Development of a Simple Sperm Cryopreservation Model Using a Chemically Defined Medium and Goat Cauda Epididymal Spermatozoa

Kundu, C.N.; Chakraborty, J.; Dutta, P; Bhattacharyya, Dipankar; Ghosh, Amitabha; Majumder, Gopal C.

doi:10.1006/cryo.2000.2230

Cited by 41 publications

(36 citation statements)

References 28 publications

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“…In the buck, removal of seminal plasma by washing the spermatozoa immediately after collection increases the percentage of live cells and their motility during storage in egg yolk or milk diluents [6] and [22] although this effect does not always occur [7]. The storability of ejaculated and washed semen is less than that of epididymal semen [5], [14] and [19], suggesting that seminal plasma contains compounds detrimental to in vitro storage.…”

Section: Discussionmentioning

confidence: 99%

Microscopic and flow cytometric semen assessment of Dutch AI-bucks: Effect of semen processing procedures and their correlation to fertility

Lievaart‐Peterson

Kappen²,

Ursem

et al. 2007

Theriogenology

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This study was done to determine the effects of processing techniques on the quality of semen from Dutch AI-bucks with the view on improving pregnancy rates after artificial insemination (AI) with liquid or frozen-thawed semen. Motility of spermatozoa was estimated under a microscope whereas the percentage live spermatozoa and the percentage live spermatozoa with intact acrosomes were determined by means of flow cytometry. Aspects of semen processing that were investigated are storage temperature of liquid semen (i), the effect of glycerol on liquid-stored semen (ii), removal of seminal plasma (iii) and type of extender (iv). The correlation between semen quality and fertility rates in inseminated does was also investigated. openUP (July 2007)The percentage motile spermatozoa in semen stored in liquid form for 72 h progressively declined over time, irrespective of whether storage occurred at 4 or 18 °C. The percentage motile spermatozoa in semen stored at 18 °C was similar to that in semen stored at 4 °C if stored for 24 h but lower if stored for 48 h. Goats differ in the sensitivity of their spermatozoa to the deleterious effects of glycerol. Neither the removal of seminal plasma nor the type of extender had any effect on semen quality before freezing but semen frozen in a Tris-citric acid-glucose (TCG) buffer with egg yolk without removal of the seminal plasma had better quality after thawing than semen frozen in another diluent or after removal of seminal plasma.Remarkably no significant correlation between fertility and membrane integrity of spermatozoa could be found. Thus, although integrity assays for spermatozoa are useful to asses resistance to semen handling, the validity of these assays for predicting fertility is questioned.

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Section: Discussionmentioning

confidence: 99%

Microscopic and flow cytometric semen assessment of Dutch AI-bucks: Effect of semen processing procedures and their correlation to fertility

Lievaart‐Peterson

Kappen²,

Ursem

et al. 2007

Theriogenology

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“…Due to its highly hydrophilic nature and low molecular weight, DMA reduces the formation of intracellular ice crystals and increases membrane permeability, thus decreasing osmotic damage [2]. The penetration of DMSO is also rapid, due to its lower molecular weight relative to glycerol [10] and DMSO has been used to successfully cryopreserve sperm [9]. However, there are few studies on the uses of DMA and DMSO for freezing boar semen.…”

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confidence: 99%

Evaluation of Different Cryoprotectants (CPAs) in Boar Semen Cryopreservation

Kim

Lee

et al. 2011

J. Vet. Med. Sci.

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ABSTRACT. This study was performed to evaluate the use of dimethylacetamide (DMA) and dimethyl sulfoxide (DMSO) in boar sperm cryopreservation. Semen from eight boars was cryopreserved following treatment with 3, 5, and 7% DMA and DMSO, and 3% glycerol (control). After thawing, sperm conventional parameters and membrane integrities were evaluated. There were no significant differences among different DMA concentrations in all evaluations. Membrane intactness were higher in 5% and 7% DMSO than 3% DMSO (P<0.05). Sperm motility of 5% DMSO was lower than that of 3% glycerol (P<0.005), and membrane intactness were lower in 5% DMA and DMSO than 3% glycerol (P<0.05). DMA and DMSO didn't improve sperm quality and glycerol remains the most useful for boar sperm cryopreservation. Glycerol (at a concentration of 3%) is the most commonly used cryoprotectant (CPA) for boar sperm [3], but it has toxic effects as well as contraceptive effects on sperm [8]. Its detrimental effects have stimulated the study of alternative CPAs such as N,N-dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO) [2,10]. Due to its highly hydrophilic nature and low molecular weight, DMA reduces the formation of intracellular ice crystals and increases membrane permeability, thus decreasing osmotic damage [2]. The penetration of DMSO is also rapid, due to its lower molecular weight relative to glycerol [10] and DMSO has been used to successfully cryopreserve sperm [9]. However, there are few studies on the uses of DMA and DMSO for freezing boar semen. Therefore, the objective of this study was to evaluate the effectiveness of DMA and DMSO as possible replacement for glycerol in boar semen cryopreservation.Three CPAs were used in this study: glycerol, DMA, and DMSO (all from Sigma-Aldrich, St. Louis, MO, U.S.A.). Glycerol was used at a final concentration of 3% as a control, and the others at final concentrations of 3, 5, and 7%. We first compared the three concentrations of DMA and DMSO (n=5), and the concentrations showing the best result for each CPA were then compared with 3% glycerol (n=8).Ejaculate from 8 Duroc boars was collected and processed according to the straw freezing procedure [1] with some modification. Sperm-rich fractions were extended in Beltsville Thawing Solution (BTS) and were slowly cooled to 15°C for 3 hr. After centrifuging, the semen pellet was resuspended in lactose-egg yolk (LEY) extender. After further cooling to 5°C for 90 min, LEY-extended semen was aliquot for treatment with each CPAs and then two parts LEY-extended semen were mixed with one part freezing extenders (LEY extender containing 1.5% Equex STM [Nova Chemical Sales, Scituate Inc., MA, U.S.A.] and the CPAs described above, v/v). The semen was loaded into 0.5-ml straws (IMV, L'Aigle, France), and held in liquid nitrogen vapor 3 cm above the liquid for 20 min. The straws were then plunged into the liquid nitrogen. After thawing the straws at 37°C for 20 sec [5], thawed semen was evaluated for sperm motility [13], morphological defect, viability by eosin-nigrosin st...

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“…Spermatozoa were cryopreserved using the procedure reported by Kundu et al (2000). Specified concentrations of dextran (w/v) (different molecular masses), glycerol (w/v) and DMSO (w/v) were prepared by dissolving the compounds in RPS medium.…”

Section: Cryopreservation Proceduresmentioning

confidence: 99%

“…Initially, as the concentration of dextran was increased, the recovery of sperm motility also increased and reached an optimum value; however, with further increases in dextran concentration, the recovery of motility decreased sharply. Of all the sugar polymers tested, 10 kDa dextran showed the highest cryoprotecting efficacy, whereas the 2000 kDa sugar polymer was the Effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium ethylene glycol (1.29 mol l -1 ) was 20% and 13%, respectively (Kundu et al, 2000). Some amino acids (for example L-alanine, glycine, L-glutamine and L-proline) offered cryoprotection (recovery of motility 10-19%) in the range 100-150 mmol l -1 .…”

mentioning

confidence: 98%

“…

A simple sperm cryopreservation model has been developed using a chemically defined medium (modified Ringer's solution; RPS) and mature goat spermatozoa from the cauda epididymidis as the model system (Kundu et al, 2000). The procedure is based on the systematic manipulation of different rates of cooling, freezing and maximum freezing temperature using a computer-controlled biofreezer.

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confidence: 99%

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Effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium

Kundu¹,

Chakrabarty²,

Dutta³

et al. 2002

Reproduction

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Experiments were carried out to investigate the cryoprotecting potential of dextrans (ranging from 10 to 2000 kDa) using a synthetic model system developed recently in this laboratory. Goat spermatozoa from the cauda epididymidis were extracted in a chemically defined medium (modified Ringer's solution) and assayed for motility using a phase-contrast microscope. The sperm cells were subjected to a freezing protocol in a computer-controlled biofreezer (cooling at 0.25 degrees C min(-1) to 5 degrees C; 5 degrees C min(-1) to -20 degrees C; 20 degrees C min(-1) to -100 degrees C) and plunged into liquid nitrogen. The frozen cells were thawed rapidly at 37 degrees C in a thermostatic waterbath. In the absence of dextran, all the spermatozoa lost their motility. The cryoprotecting efficacy of each dextran was found to be biphasic in nature. Initially, as the concentration of dextran was increased, the recovery of sperm motility also increased and reached an optimum value; however, with further increases in dextran concentration, the recovery of motility decreased sharply. Of all the sugar polymers tested, 10 kDa dextran showed the highest cryoprotecting efficacy, whereas the 2000 kDa sugar polymer was the least active. Dextrans of 10, 40, 73, 173, 252, 500 and 2000 kDa offered maximum cryorecovery of forward motility to the extent of approximately 23%, 21%, 19%, 18%, 16%, 15% and 8%, respectively. Optimum concentrations of these dextrans for cryoprotection of sperm motility were 8.42, 2.50, 1.09, 0.37, 0.31, 0.10 and 0.04 mmol l(-1), respectively. It thus appears that each dextran has a characteristic cryoprotection profile. The data show that the cryoprotecting efficacy and optimum cryoprotecting concentrations of dextrans are inversely related to their molecular mass. Dextran also served as a significant cryoprotectant in the presence of glycerol (0.87 mol l(-1)) and dimethyl sulphoxide (0.76 mol l(-1)), which are well known cryoprotectants; the action of the combined cryoprotectants was almost additive. The presence of glycerol or glycerol plus dimethyl sulphoxide caused a significant reduction (from 8.42 to 6.27 mmol l(-1)) in the optimum concentration of dextran. In the presence of the three cryoprotectants, recovery of sperm motility was as high as 58% (forward motility) and 60% (total motility).

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Development of a Simple Sperm Cryopreservation Model Using a Chemically Defined Medium and Goat Cauda Epididymal Spermatozoa

Cited by 41 publications

References 28 publications

Microscopic and flow cytometric semen assessment of Dutch AI-bucks: Effect of semen processing procedures and their correlation to fertility

Microscopic and flow cytometric semen assessment of Dutch AI-bucks: Effect of semen processing procedures and their correlation to fertility

Evaluation of Different Cryoprotectants (CPAs) in Boar Semen Cryopreservation

Effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium

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