Dataset Information

Effect of controlled and uncontrolled cooling rate on motility parameters of cryopreserved ram spermatozoa.

ABSTRACT:

Background

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including freezing temperature. The aim of this study was to determine the effects of two cooling method (controlled-rate and uncontrolled-rate) on pre-freezing and post-thaw sperm motility parameters.

Results

Ejaculates were collected using the artificial vagina from four Chal rams and three replicates of the ejaculates were diluted with a Tris-based extender and packed in 0.25 ml straws. Then, sample processed according to the two methods. Method 1: straws cooled from 37 to 5°C, at a liner rate of -0.3°C/min in a controlled-rate cooling machine (custom-built) and equilibrated at 5°C for 80 min, then the straws were frozen at rate of -0.3°C/min from 5°C to -10°C and -25°C/min from -10°C to -150°C and plunged into liquid nitrogen for storage. Method 2: straws were transferred to refrigerator and maintained at 5°C for 3 h, then the straws were frozen in liquid nitrogen vapor, 4 cm above the liquid nitrogen for 15 min and plunged into liquid nitrogen. Computer-assisted sperm motility analysis was used to analyze sperm motion characteristics.

Conclusions

Controlled rate of freezing (Method 1) significantly improve the pre-freezing and post-thaw total and progressive motility compared to uncontrolled rate (Method 2). In specific kinetic parameters, Method 1 gives significantly higher value for VSL and VCL in comparison with Method 2. There are no significant differences between the two methods for VAP and LIN. In conclusion, controlled rate of cooling conferred better cryopreserving ability to ram spermatozoa compared to uncontrolled rate of cooling prior to programmable freezing.

SUBMITTER: Ashrafi I

PROVIDER: S-EPMC3349620 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Publications

Effect of controlled and uncontrolled cooling rate on motility parameters of cryopreserved ram spermatozoa.

Ashrafi Iraj I Kohram Hamid H Naijian Hamid H Bahreini Majid M Mirzakhani Hamid H

BMC research notes 20111220

<h4>Background</h4>Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including freezing temperature. The aim of this study was to determine the effects of two cooling method (controlled-rate and uncontrolled-rate) on pre-freezing and post-thaw sperm motility parameters.<h4>Results</h4>Ejaculates were collected using the artificial vagina from four Chal rams and three replicates of t ...[more]

PMID: 22185483

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Project description:BackgroundAlthough sperm cryopreservation has been widely used in human reproductive medicine as an integral infertility management in infertility clinics and for banking sperm in sperm banks, the freezing/thawing protocols are not optimal. The freezing and thawing processes result in changes at both structural and molecular levels, some even detrimental, in human sperm when compared with fresh sperm. The change of sperm proteins after cryopreservation may play negative roles for fertilization and early embryo development. Conventionally, cryostraws (CS) and cryovials (CV) are the most widely used cryopreservation carriers (CPCs) for human sperm cryopreservation accompanied with the use of egg yolk free commercial media. However, the influence of cryopreservation on the proteomic profile of human sperm preserved with the two CPCs is unknown. Therefore the purpose of the present study was to compare the frozen-thawed motility, investigate the proteomic profile of human sperm cryopreserved with the two types of CPCs, and identify the susceptible proteins that play key roles for sperm function and fertility.MethodsThe present study compared the cryosurvival of human sperm frozen with the two different CPCs and identified the sperm proteomic changes by using the isobaric tags for relative and absolute quantification labeling technique coupled with 2D LC-MS/MS analysis after freezing and thawing.ResultsOur results indicated that sperm cryopreserved with CV showed higher values for percentage of motile sperm and forward activity rate than those with CS. Compared to fresh sperm, 434 and 432 proteins were differentially identified in human sperm cryopreserved with CS and CV, respectively.ConclusionThe proteomic profiles of human sperm are greatly affected by cryopreservation with either type of CPC. GO analysis revealed that most of the differentially identified sperm proteins enriched in the extracellular membrane-bounded organelles, cytoplasm and cytosol. In addition, 106 susceptible proteins having known identities related to sperm functions were identified. In general, cryovial seems to be the preferred CPC for human sperm cryopreservation based on the post-thaw motility parameters and the effect on sperm proteomic profiles. These results are beneficial for the insight into the understanding of the cryoinjury mechanism of sperm and the development of human sperm cryopreservation strategies.

Project description:Graphical abstractAbstractSperm cryopreservation is a valuable tool for the long-term preservation of male fertility. Thus, determining the optimal technique for isolating spermatozoa post-thaw is vital to ensure recovery of the highest quality spermatozoa with minimal iatrogenic damage. This not only enhances the chances of successful conception but also reduces the risk of genetic damage in the embryo. To address this issue, human semen samples were cryopreserved using a slow freezing protocol and Quinn's Advantage™ Sperm Freeze medium. The samples were subsequently thawed and subjected to three types of sperm isolation procedures: direct swim-up, density gradient centrifugation, and electrophoretic separation using the Felix™ device. Cryopreservation led to the anticipated loss of sperm motility and vitality in association with increases in lipid peroxidation and DNA damage. Following sperm selection, all three isolation techniques resulted in an increase in sperm motility which was particularly evident with the swim-up and Felix™ procedures. The latter also significantly improved sperm vitality. There were no differences between sperm separation techniques with respect to morphology, and mitochondrial reactive oxygen species generation remained essentially unchanged when cell vitality was taken into account. By contrast, major differences were observed in DNA integrity and lipid aldehyde formation, where Felix™ isolated cells exhibiting significantly less DNA damage than the other isolation procedures as well as lower levels of 4-hydroxynonenal formation. Electrophoretic sperm isolation, therefore, offers significant advantages over alternative separation strategies, in terms of the quality of the gametes isolated and the time taken to achieve the isolation.Lay summaryLong-term storage of sperm is vital to assisted reproductive technology because it permits the preservation of fertility that might be compromised as a result of factors such as chemotherapy or vasectomy. This goal can be achieved via cryopreservation - the freezing of cells to -196°C. When the sperm are subsequently required for conception, they must be carefully separated from the cryopreservation medium in a manner that maximizes the chances of successful conception and minimizes the risk of genetic defects in the offspring. In this paper, three isolation techniques were compared for their ability to separate ideal sperm from semen and media following cryopreservation. It was found that cryopreservation led to lower levels of motility and vitality and created higher levels of DNA and cell membrane damage. Of the three techniques compared, only cells separated on the basis of their size and electric charge (electrophoretic isolation) exhibited significantly lower levels of DNA fragmentation.

Project description:A hallmark of prion diseases or transmissible spongiform encephalopaties is the conversion of the cellular prion protein (PrP(C)), expressed by the prion gene (prnp), into an abnormally folded isoform (PrP(Sc)) with amyloid-like features that causes scrapie in sheep among other diseases. prnp together with prnd (which encodes a prion-like protein designated as Doppel), and prnt (that encodes the prion protein testis specific--Prt) with sprn (shadow of prion protein gene, that encodes Shadoo or Sho) genes, constitute the "prion gene complex". Whereas a role for prnd in the proper functioning of male reproductive system has been confirmed, the function of prnt, a recently discovered prion family gene, comprises a conundrum leading to the assumption that ruminant prnt is a pseudogene with no protein expression. The main objective of the present study was to identify Prt localization in the ram reproductive system and simultaneously to elucidate if ovine prnt gene is transcribed into protein-coding RNA. Moreover, as Prt is a prnp-related protein, the amyloid propensity was also tested for ovine and caprine Prt. Recombinant Prt was used to immunize BALB/c mice, and the anti-Prt polyclonal antibody (APPA) immune response was evaluated by ELISA and Western Blot. When tested by indirect immunofluorescence, APPA showed high avidity to the ram sperm head apical ridge subdomain, before and after induced capacitation, but did not show the same behavior against goat spermatozoa, suggesting high antibody specificity against ovine-Prt. Prt was also found in the testis when assayed by immunohistochemistry during ram spermatogenesis, where spermatogonia, spermatocytes, spermatids and spermatozoa, stained positive. These observations strongly suggest ovine prnt to be a translated protein-coding gene, pointing to a role for Prt protein in the ram reproductive physiology. Besides, caprine Prt appears to exhibit a higher amyloid propensity than ovine Prt, mostly associated with its phenylalanine residue.

Dataset Information

Effect of controlled and uncontrolled cooling rate on motility parameters of cryopreserved ram spermatozoa.

Background

Results

Conclusions

Publications

Effect of controlled and uncontrolled cooling rate on motility parameters of cryopreserved ram spermatozoa.

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