scholarly journals Effect of Addition Palmitic Acid and Vitamin E to the Tris-Egg Yolk Diluter in Canine Semen Cryopreservation

2021 ◽  
Vol 49 ◽  
Author(s):  
Marcos Antônio Celestino de Sousa Filho ◽  
Luanna Soares de Melo Evangelista ◽  
Filipe Nunes Barros ◽  
Jefferson Hallisson Lustosa da Silva ◽  
Anna Monallysa Silva de Oliveira ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Plasma Membrane ◽  
Vitamin E ◽  
Citric Acid ◽  
Palmitic Acid ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Acrosomal Membrane

Background: Canine sperm is a very delicate cell that is quite susceptible to oxidative stress since the cytoplasm is restricted and features little antioxidant reserves. Furthermore, the sperm membrane has some polyunsaturated fatty acids sensitive to lipid peroxidation, which makes it important to addition antioxidant substances to the diluter aiming at decreasing such stress to the sperm cell, particularly during seminal cryopreservation. Several antioxidants have been used in this process in some domestic animal’s species, however, the use of palmitic acid has been little reported in works on cryopreservation of semen of the canine species. Hence, this study aimed to assess the effect of addition antioxidants palmitic acid and vitamin E to the Tris-egg yolk diluter on the semen quality of dogs after thawing.Materials, Methods & Results: Samples were collected from the ejaculates of 4 adult dogs, apparently healthy, of the American Pit Bull Terrier breed of kennels in the city of Teresina, PI, places where the pre-freezing procedures of the dog’s semen were performed. The samples were diluted in Tris citric acid fructose (3.28 g Tris-hydroxymethyl-aminomethane, 1.78 g citric acid monohydrate and 1.25 g D-fructose), dissolved in 100 mL distilled water, and added 20% egg yolk and 6% glycerol, at the concentration of 100x106 sptz/mL. The semen samples were divided into 3 mL aliquots to form 3 experimental groups: G1 - Only Tris-egg yolk (Control group); G2 - Tris-egg yolk + 100 µM palmitic acid; and G3 - Tris-egg yolk + 116 µM vitamin E. Semen was collected weekly over a period of little over 2 months. After thawing, thermorresistance test (TTR) was carried out at 0, 30, 60, and 90 min to assess spermatics motility and vigor, in addition to analysis of integrity of plasma membrane, acrosomal membrane and mitochondrial activity of the sperm, using fluorescent probes. These assessments were performed out at the Animal Reproduction Biotechnology Laboratory (LBRA/UFPI). In the TTR, G2 and G3 didn´t exhibit significant results for spermatics motility or vigor when compared with the control group. The palmitic acid and vitamin E also had no significant effects on the parameters of acrosomal membrane integrity or mitochondrial activity. However, sperm cryopreserved with the addition of palmitic acid exhibited significant differences for plasma membrane integrity, providing greater protection to the sperm cells in G2.Discussion: The palmitic acid is one of the most saturated fatty acids in human semen, with reports of great proportions also in the seminal plasma of dogs. Its main role is to protect the plasma membrane from external damage, improving viability and fertility of the sperm after cryopreservation. Data is scarce in the literature on the composition of fatty acids in canine semen and regarding the use of palmitic acid as a seminal antioxidant in that species, which grants further studies aiming to investigate such valuable information for canine reproduction. It is concluded that addition palmitic acid at 100 µM concentration to the Tris-egg yolk diluter was able to preserve the integrity of the plasma membrane during the process of cryopreservation of canine semen.Keywords: dog, semen, antioxidants, cryopreservation.Descritores: cão, sêmen, antioxidantes, criopreservação.

scholarly journals Effects of the addition of docosahexaenoic acid and ?-tocopherol on quality of equine spermatozoa stored at 5°C

2020 ◽  
Vol 41 (1) ◽  
pp. 167 ◽  
Author(s):  
Breno Fernandes Barreto Sampaio ◽  
Bruno Gomes Nogueira ◽  
Maria Inês Lenz Souza ◽  
Eliane Vianna da Costa-e-Silva ◽  
Carmem Estefânia Serra Neto Zúccari
Keyword(s):  
Lipid Peroxidation ◽  
Plasma Membrane ◽  
Docosahexaenoic Acid ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Membrane Composition ◽  
Artificial Vagina

Plasma membrane composition has impact on phase transition from liquid crystal to gel state of cooled sperm cell. The incorporation of polyunsaturated fatty acids increases its fluidity and can contribute to sperm motility. The aim of this study was to compare the effect of adding docosahexaenoic acid (DHA) and ?-tocopherol (?-Toh) to the cooling extender, singly or combined, to the equine sperm parameters, submitted to cooling, up to 72 hours. Two ejaculates of ten stallions collected with artificial vagina were used, and evaluated for motility, plasma membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation, according to the following treatments: C; DHA; ?-Toh; DHA/?-Toh; EtOH 100: and EtOH 140 (corresponding to control; 10 ng mL-1 of DHA; 2 mM of ?-Toh; : 10 ng mL-1 of DHA + 2 mM of ?-Toh; 100 µL of ethanol and 140 µL of ethanol respectively). DHA treatment showed higher motility (68.2 ± 12.3; p < 0.05) when compared to control (62.1 ± 16.2), DHA/?-Toh (61.3 ± 12.7) and EtOH (58.1 ± 8.6) groups. In lipid peroxidation assay, the control group showed 2,506.2 ± 796.4 ng of MDA 108 spermatozoa-1, being significantly higher (p < 0.05) than the groups treated with DHA (2,036.0 ± 687.0), ?-Toh (1,890.8 ± 749.5) and DHA/?-Toh (1,821.1 ± 627.2). In conclusion, ?-Toh was effective in diminishing lipid peroxidation of equine sperm subjected to cooling, and DHA improved sperm motility and, in spite of being a polyunsaturated fatty acid with high susceptibility to peroxidation, reduced lipid peroxidation.

scholarly journals Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen

2019 ◽  
Vol 47 (1) ◽  
Author(s):  
Juliano Pianowski Marques Silva ◽  
André Felipe Berto de Almada ◽  
Jonathan Soares De Lima ◽  
Carlos Renato De Freitas Guaitolini ◽  
André Maciel Crespilho ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Free Radical ◽  
Beneficial Effect ◽  
Superoxide Anion ◽  
Membrane Integrity ◽  
Control Group ◽  
Study Group ◽  
Anion Formation ◽  
Progressive Motility ◽  
Acrosomal Membrane

Background: Studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from the plasma membrane. The mechanism of action of CLC is not well understood, however, it seems to involve sperm protection during the freezing and thawing process. Studies show that its use enhancing increased osmotic tolerance and reduced premature sperm capacitation reaction. In this sense, studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from the plasma membrane. Improvements were reported in the sperm parameters of buffaloes, bulls, stallions and sheep. Ram naturally present less lipids in their membrane, on average 27%, while bulls have 31%, rabbits 62%, and humans 50%. The aimed of the present study was to evaluate the use of cholesterol-loaded cyclodextrin (CLC), a commercial diluent, in the kinetics and viability of frozen and thawed ram spermatozoa. Materials, Methods & Results: Five ejaculates, from five rams of Dorper breed were collected and divided into three groups: control, 1 mg CLC and 2 mg CLC. Semen was diluted in different concentrations of CLC (0, 1, and 2 mg/120×106 spermatozoa), and incubated at room temperature (21°C) for 10 min. Samples were conditioned in 0.5 mL straws and incubated at 5°C for 4 h, exposed to LN2 vapor for 10 min and storing a cryogenic container. The parameters as spermatic kinetics, plasma membrane, acrosomal membrane (MPAI, %), and intracellular levels of superoxide anion (O2-) were evaluated. Sperm progressive motility (PM), rapid spermatozoa percentage (RAP), linearity (LIN, %), average path velocity (VAP, μm/s) and MPAI (%) were more satisfactory with the use of 1 mg compared to 2 mg (P < 0.05). In addition, 1 mg CLC showed decreased levels of superoxide anion formation (O2-), a free radical detrimental to spermatozoa (P < 0.05). The use of 2 mg of CLC reduce VAP (P < 0.05) and did not have any beneficial effect on the evaluated parameters. Discussion: Authors did not observe improvement in the parameters of progressive motility when using 1 mg of CLC in goat semen and 2 mg in bull semen with the slow freezing protocol. This differs from our work, as we found that 1 mg of CLC improved the PM parameters, but not at the concentration of 2 mg CLC. Additionally, authors verified that cyclodextrin at 3 mg concentration was effective in protecting the sperm against the deleterious effects of H2O2. They obtained superior plasma membrane motility, viability, and integrity of the CLC-treated samples compared to the control group. The superoxide anion (O2-) is a free radical formed from molecular oxygen by the addition of an electron. It is generated spontaneously, mainly in the membrane of the mitochondria, by the respiratory chain and by flavoenzimes, lipoxygenases, and cicloxygenases. In our study, we found a difference between the study group with 1 mg CLC and the control group. Thus, we suggest that CLC may have a beneficial effect in stabilizing the sperm plasma membrane. Use of CLC at a concentration of 1 mg was found to be effective for the improvement of parameters of sperm progressive motility, rapid sperm percentage, and plasma and acrosomal membrane integrity. In addition, the study group with 1 mg of CLC showed decreased levels of superoxide anion formation, a free radical detrimental to spermatozoa.

Insights into soy lecithin and egg yolk-based extenders for chilling canine spermatozoa

Zygote ◽  
2018 ◽  
Vol 27 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Andressa Dalmazzo ◽  
Daniel de Souza Ramos Angrimani ◽  
João Diego A. Losano ◽  
Carolina C. Rocha ◽  
Carlos A. B. Sobrinho ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
Compensatory Mechanism ◽  
Soy Lecithin ◽  
Sperm Analysis ◽  
Tbars Assay

SummaryThe aim of this study was to compare different concentrations of soy lecithin (LEC0.01%, LEC0.05% and LEC0.1%) with egg yolk (Control) in cooling extenders during the storage of semen at 5ºC for 5 days. Twelve dogs (n = 12) were selected, and semen was cooled and assessed after 2, 24, 48, 72, 96 or 120 h. At each time point, sperm were analyzed for kinetic patterns (using computer-assisted sperm analysis), mitochondrial activity (3′3- diaminobenzidine assay), lipid peroxidation (TBARS assay), DNA fragmentation (SCSA®) and plasma and acrosome membrane integrity (eosin/nigrosin and fast green/rose Bengal stains, respectively). The Control group (1814.4 ± 197.2) presented the highest rates of lipid peroxidation at 120 h. Conversely, progressive motility (42.8 ± 4%), linearity (45.4 ± 1%), and VAP (88 ± 3%) were higher in the Control group. In addition, there was lower mitochondrial activity in the Control group at 72 h. Therefore, our data show that lecithin used at these concentrations was not able to maintain sperm viability at as high qualities as would egg yolk. Moreover, the decrease in high mitochondrial activity and the persistence of sperm motility may indicate a compensatory mechanism in canine spermatozoa (i.e., glycolytic pathway). Furthermore, these higher lipid peroxidation indexes could indicate the necessity for future therapy using extenders and antioxidants over a long cooling time for dog sperm.

scholarly journals Penambahan alfa-tokoferol dalam pengencer susu skim - kuning telur terhadap kualitas spermatozoa domba Sapudi yang disimpan pada suhu 5°C

2020 ◽  
Vol 9 (3) ◽  
pp. 69
Author(s):  
Satya Alysa Cahya Puspita ◽  
Suherni Susilowati ◽  
Trilas Sardjito ◽  
Abdul Samik ◽  
Indah Norma Triana ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Integrity ◽  
Skim Milk ◽  
Egg Yolk ◽  
Alpha Tocopherol ◽  
Control Treatment ◽  
Control Group ◽  
Cold Temperatures ◽  
Fresh Semen

Spermatozoa in fresh semen of Sapudi ram has a limited life span. The storage of semen in cold temperatures (5 °C) is intended to prolong the spermatozoa's life. However, storage in cold temperatures can lead to increased production of reactive oxygen species (ROS). This condition reduces the quality of spermatozoa. The purpose of this study was to determine the effect of alphatocopherol supplementation in skim milk-egg yolk extender on viability, motility, and plasma membrane integrity of Sapudi ram spermatozoa. Fresh semen derived from Sapudi ram was divided into four treatment groups. Control treatment (P0): semen was added in the extender of skim milkegg yolk without alpha-tocopherol. Three other treatments: P1, P2, and P3 semen were added in skim milk-egg yolk extender with the supplementation of 0.25, 0.5, and 1 gram alpha-tocopherol/ 100 mL extender, respectively. The results showed that the viability, motility, and integrity of the spermatozoa plasma membrane decreased gradually according to the storage length. Supplementation of skim milk-egg yolk extender with 0.5 gram of alpha-tocopherol/100 mL (P2) was able to maintain spermatozoa quality longer (p <0.05) than the control group. It can be concluded that alpha-tocopherol with a concentration of 0.5 g/100 mL of skim milk-egg yolk extender effectively maintains the quality of Sapudi ram spermatozoa in storage at 5 ° C.

scholarly journals Influence of Coconut Powder Water-Based Conservation Medium (APC-102c) for Maintaining Mitochondrial Activity of Cryopreserved Ram Sperm

2019 ◽  
Vol 47 (1) ◽  
Author(s):  
Bruna Farias Brito ◽  
Bárbara Mara Bandeira Santos ◽  
Leonardo Alves Rodrigues Cabral ◽  
David Baruc Cruvinel Lima ◽  
Cristiane Clemente De Melo Salgueiro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Artificial Insemination ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Mitochondrial Activity ◽  
Plasma Membrane Integrity ◽  
Significant Difference ◽  
Motility Parameters

Background:  Semen extenders are required to protect and preserve semen, and the development of suitable extenders is key for artificial insemination. Although the use of Tris-based diluent is widespread, new diluents such as powdered coconut water have been developed for better sperm protection. One way to evaluate the effectiveness of diluents is through microscopic analyses that evaluate sperm motility, vigor, and concentration. However, these analyses are limited, and may not provide accurate results. New evaluation techniques have been studied, and one of the tests that can be used to add reliability to these analyses is mitochondrial activity evaluation, which can sum all the parameters, and provide a more accurate evaluation. Thus, the present study aimed to evaluate the efficacy of ACP-102c in cryopreserved ram semen.Materials, Methods & Results: Five semen samples were collected from two ram breeders using artificial vagina (n = 10). Each ejaculate was divided into the following two treatments: T1 - ACP-102c + 20% egg yolk + 7% glycerol and T2 - TRIS + 20% egg yolk + 7% glycerol. Extended semen samples were then packed in 0.5 mL plastic straws, subjected through the refrigeration curve up to 4°C (0.35° C/min), and equilibrated for 2 h at 4°C. Subsequently, the straws were placed at 4 cm above liquid nitrogen level (-60°C) for 15 min, immersed, and then finally stored in the liquid nitrogen at -196°C. Both fresh and thawed samples were evaluated for total and progressive sperm motility using conventional microscopy (40x), and the same evaluator on each occasion. For plasma membrane integrity (IMP), the smear staining technique with the Eosin-Nigrosin staining was used; 200 sperms were counted and classified as whole (unstained) and unhealthy (stained). Mitochondrial activity was evaluated using a cytochemical technique based on the oxidation of 3,3'-diaminobenzidine (DAB); 200 sperms were counted, and classified into four classes (I, II, III, and IV) according to the degree of coloration of the intermediate part. Fresh semen showed no significant difference (P > 0.05) between treatments with respect to motility parameters; however, T2 showed significantly inferior results regarding plasma membrane integrity. After thawing, T2 was significantly higher in sperm motility parameters compared to T1. The mitochondrial activity and plasma membrane integrity parameters did not show any significant difference between the treatments.Discussion: The TRIS-based diluent showed higher motility values than ACP-102c; however, motility rates in ACP-102c diluent, although lower, are considered satisfactory for insemination, which requires semen with minimal progressive motility of 30%. Notably, the cryopreservation protocol used in this study is the standard for TRIS-based diluent, and it is known that the optimal rate of refrigeration and cryopreservation may differ according to the composition of the storage medium; therefore, we may assume that the protocol used is not yet appropriate for the ACP-102c diluent, and further studies are required. IMP is an essential attribute for fertilization, and cryopreservation can affect the plasma membrane as observed in this study. Cryopreserved semen reduced the percentage of class I mitochondrial reaction sperms in both treatments, demonstrating that cryopreservation affects the mitochondrial activity of the intermediate portion of the sperm; however, there was no difference between treatments in thawed semen. Thus, we concluded that the ACP-102c conservation medium maintains seminal quality after thawing, and it can be used in artificial insemination processes.

Effects of Dietary Addition of Linseed Oil on the Content of Polyunsaturated Fatty Acids in Egg Yolk of Gallus domestiaus

2021 ◽  
Vol 15 (4) ◽  
pp. 565-570
Author(s):  
Suhu Duan ◽  
Zaiqiang Li ◽  
Zhenzhen Fan ◽  
Mengran Qin ◽  
Xiaoxue Yu ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Gas Chromatography ◽  
Polyunsaturated Fatty Acids ◽  
Theoretical Basis ◽  
Linseed Oil ◽  
Egg Yolk ◽  
Free Food ◽  
Control Group ◽  
Experimental Group ◽  
Group 1

In order to study the effect of dietary supplement of linseed oil on polyunsaturated fatty acids (PUFAs) in egg yolk of Gallus domestiaus, total 160 healthy Gallus domestiaus of 26-week old were randomly selected and divided into 4 groups, each of which included 40 chickens fed outdoors with an area of 63 m2 and free food and drinking water. The control group (CK) was fed the basic diet, and the experimental group was added 1, 3 and 5% of linseed oil in the fundamentals of diet (experimental group 1, 2 and 3, abbreviated as P1, P2, P3), respectively. The content of PUFA and the value of n-6 PUFA and n-3 PUFA in egg yolk were determined by gas chromatography with 24 eggs randomly selected in each group after 10th, 25th and 40th days of the commence of the experiment. We found that the levels of α-linolenic acid (C18:3n3, ALA) and docosahexaenoic acid (C22:6n3, DHA) in egg yolk were clearly higher than the CK when 3% linseed oil or 5% linseed oil were added in the diet during the three experiment periods, in which the contents of linoleic Acid (18:2n6c, LA) and arachidonic acid (C20:4n6, ARA) in egg yolk differ little between P1, P2 and P3 and the CK while the value of n-6 PUFA and n-3 PUFA in egg yolk of the P2 and P3 groups decreased significantly. The comprehensive comparisons suggested that the content of C18:3n3 and C22:6n3 in egg yolk could be clearly increased in the P2 while the value of n-6 PUFA and n-3 PUFA decreased. This study provides theoretical basis for the production of functional eggs enriched with C22:6n3 and C18:3n3.

scholarly journals Efek Penambahan Vitamin E dalam Pengencer Glukosa Fosfat terhadap Daya Tahan Hidup Spermatozoa Domba pada Suhu 5 °C

2017 ◽  
Vol 9 (1) ◽  
pp. 25
Author(s):  
Nilawati Widjaya
Keyword(s):  
Plasma Membrane ◽  
Vitamin E ◽  
Analysis Of Variance ◽  
Membrane Integrity ◽  
Plasma Membrane Integrity ◽  
Glucose Phosphate ◽  
Randomized Design ◽  
Completely Randomized Design

<p class="p1">The purpose of this study was to know the effect of vitamin E addition in glucose phosphate diluent on the survival of sheep spermatozoa at 5 °C. The xperiment used 3 rams. The design used was Completely Randomized Design (CRD) with 5 treatments and 4 replicates, each replicate with 3 ejaculate cement of sheep. The treatments were the dose of Vitamin E with E<span class="s1">0 </span>= 0 μg/ml, E<span class="s1">1 </span>= 1 μg/ml, E<span class="s1">2 </span>= 4 μg/ml, E<span class="s1">3 </span>= 7 μg/ml, and E<span class="s1">4 </span>= 10 μg/ml. Variables measured were motility, live spermatozoa and plasma membrane integrity. Data were analyzed by using analysis of variance according to Steel and Torrie (1981). The result showed that treatment of vitamin E with a dose of 1 μg/ml - 10 μg/ml l had no significant effect on motility, percentage live spermatozoa and membrane integrity of sheep spermatozoa on day two (P&gt; 0.05).</p>

scholarly journals Integrity of the plasma membrane, the acrosomal membrane, and the mitochondrial membrane potential of sperm in Nelore bulls from puberty to sexual maturity

2016 ◽  
Vol 68 (3) ◽  
pp. 620-628 ◽  
Author(s):  
L.S.L.S. Reis ◽  
A.A. Ramos ◽  
A.S. Camargos ◽  
E. Oba
Keyword(s):  
Plasma Membrane ◽  
Mitochondrial Membrane ◽  
Sexual Maturity ◽  
Wave Motion ◽  
Membrane Integrity ◽  
Mitochondrial Potential ◽  
Plasma Membrane Integrity ◽  
Scrotal Circumference ◽  
Acrosomal Membrane ◽  
Sperm Membrane

ABSTRACT This study evaluated the plasma membrane integrity, acrosomal membrane integrity, and mitochondrial membrane potential of Nelore bull sperm from early puberty to early sexual maturity and their associations with sperm motility and vigor, the mass motility of the spermatozoa (wave motion), scrotal circumference, and testosterone. Sixty Nelore bulls aged 18 to 19 months were divided into four lots (n=15 bulls/lot) and evaluated over 280 days. Semen samples, collected every 56 days by electroejaculation, were evaluated soon after collection for motility, vigor and wave motion under an optical microscope. Sperm membrane integrity, acrosomal integrity, and mitochondrial activity were evaluated under a fluorescent microscope using probe association (FITC-PSA, PI, JC-1, H342). The sperm were classified into eight integrity categories depending on whether they exhibited intact or damaged membranes, an intact or damaged acrosomal membrane, and high or low mitochondrial potential. The results show that bulls have a low amount of sperm with intact membranes at puberty, and the sperm show low motility, vigor, and wave motion; however, in bulls at early sexual maturity, the integrity of the sperm membrane increased significantly. The rate of sperm membrane damage was negatively correlated with motility, vigor, wave motion, and testosterone in the bulls, and a positive correlation existed between sperm plasma membrane integrity and scrotal circumference. The integrity of the acrosomal membrane was not influenced by puberty. During puberty and into early sexual maturity, bulls show low sperm mitochondrial potential, but when bulls reached sexual maturity, high membrane integrity with high mitochondrial potential was evident.

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