Relationship between the bacterial ocular surface microbiota and outcomes for cats with feline herpesvirus type 1 ocular surface disease.

OBJECTIVE: Feline herpesvirus 1 (FHV-1) causes ocular surface disease in domestic cats. The purpose of this study was to assess the relationship between bacterial ocular surface microbiota and outcomes for cats with FHV-1 ocular surface disease. ANIMALS STUDIED: Twenty-two shelter-housed cats with confirmed FHV-1 ocular surface disease. PROCEDURES: Animals were grouped according to FHV-1 shedding and ocular clinical scores following intervention: worsened outcome (WorOut, n = 11) or improved outcome (ImpOut, n = 11). Scoring and conjunctival sampling were completed on Days 1 and 8 of twice daily antiviral treatment. Bacterial DNA was extracted and submitted for 16S rRNA gene sequencing. Real-time polymerase chain reaction was performed for selected bacterial species. Overall DNA concentration between groups was assessed. RESULTS: Bacterial microbiota relative abundance composition was significantly different between ImpOut and WorOut groups (weighted UniFrac p = .006). Alpha diversity was significantly higher in the ImpOut group compared with the WorOut group (Shannon p = .042, Simpson's p = .022, Pielou's p = .037). Differences in the relative abundance of various phyla and species were detected between groups. Total DNA concentration was higher in the WorOut group compared with the ImpOut group (p = .04). Feline GAPDH (p = .001) and Bilophila wadsworthia (p = .024) copy number was significantly higher in the ImpOut group compared with the WorOut group. CONCLUSIONS: The results highlight the important relationship between the bacterial ocular surface microbiota and FHV-1 infection outcomes in cats treated with antiviral medications. Low bacterial species diversity, higher overall DNA (presumed predominantly bacterial) load, and certain bacterial phyla/species were associated with poor outcomes for cats with FHV-1 ocular disease.

Validation of a novel rebound tonometer (Tono-Vera® Vet) in normal ex vivo rabbit eyes.

OBJECTIVE: The purpose of this study was to validate the use of the Reichert Tono-Vera® Vet tonometer rabbit setting in normal ex vivo rabbit eyes and to compare the rabbit setting to the dog, cat, and horse settings of this tonometer. PROCEDURE: Six freshly enucleated normal rabbit eyes were cannulated and connected to a fluid reservoir and physiologic monitor. Triplicate measurements were obtained with the four available settings: dog, cat, horse, and rabbit at various intraocular pressures (IOP) ranging from 5 to 80 mmHg. Bland-Altman analysis was utilized to determine bias and 95% limits of agreement for each setting. RESULTS: Linear regression equations for the dog, horse, cat, and rabbit settings were y = 0.8101x + 2.5058, y = 0.7594x - 3.4673, y = 0.6635x + 0.3021, and y = 0.8935x + 1.3295, respectively. All settings demonstrated strong positive linear trends (dog r2 = 0.9644, horse r2 = 0.9456, cat r2 = 0.9309, and rabbit r2 = 0.9558). Bland-Altman plots revealed that the average bias and 95% limits of agreement (mmHg) were -4.73, -12.65, -12.86 and -2.73 and (-15.31, 5.86), (-29.03, 3.74), (-25.67, -0.05), and (-12.21, 6.76) for the dog, horse, cat, and rabbit settings, respectively. CONCLUSION: The Tono-Vera® Vet rabbit setting provided the most accurate and precise measurements compared with the other settings, but slightly underestimated actual IOP, especially as IOP was increased. This tonometer, using the rabbit setting, is likely to be appropriate for the estimation of IOP in rabbits with the appropriate correction formula applied.

Surveillance for feline herpesvirus type 1 mutation and development of resistance in cats treated with antiviral medications.

Feline herpesvirus type 1 (FHV-1) commonly causes ocular surface disease in cats and is treated with antiviral medications targeting viral DNA polymerase (UL30/42). Herein, we describe a method to assess the FHV-1 genome for mutation development and to assess the functional impact of mutations, if present. Fourteen shelter-housed domestic cats with FHV-1 ocular surface disease were assigned to one of four treatment groups: placebo (n = 3), cidofovir 0.5% ophthalmic solution (n = 3), famciclovir oral solution (n = 5), or ganciclovir 0.15% ophthalmic solution (n = 3). Swabs were collected before (day 1) and after (day 8) 1 week of twice-daily treatments to isolate viable FHV-1. Viral DNA was extracted for sequencing using Illumina MiSeq with subsequent genomic variant detection between paired day 1 and day 8 isolates. Plaque reduction assay was performed on paired isolates demonstrating non-synonymous variants. A total of 171 synonymous and 3 non-synonymous variants were identified in day 8 isolates. No variants were detected in viral UL23, UL30, or UL42 genes. Variant totals were not statistically different in animals receiving antiviral or placebo (p = 0.4997). A day 8 isolate from each antiviral treatment group contained a single non-synonymous variant in ICP4 (transcriptional regulator). These 3 isolates demonstrated no evidence of functional antiviral resistance when IC50 was assessed. Most (10/14 pairs) day 1 and 8 viral isolate pairs from the same host animal were near-identical. While functional variants were not detected in this small sample, these techniques can be replicated to assess FHV-1 isolates suspected of having developed resistance to antiviral medications.

Calibration of a novel rebound tonometer (Tono-Vera® vet) in the porcine eye.

OBJECTIVE: Porcine models of ocular disease are becoming increasingly utilized. A recently commercialized ocular tonometer, the Reichert Tono-Vera® Vet, has not been evaluated for use in pigs. The purpose of this study was to calibrate this device for use in porcine eyes and to determine which settings are most appropriate for use in pigs. PROCEDURE: The anterior chambers of five freshly enucleated normal porcine eyes were cannulated then connected to a reservoir of balanced salt solution and a physiologic monitor. Triplicate measurements were obtained with the four available settings: dog, cat, horse, and rabbit at intraocular pressures ranging from 5- to 80 mmHg. Bland-Altman analysis was utilized to determine bias and 95% limits of agreement for each setting. RESULTS: There was a strong positive linear regression trend for all settings (dog r2  = 0.986, horse r2  = 0.947, cat r2  = 0.977, and rabbit r2  = 0.982). The linear regression equations for the dog, horse, cat, and rabbit setting were y = 1.0168x - 2.6128, y = 0.8743x - 3.4959, y = 0.9394x - 7.3188, and y = 1.1082x - 3.4077. The average bias and 95% limits of agreement for dog, horse, cat, and rabbit settings were - 2.00, -8.32, -9.58, and 0.57 mmHg, and (-7.52, 3.53), (-19.00, 2.37), (-16.66, -2.50), and (-7.79, 8.93), in mmHg. CONCLUSION: The Tono-Vera® Vet dog setting was most accurate and precise setting compared to true intraocular pressures. This setting is likely to be appropriate for in vivo use in pigs, with the appropriate correction formula applied.

Effect of multiple head positions on intraocular pressure in healthy, anesthetized horses during hoisting.

OBJECTIVE: To evaluate changes in intraocular pressure (IOP) with variable head position in healthy, anesthetized horses in hoisted inversion and to assess the influence of various cofactors (age, sex, body weight, body condition score, and neck length) on IOP changes during hoisting. ANIMALS STUDIED: Seventeen healthy adult horses without significant ocular abnormalities. PROCEDURES: Subjects were administered intravenous xylazine/butorphanol premedication and ketamine/midazolam induction with xylazine/ketamine boluses for anesthetic maintenance. While hoisted, IOP was measured in triplicate for each eye via rebound tonometry (TonoVet) at neutral head position (ie, eyes level with the withers), at multiple 5 cm increments above and below neutral (-20 cm through +20 cm) using foam pads for head support, and with eyes above heart level via manual support. RESULTS: In hoisted positions, IOP ranged from 18 to 51 mmHg. Intraocular pressure significantly decreased with head position elevated ≥+15 cm from neutral and significantly increased when lowered ≤-5 cm from neutral. Neck length significantly influenced IOP (P = .0328) with linear regression indicating a median (range) increase of 0.244 (0.034-0.425) mmHg in IOP for every 1 cm increase in neck length. Age, sex, breed, body weight, body condition score, and eye (OD vs OS) did not significantly influence IOP. Intraocular pressure only varied significantly between eyes at +10 cm above neutral (OS > OD, 1.7 ± 0.6 mm Hg, P = .0044). CONCLUSIONS: Intraocular pressure in healthy, anesthetized horses varies with head position during hoisting; increased neck length may be associated with larger changes in IOP during hoisting.

In vitro efficacy of ganciclovir against feline herpesvirus type 1 and assessment of ocular tolerability in healthy cats.

OBJECTIVES: Feline herpesvirus-1 (FHV-1) is a prevalent cause of ocular disease in cats and limited topical options for treatment currently exist. The first objective of this study was to confirm the efficacy of ganciclovir against FHV-1 in vitro. The second objective was to assess the safety and ocular tolerability of topically applied ganciclovir eye gel (GEG) in healthy cats. METHODS: FHV-1 was used to infect tissue culture wells covered in maximally confluent Crandall-Rees feline kidney cells prior to the addition of three molarities of ganciclovir (8.9 µM, 17.8 µM and 89 µM) before being incubated for 48 h. Ganciclovir efficacy in vitro was then assessed using standard plaque reduction assay. Commercially available GEG (0.15%) was applied q8h to one randomly chosen eye of four healthy cats for 7 days. Commercially available lubricating eye gel (LEG) was applied to the opposite eye q8h. Complete blood counts (CBCs), blood chemistry panels (CHEM) and urinalysis (UA) were performed on all cats before and after the study period. Ocular lesions were assessed daily using a standardized scheme. RESULTS: Ganciclovir led to a significant reduction in FHV-1 plaque number, area and diameter at all tested molarities in vitro. The highest molarity assessed (89 µM) caused a 100% reduction in viral plaque number. There was no significant difference in ocular lesion scores between eyes receiving GEG and LEG. Animals remained healthy throughout the study period with CBC, CHEM and UA showing no clinically significant alterations. CONCLUSIONS AND RELEVANCE: Based on the in vitro results, ganciclovir appears to be effective against FHV-1 in vitro. When applied q8h as a commercial 0.15% gel to a small group of cats with normal eyes, this medication was well tolerated. Taken together, these data suggest this medication warrants further investigation in cats with ocular disease caused by FHV-1.

Ocular findings in a group of healthy captive leopard geckos.

OBJECTIVE: Leopard Geckos (Eublepharis macularius) are popular pets and can be affected by a range of ocular disorders. Our objective was to report ocular findings in a group of healthy captive leopard geckos and to establish reference ranges for commonly performed ocular diagnostic tests. ANIMALS STUDIED: Twenty-six healthy male geckos aged 1 year old (n = 4) and >2 years old (n = 22). PROCEDURES: All animals underwent ophthalmic examination, corneal esthesiometry, modified Schirmer tear test (mSTT), rebound tonometry, conjunctival bacterial aerobic and fungal culture, and measurement of ocular dimensions. Student's t test was used to compare values of corneal esthesiometry, tonometry and mSTT between groups. Multiple correlations were assessed by Pearson correlation coefficient. RESULTS: All animals had a normal ocular examination. Tear production as measured with a mSTT (mean ± SD) technique was 3.1 ± 1.3 mm/min and tonometry values (mean ± SD) were 8.2 ± 1.7 mm Hg. Corneal touch threshold (median, range) was 4.4 cm, 2.5-5.0. Younger animals had a significantly increased corneal sensitivity compared to older animals (P = .0383). Results of culture showed no growth for fungal organism in any animals. Conjunctival bacterial isolation rates were low, with only 7/26 samples positive for nine bacterial species. CONCLUSIONS: Leopard geckos are amenable to ophthalmic examination and ocular diagnostic database testing with minimal manual restraint.

Inter-user and intra-user variation of two tonometers in horses.

BACKGROUND: It is currently unknown which of the two devices most commonly used in equine ophthalmology for intraocular pressure (IOP) estimation demonstrates the lowest inter-user and intra-user variation. OBJECTIVES: To assess the inter-user and intra-user variation of two tonometers in sedated and unsedated horses. STUDY DESIGN: Randomised masked cross-over trial. METHODS: Four examiners used the rebound (ICare® TonoVet) and applanation (TonoPen® ) tonometers to measure the intraocular pressure (IOP) in triplicate in 10 normal horses before and after sedation with xylazine. For inter-user variation, coefficient of variation (CV) values were calculated from the mean of each examiner for each condition combination. For intra-user variation, CV values were calculated from the individual measurements of each examiner for each condition combination. CV values were also assessed in relation to other variables using ANOVA. RESULTS: The rebound tonometer was found to have lower inter-user (15.4% vs 21.7%, P = .01) and intra-user (9.1% vs 16.1%, P < .0001) variation in unsedated horses and lower intra-user (8.4% vs 14.7%, P < .0001) variation in sedated horses than the applanation tonometer. Both instruments had similar inter-user variation in sedated horses. For the rebound tonometer, sedation did not affect inter-user or intra-user variation, but for the applanation tonometer inter-user variation was lowest while horses were sedated (16.0% vs 21.7%, P = .03). No other variable assessed was found to have an effect on IOP. MAIN LIMITATIONS: No animals with ocular disease were included in this study. CONCLUSIONS: The rebound tonometer may be the preferred instrument to minimise intra-user and inter-user variation for IOP measurement in unsedated horses. The rebound tonometer is also likely to be the preferred instrument to minimise intra-user variation in sedated horses. If the applanation tonometer is used to perform IOP measurement in horses, it is recommended that this is performed while horses are sedated to minimise inter-user variation for this instrument.

Advances in Diagnostics and Treatments in Horses and Foals with Gastric and Duodenal Ulcers.

Equine gastric ulcer syndrome (EGUS) primarily describes ulceration in the terminal esophagus, nonglandular squamous mucosa, glandular mucosa of the stomach, and proximal duodenum. EGUS is common in all breeds and ages of horses and foals. This article focuses on the current terminology for EGUS, etiologies and pathogenesis for lesions in the nonglandular and glandular stomach, diagnosis, and a comprehensive approach to the treatment and prevention of EGUS in adult horses and foals.

Alternative oil extraction methods from Echium plantagineum L. seeds using advanced techniques and green solvents.

The edible oil processing industry involves large losses of organic solvent into the atmosphere and long extraction times. In this work, fast and environmentally friendly alternatives for the production of echium oil using green solvents are proposed. Advanced extraction techniques such as Pressurized Liquid Extraction (PLE), Microwave Assisted Extraction (MAE) and Ultrasound Assisted Extraction (UAE) were evaluated to efficiently extract omega-3 rich oil from Echium plantagineum seeds. Extractions were performed with ethyl acetate, ethanol, water and ethanol:water to develop a hexane-free processing method. Optimal PLE conditions with ethanol at 150 °C during 10 min produced a very similar oil yield (31.2%) to Soxhlet using hexane for 8 h (31.3%). UAE optimized method with ethanol at mild conditions (55 °C) produced a high oil yield (29.1%). Consequently, advanced extraction techniques showed good lipid yields and furthermore, the produced echium oil had the same omega-3 fatty acid composition than traditionally extracted oil.

Ultrasonic Removal of Mucilage for Pressurized Liquid Extraction of Omega-3 Rich Oil from Chia Seeds (Salvia hispanica L.).

Chia (Salvia hispanica L.) seeds contain an important amount of edible oil rich in omega-3 fatty acids. Fast and alternative extraction techniques based on polar solvents, such as ethanol or water, have become relevant for oil extraction in recent years. However, chia seeds also contain a large amount of soluble fiber or mucilage, which makes difficult an oil extraction process with polar solvents. For that reason, the aim of this study was to develop a gentle extraction method for mucilage in order to extract chia oil with polar solvents using pressurized liquids and compare with organic solvent extraction. The proposed mucilage extraction method, using an ultrasonic probe and only water, was optimized at mild conditions (50 °C and sonication 3 min) to guarantee the omega-3 oil quality. Chia oil extraction was performed using pressurized liquid extraction (PLE) with different solvents and their mixtures at five different extraction temperatures (60, 90, 120, 150, and 200 °C). Optimal PLE conditions were achieved with ethyl acetate or hexane at 90 °C in only 10 min of static extraction time (chia oil yield up to 30.93%). In addition, chia oils extracted with nonpolar and polar solvents by PLE were analyzed by gas chromatography-mass spectrometry (GC-MS) to evaluate fatty acid composition at different extraction conditions. Chia oil contained ∼65% of α-linolenic acid regardless of mucilage extraction method, solvent, or temperature used. Furthermore, tocopherols and tocotrienols were also analyzed by HPLC in the extracted chia oils. The mucilage removal allowed the subsequent extraction of the chia oil with polar or nonpolar solvents by PLE producing chia oil with the same fatty acid and tocopherol composition as traditional extraction.

Critical Role of Different Immobilized Biocatalysts of a Given Lipase in the Selective Ethanolysis of Sardine Oil.

Different immobilized derivatives of two lipases were tested as catalysts of the synthesis of ethyl esters of omega-3 fatty acids during the ethanolysis of sardine oil in solvent-free systems at 25 °C. Lipases from Thermomyces lanuginosus (TLL) and Lecitase Ultra (a phospholipase with lipolytic activity) were studied. Lipases were adsorbed on hydrophobic Sepabeads C18 through the open active center and on an anion-exchanger Duolite with the active center exposed to the reaction medium. TLL-Sepabeads derivatives exhibit a high activity of 9 UI/mg of immobilized enzyme, and they are 20-fold more active than TLL-Duolite derivatives and almost 1000-fold more active than Lipozyme TL IM (the commercial derivative from Novozymes). Lecitase-Sepabeads exhibit a high selectivity for the synthesis of the ethyl ester of EPA that is 43-fold faster than the synthesis of the ethyl ester of DHA.

Fate of orally administered radioactive fatty acids in the late-pregnant rat.

To investigate the biodisponibility of placental transfer of fatty acids, rats pregnant for 20 days were given tracer amounts of [(14)C]palmitic (PA), oleic (OA), linoleic (LA), α-linolenic (LNA), or docosahexaenoic acid (DHA) orally and euthanized at 0.5, 1.0, 2.0, or 8.0 h thereafter. Maternal plasma radioactivity in lipids initially increased only to decline at later times. Most of the label appeared first as triacylglycerols (TAG); later, the proportion in phospholipids (PhL) increased. The percentage of label in placental lipids was also always highest shortly after administration and declined later; again, PhL increased with time. Fetal plasma radioactivity increased with time, with its highest value at 8.0 h after DHA or LNA administration. DHA initially appeared primarily in the nonesterified fatty acids (NEFA) and PA, OA, LA, and LNA as TAG followed by NEFA; in all cases, there was an increase in PhL at later times. Measurement of fatty acid concentrations allowed calculation of specific (radio)activities, and the ratio (fetal/maternal) of these in the plasmas gave an index of placental transfer activity, which was LNA > LA > DHA = OA > PA. It is proposed that a considerable proportion of most fatty acids transferred through the placenta are released into the fetal circulation in the form of TAG.

Parathyroid hormone-related protein induces hypertrophy in podocytes via TGF-beta(1) and p27(Kip1): implications for diabetic nephropathy.

BACKGROUND: Hypertrophy of podocytes is characteristic in diabetic nephropathy (DN). Previously, we observed the upregulation of parathyroid hormone-related protein (PTHrP) and its receptor PTH1R, in experimental DN, associated with renal hypertrophy. Herein, we test the hypothesis that PTHrP participates in the mechanism of high glucose (HG)-induced podocyte hypertrophy. METHODS: On mouse podocytes, hypertrophy was assessed by protein content/cell and [H(3)]leucine incorporation. Podocytes were stimulated with HG (25 mM), PTHrP(1-36) (100 nM), angiotensin II (AngII) (100 nM) or TGF-beta(1) (5 ng/mL) in the presence or absence of PTHrP-neutralizing antibodies (alpha-PTHrP), the PTH1R antagonist JB4250 (10 microM), PTHrP silencer RNA (siRNA) or TGF-beta(1) siRNA. Protein expression was analysed by western blot and immunohistochemistry. RESULTS: HG-induced hypertrophy was abolished in the presence of either alpha-PTHrP or PTHrP siRNA. This effect was associated with an inhibition of the upregulation of TGF-beta(1) and p27(Kip1). JB4250 also inhibited HG-induced p27(Kip1) upregulation. Interestingly, whilst HG and AngII were unable to stimulate the expression of p27(Kip1) on PTHrP siRNA-transfected podocytes, TGF-beta(1) was still able to upregulate p27(Kip1) in these cells. Moreover, HG and PTHrP-induced hypertrophy as well as p27(Kip1) upregulation were abolished on TGF-beta(1) siRNA-transfected podocytes. Furthermore, the glomeruli of transgenic PTHrP-overexpressing mice showed a constitutive overexpression of TGF-beta(1) and p27(Kip1) to a degree similar to that of diabetic animals. CONCLUSIONS: PTHrP seems to participate in the hypertrophic signalling triggered by HG. In this condition, AngII induces the upregulation of PTHrP, which might induce the expression of TGF-beta(1) and p27(Kip1). These findings provide new insights into the protective effects of AngII antagonists in DN, opening new paths for intervention.

Effect of diet supplementation with sunflower oil on milk production, fatty acid profile and ruminal fermentation in lactating dairy ewes.

The aim of this research was to enhance the nutritional quality of ewe milk fat by increasing potentially healthy fatty acids (FA) through diet supplementation with unprotected oil rich in linoleic acid, and without detrimental effects on animal performance. Twenty-four ewes were assigned to two high concentrate diets, control or supplemented with 6% sunflower oil (SO), for 4 weeks. No differences between treatments were found in milk production and dry matter intake. Although the SO diet increased milk fat percentage and tended to reduce milk protein concentration, it did not affect milk fat, protein or total solid yield. Most of the modifications in milk FA composition were addressed toward a potentially healthier profile: a decrease in C12:0 to C16:0 and a remarkable increase in the contents of cis-9 trans-11 C18:2 (from 0.94 to 3.60 g/100 g total FA) and trans-11 C18:1 (from 2.23 to 8.61 g/100 g total FA). Furthermore, the levels reached were maintained throughout the period monitored. However, the SO diet increased other trans C18:1 isomer percentages, too. The lack of differences between treatments in the in vitro ruminal fermentation parameters, studied with batch cultures of rumen microorganisms, would indicate no negative effects on ruminal fermentation.

Gas chromatography and silver-ion high-performance liquid chromatography analysis of conjugated linoleic acid isomers in free fatty acid form using sulphuric acid in methanol as catalyst.

This study used GC and silver-ion HPLC to examine the effects of temperature and time on methylation of individual and mixtures of conjugated linoleic acid (CLA) isomers in free fatty acid form using sulphuric acid as catalyst. In the conditions tested (temperatures between 20 and 50 degrees C and times between 10 and 60 min) methylation was complete while avoiding isomerization of conjugated dienes and the formation of artefacts that could interfere with chromatographic determinations. An analytical method using solvent extraction of the lipids followed by selective elution of the free fatty acids from aminopropyl bonded phase columns and methylation with H(2)SO(4) in mild conditions was then applied to determine the CLA isomers in free fatty acid form in rumen fluid, and the results were evaluated.

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Conjugated linoleic acid in ewe milk fat.

Ewe milk fat from five different herds was studied to determine the content of conjugated linoleic acid (CLA) isomers. Research was carried out by combining gas chromatography-mass spectrometry (GC-MS) of fatty acid methyl esters (FAME) and 4,4-dimethyloxazolyne derivatives (DMOX) with silver ion-high performance liquid chromatography (Ag+-HPLC). Reconstructed mass spectral profiles of CLA characteristic ions from DMOX were used to identify positional isomers and Ag+-HPLC to quantify them. Total CLA content varied from 0.57 to 0.97 g/100 g of total fatty acids. FAME and DMOX were separated into a complex mixture of minor isomers and major rumenic acid (9-cis 11-trans C18:2) by GC-MS using a 100-m polar capillary column. Rumenic acid would represent more than 75% of total CLA. 11-trans 13-trans, 11-13 cis/trans plus trans/cis and 7-9 cis/trans plus trans/cis were the main CLA isomers after rumenic acid. Minor amounts of 8-10 and 10-12 C18:2 isomers were also found. Although most of the isomers were present in each herd's milk, differences in content were observed for some CLA species.

Changes in the milk and cheese fat composition of ewes fed commercial supplements containing linseed with special reference to the CLA content and isomer composition.

A study was carried out to increase the CLA contents in ewes' milk fat under field conditions by dietary means and to investigate the extent of the changes and consequences for milk processing and cheese quality. During a 3-mon period, ewes' bulk milk samples were collected every week from two different herds. For the first 4 wk the ewes were fed a conventional diet. Then the following 6 wk a supplement enriched in alpha-linolenate (whole linseed) was incorporated into the ovine diet. Finally, in the last 3 wk the feeding was the same as in the first 4 wk. The FA profile in milk fat was monitored by GC, and the distribution of CLA isomers was thoroughly tested by combining GC-MS of 4,4-dimethyloxazoline derivatives (DMOX) with silver ion-HPLC (Ag(+)-HPLC) of FAME. Reconstructed mass spectral profiles of CLA characteristic ions from DMOX were used to identify positional isomers, and Ag(+)-HPLC was used to quantify them. An increase in total CLA in milk fat was observed, and total CLA remained elevated during the weeks of enriched alpha-linolenate feeding. In our experimental conditions there was a linear relationship between trans-vaccenic acid (trans-11-octadecenoic acid; trans-11 18:1) and 9-cis,11-trans CLA in ewes' milk fat. Concerning the CLA isomer profile, increases in the 11,13- and 12,14-18:2 positional isomers were considerable when linseed was included in the diet. Organoleptic characteristics of cheeses made with CLA-enriched milk did not substantially differ from those made with nonsupplemented ewes' milk. CLA total content and isomer profile did not change during ripening.