Genetic parameters of colostrum traits in Holstein dairy cows.

The main objective of this study was to assess the genetic background of colostrum yield and quality traits after calving in Holstein dairy cows. The secondary objective was to investigate genetic and phenotypic correlations among laboratory-based and on-farm-measured colostrum traits. The study was conducted in 10 commercial dairy herds located in northern Greece. A total of 1,074 healthy Holstein cows with detailed pedigree information were examined from February 2015 to September 2016. All cows were clinically examined on the day of calving and scored for body condition. All 4 quarters were machine-milked, and a representative and composite colostrum sample was collected and examined. Colostrum total solids (TS) content was determined on-farm using a digital Brix refractometer. Colostrum fat, protein, and lactose contents were determined using an infrared milk analyzer, and energy content was calculated using National Research Council (2001) equations. Dry period length (for cows of parity ≥2), milk yield of previous 305-d lactation (for cows of parity ≥2), age at calving, parity number, season of calving, time interval between calving and first colostrum milking, and milk yield were recorded. Each trait (colostrum yield and quality traits) was analyzed with a univariate mixed model, including fixed effects of previously mentioned factors and the random animal additive genetic effect. All available pedigrees were included in the analysis, bringing the total animal number to 5,662. Estimates of (co)variance components were used to calculate heritability for each trait. Correlations among colostrum traits were estimated with bivariate analysis using the same model. Mean percentage (±SD) colostrum TS, fat, protein, and lactose contents were 25.8 ± 4.7, 6.4 ± 3.3, 17.8 ± 4.0, and 2.2 ± 0.7%, respectively; mean energy content was 1.35 ± 0.3 Mcal/kg and mean colostrum yield was 6.18 ± 3.77 kg. Heritability estimates for the above colostrum traits were 0.27, 0.21, 0.19, 0.15, 0.22, and 0.04, respectively. Several significant genetic and phenotypic correlations were derived. The genetic correlation of TS content measured on-farm with colostrum protein was practically unity, whereas the correlation with energy content was moderate (0.61). Fat content had no genetic correlation with TS content; their phenotypic correlation was positive and low. Colostrum yield was not correlated genetically with any of the other traits. In conclusion, colostrum quality traits are heritable and can be amended with genetic selection.

Protective effect of crocetin on bovine spermatozoa against oxidative stress during in vitro fertilization.

Oxidative stress is one of the major factors that contribute to poor semen quality and low rates of in vitro fertilization. Crocetin, a main constituent of saffron (Crocus sativus L.) possesses potent antioxidant activity, by scavenging reactive oxygen species (ROS) and/or enhancing the activity of intracellular antioxidant enzymes. The aim of this study was to investigate, for the first time, the effect of crocetin on the quality characteristics of bull spermatozoa and fertilization rate. For this reason, frozen/thawed bovine spermatozoa were incubated with crocetin (1, 2.5, and 5 µm), for 120 or 240 min, in the presence of a negative control, and evaluated in terms of motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation. In order to evaluate the impact of crocetin on cleavage and blastocyst rate, the compound was added in the IVF medium at the previously identified optimal concentration (2.5 µm). The results indicate that incubation of spermatozoa with 2.5 µm of crocetin resulted in a statistically significant lower production of superoxide anion and hydrogen peroxide, lower lipid peroxidation, and in better maintenance of motility parameters, viability, and acrosomal integrity, with a very small number of cells with DNA fragmentation, compared to the other groups (p < 0.05). The presence of crocetin (2.5 µm) in the fertilization medium also resulted in a significant increase in acrosome-reacted spermatozoa and blastocyst production, compared to the control group (p < 0.01). These data indicate that crocetin (2.5 µm) positively affects bovine sperm quality characteristics during a 240-min incubation and improves its fertilizing ability, directly and/or indirectly, by regulating ROS concentration and lipid peroxidation.

Antioxidant effect of crocin on bovine sperm quality and in vitro fertilization.

Reactive oxygen species (ROS) production above critical levels affects the genetic and functional integrity of spermatozoa by causing oxidative stress. Spermatozoa are susceptible to oxidative stress in terms of motility and fertilization capacity. Crocin (crocetin di-gentiobiose ester), a main constituent of Crocus Sativus L. (saffron), is known for its antioxidant activity by scavenging ROS, especially superoxide anion. The aim of the present study is to evaluate the effect of crocin on the quality characteristics of spermatozoa and fertilization rate. Frozen-thawed and washed spermatozoa from four different bulls were incubated with three different concentrations of crocin (0.5, 1, and 2 mM), for 120 and 240 minutes, in the presence of a negative control, and were evaluated in terms of motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation. The most potent concentration of crocin (1 mM) was also added in the fertilization medium to test its impact on fertilization outcome. The results indicate that the incubation of spermatozoa with 1 mM of crocin resulted in a statistically significant lower production of ROS, lower lipid peroxidation and in better maintenance of motility, viability, and acrosomal integrity, with a very small number of fragmented cells, compared to the control and the other treated groups (P < 0.05). Crocin concentration of 1 mM resulted in a significant increase of blastocyst rate, compared to the control group (P < 0.01). These data indicate that crocin (1 mM) improves bovine sperm quality and its fertilization capability, directly and/or indirectly, by modulating ROS concentration.

Olive leaves (Olea europea L.) and α-tocopheryl acetate as feed antioxidants for improving the oxidative stability of α-linolenic acid-enriched eggs.

Ninety-six brown Lohmann laying hens were equally assigned into four groups with six replicates. Hens within the control group were fed a corn-soybean-based diet supplemented with 4% linseed oil. Two other groups were given the same diet further supplemented with 5 or 10 g ground olive leaves/kg feed, while the diet of the fourth group was further supplemented with 200 mg α-tocopheryl acetate/kg. Supplementing diets with olive leaves had no effect on egg production, feed intake and egg traits. Eggs collected 28 days after feeding the experimental diets were analysed for lipid hydroperoxides and malondialdehyde (MDA) content, fatty acid profile, α-tocopherol concentrations and susceptibility to iron-induced lipid oxidation. Olive leaves were also analysed for total and individual phenolics, and total flavonoids, whereas their antioxidant capacity was determined using both the DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2-azinobis3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity assays. Results showed that neither α-tocopheryl acetate nor olive leaves supplementation exerted (p>0.05) any effect on the fatty acid composition of n-3 eggs. Supplementing the diet with 5 g olive leaves/kg had no (p>0.05) effect on the hydroperoxide levels of n-3 eggs, while supplementing with 10 g olive leaves/kg or 200 mg α-tocopheryl acetate/kg, the lipid hydroperoxide levels were reduced (p≤0.05) compared to control. However, although hydroperoxides were reduced, MDA, a secondary lipid oxidation product, was not affected (p>0.05). Iron-induced lipid oxidation increased MDA values in eggs from all groups, the increase being higher (p≤0.05) in the control group and the group supplemented with 5 g olive leaves/kg. The group supplemented with 10 g olive leaves/kg presented MDA values lower (p≤0.05) than the control but higher (p≤0.05) than the α-tocopheryl acetate group, which presented MDA concentrations lower (p≤0.05) than all other experimental diets at all incubation time points.

Effect of supplementation of the laying hen diet with olive leaves (Olea europea L.) on lipid oxidation and fatty acid profile of α-linolenic acid enriched eggs during storage.

1. The aim of this study was to evaluate the effect of supplementation of the layer diet with olive leaves (Olea europea L.) on lipid oxidation and fatty acid profile of α-linolenic acid enriched eggs during refrigerated storage, and to compare this effect with α-tocopheryl acetate supplementation. 2. A total of 72 brown Lohmann laying hens, equally allocated to 3 groups, were fed on diets supplemented with 40 g/kg linseed oil, or linseed oil and olive leaves at 10 g/kg or linseed oil and α-tocopheryl acetate at 200 mg/kg. Collected eggs were analysed for fatty acid profile and lipid oxidation either fresh or following 60 d storage at 4°C. 3. Results showed that olive leaves or α-tocopheryl acetate supplementation reduced lipid hydroperoxide concentration in fresh eggs but had no effect on their fatty acid profile and malondialdehyde (MDA) content compared to controls. 4. Refrigerated storage for 60 d decreased the proportions of PUFAs but increased those of MUFAs in eggs from the control diet, whilst it had no effect on the fatty acid composition of eggs from the diets supplemented with olive leaves or α-tocopheryl acetate, which in turn showed decreased concentrations of lipid hydroperoxides and MDA.

Lipid oxidation of stored eggs enriched with very long chain n-3 fatty acids, as affected by dietary olive leaves (Olea europea L.) or α-tocopheryl acetate supplementation.

The antioxidant potential of dietary olive leaves or α-tocopheryl acetate supplementation on lipid oxidation of refrigerated stored hen eggs enriched with very long-chain n-3 fatty acids, was investigated. Ninety-six brown Lohmann laying hens, were equally assigned into three groups. Hens within the control group were given a typical diet containing 3% fish oil, whereas other groups were given the same diet further supplemented with 10 g ground olive leaves/kg feed or 200mg α-tocopheryl acetate/kg feed. Results showed that α-tocopheryl acetate or olive leaves supplementation had no significant effect on the fatty acid composition and malondialdehyde (MDA) levels of fresh eggs but reduced their lipid hydroperoxide levels compared to controls. Storage for 60 d decreased the proportions of polyunsaturated fatty acids (PUFAs) but increased those of monounsaturated fatty acids (MUFAs) in eggs from the control group, while had no effect on the fatty acid composition of the eggs from the other two groups, which showed decreased levels of lipid hydroperoxides and MDA. Therefore, the very long chain n-3 PUFAs in eggs were protected from undergoing deterioration partly by olive leaves supplementation and totally by α-tocopheryl acetate supplementation. In addition, incorporating tocopherols into eggs might also provide a source of tocopherols for the human diet.

Tissue residue depletion of oxytetracycline after repeated intramuscular administration of Oxysentin 100 in sheep.

The depletion profile of oxytetracycline was studied in healthy sheep after intramuscular administration of Oxysentin 100, given at a dose of 10 mg oxytetracycline per kg body weight once daily for 5 consecutive days. Five medicated sheep were slaughtered at 0, 2, 4, 6, 9 and 12 days postmedication, and injection site, muscle, fat, liver and kidney tissues were sampled and analysed using a liquid chromatographic method, which was fully validated for oxytetracycline and 4-epi-oxytetracycline. At day 0 postmedication, the concentrations of oxytetracycline marker residue (sum of oxytetracycline and 4-epi-oxytetracycline) in all tissues examined were at the mg/kg level. At day 2 postmedication, the concentrations of oxytetracycline marker residue in all injection site and kidney samples examined were higher than the corresponding maximum residue limits (MRLs) established by the European Union, while the concentrations in muscle and liver tissues of two and three out of five animals examined, respectively, were below the corresponding MRLs. At days 4 and 6 postmedication, concentrations of oxytetracycline marker residue above the MRLs were found only in the injection site, whereas at day 9 postmedication, all observations were below the corresponding MRLs.

Assimilation of cholesterol by yeast strains isolated from infant feces and Feta cheese.

Eight yeast strains isolated from infant feces and the traditional Greek Feta cheese, selected for their probiotic properties, were tested along with a commercially available strain of Saccharomyces boulardii for their ability to remove cholesterol from a growth medium (yeast extract glucose peptone broth) supplemented with 0.3% Oxgall. The amount of cholesterol removed during 72 h of growth at 37 degrees C revealed significant variations among the yeast strains examined. Two isolates from infant feces, namely Saccharomyces cerevisiae KK1 and Isaatchenkia orientalis KK5.Y.1 and one isolate from Feta cheese, namely S. cerevisiae 832, along with the commercial strain S. boulardii, were able to remove cholesterol from the growth medium after 48 h of incubation at 37 degrees C. However, Saccharomyces strains proved to be able to remove cholesterol even after 24 h of growth at 37 degrees C. The cholesterol removed from the growth medium was not metabolically degraded but was rather assimilated into the yeast cells. The ability to assimilate cholesterol in vitro and to tolerate low pH levels, gastric juice, and bile indicate that S. cerevisiae 832, and especially S. cerevisiae KK1 and I. orientalis KK5.Y.1 (being more bile and gastric juice tolerant because of their human origin) may be promising candidate strains for use as probiotics.

Effect of dietary oregano essential oil on performance of chickens and on iron-induced lipid oxidation of breast, thigh and abdominal fat tissues.

1. We studied the effect of dietary oregano essential oil (50 and 100 mg/kg of feed) on the performance of broilers, and determined the susceptibility of the resulting broiler meat to iron-induced lipid oxidation. 2. Performance of the birds was unaffected by the experimental diets. Therefore, dietary oregano oil exerted no growth-promoting effect on broilers. 3. Iron-induced lipid oxidation showed that as oregano oil increased in the diet, malondialdehyde values decreased in tissue samples, suggesting that the oil, particularly at 100 mg/kg of feed, exerted an antioxidant effect on chicken tissues. 4. Dietary alpha-tocopheryl acetate supplementation at 200 mg/kg of feed displayed greater antioxidant activity than oregano oil at either supplementation rate. 5. Thigh muscle was more susceptible to oxidation than breast muscle, although the former contained alpha-tocopherol at higher concentration. Muscle alpha-tocopherol is an important factor influencing lipid oxidation, but the influence of polyunsaturated fatty acids and content of pro-oxidants must be taken into consideration too.

The effect of dietary oregano essential oil on lipid oxidation in raw and cooked chicken during refrigerated storage.

The antioxidative effect of dietary supplementation with oregano essential oil on susceptibility of raw and cooked breast and thigh muscle meat of chickens to lipid oxidation during refrigerated storage for 9 days was investigated. Day-old chickens (n=80) were randomly divided into four groups and fed a basal diet containing 30 mg α-tocopheryl acetate kg(-1) feed as control, or basal diet plus 200 mg α-tocopheryl acetate kg(-1) feed, or basal diet plus 50 or 100 mg oregano essential oil kg(-1) for 38 days prior to slaughter. Lipid oxidation was assessed by monitoring malondialdehyde (MDA) formation in raw and cooked meat during 0, 3, 6 and 9 days of refrigerated storage, using the thiobarbituric acid (TBA) assay and third-order derivative spectrophotometry. Results showed that dietary oregano essential oil supplementation exerted antioxidative effects, the supplementation being most effective in retarding lipid oxidation in stored raw and cooked meat at the 100 mg oregano essential oil kg(-1) feed. However, dietary α-tocopheryl acetate supplementation at 200 mg kg(-1) feed displayed greater antioxidant activity than oregano treatments. Thigh muscle was more susceptible to oxidation compared to breast muscle in all treatments, although the former tissues contained α-tocopherol at markedly higher levels.

Rapid gas chromatographic method for simultaneous determination of cholesterol and alpha-tocopherol in eggs.

A new method was developed for simultaneous determination of cholesterol and alpha-tocopherol in eggs. It involves rapid and simple sample preparation accomplished in one tube and chromatographic separation that does not require derivatization of analytes. Total analysis time per sample is 40 min. Labor, cost, and use of hazardous chemicals are minimized. To ensure selectivity, accuracy, and precision, critical analytical parameters were investigated. Overall recoveries were 98.8 and 99.2% for cholesterol and alpha-tocopherol, respectively. Linearity was acceptable for both analytes (r = 0.9964 for cholesterol and 0.9996 for alpha-tocopherol) in the fortification range examined. Precision data based on within-day and between-days variation gave overall relative standard deviations of 2.0% for cholesterol and 7.0% for alpha-tocopherol. The method was applied successfully for quantitation of cholesterol and alpha-tocopherol in eggs.

Rapid determination of cholesterol in milk and milk products by direct saponification and capillary gas chromatography.

A simple method is described for the determination of cholesterol in milk and milk products. Samples (0.2 g) are saponified in capped tubes with 0.5 M methanolic KOH solution by heating for 15 min at 80 degrees C. Water is added to the mixtures, and the unsaponifiable fractions are extracted with hexane to be further analyzed by capillary gas chromatography. Because of the rapid sample preparation and gas chromatographic procedures, a single sample can be analyzed in 30 min. Overall recovery was 98.6%, and the linearity was excellent for the fortification range examined. Precision data that were based on the variation within and between days suggested an overall relative standard deviation value of 1.4%. The method has been successfully applied to quantitate cholesterol in a variety of milk products.

Albendazole-related drug residues in milk and their fate during cheesemaking, ripening, and storage.

The level and nature of the albendazole residues in milk of treated cows were determined as a function of the time of milking (12-h intervals), and the fate of those residues during cheesemaking, ripening, and storage was examined when the obtained milk was used for making Teleme cheese. Ion-pair liquid chromatographic analysis with fluorescence detection showed that the albendazole sulfoxide metabolite reached its maximum (523 +/- 199 micrograms/kg) at the 1st milking and declined below the detection limit by the 4th milking. The sulfone metabolite attained its highest level (812 +/- 99 micrograms/kg) more slowly (at the 2nd milking) and declined below detection limit by the 13th milking. The 2-aminosulfone metabolite, which was present in the milk obtained at the 1st milking, reached its maximum (128 +/- 36 micrograms/kg) at the 3rd milking, and slowly declined to a level below detection limit by the 15th milking. Whey and cheese analysis revealed that about 70% of each major metabolite initially present in milk could be distributed in the whey. The remaining 30% occurred in the cheese at residue levels higher than those initially present in the milk of the 1st or 2nd milking (688 versus 445 or 450 versus 230 micrograms/kg for albendazole sulfoxide; 890 versus 608 or 1502 versus 783 micrograms/kg for albendazole sulfone; 19 versus 15 or 161 versus 105 micrograms/kg for albendazole 2-aminosulfone). Ripening and storage of the cheeses made from milks from the 1st or 2nd milkings results in a decrease of the sulfoxide metabolite (to 225 or 206 micrograms/kg), an increase of the sulfone metabolite (to 1,181 or 1,893 micrograms/kg), and no effect on the 2-aminosulfone metabolite.

Lipid peroxidation of rat myocardial tissue following daunomycin administration.

Daunomycin-induced cardiotoxicity has been regarded to be the result of oxygen-mediated lipid peroxidation of cell membranes. The aim of the present work was to evaluate the extent of lipid peroxidation in rat heart after administration of this anticancer drug and, further, to examine possible activation of some endogenous antioxidant defense systems. Myocardial tissue from both control and drug-treated rats was tested for lipid peroxidation using a selective third-order derivative method that is based on the analysis of the free malondialdehyde produced. Determination of reduced/oxidized glutathione levels and measurement of the activity of DT-diaphorase, glutathione-S-transferase, glutathione reductase, glucose-6-phosphate dehydrogenase and NADPH-cytochrome P-450 reductase were also carried out using literature methods. Significant increase of malondialdehyde content, and DT-diaphorase and glutathione-S-transferase activities were found in myocardial tissue from daunomycin-treated rats. On the other hand, reduced and oxidized glutathione levels were significantly decreased while the activity of glutathione reductase, glucose-6-phosphate dehydrogenase and NADPH-cytochrome P-450 reductase remained unchanged after daunomycin administration. The results of the present study give further evidence that daunomycin can induce lipid peroxidation in heart. However, additional experimentation is needed in order to delineate the molecular details of this process as well as of the mechanisms evolved to limit it.

Determination of the marker residue of albendazole in cheese by ion-pair liquid chromatography and fluorescence detection.

A liquid chromatographic method is described that allows quantitative determination of the marker residue of albendazole in cheese. Samples were extracted with acetonitrile, and the extracts were defatted with hexane, evaporated to dryness, reconstituted in ethyl acetate, and purified by partitioning with phosphate buffer. Separation of the sulfoxide, sulfone, and 2-aminosulfone metabolites that constitute the marker residue of albendazole 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 of 290 and 320 nm, respectively. Overall recoveries ranged from 73.7 to 84.9%. Precision data based on variation within and between days suggested overall values for relative standard deviation of 3.0 to 3.9%. The sensitivity of the method permitted low limits of detection, particularly for the sulfone and 2-aminosulfone metabolites.

Trace analysis of albendazole and its sulphoxide and sulphone metabolites in milk by liquid chromatography.

Analytical methodology for determination of albendazole and its sulphoxide and sulphone metabolites in milk at levels down to 2-5 ng/ml has been developed. Extraction was carried out with ethyl acetate under alkaline conditions, and extracts were analyzed on a silica-based C18 column in the presence of positively-charged pairing ions. Accuracy data showed overall recoveries ranged from 78.4% to 100%, whereas precision data, based on within and between-day variation, suggested overall precision values better than 4.9%. The method was successfully applied to determine residues in milk of a dairy cow orally given albendazole.

Rapid quantitative screening assay of trace benzimidazole residues in milk by liquid chromatography.

A simple, rapid, and sensitive liquid chromatographic (LC) assay for quantitative screening of albendazole 2-aminosulfone, albendazole sulfoxide, oxibendazole, oxfendazole, albendazole sulfone, p-hydroxyfenbendazole, albendazole, mebendazole, fenbendazole sulfone, and fenbendazole residues in milk was developed. Samples are made basic (pH 10) and extracted with ethyl acetate. Extracts are partitioned with water, evaporated to dryness, reconstituted with mobile phase, and analyzed isocratically by ion-pair reversed-phase LC at 292 nm. Overall recoveries ranged from 79 to 100%. Linearity was excellent in the fortification range examined (5.3-200 ng/mL). Precision data, based on within- and between-days variations, suggested an overall relative standard deviation of 2.0 to 5.8%. The method was successfully used to quantitate albendazole and fenbendazole and metabolites in milk from 2 drug-treated dairy cows.

Rapid determination of amphotericin B in serum and urine by third-order derivative spectrophotometry.

A derivative spectrophotometric method for rapid monitoring of amphotericin B in serum and urine down to 30 ng/mliters is described. Samples are treated with acetonitrile, and amphotericin B is directly quantified in the crude extracts on the basis of the intensity of the peak that appears at 402 nm when the normal absorption spectrum is submitted to third-order derivative processing. Accuracy data suggested recoveries in the range of 84.3-94.9% for serum and 85.6-93.4% for urine. The precision of the method was better than 11.3% for serum and 9.2% for urine when samples contained as low as 29.6 ng/mliters of amphotericin B. Ease of applicability, short analysis time, low cost, and reliability are the main advantages of the method.

Rapid spectrophotometric method for analyzing natamycin in cheese and cheese rind.

A rapid spectrophotometric method for determining natamycin in cheese and cheese rind has been developed. Samples are homogenized with acidified aqueous acetonitrile and homogenates are filtered. Natamycin is directly quantitated in filtered extracts on the basis of the characteristic third-derivative trough at 322.6 nm. Additional cleanup of extracts is not required because derivative transformation of the conventional analytical band at around 319 nm eliminates spectral interferences from other compounds. The analysis is simple and can be completed in 6 min. The equipment is easily accessible because most modern spectrophotometers allow instant generation of derivative spectra. The method needs small amounts of solvents and has good analytical characteristics. Overall recovery was 98.4 +/- 0.7%, and linearity was excellent (r = 0.9998) in the range examined (0.5-20 mg/kg). Quantitation and detection-limits were estimated at 0.5 and 0.25 mg/kg, respectively. Precision statistics based on within-day and between-days variations suggest an overall relative standard deviation of 1.4%.

Rapid ion-pair liquid chromatographic method for the determination of fenbendazole in cows' milk.

A simple and rapid methodology for the analysis of fenbendazole residues in cows' milk, at levels down to 3 ng ml-1, has been developed. Samples are extracted with acetonitrile and centrifuged. The supernatant is de-fatted with isooctane, and mixed with dichloromethane. The separated aqueous layer is discarded, while the bottom organic layer is washed with a phosphate buffer (pH 10) and evaporated to dryness. The residue is dissolved in the mobile phase and analysed by ion-pair reversed-phase liquid chromatography, using octanesulfonate as the ion-pair reagent. Over-all recovery was found to be 99.0%, linearity was excellent and precision data based on within- and between-day variation suggested an over-all variation of 2.0%.