by spectrophotometry in a 24 well plate in the presence of Acetyl-CoA, Malonyl-CoA, and NADPH. To assess the impact of SNP induced amino acid changes in FASN protein structure, in-silico simulations were performed. Our results indicated that FASN g.17924A>G SNP induces a change in the enzyme activity in subcutaneous adipose tissue, which is higher when the AA genotype is present and lower in the presence of the AG genotype. The in-silico analysis of the amino acid substitution shows that there was a structural change in the dimeric form of the protein between genotypes. Moreover, the global energy between subunits is lower and more favorable when the AA genotype is present and higher and less favorable for the AG genotype. It was also found that the fatty acid profile of subcutaneous adipose tissue was affected when the AG genotype was present, decreasing the C16:0 fatty acid levels and increasing the C18:0 fatty acid levels. The FASN g.17924A>G SNP alters the FASN enzyme structure and activity, leading to a variation in the fatty acid composition of subcutaneous adipose tissue in Holstein Friesian steers. Implications: This SNP could be considered as a tool to improve the fat deposition or marbling and the fatty acid profile in cattle.