A Comparison of White and Yellow Seminal Plasma Phosphoproteomes Obtained from Turkey (Meleagris gallopavo) Semen.

Seminal plasma is rich in proteins originating from various male reproductive organs. The phosphorylation of these proteins can significantly impact sperm motility, capacitation, and acrosome reaction. Phosphoproteomics identifies, catalogues, and characterizes phosphorylated proteins. The phosphoproteomic profiling of seminal plasma offers valuable insights into the molecular mechanisms that influence semen quality and male fertility. Thus, the aim of this study was a phosphoproteomic analysis of white and yellow turkey seminal plasma. The experimental material consisted of 100 ejaculates from BIG-6 turkeys between 39 and 42 weeks of age. The collected white and yellow turkey seminal plasmas were analyzed for total protein content; the activity of selected enzymes, i.e., alkaline phosphatase (ALP), acid phosphatase (ACP), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT); and the content of reduced glutathione (GSH) and malondialdehyde (MDA). Phosphoproteins were isolated from white and yellow seminal fluids, and the resulting protein fractions were separated by SDS-PAGE and Western blotting. Phosphorylated residues were immunodetected, and the isolated phosphoproteins were identified (nano LC-MS/MS). Yellow seminal plasmas were characterized by higher levels of total protein, GSH, and MDA, as well as higher levels of ALP, ACP, and GPx activity. There were no significant differences in the activity of SOD and CAT. A total of 113 phosphoproteins were identified in turkey seminal fluids. The functional analysis demonstrated that these phosphoproteins were mainly involved in oocyte fertilization, organization and metabolism of the actin cytoskeleton, amplification of the intracellular signal transduction pathway, general regulation of transport, vesicular transport, proteome composition of individual cellular compartments, and the organization and localization of selected cellular components and macromolecules. Increased phosphorylation of the fractions containing proteins encoded by SPARC, PPIB, TRFE, QSOX1, PRDX1, PRDX6, and FASN genes in white plasmas and the proteins encoded by CKB, ORM2, APOA1, SSC5D, RAP1B, CDC42, FTH, and TTH genes in yellow plasmas was observed based on differences in the optical density of selected bands. The obtained results indicate that the phosphorylation profiles of turkey seminal plasma proteins vary depending on the type of ejaculate.

Changes in the Morphology and Antioxidant Status of European Red Deer Sperm Stored in the Epididymides and in a Liquid State.

The aim of this study was to evaluate the motility, morphology, and antioxidant status of European red deer sperm stored in a liquid state (variant I) and in the epididymides (variant II). Spermatozoa were harvested post-mortem from the cauda epididymis. Sperm samples in both variants were stored for up to six days (D6) at 5 °C. Spermatozoa were assessed for motility, viability, morphology, activity of antioxidant enzymes (superoxide dismutase, SOD; glutathione peroxidase, GPx; catalase, CAT), and lipid peroxidation (malondialdehyde, MDA, content). Sperm samples were analyzed on storage days 0, 2, 4, and 6 (D0-D6). Storage time and storage method significantly (p ≤ 0.05) influenced the examined variables. On D2, a decrease in motility and acrosomal integrity was observed in both storage variants, whereas a decrease in viability and an increase in MDA content were noted in spermatozoa stored in the epididymides. On D4, higher values of SOD and GPx activity and MDA content were noted in variant I than in variant II. Catalase activity was very low. GPx is the key enzyme that participates in the reduction of hydrogen peroxide in sperm cells. Spermatozoa stored in a liquid state were characterized by higher motility and viability, improved morphology and antioxidant status than those stored in the epididymides; therefore, liquid storage is more recommended for short-term preservation of epididymal spermatozoa.

The effect of solarium light therapy on selected biological and biochemical parameters of peripheral blood in young and old horses.

The aim of the study was to assess the impact of solarium light therapy on selected biological and biochemical parameters of peripheral blood in recreational horses. The study involved 10 horses divided into two groups of young (aged 5 to 7 years) and old (aged 14 to 19 years) individuals. All animals participated in light therapy sessions every other day. Blood was sampled three times during the study: before the treatment, after five light sessions, and after ten light sessions. Morphological parameters, the activity of antioxidant enzymes, TAS values, and the levels of glutathione (GSH), vitamin D3, vitamin C, and malondialdehyde (MDA) were measured in the whole blood. Light therapy contributed to an increase in MCV, HDW, MCVr, CHr and MPV indices, and simultaneously a decrease in the basophil counts, MCHC, RDW and CHCMr indices in both groups of horses (p ≤ 0.05). At the same time reticulocytes fell in older whereas white blood cells and monocytes counts expanded in younger individuals. The treatment also increased the activity of glutathione reductase (GR) and glutathione peroxidase (GPx) in young but decreased the activity of mentioned enzymes in blood plasma of old horses. The total antioxidant status (TAS) of the blood plasma rose progressively, whereas GSH levels declined in all individuals. Moreover, vitamin D3 levels did not change, whereas vitamin C levels gradually decreased during the experiment. The therapy also helped to reduce levels of MDA in the blood plasma, especially of older horses (p ≤ 0.05). In turn, GPx and GR activities as well as MDA levels significantly declined, whereas GSH levels notably elevated in erythrocytes (p ≤ 0.05). Solarium light therapy appears to have a beneficial impact on the morphological parameters and antioxidant status of blood in recreational horses in the winter season. However, the observed results could in part be attributed to the natural physiological adaptation of each individual organism to the treatment.

A comparison of the biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state at 5 °C.

The biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state were compared. Spermatozoa were collected from the epididymides harvested post-mortem. In the first variant, spermatozoa were diluted in two extenders (Bovidyl® and Salomon's), and were stored at 5 °C for up to 144 h. In the second variant, spermatozoa were stored in the epididymides at 5 °C for up to 144 h, and then diluted in the same extenders. Biological properties were evaluated after 0, 48, 96, and 144 h of storage. Sperm motility parameters were determined in the CASA system. Plasma and acrosomal membrane integrity, mitochondrial activity, apoptotic changes, and DNA integrity were assessed by the fluorescence method. Most variables were significantly influenced by the storage method and time. At 144 h, differences (P ≤ 0.05) in sperm parameters were observed between storage variants. Total motility (TMOT), plasma membrane integrity, and mitochondrial activity decreased below 50% of baseline values in the spermatozoa stored in the epididymides, but remained above 70% of baseline values in the spermatozoa stored in a liquid state. The compared storage variants did not differ in TMOT, mitochondrial activity, or the percentage of viable spermatozoa without apoptotic-like changes up to 96 h of storage, regardless of the applied extender. The earliest significant changes were noted in sperm motility parameters. In conclusion, European red deer spermatozoa can be stored in the epididymides at 5 °C for up to 96 h, but their biological parameters are more effectively preserved during liquid storage.

Retinoic acid alters metalloproteinase action in red deer antler stem cells.

Metalloproteinases (MMP)s regulate developmental processes, control angiogenesis and wound healing, participate in the formation of immune receptors, and are expressed in stem cells. Retinoic acid (RA) is a potential modulator of these proteinases. The aim was to determine (1) MMPs' action in antler stem cells (ASCs) before and after differentiation into adipo-, osteo-, and chondrocytes and (2) the effect of RA on modifying MMP action in ASCs. Antler tissue from pedicle was collected approximately 40 days after antler casting, post mortem from healthy breeding five year old males (N = 7). The cells were isolated from the pedicle layer of periosteum after skin separation and cultured. The pluripotency of the ASCs was evaluated by mRNA expression for NANOG, SOX2, and OCT4. ASCs were stimulated with RA (100nM) and differentiated for 14 days. The MMP (1-3) and TIMP(1-3) (tissue inhibitor of MMPs) mRNA expression was determined in the ASCs, their concentrations in the ASCs and the medium after RA stimulation as well as profiles of mRNA expression for MMPs: 1-3 and TIMPs: 1-3 during differentiation of ASC to osteocytes, adipocytes and chondrocytes. RA increased MMP-3 and TIMP-3 mRNA expression and output (P < 0.05) and not influenced on MMP-1 and TIMP-1 mRNA expression and output in ASC (P > 0.05). Depending on differentiation of ASC to osteocytes, adipocytes or chondrocytes, MMPs`and TIMPs`expression profile fluctuates for all studied proteases and its inhibitors. The studies demand continuation considering the role of proteases in stem cells physiology and differentiation. The results may be relevant for the study of cellular processes during the cancerogenesis of tumor stem cells.

Influence of the Season and Region Factor on Phosphoproteome of Stallion Epididymal Sperm.

Epididymal maturation can be defined as a scope of changes occurring during epididymal transit that prepare spermatozoa to undergo capacitation. One of the most common post-translational modifications involved in the sperm maturation process and their ability to fertilise an oocyte is the phosphorylation of sperm proteins. The aim of this study was to compare tyrosine, serine, and threonine phosphorylation patterns of sperm proteins isolated from three subsequent segments of the stallion epididymis, during and out of the breeding season. Intensities of phosphorylation signals and phosphoproteins profiles varied in consecutive regions of the epididymis. However, significant differences in the phosphorylation status were demonstrated in case of endoplasmic reticulum chaperone BiP (75 and 32 kDa), protein disulfide-isomerase A3 (50 kDa), nesprin-1 (23 kDa), peroxiredoxin-5 (17 kDa), and protein bicaudal D homolog (15 kDa) for season x type of phosphorylated residues variables. Significant differences in the phosphorylation status were also demonstrated in case of endoplasmic reticulum chaperone BiP and albumin (61 kDa), protein disulfide-isomerase A3 (50 kDa), and protein bicaudal D homolog (15 kDa) for region x type of phosphorylated residues variables.

Influence of Zinc and Manganese Nanoparticles on Selected Parameters of Turkey Spermatozoa Stored in a Liquid State at 4 °C.

The aim of this study was to determine the effect of semen extender supplementation with 25 or 50 µM of zinc nanoparticles (ZnNPs) or manganese nanoparticles (MnNPs) on turkey spermatozoa preserved in a liquid state. Twenty turkey ejaculates were obtained from twenty healthy males. The collected semen was preserved at 4 °C for 48 h with or without NPs. Selected qualitative and quantitative parameters of sperm (motility, plasma membrane activity, mitochondrial membrane potential (MMP) and the percentage of sperm demonstrating NO and SOD activity) were examined after 2, 24 and 48 h of storage. Sperm motility and MMP decreased in semen preserved with ZnNPs at each time point of the analysis. However, all spermatozoa remained viable throughout storage. In contrast, membrane integrity and mitochondria activity (p ≤ 0.05) increased, and the highest SOD activity (p ≤ 0.05) was observed in semen preserved with MnNPs. The addition of MnNPs to the semen extender could potentially improve the parameters of turkey semen during prolonged storage.

The activity of N-acetyl-ß-hexosaminidase in boar seminal plasma is linked with semen quality and its suitability for cryopreservation.

The determination of sperm cryotolerance is an important step in the process of developing optimal techniques for the storage of boar semen. The objective of this study was to determine individual proteome variations in boar seminal plasma and spermatozoa and establish their influence on the cryotolerance of ejaculate. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the presence of protein with estimated molecular weight of 90 kDa in sperm extracts from ejaculates of selected boars. In all cases, dialysis performed at the initial stage of cryopreservation effectively removed the protein from sperm cells. The protein had an affinity for Zn(2+) ions. Mass spectrometry revealed similarities between the discussed protein and the ß subunit of N-acetyl-ß-hexosaminidase (ß-HEX). Seminal plasma ß-HEX was purified 252-fold with approximately 27% recovery and specific activity of 1800 U/mg of protein. Enzyme activity in fresh seminal plasma was correlated with superoxide dismutase activity (r = -0.42, P < 0.05), glutathione peroxidase activity (r = -0.42, P < 0.05), mitochondrial function (r = 0.31, P < 0.05), glutathione content (r = 0.34, P < 0.05), total protein content (r = 0.42, P < 0.05), and total oxidant status of seminal plasma (r = 0.37, P < 0.05). After thawing, ß-HEX activity in seminal plasma was negatively correlated with the total motile sperm count (r = -0.33, P < 0.05), plasma membrane integrity (r = -0.31, P < 0.05), and lipid peroxidation (r = 0.33, P < 0.05). The observed correlations indicate that lower levels of ß-HEX activity in boar seminal plasma are linked with higher quality of sperm after thawing. Based on those observations, the ejaculates were divided into two groups characterized by low (<20,000 U/L) and high (>20,000 U/L) levels of ß-HEX activity in seminal plasma. In plasma with high ß-HEX activity, spermatozoa were characterized by lower plasma membrane integrity (84.7%, P < 0.05). Higher glutathione levels (1250.3 µM), higher total protein content (50 mg/mL), and higher total oxidant status (6.82-µmol H2O2 Equiv/L) were also observed (P < 0.05). After thawing, lower sperm motility (20.4%), lower plasma membrane integrity (41.7%), and higher lipid peroxidation (30.9-nM malondialdehyde/10(8) spermatozoa/h) were reported in ejaculates with high seminal plasma ß-HEX activity. The results of this study indicate that ß-HEX activity in seminal plasma is a useful indicator in preliminary evaluations of boar sperm cryotolerance.

Superoxide dismutase (SOD) in boar spermatozoa: purification, biochemical properties and changes in activity during semen storage (16°C) in different extenders.

The antioxidant system in semen is composed of enzymes, low-molecular weight antioxidants and seminal plasma proteins. Loss of enzymatic activity of superoxide dismutase (SOD) during semen preservation may cause insufficient antioxidant defense of boar spermatozoa. The aim of this study was to isolate and characterize SOD molecular forms from spermatozoa and to describe changes in SOD activity in boar sperm during preservation at 16°C. Sperm extracts were prepared from fresh or diluted semen and used for SOD purification or activity measurement. Ion-exchange chromatography and gel filtration was used to purify SOD molecular forms. BTS, Dilu Cell, M III and Vitasem were used as diluents for 5-day storage of semen at +16°C. The molecular form of SOD released from spermatozoa after cold shock and homogenization had a molecular weight of approximately 67kDa. The activity of the SOD form was the highest at pH 10 within the temperature range between 20 and 45°C. The enzymatic activity of form released after cold shock was inhibited by H2O2 and diethyldithiocarbamate (DDC; by 65 and 40%, respectively). The SOD form released by homogenization was inhibited by H2O2 and DDC (40%). The molecular form released after urea treatment was a 30kDa protein with maximum activity at 20°C and pH 10. Enzymatic activity of this form was inhibited by H2O2 by 35%, DDC by 80% and 2-mercaptoethanol by 15%. The antigenic determinants of SOD isolated from boar seminal plasma and spermatozoa were similar to each other. Susceptibility of spermatozoa to cold shock increased during storage, but the differences between extenders were statistically non-significant.