Development of a Novel Pharmaceutical Formula of Nanoparticle Lipid Carriers of Gentamicin/α-Tocopherol and In Vivo Assessment of the Antioxidant Protective Effect of α-Tocopherol in Gentamicin-Induced Nephrotoxicity

Gentamicin is a potent antibiotic with a nephrotoxicity drawback which limits its use. D-α-tocopherol polyethylene glycol succinate (α-tocopherol) is widely used as a surfactant and have potent antioxidant properties. This study aimed to assess the protective effect of α-tocopherol on gentamicin-induced nephrotoxicity by loading gentamicin on nanostructured lipid carriers (NLC). In vivo, the product was administered intravenously to three groups of rabbits (control, gentamicin and gentamicin/α-tocopherol NLC) for 10 consecutive days. Blood was collected on days 1, 5 and 10 to assess renal function. A significant difference in all plasma parameters related to kidney function were observed in the gentamicin group compared to the control by day 5 and 10, confirming the nephrotoxicity effect. On the other hand, the same parameter levels of the NLC group were significantly different compared to the gentamicin group, confirming the protective effect on kidney function. Gentamicin also caused significant decreases in plasma levels of glutathione sulfhydryl (GSH) and superoxide dismutase (SOD) activity. However, gentamicin-α-tocopherol NLC significantly elevates both plasma levels of GSH as well as SOD activity. The present work indicates that, loading of gentamicin on NLC by using α-tocopherol, is an innovative strategy to protect against aminoglycoside-induced nephrotoxicity due to its antioxidant activity.

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Protective Effect of Coconut Oil Meal Phenolic Antioxidants against Macromolecular Damage: In Vitro and In Vivo Study

Journal of Chemistry ◽

10.1155/2020/3503165 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

A. N. Karunasiri ◽

C. M. Senanayake ◽

H. Hapugaswatta ◽

N. Jayathilaka ◽

K. N. Seneviratne

Keyword(s):

Oxidative Stress ◽

Protective Effect ◽

Stress Responses ◽

Coconut Oil ◽

Oral Feeding ◽

Phenolic Antioxidants ◽

Significant Difference ◽

Macromolecular Damage

Coconut oil meal, a cheap by-product of coconut oil production, is a rich source of phenolic antioxidants. Many age-related diseases are caused by reactive oxygen species- (ROS-) induced damage to macromolecules such as lipids, proteins, and DNA. In the present study, the protective effect of the phenolic extract of coconut oil meal (CMPE) against macromolecular oxidative damage was evaluated using in vitro and in vivo models. Sunflower oil, bovine serum albumin (BSA), and plasmid DNA were used in the in vitro study, and thiobarbituric acid reactive substances (TBARS), protein carbonyl, and nicked DNA were evaluated as oxidation products. The inhibitory effect of CMPE against H2O2-induced macromolecular damage was evaluated using cultured HEp-2 cells. The results indicate that CMPE inhibits macromolecular damage both in vitro and in vivo. In addition, CMPE regulates redox status of HEp-2 cells under oxidative stress conditions by maintaining higher reduced glutathione levels. There was no significant difference in the expression of glutathione peroxidase in stressed and unstressed cells suggesting that CMPE regulates the cellular oxidative stress responses without affecting the expression of oxidative stress response genes. Oral feeding of Wistar rats with CMPE improves the serum and plasma antioxidant status without causing any toxic effects.

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Protective effects of erdosteine on rotenone-induced oxidant injury in liver tissue

Toxicology and Industrial Health ◽

10.1191/0748233704th208oa ◽

2004 ◽

Vol 20 (6-10) ◽

pp. 141-147 ◽

Cited By ~ 20

Author(s):

Alpaslan Terzi ◽

Mustafa Iraz ◽

Semsettin Sahin ◽

Atilla Ilhan ◽

Nuri Idiz ◽

...

Keyword(s):

Lipid Peroxidation ◽

Liver Tissue ◽

Antioxidant Properties ◽

Albino Rats ◽

Protective Agent ◽

Protective Effects ◽

Oxidant Injury ◽

Sod Activity ◽

Significant Difference ◽

Wistar Albino Rats

Rotenone, an insecticide of botanical origin, causes toxicity through inhibition of complex I of the respiratory chain in mitochondria. This study was undertaken to determine whether rotenone-induced liver oxidant injury is prevented by erdosteine, a mucolytic agent showing antioxidant properties. There were four groups of Male Wistar Albino rats: group one was untreated as control; the other groups were treated with erdosteine (50 mg/kg per day, orally), rotenone (2.5 mg/mL once and 1 mL/kg per day for 60 days, i.p.) or rotenone plus erdosteine, respectively. Rotenone treatment without erdosteine increased xanthine oxidase (XO) enzyme activity and also increased lipid peroxidation in liver tissue P < 0.05). The rats treated with rotenone plus erdosteine produced a significant decrease in lipid peroxidation and XO activities in comparison with rotenone group PB / 0.05). Erdosteine treatment with rotenone led to an increase in catalase (CAT) and superoxide dismutase (SOD) activities in comparison with the rotenone group PB / 0.05). There was no significant difference in nitric oxide (NO) level between groups. There were negative correlations between CAT activity and malondialdehyde (MDA) level (r= -0.934, P <0.05) with between CAT and SOD activities (r= -0.714, P <0.05), and a positive correlation between SOD activity and MDA level (r= 0.828, P <0.05) in rotenone group. In the rotenone plus erdosteine group, there was a negative correlation between XO activity and NO level in liver tissue (r= -0.833, P -0.05). In the light of these findings, erdosteine may be a protective agent for rotenone-induced liver oxidative injury in rats.

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Antioxidant activity of ethanolic extract of three Selaginella species from Java Island, Indonesia

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d201234 ◽

2019 ◽

Vol 20 (12) ◽

Author(s):

M Miftahudin ◽

Rini Hasibuan ◽

Tatik Chikmawati

Keyword(s):

Oxidative Stress ◽

Lipid Peroxidation ◽

Antioxidant Activity ◽

Antioxidant Activities ◽

Antioxidant Properties ◽

Ethanolic Extract ◽

Ethanol Extract ◽

Sod Activity ◽

Different Doses

Abstract. Miftahudin, Hasibuan RS, Chikmawati T. 2019. Antioxidant activity of ethanolic extract of three Selaginella species from Java Island, Indonesia. Biodiversitas 20: 3715-3722. Three Selaginella species, S. ornata, S. plana, and S. willdenowii, from Java Island, Indonesia, have been known to have antioxidant properties; however, in vivo antioxidant activities of these species have not been reported. This research aimed to evaluate the in vivo antioxidant activity of ethanolic extract of three Selaginella species. The 70% ethanol extract of three Selaginella species at four different doses was administered to mice one day before being treated with oxidative stress. The liver tissue of mice treated with or without oxidative stress was analyzed their lipid peroxidation by measuring MDA concentration and Superoxide Dismutase (SOD) activities. The results showed that there were variations in antioxidant activity among the three Selaginella species. In general, the dose of 0.3 g extract kg-1 BW has been able to reduce lipid peroxidation and increase SOD activity. The administration of S. ornata extract to the mice at 1.2 g extract kg-1 BW reduced the MDA concentration to the lowest level, but the same dose of two other Selaginella extracts caused toxic effects in mice. The antioxidant activities of S. ornata and S. plana were better than that of S. willdenowii extract, and among those species, S. ornata has the best antioxidant activity.

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Platelet-Dependent Thrombin Generation Induced By ADP or Activated Factor X Does Not Contribute to Increased In Vivo Thrombin Formation in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2018-99-118487 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4336-4336

Author(s):

Christina Berens ◽

Heiko Rühl ◽

Jens Müller ◽

Johannes Oldenburg ◽

Peter Brossart ◽

...

Keyword(s):

Plasma Levels ◽

Research Funding ◽

Thrombin Generation ◽

Myeloproliferative Neoplasms ◽

Final Concentration ◽

Factor X ◽

Prothrombinase Complex ◽

Significant Difference ◽

Thrombin Formation

Abstract Introduction: Myeloproliferative Neoplasms (MPN), including the clinical entities Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF), are characterized by an increased thrombotic risk, the pathomechanisms of which are not well-understood. It has been suggested that an increased sensitivity of platelets to adenosin diphosphate (ADP) contributes to the hypercoagulable state in PV and ET through increased thrombin generation. In the present study we analyzed plasma levels of thrombin and platelet-dependent thrombin generation in MPN patients with an additional focus on prothrombin activation by the prothrombinase complex. Methods: A total of 33 blood samples were obtained from patients with MPN (PV, n=18; ET, n=5; PMF, n=10) and from 33 healthy blood donors that served as controls. In vitro thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) was assessed using the Calibrated Automated Thrombogram (CAT) assay. To induce thrombin generation either ADP (1 µmol/L final concentration) or activated factor X (FXa, 10 ng/mL final concentration) were applied. To further characterize the MPN-associated hypercoagulable state in vivo, plasma levels of free thrombin were measured using an oligonucleotide-based enzyme capture assay (OECA). Prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and D-dimer were measured additionally. Results: In PRP of MPN patients a slightly higher ADP-induced peak thrombin concentration (Cpeak) was observed than in the healthy controls, with 106 (79-130) vs. 84 (65-110) nmol/L (median and interquartile range, p=.026). There was no statistically significant difference in the ADP-induced endogenous thrombin potential (ETP) in MPN patients (1445, 1194-1643 nmol/L·min) compared with the controls (1417, 1258-1814 nmol/L·min). There was no statistically significant difference in the FXa-induced Cpeak and ETP between MPN patients and controls, with 106 (79-127) vs. 97 (82-128) nmol/L, and 1424 (1165-1560) vs. 1641 (1193-1841) nmol/L·min, respectively. With 0.68 (<0.46-1.20) pmol/L, plasma levels of free thrombin were significantly higher (p=.025) in MPN patients than in the control group, in which median thrombin levels were below the limit of detection. Plasma levels of F1+2 and TAT were also higher in the MPN group than in healthy controls, with 0.31 (0.17-0.50) vs. 0.18 (0.13-0.25) nmol/L (p=.002) and 4.36 (2.53-6.76) vs. 2.36 (<2.00-2.68) ng/mL (p=.003), respectively. Conclusion: Increased plasma levels of thrombin, F1+2, and TAT indicate enhanced in vivo thrombin formation in MPN patients. Despite a slightly increased ADP sensitivity of MPN-platelets, the total amount of thrombin generated in PRP from MPN patients is not increased. This makes it unlikely that a 'hyperreactivity' of MPN platelets, resulting in increased activities of the prothrombinase complex on the platelet surface, contributes to the increased thrombin formation in MPN patients. Disclosures Berens: Shire: Research Funding; Biotest: Research Funding; Pfizer: Research Funding; Sanofi Genzyme: Research Funding; CSL-Behring: Research Funding. Rühl:Shire: Research Funding; Swedish Orphan Biovitrum: Consultancy, Research Funding; Grifols: Research Funding; Sanofi Genzyme: Research Funding; CSL-Behring: Research Funding. Müller:Swedish Orphan Biovitrum: Consultancy, Research Funding. Oldenburg:Roche: Honoraria, Research Funding; Grifols: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Shire: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Swedish Orphan Biovitrum: Honoraria, Research Funding.

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Novel nanotech antioxidant cocktail prevents medical diagnostic procedures ionizing radiation effects

Scientific Reports ◽

10.1038/s41598-021-84596-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Miguel Gorenberg ◽

Abed Agbarya ◽

David Groshar ◽

Ilya Volovik ◽

Ofir Avitan ◽

...

Keyword(s):

Ionizing Radiation ◽

Protective Effect ◽

Radiation Effects ◽

Diagnostic Procedures ◽

Peripheral Blood Lymphocytes ◽

Dna Lesions ◽

Control Group ◽

The Novel ◽

Significant Difference

AbstractIonizing radiation (IR) exposure results in oxidative damage causing cytotoxic and genotoxic effects. Double-strand breaks (DSBs) are considered the most significant DNA lesions induced by ionizing radiation. The present study evaluates the radio protective effect of a novel antioxidant cocktail through quantification of DSB in peripheral blood lymphocytes (PBL) in vivo. The study included 16 consecutive patients who were divided into 2 groups, 6 patients received the novel antioxidant cocktail and 10 control patients. Blood samples were drawn from the patients undergoing bone scan, before the injection of the 99mTc MDP tracer and 2 h after the injection. Quantification of the IR damage was done by Immunofluorescence analysis of the phosphorylated histone, γ-H2AX, used to monitor DSB induction and repair in PBL. The radiation effect of the control group was measured by 2 variables, the average DBSs foci per nucleus and the percent of the DSB bearing cells in PBL. The findings showed a significant increase in the DSBs after isotope injection with an average increment of 0.29 ± 0.13 of foci/nucleus and 17.07% ± 7.68 more DSB bearing cells (p < 0.05). The cocktail treated group showed a lower difference average of − 2.79% ± 6.13 DSB bearing cells. A paired t-test revealed a significant difference between the groups (p < 0.005) confirming the cocktail’s protective effect. The novel anti-oxidant treatment decreases the oxidative stress-induced DNA damage and can be considered as a preventative treatment before radiation exposure.

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Treatment of Acute Kidney Injury Using a Dual Enzyme Embedded Metal-organic Framework-mediated Cascade That Catalyzes in Vivo ROS Scavenging

10.21203/rs.3.rs-502119/v1 ◽

2021 ◽

Author(s):

Xinyue Hou ◽

Jianxiang Shi ◽

Jie Zhang ◽

Zhigang Wang ◽

Sen Zhang ◽

...

Keyword(s):

Acute Kidney Injury ◽

Protective Effect ◽

Metal Organic Framework ◽

Antioxidant Properties ◽

Kidney Injury ◽

Intracellular Enzyme ◽

Metal Organic ◽

Enzyme Delivery ◽

Organic Framework

Abstract Background: Significant advances have been made in recent years to utilize natural enzymes with antioxidant properties for the treatment of acute kidney injury (AKI). However, these enzymes have limited clinical utility due to their limited cellular uptake, poor pharmacokinetic properties, and suboptimal stability. We prepared a novel biomimetic mineralization approach to overcome these limitations.Results: Catalase (CAT) and superoxide dismutase (SOD) were encapsulated in a zeolitic imidazolate framework-8 (ZIF-8), then this SOD@CAT@ZIF-8 complex was anchored with MPEG2000-COOH to yield an MPEG2000-SOD@CAT@ZIF-8 (PSCZ) composite. This composite was then utilized as a stable tool with antioxidant properties for the integrated cascade-based treatment of AKI, remarkably improved intracellular enzyme delivery. The PSCZ composite showed a significantly relieve ability of H2O2 induced ROS and prevented apoptosis in HEK293 cells. In vivo, this composite showed a protective effect in the cisplatin-induced AKI mouse model. In addition, PSCZ treatment mitigated renal dysfunction and histological injury, like tubulointerstitial infiltration. RNA sequencing results further confirmed the protective effect of PSCZ and the potential molecular mechanism was revealed. PSCZ treatment reduced renal inflammation and inflammatory gene expression and ameliorated increased apoptosis and inflammatory infiltration of renal tubules. Moreover, PSCZ administration regulated renal injury via the p53 signaling pathway. These renoprotective effects of PSCZ treatment were more potent in CP-AKI mice than those of SOD or CAT alone. This dual-enzyme-embedded metal-organic framework was able to scavenge reactive oxygen species synergistically and effectively. Conclusion: PSCZ can remarkably improve intracellular enzyme delivery, and it can be used as a stable tool with antioxidant properties for the integrated cascade-based treatment of AKI. The ZIF-8-based “armor plating” represents an effective means of shielding enzymes with improved therapeutic utility to guide the precision medicine-based treatment of AKI.

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Astaxanthin as Potential Antioxidant Agent Protects from Accumulation of Heavy-metals in Brain

Journal of agricultural, environmental and veterinary sciences - مجلة العلوم الزراعية والبيئية والبيطرية ◽

10.26389/ajsrp.m090617 ◽

2017 ◽

Vol 1 (2) ◽

Author(s):

Mohammed Said Moosa Al-Bulish, Changhu Xue, Mostafa I. Waly,

Keyword(s):

Oxidative Stress ◽

Heavy Metals ◽

Hydrogen Peroxide ◽

Protective Effect ◽

Neurological Disorders ◽

Antioxidant Properties ◽

Body Burden ◽

Antioxidant Potential ◽

Antioxidant Agent

Humans are increasingly exposed to heavy-metals from food, water, medicine, vaccines, and cosmetics. The toxicity of heavy-metals in humans is briefly summarized, links the possible causal relationships between a high heavy-metals body burden and a number of neurological disorders including Alzheimer’s, Parkinson and Autism disorders. This study aimed to assess the antioxidant properties of Astaxanthin (ASTA) to determine the effect of orally administered ASTA capability of restrict accumulation and toxicity of heavy-metals in brain of rats. It also, assess against Hydrogen peroxide induced oxidative stress and antioxidant potential properties of ASTA with comparing the affectivity of 5% and 10% of ASTA in increased glutathione-recycling enzymes (GPx oxidation). A significant change was observed as increased glutathione-recycling enzymes (GPx oxidation) of rats and showed a protective effect against accumulation of Aluminum(AL) in rat’s brain tissues. The results of this in-vivo study demonstrated that ASTA 10% is more can affective in restriction of accumulation and toxicity of Al in rate brain and its contented can protects against oxidative-stress.

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Enhanced Skin Performance of Emulgel vs. Cream as Systems for Topical Delivery of Herbal Actives (Immortelle Extract and Hemp Oil)

Pharmaceutics ◽

10.3390/pharmaceutics13111919 ◽

2021 ◽

Vol 13 (11) ◽

pp. 1919

Author(s):

Vanja M. Tadić ◽

Ana Žugić ◽

Milica Martinović ◽

Milica Stanković ◽

Svetolik Maksimović ◽

...

Keyword(s):

Essential Fatty Acids ◽

Antioxidant Properties ◽

Skin Hydration ◽

Omega 3 ◽

Healthy Human ◽

Significant Difference ◽

Omega 6 ◽

Anti Inflammatory ◽

Hemp Oil

Immortelle, as rich source of chlorogenic acid and the phloroglucinol alpha-pyrone compound arzanol, possesses anti-inflammatory and antioxidant properties, affects cell regeneration, and has positive effect on many skin conditions. Hemp oil, characterized by a favorable omega-6 to omega-3 ratio, as well as an abundance of essential fatty acids and vitamin E, participates in immunoregulation and also act as an anti-inflammatory. In the present study, we examined the effect on the skin of creams and emulgels with immortelle extract and hemp oil, by comparing them to placebo samples and a non-treated control. A long-term in vivo study of biophysical skin characteristics, which lasted for 30 days, was conducted on 25 healthy human volunteers. Measured parameters were electrical capacitance of the stratum corneum, trans-epidermal water loss (TEWL), and skin pH and erythema index. Further, a sensory study was carried out in which the panelists had to choose descriptive terms for sensory attributes in questionnaire. The results showed that application of all preparations led to increase of skin hydration and TEWL reduction, while the skin was not irritated, and its normal pH was not disrupted. This study also showed importance of the carrier. Not only were emulgels described by panelists as preparations with better sensory properties, there was a significant difference between the skin hydration effect of emulgel with immortelle extract and hemp oil compared to the placebo emulgel, which was not the case with creams. Such findings indicated enhanced delivery of herbal active substances from emulgel compared to the cream.

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Antioxidant Properties and Protective Effect of Aqueous Anti-Ulcer Drug (AQAUD) against Aspirin-induced Gastric Ulcers in Albino Rats

Asian Journal of Biochemistry, Genetics and Molecular Biology ◽

10.9734/ajbgmb/2020/v3i230081 ◽

2020 ◽

pp. 9-21

Author(s):

B. A. Mba ◽

C. S. Alisi ◽

A. C. Ene

Keyword(s):

Protective Effect ◽

Antioxidant Properties ◽

Toxicity Testing ◽

Antioxidant Potential ◽

Gastric Ulcers ◽

Albino Rats ◽

Protective Effects ◽

Sod Activity ◽

Group I

Aim: The aim of this study is to evaluate the antioxidant properties and protective effects of aqueous anti-ulcer drug (AQAUD) against aspirin-induced gastric ulcer in albino rats. Methods: In this study, 30 male albino rats were divided into 5 groups of 6 each. Rats in group I served as normal control and received food and water. Animals in group II received food and water in addition to aspirin (400 mg/kg.b.wt) orally on the 14th day. Rats in groups III, IV and V received “AQAUD” (250 mg/kg.b.wt), (500 mg/kg.b.wt) and Omeprazole (20 mg/kg.b.wt) respectively for 14 days and aspirin (400 mg/kg.b.wt) orally on the 14th day. In vitro antioxidant property of “AQAUD” was assessed by its nitric oxide and hydroxyl radicals scavenging properties. The ulcer protective effect of “AQAUD” was assessed by determining the free and total acidity, ulcer index and % protection in the stomach content. The antioxidant potential in animals was evaluated by determining the concentrations of malondialdehyde and reduced glutathione. Superoxide dismutase and catalase activities were assayed in the stomach homogenates to further assess antioxidant potential. Total phenolics and flavonoid compounds were quantified to know the antioxidant content. Histopathological assessment of the gastric mucosa was used to assess the protective potentials of “AQAUD”. Data were analyzed using Statistical Package for Social Science (SPSS) version 21. Results: The results revealed that free acidity and ulcer indexes were significantly (p<0.05) reduced by “AQAUD”. There was a significant decrease in SOD activity of the stomach homogenates when compared to the aspirin group, with values for “AQAUD” 250 mg/kg.b.wt and “AQAUD” 500 mg/kg b.wt as 37.24±5.39ux10-2/mg protein and 23.64±2.91ux10-2/mg protein respectively. Result of acute toxicity testing showed that “AQAUD” is generally safe up to 5000 mg/kg b.wt. Conclusion: The results revealed that treatment with aspirin caused loss of gland architecture with erosion of epithelial layer, but AQAUD treatment ameliorated the effect of aspirin administration. The study revealed that “AQAUD” has considerable antioxidant potentials and can effectively protect against gastric ulcers.

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Cryopreservation of canine semen: the effect of two extender variants on the quality and antioxidant properties of spermatozoa

Polish Journal of Veterinary Sciences ◽

10.2478/v10181-012-0109-0 ◽

2012 ◽

Vol 15 (4) ◽

pp. 721-726 ◽

Cited By ~ 11

Author(s):

R. Strzeżek ◽

M. Koziorowska-Gilun ◽

M. Stawiszyńska

Keyword(s):

Plasma Membrane ◽

Antioxidant Properties ◽

Membrane Integrity ◽

Egg Yolk ◽

Biological Properties ◽

Sod Activity ◽

Significant Difference ◽

Sperm Plasma Membrane ◽

Hen Egg Yolk ◽

Hen Egg

Abstract The aim of this study was to determine the effect of two variants of Tris-citric acid-fructose (TCF) extender containing whole hen egg yolk (TCF-HEY) and lyophilized lipoprotein fractions extracted from ostrich egg yolk (TCF-LPFo) on selected biological properties of cryopreserved sperm cells. Post-thaw percentage of motile sperm (MOT) was significantly higher (P<0.05) for TCF-HEY extender (66.3 ± 3.2%) than for TCF-LPFo extender (52.4 ± 3.4%). Moreover, there was no significant difference in the percentage of sperm with progressive motility (PMOT). Both diluents effectively preserved sperm plasma membrane integrity and mitochondrial function. However, it was observed that cryopreservation impaired the functionality of antioxidant sperm enzymes. The above was manifested by reduced SOD activity, in particular in samples preserved in the TCF-HEY extender, as well as decreased GPx activity. Both diluents inhibited the rate of lipid peroxidation in sperm plasma membrane during freezing-thawing. Our results suggest that LPFo is a satisfactory alternative to hen egg yolk in the extender used for canine sperm cryopreservation.

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