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      Adición de metil-β-ciclodextrina cargada de colesterol sobre la criopreservación de semen de toros Holstein Friesian Translated title: Addition of methyl-β-cyclodextrin loaded with cholesterol (CLC) on the cryopreservation of semen of Holstein Friesian bulls

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          Abstract

          Se evaluó el efecto de la adición de metil-β-ciclodextrina cargada con colesterol (CLC) sobre la criopreservación de semen de toros Holstein Friesian a través de la valoración de las características seminales pre y pos-congelación. Se utilizaron cuatro toros de 3-4 años, obteniéndose 28 eyaculados, que fueron divididos en tres alicouotas, donde se agregó el CLC: 0, 1.5 y 2 mg de CLC por cada 120 millones de espermatozoides (T1, T2 y T3, respectivamente) a 37 °C y fueron incubados por 15 minutos. Las muestras fueron refrigeradas a 5 °C durante 18 horas para su estabilización. La congelación del semen se hizo en pajillas de 0.5 ml usando vapores de nitrógeno por 7 minutos y luego sumergidas en nitrógeno líquido. No se encontraron diferencias estadísticas entre tratamientos en todas las características del semen refrigerado. Los resultados pos-descongelación revelaron mejoras significativas en los tratamientos con colesterol, obteniendo una motilidad total del 67.73, 79.59 y 74.73%; integridad de membrana de 46.89, 60.43 y 55.06% y vitalidad de 63.26, 75.84 y 70.53% para T1, T2 y T3, respectivamente (p<0.05) No hubo diferencias significativas entre tratamientos con respecto a las anormalidades morfológicas.

          Translated abstract

          The effect of the addition of cholesterol-loaded methyl-β-cyclodextrin (CLC) on the semen cryopreservation of Holstein Friesian bulls was assessed through the assessment of pre and post-freezing seminal characteristics. Four bulls of 3-4 years of age were used and 28 ejaculates were collected. The ejaculates were divided into three aliquots, where the CLC was added: 0, 1.5 and 2 mg of CLC per 120 million sperm cells (T1, T2 and T3 respectively) at 37 °C and incubated for 15 minutes. The samples were refrigerated at 5 °C for 18 hours for stabilization. Semen was frozen in 0.5 ml straws using nitrogen vapours for 7 minutes and then immersed in liquid nitrogen. No statistical differences were found between treatments in all the characteristics of the refrigerated semen. The post-thawing results revealed significant improvements in the cholesterol treatments, obtaining a total motility of 67.73, 79.59 and 74.73%; membrane integrity of 46.89, 60.43 and 55.06%, and vitality of 63.26, 75.84 and 70.53% for T1, T2 and T3, respectively (p<0.05). There were no significant differences between treatments with respect to morphological abnormalities.

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          The hypoosmotic swelling test: Its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane.

          The objective of this study was to determine the effectiveness of the hypoosmotic swelling (HOS) test together with the supravital test as a means of evaluating the functional integrity of frozen-thawed bovine sperm membrane. A solution consisting of equal parts of fructose and sodium citrate was prepared and the osmolality varied from 50 to 300 mOsm/L. From these various solutions under study, the 100 mOsm/L solution resulted in a maximal number of clearly identifiable swollen spermatozoa. The results from the supravital test indicated that the HOS solution preserved the integrity and prevented excessive lysis of the sperm membrane during the assay. A good correlation was found between the percentage of motile spermatozoa and spermatozoa that reacted to the HOS test (r = 0.73) and between the percentage of sperm with intact membranes and HOS reactive sperm (r = 0.81). Spermatozoa showing swelling of the entire tail region accounted for more than 60% of the total swelling for the HOS solution at 100 mOsm/L. The results obtained in this study indicate that frozen-thawed bovine spermatozoa did react to the HOS test. This technique could prove useful in studies involving the function of the sperm membrane and could possibly predict the sperm's ability to fertilize.
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            Effects of cryopreservation procedures on sperm membranes.

            Empirical approaches to semen cryopreservation have resulted in the production of young in a broad range of species. However, acceptable levels of fertility in most domestic animal species has not been achieved. In this review, an attempt has been made to describe the complexity of the sperm plasma membrane and the many steps in a cryopreservation procedure where membrane perturbations can occur. Improvement in sperm cryopreservation procedures will require a careful consideration of the complexity of the sperm plasma membrane, the interaction of its components and the influence of cooling, freezing and thawing on these interactions.
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              Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival.

              Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sperm were maintained after thawing when 1.5 mg CLC was added to sperm from stallions whose sperm do not survive freezing well, compared to control sperm from those same stallions (67% vs. 50%; P<0.05). Addition of CLCs increased the percentages of membrane intact sperm surviving cryopreservation compared to untreated sperm for all stallions (P<0.05). The amount of cholesterol that incorporated into the membranes of the sperm cells increased in a polynomial fashion (R2=0.9978) and incorporated into all sperm membranes. In addition, there was a significant loss of cholesterol from sperm membranes after cryopreservation; however, addition of CLCs to sperm prior to cryopreservation maintained higher cholesterol levels in the sperm after freezing and thawing than untreated sperm (P<0.05). Addition of CLCs also resulted in more sperm binding to the zona pellucida of bovine oocytes after cryopreservation than control sperm (48 vs. 15; P<0.05). In conclusion, CLCs improved the percentage of post-thaw viability in equine sperm as well as increased the number of sperm that bind to zona pellucida. Addition of CLCs to stallion sperm prior to cryopreservation is a simple procedure that increases the cryosurvival of cells.
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                Author and article information

                Journal
                rivep
                Revista de Investigaciones Veterinarias del Perú
                Rev. investig. vet. Perú
                Universidad Nacional Mayor de San Marcos. Facultad de Medicina Veterinaria (Lima, , Peru )
                1609-9117
                October 2019
                : 30
                : 4
                : 1611-1618
                Affiliations
                [02] Lima orgnameUniversidad Nacional Agraria La Molina orgdiv1Banco Nacional de Semen Perú
                [03] Lima orgnameUniversidad Nacional Agraria La Molina orgdiv1Facultad de Zootecnia orgdiv2Departamento de Producción Animal Perú
                [01] Lima orgnameUniversidad Nacional Agraria La Molina orgdiv1Facultad de Zootecnia Perú
                Article
                S1609-91172019000400022 S1609-9117(19)03000400022
                10.15381/rivep.v30i4.17159
                f08781da-3beb-405b-a0b8-13c2a6f29aeb

                This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

                History
                : 18 January 2019
                : 01 October 2019
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 25, Pages: 8
                Product

                SciELO Peru

                Categories
                Artículos primarios

                semen,toro,ciclodextrina,colesterol,criopreservación,bull,cyclodextrin,cholesterol,cryopreservation

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