Differential effects of propionate or β-hydroxybutyrate on genes related to energy balance and insulin sensitivity in bovine white adipose tissue explants from a subcutaneous and a visceral depot

Ruminants rely on short-chain fatty acids (SCFA) as principal energy source. Herein, we compared the effects of propionate, β-hydroxybutyrate (BHB) and insulin on mRNA abundance of energy balance-related genes by short-term incubation (4 h) in bovine subcutaneous (SC) and retroperitoneal (RP) adipose tissue (AT) explants in vitro. Propionate either significantly (p < 0.05), or as a trend (p ≤ 0.1) affected mRNA abundance of genes such as adiponectin system in both depots in treated samples versus controls. Propionate increased adiponectin receptor 1 (AdipoR1) and AdipoR2 mRNA only in SC AT. β-hydroxybutyrate decreased mRNA abundance of adiponectin and AdipoR1 in SC AT as a trend. The mRNA abundance of free fatty acid receptor 2/3 (FFAR2/3) and other genes of interest (GOI) was increased during differentiation in bovine preadipocyte culture. The mRNA abundance of all the GOI remained unchanged after short-term insulin stimulation. In total, propionate, BHB or insulin during short-term treatment exert divergent effects on the mRNA abundance of GOI in both depots in vitro. Our results indicate that the bovine adiponectin system might be more sensitive to propionate than to BHB. We demonstrated the presence of FFAR2/3 mRNA not only in both AT depots but also in differentiating preadipocytes isolated from bovine SC AT. Therefore, we established that SCFA are able to exert insulin-independent effects on bovine adipose tissue, which might be independent from propionate uptake-related events.     

β‐Carotene is incorporated or mobilized along with triglycerides in bovine adipose tissue in response to insulin or epinephrine

Pasture fed cattle ingest substantial amounts of β‐carotene (β‐C). Not all of the carotenoid compound is transformed into vitamin A, but the surplus is deposited in adipose tissue (AT). The mechanisms of β‐C incorporation and mobilization are unknown. Two experiments were conducted using explants from bovine AT cultured in vitro. First, β‐C incorporation by explants from three animals was examined with different β‐C concentrations (0, 1, 5 and 20 μm ) and different times of incubation (every 5 h up to 25 h). The data showed a significant increase of β‐C concentration in explants only for 20 μm β‐C. Secondly, effects of insulin and epinephrine on β‐C and triglyceride (TG) contents of explants were studied. Explants from six animals were incubated with either hormone and 0 or 20 μm β‐C for 20 h. Both TG and β‐C contents were affected positively by insulin and negatively by epinephrine. Interestingly, changes in ratios of β‐C/TG (hormone vs. control) were similar (1.7 × 10^?3 and 1.8 × 10^?3), respectively, for insulin and epinephrine, indicating that β‐C level is directly related to TG content. We also report the presence of mRNA for β‐C 15, 15′ oxygenase in bovine AT. The in vitro culture system using explants from bovine AT is a promising model to investigate factors that might affect the accumulation and metabolism of β‐C.     

Identification of Stably Expressed Reference Genes for RT‐qPCR Data Normalization in Defined Localizations of Cyclic Bovine Ovaries

Ovaries are highly complex organs displaying morphological, molecular and functional differences between their cortical zona parenchymatosa and medullary zona vasculosa, and also between the different cyclic luteal stages. Objective of the present study was to validate expression stability of twelve putative reference genes (RGs) in bovine ovaries, considering the intrinsic heterogeneity of bovine ovarian tissue with regard to different luteal stages and intra‐ovarian localizations. The focus was on identifying RGs, which are suitable to normalize RT‐qPCR results of ovaries collected from clinical healthy cattle, irrespective of localization and the hormonal stage. Expression profiles of twelve potential reference genes (GAPDH, ACTB, YWHAZ, HPRT1, SDHA, UBA52, POLR2C, RPS9, ACTG2, H3F3B, RPS18 and RPL19) were analysed. Evaluation of gene expression differences was performed using genorm , normfinder , and bestkeeper software. The most stably expressed genes according to genorm , normfinder and bestkeeper approaches contained the candidates H3F3B, RPS9, YWHAZ, RPS18, POLR2C and UBA52. Of this group, the genes YWHAZ, H3F3B and RPS9 could be recommended as best‐suited RGs for normalization purposes on healthy bovine ovaries irrespective of the luteal stage or intra‐ovarian localization.     

Development of an in vitro oviduct epithelial explants model for studying sperm–oviduct binding in the buffalo

In this study, we developed an in vitro model for studying sperm–oviduct binding in the buffalo. Oviduct explants were prepared by overnight culture of epithelial cells in TCM‐199 medium under 5% CO₂ at 38.5 °C. Cryopreserved spermatozoa from buffalo bulls (n = 4) were incubated with the oviduct explants, and the sperm–oviduct explants complex was stained with JC‐1. The effect of sperm concentration (2, 3 and 4 million), size of the oviduct explants (<0.2, 0.2–0.3, 0.3–0.4 and >0.4 mm²) and time of incubation (1 hr and 4 hr) on binding index (BI—number of sperm bound to unit area of explants) was studied. No significant difference was observed in the BI among <0.2, 0.2–0.3 and 0.3–0.4 mm² size of explants; however, the BI decreased significantly (p < .05) when the size of explants exceeded 0.4 mm². The BI decreased significantly (p < .05) when the sperm concentration was increased to 4 million, while the duration of incubation did not have any significant effect on the BI. The interaction of bulls with explants size, sperm concentration and incubation time was not significant. The developed assay has the potential to be used as an in vitro model for studying sperm–oviduct binding in the buffalo.     

ORIGINAL ARTICLE: Changes in composition a metabolism of caecal microbiota in rats fed diets supplemented with copper‐loaded chitosan nanoparticles

The study was conducted to investigate the effect of copper‐loaded chitosan nanoparticles on the composition and metabolism of the caecal microbiota in rats. Forty male Sprague–Dawley (SD) rats (average body weight of 82 ± 5 g) were randomly assigned to five groups (n = 8). The dietary treatments were: (i) basal diet, (ii) basal diet + 80 mg/kg BW CuSO₄, (iii) basal diet + 80 mg/kg BW chitosan (CS‐I), (iv) basal diet + 80 mg/kg BW copper‐loaded chitosan nanoparticles (CSN‐I) and (v) basal diet + 160 mg/kg BW copper‐loaded chitosan nanoparticles (CSN‐II). The trial lasted 21 days. The results showed that compared with control, Average day gain (ADG) of group CSN‐I and CSN‐II increased (p < 0.05). No significant difference was found in CuSO₄ or CS‐I‐treated groups (p > 0.05). There were no effects of these treatments on average day feed intake (ADFI) of rats (p > 0.05). The counts of Bifidobacteria and Lactobacillus in group CSN‐II were higher than those of the control group (p < 0.05), while the counts of total aerobes, total anaerobes, Salmonella, Clostridium and coliform were lower than those of the control (p < 0.05). The activity of β‐glucuronidase in group CSN‐I or CSN‐II was significantly depressed (p < 0.05), while the activities of α‐galactosidase and β‐galactosidase were enhanced significantly (p < 0.05). The pH of the caecum digesta and the concentration of propionate and butyrate in group CSN‐I and CSN‐II were higher than those of the control group (p < 0.05). No significant difference was found in these parameters in CuSO₄ or CS‐I‐treated groups (p > 0.05). The results indicate that the microbiota and environment of caecum are beneficially changed by the administration of copper‐loaded CSN.     

Proposal of screening method for intestinal mucus adhesive lactobacilli using the enzymatic activity of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH)

Adhesion tests are complex, time‐consuming and expensive, while the most important criterion for a probiotic lactobacilli is the ability to adhere to the human intestine. Thirty lactobacilli isolates from human intestinal tissues were measured for cell surface glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) activity using a microtiter plate screening method. GAPDH activities were detected in 21 out of 30 samples from 12 h cultures and in all samples from 18 h cultures. This suggests GAPDH is universally expressed on the bacterial cell surfaces from many lactobacilli. A statistically significant positive correlation was shown between GAPDH activity and adhesion using the BIACORE adhesion assay (P < 0.01). The new screening method using GAPDH enzymatic activity without an adhesion test may be possible due to the significant positive correlation of GAPDH activity with adhesion of lactobacilli derived from the human intestine.     

Evaluating the rumen‐protected lysine stability in forage‐based total mixed rations in vitro and determining the lysine Brix value

Three rumen‐protected lysine (RPL) products (AjiPro^®‐L, LysiPEARL^TM, and Feedtech Bypass Lysine^TM: A, B, and C, respectively) were tested for stability in two forage‐based total mixed rations (TMR1, 41.3% dry matter (DM), and TMR2, 49.5% DM) (experiment 1) and for Brix value (experiment 2). In experiment 1, each RPL product (2 g each) and TMR diet (200 g) were mixed and stored in plastic bags at 20°C for 0, 1, 3, 6, 12, 24, and 48 hr. In experiment 2, each RPL product (2 g) was dispensed into ion‐exchanged water (20 ml) and kept at 20°C for 0, 0.5, 1, 3, 6, 12, 24, and 48 hr. At each time point, free lysine (Lys) content and Brix values of extracts were measured, and Lys release (LR, %) was calculated. All RPL products LR% varied with varying diets DM and increased with increasing of time exposed to diets; it was highest in C, followed by B, and then A. Water LR% positively correlated with that from diets and with Brix values of Lys dissociated in water. Our results indicated that Lys dissociation from RPL products is affected by diet DM content. Brix value may be used as a potential marker for RPL protection efficacy.     

Study on some risk factors and effects of bovine ketosis on dairy cows from the Galicia region (Spain)

The study was designed to determine the relation between β‐hydroxybutyrate (BHB) concentrations in milk from dairy cows after calving and the length of the previous lactation, the dry period and the 305‐day normalized production, and to assess the influence of BHB concentrations on culling and test‐day milk productions and somatic cell counts (SCC) throughout the lactation that followed the BHB measurement. The data used in the study were obtained from 59 187 cows in the Galicia region (Spain). BHB determination was performed using Fourier‐transformed infrared spectrometry from the milk samples collected from each cow on the first post‐partum test day. For statistical analysis, the following methods were applied: (i) ordinal regression to assess the effect of the length of the previous lactation, the dry period and the 305‐day normalized milk production on milk BHB, (ii) a Cox model to estimate the influence of the BHB concentration on risk of culling (overall and for a variety of reasons) and (iii) linear regression to assess the link between BHB and the milk yield and SCC obtained from each of the tests day performed throughout lactation. The probability of having higher BHB concentrations increased when the length of the previous lactation (p = 0.006), the dry period (p = 0.003) and the 305‐day normalized milk yield (p = 0.005) increased. However, the slight increase observed (especially for the case of the dry period and the 305‐day milk yield) would not justify that measures be implemented to reduce these traits. Higher concentrations of BHB led to an increased risk of culling due to ‘death’ (p ≤ 0.001) and ‘urgent slaughter’ (p ≤ 0.002) (both causes of involuntary culling). It also led to a reduction in milk production (p < 0.001) and an increase in SCC (p < 0.001) in the post‐partum; from that moment onward (including peak lactation), there were no differences in those two parameters depending on the BHB levels.     

Correlation of Whole Blood Concentrations of Acetoacetate, β-Hydroxybutyrate,Glucose and Milk Yield in Dairy Cows as Studied Under Field Conditions

The whole blood concentrations of acetoacetate (AA concn), β-hydroxybutyrate (BHB concn) and glucose (gluc concn) of 662 Ayrshire and Friesian dairy cows were measured and their milk yield during the indoor-housing period was recorded. Simple correlations among these parameters were evaluated. The correlation between the AA concn and the BHB concn (r = 0.869) was statistically highly significant (P < 0.001), as were the correlations of the logarithmic value of the AA concn with the gluc concn (r = —0.471) and with the milk yield (r = 0.259), and the correlation between the BHB concn and the glue concn (r = —0.288). The milk yield was found also to be associated with the BHB concn and the glue concn (P< 0.001). The associations between each pair of blood parameters were highly significant, too (P < 0.001). The AA concn was taken to be at least as good an indicator of the energy status of dairy cows as the BHB concn and the gluc concn. The AA concn is not diet-derived like the BHB concn and not as stress-sensitive as the blood concentration of free fatty acids and its diurnal variation is not as wide as the BHB concn and the gluc concn in subclinically ketotic cows. Thus the AA concn may be used as a proxy of the energy status and the ketotic stage of dairy cows under field conditions.Key words: acetoacetate, ß-hydroxy butyrate, glucose, energy status, dairy cattle     

In vitro enantioselective pharmacodynamics of Carprofen and Flunixin‐meglumine in feedlot cattle

The activity of the anti‐inflammatory agents Flunixin‐meglumine (FLU), RS (±) Carprofen (CPF) and S (+) CPF on bovine cyclooxygenases (COXs) has been characterized in feedlot calves using an in vitro whole blood model. The drugs showed equivalent efficacy in their inhibitory activity on COXs, and the rank order of potency for both COX‐1 and COX‐2 inhibition was FLU > S (+) CPF > RS (±) CPF. Our results indicated that FLU is a nonselective inhibitor of bovine COXs, whereas RS (±) CPF and S (+) CPF exhibited different degrees of preferential inhibition of COX‐2 isoenzyme. The rank order of IC₅₀ COX‐1: IC₅₀ COX‐2 potency ratios was in fact S (+) CPF (51.882) > RS (±) CPF (13.964) > FLU (0.606), and the calculated percentage inhibition of COX‐1 corresponding to COX‐2 inhibition values comprised between 80% and 95% was comprised between 57.697 and 79.865 for FLU, 33.373 and 51.319 for RS (±) CPF, and 0.230 and 4.622 for S (+) CPF, respectively. These findings are discussed in relation to the prediction of the clinical relevance of COX inhibition by the test drugs in cattle.     

Metabolic parameters and their relationship to energy balance in multiparous Simmental,Brown Swiss and Holstein cows in the periparturient period as influenced by energy supply pre‐ and post‐calving

A study was conducted to evaluate the effects of three energy supply (E) levels [low (L), medium (M), high (H)], both pre‐partum (PRE) and post‐partum (POST), and their interactions on metabolic parameters and energy balance (EB) in dairy cows of three breeds. In both phases, E levels applied to a total of 81 multiparous cows of breeds Simmental (SI), Brown Swiss (BS) and Holstein–Friesian (HF; n = 27 for each breed) were 75%, 100% and 125% of recommendations of the German Society of Nutrition Physiology, using a 3 × 3 factorial arrangement of treatments. During the pre‐calving period, serum concentrations of non‐esterified fatty acids (NEFA) were higher for L_PRE cows, and glucose concentrations were elevated for H_PRE cows. During the lactation period, NEFA concentrations were greatest for treatment L_POST. Mean concentrations of β‐hydroxybutyrate (BHB) were highest for cows of the L_POST treatment, intermediate for M_POST and lowest for H_POST. Glucose concentrations were lower for L_POST cows. SI cows had lower BHB concentrations both pre‐ and post‐calving and higher glucose concentrations during early lactation than the other breeds. BHB concentration POST was highest for BS cows. Restricted feeding PRE resulted in a better energy status of cows fed above energy requirements POST (E_PRE × E_POST interaction). HF cows had a higher EB pre‐calving, whereas SI cows had a less negative EB during early lactation, compared with the other breeds respectively. Correlations of serum NEFA, BHB and glucose concentrations with EB were strongest during the transition period. Results suggest that controlling energy intake during the dry period might be advantageous for the energy status of dairy cows after calving, whereas energy restriction in early lactation leads to metabolic stress. Evidence is provided of a clear relationship between EB and the blood metabolites NEFA and BHB, especially in the transition period.     

Effect of γ‐aminobutyric acid on growth performance and immune function in chicks under beak trimming stress

This experiment was undertaken to examine the effect of beak trimming stress on the growth performance and immune system, and to consider possible roles of γ‐aminobutyric acid (GABA) in this stress response. Results showed that body weight, feed intake and relative spleen weight were significantly increased by GABA at 80 mg/kg (P < 0.05) under beak trimming stress, whereas the relative organ weights of the bursa of fabricius and thymus were not significantly affected (P > 0.05). Adrenocorticotropic hormone concentration in serum was highest for chicks fed the GABA‐deficient water and was significantly decreased by the supplement of GABA at days 1, 3 and 5 after beak trimming (P < 0.05). The supplement of GABA significantly increased the proportions of CD4⁺ and CD8⁺ lymphocytes, especially at the dose of 60 mg/kg (P < 0.05). The levels of interleukin (IL)‐1β, lipopolysaccharide‐induced tumor necrosis factor‐α and IL‐6 in serum were significantly decreased by GABA at 80 mg/kg (P < 0.05). All the three cytokines expressed in the spleen were significantly decreased by GABA at 80 mg/kg when birds were under beak trimming stress (P < 0.05). It is concluded that beak trimming suppressed the immune response of chicks, whereas the immune response of chicks could be improved by GABA supplementation.     

FC‐39 Successful management of canine atopic dermatitis using a plant extract: a randomized,double‐blinded,placebo‐controlled trial

This study evaluated the efficacy of Phytopica^TM, a proprietary blend of standardised plant extracts, in canine atopic dermatitis (AD). One hundred twenty dogs with perennial AD were recruited on the basis of history and clinical signs, and a positive intradermal allergen test or rFcεRIα serology to perennial allergens. Other pruritic dermatoses were eliminated by antimicrobial treatment, skin scrapings, Sarcoptes serology, flea control and a 6‐week food trial. Exclusion criteria included antimicrobial therapy within 7 days, antihistamines within 14 days, oral/topical glucocorticoids or cyclosporin within 28 days, and parenteral glucocorticoids, essential fatty acids or immunotherapy within 56 days of entry into the study. Dogs [minimum Canine Atopic Dermatitis Extent and Severity Index (CADESI) = 25] were randomly allocated to receive placebo, 100, 200 or 400 mg/kg Phytopica^TM daily for 12 weeks. Their CADESI was assessed every 4 weeks. A modified intention‐to‐treat population was analysed. The mean reductions in CADESI scores at the end of treatment compared to baseline were 4.4% (100 mg/kg; n = 30), 23.4% (200 mg/kg; n = 29, P < 0.01), 8.5% (400 mg/kg; n = 29) and 3.9% (placebo; n = 29). For more severely affected dogs (minimum CADESI ≥ 50 at baseline), there was significant reduction in mean CADESI score (29.3%, P = 0.038) only in the 200 mg/kg treatment group (n = 14). In conclusion, this study demonstrates that Phytopica^TM is an effective nonsteroidal treatment for canine AD. Funding: Phytopharm plc.     

In vitro fermentative characteristics of ruminant diets supplemented with fibrolytic enzymes and ranges of optimal endo‐β‐1,4‐glucanase activity

Effectiveness of fibrolytic enzymes supplementing a range of forage to concentrate (F:C) diets was assessed with goat (G) or cow (C) inoculum using the gas production (GP) technique. Four F:C diets were evaluated: forage (1:0), high forage (0.7:0.3), medium forage (0.5:0.5) and low forage (0.3:0.7) diets, supplemented or not with Promote^TM (PRO) at 1 or 2 ml/kg dry matter (DM). The GP kinetic was different between F:C (1:0 < 0.7:0.3 < 0.5:0.5 < 0.3:0.7) and inoculum. Responses to enzyme were positively related to forage level and differed with inoculum. The neutral detergent fibre and acid detergent fibre degradation were depressed by the concentrate in the substrates fermented with C and were not altered or even enhanced in G sets. Results confirm that increasing starch proportion modified the pattern of microbial fermentation, while no influences were detected in the improvement of cell wall degradation with fibrolytic enzymes. Another in vitro experiment was conducted to investigate factors by which endo‐β‐1,4‐glucanase activity (EA) of PRO is compromised in a factorial design (3 × 4 × 3) for three pH (4.0, 5.5 and 6.5), four temperatures (30, 40, 50 and 70 °C) and three doses (1, 2 and 3 ml/kg DM of substrate). Maximum EA were obtained for pH 4.0, 50 °C and 3 ml/kg DM. Optimal conditions for PRO proved to be outside the normal ranges in ruminal environment.     

Lung toxicity of a vapor-grown carbon fiber in comparison with a multi-walled carbon nanotube in F344 rats

Carbon fibers have excellent physicochemical and electrical properties. Vapor-grown carbon fibers are a type of carbon fibers that have a multi-walled carbon tube structure with a high aspect ratio. The representative vapor-grown carbon fiber, VGCF^TM-H, is extremely strong and stable and has superior thermal and electrical conductivity. Because some high-aspect-ratio multi-walled carbon nanotubes (MWCNTs) have been reported to have toxic and carcinogenic effects in the lungs of rodents, we performed a 13-week lung toxicity study using VGCF^TM-H in comparison with one of MWCNTs, MWNT-7, in rats. Male and female F344 rats were intratracheally administered VGCF^TM-H at doses of 0.2, 0.4, and 0.8 mg/kg bw or MWNT-7 at doses of 0.4 and 0.8 mg/kg bw once a week for 8 weeks and then up to week 13 without treatment. The lung burden was equivalent in the VGCF^TM-H and MWNT-7 groups; however, the lung weight had increased and the inflammatory and biochemical parameters in the broncho-alveolar lavage fluid and histopathological parameters, including inflammatory cell infiltration, alveolar type II cells proliferation, alveolar fibrosis, pleural fibrosis, lung mesothelium proliferation, and diaphragm fibrosis, were milder in the VGCF^TM-H group than in the MWNT-7 group. In addition, the proliferating cell nuclear antigen (PCNA)-positive index in the visceral and pleural mesothelium was significantly higher in the MWNT-7 group than in the controls, but not in the VGCF^TM-H group. Thus, the results of this study indicate that the lung and pleural toxicities of VGCF^TM-H were less than those of MWNT-7.     

Effect of Megasphaera elsdenii NCIMB 41125 dosing on rumen development,volatile fatty acid production and blood β‐hydroxybutyrate in neonatal dairy calves

Thirty calves were randomly assigned to two treatments and fed until weaning [42 days (d) of age]. Treatments were a control group (n = 15), which did not receive Megasphaera elsdenii (Me0) and a M. elsdenii group, which received a 50‐ml oral dose of M. elsdenii NCIMB 41125 (10⁸ CFU/ml) at day 14 day of age (Me14). Calves were given colostrum for the first 3 day followed by limited whole milk feeding. A commercial calf starter was offered ad libitum starting at day 4 until the end of the study. Fresh water was available throughout the study. Feed intake and growth were measured. Blood samples were collected via jugular venipuncture to determine β‐hydroxybutyrate (BHBA) concentrations. Fourteen male calves (seven per group) were euthanised on day 42 and digestive tracts harvested. Reticulo‐rumen weight was determined and rumen tissue samples collected from the cranial and caudal sacs of the ventral and dorsal portions of the rumen for measurements of papillae length, papillae width and rumen wall thickness. Dosing with M. elsdenii NCIMB 41125 improved starter dry matter intake (DMI), weaning body weight (BW) and tended to improve average daily gain. Calves in Me14 group had greater plasma BHBA concentration than Me0‐calves during the last 3 weeks of the trial and had at day 42 greater reticulo‐rumen weight, papillae width and papillae density compared to Me0. No differences in rumen wall thickness or papillae length were observed between the two groups. Total volatile fatty acids, acetate and propionate production did not differ between treatments, but butyrate production was greater in Me14 than Me0. Dosing M. elsdenii NCIMB 41125 showed benefit for calves with improved feed intake and rumen development suggesting increased epithelium metabolism and improved absorption of digestive end products.     

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.     

Ovine sperm DNA oxidation quantification using an 8‐OHdG immunodetection assay

Our aim was to optimize 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) immunodetection in order to detect DNA damage caused by oxidative stress that may not be detected by other DNA integrity analysis techniques, especially due to the high compaction of DNA in ruminants. Semen samples from 6 rams were cryopreserved. After thawing, samples were subjected to the DNA oxidation quantification using an 8‐OHdG immunodetection assay by flow cytometry. We have evaluated two different incubation times (30 min vs. overnight) at 4°C of the primary antibody (monoclonal anti‐8‐OHdG antibody). We have also compared the results of this technique with the sperm chromatin structure assay (SCSA^®). The analysis revealed that there were no significant differences (p > .05) between different incubation times. However, overnight incubation seems to cause more non‐specific binding of the secondary antibody. Significant differences (p < .05) between subjects and oxidation controls (8 M H₂O₂/800 μM FeSO₄?7H₂O) were evident. We can conclude that the 8‐OHdG immunodetection assay for DNA oxidation quantification of ram sperm can be performed subjecting sperm samples to a very high oxidative treatment.