The addition of crocin in the freezing medium extender improves post-thaw semen quality.

Semen cryopreservation is arguably the most important method or technique contributing to the advancement of modern animal production. However, the quality of sperm after thawing is still highly variable. The addition of antioxidant compounds to the freezing medium has been used customarily to counteract the harmful effects of Reactive Oxygen Species (ROS) that are produced during the freeze/thaw process. Crocin, a potent antioxidant, improves the fertilizing capacity of spermatozoa. In this study, we evaluated the potential of crocin (0, 0.5 and 1 mM) as an extender additive to diminish the damaging effects of cryopreservation on bovine spermatozoa. Post-thaw semen quality was assessed by means of motility, viability and lipid peroxidation (LPO). We further investigated the effect of crocin supplementation upon freezing on sperm quality parameters during their incubation at 37°C for up to 2 hr. Overall, the data assessment indicates that crocin facilitated a general improvement of the quality of freeze/thawed spermatozoa, under the present experimental conditions. Crocin (1 mM) maintained a higher percentage of alive spermatozoa with intact acrosome with rapid and progressive motility, compared to the control extender. Moreover, the spermatozoa cryopreserved in the presence of crocin exhibited higher values in CASA kinematic parameters (VCL, VSL, VAP, ALH) immediately after thawing. Furthermore, the positive effect of crocin on motility parameters was also sustained over a period of 2 hr incubation at 37°C. This effect of crocin may be attributed to the observed inhibition of LPO during the incubation period. Thus, the results indicate that the addition of crocin (especially at a final concentration of 1 mM) in the freezing extender medium may benefit the preservation of the quality parameters of spermatozoa that are compromised by the freeze/thaw heat shock and the stress during handling for IVF or artificial insemination.

Effects of Dietary Supplementation of Salvia officinalis L. in Organic Laying Hens on Egg Quality, Yolk Oxidative Stability and Eggshell Microbiological Counts.

Aromatic plants of Labiatae family are used in poultry diets because of their antimicrobial and antioxidant activity. The notion is that hens raised in organic systems face several health and environmental challenges. Hence, the objective here was to assess hens' performances and the quality of their eggs in such systems following a dietary supplementation of Salvia officinalis L. in powder form. The experiments were conducted over two successive years (1 and 2). They lasted 16 weeks each and involved 198 laying hens aged 40 weeks old randomly assigned to three groups: Con (control diet), Sal-0.5%, and Sal-1.0% (diets supplemented with Salvia officinalis L. at 0.5% or 1.0%, respectively). The malondialdehyde (MDA) levels in egg yolks in year 2 were lower in both Sal-0.5% and Sal-1.0% compared to the Con (p < 0.05). The total number of Enterobacteriaceae in eggshells were lower in Sal-1.0% compared to the Con (p < 0.05) in both years. The results suggest that a dietary supplementation of Salvia officinalis L. at 1.0% improves the antioxidant status and reduces the microbial load of eggs produced in organic systems.

Evaluation of Factors Affecting Colostrum Quality and Quantity in Holstein Dairy Cattle.

The objective of this study was to conduct a large-scale investigation of colostrum composition and yield and an evaluation of factors affecting them. In this study, 1017 clinically healthy Holstein cows from 10 farms were used. The colostrum TS were measured using a digital Brix refractometer. Fat, protein and lactose content were determined using an infrared Milk Analyzer. Statistical analysis was conducted using a series of univariate general linear models. The mean (±SD) percentage of colostrum fat, protein, lactose and TS content were 6.37 (3.33), 17.83 (3.97), 2.15 (0.73) and 25.80 (4.68), respectively. Parity had a significant positive effect on the protein and TS content and a negative one on fat content. The time interval between calving and colostrum collection had a significant negative effect on the fat, protein and TS contents and a positive one on lactose. Colostrum yield had a significant negative effect on the protein and TS content, and it was affected by all factors considered. In addition to TS, the evaluation of the colostrum fat content appears essential when neonates' energy needs are considered. The Brix refractometer, an inexpensive and easy to use devise, can be used effectively in colostrum quality monitoring.

Synergistic, bacteriostatic effect of propolis and glycerol against Listeria monocytogenes in chocolate milk under refrigerated storage.

Propolis ethanolic extracts, with or without glycerol, were added into pasteurized, non-fat chocolate milk, which was artificially contaminated with Listeria monocytogenes. The addition of propolis ethanolic extracts dissolved into glycerol led to a definite anti-listerial effect in milk stored at 4 ℃, with both propolis concentrations tested (2 or 4 mg of dry propolis ethanolic extract per milliliter of chocolate milk) leading to inhibition of L. monocytogenes growth throughout 20 days of storage. The combined addition of propolis ethanolic extracts with glycerol was also effective in significantly reducing the rate of growth of L. monocytogenes in chocolate milk stored under improper (10 ℃) refrigeration storage conditions (more than five-fold increase in the generation time of L. monocytogenes compared to control trials). Finally, the combined addition of a deodorized propolis ethanolic extract with glycerol resulted in a significant anti-listerial effect upon storage of contaminated milk at 4 ℃ (more than three-fold increase in the generation time of L. monocytogenes compared to controls) and in a smaller anti-listerial effect upon milk storage at 10 ℃ (two-fold increase in the generation time of the pathogen compared to controls). Of note, chocolate milk containing deodorized propolis ethanolic extract and glycerol received a positive consumer acceptability score on the nine-point hedonic scale (median acceptability score of "7"). Hence, propolis may possess a promising role as a natural anti-listerial preservative in dairy drinks.

Modifications of Own Mothers' Milk Fortification Protocol Affect Early Plasma IGF-I and Ghrelin Levels in Preterm Infants. A Randomized Clinical Trial.

The aim was to investigate the effect of two own mother's milk (OMM) fortification protocols on (a) IGF-I and ghrelin plasma levels at 35 post-conceptional weeks (PCW, T2) and whether this effect is maintained after elimination of the differences in OMM fortification, and (b) growth until 12 months corrected age. Forty-eight OMM-fed preterm infants (GA 24-32 weeks) were randomly allocated to the fixed-fortification (FF) group (n = 23) and the protein-targeting fortification (PTF) group (n = 25) targeting the recommended daily protein intake (PI). Plasma IGF-I and ghrelin were assessed at 35 (T2) and 40 (T3) PCW while growth was longitudinally assessed until 12 months corrected age. PTF group had lower IGF-I and higher ghrelin than FF group at T2, while receiving lower daily protein and energy amounts. PI correlated positively to T2-IGF-I and inversely to T3-ghrelin while energy intake (EI) correlated inversely to T2- and T3-ghrelin. Group and PI were independent predictors of adjusted T2-IGF-I, while group and EI were predictors of adjusted and T2-ghrelin. Growth parameter z-scores were comparable between groups up to 12 months corrected age. Modifications of OMM fortification have a transient effect on early plasma IGF-I and ghrelin levels in preterm infants in a way consistent with the previously recognized protein-energy/endocrine balance, indicating a potential programming effect.

Inhibitory activity of propolis against Listeria monocytogenes in milk stored under refrigeration.

Propolis is a natural bee-product with documented antimicrobial properties in vitro. The objective of this study was to develop a protocol for adding propolis into milk and to determine whether the addition of propolis can confer anti-listerial activity during the storage of milk under optimal or improper refrigeration conditions. Upon dissolving propolis ethanolic extract (PEE) into glycerol, the PEE-glycerol mixture contained no visible insoluble particles and could be dispersed evenly into milk, without leaving any insoluble material. PEE, with or without glycerol, was added into extended shelf-life milk, artificially contaminated with Listeria monocytogenes. The addition of PEE dissolved into glycerol resulted in a pronounced and dose-dependent anti-listerial effect in milk stored at 4 °C, with the higher concentration tested (4 mg of dry PEE per mL of milk) resulting in complete inhibition of L. monocytogenes growth throughout 30 days of storage. The combination of PEE with glycerol was also effective in significantly reducing the growth rate of the pathogen in milk stored under improper refrigeration (10 °C). Based on a patented PEE-deodorization protocol, the addition of deodorized PEE into milk resulted in a product with average consumer acceptability. However, the PEE deodorization process resulted in reduction or even complete removal of propolis constituents with known antibacterial activity, with a concomitant significant reduction in its anti-listerial effect. Nonetheless, the data presented in this manuscript highlight the strong anti-listerial potential of propolis in milk and suggest that, upon further research on its deodorization and standardization, there may be room for the application of propolis as a natural preservative in dairy beverages.

Rapid ion-pair liquid chromatographic method for the determination of fenbendazole marker residue in fermented dairy products.

A simple, rapid and sensitive liquid chromatographic method that allows for the quantitative determination of fenbendazole residues in fermented dairy products is described. Samples were extracted with a mixture of acetonitrile-phosphoric acid and the extracts were defatted with hexane to be further partitioned into ethyl acetate. The organic layer was evaporated to dryness and the residue was reconstituted in mobile phase. Separation of fenbendazole and its sulphoxide, sulphone, and p-hydroxylated metabolites was carried out isocratically with a mobile phase containing both positively and negatively charged pairing ions. Overall recoveries ranged from 79.8 to 88.8%, while precision data, based on within and between days variations, suggested an overall relative standard deviation of 6.3-11.0%. The detection and quantification limits were lower than 9 and 21µg/kg, respectively. The method has been successfully applied to quantitate fenbendazole residues in Feta cheese and yoghurt made from spiked and incurred ovine milk.

Effect of olive leaf (Olea europea L.) extracts on protein and lipid oxidation of long-term frozen n-3 fatty acids-enriched pork patties.

Our previous study has demonstrated the protective effects of olive leaf extracts on the oxidation of pork patties from n-3 fatty acid-enriched meat during refrigerated storage. The target of the present study was to examine these effects during frozen storage. Results showed that frozen storage accelerated (P=0.05) both lipid and protein oxidation in pork patties, but an addition of olive leaf extract at 200mg gallic acid equivalent/kg improved sensory attributes by delaying oxidation of lipids (reduction (P=0.05) of conjugated dienes, hydroperoxides and malondialdehyde), and of proteins (reduction (P=0.05) of protein carbonyls and inhibition (P=0.05) of the decrease of protein sulfhydryls).

Effect of olive leaf (Olea europea L.) extracts on protein and lipid oxidation in cooked pork meat patties enriched with n-3 fatty acids.

BACKGROUND: The effect of olive leaf extracts on lipid and protein oxidation of cooked pork patties refrigerated stored for 9 days was evaluated. Patties were prepared from longissimus dorsi muscle of pigs, and dietary supplemented with linseed oil. RESULTS: Results showed that dietary linseed oil modified the fatty acid composition of pork patties by increasing (P ≤ 0.05) n-3 (α-linolenic acid) and decreasing (P ≤ 0.05) n-6 (linoleic acid) fatty acids. Olive leaf extracts at supplementation levels of 200 and, especially, of 300 mg gallic acid equivalents kg⁻¹ meat, delayed lipid oxidation by reducing (P ≤ 0.05) both primary (conjugated dienes and hydroperoxides) and secondary (malondialdehyde) oxidation products. They also inhibited protein oxidation in a concentration-dependent manner by reducing (P ≤ 0.05) protein carbonyls and increasing (P ≤ 0.05) protein sulfhydryls. In addition, they improved sensory attributes of the n-3 enriched patties. CONCLUSION: Results suggested that olive leaf extracts might be useful to the meat industry as an efficient alternative to synthetic antioxidants.

Olive leaves (Olea europaea L.) versus α-tocopheryl acetate as dietary supplements for enhancing the oxidative stability of eggs enriched with very-long-chain n-3 fatty acids.

BACKGROUND: Ninety-six brown Lohmann laying hens were equally assigned into four groups with six replicates. Hens within the control group were given a corn/soybean-based diet supplemented with 30 g kg(-1) fish oil. Two other groups were given the same diet further supplemented with olive leaves at 5 (OL5) and 10 (OL10) g kg(-1) respectively, while the diet of the fourth group was supplemented with α-tocopheryl acetate (TOC) at 200 mg kg(-1). Eggs were analysed for lipid hydroperoxide and malondialdehyde (MDA) contents, fatty acid profile, α-tocopherol content and susceptibility to iron-induced lipid oxidation. RESULTS: Neither OL nor TOC supplementation affected (P>0.05) the fatty acid composition. Dietary supplementation with OL10 or TOC reduced (P≤0.05) the lipid hydroperoxide content but exerted no (P>0.05) effect on the MDA content of fresh eggs compared with controls. Eggs submitted to iron-induced lipid oxidation from the OL5 group presented higher (P≤0.05) MDA levels than the control but lower (P≤0.05) than the OL10 group. Eggs from the TOC group presented lower (P≤0.05) MDA levels compared with all groups at all incubation time points. CONCLUSION: The results of this study suggested that dietary supplementation with both OL10 and TOC could protect n-3 fatty acids in eggs from deterioration.

Lipid and protein oxidation of α-linolenic acid-enriched pork during refrigerated storage as influenced by diet supplementation with olive leaves (Olea europea L.) or α-tocopheryl acetate.

The objective of this study was to evaluate the effect of diet supplementation with olive leaves or α-tocopheryl acetate on lipid and protein oxidation of raw and cooked n-3 enriched-pork during refrigerated storage. Enrichment of pork with α-linolenic acid through diet supplementation with linseed oil enhanced (p≤0.05) lipid oxidation in both raw and cooked chops but had no effect (p>0.05) on protein oxidation during refrigerated storage while decreasing (p≤0.05) the sensory attributes of cooked pork. Diet supplementation with olive leaves or α-tocopheryl acetate had no effect (p>0.05) on the fatty acid composition of pork but decreased (p≤0.05) lipid oxidation while exerting no effect (p>0.05) on protein oxidation in both raw and cooked α-linolenic acid-enriched chops stored and chilled for 9 days. Moreover, olive leaves and α-tocopheryl acetate supplemented at 10 g/kg and 200mg/kg diet, respectively, exerted (p≤0.05) a beneficial effect on the sensory attributes of cooked α-linolenic acid-enriched pork chops.

Endosulfan-induced lipid peroxidation in rat brain and its effect on t-PA and PAI-1: ameliorating effect of vitamins C and E.

Endosulfan provokes systemic toxicity in mammals and induces reactive oxygen species (ROS) and lipid peroxidation (LPO). The brain is susceptible to LPO and several studies implicate ROS and LPO in CNS diseases. Tissue plasminogen activator (t-PA) has been accredited with plasminogen-dependent roles in the CNS, as well as plasminogen-independent functions. The aim of this study was to investigate the activities of t-PA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1) in the adult rat brain, after subchronic endosulfan treatment. Furthermore, the potency of vitamins C and E to attenuate these effects was explored. Endosulfan was administered in Wistar rats either alone or with vitamin C and/or vitamin E. The induced oxidative stress was manifested by induction of LPO as determined by higher malondialdehyde levels. This was accompanied by elevation of t-PA and PAI-1 activities. Vitamins E and C, both well-known for their antioxidant properties, substantially acted in a preventive way and protected the brain from these effects.

Liquid chromatographic determination and depletion profile of oxytetracycline in milk after repeated intramuscular administration in sheep.

A simple, rapid and specific ion-pair liquid chromatographic method for the routine determination of the marker residue of oxytetracycline in sheep milk, at levels as low as 20 microg/kg, has been developed. Milk samples were acidified and extracted with acetonitrile. The extracts were purified by treatment with ammonium sulphate and concentrated into diluted phosphoric acid. Separation was carried out isocratically on a Nucleosil C(18) column using a mobile phase that contained both positively and negatively charged pairing ions. The in-house validated method gave overall recoveries and overall relative standard deviations better than 86% and 4.6%, respectively. The method was successfully applied to study the depletion of oxytetracycline in sheep milk and to estimate the withdrawal period after intramuscular administration of a commercial oxytetracycline formulation.

A new liquid chromatographic method for routine determination of oxytetracycline marker residue in the edible tissues of farm animals.

A simple, rapid, and specific ion-pair liquid chromatographic method for routine determination of the marker residue of oxytetracycline (OTC), namely OTC and 4-epi-oxytetracycline (4-epiOTC), in edible animal tissues (muscle, liver, kidney, and fat) has been developed. Minced tissue samples were acidified at pH 2.7 with 2 mol L(-1) sulfuric acid and extracted with acetonitrile. The extracts were purified by treatment with ammonium sulfate solution and concentrated into 0.1 mol L(-1) phosphoric acid. Baseline separation was carried out isocratically on a Nucleosil 100-5 C(18), 5-microm column using an acetonitrile-0.01 mol L(-1) disodium hydrogen phosphate (20:80, v/v) mobile phase that contained both positively (tetrabutylammonium) and negatively (octanesulfonate) charged pairing ions and EDTA, and was adjusted to pH 3.8. Detection was by UV at 370 nm. The method was fully validated according to Commission Decision 2002/657/EC. Overall recoveries were better than 82.6% and overall relative standard deviation was better than 6% for all the tissues examined. The good analytical characteristics of the method allowed limits of quantification as low as 30 ng g(-1) for muscle and fat and 50 ng g(-1) for liver and kidney, for both OTC and 4-epiOTC, to be realized. The method was successfully used to determine the OTC marker residue in tissues of two sheep intramuscularly administered a commercial OTC formulation.

Effect of rosemary extract, chitosan and α-tocopherol on lipid oxidation and colour stability during frozen storage of beef burgers.

The effect of rosemary extract, chitosan and α-tocopherol, added individually or in combination, on lipid oxidation and colour stability of frozen (-18°C) beef burgers stored for 180 days was investigated. The burgers' lipid oxidation and appearance were evaluated through measurement of primary (conjugated dienes and peroxide value) and secondary (malondialdehyde) oxidation products, together with visual assessment and instrumental measurement of colour. Chitosan alone and in combination with either rosemary or α-tocopherol had the best antioxidative effect (P⩽0.05) compared to individual use of rosemary or α-tocopherol, while the best results were obtained with the combination of chitosan and rosemary. The differences of antioxidative effects, however, between individual use of rosemary or α-tocopherol as compared to the controls were also significant (P⩽0.05). Chitosan added individually or in combination with either rosemary or α-tocopherol had also a noteworthy effect on the burgers' appearance as it contributed to red colour retention for a much longer period (P⩽0.05) compared all other treatments and the controls. In conclusion, the best antioxidative effects were obtained with the combination of chitosan and rosemary extract.

Highly sensitive ion pair liquid chromatographic determination of albendazole marker residue in animal tissues.

A simple, rapid, and highly sensitive ion pair liquid chromatographic method for the determination of albendazole sulfoxide, albendazole 2-aminosulfone, and albendazole sulfone, which constitute the marker residue of albendazole in animal tissues (muscle, fat, liver, and kidney), is described. Tissue samples were extracted with acetonitrile, and the extracts were partitioned, as ion pairs, into dichloromethane. The organic layer was evaporated to dryness, and the residue was reconstituted in phosphate buffer and extracted with ethyl acetate. Separation was carried out isocratically with a mobile phase containing both positively and negatively charged pairing ions. Detection was performed fluorometrically, with excitation and emission wavelengths set at 290 and 320 nm, respectively. Overall recoveries were better than 76%, and the overall relative standard deviation was better than 7.3% in all tissues examined. The limits of quantification were 20, 1, and 0.5 ng/g for sulfoxide, 2-aminosulfone, and sulfone metabolites, respectively. The method was successfully applied to determine residues in tissues of two sheep orally administered an albendazole formulation.