Nonbeneficial effects of glycerol on the oocyte penetrating capacity of cryopreserved and incubated human spermatozoa

doi:10.1016/0011-2240(85)90154-3

Cryobiology

Volume 22, Issue 5, October 1985, Pages 434-437

https://doi.org/10.1016/0011-2240(85)90154-3 Get rights and content

Abstract

The effect of cryopreservation on human spermatozoa in the presence or absence of glycerol was assessed by using sperm motility, functional integrity of sperm membrane, and denuded hamster oocyte penetration tests. Glycerol treated cryopreserved spermatozoa yielded a significantly higher (P < 0.01) percentage of motile sperm and percentage of sperm with functionally intact membrane immediately after thawing than the spermatozoa not treated with glycerol but cryopreserved. However, no significant difference was observed between these cryopreserved spermatozoa (either treated or untreated with glycerol) on the percentage of motile sperm and the rate of oocyte penetration when the sperm were washed and incubated for 2 hr in a medium containing no glycerol. Thus, it appears glycerol may not be beneficial, since cryopreservation of spermatozoa either treated or untreated with glycerol essentially yields similar oocyte-penetrating capacity of sperm.

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Cited by (26)

The cryoprotective effect of Guar gum co-supplemented with ethylene glycol and glycerol in Simmental bull semen
2023, Animal Reproduction Science
Sperm cryopreservation often reduces sperm quality by forming of intra- and extracellular ice crystals. Various compounds widely used to counteract this effect. The guar gum was considered as an extracellular cryoprotective substance. The present study evaluated the impact of the co-supplementation of guar gum with ethylene glycol or glycerol in the cryopreservation of bull sperm. Four ejaculates from 4 bulls were pooled and divided into ten groups consisting of 4 controls (glycerol 6%, ethylene glycol 6%, glycerol 3.5%, and ethylene glycol 3.5%, and six treatment groups including guar gum in 0.001% and 0.002% alone and or co-supplemented either with 3.5% glycerol or 3.5% ethylene glycol and frozen in liquid nitrogen. The sperm motility, viability, plasma membrane and DNA integrity, apoptotic-like changes, antioxidant capacity (TAC), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities evaluated. The groups contained 3.5% glycerol + 0.001% guar gum, 3.5% ethylene glycol + 0.001% guar gum, and 0.001% guar gum alone showed higher values for live sperm, antioxidant enzymes, membrane integrity, mitochondrial membrane potential (MMP), fertilization, cleavage, and blastocyst rates; and lower values for apoptotic-like changes, H2O2 level, and DNA damage than the control groups. In conclusion, adding guar gum to the bull sperm diluent either alone or combined with glycerol or ethylene glycol ameliorated sperm viability and kinematic parameters and antioxidant capacity while reducing DNA damage and apoptotic-like changes. Guar gum also has improved embryo development. Due to its cost-effectiveness and physicochemical properties, guar gum is a promising supplement for bull sperm cryopreservation.
Comparing ethylene glycol with glycerol for cryopreservation of buffalo bull semen in egg-yolk containing extenders
2011, Theriogenology
Citation Excerpt :
However, glycerol has osmotic and toxic effects on the plasma membrane and metabolism of cryopreserved cells [12]. It is responsible for the disorganization of sperm plasma membrane [18,19] and reducing motility and fertilizing ability [20]. Higher concentrations of glycerol lead to cell death [21].
The objective of this work was to evaluate the possibility of substituting glycerol for ethylene glycol when cryopreserving buffalo semen. Semen of eight buffalo bulls was mixed, pooled, and frozen in one of these four diluents: centrifuged Tris egg yolk glycerol; centrifuged Tris egg yolk ethylene glycol; centrifuged Milk egg yolk glycerol; or centrifuged Milk egg yolk ethylene glycol. Semen quality parameters assessed after thawing were motility, survivability, livability, sperm abnormality, acrosome integrity, and plasma membrane integrity. Conception rate and pregnancy rate were calculated after insemination of 104 buffaloes by straws of different groups (26 female/extender). Improvement in livability, sperm abnormality, acrosome integrity, plasma membrane integrity, conception rate, and pregnancy rate were seen when using ethylene glycol to replace glycerol when freezing buffalo bull semen in centrifuged TRIS egg yolk 61.15 ± 0.73, 24.85 ± 0.41, 69.10 ± 0.81, 71.75 ± 0.72, 46.2%, and 46.2%, respectively, followed by centrifuged milk egg yolk extenders.
Storage of Chinchilla lanigera semen at 4°C for 24 or 72 h with two different cryoprotectants
2001, Cryobiology
The objectives of this study were to: (a) test the functional activity of Chinchilla lanigera spermatozoa suspended in either glycerol or ethylene glycol, cooled to 4°C, and stored for 24 or 72 h and (b) investigate, after these cooling periods, the effects of incubating sperm at 37°C (for 4 h) upon sperm functional activity. The ejaculate was mixed with the cryoprotectant medium (at 1 M final concentration) and cooled to 4°C. After warming, sperm motility, sperm viability, hypoosmotic swelling test results, and acrosomal integrity were significantly higher for samples containing ethylene glycol than for those in glycerol, stored for 24 or 72 h, and then assayed after 0 or 4 h incubation at 37°C. A significant reduction of sperm motility and viability was detected only when the glycerol cryoprotectant agent was employed, compared to the fresh samples. These results clearly indicate that under our experimental conditions, ethylene glycol is a better protectant for sperm storage than glycerol.
Sperm membrane functional integrity and response of frozen-thawed bovine spermatozoa during the hypoosmotic swelling test incubation at varying temperatures
1997, Theriogenology
The objective of this study was to assess the sperm membrane integrity and permeability of frozen-thawed bovine spermatozoa, processed at varying temperatures during and after thawing, by exposing the spermatozoa to standardized hypoosmotic conditions. The hypoosmotic swelling (HOS) test was employed to measure changes in sperm membrane functional status and permeability. Frozen specimens (from 5 bulls) were thawed at 37h °C for 10 sec and transferred to a water bath at 37 (Aliquot 1), 21 (Aliquot 2) or 5 °C (Aliquot 3) to complete thawing (1 to 2 min). The specimens were maintained and processed at these temperatures for additional 5 to 10 min. Specimens were slowly diluted 1:1 (v/v) and washed with Ham's F-10 media containing 3% (w/v) BSA. The HOS test was performed by adding 0.1 ml of the sperm specimen to 1.0 ml of a 100 mOsm/L HOS diluent. The following treatments were performed: 1) Aliquot 1 (control), specimens were incubated in HOS solutions at 37 °C for 5 min; 2) Aliquot 2, specimens were incubated in HOS solutions at 21 or 37 °C for 5 min; and 3) Aliquot 3, specimens were incubated in HOS solutions at 5 or 37 °C for 5 min. Samples were obtained from the sperm specimen-HOS diluent mixtures at 1 min intervals (during the 5 min incubation period), fixed and assessed for sperm swelling patterns. The sperm response to the HOS test for specimens processed at temperatures below 37 °C was higher when samples were incubated in HOS diluents at 37 °C. This finding indicates that the potential for sperm swelling (measurement of sperm membrane functional status) can be maintained when spermatozoa are processed at temperatures below 37 °C. The highest response to the HOS test was observed in spermatozoa processed at 21 °C and incubated in a HOS solution at 37 °C. The response to the HOS test was superior to the one observed in specimens maintained and processed at 37 °C throughout. Thawing of spermatozoa at 37 °C, followed by processing at 21 °C seems to reduce the negative effects associated with osmotic shock and results in the preservation of the sperm membrane functional status during the in vitro handling of frozen-thawed bovine spermatozoa.
Thawing and processing of cryopreserved bovine spermatozoa at various temperatures and their effects on sperm viability, osmotic shock and sperm membrane functional integrity
1996, Theriogenology
The objective of this study was to evaluate the effects of thawing and processing temperatures on post-thaw sperm viability, occurrence of osmotic shock and sperm membrane functional status. The occurrence of osmotic shock, characterized by increased spermatozoa with coiled tails, eventually results in reduced sperm viability and sperm membrane integrity. The effects of different thawing temperatures were assessed by thawing frozen specimens at 37, 21 or 5 °C for 1 to 2-min, followed by processing at these temperatures. A subset of frozen specimens were thawed at 37 °C for 10 to 15-sec and transferred to a water bath at 21 or 5 °C for 1 to 2-min to complete thawing, followed by processing at these temperatures. Sperm processing (washing) consisted of dilution, centrifugation and resuspension to remove glycerol from the medium and to gradually return the spermatozoa to isotonic conditions. Post-thawed specimens (0.5 mL) were slowly diluted 1:1 (v/v) at a rate of 0.1 mL/min, centrifuged, and resuspended to 0.5 mL (37 °C). Diluted specimens were equilibrated for 1 to 2-min after dilution and for 5-min after resuspension. The specimens were then incubated for 2-h (37 °C) and assessed at 60-min intervals for the percentage of motility, for progressive motility (Grades 0 to 4), for the percentage of spermatozoa with coiled tails, and for the percentage of swollen spermatozoa. The percentage of swollen spermatozoa (measurement of sperm membrane integrity) was assessed by exposing spermatozoa to a modified hypoosmotic swelling (HOS) test. The results obtained seem to indicate that physiological thawing and processing temperatures (37 °C) are required to maintain sperm motility. However, thawing and processing at lower temperatures (< 37 °C) seems to prevent the occurrence of osmotic shock and to maintain sperm membrane functional integrity. In this study, thawing at 37 °C (10 to 15-sec) and transfer to a water bath at 21 °C (1-min) to complete thawing, followed by processing at 21 °C, yielded better results in terms of increased sperm viability, reduced occurrence of osmotic shock and higher reactivity to the HOS test.
Cold- and cryopreservation of dog liver and kidney slices
1996, Cryobiology
The use of tissue slices in culture could decrease the number of animals used in health-related research and decrease experimental variation. This reduction may come about particularly if the methods of cold- and cryopreserving tissue slices are perfected, and one can conduct sequentialin vitroexperiments into xenobiotic metabolism, organ-specific toxicity, or organ-specific biochemical processes with tissue slices. With this goal in mind, dog liver and kidney slices were placed in cold storage at 0°C using Viaspan (UW), Euro–Collins (EC), Sacks + prostacyclin (SP), and V-7 (V7) cold-preservation solutions for 10 days. Viability was assessed each day by measuring K⁺content and protein synthesis after 4 h of incubation in Waymouth + 10% fetal calf serum (FCS). Dog liver slices can be cold-preserved in V7 for up to 7 days using K⁺retention as the viability criterion but only up to 4 days using protein synthesis. Dog kidney slices can be cold-preserved in UW, EC, and V7 for up to 10 days using K⁺retention, but only V7 could maintain protein synthesis for 10 days. Cryopreserved dog liver and kidney slices retained 63–68% of control viability after 4 h of incubation in FCS. The cryopreservation regimen included using 10% dimethyl sulfoxide in FCS as the cryoprotectant, a freezing rate of 0.5°C/min for liver slices and 12°C/min for kidney slices, and thawing in 37°C FCS. Continued development of cold- and cryopreserving tissue slices could reduce the numbers of animals used and provide accurate and reproducible data.

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²: Present address: Department of Obstetrics & Gynecology, University of Bonn, S-Frend-Str 25, Bonn, F.R. Germany.

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Nonbeneficial effects of glycerol on the oocyte penetrating capacity of cryopreserved and incubated human spermatozoa

Abstract

Urology

Fertil. Steril

Fertil. Steril

Fundamentals of the preservation of spermatozoa

Transport and survival of spermatozoa in the female reproductive tract

Ultrastructural categorization of human spermatozoa cryopreserved in glycerol and in TESTCY

Comparison of glycerol and a zwitter ion buffer system as a cryoprotective media for human spermatozoa: Effect on motility, penetration of zona-free hamster oocytes and acrosin/proacrosin

J. Androl

Effect of glycerol and cryopreservation on oocyte penetration by human spermatozoa

Andrologia

Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics

J. Reprod. Fertil