Yolk Plasma Research Articles

The effect of CaCl2 on water migration, rheological properties, aggregation behavior and protein structure in rapidly salted separated egg yolk plasma and granules

This research investigated the effects of CaCl2 on the aggregation behavior and protein structure of egg yolk plasma and granules after fast salting. The addition of CaCl2 to the salt solution decreased T23, D [4,3] and the absolute value of the zeta potential by 6.71%, 3.66%, and 3.15%, respectively, while increasing T22 by 15.85% in egg yolk plasma. Moreover, adding CaCl2 also increased the apparent viscosity and G' value of egg yolk plasma. On the other hand, the addition of CaCl2 decreased the T22, T23, D [4,3], and absolute value of the zeta potential of egg yolk granules by 56.53%, 6.71%, 6.02%, and 34.27%, respectively. Furthermore, the addition of CaCl2 increased the number of β-turns by 51.22%, whereas it decreased the number of β-sheets by 26.55% in egg yolk plasma protein. The α-helices, β-turns, and β-sheets of egg yolk granule protein decreased by 6.58%, 3.58%, and 6.96%, respectively. Additionally, the addition of CaCl2 can increase the degree of λmax redshift in egg yolk plasma and decrease the degree of λmax redshift in egg yolk granules. Overall, the addition of CaCl2 can change the aggregation mode of proteins in egg yolk plasma and granules, improving the quality of salted egg yolk products.

Research Note: Comprehensive proteomic, phosphoproteomic, and N-glycoproteomic analysis of chicken egg yolk plasma

Chicken egg yolk plasma (EYP), the supernatant fraction of egg yolk obtained by water dilution and centrifugation, is a rich source of various bioactive substances and a significant bearer of yolk-emulsifying properties. This study utilized proteomics to conduct a comprehensive and in-depth analysis of both common and modified EYP proteins (phosphorylated proteins and N-glycosylated proteins). Total of 208 proteins were identified in EYP, including 42 phosphorylated proteins with 137 phosphorylation sites and 150 N-glycoproteins with 332 N-glycosylation sites. Among the phosphorylation sites, tyrosine accounted for 80.6%, while the N-glycosylation sites predominantly featured "N-X-T" motifs, accounting for 58.7%. Functional enrichment analysis revealed that most proteins were involved in regulating enzyme activity and inhibition with a particular focus on modulating peptidase activity. Notably, vitellogenins-2 (30 phosphorylation sites, 9 N-glycosylation sites) and apolipoprotein B (10 phosphorylation sites, 56 N-glycosylation sites) were the 2 proteins with the most modification sites. Additionally, EYP was found to contain the highly N-glycosylated complement proteins C3 and C4. These findings provide new insights into the protein composition of EYP and its roles in chicken embryo development and immune defense, offering a theoretical foundation for the application of EYP in various fields.

Rheology of heat-set pudding (emulsion gel) and O/W interface: effects of interaction between tea catechins and egg yolk plasma at O/W interface

Herein, the effects of tea catechins (TCs) on the rheological properties of a model heat-set pudding (emulsion gel) were investigated. A combination of skimmed milk powder, egg white powder, egg yolk, egg yolk plasma, egg yolk granules, coconut oil, and TCs was used as the raw material for the model pudding. Viscoelastic measurements showed that the storage modulus G′ of the TC-containing model pudding in the range of 10°C–15 °C (each solid fat content in coconut oil ranged from 79% to 71%) was approximately 10-fold higher than that of the model pudding without TCs. Furthermore, a very high Gʹ could be obtained when egg yolk plasma and coconut oil were used in the raw material for the TC-containing model pudding at 10°C–15 °C. Additionally, the results of interfacial tension and viscoelasticity measurements at the oil-water interface suggested that the interfacial structure changes due to the interaction between egg yolk plasma and TCs contributed to a very high G′ for the TCs-containing model pudding in the range of 10°C–15 °C.

Foaming and structural properties of egg yolk components microgel particles and their adsorption characteristics at the air-water interface

In this study, microgel particles of egg yolk and its components were constructed by inducing and modifying their structure via the high-pressure homogenization (HPH). In order to compare their structural characterization and reveal their adsorption mechanism at the air-water interface, yolk components were also combined with egg white protein (EWP) to achieve their microgels for investigation. Results suggested that after HPH, foam stabilities of egg yolk protein microgel (EYM) and egg yolk plasma microgel (EPM) were significantly improved, whereas no significance occurred on egg yolk granules microgel (EGM). Interestingly, after HPH, the adsorption capacity of the microgels from combination of yolk components and EWP exhibited a significant increasing trend. However, their interfacial adsorption stability was poor. Fluorescence spectrum and surface tension revealed that more hydrophilic groups and lower surface tension would occur on EY and EP after HPH, whilst no significant effects would happen on EG, which was consistent with the results of foaming characteristics. SDS-PAGE exhibited that there was no obvious change in the protein bands of egg yolk and its components, indicating that egg yolk and component proteins did not undergo covalent crosslinking or degradation with HPH treatment. In summary, our study prepares for the first time of the microgel particles from egg yolk components for stabilizing the air-water interface. In addition, we have revealed the relationship between the physicochemical properties, microstructures and interfacial adsorption properties of the microgel particles from egg yolk components, and have explored their mechanisms in depth.

Albumen and Yolk Plasma Peptidomics for the Identification and Quantitation of Bioactive Molecules and the Quality Control of Hen Egg Products.

Hen eggs and the correspondingfood products are essential components of human diet. In addition to supplying basic nutrients, they contain functional peptides that are released in vivo within the intact raw material following physiological proteolytic events affecting specific proteins or derive from technological processing of albumen and yolk fractions as a result of the dedicated use of proteases from plant and microbial sources. Besides their potential importance for functional applications, peptides released under physiological conditions in intact egg can be used as markers of product storage and deterioration. Therefore, characterization and quantitation of peptides in egg and egg-derived products can be used to implement evaluation of potential bioactivities as well as to assess food product qualitative characteristics. Here, we provide dedicated information on extraction, identification, and quantitative analysis of peptides from albumen and yolk plasma; nano-liquid chromatography-mass spectrometry combined with bioinformatic analysis of resulting raw data by different software tools allowed to assign molecules based on database searching and to evaluate their relative quantity in different samples.

Research Note: Comparison of water-soluble metabolites in egg yolk, yolk granules, and yolk plasma based on quantitative metabolomic analysis

Research Note: Comparison of water-soluble metabolites in egg yolk, yolk granules, and yolk plasma based on quantitative metabolomic analysis

Assessment of whole egg fractionation by tangential flow filtration: The problem of low-density lipoprotein aggregates

Eggs are a compelling source of bioactive compounds. Membrane separation is a green technology that allow components to be obtained while preserving their biological activity. The objective of this study was to assess the application of membrane separation technology to egg products. Whole egg plasma, egg yolk plasma and egg white were tangential flow filtrated evaluating the effect of the pore size and membrane materials. Transmission of dry matter and proteins from feed to filtrate was noticed in all the processes. Nevertheless, cholesterol and carotenoids were not detected at filtrates from 750 kDa to 0.2 µm when mixed ester or polyethersulfone were the membrane materials. The interactions between egg yolk and egg white components difficult the separation. Cholesterol was part of low-density lipoproteins. These lipoproteins together with carotenoids were filtrated with modified polyethersulfone at a minimum pore size of 0.2 µm. High uncertainty was obtained due to low-density lipoproteins aggregation. Three kinds of aggregates can justify these results: low-density lipoproteins aggregated themselves, low-density lipoproteins bound to ovomucin and other smaller egg white proteins (<750 kDa), and low-density lipoproteins bound only to smaller egg white proteins. The detected chelate effect of egg white proteins could be important in vivo. Therefore, research should be led to assess the influence of cooking method and digestion of the different egg components on the bioaccessibility of nutrients and the generation of bioactive peptides. Moreover, the filtration processes analysed herein may be useful techniques for liquid food matrix studies.

Egg yolk plasma enriched with β-carotene through the diet of laying hens and adding it to the extender improves the quality of frozen semen in Arabic stallions.

Equine semen cryopreservation is one of the major procedures for the genetic conservation of rare and endangered genotypes. The current study was conducted to evaluate the effect of egg yolk plasma (EYP) enriched with β-carotene as an antioxidant supplement on INRA-96 extender regarding freezing Arabic stallion sperm. For this purpose, β-carotene various concentrations were utilized as a supplementary ingredient in formulating the diets of laying hens. Birds were randomly divided into four groups, fed with 0 (control), 500, 1000 and 2000 mg/kg in a supplemented diet with β-carotene. Subsequently, various variants of enriched extender (INRA-96 + 2.5% glycerol [G]) were gained by adding 2% EYP from four treatment groups. The sperm characteristics, including motility, viability, morphology, plasma membrane integrity (HOS test), lipid peroxidation (MDA) and DNA fragmentation, were evaluated after thawing. According to the results obtained in this study, the addition of EYP from T2 and T4 (500 and 2000 mg/kg of β-carotene in hens' diet) to the extender (INRA-96 + 2.5% G) leads to an increase in total motility (50.50% and 49.49%, respectively), progressive motility (32.6% and 31.8%, respectively), viability (68.7% and 66.1%, respectively) and plasma membrane integrity (57.7% and 50.6%, respectively). Moreover, lipid peroxidation (1.3 and 1.4nmol/mL, respectively) and DNA fragmentation (8.6% and 9.9%, respectively) were diminished using the mentioned treatments. However, sperm morphology was not affected by the treatments. In the current study, we concluded that the optimal concentration of β-carotene in the laying hen's diet (500 mg/kg) could reveal the best results about sperm quality. So, EYP enriched with β-carotene acts as a valuable natural and safe supplementary material that could be exploited for enhancing stallion sperm quality in cryopreservation conditions.

The effect of varying concentrations of chicken plasma egg yolk and glycerol on the viability of canine sperm following short and long-term preservation.

Plasma egg yolk (PEY), due to simple preparation and easier access, could be a suitable alternative to raw egg yolk for preserving canine semen. The present study investigated suitable concentrations of PEY and glycerol for preservation of canine semen. Semen was collected by digital manipulation (seven replicates from four dogs). Following initial raw semen evaluation, the semen was diluted in a tris-based extender supplemented with varying concentrations of chicken PEY (0, 20, and 40% v/v) and glycerol (3%; v/v). After cooling the specimen to 4°C within 1 h, the specimens were diluted with an equal volume of freezing extender consisting of similar concentrations of chicken PEY and 0 and 7% glycerol to reach the final glycerol concentration of 1.5 and 5% for short-term storage of canine semen. Samples with different concentrations of PEY and 5% glycerol were frozen. The sperm viability parameters including total motility, progressive forward motility, plasma membrane integrity, and live percentage of sperm were assessed following short and long-term storage. Sperm viability parameters of semen extended in an extender supplemented with 20 or 40% chicken PEY with either 1.5 or 5% glycerol remained superior until 72 h after semen collection compared to the specimen that did not receive any PEY (P<0.05). Post-thaw sperm viability was also greater in samples extended in extender supplemented with either 20 or 40% PEY compared to 0% PEY. Tris-based extender supplemented with either 20% chicken PEY could be suitable for short and long-term preservation of canine semen.

Application of UV-VIS-NIR spectroscopy in membrane separation processes for fast quantitative compositional analysis: A case study of egg products

Membrane separation technology is achieving broad applications in research, food and pharmaceutical industry. In this study, the application of UV-VIS-NIR spectroscopy to quantify the composition of the fractions obtained in a membrane separation process was assessed. An egg membrane filtration was employed as a case study to develop predictive models for a rapid determination of dry matter, protein and cholesterol composition in the feed and the fractions (retentate and filtrate). Whole egg plasma and egg yolk plasma obtained by centrifugation, and egg white were tangential filtrated using different pore size and membrane materials. Transmittance UV-VIS-NIR spectra showed significant differences between samples. Cholesterol, dry matter and proteins were predicted with UV-VIS-NIR spectroscopy with good statistical significance (RPDEV over 3). UV-VIS-NIR spectroscopy combined with chemometric tools demonstrates to be an efficient green method for composition analysis in the membrane filtration process. It is reliable, non-destructive, quick and environmentally friendly. Moreover, it can be applied on-line for real-time quantitative compositional analysis.

Evaluation of Chicken Egg Yolk Plasma and Low-Density Lipoprotein Alone or Enriched with Ewe or Cow Skim Milk in Tris-Citric Acid-Based Diluent for Cryostorage of Ram Semen.

The main purpose of the current study was to find suitable and optimum levels of protectants among chicken egg yolk plasma (CEYP) and low-density lipoproteins (LDLs), alone or supplemented with ewe or cow skim milk, for cryopreservation of ram semen. In Experiments 1 and 2, the CEYP (28%) freezing extender was enriched with ewe or cow milk (2.5%, 5%, 10%, or 20%; v/v), respectively. In Experiments 3 and 4, the semen extender was prepared by varying the amounts of fresh or lyophilized LDL (lyo-LDL), respectively. Finally, ewe or cow skim milk was added to the freshly extracted LDL extender and the quality of frozen-thawed semen was examined (Experiments 5 and 6). Kinematics of spermatozoa (assessed using a computer-assisted sperm analysis system), viability, functionality of the plasma membrane, and levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) were evaluated. Results revealed that addition of ewe or cow skim milk (5%, 10%, or 20%; v/v) to the CEYP diluent enhanced kinematics, viability, and membrane integrity of spermatozoa compared with the control (p < 0.05). Moreover, fresh LDL diluent was more effective than lyo-LDL in the cryosurvival of ram spermatozoa. In addition, enrichment of fresh LDL diluent with ewe or cow skim milk improved different variables of spermatozoa compared with the control (p < 0.05). Levels of MDA and TAC were not affected by adding ewe or cow milk to the diluents (p > 0.05). In conclusion, enrichment of fresh LDL extenders with ewe or cow milk also is proposed as an approach to preserve ram semen quality against cold shock and cryodamage injuries.

Quantitative lipidomic analysis of egg yolk, yolk granule, and yolk plasma

Chicken egg yolk (EY) can be divided into yolk granule (EYG) and yolk plasma (EYP); they develop individually based on their remarkably different nutritional compositions and properties. However, the differences in lipid profiles between EYG and EYP have not been fully revealed. In this study, quantitative lipidomic analyses of EY, EYG, and EYP were implemented through a liquid chromatography-tandem mass spectrometry strategy. The results showed a total of 702 identified lipid species, mainly including 136 triglycerides, 112 phosphatidylcholines, 104 phosphatidylethanolamines, 50 lysophosphatidylcholines, and 41 diglycerides. A quantitative comparison showed that phospholipids were the representative differentially abundant lipids in the contrast of EY and EYG, EY and EYP; while triglycerides were the most representative differentially abundant lipids between EYG and EYP. Docosahexaenoic and eicosapentaenoic acids mainly exist in the phospholipids of EY. The research results provide important information for the systematic understanding of the lipid composition of EY, EYG, and EYP, and provide insights into the differential development of EYG and EYP.

Probe ultrasonification of egg yolk plasma forms low-density lipoprotein nanoparticles that efficiently protect canine semen during cryofreezing

Around the world, many couples have turned to in vitro fertilization as a viable solution to fertility issues. Low-density lipoprotein (LDL) is a protein best known for transporting fat molecules throughout the body, but it has also been shown to protect sperm cells during cryopreservation due to its micellar structure. In the present study, we aimed to evaluate different protocols for the preparation of nanoparticles from egg yolk plasma (EYP) containing LDL to improve the viability of cryopreserved canine semen. EYP was subjected to three distinct treatments: ultrasonification in an ultrasound bath at 40 kHz for 30 min (LDL-B); ultrasonification via an ultrasound probe at 50% amplitude for 30 min (LDL-P); and high-pressure homogenization at 10,000 PSI for six cycles (LDL-H). Sperm quality was assessed after thawing using computer-assisted sperm analysis and flow cytometry. The results revealed that compared to the EYP control, the LDL-P formulation presented significantly higher efficiency (p < 0.05) in maintaining total and progressive sperm motility, sperm membrane integrity and fluidity, and levels of intracellular reactive oxygen species. The LDL-P nanoparticles had an average size of approximately 250 nm, a PDI value of 0.3, and −1.15 mV of zeta potential, which are very important because it is an indicator of the stability of a colloidal dispersion. Therefore, we conclude that ultrasonication of EYP using a probe is an efficient method for the preparation of LDL nanoparticles that would enhance the cryoprotection of semen during freezing.

Effect of α-Tocopherol Supplementation in Rooster Semen on Sperm Quality Parameters during in-Vitro Storage at 4°C

The purpose of this study was to evaluate the probable effects of the α-tocopherol (Vitamin E) addition in different levels to the Tris based egg yolk plasma (T-EYP) extender in semen of Rhode Island Red roosters on spermatozoa quality during storage of semen at 4°C for 0, 24,48 and 72h. The collected semen from roosters was mixed together and extended with T-EYP. The extended pooled semen was divided into 5 treatments (T). T1 was a control group without any vitamin E addition. For T2 to T5 groups 0.5%, 1%, 2% and 3% vitamin E (w/v) was added respectively. Treatments were evaluated for sperm motility, sperm viability, morphological defect, membrane integrity and acrosome integrity after 0, 24, 48 and 72 h of incubation at 4°C. The evaluations of spermatozoa immediately after semen collection, revealed no significant differences among values the treatment groups, whereas after incubating the treatments for different spans of time, the sperm progressive motility and viability rates for groups supplemented with vitamin E were significantly (P&lt;0.05) higher than that of the control group. In addition, morphological defect rates of spermatozoa in the groups supplemented with different levels of α-tocopherol were significantly (P&lt;0.05) lower than the control group. It was found that 0.5% level of α-tocopherol in extended rooster semen improved the sperm motility, viability plus to reduce the morphological defect rates of the spermatozoa up to 72 h storage time at 4°C.

Effects of Linolenic Acid supplementation on Seminal Attributes of Labrador during in-vitro Storage at 4°C

The purpose of this study was to evaluate the probable effects of the linolenic acid addition in different levels to the Tris based egg yolk plasma (T-EYP) extender in Labrador dog semen on spermatozoa quality during storage of semen at 4°C for 0, 24,48 and 72 hours. The collected semen from all dogs was mixed together and diluted with T-EYP. The diluted pooled semen was divided into 5 treatments (T). T1 was a control group without any linolenic acid addition. For T2 to T5 groups 0.5 ng/ml , 1.5 ng/ml, 2.5 ng/ml and 3.5 ng/ml linolenic acid were added respectively. Treatments were evaluated for sperm motility, sperm viability, morphological defect, acrosome integrity and membrane integrity after 0, 24, 48 and 72 hours of incubation at 4°C. The evaluations of spermatozoa immediately after semen collection, were revealed no significant differences among values of treatment groups, whereas after incubating the treatments for different spans of time, the sperm progressive motility and viability rates for groups supplemented with linolenic acid were significantly (P&lt;0.05) higher than that of the control group. Motility, viability and acrosome integrity were higher in 2.5ng/ml linolenic acid concentration. According to the results of this study we conclude that, the most excellent level of linolenic acid for supplementation to the extended semen of dog in order to improve the sperm motility and viability plus to reduce the morphological defect rates of the spermatozoa up to 72 hours storage time at 4°C is 2.5 ng/ml.

INRA96 Supplemented With Phospholipids Liposomes, A Promising Approach for Stallion Sperm Chilling

Among biotechnologies of reproduction in the equine species, artificial insemination remains the most used technology especially for cooled transported sperm. Although the use of INRA96 extender has demonstrated its efficiency for long-term sperm storage at 4°C or 15°C, some stallions (“bad coolers”) are excluded from such technology.Some years ago, we demonstrated that liposomes produced from egg yolk (EY) phospholipids could be an alternative to egg yolk plasma in stallion freezing extenders. To develop a new extender for sperm chilling, we evaluated the protective effect of liposomes produced from EY phospholipids on stallion sperm storage at 4°C.The sperm of stallions from two studs was diluted in INRA96 extender (as control) or an experimental extender (EE) composed of INRA96 supplemented with liposomes of EY phospholipids. After 24H (D1), 72H (D3), and 6 days (D6) or 7 days (D7), motility parameters were evaluated using Computer Assisted Semen Analyzer.Our results demonstrated that total and progressive motility decreased significantly after dilution and storage in INRA96 between D1 and D3 (P < .05) while no significant decrease was observed between D1 and D3 with EE. Regarding VAP parameter, no significant difference was observed between extenders except at D7 in stud 2.Moreover, total and progressive motility were maintained at a significantly higher level (D3, D6, D7) when sperm was stored in EE compared to INRA96.These promising results demonstrate that the supplementation of INRA96 extender with egg-yolk phospholipids liposomes allows a higher protection to stallion sperm cells.

Comparative Study on Foaming Properties of Egg White with Yolk Fractions and Their Hydrolysates.

As an excellent foaming agent, egg white protein (EWP) is always contaminated by egg yolk in the industrial processing, therefore, decreasing its foaming properties. The aim of this study was to simulate the industrial EWP (egg white protein with 0.5% w/w of egg yolk) and characterize their foaming and structural properties when hydrolyzed by two types of esterase (lipase and phospholipase A2). Results showed that egg yolk plasma might have been the main fraction, which led to the poor foaming properties of the contaminated egg white protein compared with egg yolk granules. After hydrolyzation, both foamability and foam stability of investigated systems thereof (egg white protein with egg yolk, egg white protein with egg yolk plasma, and egg white protein with egg yolk granules) increased significantly compared with unhydrolyzed ones. However, phospholipids A2 (PLP) seemed to be more effective on increasing their foaming properties as compared to those systems hydrolyzed by lipase (LP). The schematic diagrams of yolk fractions were proposed to explain the aggregation and dispersed behavior exposed in their changes of structures after hydrolysis, suggesting the aggregated effects of LP on yolk plasma and destructive effects of PLP on yolk granules, which may directly influence their foaming properties.

Comparing Glycerol with Dimethylformamide and Combination for Cryopreservation of Pug Dog Semen in Egg Yolk Plasma Containing Extender

Background: In order to find an alternative cryoprotectant for canine semen, different cryoprotectants, as dimethyl sulphoxide and ethylene glycol, dimethyle formamide either alone or in combination with glycerol have been evaluated on pooled semen of different breeds with different opinions. Methods: In the current study the effect of glycerol, DMF and their combination in Tris-citric acid-fructose egg yolk plasma extender on motility, viability, plasma membrane integrity (PMI), acrosome integrity (AI), inner mitochondrial membrane potential (IMMP), reactive oxygen species (ROS) and antioxidant enzymes in frozen-thaw semen of pug breed was compared. Result: Values for motility, viability, (PMI), AI were significantly (p less than 0.05) high in TCFEYP-G compared to TCFEYP-DMF and TCFEYP-G+DMF at post thaw. However, HIMMP and MIMMP/MDA were non-significantly (P greater than 0.05) high and low in TCFEYP-G compared to TCFEYP-DMF and TCFEYP- G + DMF at post thaw, respectively. MDA concentration was significantly (p less than 0.05) low in TCFEYP-G compared to TCFEYP-DMF and TCFEYP-G + TCFEYP-DMF extenders. However, H2O2, scavenging capacity of spermatozoa, cryopreserved in TCFEYP-G was significantly (p less than 0.05) higher than in TCFEYP-DMF and TCFEYP-G + TCFEYP-DMF. However, there was no significant (p greater than 0.05) difference in superoxide free radical scavenging activity and nitrite concentration of spermatozoa among the three extenders. Activity of GPX, SOD and Catalase was also significantly (p less than 0.05) higher in TCFEYP-G compared to TCFEYP-DMF and TCFEYP-G+DMF. However, values were reverse for GRE activity. This study concludes that glycerol is a healthier cryoprotectant to protect canine spermatozoa from cryo-injury during freezing-thawing process.

Effect of egg yolk-based extender and seminal plasma removal on sperm viability of cooled donkey semen

Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P&lt;0.05) when semen was extended with 2% of egg yolk and seminal plasma was removed. Membrane integrity was affected (P&lt;0.05) in centrifuged samples. In conclusion, the obtained results suggest that there is no difference in sperm kinetics and membrane integrity when 1% or 2% of egg yolk was added to the Equiplus(R) extender. Also, the removal of seminal plasma by centrifugation did not have any beneficial effect on cooled donkey semen. Further studies are needed to relate these results with in vivo fertility tests with cooled donkey semen.

Using egg yolk in a TRIS-Equex STM paste extender for freezing of dog semen is superior to egg yolk plasma, also after addition of lecithin and catalase

We compared the results of using egg yolk plasma (EYP) instead of egg yolk (EY) in a TRIS-based Equex STM Paste freezing extender system for dog semen [25]. We also tested whether the addition of lecithin and catalase to the EYP extenders would improve results. Fractionated semen collection was done in 17 stud dogs and the sperm rich fraction diluted with different extenders in 2 steps: (I) TRIS-fructose-citric acid extender (TRIS) containing 20% egg yolk (EY) and 3% glycerol [25], (II) TRIS containing 20% egg yolk plasma (EYP) and 3% glycerol, and (III) TRIS containing 20% EYP and 0.8% lecithin (EYP-L) and 3% glycerol. After equilibration the second dilution step was done: samples with (I) were diluted with TRIS-EY with 7% glycerol and 1% Equex STM paste [25]; samples with (II) and (III) were divided in 2 aliquots each, and one part diluted with TRIS-EYP or TRIS-EYP-L, both containing 7% glycerol and 1% Equex STM paste, and the other one part with the same extenders containing additionally 300 I.U./mL catalase. After freezing and thawing, samples were analyzed by CASA and sperm chromatin structure assay (SCSA); reactive oxygen species (ROS), degree of apoptosis and zona binding ability were determined. Semen samples with TRIS-EY with a final concentration of 5% glycerol and 0.5% Equex STM paste [25] showed best post thaw progressive motility (P), most intact cells, lowest percentage of ROS, acrosome damages, dead and apoptotic cells. Curvilinear velocity (VCL), DNA fragmentation, morphological abnormalities and zona binding ability did not differ between groups. Replacement of egg yolk by EYP increased the ROS and late apoptotic cells. Addition of lecithin and catalase to EYP containing extenders decreased motility and increased complete apoptosis. We conclude that egg yolk is superior to EYP in the here investigated extenders. The TRIS-based extender [25] with EYP could not be improved by addition of lecithin and catalase; however, in-vivo fertilization capacity of the here examined extenders remains to be investigated.