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.
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.
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