Lipopolysaccharide (LPS)-induced inflammation impairs sperm function; however, its impact on ejaculated rabbit sperm remains unexplored. This dose-response study aims to determine the LPS concentration that negatively affects sperm motility in vitro, while also providing the first identification of TLR4 localization on rabbit spermatozoa. Additionally, it evaluates malondialdehyde (MDA) levels in seminal plasma as an indicator of oxidative stress. Sperm motility was analyzed using computer-assisted sperm analysis (CASA) after incubation with increasing LPS concentrations (0, 50, 100, 200, 400, 600, and 800 µg/mL) at multiple time points (0, 1, 2, and 4 h). LPS doses ≥ 400 µg/mL significantly reduced progressive and non-progressive motility, as well as curvilinear velocity (all p < 0.001), while increasing the proportion of static spermatozoa (p < 0.05). Receiver operating characteristic (ROC) analysis identified 300 µg/mL as the threshold dose for motility decline. Immunofluorescence revealed TLR4 localization in the midpiece of sperm tails, with weak labeling in control samples and a marked increase after 4 h of incubation with 400 μg/mL LPS. MDA levels were assessed using the thiobarbituric acid reactive substances (TBARS) assay with a colorimetric kit, showing no significant effect of LPS treatment. No correlation was found between MDA and other semen parameters. ccThese findings identify TLR4 on rabbit sperm for the first time and establish a threshold LPS dose for future in vitro studies.

To the best of our knowledge, no studies in the literature have reported on the relationship between the pregnancy-associated glycoprotein (PAG) concentration and undernutrition during pregnancy in sheep. Therefore, the aim of this study was to investigate the effects of undernutrition on the blood concentration of PAGs in pregnant ewes carrying single and multiple fetuses, undergoing either dietary energy restriction or receiving 100% of their energy requirements during the period of maximal placenta growth. From d 24 to 100 of pregnancy, the ewes were fed ryegrass hay and two different iso-proteic concentrates, fulfilling either 100% of the ewes’ energy requirements (control group; n = 30, 14 singleton pregnancies, 16 multiple pregnancies) or only 50% (feed-restricted group; n = 29; 11 singleton pregnancies, 18 multiple pregnancies). Blood samples were collected from all the ewes the day before intravaginal sponge insertion (T0) and, thereafter, every 30 days starting from d 24 of gestation (T1) until d 30 after parturition (T6), to determine the PAG, progesterone, NEFA, total protein, and glucose concentrations. Our results showed that PAG concentrations are not only influenced by the number of fetuses, but also by the ewe’s diet. In particular, the PAG marginal means were higher in the feed-restricted group than in the control group (7.8 ± 0.6 and 10.8 ± 0.9 ng/mL for the control and feed-restricted groups, respectively; p = 0.040). This finding confirms that a dietary restriction between d 24 and d 100 of gestation leads to a growth in placental functions as inferred from the increase in the concentration of the PAG levels. Therefore, the measurement of PAG concentrations, in addition to serving as a marker of pregnancy, can also serve as a marker of placental functionality.

Corticosterone (CCS) concentration in chicken skeletal muscle could be a potential Animal-Based Measure that could give ex-post indications of adaptive responses to different farming systems and management practices. The aim of the present trial was to measure CCS concentrations in chicken skeletal muscle and assess their relationship with total plasma CCS concentrations. Blood and muscle samples were recovered respectively at the beginning and the end of an industrial slaughter line from 48 identified female broilers aged 84 d. Muscle juice samples were obtained by high-speed centrifugation, and analytical validation of a commercial CCS EIA kit has been carried out. Regarding repeatability (precision), intra-assay coefficient of variation (CV, n = 12 replicates) resulted 4.2 %. Inter-assay CV (2 replicates in n = 4 assay sessions) was 11.9 %. The line resulting from serial dilutions of a pooled muscle juice sample has been compared with the calibrators line for parallelism and with the expected values line for linearity. The slopes of the obtained and calibrators lines were not different (F = 4.082, P = 0.090). A strong correlation was found between the observed and expected concentrations (Pearson correlation coefficient, r = 0.991, P < 0.001). The mean RR (recovery rate, accuracy) of different quantities of hormone added to the pooled muscle juice was 90.0 ± 1.6 %. The mean CCS concentration in muscle juice samples was 455.1 ± 46.8 pg/mL (range from 47.5 to 1,340.0 pg/mL). The mean total plasma CCS concentration was 11.2 ± 0.4 ng/mL (range 7.0 to 19.5 ng/mL). The mean ratio of individual muscle:plasma CCS concentration was 0.040 ± 0.004, ranging from 0.005 to 0.133. Muscle and plasma CCS levels were moderately correlated (r = 0.324, P = 0.025). It is concluded that this EIA kit can provide reliable results for measuring CCS concentrations in juice obtained by high-speed centrifugation of chicken muscle, directly without previous extraction. Such a method can be a further tool for the assessment of animal physiological responses, stress, and welfare.

Storage of Blood units determines the accumulation of harmful substances, such as malondialdehyde (MDA) and free hemoglobin (fHb). These may lead to several complications, including cardiovascular, neurodegenerative, and metabolic disorders in recipients. The objective of this study was to evaluate the concentrations of MDA and fHb in canine leukoreduced (LR) and non-leukoreduced (NLR) packed red blood cells (pRBC) during the storage period of six weeks. Blood samples were collected from six healthy adult Weimaraner dogs (three females and three males). Whole blood was stored in citrate-phosphate-dextrose saline-adenine-glucose-mannitol additive solution (CPD-SAGM) bags and, for each donor, two pRBC units (one NLR and one LR) were produced and stored at 4 °C for 42 days. Samples were collected on days 0, 7, 14, 21, 28, 35, and 42, and analyzed for malondialdehyde (MDA) using a canine-specific ELISA method, and for free hemoglobin (fHb) using the Harboe direct spectrophotometric method. The results demonstrated a statistically significant reduction in MDA accumulation in LR-pRBC compared to NLR-pRBC blood units and lower values of fHb in LR at T6. However, no significant difference in fHb levels were demonstrated. These findings suggest that leukoreduction may limit oxidative stress during blood storage, reducing the potential adverse effects of transfusions related to oxidative damage.

The aim of this study was to investigate the interferon tau (IFNt) concentration in the peripheral maternal blood during the early phase of pregnancy in buffalo cows and improve the knowledge on the physiological importance of circulating IFNt, evaluating the possible interaction with pregnancy-associated glycoproteins (PAGs) and progesterone (P4). Blood samples were taken from buffalo cows on day 0 (day of AI), 7, 14, 18, 28, and 40 post insemination for the IFNt, PAG, and P4 analysis and to determine the IFNt mRNA expression. The animals were categorized ex post into Pregnant, Non-pregnant and Embryo mortality groups. The interferon value was influenced by group (p = 0.003), being always higher in pregnant buffalo cows than in non-pregnant ones, while the embryo mortality group showed intermediate values between those for pregnant and non-pregnant animals. The mRNA expression of IFNt was not influenced by groups or any time points. The regression analysis that included IFNt as the independent variable showed that PAGs, from day 18 (p < 0.01), and P4, from day 28 (p < 0.05), were positively associated with IFNt values. The close associations among IFNt, PAGs and P4 demonstrate that all three molecules work together for fetal–placental well-being and pregnancy support. Unfortunately, the great individual variability in circulating IFNt makes this analysis unsuitable for early pregnancy diagnosis.

This study validated immunoassays for measuring circulating DHEA and DHEA-S in broiler plasma and compared the two biomarkers. A commercially available RIA kit for DHEA and a species-independent ELISA kit for DHEA-S were tested against each other and available literature data. Blood samples were collected from 68 female broilers at slaughter. Most sample concentrations were close to the lower calibration range. DHEA concentrations ranged from 52 to 354 pg/mL, whereas DHEA-S values ranged from 76 to 7320 pg/mL. Both assays showed satisfactory validation results, including good precision (intra- and inter-assay CV ≤ 15%), accuracy (recovery ≥ 99%), linearity (R² > 0.9; run test p > 0.1), and parallelism between calibrator curves and sample dilutions (ANCOVA p > 0.1). However, the DHEA-S assay showed high cross-reactivity with DHEA (160%), making quantitative DHEA-S determination unreliable; therefore, values should be interpreted as an estimate of combined DHEA/DHEA-S concentrations. Consequently, the higher concentrations measured with the DHEA-S ELISA compared with the DHEA RIA (Bland–Altman bias: −525.8 pg/mL) do not indicate a true predominance of DHEA-S. Nevertheless, both assays may represent useful tools for comparative evaluations among samples, although they do not provide reliable information on the physiological interconversion between the two steroids.