scholarly journals Effect of Oral Administration of Selenium and Vitamin E on the Quality of Fresh, Refrigerated and Frozen Semen in French Bulldog Breed Dogs

2017 ◽  
Vol 45 (1) ◽  
pp. 7
Author(s):  
Marcelo George Mungai Chacur ◽  
Mariana Grandis Ripari de Souza ◽  
Camila Dutra de Souza ◽  
Camila Pires Cremasco
Keyword(s):  
Vitamin E ◽  
Citric Acid ◽  
Oral Administration ◽  
Liquid Nitrogen ◽  
Sperm Concentration ◽  
Oral Supplementation ◽  
Frozen Semen ◽  
French Bulldog ◽  
Fresh Semen

Background: New methodologies have been developed seeking to maximize pregnancy rate in female dogs created in commercial kennels, and also in order to maintain the quality of canine semen after dilution, refrigeration or freezing. One of the main factors that generate damage to sperm is oxidative stress, to minimize sperm damage, selenium and antioxidants like vitamin E are administered, by oral administration, seeking to improve the quality of semen. The objective was to study the effect of vitamin E and selenium, by oral administration, in the quality of fresh, refrigerated and frozen semen in adult dogs French Bulldog breed.Materials, Methods & Results: Semen samples were collected from 5 adult dogs, French Bulldog breed, being 2 semen drawing before the daily oral supplementation with vitamin E and selenium (ESE®) and semen drawing at 20, 40 and 60 days after the beginning of oral supplement. The ejaculated samples were diluted in TRIS - fructose citric acid (3.28 g TRIS-hydroxy-methyl-amino-methane, 1.78 g of citric acid monohydrate and 1.25 g of D - fructose, dissolved in 100 mL of distilled water and added of 20% egg yolk and 6% of glycerol. The characteristics evaluated in fresh semen were: volume (mL), color, appearance, concentration (x106 / mL), sperm motility (%), sperm strength (1 to 5) and morphology (%). For refrigerated and frozen semen were analyzed: sperm motility (%), sperm strength (1-5) and morphology (%). Diluted semen samples were centrifuged at: 1500 g/10 min and “pellets” formed by sperm of each ejaculated, detached from the tube wall were diluted homogeneously in the diluent TRIS type up to the final volume of 1.5 mL. After that, packaged in 0.5 mL French straws, kept under refrigeration at 5ºC/4 h, placed in nitrogen vapor at -120ºC/15 min, and dipped in liquid nitrogen at -196ºC and then stored on identified rachis and stored in liquid nitrogen container until the time of thawing in  water bath at 37°C/30 s for semen microscopic analysis. Data from fresh, refrigerated and frozen semen were statistically analyzed by analysis of variance and the average compared by 5% of Tukey test. Fresh semen sperm concentration differed (P < 0.05) between the samples, rising after 40 days after the beginning of oral supplementation with selenium and vitamin E. For the spermatic strength, better score (P < 0.05) was observed at collection 4, in 40 days after the beginning of oral supplementation to dogs. For fresh and refrigerated semen, the total defects, defects of head, acrosome and tail did not differ (P > 0.05) between the samples. Total sperm defects and minor head and tail defects did not differ (P > 0.05) between the samples in post-thawing. Regarding the acrosome defects after thawing, there was a significant reduction (P < 0.05) in samples performed 40 and 60 days after the beginning of oral supplementation with selenium and vitamin E.Discussion: Attention should be paid for what purpose the extenders within the refrigeration or freezing biotech will be used. The managed supplement, by oral administration, containing selenium and vitamin E, influenced beneficially raising the sperm concentration in fresh semen and decreasing the acrosome defects in frozen semen. Oral administration of supplementation with selenium and vitamin E is recommended for improving the quality of fresh and frozen semen in dogs.

scholarly journals Influence of the Addition of Vitamin E in Extenders in the Quality of Fresh Semen Diluted, Refrigerated and Frozen Semen in Dogs of French Bulldog Breed

2017 ◽  
Vol 45 (1) ◽  
pp. 6
Author(s):  
Marcelo George Mungai Chacur ◽  
Mariana Grandis Ripari de Souza ◽  
Camila Dutra de Souza ◽  
Camila Pires Cremasco
Keyword(s):  
Vitamin E ◽  
Citric Acid ◽  
Liquid Nitrogen ◽  
Artificial Insemination ◽  
Sperm Head ◽  
Cooling Curve ◽  
Frozen Semen ◽  
French Bulldog ◽  
Fresh Semen

Background: Researches have been conducted in order to maintain the quality of the fresh semen which is diluted, refrigerated or frozen in liquid nitrogen for artificial insemination purposes in dogs. The semen biotechnology cooperates with the development of new formulations of types of extenders which minimize the death of the sperms due to thermal stress during temperature reduction of the refrigeration and freezing curves of the semen. The objective was to study the influence of the addition of vitamin E in types of extenders in the quality of the fresh, refrigerated and frozen semen in dogs of French Bulldog breed.Materials, Methods & Results: Semen samples by digital manipulation were performed on 5 adult dogs, French bulldog breed, five on each dog, totaling 25 ejaculated. The characteristics evaluated in fresh semen were: Volume (mL), color, aspect, concentration (x106/mL), sperm motility (%), vigor (1-5) and sperm morphology (%). For refrigerated and frozen semen, motility (%), vigor (1-5) and morphology (%) were analyzed. The ejaculated ones were fractionated in 4 equal parts and diluted in the ratio 1: 1 in the following extenders: 1 – TRIS - Fructose Citric acid + 200 mM of vitamin E; 2 – TRIS - Fructose Citric acid; 3 - coconut water (ACP-106®) + 200 mM of vitamin E; and 4 – coconut water (ACP-106®). The four aliquots of semen, diluted in the four respective extenders were centrifuged at 1500 g/10 min and the “pellets” formed of sperm from every ejaculated, detached from the tubes wall were diluted homogeneously with the four extenders to the volume of 1.5 mL and filled into 0.5 mL French straws kept under refrigeration at 5oC/4 h after placed in a nitrogen vapor at -120ºC/15 min, and immersed in liquid nitrogen at -196ºC in the sequence stored in identified racks and stored in liquid nitrogen container until the time of thawing in a water bath at 37ºC/30 s for microscopic semen analysis. Data from fresh, refrigerated and frozen semen were statistically analyzed by analysis of variance and the average compared by Tukey (P < 0.05). For fresh semen diluted in the four extenders, in pre-cooling curve, there was a significant difference (P < 0.05) for defects in the sperm head, between TRIS + vit. E (7.59 ± 4.01%) and TRIS (10.48 ± 5.42%). In the post-cooling curve to 5ºC/4 h, for the four extenders, there was no difference (P > 0.05) between the evaluated characteristics. For frozen semen with TRIS and thawed at 37ºC/30 s, there was difference (P < 0.05) for the major sperm defects, being the top average (26.62 ± 5.52%) compared to the other three extenders. For minor sperm defects in frozen semen with TRIS, there was difference (P < 0.05) with a lower percentage of incidence (16.23 ± 2.02%) compared to other extenders. There was difference (P < 0.05) with a significant increase of total defects in frozen semen with the extender ACP + vit. E, compared to other extenders.Discussion: Attention should be paid for what purpose the extenders within the refrigeration or freezing biotech will be used. In the present study, we found that the microscopic analysis of the spermatic motility and vigor in frozen semen with the ACP extender is hampered due to the lower transparency of this extender in relation to the TRIS extender. We conclude that the TRIS + vit. E extender it is the most recommended to dilute the fresh semen for the purpose of immediate artificial insemination due to lower presence of the sperm head defects. For refrigeration, the four extenders are recommended, with similarity in semen characteristics maintenance. For frozen semen the indicated extenders are the TRIS, TRIS + vit. E, and the extender ACP. The addition of vit. E in these extenders did not provide improvement of refrigerated and frozen semen, with optional use of it.

scholarly journals Individual variation in fresh and frozen semen of Bali bulls (Bos sondaicus)

2020 ◽  
Vol 13 (5) ◽  
pp. 840-846
Author(s):  
R. Indriastuti ◽  
M. F. Ulum ◽  
R. I. Arifiantini ◽  
B. Purwantara
Keyword(s):  
Dna Fragmentation ◽  
Sperm Motility ◽  
Individual Variation ◽  
Sperm Concentration ◽  
Individual Factors ◽  
Abnormal Morphology ◽  
Frozen Semen ◽  
Semen Volume ◽  
Fresh Semen

Aim: This study aimed to analyze the individual factors influencing the sperm quality of Bali bulls at Baturiti Artificial Insemination (AI) center. Materials and Methods: Semen that was ejaculated from nine Bali bulls was collected using artificial vaginas (n=5/bull). Semen ejaculates were evaluated immediately after collection to measure the quality of the fresh semen, including semen volume, sperm concentration, progressive motility, membrane integrity (MI), and abnormal morphology. Frozen semen was evaluated for progressive sperm motility, concentration, viability, MI, abnormal morphology, and deoxyribonucleic acid (DNA) fragmentation. Other secondary data, focusing on semen quantity (semen volume and sperm concentration), were also collected from frozen the semen production data of the Baturiti AI center from 2017 to 2019. Data were analyzed statistically using a completely randomized design, and one-way analysis of variance was applied to find differences among individual bulls. Results: Significant differences (p<0.05) were found among the bulls in semen volume, sperm motility, concentration, and MI of the fresh semen. Significant differences (p<0.05) were also found among the bulls in sperm motility, viability, MI, abnormal morphology, and DNA fragmentation of the frozen semen. Conclusion: Individual variation in all the tested sperm parameters of the fresh semen of Bali bulls, except sperm viability and abnormalities, was noted. Similarly, individual variation in all the tested sperm parameters in frozen semen, except sperm concentration, was noted. Therefore, individual factors can be used for selecting a superior bull in Bali cattle.

scholarly journals THE QUALITY OF BOAR FROZEN SEMEN DILUTED IN BTS® AND MII® WITH DIFFERENT CRYOPROTECTANT SUPPLEMENTED WITH SODIUM DODECYL SULPHATE

2017 ◽  
Vol 11 (1) ◽  
pp. 6-10
Author(s):  
Nancy Diana Frederika Katerina Foeh ◽  
Raden Iis Arifiantini ◽  
Tuty Laswardi Yusuf
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Sodium Dodecyl Sulphate ◽  
Semen Quality ◽  
Sodium Dodecyl ◽  
Sperm Concentration ◽  
Sperm Abnormalities ◽  
Semen Collection ◽  
Frozen Semen

This research was aimed to study the effect of administration of glycerol and dimetilacetamida (DMA) in BTS® and MIII®extender supplemented with sodium dodecyl sulphate (SDS) on boar frozen semen. A number of four boars were used in this study for semen collection (n=20). The collected semen was evaluated both macroscopically and microscopically. In this study, only the semen that demonstrated>70% sperm motility, >200.106/mL sperm concentration, and<20% sperm abnormalities were used and divided into eight tubes. A number of 4 tubes were diluted with 5 mL of BTS, and the rest with 5 mL MIII. The sampel was stored at 20-22° C for 2 hours, followed by centrifugation for 15 minutes (at 2000 rpm), and taken of pellet with 1 ml supernatant. The pellet that was resulted from centrifugation using BTS, then re-diluted with BTS-glycerol 5% (BTSG), BTS DMA 5% (BTSD), BTS-glycerol 5% and SDS (BTSG-S), BTS-DMA 5% and SDS (BTSD-S). Four other pellets that were centrifuged with MIII also re-diluted with MIII-glycerol 5% (MIIIG), MIII-DMA 5% (MIIID), MIII-glycerol 5% and SDS (MIIIG-S), MIII-DMA 5% and SDS (MIIID-S). Next, all of diluted semen were inserted into 0.5 mL straw and equilibrated for 2 hours (4° C), then frozen and stored in liquid nitrogen. The evaluation of frozen semen quality was conducted at 24 hours after frozen. The result of this study showed that post-thawing motility of spermatozoa in BTSD-S (40.17±0.2%) was found higher (P<0.05) compared to seven other dilution processes. Therefore, it is concluded that the concentration of 5% DMA that supplemented with SDS in BTS dilution much better for maintaining boars frozen semen quality.

scholarly journals Effect of dietary supplementation with omega-3 and -6 on fresh and frozen/thawed sperm quality of dogs

2017 ◽  
Vol 38 (5) ◽  
pp. 3069 ◽  
Author(s):  
Ana Carolina Rodrigues ◽  
Camila Montanari Ruiz ◽  
Carla Daniela Dan De Nardo ◽  
Gabriele Barros Mothé ◽  
Fabiano Martinez Rossi ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Dietary Supplementation ◽  
Reproductive Efficiency ◽  
Omega 3 ◽  
Oral Supplementation ◽  
Animal Reproduction ◽  
Fresh Semen

For years, fatty acids have been recommended as a dietary supplement to improve canine hair. For animal reproduction, supplementation with omegas has been used to increase the reproductive efficiency and conception rate, but few studies have been conducted in dogs. The aim of this study was to evaluate the effects of daily dietary supplementation with omega-3 and -6 on the quality of fresh and frozen/thawed semen in canines. Semen was collected from seven dogs and evaluated for sperm motility, vigor, concentration, and morphology. The 17-week study included 119 ejaculates and was divided according to oral supplementation with omega-3 and -6: M1 (1st-5th week) or pre-supplementation; M2 (6th-9th week) and M3 (10th-13th week) or during supplementation; and M4 (14th-17th week) or post-supplementation. After analysis, the semen was frozen and then revaluated both immediately and 30 minutes (at 37° C) after thawing. Supplementation with omegas increased sperm motility, vigor, and concentration; however, supplementation had no influence on semen freezability. In addition, there was no improvement in sperm motility after supplementation when the thawed cells were maintained at 37° C for 30 minutes. We concluded that dietary supplementation with omega-3 and -6 for 4 to 8 weeks can improve the quality of fresh semen, although it has no effect on the freezability of canine semen.

scholarly journals Evaluating the ability of semen cryopreservation of Blance Blue Belge bull cattle in Vietnam

2021 ◽  
Vol 19 (2) ◽  
pp. 237-244
Author(s):  
Nguyen Huu Duc ◽  
Pham Thu Giang ◽  
Tran Thi Binh Nguyen ◽  
Nguyen Thi Mai ◽  
Bui Dai Phong
Keyword(s):  
Sperm Motility ◽  
Sperm Concentration ◽  
Joint Stock Company ◽  
Stock Company ◽  
Cattle Breeding ◽  
Semen Cryopreservation ◽  
Semen Collection ◽  
Frozen Semen ◽  
Semen Volume ◽  
Fresh Semen

The objective of this study was to determine the semen cryopreservation capacity of BBB bulls in Hanoi-Vietnam. Research conducted on the fresh semen collected from 05 BBB bulls. Results showed that semen color was normal (milky white, ivory white, ivory yellow), semen volume ranged from 6.35 mL to 7.48 mL (P <0.05), initial motility of semen ranged from 80.53% to 82.92% (P <0.05), sperm concentration in semen  ranged from 1.02 x 109 sperms/ml to 1.12 x 109 sperms/mL (P <0.05), abnormal sperm ratio ranged from 6.45% to 8.12% (P <0.05), alive sperm ratio ranged from 76.34% to 82.97% (P <0.05), sperm motility after thawing from straw semen ranged from 71.33% to 75.92% (P<0.05). In conclusion, successfully semen collection from 05 breeding BBB bulls at Hanoi Cattle Breeding Joint Stock Company, semen samples had normal color and good quantity and quality, suitable for production of frozen semen; and semen cryopreservation of straws of the 05 bull BBB semen mentioned at -196oC, sperm motility after freezing-thawing reached the economic and technical norms of 675/2014 of the Ministry of Agriculture and Rural Development.

Effect of different extenders on quality of frozen-thawed boar semen

2015 ◽  
Vol 49 (6) ◽  
Author(s):  
S Frydrychová ◽  
A Lustyková ◽  
E Václavková ◽  
J Lipenský ◽  
M Rozkot
Keyword(s):  
Sperm Motility ◽  
Aspartate Aminotransferase ◽  
Sperm Concentration ◽  
Holding Time ◽  
Holding Period ◽  
Semen Volume ◽  
Significant Difference ◽  
Boar Semen ◽  
Fresh Semen

The objective of this study was to investigate the effect of using different extenders <italic>viz.</italic> Androhep, Safecell Plus and SUS during cryopreservation on quality of frozen-thawed boar semen. Semen volume, sperm motility, sperm concentration, percentage of morphologically abnormal spermatozoa, total number of spermatozoa per ejaculate and activity of the enzyme aspartate aminotransferase (AST) were assessed in fresh semen collected from 39 fertile AI boars. Semen from each boar was divided into three portions and diluted 1:1.5 in extender Androhep, Safecell Plus and SUS and keep at 17°C for 15-h holding time before cryopreservation. Then sperm was cryopreserved. Straws were thawed in a water bath at 38°C for 40s and post-thaw sperm motility with AST activity was assessed. Significant difference in post-thaw sperm motility was found between extender Androhep and Safecell Plus (P<0.05). AST activity did not differ significantly between tested extenders (P>0.05). In conclusion, the results of the study indicate that using Safecell Plus extender during holding period before cryopreservation significantly affected post-thaw sperm motility.

scholarly journals Effect of Selenium and Vitamin E Supplementation on Semen Quality in Dogs with Lowered Fertility

2015 ◽  
Vol 59 (1) ◽  
pp. 85-90 ◽  
Author(s):  
Anna Domosławska ◽  
Sławomir Zduńczyk ◽  
Wojciech Niżański ◽  
Andrzej Jurczak ◽  
Tomasz Janowski
Keyword(s):  
Vitamin E ◽  
Semen Quality ◽  
Sperm Count ◽  
Sperm Concentration ◽  
Control Group ◽  
Organic Selenium ◽  
Healthy Dogs ◽  
Manual Manipulation ◽  
Motility Parameters

Abstract Thirty clinically healthy dogs with poor semen quality were used in the study. Fifteen dogs were supplemented daily with selenium (0.6 mg/kg organic selenium from yeast) and vitamin E (5 mg/kg) per os for 60 d. The control group (15 dogs) was not supplemented. Semen was collected from all dogs by manual manipulation on days 0, 30, 60, and 90. The sperm concentration and motility parameters were evaluated with a Hamilton Thorne sperm analyser, version IVOS 12.3. For the assessment of sperm morphology, Diff-Quik stain was used. The percentage of live and dead spermatozoa was estimated on dried smears stained with eosin-nigrosin. The concentration of spermatozoa, most motility parameters determined (PMOT, VSL, VCL, ALH, BCF, RAPID, MEDIUM, SLOW, and STATIC), and the percentage of spermatozoa morphologically normal and live increased significantly (P < 0.05) after 60 d of supplementation. In the control group, there were no changes in motility parameters while the concentration and total sperm count decreased over the duration of the study. In conclusion, supplementation with selenium and vitamin E for 60 d can improve the quality of semen in dogs with lowered fertility.

scholarly journals Effect of preservation time on the quality of frozen semen in indigenous rams

2015 ◽  
Vol 44 (1) ◽  
pp. 10-15 ◽  
Author(s):  
BBA Mahmuda ◽  
Azizun Nesa ◽  
BF Zohara ◽  
MGS Alam ◽  
FY Bari
Keyword(s):  
Semen Quality ◽  
Egg Yolk ◽  
Volume Density ◽  
Normal Morphology ◽  
Mean Values ◽  
Frozen Semen ◽  
Significant Difference ◽  
The Mean ◽  
Fresh Semen

The study was carried out to observe the effects of preservation time on the quality of frozen semen of indigenous rams. Semen was collected using AV once a week from 4 rams. Tris based with 10% egg yolk and 7% glycerol extender was used to extend and freezing the semen. Fresh semen was evaluated for volume, density, mass motility and concentration, and mean values were observed as 0.8±0.2ml, 3.0±0.3, 3.2±0.7, 3.9±0.7×109/ml, respectively. Significant difference (p<0.05) was found for all the parameters among the rams. Mean values of motility, viability and normal morphology percentages were 83.3±4.3%, 88.2±4.4%, 84.2±3.5% in fresh semen while those of chilled semen at 40C were 74.7±2.3, 78.8±4.9 and 79.2±2.9%, respectively. For all the parameters, significant (p<0.05) difference was found among the rams. Frozen sperm motility was observed after thawing at 39-400C for 14-15 seconds. The mean motility, viability and normal morphology percentages after freezing for 24hrs, 7, 15 and 30 days of duration were 39.8±3.1, 41.1±4.3, 40.1±4.1 and 39.4±2.9%; 44.5±2.5, 45.3±2.8, 44.6±2.8 and 43.9±2.8%; 71.0±2.0, 71.7±1.5, 70.7±1.7 and 70.3±1.8%, respectively and values did not decrease significantly (p>0.05) with the increasing time of preservation. Non significantly decrease of the semen quality with advance of preservation time indicates the suitability of the protocol used for freezing of indigenous ram semen in Bangladesh.DOI: http://dx.doi.org/10.3329/bjas.v44i1.23113            Bang. J. Anim. Sci. 2014. 44 (1): 10-15

scholarly journals The effect of freezing on cryosurvival of yak sperm

2018 ◽  
Vol 15 (2) ◽  
pp. 215-218
Author(s):  
S Deori
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Artificial Insemination ◽  
Sperm Concentration ◽  
Egg Yolk ◽  
Mean Values ◽  
Frozen Semen ◽  
Sperm Abnormality ◽  
Semen Characteristics ◽  
Live Sperm

A study was carried out to study the effect of freezing on cryosurvival of yak semen. Artificial insemination in yak is still in infancy. Semen cryopreservation and use of artificial insemination can be applied in yak husbandry for conservation and rapid multiplication of superior germplasm. Semen was collected from four adult yak bulls using artificial vagina method managed under uniform conditions. A total of 40 ejaculates comprising of 10 ejaculates each bull were collected following twice a week schedule and evaluated for fresh semen characteristics. The fresh yak semen characteristics viz. ejaculate volume (ml), mass activity (0-4), initial sperm motility (%), sperm concentration (x 106/ml), live sperm (%), sperm abnormality (%) and intact acrosome (%) were 3.10 ± 0.18, 3.53 ± 0.96, 83.89 ± 2.87, 1180.22 ± 42.32, 77.63 ± 4.23, 8.45 ± 3.33 and 93.61 ± 3.78 respectively. The ejaculates were diluted (1:10) with Tris extender consisting of 6.4 ml glycerol and 20 ml of fresh egg yolk. Straws were equilibrated at 5°C for 4 hours followed by exposure to liquid nitrogen vapour for 10 minutes and finally transferred to liquid nitrogen container for storage. The cryosurvival rate was studied after 7 days of storage in liquid nitrogen. The frozen semen was thawed in warm water (37°C) for 30 seconds for evaluation. Mean values of postthaw sperm motility (%), live sperm (%) and intact acrosome (%) in yaks were 55.67 ± 4.67, 65.62 ± 3.23 and 89.26 ± 3.67 respectively. In conclusion, yak semen has a better cryosurvival while freezing in tris extender with 6.4 per cent glycerol and 20 per cent egg yolk following an equilibration period of 4h.SAARC J. Agri., 15(2): 215-218 (2017)

scholarly journals In vitro addition of docosahexaenoic acid improves the quality of cooled but not frozen–thawed stallion semen

2017 ◽  
Vol 29 (10) ◽  
pp. 2021
Author(s):  
D. M. Silva ◽  
S. A. Holden ◽  
A. Lyons ◽  
J. C. Souza ◽  
S. Fair
Keyword(s):  
Lipid Peroxidation ◽  
Vitamin E ◽  
Docosahexaenoic Acid ◽  
Membrane Fluidity ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Acrosome Integrity

The aim of the present study was to assess the effect of the addition of docosahexaenoic acid (DHA) on the in vitro quality of cooled and frozen–thawed stallion semen. In Experiment 1, semen from 10 stallions was collected (three ejaculates per stallion). Semen was diluted to 100 × 106 spermatozoa mL–1 with 0.02 mM vitamin E (VE) and 0, 1, 10 or 20 ng mL–1 DHA and frozen. Semen was thawed and total motility (TM), rapid progressive motility (PM), acrosome integrity, membrane fluidity and morphology were assessed. In Experiment 2, semen from three stallions was collected (three ejaculates per stallion) and frozen as in Experiment 1, but VE and DHA were added after thawing. TM and PM were assessed at 30, 60 and 120 min and viability, acrosome integrity and membrane fluidity were evaluated at 30 min. In Experiment 3, semen from five stallions was collected (one to three ejaculates per stallion), diluted to 20 × 106 spermatozoa mL–1 and stored at 4°C. After 1, 24, 48 and 72 h, TM, PM, viability, membrane fluidity and lipid peroxidation were assessed. The addition of DHA had no effect on frozen semen (Experiments 1 and 2) but improved TM, PM and membrane fluidity in cooled stallion semen.

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