Comparison of Uterine Involution and the Resumption of Ovarian Cyclicity between Lame and Sound Holstein Cows.

The aim of the study was to examine the effect of lameness and energy status on the involution of the uterus and the resumption of ovarian cyclicity in dairy cows. Lame (lameness score of four and the presence of hoof lesions, n = 22) and sound (normal gait and the absence of hoof lesions, n = 25) multiparous cows with healthy puerperium were enrolled simultaneously in the study and were monitored from day 10 antepartum (ap) to day 50 post-partum (pp). Ultrasonography of the cervix, the formerly gravid uterine horn and the ovarian structures was performed on d 8, 11, 14, 23, 30, and 42 pp. Blood sampling for progesterone, ß-hydroxybutyrate (BHBA), and non-esterified fatty acids (NEFAs) was used to assess cyclicity and energy status. Lame compared to sound cows had higher NEFA concentrations on day 14 pp (0.54 ± 0.05 vs. 0.37 ± 0.05, respectively, p = 0.005), delayed involution of the cervix and the formerly pregnant uterine horn (p = 0.0003 and p = 0.02, respectively), lower ovulation rates within the experimental period (63.6% vs. 88%, respectively, p = 0.05), and higher rates of atresia or cyst formation on day 50 pp (36.4% vs. 12%, respectively, p = 0.05). Independently of lameness status, cows with high NEFA concentrations had lower ovulation rates within the experimental period (65.5% vs. 94.4%, p = 0.02), lower normal ovarian activity on day 50 pp (58.6% vs. 88.9%, p = 0.03), and higher rates of atresia or cyst formation on day 50 pp (34.5% vs. 5.6%, p = 0.02) compared to cows with optimal NEFA concentrations. Furthermore, an interaction between lameness and increased NEFA concentrations was observed regarding the ovulation rate within the experimental period and the percentage of atresia or cyst formation on day 50 pp. Sound cows with low NEFA levels had the lowest mean cervical diameter compared to cows with lameness (both with elevated and optimal NEFA concentrations, p = 0.009 and p = 0.002, respectively). Conclusively, lameness during puerperium negatively affected ovarian function and uterine involution. These effects were exacerbated (through interaction or cumulation) in relation to elevated NEFA concentrations.

Ovarian and Energy Status in Lame Dairy Cows at Puerperium and Their Responsiveness in Protocols for the Synchronization of Ovulation.

The purpose of this study was to assess the ovarian and energy status of multiparous lame dairy cows at the end of puerperium and investigate their responsiveness to estrous synchronization treatment regimens. Initial lameness scoring was performed at 28 ± 5 and 37 ± 5 d post partum, followed by lesion documentation and treatment. Cows were blocked by lameness severity and were randomly allocated to an estrous synchronization treatment regimen with seven days of progesterone supplementation (group LP, n = 26) or with an administration of PGF2α twice, 14 d apart (group LC, n = 26). Non-lame cows served as controls (group C, n = 27) and the same treatment regimen was imposed as that for group LC. Twelve days after estrous presynchronization, an Ovsynch treatment regimen and timed AI were imposed. Ultrasonography of the ovaries and blood sampling for progesterone were used to assess cyclicity status, whereas ß-hydroxybutyrate (BHBA) and non-esterified fatty acids (NEFA) were used to assess energy status. Lame cows were to a greater proportion non-cycling (36.5% vs. 11.1%; p = 0.02), had greater overall NEFA concentrations (0.32 ± 0.02 vs. 0.26 ± 0.02 mEq/L; p = 0.02) and a greater incidence of elevated NEFA concentrations (53.9% vs. 29.6%, p = 0.04) compared to control cows. However, no interaction between energy and lameness status was evident regarding non-cycling cows. The percentage of cows responding to the presynchronization, synchronization and ovulating did not differ between groups LP, LC, and C. The first-service conception rate (FSCR) tended to be greater for group C (37.0%) compared to group LP (16.0%; p = 0.08). Long-term reproductive performance did not differ between lame and control cows, although culling rates did (21.2% vs. 0%, respectivly; p = 0.01). The severity of lameness had an effect on culling rates (30.6% vs. 0% for cows with marked vs. moderate lameness; p = 0.01), whereas the type of lesion largely explained poor reproductive performance (FSCR 13.9% vs. 40.0% for cows with claw horn disruptions vs. infectious lesions; p = 0.04). Conclusively, cows that were lame during puerperium are at a greater risk of not cycling irrespective of energy status. Treatment regimens for the synchronization of ovulation seem to be efficient at resuming ovarian cyclicity. Marked lameness was detrimental to survivability, whereas cows with claw horn lesions had compromised reproductive capacity.

The Role of Ewes' Udder Health on Echotexture and Blood Flow Changes during the Dry and Lactation Periods.

The objective of the current study was to investigate the echotextural and hemodynamic changes of ewes entering the dry period with or without subclinical mastitis. B-mode and color Doppler ultrasonography were applied to 12 Chios ewes (6 with healthy udders (group A) and 6 with subclinical mastitis (group B)) before the dry period, during the dry period (the involution phase, steady state, and transition phase), and postpartum. The color Doppler of the mammary arteries was used to evaluate them according to the pulsatility index (PI), resistive index (RI), end-diastolic velocity (EDV), time-averaged maximum velocity (TAMV), blood flow volume (BFV), and artery diameter (D). Udder parenchyma images, analyzed by Echovet v2.0, were used to evaluate the mean value (MV), standard deviation (SD), gradient mean value (GMV), gradient variance (GV), contrast (Con), entropy (Ent), gray value distribution (GVD), run length distribution (RunLD), and long run emphasis (LRunEm). In the involution phase, the PI was higher in group B compared to group A (p ≤ 0.05). The PI and RI were higher postpartum, whereas the EDV, TAVM, and D were higher in the transition phase (p ≤ 0.05). Neither the period nor the ewe group affected the MV, SD, GMV, GV, Con, and GVD values (p ≤ 0.05). In the steady state, the LRunEm was higher in group B, but postpartum, it was higher in group A (p ≤ 0.05). In conclusion, B-mode and Doppler can reveal differences (i) between healthy ewes and ewes with subclinical mastitis and (ii) among the different periods studied. Further research is needed on the blood flow and echotexture indices of the udders of ewes with unilateral subclinical mastitis.

The Use of Animal's Body, Scrotal Temperature and Motion Monitoring in Evaluating Boar Semen Production Capacity.

Biomedical measurements by specialized technological equipment have been used in farm animals to collect information about nutrition, behavior and welfare. This study investigates the relation of semen quality (CASA analysis, viability, morphology, membrane biochemical activity and DNA fragmentation) with boar behavior during ejaculation. Sensors were placed on the boar's body. Movement features were collected using an inertial measurement unit (IMU), comprising an accelerometer, a gyroscope and a magnetometer. Boar, scrotal and dummy temperatures were measured by an infrared (IR) camera and an IR thermometer, while the face salivation of the boar was recorded by a moisture meter (also based on IR technology). All signals and images were logged on a mobile device (smartphone or tablet) using a Bluetooth connection and then transferred wirelessly to the cloud. The data files were then processed using scripts in MATLAB 2021a (MathWorks, Natick, Massachusetts) to derive the necessary indices. Ninety-four ejaculates from five boars were analyzed in this study. The statistical analysis was performed in the Statistics and Machine Learning Toolbox of MATLAB 2021a using a linear mixed effects model. Significant and strong negative correlations (R2 > 0.5, p ≤ 0.05) were observed between boar, dummy and scrotal temperature with the progressive, rapid and slow movement of spermatozoa, VCL (curvilinear velocity), VSL (straight line velocity) and ALH (amplitude of lateral head displacement) kinematics. The volume of the ejaculate was correlated with the scrotal and dummy temperature. Dummy's temperature was negatively correlated with BCF (beat/cross-frequency), viability and total time of ejaculation, while it was positively correlated with abnormal morphology. Body temperature was negatively correlated with BCF. Positive correlations were noticed between VAP (average path velocity) and total time of ejaculation with body acceleration features, as well as between the overall dynamic body acceleration (ODBA) and total time of ejaculation. In conclusion, the use of biomedical sensors can support the evaluation of boar sperm production capacity, providing valuable information about semen quality.

Varicocele in an Adult Ram: Histopathological Examination and Sperm Quality Evaluation.

Varicocele is a common pathological condition of testis that is related to male fertility problems. A 3-year age Chios ram had an abnormally enlarged scrotal area, was excluded from reproductive duties, and was euthanized with the owners' permission. The main pathological finding was the presence of bilateral multinodular spermatic cord enlargement with laminated vascular thrombi. Histopathological examination revealed commonly mineralized thrombi within the lumen of veins of the pampiniform plexus, inflammation and testicular degeneration. The epididymides were transported to the laboratory and each cauda region was sliced and washed (8 mL water for injection/epididymis), and the epididymal sperm samples were collected. Sperm motility variables (CASA), viability (eosin-nigrosine), morphology (SpermBlue®), and DNA integrity (Acridine Orange Test, AOT) were assessed. The total and progressive motility were low in semen samples of both sides (30.00% and 1.00% vs. 42.60% and 2.50% for left and right epididymis, respectively). Low viability values were observed for both sides (26.00% vs. 23.00% for left and right epididymis, respectively), while sperm morphological abnormalities were within normal limits. No sperm with DNA damage were detected. The results of this case report indicate that varicocele is associated with testis dysfunction and degradation of ram semen quality, mainly affecting motility and kinematics.

Investigating Visual Monitoring of the Scrotum as a Supplementary Tool for Boar Semen Quality Evaluation.

Farm animals behavior research uses video cameras, mainly for visual observation and recording. The purpose of this feasibility study was to enrich the predictable methods of boar semen production capacity by correlating sperm variables with the scrotal contractions (SC) frequency and intensity. A video camera was used to record the reaction of the scrotum during ejaculation. The respective collected ejaculates were evaluated and semen parameters, such as viability, morphology, membranes functional integrity and kinematics, were determined. The camera recorded the scrotal contractions/relaxations and the video was handled by the Image Processing Toolbox of Matlab (Mathworks Inc., Natick, MA, USA). The SC intensity was verified as a percentage change in the scrotum size among the video frames of maximum contraction and relaxation. The archived data from the frames were analyzed statistically, using a linear mixed effects model that involved sperm assessed parameters. Correlations of the SC intensity with the average path velocity, VAP (R2 = 0.591, p = 0.043) and with the percentage of the cytoplasmic droplets (R2 = 0.509, p = 0.036) were noticed. Previous studies reported the positive correlation of VAP with the number of live-born piglets. In conclusion, video monitoring of the boar scrotal function during ejaculation is useful, but more research is needed to establish its appropriateness as a supplementary method for the prognosis of boar ability to produce high-quality semen.

Effect of Boar Sperm Proteins and Quality Changes on Field Fertility.

This study aimed to evaluate boar sperm characteristics and proteins, in relation to their importance regarding in vivo fertility. Sixty-five ejaculates were used and 468 sows (parity ≥ 2) were inseminated. Sperm CASA kinetics, morphology, viability, DNA fragmentation, mitochondrial membrane potential, sperm membrane biochemical activity (HOST) and sperm proteins (Heat Shock Protein 90-HSP90, glutathione peroxidase-5-GPX5, Osteopontin 70-OPN70) were assessed and related to field fertility (number of live-born piglets-NLBP, litter size ≥ 12 piglets-LS, farrowing rate-FR). Statistical analysis was conducted with simple and multiple regression models. Simple regression analysis showed that immotile sperm (IM) significantly affected the NLBP and LS, explaining 6.7% and 6.5% of their variation, respectively. The HOST positive spermatozoa significantly affected the NLBP and LS, explaining 24.5% and 7.8% of their variation, respectively. Similarly, sperm with activated mitochondria significantly affected the NLBP, explaining 13.5% of its variation. Moreover, the OPN70 affected LS and FR, explaining 7.5% and 10.8% of their variation, respectively. Sperm GPX5 protein affected FR, explaining 6.7% of its variation. Multiple regression analysis showed that the combination of IM and/OPN70 explains 13.0% of the variation regarding LS, and the combination of GPX5 and OPN70 explains 13.6% of the variation regarding FR. In conclusion, the estimation of parameters IM, membrane biochemical activity, mitochondrial membrane potential, OPN and GPX5 can provide useful information regarding semen doses for field fertility.

Efficiency of three boar sperm enrichment techniques.

The objective of the study was to investigate the efficiency of three enrichment methods to separate boar spermatozoa. Twenty-four ejaculates from 12 boars (2 ejaculates/boar) were extended (30 × 106 spermatozoa/mL) in commercial Beltsville Thawing Solution. Each semen sample was processed with glass wool column (GW) and glass beads (GB) filtration and with the single-layer centrifugation (SLC) technique. Semen samples before (control; C) and after treatment were evaluated for sperm CASA motility/kinetics and concentration, viability, morphology and chromatin integrity. Data were analysed with mixed models. The concentration of total and motile spermatozoa was significantly decreased after treatment in groups GW and SLC, but not in group GB. Group GW showed increased values of WOB compared with both groups C and GB. Group GB showed greater values of rapid movement spermatozoa and lower values of slow movement spermatozoa compared with group C. In group SLC, higher values of VSL, LIN and STR were observed compared with group C. In conclusion, all techniques under examination enhanced various CASA variables. Based on our results, the GB method is a promising alternative separation technique for boar sperm and deserves further research regarding swine in vitro fertilization.

Toxic and Microbiological Effects of Iron Oxide and Silver Nanoparticles as Additives on Extended Ram Semen.

The aim of the study was to investigate the effect of iron oxide (Fe) and silver (Ag) nanoparticles (NPs) on ram semen. A skim milk extender without antibiotics was used as a diluent of 21 ejaculates (8 rams; 2-3 ejaculates/ram). The groups of control (C; semen without NPs), Fe NPs (3.072 mg Fe3O4/mL semen), and Ag NPs (2.048 mg Ag-Fe/mL semen) were incubated (15 °C; 30 min), and then a magnetic field was used for NPs' removal. Standard microbiological procedures were performed for all groups. Post-treated samples were stored (15 °C) for 24 h, and sperm variables (kinetics by computer assisted sperm analysis (CASA); viability; morphology; HOST; DNA integrity) were evaluated at 6 and 24 h. Semen data were analyzed by a mixed model for repeated measures and microbiological data with Student's t-test for paired samples. At 6 h of storage, VCL and rapid movement-spermatozoa, and at 24 h, total/progressive motility and amplitude of lateral head displacement (ALH) were significantly decreased in group Ag compared to control. In group Fe, progressive/rapid movement-spermatozoa were significantly lower compared to control after 24 h of storage. Only in group Ag was a significant reduction of total bacterial count revealed. In conclusion, the examined Fe NPs demonstrated slight antibacterial effect, while the examined Ag NPs provided higher antibacterial properties accompanied by cytotoxicity.

Effect of Toxoplasma gondii on Ram Sperm Quality After Experimental Infection.

The aim of this study was to investigate the effect of experimental Toxoplasma gondii infection on ram sperm quality. Five months old, pre-pubertal, rams were divided into four groups (n = 8 per group). Group A was the control group; the remaining animals received per os (p.o.) 5000 oocysts per ram. Group B did not receive treatment post-infection (p.i.). Group C received sulphadimidine (intermuscular injection (i.m.) 33 mg/kg for eight days; every 48 hrs) two months p.i. and Group D received the same drug twice (24 hours p.i. and two months later). Blood samples were collected every 15 days to detect serum immunoglobulin G (IgG). Epididymal sperm samples were analyzed for concentration, kinetics, morphology/viability, functional membrane integrity, DNA integrity, and the presence of parasite DNA. Histopathological examination was performed on the testes. The IgG titres in infected groups raised two weeks p.i. and remained high for four months. Higher values were noticed in viability and functional membrane integrity in positive spermatozoa in the control group compared to other groups, level of significance p < 0.05. Abnormal sperm was higher in groups C and D vs. A and C vs. B (p < 0.05). T. gondii DNA was detected in three sperm samples of the infected rams (12.5%). Histopathology revealed similar findings with little variation among all infected groups, characterized mostly by increased interstitial connective tissue, non-purulent inflammation, and presence of seminiferous tubules with spermatogenic cell depletion, which increased gradually from D to C and B groups. In conclusion Toxoplasmosis in pre-pubertal age negatively affected mature ram sperm quality, while sulphadimidine administration failed to alter this.

Iron Oxide Nanoparticles as an Alternative to Antibiotics Additive on Extended Boar Semen.

This study examined the effect of Fe3O4 nanoparticles on boar semen. Beltsville thawing solution without antibiotics was used to extend ejaculates from 5 boars (4 ejaculates/boar). Semen samples of control group (C) and group with Fe3O4 (Fe; 0.192 mg/mL semen) were incubated under routine boar semen storage temperature (17 °C) for 0.5 h and nanoparticles were removed by a magnetic field. Before and after treatment, aliquots of all groups were cultured using standard microbiological methods. The samples after treatment were stored (17 °C) for 48 h and sperm parameters (computer-assisted sperm analyzer (CASA) variables; morphology; viability; hypo-osmotic swelling test (HOST); DNA integrity) were evaluated at storage times 0, 24, 48 h. Semen data were analyzed by a repeated measures mixed model and microbial data with Student's t-test for paired samples. Regarding CASA parameters, Fe group did not differ from C at any time point. In group C, total motility after 24 h and progressive motility after 48 h of storage decreased significantly compared to 0 h. In group Fe, linearity (LIN) after 48 h and head abnormalities after 24 h of storage increased significantly compared to 0 h. The microbiological results revealed a significant reduction of the bacterial load in group Fe compared to control at both 24 and 48 h. In conclusion, the use of Fe3O4 nanoparticles during semen processing provided a slight anti-microbiological effect with no adverse effects on sperm characteristics.

Urine protein-to-creatinine ratio in cattle with subclinical renal disease.

BACKGROUND: Urinalysis is not routinely used in bovine medicine, and there is no evidence as to whether urine protein-to-creatinine ratio (UPC) could be used for the diagnosis of renal diseases in cattle. OBJECTIVE: The goal of the study was to determine alterations in UPCs observed with different subclinical renal diseases in clinically healthy cattle and to investigate whether UPC can efficiently differentiate cattle with and without subclinical renal pathology. METHODS: Kidney and urine samples from 57 clinically healthy adult dairy (44) and beef (13) cattle were collected after slaughter. Urinary protein and creatinine concentrations were measured in an automatic analyzer, and urinary-specific gravity (USG) was measured using a temperature compensated refractometer. Kidney samples underwent histopathologic examination, and the cattle were classified as NL (no renal lesion) and L (lesions detected even in one kidney). Based on USG, the cattle were divided into the Normal USG (≥1.020) and Low USG (<1.020) groups. The cattle with either histopathologic lesions or low specific gravities were considered to have renal disease. RESULTS: Renal lesions were detected in 37 cattle. UPC values were significantly affected (P < .05) by USG values, and not by the type of lesion detected, breed, or age, and their interactions (P > .05). The analysis revealed that a UPC of ≥0.19 provided an optimal cut-off point for the differentiation between normal animals and those with renal disease with 66.0% sensitivity and 90% specificity. CONCLUSIONS: The UPC calculation is a useful tool for the differentiation of normal cattle and those with renal disease. A UPC of less than 0.19 is associated with the absence of renal damage, whereas higher values raise suspicion for renal disease.

Method agreement between three different chambers for comparative boar semen computer-assisted sperm analysis.

The computer-assisted sperm analysis (CASA) has become a standard laboratory tool. Although it contributes a lot to the objective sperm motility assessment, its measurements may be affected by many factors. The aim of the study was to evaluate the effect of chamber on boar semen CASA results. Totally, 100 extended (30 × 106  sperm/ml) boar semen samples were analysed by CASA. Each sample was evaluated using Makler, Leja 4 chamber 20 µm and conventional glass slide/coverslip chambers (MC, LC and GSC, respectively). The differences in values between MC and LC and between MC and GSC were significantly positive (higher values for MC compared with LC and GSC) for total motility, progressive, rapid movement, VCL, VSL, VAP, STR and hyperactive, thus indicating a systematic effect. Between LC and GSC, the differences in many parameters (non-progressive, progressive, slow, LIN, STR, hyperactive) were evenly distributed around zero, while in all other parameters the differences were significantly positive (higher values for LC compared with GSC), except for medium movement. Based on the estimated intraclass correlation coefficients, the method agreement between MC and LC and between LC and GSC was overall moderate to good, depending on the parameter; nonetheless, it was poor between MC and GSC. The limits of agreement between methods can vary considerably depending on the parameter and should be considered when comparisons between CASA measurements of different andrology laboratories or studies have to be performed.

Effects of testicular hemodynamic and echogenicity changes on ram semen characteristics.

This study investigated correlations among ram semen quality parameters, testicular vascularity and echotexture. Measurements were performed biweekly for a year, before (BE) and after ejaculation (AE), on seven Chios breed rams [three rams (group A) and four rams (group B) of 2-6 and 9-13 years old, respectively], considering breeding (1) and non-breeding period (2). Hemodynamics [Pulsatility Index (PI), Resistive Index (RI)] were evaluated in each testis. Images of testicular parenchyma were analysed to evaluate echogenicity [Mean Value (MV), Contrast (Con), Gray Value Distribution (GVD), Run Length Distribution (RunLD), Long Run Emphasis (LRunEm)]. Semen volume (V), concentration (C), kinetics (CASA), morphology (Sperm Blue), viability (Eosin-Nigrosin) and DNA fragmentation (Acridine Orange) were assessed. In group A/2, semen V was correlated to left testis PI and right testis RI, BE (r = 0.4, p = 0.013/r = 0.6, p < 0.001, respectively), and to right testis PI and RI, AE (r = 0.5, p < 0.001). In A/1/AE, semen C was correlated to left testis PI (r = -0.5, p = 0.003). In group A/1/BE, RI of both testes was correlated to total and tail abnormalities (left: r = -0.5, p = 0.008/ p = 0.004, respectively - right: r = -0.4/r = -0.5, p = 0.01/ p = 0.008, respectively). In group B/1/BE, sperm tail and total abnormalities were correlated to left testis Con (r = 0.5, p = 0.02/ p < 0.001, respectively), whereas in A/2/BE, they were correlated to left testis LRunEm (r = -0.8/r = -0.7, p < 0.001, respectively). In group A/1/BE, DNA damage was correlated to right testis LRunEm (r = -0.6, p < 0.001) and tail abnormalities to left testis PI (r = -0.5, p = 0.03). In group B/1/AE, right testis LRunEm, GVD, RunLD, were correlated to sperm DNA integrity (r = -0.9, p < 0.001). Immotile spermatozoa and viability were correlated to left testis Con in group B/2/AE (r = -0.6/r = 0.6, p < 0.001) and to right testis MV in group A/1/AE (r = -0.6/r = 0.6, p < 0.001). In conclusion, alterations in both ram testes blood flow and echotexture seem to be moderately correlated with sperm assessed parameters, depending on season and ram age.

Effect of aflatoxin B1 on blood serum oestradiol-17ß and progesterone concentrations during the luteal phase and the synchronized oestrus of goats.

The effect of prolonged aflatoxin B1 (AFB1) administration on blood serum oestradiol-17ß and progesterone concentrations in goats during the luteal phase and the synchronized oestrus was investigated. Thirty-six Greek indigenous primiparous goats were used, during the oestrus period; 12 goats received, per os, 50 µg (treated group T50) and 12 goats received 100 µg (treated group T100) AFB1/day/head, respectively, for approximately 1.5 month, while 12 goats served as controls (C). On day 36 of the experiment, each goat was injected, i.m, 0.5 ml prostaglandin F2α (PGF2α). Blood samples were collected from each goat twice a week, before PGF2α injection, as well as every 4 hours from the onset to the end of the synchronized oestrus. Oestradiol-17ß and progesterone concentrations in blood serum were determined using radioimmunoassay. During the whole luteal(s) phase(s), linear regression analysis revealed a significant negative dependence (P < 0.05) of oestradiol-17ß and a significant positive dependence (P < 0.05) of progesterone over group (C = 0, T50 = 50, T100 = 100), in a dose dependent manner. During the synchronized oestrus, multiple linear regression analysis revealed a significant negative dependence (P < 0.05) of oestradiol-17ß, as well as a significant positive dependence (P < 0.05) of progesterone over group (C = 0, T50 = 50, T100 = 100) and over time (hours, from the onset to the end of the synchronized oestrus). No significant differences were noticed among the three groups, regarding the body weight of the goats from the onset to the end of AFB1 administration, the occurrence or the duration of the synchronized oestrus presented by the goats (P > 0.05). In conclusion, prolonged AFB1 administration at doses of 100 or even of 50 µg/day/head changes the hormonal pattern in blood during the luteal phase and the synchronized oestrus of goats, being in oestrus period.

The effect of antioxidant agents' addition and freezing method on quality parameters of frozen thawed ram semen.

The aim of this study was to evaluate the effect of antioxidant agents and freezing methods on the ability of ram sperm to preserve its post-thaw quality characteristics. Six Chios rams were subjected to 52 weekly semen collections. Each ram was used as semen donor for freezing experiments once every 2 weeks. Equal number of good quality spermatozoa from each ejaculate (concentration ≥1 × 109 spermatozoa/ml, motility ≥70%, motility score ≥3.5) were pooled. Three equal aliquots of the pooled sample were diluted using three different fractions of a milk-based and glycerol extender (control, quercetin-enriched, α-tocopherol-enriched). Three freezing methods were applied (slow and fast freezing rate in a programmable freezer, vapors of liquid nitrogen) in every aliquot. Sperm aliquots were tested before freezing, immediately after thawing and after 3 h of incubation at 37 °C. Sperm motility (%) was evaluated microscopically. The percentage of membrane and acrosome-intact spermatozoa (IL%) as well as the percentage of membrane-intact and acrosome-reacted spermatozoa (ARL%) were determined by eosin-nigrosin stain. Furthermore, the percentage of hypo-osmotic swelling (HOS) test-positive spermatozoa was estimated. The results revealed no beneficial effect of the antioxidant treatment on the parameters of post-thaw semen (P > 0.05). However, the slow freezing rate method was more beneficial regarding motility, IL, ARL and HOS-positive spermatozoa compared to the other methods. In conclusion, the antioxidant agents used in this study failed to protect sperm against cryopreservation stress; however, the choice of the appropriate freezing method could contribute to the improvement of post-thaw ram sperm quality.

Evaluation of the in-field efficacy of oregano essential oil administration on the control of neonatal diarrhea syndrome in calves.

The objective of this study was to evaluate under field conditions, whether daily administration of oregano essential oil is effective in preventing and/or diminishing the severity of neonatal diarrhea syndrome in calves aged less than 15days. Ninety-one newborn calves from three dairy farms were assigned into two groups; "Eco" group (n=46) calves were drenched with Greek oregano (Origanum vulgare ssp. Hirtum) essential oil (ECODIAR® liquid 5%) at the dose of 12.5mg/kg body weight once per day until the age of 10days. "Conts" group (n=45) calves were left untreated and served as controls. All animals were monitored daily for the incidence of diarrhea until the age of 15days and their fecal score was recorded. Fecal samples were collected on days 3, 6 and 10 for microbiological and parasitological evaluation. Average fecal score throughout the experiment, incidence of diarrhea, duration and severity of diarrhea episodes were significantly lower in Eco group compared to the controls. Daily administration of oregano essential oil in calves for the first 10days of their life effectively diminishes the severity of naturally acquired diarrhea under field conditions and, under certain hygiene practices, possess a preventive effect against neonatal diarrhea syndrome.

Comparison of biuret and refractometry methods for the serum total proteins measurement in ruminants.

BACKGROUND: Determination of serum total protein concentration is commonly performed by the biuret method. Refractometric measurement is a faster and less expensive alternative but its accuracy has not been determined in ruminants. OBJECTIVE: The purpose of the study was to compare the serum total protein concentrations in cattle, sheep, and goats measured by the biuret method with those obtained by refractometry. METHODS: Serum total protein concentration was determined in 120 cattle, 67 sheep, and 58 goat blood samples refractometrically and with the biuret method. The data were analyzed with a paired samples t-test, and Passing and Bablok regression equations and Bland and Altman plots were generated. RESULTS: There was a strong linear relationship between the total protein values determined with the refractometer and the biuret method in cattle, sheep, and goats. The statistical accuracy, which represents a bias correction factor that measures the deviation of the best-fit line from the 45° line through the origin, was 90.63% for cattle, 93.05% for sheep, and 91.76% for goats. The mean protein values determined with the refractometer were significantly lower than those measured with the biuret method in cattle and goats (P < .05) but not in sheep (P > .05). CONCLUSIONS: The evaluated refractometer was sufficiently accurate for the determination of serum total proteins in cattle, sheep, and goats, although it cannot be used interchangeably with the biuret method. The RIs should be corrected for negative bias based on the created equations.