Krzysztof Kozłowski

Effect of organic and inorganic forms of selenium in diets on turkey semen quality.

The effects of Se supplementation and its organic or inorganic form on semen quantitative parameters (ejaculate volume, sperm concentration, and total number of sperm) and biochemical parameters of seminal plasma (protein concentration, acid phosphatase activity, superoxide dismutase activity, and total antioxidant capacity) were investigated over a 25-wk reproductive season. Additionally, DNA fragmentation and motility characteristics of turkey spermatozoa were measured. The parameters of turkey semen in relation to yellow semen syndrome were also determined. Twenty-four males (Big 6) were divided into 3 experimental groups differing in form of Se supplementation (no Se supplementation, 0.3 mg/kg of inorganic Se from sodium selenite and 0.3 mg/kg of organic Se from Sel-Plex, Alltech Inc., Nicholasville, KY). Dietary Se supplementation enhanced the sperm concentration and total number of sperm and did not influence the antioxidative properties of turkey seminal plasma and most biochemical parameters. Only seminal plasma acid phosphatase activity was increased in turkeys fed inorganic Se. The main sperm DNA fragmentation parameters were not affected by dietary Se. The highest percentage of motile spermatozoa (85%) was recorded for the semen of turkeys fed organic Se. Values of the biochemical parameters (acid phosphatase, superoxide dismutase, total antioxidant capacity) of seminal plasma increased during the reproductive season. Yellow semen was characterized by increased biochemical parameters and decreased spermatozoa motility characteristics. However, the percentage of motile spermatozoa did not differ between white and yellow semen. Organic Se seemed to be the preferred form of diet supplementation in comparison with inorganic Se. Biochemical parameters of semen and spermatozoa motility parameters appear to be useful for evaluating the effect of age on semen quality. Monitoring the DNA fragmentation of spermatozoa at the end of the reproductive season could be a useful tool for monitoring turkey semen quality. Increased superoxide dismutase activity can be used as an indicator of yellow semen. A decline in the quality of yellow semen can be related to a decrease in the spermatozoa motility parameters of turkeys.

Isolation and Characterization of an Ovoinhibitor, a Multidomain Kazal-Like Inhibitor from Turkey (Meleagris gallopavo) Seminal Plasma

ABSTRACT Turkey seminal plasma contains three serine proteinase inhibitors. Two of them, with low molecular masses (6 kDa), were identified as single-domain Kazal-type inhibitors responsible for regulating acrosin activity. Our experimental objective was to isolate and characterize the inhibitor with the high molecular weight from turkey seminal plasma. The inhibitor was purified using hydrophobic interaction and affinity chromatography. Pure preparations of the inhibitor were used for identification by mass spectrometry, for determination of physicochemical properties (molecular weight, pI, and content and composition of the carbohydrate component), for kinetic studies, and for antibacterial tests. Gene expression and immunohistochemical detection of the inhibitor were analyzed in the testis, epididymis, and ductus deferens. The inhibitor with a high molecular weight from turkey seminal plasma was identified as an ovoinhibitor, which was found in avian semen for the first time. The turkey seminal plasma ovoinhibitor was a six-tandem homologous Kazal-type domain serine proteinase inhibitor that targeted multiple proteases, including subtilisin, trypsin, and elastase, but not acrosin. Our results suggested that hepatocyte growth factor activator was a potential target proteinase for the ovoinhibitor in turkey seminal plasma. The presence of the ovoinhibitor within the turkey reproductive tract suggested that its role was to maintain a microenvironment for sperm in the epididymis and ductus deferens. The turkey seminal plasma ovoinhibitor appeared to play a significant role in an antibacterial semen defense against Bacillus subtilis and Staphylococcus aureus.

Proteomic analysis of white and yellow seminal plasma in turkeys (Meleagris gallopavo)

Yellow semen syndrome (YSS) is endemic within domestic turkey populations. Yellow semen is of lower quality and, when used for insemination, results in reduced fertility and hatchability. Little is known about the etiology of YSS. The aim of this study was to compare the proteome of white and yellow seminal plasma of turkeys using 1) 2-dimensional difference gel electrophoresis (2D-DIGE) to quantify seminal plasma proteins and 2) matrix-assisted laser desorption/ionization mass spectrometry to identify the proteins that are differentially abundant in white and yellow seminal plasma. A total of 49 protein spots (30 upregulated and 19 downregulated) were differentially expressed in yellow seminal plasma compared with white seminal plasma. Transthyretin and serum albumin-like showed a 3-fold increase in seminal plasma from males with YSS, and the latter was validated using Western blot analysis. A 3-fold increase was observed for hemopexin-like and immunoglobulin light chain V-J-C region. Pantetheinase-like showed a 1.3-fold increase. Ovotransferrin, hepatocyte growth factor activator, cysteine-rich secretory protein 3-like, and ferritin heavy chain-like showed a significant decrease (at least a 1.3-fold decrease) in yellow semen. Further studies are necessary to evaluate the precise function of the above-mentioned proteins in YSS and to establish quality markers of turkey semen to predict the reproductive potential of individual turkeys.

The effect of a probiotic preparation containing Bacillus subtilis ATCC PTA-6737 on egg production and physiological parameters of laying hens

Abstract A total of 288 Lohmann Brown laying hens were randomly divided into two groups (9 replicates of 16 birds each). The hens were housed in three-tier battery cages for 26 weeks, including a two-week pre-laying period. All birds were fed iso-nitrogenous and iso-caloric diets, and had free access to water. Control group (C) hens were fed a basal diet, and experimental group (BS) hens received a basal diet supplemented with a commercial probiotic preparation of Bacillus subtilis ATCC PTA- 6737 at 1×108 CFU/kg feed. The number and weight of eggs laid, feed intake, feed conversion, egg quality, the fatty acid profile and cholesterol content of yolk lipids, and selected blood biochemical parameters of hens were determined throughout the experiment. No significant differences (P>0.05) were noted between the groups in average egg weight, laying performance (%), daily feed intake or feed conversion. Eggs laid by BS group hens received significantly higher scores for yolk color (Roche yolk color fan) and albumen quality (Haugh units), and they were characterized by a significant improvement in shell thickness and breaking strength (P<0.05) in comparison with eggs laid by control group hens. No significant differences (P>0.05) were observed between the groups in the fatty acid profile of yolk lipids, except for a significant increase (P<0.05) in oleic acid concentrations in group BS. No significant differences (P>0.05) were noted between the groups in selected blood biochemical parameters of laying hens. Group BS eggs had a significantly lower (P<0.05) cholesterol content of yolk lipids. It can be concluded that a probiotic preparation containing Bacillus subtilis ATCC PTA-6737 had a beneficial influence on selected performance parameters of laying hens, egg quality and the cholesterol content of yolk lipids.

Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma.

A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases. It is of interest if the single-domain Kazal-type inhibitor controls the activity of turkey seminal plasma proteinases. Pure preparations of the Kazal-type inhibitor and anti-Kazal-type inhibitor monospecific immunoglobulin Gs were used as ligands in affinity chromatography for proteinase isolation from turkey seminal plasma. Gene expression and the immunohistochemical detection of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. The hepatocyte growth factor activator (HGFA) was identified in the binding fraction in affinity chromatography. Hepatocyte growth factor activator activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner. This protease was a primary physiological target for the single-domain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma, and phosphorylation was the primary posttranslational modification of HGFA. In addition to HGFA, acrosin was a target proteinase for the single-domain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and was not present as a free enzyme. The single-domain Kazal-type inhibitor was specific for the reproductive tract. The germ cell-specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis; secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in the changes in sperm membranes during maturation and in the maintenance of the microenvironment in which sperm maturation occurred and sperm was stored. The role of HGFA in these processes remains to be established.

Enhancing The Nutritional Value of Poultry Feedstuffs Using The Example of Rapeseed Products – A Review

Abstract This paper outlines the main goals and methods for improving the nutritional value of poultry diets. The benefits of various processing techniques are demonstrated using the example of rapeseed and rapeseed by-products, i.e. rapeseed cake and rapeseed meal. The progress made in plant breeding in the past decades led to a significant reduction in the content of anti-nutritional factors and ingredients which reduce the nutritional value of feed. Rapeseed by-products have become a valuable source of protein for feedstuffs, and they can be safely used in poultry rations at high inclusion rates. Mechanical treatments, such as rapeseed hulling, and enzyme supplementation (phytases, carbohydrases) also significantly increase the nutritional value of feed ingredients. Further research is needed to tap into the new opportunities for improving the nutritional value of feedstuffs

Marking and return method for evaluating the effects of stocking larval vendace, Coregonus albula (L.), into Lake Wigry in 2000-2001

Marking and return method for evaluating the effects of stocking larval vendace, Coregonus albula (L.), into Lake Wigry in 2000-2001 In 2000 and 2001, larval vendace, Coregonus albula (L.), were marked and released into Lake Wigry. The larvae were immersed in alizarin red S. Of the 19.2 million vendace larvae released in 2000, 2 million were marked (10.4% of the overall number of fish released), and in 2001 of the 18.8 million fish released, 7 million were marked (47.8%). In subsequent years, otoliths were excised from caught vendace and the number of them with alizarin marks was determined. It was assumed that all unmarked specimens came equally from natural spawning and stocking assuming that survival is equal in both forms of recruitment. It was confirmed that 82.4% of the vendace caught from the 2000 generation originated from stocking, while this figure was 64.2% of all specimens caught from the 2001 generation. Lake Wigry hosts the most abundant vendace population in Poland, and this stock spawns on a massive scale annually. Even so, the study described herein provides evidence of just how important systematic stocking is to the maintenance of the vendace population in this lake. Zastosowanie metody znakowanie - zwroty do oceny efektów zarybień jeziora Wigry larwami sielawy, Coregonus albula (L.) w latach 2000-2001 W roku 2000 i 2001 przeprowadzono znakowanie w alizarynie S larw sielawy, Coregonus albula (L.), przeznaczonych do zarybienia jeziora Wigry. Spośród 19,2 mln larw sielawy w 2000 roku poznakowano 2 mln (10,4% całkowitej ilości wpuszczonych ryb) a w roku następnym z 18,8 mln ryb, poznakowano 7 mln (47,8%). W kolejnych latach od wszystkich odłowionych sielaw pobierano otolity i ustalano ile z nich posiadało znaczek alizarynowy. Założono, że wszystkie osobniki niepoznakowane pochodzą, w równym stopniu, tak z naturalnego tarła, jak i z zarybień, przyjmując podobną przeżywalność ryb z obu źródeł rekrutacji. Stwierdzono, że w odłowach sielaw z pokolenia 2000 ryby z zarybień stanowiły 82,4%, a z pokolenia 2001 64,2% wszystkich odłowionych osobników. Jezioro Wigry jest zbiornikiem o największej liczebnie populacji sielawy w Polsce, w którym co roku odbywa się jej masowe tarło. Jednak wyniki opisywanych badań świadczą o niezbędności systematycznych zarybień dla utrzymania populacji sielawy w jeziorze.

Proteomic identification of turkey (Meleagris gallopavo) seminal plasma proteins1,2

ABSTRACT SDS‐PAGE combined with liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and 2‐dimensional electrophoresis (2DE) combined with matrix‐assisted laser desorption/ionization time of flight/time of flight mass spectrometry (MALDI TOF/TOF) were applied to characterize the turkey seminal plasma proteome. LC‐MS/MS led to the identification of 175 proteins, which were classified according to their function and to corresponding biochemical pathways. Using 2DE and MALDI TOF/TOF, 34 different turkey seminal plasma proteins could be identified, of which 20 were found in more than one spot, indicating different proteoforms of these proteins. For validation, antibodies against turkey albumin and ovoinhibitor as well as sperm acrosin were used in 2DE Western blots experiments. The bioinformatic analysis of the results indicates that turkey seminal plasma proteins may be involved in regulation of lipid metabolism [liver X receptor/retinoid X receptor (LXR/RXR) activation and farnesoid X receptor/retinoid X receptor (FXR/RXR) activation pathways)], endocytic entry of proteins and lipids at the plasma membrane (clathrin‐mediated endocytosis pathway), and defense against pathogens (acute phase response signaling pathway) and energy production (glycolysis and gluconeogenesis). Moreover, a comparative meta‐analysis of seminal plasma proteomes from other species indicated the presence of proteins specific for avian reproduction, but distinct differences between turkey and chicken seminal plasma proteomes were detected. The results of our study provide basic knowledge of the protein composition of turkey seminal plasma highlighting important physiological pathways which may play crucial roles in the sperm environment after ejaculation. This knowledge can be the basis to further develop procedures improving the reproduction of farmed turkeys.

Effect of dialysis on the proacrosin/acrosin system and motility of turkey (Meleagris gallopavo) spermatozoa during liquid storage

1. The effect of dialysis on the proacrosin/acrosin system and motility of turkey spermatozoa were examined after 24 and 48 h of liquid storage at 4°C. 2. Fifteen pools of semen diluted in extender were dialysed against Clemson Turkey Semen Diluent (dialysed semen) or stored in aerobic conditions (undialysed semen). Semen quality was assessed by measuring spermatozoa motility, amidase activity of spermatozoa suspension, spermatozoa extract and seminal plasma and anti-trypsin activity of seminal plasma. 3. Extracted amidase activity of dialysed semen was lower than undialysed by 28%. Higher values for speed parameters of spermatozoa were found in dialysed semen in comparison to undialysed, for example, 81.6 µm/s versus 75.0 µm/s for straight-line velocity (VSL), 114.7 µm/s versus 110.3 µm/s for curvilinear velocity (VCL) and 86.6 µm/s versus 79.8 µm/s for average path velocity (VAP). 4. It was concluded that dialysis caused lower amidase activity of spermatozoa and increased speed parameters of progressively motile turkey spermatozoa during storage. Lower extracted amidase activity of dialysed semen reflected better membrane integrity of dialysed semen and suggests that the proacrosin/acrosin system of dialysed spermatozoa is less susceptible to activation compared to undialysed semen.

The effects of fermentation and enzymatic treatment of pea on nutrient digestibility and growth performance of broilers

The present study examined the impacts of native, fermented or enzymatically treated peas (Pisum sativum L.) inclusion in broiler diets, on growth performance and nutrient digestibility. For the fermentation process, Madonna pea was mixed with water (1/1) containing 2.57×108 Bacillus subtilis (GalliPro®) spores/kg pea and then, incubated for 48 h at 30 °C. For the enzymatic treatment process, the used water for dough production contained three enzymes, AlphaGalTM (α-galactosidase), RONOZYME® ProAct and VP (protease and pectinases respectively - DSM, Switzerland) and the pea dough incubated for 24 h at 30°C. Nine corn-wheat-soybean diets were formulated by supplying 10%, 20% and 30% of the required CP with either native, fermented or enzymatically treated peas. Performance was recorded weekly and at the end of the experiment (day 35), apparent ileal digestibility (AID) of CP, amino acids (AA), crude fat, starch, Ca, P and K were determined. Data were subjected to ANOVA using GLM procedure with a 3×3 factorial arrangement of treatments. Both processes reduced α-galactosides, phytate, trypsin inhibitor activity and resistant starch in peas. Increasing levels of pea products up to 300 g/kg diet, reduced BW gain and feed intake (P⩽0.05). Broilers fed diets containing enzymatically treated pea had the best feed conversion ratio at day 35. Different types of pea product and their inclusion levels had no effect on AID of all nutrients. The interaction between type of the pea products and inclusion levels was significant for AID of starch. For native pea diets, 10% group showed similar AID of starch to 20% native pea but it had higher AID than 30% native pea. For fermented and enzymatically treated groups, all three levels displayed similar AID of starch. In conclusion, enzymatic treatment and fermentation could improve the nutritional quality of pea. Inclusion of enzymatically treated pea in broiler diets could improve broiler performance compared with other pea products while, it displayed neither positive nor negative impact on nutrient digestibility. The present findings indicate the feasibility of these processes, particularly enzymatic treatment, for improving the nutritional quality of pea as a protein source for broiler nutrition.