Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

Review: Effect of Experimental Diets on the Microbiome of Productive Animals.

The microorganisms that inhabit the gastrointestinal tract are responsible for multiple chains of reactions that affect their environment and modify the internal metabolism, their study receives the name of microbiome, which has become more relevant in recent years. In the near future, the challenges related to feeding are anticipated to escalate, encompassing the nutritional needs to sustain an overpopulated world. Therefore, it is expected that a better understanding of the interactions between microorganisms within the digestive tract will allow their modulation in order to provide an improvement in the immune system, feed efficiency or the promotion of nutritional characteristics in production animals, among others. In the present study, the main effects of experimental diets in production animals were described, emphasizing the diversity of the bacterial populations found in response to the diets, ordering them between polygastric and monogastric animals, and then describing the experimental diets used and their effect on the microorganisms. It is hoped that this study will help as a first general approach to the study of the role of the microbiome in production animals under different diets.

Maqui (Aristotelia chilensis (Mol.) Stuntz): A Natural Antioxidant to Improve Quality of Meat Patties.

Aristotelia chilensis is an endemic shrub of the South Pacific with high concentrations of bioactive compounds in its leaves and, therefore, it is highly valued. The effect of Aristotelia chilensis leaf powders (maqui leaf powders; Ma) on the quality and shelf life of beef patties during 7 days of storage was investigated. Five beef patties treatments were prepared: (1) Control without antioxidants (CT); (2) Beef patties with synthetic antioxidants plus color (250 mg/kg) (PL); (3) Beef patties with 500 ppm of maqui leaf powders (Ma500); (4) Beef patties with 1000 ppm of maqui leaf powders (Ma1000); and (5) Beef patties with 2000 ppm of maqui leaf powders (Ma2000). The quality of the beef patties was evaluated on day 0 and day 7 of storage by physicochemical analysis (moisture, ash and lipid content, color, pH, fatty acid profile and lipid oxidation) and organoleptic analysis. The addition of maqui leaf powders did not produce changes in the proximate composition of the beef patties. The pH for all treatments showed a range of 5.50−5.75 and significant differences (p < 0.05) were observed at the beginning and end of storage. The pH of the control beef patties increased during storage while the pH of the beef patties with synthetic and natural antioxidants decreased. Redness (a*) was the color indicator that was mostly affected by the inclusion of 1000 ppm and 2000 ppm powders. High lipid oxidation was observed in control samples on the seventh day of storage due to the high percentage of fat used in the formulation and the absence of any antioxidant. However, the Ma500, Ma1000, and Ma2000 treatments presented the lowest lipid oxidation rates (42.05%, 40.29%, and 43.14%, respectively) in comparison with the synthetic antioxidant (52.23%). This lipid inhibition is related to the strong antioxidant activity (29.75 µg/mL IC50 DPPH) of the maqui leaf powder due to its high content of total polyphenols (148.76 mg GAE/g), mainly characterized by having great amounts of hydroxybenzoic acids (82.5 mg GAE/g), flavonoids (7.1 mg QE/g), and hydroxycinnamic acids (3.7 mg CAE/g). Although minimal variations were observed in some individual fatty acids, and despite the trend to decrease MUFA and increase SFA with the maqui leaf powder addition, these differences were minimal and, according to the nutritional indices results, without any influence on the nutritional quality of the beef patties. The organoleptic analysis showed that the addition of maqui leaf powders did not affect the general acceptability of the new formulations. This study reports for the first time the substitution of synthetic antioxidants with Aristotelia chilensis leaves extract. Based on the results, it can be concluded that this ingredient can be used as an alternative for the production of raw meat products with clean labels.

Analysis of Muscle Lipidome in Juvenile Rainbow Trout Fed Rapeseed Oil and Cochayuyo Meal.

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.

Natural Antioxidants from Endemic Leaves in the Elaboration of Processed Meat Products: Current Status.

During the last few years, consumers' demand for animal protein and healthier meat products has increased considerably. This has motivated researchers of the meat industry to create products that present healthier components while maintaining their safety, sensory characteristics, and shelf life. Concerning this, natural plant extracts have gained prominence because they can act as antioxidants and antimicrobials, increasing the stability and shelf life of processed meat products. It has been observed that the leaves of plant species (Moringa oleifera, Bidens pilosa, Eugenia uniflora, Olea europea, Prunus cerasus, Ribes nigrum, etc.) have a higher concentration and variety of polyphenols than other parts of the plants, such as fruits and stems. In Chile, there are two native berries, maqui (Aristotelia chilensis) and murtilla (Ugni molinae Turcz), that that stand out for their high concentrations of polyphenols. Recently, their polyphenols have been characterized, demonstrating their potential antioxidant and antimicrobial action and their bioactive action at cellular level. However, to date, there is little information on their use in the elaboration of meat products. Therefore, the objective of this review is to compile the most current data on the use of polyphenols from leaves of native plants in the elaboration of meat products and their effect on the oxidation, stability, and organoleptic characteristics during the shelf life of these products.

Effect of exogenous lipids on cryotolerance of Atlantic salmon (Salmo salar) spermatozoa.

The development of semen cryopreservation strategies is necessary to improve the semen storage technologies of species of great commercial interest for aquaculture. Recent studies demonstrate that lipids play an important role in the fertility and cryotolerance of fish gametes. This study investigated the effect of exogenous lipids in the freezing medium on the post-thaw functional parameters of Salmo salar spermatozoa. Semen samples (n = 12) were incubated in standard extender supplemented with different concentrations of oleic acid (OA, C18:1n9), linoleic acid (LA, C18:2n6), arachidonic acid (ARA, C20:4n6) and cholesterol-loaded cyclodextrin (CLC). Post-thaw motility, membrane integrity, mitochondrial membrane potential (ΔΨm), superoxide anion (O2•-) and fertility rates were analyzed. The results revealed that the semen incubated with 0.003 mmol/L OA increased the motility (~7%) and ΔΨm (~2%) (P < 0.05), but membrane integrity and fertility were not increased. The addition of 0.003 mmol/L LA increased the motility (~4%) and all LA extenders increased the ΔΨm (P < 0.05); however, LA increased the O2•- levels and decreased the membrane integrity and fertility (P < 0.05). Semen incubated with ARA improved sperm motility (~5%), membrane integrity (~10.5%) and fertility rates (~11%) (P < 0.05). The maximum improvement in post-thaw sperm functionality was observed by adding 0.003 mmol/L ARA. In contrast, sperm quality parameters and fertility were decreased by the CLC addition (P < 0.05). This study showed that ARA could be considered as an additive for semen cryopreservation and could be relevant in the reproductive process and reproductive management of Salmo salar.

Effect of in vitro cold acclimation of Deschampsia antarctica on the accumulation of proteins with antifreeze activity.

Deschampsia antarctica has managed to colonize the maritime Antarctic. One of the main factors associated with its tolerance to low temperatures is the presence of apoplastic proteins with antifreeze activity. This work focuses on the effect of cold acclimation of D. antarctica on the accumulation of apoplastic proteins with antifreeze activity. Antifreeze proteins present in apoplastic extracts were purified by ice affinity purification, and their identity was determined by protein sequencing. D. antarctica plants were subjected to 22 days of cold acclimation at 4 °C. The highest content of apoplastic proteins with antifreeze activity was obtained at between 12 and 16 days of acclimation. Protein sequencing allowed their identification with >95% probability. Percentage coverage was 74% with D. antarctica ice recrystallization inhibition protein 1 (DaIRIP1) and 55% with DaIRIP3. Cold acclimation of D. antarctica improved the yield of apoplastic proteins, and resulted in an increase in the antifreeze activity of apoplastic extracts. An in silico analysis suggested that the fluctuations presented by the three-dimensional structures of DaIRIPs help to explain the presence of certain DaIRIPs in apoplastic extracts under the cold acclimation conditions evaluated.

Changes in Atlantic salmon (Salmo salar) sperm morphology and membrane lipid composition related to cold storage and cryopreservation.

The cold storage and cryopreservation of semen decrease sperm quality. Morphological and biochemical analyses of spermatozoa provide valuable information for the optimization of storage protocols to obtain a sufficient number of spermatozoa for in vitro fertilization. The aim of this study was to evaluate the morphology and lipid composition of Atlantic salmon (Salmo salar) spermatozoa after storage at 4 °C and cryopreservation. Semen samples were obtained by stripping. One aliquot was stored at 4 °C for 7 days, and another aliquot was cryopreserved. The morphology and ultrastructure were analysed using electron microscopy. The lipid composition was analysed by gas chromatography and a commercial kit. After cold storage, the mitochondrion was the most affected component; however, plasma membrane rupture and detachment of the flagellum were also observed. Morphological abnormalities were greater in cryopreserved spermatozoa. The head and mid-piece were dehydrated, sperm membranes were vesiculated, and alterations of mitochondria were observed. After cold storage and cryopreservation, there were less polyunsaturated and omega-3 fatty acids. Furthermore, there was an increase in saturated fatty acids and decrease in cholesterol concentration after cryopreservation (P < 0.05). Based on the results, cryopreservation drastically damaged sperm membranes; the cryogenic damage was associated with membrane lipid composition alterations. The sperm membranes were affected less by cold storage but there was also a decrease of some lipids; therefore, there is a need for improvement in cold storage processes to decrease structural damage of spermatozoa so that semen cryopreservation can be effectively used in the salmon industry.

Dietary inclusion of fish oil changes the semen lipid composition but does not improve the post-thaw semen quality of ram spermatozoa.

The aim of this study was to investigate the effects of dietary fish oil (FO) time-response on the fatty acid profile, cholesterol levels and sperm cryosurvival in ram semen. Criollo Araucano rams were randomly assigned to two groups (n=4) according to the type of supplementation: a control group without FO and a supplemented group fed a diet with 3% FO for 8 weeks. The semen lipid profile and post-thaw sperm quality were analyzed at weeks 0 (pre-supplementation), 4, 8, 12 and 16 (post-supplementation) to evaluate the effects of FO supplementation by time interaction. Post-thaw sperm quality was determined by CASA and flow cytometry. In spermatozoa, the supplemented group increased the linoleic acid (C18:2n6c) and docosahexaenoic acid (DHA; C22:6n3) with levels higher at week 16 (P<0.05). The effect of FO on cholesterol concentration in sperm was significant at the end of the experiment (week 16). In seminal plasma, statistical differences of butyric acid (C4:0), palmitic acid (C16:0), stearic acid (C18:0), eicosatrienoic acid (C20:3n3) and DHA were observed at week 12. The cholesterol concentration was not affected by dietary treatments (P>0.05). However, the post-thaw sperm quality of the FO treatment group decreased. Motility percentage decreased 50% and spermatozoa with permeable plasma membrane and reacted acrosome were higher (63%) at week 16 than the control group. These results showed that DHA was effectively incorporated into semen through dietary supplementation with FO, but evaluations of post-thaw sperm quality confirm alteration specificity related to the structure of the lipid bilayer.

Polyunsaturated fatty acid induces cardioprotection against ischemia-reperfusion through the inhibition of NF-kappaB and induction of Nrf2.

The mechanistic evidence to support the cardioprotective effects of polyunsaturated fatty acids (PUFA) are controversial. The aim was to test cardioprotective mechanisms induced by PUFA supplementation against cardiac ischemia-reperfusion (IR) injury. Ten-week-old male Wistar rats (225 ± 14 g, n = 14) were divided in two groups: rats without supplementation ( n = 7) and a PUFA group, supplemented by PUFA (0.6 g/kg/day; DHA:EPA = 3:1) for eight weeks ( n = 7). Hearts were perfused with Krebs-Henseleit buffer for 20 min (control conditions); others were subjected to control conditions, 30 min of global ischemia and 120 min of reperfusion (IR group). Infarct size (IS) and left ventricular developed pressure (LVDP) were measured at 120 min of reperfusion. Oxidative stress biomarkers (TBARS, total carbonyls), antioxidant status (CAT, catalase; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase activity and GSH/GSSG ratio), myeloperoxidase activity, ATP levels and nuclear transcription factor erythroid 2-related factor 2 (Nrf2) and nuclear factor kappaB (NF-κB) were determined in both experimental conditions. At the end of reperfusion, hearts supplemented with PUFA showed lower IS and a higher LVDP compared with the nonsupplemented rats. Hearts in the group supplemented with PUFA showed lower levels of oxidative stress markers and higher antioxidant activity, decreased MPO activity and NF-κB and Nrf2 activation compared with the nonsupplemented group. Cardioprotective effects of PUFA are exerted through induction of anti-inflammatory and antioxidant mechanism at tissue level.