A New Method of Assessing Sheep Red Blood Cell Types from Their Morphology.

Data concerning the morphometric parameters of sheep red blood cells (RBCs) obtained using computer-assisted image analysis have not yet been investigated, and there are no data on any analyses of ovine RBC subpopulations based on their morphometric parameters. The aims of this study are to determine the values of RBC haematological and morphometric size and shape parameters, to form groups according to the obtained values of haematological parameters; to determine the differences in RBC morphometric parameters between the formed groups, and to determine RBC subpopulations and their respective proportions in the formed groups. Thirty-six blood samples were collected from the jugular vein of clinically healthy Lika pramenka sheep, aged between 2 and 5 years. Haematological parameters including haemoglobin (HGB), haematocrit (HTC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and RBC distribution width were analysed using a haematology analyser. Haematological parameters were categorized into two groups: those with lower values or values below the physiological range (Groups 1) and groups with higher values or values above the physiological range (Groups 2). Morphometric parameters of RBCs were determined from stained blood smears using SFORM, a computer-assisted program. Significantly higher values of RBC area, outline, convex, minimal and maximal radius, as well as length and breadth were established in Groups 2 compared to Groups 1 of HGB, HCT, MCV, MCH, and MCHC, respectively. Based on the morphometric parameters of RBCs, three RBC subpopulations were obtained using principal component and cluster analysis: ES 1-the smallest and most elongated RBCs, ES 2-the biggest and most rounded RBCs, and ES 3-average size and shape RBCs. Significantly higher proportions of ES 2 and ES 3 subpopulations, as well as a significantly lower proportion of ES 1 subpopulation, were established in Groups 2 compared to Groups 1 of HGB, HTC, MCV, and MCH, respectively. It can be concluded that ovine RBC subpopulations, based on their morphometric parameters, can be obtained by using computer-assisted image analysis of RBC morphometry and multivariate statistical methods, including principal component and cluster analysis. RBC morphometry, including classification into subpopulations, could serve as a basis for future possibilities in the diagnostic interpretation of anaemic syndromes in veterinary medicine, especially in normocytic, macrocytic, and microcytic anaemias in sheep.

Exogenous melatonin influences distribution of French Alpine buck spermatozoa in morphometrically distinct subpopulations during the non-breeding season.

The aim of this study was to establish subpopulations of spermatozoa in bucks using the principal component (PC) and cluster analysis according to morphometric head and tail variables, and to determine differences in proportions of subpopulations between exogenous melatonin-treated and control bucks. The bucks (n = 12) were assigned to two groups comprising six bucks each. By the end of March, four melatonin implants were inserted in the bucks in the experimental group. Semen was collected weekly using an artificial vagina from March to May (the non-breeding season). Analyses were performed in stained smears by SFORM computer-assisted program for eight head and five tail variables. The PC analysis revealed four components with the most important value for each (head outline, head ellipticity, mid-piece length and width). Cluster analysis indicated there were three subpopulations (average-sized spermatozoa- C_1; small and less-elliptic - C_2; big and elliptic - C_3). Melatonin-treated bucks had a greater proportion of C_1 spermatozoa and a lesser proportion of C_2 spermatozoa during May (P < 0.05). To the best of our knowledge, this is the first study where PC and cluster analyses were performed on buck semen with head and tail variables analyzed together, in the same analysis, to evaluate spermatozoa population. Also, this is the first analysis of morphometric variables for assessing the influence of melatonin on spermatozoa subpopulations. The positive effect of melatonin on the proportions of spermatozoa in subpopulations could have been a consequence of the decreasing the proportion of the subpopulation with the least head and tail sizes and ellipticity.

Antioxidant protection and lipid peroxidation in testes and different parts of epididymis in boars.

The antioxidative ability of testes and epididymis to protect spermatozoa from detrimental effects of oxidation processes induced by excessive generation of reactive oxygen species has not been previously studied in detail in boar reproductive tissues. The aim of this study was to investigate differences in level of antioxidative protection, intensity of lipid peroxidation, and values of biochemical parameters in testes and different parts of epididymis in sexually mature boars. The study was performed on five Swedish landrace boars from the same litter aged 10 months kept under same ambient conditions. After slaughtering performed at the end of November; tissue samples of testes and the head, body and tail of epididymis were taken. The activity of glutathione peroxidase, glutathione reductase, superoxide dismutase, γ-glutamyltransferase, acid phosphatase, and lactate dehydrogenase, and concentrations of triacylglycerol, phospholipids, cholesterol, free fatty acids were determined in obtained supernatants from homogenized tissues spectrophotometrically; the concentration of malondialdehyde was determined by high-performance liquid chromatography. Significantly higher activities of glutathione peroxidase and glutathione reductase (P < 0.05) were found in testes compared with epididymis. In testes, a significantly higher activity of superoxide dismutase was found than in the head and tail of epididymis (P < 0.05). Malondialdehyde concentration in head of epididymis was significantly higher than in testes, or the body and tail of epididymis (P < 0.05). Simultaneously, malondialdehyde concentration in testes was significantly higher than in body and tail of epididymis (P < 0.05). In tail of epididymis, significantly higher activities of γ-glutamyltransferase, acid phosphatase, and lactate dehydrogenase were recorded than in testes. Significantly lower concentrations of triacylglycerol and free fatty acids were recorded in epididymis tail in comparison to epididymis head (P < 0.05). It could be concluded that high activities of antioxidative enzymes in testes of boars are essential for the appropriate protection of spermatozoa and cells of testes tissue against oxidative damages. The tissues of testes and epididymis head in boars were more susceptible to lipid peroxidation. Results of the present study indicated physiological importance of antioxidative enzymes in reproductive system in boars, and thus may serve for better understanding the mechanisms of male infertility.

Influence of different periods of the year and age on the parameters of antioxidative status and oxidative stress in the blood serum of breeding bulls.

The sources of variations that may cause physiological differences between blood serum biochemistry parameters of bulls have not been investigated in detail. Aim of the present study was to establish influence of different periods of the year and the age of breeding bulls on parameters of antioxidative status and oxidative stress in their serum and to correlate these monitored variables. Research was performed on two groups, each comprising 9 Simmental bulls: a younger group (YB) (aged 2-4 years) and older one (OB) (aged 5-10 years). Blood samples for biochemical analyses were collected from jugular vein in cold (CP) and warm periods (WP) of the year. Reduced glutathione (GSH), uric acid (UA), total protein (TP), albumin (ALB), thiobarbituric acid reactive substance (TBARS), and protein carbonyl content (PCC) serum concentration were determined, as well as activities of selenium-dependent glutathione peroxidase (Se-GSH-Px), total superoxide dismutase (TSOD), manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD) and catalase (CAT). Serum values of SeGSH-Px, MnSOD, UA and TP in OB were significantly higher compared to those in YB during CP of the year. Significantly higher PCC concentration in serum of YB and OB were established in CP of the year than in WP. TBARS serum concentration in YB was significantly higher in comparison to that in OB during CP of the year. It can be concluded that both OB and YB show a great sensitivity to climate condition alterations during CP in comparison to WP of the year and that YB show even greater sensitivity.

Differences in seminal plasma and spermatozoa antioxidative systems and seminal plasma lipid and protein levels among boar breeds and hybrid genetic traits.

The objectives of this study were to determine the influence of breed and hybrid genetic traits of boars on lipid and protein concentrations and antioxidative system variables in seminal plasma (SP) and spermatozoa and their correlations with semen quality variables. Semen samples from 27 boars: Swedish Landraces (SL), German Landraces (GL), Large Whites (LW), Pietrains (P) and Pig Improvement Company hybrids (PIC-hybrid), aged from 1.5 to 3 years old, were collected. SP was spectrophotometrically analyzed to determine total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triacylglycerol (TAG), total protein (TP), albumin, and zinc concentrations. The antioxidative system in SP and spermatozoa was established spectrophotometrically by determining total antioxidative status (TAS), total superoxide dismutase (TSOD) and glutathione peroxidase (GSH-Px) parameters, as well as copper-zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) activity in spermatozoa. The hybrid boars had higher (P<0.05) SP concentrations of: TC, LDL-C and TAG than P and GL; HDL-C than P, GL and SL; and TP than P and LW. PIC-hybrid had lower values (P<0.05) in spermatozoa of: TAS and CuZnSOD than SL; TSOD and GSH-Px than SL and P; and MnSOD than SL and LW. Differences in SP and spermatozoa antioxidative system variables and the significant differences in SP protein and lipid variables exist among boars of different breeds and hybrid. Novel data and observed differences in semen variables among boar breeds and hybrids and their correlations with semen quality parameters in this study could contribute to better assessment of boar semen quality.

Influence of boar breeds or hybrid genetic composition on semen quality and seminal plasma biochemical variables.

The enzyme concentrations of seminal plasma are important for spermatozoa metabolism and function in boars. The need has arisen for introducing a biochemical evaluation of semen, along with the usual standard semen analyses. There are no data on the influence of boar breeds on the seminal plasma biochemical variables investigated in this study. Therefore, the objective was to determine the influence of breed and hybrid genetic composition of boars on semen quality and seminal plasma biochemical variables. Semen samples of 27 boars (Swedish Landrace, German Landrace, Large White, Pietrain and Pig Improvement Company hybrid-PIC-hybrid), aged between 1.5 and 3 years, were collected. After evaluation of semen quality, the seminal plasma was separated from the spermatozoa by centrifugation of semen. The seminal plasma was subjected to spectrophotometric analysis to determine alkaline phosphatase (ALP), acid phosphatase (ACP), γ-glutamyltransferase (GGT), creatine kinase (CK) and lactate dehydrogenase (LDH) and to atomic absorption spectrophotometric analysis to measure the concentration of calcium and magnesium. Conventional semen quality variables differed depending on breed and PIC-hybrid genetic composition, though these differences were typically insignificant. In the seminal plasma, significant differences were determined in enzyme activity (ALP, GGT, CK and LDH) and in calcium concentration among boars of different breeds. There are, therefore, differences in semen quality and significant differences in the seminal plasma biochemical variables among boars of different breeds and PIC-hybrid genetic composition. The data and differences in semen variables detected in the present study provide knowledge for enhancing evaluation and monitoring of boar reproductive potential, semen quality and explain the potential causes of boar infertility.

Metabolic and oxidative status of Saanen goats of different parity during the peripartum period.

The aim of this study was to research changes in metabolic and antioxidative status of Saanen goats of different parity occurring during the peripartum period. Blood samples were taken on 10-7 and 3-1 d prepartally and 1-3, 14 and 28 d postpartally from goats allocated in three groups according to their parity: primiparous (PRIM), goats that kidded the 2nd or 3rd time (MID), and goats that kidded 4 or more times (MULTI)). Metabolic profile parameters (non-esterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), glucose, triglycerides, albumin and urea) and indicators of oxidative stress ((superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA)) were determined. Intense metabolic changes associated with late pregnancy and onset of lactation were pronounced the most in MULTI goats that also had the biggest litter per goat. Significant differences were found in metabolic parameters NEFA, BHB, glucose, triglycerides within groups during peripartum period, as well as between them (the effect of parity). MDA concentrations were indicative of increased lipid peroxidation around parturition, especially pronounced in MULTI group 1-3 d prepartally, when the highest GSH-Px/SOD ratio was also found. Postpartally, antioxidant enzymes ratio in MID and MULTI group decreased while MDA concentrations remained high, suggesting antioxidant system inefficiency. Significant time × group interaction was observed for most of the parameters. The obtained results show that the goats of higher parity display higher levels of metabolism intensity and consequently, varying levels of oxidative stress during the peripartum period. Further studies should determine applicability of NEFA and BHB in periparturient metabolic profiling in dairy goats as well as establish normal ranges and cut-off levels for these biomarkers.

Biochemical properties of bull spermatozoa separated in iodixanol density solution.

Bull spermatozoa samples contain variable portion of motile and normal morphology spermatozoa along with spermatozoa incapable of fertilization due to their pathologic changes. As semen quality is influenced by biochemical and morphological characteristics of all spermatozoa, the aim of the study was to separate spermatozoa in discontinuous iodixanol density gradient solution and to determine their cholesterol, phospholipid, triacylglycerol and lipid peroxide concentrations and creatine kinase activity. The study was performed in winter and included seven Simmental bulls aged 1.5-3.5 years. Semen samples were collected by use of artificial vagina. Upon evaluation of semen quality (volume, concentration and progressive sperm motility), the samples were centrifuged in iodixanol density solution to obtain two sperm fractions. The two fractions included sperms with progressive motility greater than 90% and less than 20%, respectively. A statistically significantly higher lipid peroxide concentration was determined in sperm fraction with <20% progressive motility. Different sperm subpopulations can be obtained by separating bull spermatozoa in different iodixanol density gradient solutions, while monitoring their biochemical properties can help assess the sperm quality.

Effects of fasting and refeeding on the antioxidant system in cockerels and pullets.

The effect of fasting and refeeding on total antioxidant status (TAS), glutathione peroxidase (GSH-Px) activity and concentration of some non-enzymatic antioxidant compounds was studied in cockerels and pullets. Blood was collected before and after 48-h fasting and 24 h after refeeding. In cockerels, fasting resulted in a significant decrease of TAS and uric acid concentration. After refeeding, the concentration of TAS remained significantly lower as compared to the control level. At the same time, blood plasma level of total lipids increased in comparison to the control and post-fasting values. In pullets, fasting resulted in a significant decrease of whole blood haemolysate GSH-Px activity and blood plasma concentrations of albumin and uric acid. Simultaneously, a significant increase in total lipids and cholesterol was obtained. In pullets, refeeding resulted in a further decrease of TAS to undetectable values, a significant decrease of blood plasma cholesterol, and a significant increase of GSH-Px in the whole blood haemolysate and in blood plasma uric acid content. The results indicate that fasting has a negative impact on the antioxidant defence system of the blood, which leads to a reduced resistance to oxidative stress in both cockerels and pullets. However, pullets seem to be more susceptible to fasting-provoked oxidative stress than cockerels.