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Fulltext Effects of cysteine and ascorbic acid in freezing extender on sperm characteristics and level of enzymes in post-thawed stallion semen

Alamaary MS, Haron AW, Hiew MWH, Ali M

Vet Med Sci, 2020 11;6(4):666-672.
PMID: 32602662 DOI: 10.1002/vms3.315

Present study aimed to investigate the effect of adding antioxidants, cysteine and ascorbic acid on the levels of glutamic oxaloacetic transaminase (GOT), glutamic-pyruvate (GPT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (GGT) enzymes of post-thawed stallion sperm. Ten ejaculates were collected each from four healthy stallions and cryopreserved using HF-20 freezing extender containing either 0 mg/ml cysteine or ascorbic acid, 0.5 mg/ml cysteine and 0.5 mg/ml ascorbic acid. All samples in freezing extender containing cysteine or ascorbic acid or none of them were assessed for sperm motility, viability, plasma membrane integrity, morphology and enzymes concentration. The ALP, LDH and GGT were significantly higher in 0-group compared with cysteine and ascorbic acid groups. The sperm motility of frozen-thawed semen with 0-group was significantly better compared with cysteine and ascorbic acid groups. The variation on viability, sperm membrane integrity and morphology were insignificant between all treated groups. Therefore, these enzymes were reduced when using antioxidants in the freezing extender. Results of the present study suggest that concentration of ALP, LDH and GGT enzymes could be used as parameters for prediction of frozen-thawed stallion semen.

Matched MeSH terms: Cryoprotective Agents/pharmacology*
Comparative characteristics of spermatozoa harvested and cryopreserved in culture and cryoprotectant media with or without donor serum proteins

Ata'Allah GA, Adenan NAM, Razali N, Palaniappan K, Saad R, Idris SK, et al.

Reprod Biol, 2017 Jun;17(2):172-179.
PMID: 28511996 DOI: 10.1016/j.repbio.2017.04.004

The objectives of this study is to evaluate the efficacy of protein-free media in the preparation, holding and crypreservation of spermatazoa for use in ART. Normozoospermic semen samples (N=71) were used to compare the effects of media on the survival and quality of spermatozoa when washed and cultured with different media with and without added proteins at 4°C, 15°C, 22°C and 37°C for 0, 4-7 and 24h. Survival and quality of spermatozoa were assessed after freeze-thaw with synthetic cryoprotectant with and without proteins. Ethics/IRB approval was obtained (Ref. 1073.52). Spermatozoa parameters were similar in all media after washing and culture for 24h. Post-thaw survival and quality of spermatozoa was not significantly different 24h after thawing of samples frozen in all cryoprotectant medium. In conclusion synthetic protein-free culture and cryoprotectant media are equal in efficacy to protein-containing media in culture and cryopreservation of spermatozoa . Use of these synthetic media are anticipated to significantly reduce the risk, potentially associated with conventional protein-containing media, of transmission of disease and possibly harmful undeclared proteins to the patient, baby and the healtcare worker. Synthetic media also ensure consistency of quality between batches of media.

Matched MeSH terms: Cryoprotective Agents/pharmacology*
Impact of Eurycoma longifolia extract on DNA integrity, lipid peroxidation, and functional parameters in chilled and cryopreserved bull sperm

Baiee FH, Wahid H, Rosnina Y, Ariff O, Yimer N, Jeber Z, et al.

Cryobiology, 2018 02;80:43-50.
PMID: 29269043 DOI: 10.1016/j.cryobiol.2017.12.006

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental-Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris-egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen-thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p 

Matched MeSH terms: Cryoprotective Agents/pharmacology*
Cryopreserved mesenchymal stromal cell treatment is safe and feasible for severe dilated ischemic cardiomyopathy

Chin SP, Poey AC, Wong CY, Chang SK, Teh W, Mohr TJ, et al.

Cytotherapy, 2010;12(1):31-7.
PMID: 19878080 DOI: 10.3109/14653240903313966

Bone marrow (BM) mesenchymal stromal cells (MSC) represent a novel therapy for severe heart failure with extensive myocardial scarring, especially when performed concurrently with conventional revascularization. However, stem cells are difficult to transport in culture media without risk of contamination, infection and reduced viability. We tested the feasibility and safety of off-site MSC culture and expansion with freeze-controlled cryopreservation and subsequent rapid thawing of cells immediately prior to implantation to treat severe dilated ischemic cardiomyopathy.

Matched MeSH terms: Cryoprotective Agents/therapeutic use
Fulltext Cytoprotective and enhanced anti-inflammatory activities of liposomal piroxicam formulation in lipopolysaccharide-stimulated RAW 264.7 macrophages

Chiong HS, Yong YK, Ahmad Z, Sulaiman MR, Zakaria ZA, Yuen KH, et al.

Int J Nanomedicine, 2013;8:1245-55.
PMID: 23569374 DOI: 10.2147/IJN.S42801

Liposomal drug delivery systems, a promising lipid-based nanoparticle technology, have been known to play significant roles in improving the safety and efficacy of an encapsulated drug.

Matched MeSH terms: Cryoprotective Agents/pharmacology*; Cryoprotective Agents/chemistry
Cryopreservation of Citrus madurensis embryonic axes by vitrification: importance of loading and treatment with vitrification solution

Cho EG, Hor YL, Kim HH, Rao VR, Engelmann F

Cryo Letters, 2002 Sep-Oct;23(5):317-24.
PMID: 12447491

This paper investigates the importance of loading and treatment with a vitrification solution on the survival of Citrus madurensis embryonic axes cryopreserved using a vitrification protocol. Among the seven different loading solutions tested, the solution containing 2 M glycerol + 0.4 M sucrose was the most efficient. Of the six vitrification solutions tested, the PVS2 vitrification solution, applied for 20 min at 25 degree C or for 60 min at 0 degree C, ensured the highest survival. A three-step vitrification protocol, involving the treatment of embryonic axes at 0 degree C with half strength PVS2 solution for 20 min then with full strength PVS2 for an additional 40 min was more efficient than a two-step protocol that involved treatment of axes directly with full strength PVS2 solution for 60 min. After rapid immersion in liquid nitrogen, rapid rewarming, unloading in a 1.2 M sucrose solution for 20 min, culture on solid medium with 0.3 M sucrose for 1 day and growth recovery for 4 weeks on standard medium, survival of C. madurensis embryonic axes reached 85 % following the three-step process, compared with 70 % for the two-step process.

Matched MeSH terms: Cryoprotective Agents/pharmacology*
Cryopreservation of Citrus madurensis embryonic axes encapsulation-dehydration

Cho EG, Hor YL, Kim HH, Rao VR, Engelmann F

Cryo Letters, 2002 Sep-Oct;23(5):325-32.
PMID: 12447492

In this paper, we demonstrate that C. madurensis embryonic axes can withstand cryopreservation using the encapsulation-dehydration technique. Up to 57.5 % survival was achieved using a standard encapsulation-dehydration protocol, which included pregrowth of encapsulated axes for 16 h in medium containing 0.8 M sucrose + 1 M glycerol, desiccation of beads to around 30 % moisture content (fresh weight basis) followed by rapid freezing. A slightly higher survival percentage (65 %) was obtained using a modified encapsulation-dehydration protocol, which included pretreatment of axes with 2 M glycerol + 0.6 M sucrose for 1 h, concomitantly with their encapsulation in 3 % calcium alginate beads, followed by desiccation of the beads to around 30 % moisture content.

Matched MeSH terms: Cryoprotective Agents/pharmacology
Fulltext Histological and mechanical evaluation of antifreeze peptide (Afp1m) cryopreserved skin grafts post transplantation in a rat model

Ibrahim SM, Kareem OH, Saffanah KM, Adamu AA, Khan MS, Rahman MBA, et al.

Cryobiology, 2018 06;82:27-36.
PMID: 29679551 DOI: 10.1016/j.cryobiol.2018.04.012

The objective of this study was to evaluate the use of Afp1m as a cryopreservative agent for skin by examining the transplanted skin histological architecture and mechanical properties following subzero cryopreservation. Thirty four (34) rats with an average weight of 208 ± 31 g (mean ± SD), were used. Twenty four (n = 24) rats were equally divided into four groups: (i) immediate non-cryopreserved skin autografts (onto same site), (ii) immediate non-cryopreserved skin autografts (onto different sites), (iii) skin autografts cryopreserved with glycerol for 72 h and (iv) skin autografts cryopreserved with Afp1m for 72 h at -4 °C. Rounded shaped full-thickness 1.5-2.5 cm in diameter skin was excised from backs of rats for the autograft transplantation. Non-cryopreserved or cryopreserved auto skin graft were positioned onto the wound defects and stitched. Non-transplanted cryopreserved and non-cryopreserved skin strips from other ten rats (n = 10) were allowed for comparative biomechanical test. All skin grafts were subjected to histological and mechanical examinations at the end of day 21. Histological results revealed that tissue architecture especially the epidermal integrity and dermal-epidermal junction of the Afp1m cryopreserved skin grafts exhibited better histological appearance, good preservation of tissue architecture and structural integrity than glycerolized skin. However, there was no significant difference among these groups in other histological criteria. There were no significant differences among the 4 groups in skin graft mechanical properties namely maximum load. In conclusion, Afp1m were found to be able to preserve the microstructure as well as the viability and function of the skin destined for skin transplantation when was kept at -4 °C for 72 h.

Matched MeSH terms: Cryoprotective Agents/pharmacology*
Biochemical analyses of Dendrobium Sabin Blue PLBs during cryopreservation by vitrification

James Antony JJ, Zakaria S, Zakaria R, Anak Ujang J, Othman N, Subramaniam S

Physiol Mol Biol Plants, 2019 Nov;25(6):1457-1467.
PMID: 31736548 DOI: 10.1007/s12298-019-00703-2

Dendrobium Sabin Blue is an important orchid hybrid that has been grown extensively as cut flower, potted plant and is also popular for its deep purplish blue flowers. The most efficient long term conservation method of this hybrid is through cryopreservation. Cryopreservation involving the vitrification method consists of explants exposure to highly concentrated cryoprotective solution followed by freezing rapidly in liquid nitrogen. However, these treatments involved highly concentrated cryoprotectant that could incur toxicity to the explants. Hence, cryopreservation protocol requires biochemical analyses in understanding the damages or injuries occurred during cryopreservation treatments. In this study, biochemical analyses revealed a general reduction in chlorophyll, carotenoid and porphyrin content to 0.40 µg/g F W (thawing stage), 31.50 µg/g F W unloading stage and 2230.41 µg/g F W (thawing stage), respectively in comparison to the control treatments. In addition, increased level in proline content were obtained at different cryopreservation stages with highest level (5.42 µmole/g F W) recorded at the PVS2 dehydration stage. Fluctuated outcomes were obtained in catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POX) enzyme activities in PLBs exposed to different cryopreservation stages. Lowest values recorded for CAT enzyme activity were obtained at the dehydration stage (3.94 U/g). Lowest POX enzyme activities were obtained at the dehydration (122.36 U/g) and growth recovery (106.40 U/g) stages. Additionally, lowest APX enzyme activities values were recorded at the thawing (7.47 U/g) and unloading (7.28 U/g) stages. These have contributed to low regeneration of Dendrobium Sabin Blue protocorm like bodies (PLBs) following cryopreservation. Hence, in the future experimental design, exogenous antioxidant could be included in the cryopreservation procedures to improve the existing protocol.

Matched MeSH terms: Cryoprotective Agents
Timing of The First Zygotic Cleavage Affects Post-Vitrification Viability of Murine Embryos Produced In Vivo

Jusof WH, Khan NA, Rajikin MH, Satar NA, Mustafa MF, Jusoh N, et al.

Int J Fertil Steril, 2015 07 27;9(2):221-9.
PMID: 26246881

BACKGROUND: Timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early (EC) have been shown to exhibit higher develop- mental viability compared to those that cleaved at a later period (LC). However, the vi- ability of EC embryos in comparison to LC embryos after vitrification is unknown. The present study aims to investigate the post-vitrification developmental viability of murine EC versus LC embryos.
MATERIALS AND METHODS: In this experimental study, female ICR mice (6-8 weeks old) were superovulated and cohabited with fertile males for 24 hours. Afterwards, their ovi- ducts were excised and embryos harvested. Embryos at the 2-cell stage were catego- rized as EC embryos, while zygotes with two pronuclei were categorized as LC embryos. Embryos were cultured in M16 medium supplemented with 3% bovine serum albumin (BSA) in a humidified 5% CO2atmosphere. Control embryos were cultured until the blastocyst stage without vitrification. Experimental embryos at the 2-cell stage were vitri- fied for one hour using 40% v/v ethylene glycol, 18% w/v Ficoll-70 and 0.5 M sucrose as the cryoprotectant. We recorded the numbers of surviving embryos from the control and experimental groups and their development until the blastocyst stage. Results were analyzed using the chi-square test.
RESULTS: A significantly higher proportion of EC embryos (96.7%) from the control group developed to the blastocyst stage compared with LC embryos (57.5%, P<0.0001). Similarly, in the experimental group, a significantly higher percentage of vitrified EC embryos (69.4%) reached the blastocyst stage compared to vitrified LC embryos (27.1%, P<0.0001).
CONCLUSION: Vitrified EC embryos are more vitrification tolerant than LC embryos. Prese- lection of EC embryos may be used as a tool for selection of embryos that exhibit higher developmental competence after vitrification.

Matched MeSH terms: Cryoprotective Agents
Fulltext α-Linolenic acid supplementation in BioXcell® extender can improve the quality of post-cooling and frozen-thawed bovine sperm

Kaka A, Wahid H, Rosnina Y, Yimer N, Khumran AM, Sarsaifi K, et al.

Anim. Reprod. Sci., 2015 Feb;153:1-7.
PMID: 25544152 DOI: 10.1016/j.anireprosci.2014.12.001

The present study was conducted to determine the effects of supplementing α-linolenic acid (ALA) into BioXcell(®) extender on post-cooling, post-thawed bovine spermatozoa and post thawed fatty acid composition. Twenty-four semen samples were collected from three bulls using an electro-ejaculator. Fresh semen samples were evaluated for general motility using computer assisted semen analyzer (CASA) whereas morphology and viability with eosin-nigrosin stain. Semen samples extended into BioXcell(®) were divided into five groups to which 0, 3, 5, 10 and 15 ng/ml of ALA were added, respectively. The treated samples were incubated at 37°C for 15 min for ALA uptake by sperm cells before being cooled for 2 h at 5°C. After evaluation, the cooled samples were packed into 0.25 ml straws and frozen in liquid nitrogen for 24 h before thawing and evaluation for semen quality. Evaluation of cooled and frozen-thawed semen showed that the percentages of all the sperm parameters improved with 5 ng/ml ALA supplement. ALA was higher in all treated groups than control groups than control group. In conclusion, 5 ng/ml ALA supplemented into BioXcell(®) extender improved the cooled and frozen-thawed quality of bull spermatozoa.

Matched MeSH terms: Cryoprotective Agents/pharmacology*
Alpha-linolenic acid supplementation in tris extender can improve frozen-thawed bull semen quality

Kaka A, Wahid H, Rosnina Y, Yimer N, Khumran AM, Behan AA, et al.

Reprod. Domest. Anim., 2015 Feb;50(1):29-33.
PMID: 25366298 DOI: 10.1111/rda.12445

The study was conducted to evaluate the effects of α-linolenic acid (ALA) on frozen-thawed quality and fatty acid composition of bull sperm. For that, twenty-four ejaculates obtained from three bulls were diluted in a Tris extender containing 0 (control), 3, 5, 10 and 15 ng/ml of ALA. Extended semen was incubated at 37°C for 15 min, to allow absorption of ALA by sperm cell membrane. The sample was chilled for 2 h, packed into 0.25-ml straws and frozen in liquid nitrogen for 24 h. Subsequently, straws were thawed and evaluated for total sperm motility (computer-assisted semen analysis), membrane functional integrity (hypo-osmotic swelling test), viability (eosin-nigrosin), fatty acid composition (gas chromatography) and lipid peroxidation (thiobarbituric acid-reactive substances (TBARS)). A higher (p < 0.05) percentage of total sperm motility was observed in ALA groups 5 ng/ml (47.74 ± 07) and 10 ng/ml (44.90 ± 0.7) in comparison with control (34.53 ± 3.0), 3 ng/ml (34.40 ± 2.6) and 15 ng/ml (34.60 ± 2.9). Still, the 5 ng/ml ALA group presented a higher (p < 0.05) percentage of viable sperms (74.13 ± 0.8) and sperms with intact membrane (74.46 ± 09) than all other experimental groups. ALA concentration and lipid peroxidation in post-thawed sperm was higher in all treated groups when compared to the control group. As such, the addition of 5 ng/ml of ALA to Tris extender improved quality of frozen-thawed bull spermatozoa.

Matched MeSH terms: Cryoprotective Agents*
Fulltext Pre-grafting histological studies of skin grafts cryopreserved in α helix antarctic yeast oriented antifreeze peptide (Afp1m)

Khan MS, Ibrahim SM, Adamu AA, Rahman MBA, Bakar MZA, Noordin MM, et al.

Cryobiology, 2020 02 01;92:26-33.
PMID: 31580830 DOI: 10.1016/j.cryobiol.2019.09.012

A number of living creatures in the Antarctic region have developed characteristic adaptation of cold weather by producing antifreeze proteins (AFP). Antifreeze peptide (Afp1m) fragment have been designed in the sequence of strings from native proteins. The objectives of this study were to assess the properties of Afp1m to cryopreserve skin graft at the temperature of -10 °C and -20 °C and to assess sub-zero injuries in Afp1m cryopreserved skin graft using light microscopic techniques. In the present study, a process was developed to cryopreserve Sprague-Dawley (SD) rat skin grafts with antifreeze peptide, Afp1m, α-helix peptide fragment derived from Glaciozyma antractica yeast. Its viability assessed by different microscopic techniques. This study also described the damages caused by subzero temperatures (-10 and -20 °C) on tissue cryopreserved in different concentrations of Afp1m (0.5, 1, 2, 5 and 10 mg/mL) for 72 h. Histological scores of epidermis, dermis and hypodermis of cryopreserved skin grafts showed highly significant difference (p 

Matched MeSH terms: Cryoprotective Agents
Fulltext Ascorbate peroxidase activity of Aranda Broga Blue orchid protocorm-like bodies (plbs) in response to PVS2 cryopreservation method

Khor, Soo Ping, Rahmad Zakaria, Subramaniam, Sreeramanan

Trop Life Sci Res, 2016;27(11):139-143.
MyJurnal

Throughout the cryopreservation process, plants were exposed to a series of
abiotic stresses such as desiccation and osmotic pressure due to highly concentrated
vitrification solution. Abiotic stress stimulates the production of reactive oxygen species
(ROS) which include hydrogen peroxide, superoxide radicals, and singlet oxygen. Higher
production of ROS may lead to oxidative stress which contributes to the major injuries in
cryopreserved explants. Antioxidant enzymes in plant such as ascorbate peroxidase
(APX) can protect plants from cell damage by scavenging the free radicals. This study was
determined based on APX enzyme activity of Aranda Broga Blue orchid’s protocorm-like
bodies (PLBs) in response to PVS2 (Plant Vitrification Solution 2) cryopreservation
treatments at different stages. PLBs that were precultured at 0.25 M sucrose for 3 days
were subjected to vitrification cryopreservation method. Results obtained showed that the
highest APX activity was achieved at PVS2 cryoprotectant treatment prior liquid nitrogen
(LN) storage. This phenomenon indicating that accumulation of osmotic and dehydrating
stress throughout the cryopreservation treatment resulted in oxidative burst which in turn
leads to higher APX activity in order to control the excess production of ROS. To
conclude, PVS2 treatment was revealed as the most detrimental step throughout
cryopreservation treatment. Thus, this research also suggested that exogenous
antioxidant such as ascorbic acid can be added throughout cryopreservation procedure
especially at PVS2 treatment in the future experiments to aid in regrowth of cryopreserved
explants by reducing oxidative stress.

Matched MeSH terms: Cryoprotective Agents
Enhancement of viability of a probiotic Lactobacillus strain for poultry during freeze-drying and storage using the response surface methodology

Khoramnia A, Abdullah N, Liew SL, Sieo CC, Ramasamy K, Ho YW

Anim Sci J, 2011 Feb;82(1):127-35.
PMID: 21269371 DOI: 10.1111/j.1740-0929.2010.00804.x

A rotatable central composite design (CCD) was used to study the effect of cryoprotectants (skim milk, sucrose and lactose) on the survival rate of a probiotic Lactobacillus strain, L. reuteri C10, for poultry, during freeze-drying and storage. Using response surface methodology, a quadratic polynomial equation was obtained for response value by multiple regression analyses: Y = 8.59546-0.01038 X(1)-0.09382 X(2)-0.07771 X(3)-0.054861 X(1)(2)-0.04603 X(3)(2)-0.10938 X(1)X(2). Based on the model predicted, sucrose exerted the strongest effect on the survival rate. At various combinations of cryoprotectants, the viability loss of the cells after freeze-drying was reduced from 1.65 log colony forming units (CFU)/mL to 0.26-0.66 log CFU/mL. The estimated optimum combination for enhancing the survival rate of L. reuteri C10 was 19.5% skim milk, 1% sucrose and 9% lactose. Verification experiments confirmed the validity of the predicted model. The storage life of freeze-dried L. reuteri C10 was markedly improved when cryoprotectants were used. At optimum combination of the cryoprotectants, the survival rates of freeze-dried L. reuteri C10 stored at 4°C and 30°C for 6 months were 96.4% and 73.8%, respectively. Total viability loss of cells which were not protected by cryoprotectants occurred after 12 and 8 weeks of storage at 4°C and 30°C, respectively.

Matched MeSH terms: Cryoprotective Agents
Effect of type and concentration of cryoprotectant on the motility, viability, and fertility of climbing perch Anabas testudineus Bloch, 1792 (Pisces: Anabantidae) sperm

Maulida S, Eriani K, Fadli N, Kocabaş FK, Siti-Azizah MN, Wilkes M, et al.

Theriogenology, 2023 Apr 15;201:24-29.
PMID: 36822040 DOI: 10.1016/j.theriogenology.2023.02.014

The climbing perch, Anabas testudineus is a freshwater fish that has economic value in Indonesia. It is cultured in the country, but the breeding technology, specifically sperm storage, is not well developed. Sperm cryopreservation is one of the preservation methods that need to be developed to support fish breeding technology. The type of cryoprotectants and its concentration are species-dependent and determines the success of this approach. Therefore, this study is aimed at determining the optimal type and concentration of cryoprotectant for sperm cryopreservation of A. testudineus. Four separate study series were performed, each of which evaluated one type of cryoprotectant at five concentration levels. The cryoprotectants used were DMSO, methanol, glycerol, and ethanol, and the tested concentrations were 0%, 5%, 10%, 15%, and 20%, which were combined with 5% egg yolks. Each treatment was conducted with three replications. The results showed that the type of cryoprotectant and its concentration significantly affected sperm motility, viability, and fertility of climbing perch (P

Matched MeSH terms: Cryoprotective Agents/pharmacology
Effect of ascorbic acid concentrations, methods of cooling and freezing on Boer goat semen cryopreservation

Memon AA, Wahid H, Rosnina Y, Goh YM, Ebrahimi M, Nadia FM

Reprod. Domest. Anim., 2013 Apr;48(2):325-30.
PMID: 22909427 DOI: 10.1111/j.1439-0531.2012.02155.x

To improve the Boer goat semen quality during cryopreservation process, three experiments were carried out to investigate the effect of (i) different concentration of ascorbic acid supplementation (ii) rate of cooling with chilled semen characteristics and (iii) method of freezing on post-thaw Boer goat sperm using Tris-based extender. Ascorbic acid at 8.5 mg/ml improved the sperm parameters (motility, integrity of membrane and acrosome, morphology and viability), compared to control in cooled samples (p < 0.05). With regard to other concentrations and post-thawed parameters, ascorbic acid at 2.5-8.5 mg/ml led to higher percentages of sperm motility and integrities of membrane and acrosome when compared to control (p < 0.05). Slow cooling rises to higher percentages of sperm motility, acrosome integrity and viability, in comparison with fast cooling, in terms of cooled and frozen samples (p < 0.05). Programmable freezing method produced the higher percentages of sperm motility, integrities of membrane and acrosome and viability when compared to the freezing method of polystyrene box during goat sperm freezing (p < 0.05). In conclusion, chilled and post-thawed sperm quality of Boer goat was improved when a Tris-based extender supplemented with ascorbic acid was used at stages of different cooling rates and freezing methods.

Matched MeSH terms: Cryoprotective Agents/pharmacology*
Effect of antioxidants on post thaw microscopic, oxidative stress parameter and fertility of Boer goat spermatozoa in Tris egg yolk glycerol extender

Memon AA, Wahid H, Rosnina Y, Goh YM, Ebrahimi M, Nadia FM

Anim. Reprod. Sci., 2012 Dec;136(1-2):55-60.
PMID: 23182473 DOI: 10.1016/j.anireprosci.2012.10.020

This study was conducted to determine the effect of antioxidants on standard semen parameters, lipid peroxidation and fertility of Boer goat semen after cryopreservation. Ejaculates from four bucks were collected, evaluated and pooled at 37°C. The pooled semen was diluted with Tris citric acid fructose for washing. Semen samples, which were diluted with a Tris-based extender containing the antioxidant ascorbic acid (8.5mg/ml), butylated hydroxytoluene (2mM), cysteine (5mM) and hypotaurine (10mM) and an extender without antioxidant supplementation were cooled to 4°C and frozen in 0.25 straws with programmable freezer and finally stored in liquid nitrogen. Data (10 replicates) were analyzed by one-way analysis of variance. Mean (±SEM) progressive motility was significantly higher in ascorbic acid than other supplement groups and control samples (P>0.05). Best values were observed in ascorbic acid followed by BHT, cysteine, and hypotaurine. Antioxidant supplementation in extender showed significant (P<0.05) better values than the control group for sperm membrane integrity, acrosome integrity and viability. The ability of antioxidants to reduce the lipid peroxidation (LPO) after freeze thawing was measured by the formation of malondialdehyde (MDA) using the thiobarbituric acid method. Results showed that addition of antioxidants significantly reduced the rate of LPO in comparison to control (P<0.05). Ascorbic acid exhibited better values (1.27±0.28), than butylated hydroxytoluene, cysteine and hypotaurine 1.32±0.42, 2.27±0.16 and 2.38±0.17 respectively, which are significantly better than control (3.52±0.54). Higher pregnancy rate was observed with ascorbic acid followed by butylated hydroxtolune, hypotaurine and cysteine. However, differences in the fertility rate were non-significant with hypotaurine, cysteine and control groups.

Matched MeSH terms: Cryoprotective Agents/pharmacology
Fulltext Vitrification of Blastocyst Murine embryos affects PI3K pathway by modulating the expression of XIAP and S6K1 proteins

Mohd Fazirul, M., Sharaniza, A.R., Norhazlin, J.M.Y., Wan Hafizah, W.J., Razif, D., Froemming, G.R.A., et al.

Pertanika Journal of Science & Technology, 2017;25(108):187-198.
MyJurnal

Cryopreservation by vitrification has been widely used in Assisted Reproductive Technology (ART) to preserve embryos for an extended period of time. However, the effect of vitrification on development of the embryos is lacking. Therefore, understanding on vitrification effects on embryonic proteins, especially those involved in preimplantation development is crucial to provide high quality embryos for further usage. In this study, XIAP and S6K1 protein expressions following vitrification was investigated, since they have been implicated in diverse cellular processes including cell growth, migration, proliferation, differentiation, survival and development of preimplantation embryos via the PI3K pathway. Embryos were obtained from superovulated female ICR mice which were mated with fertile males. The embryos were harvested at the 2-cell stage and cultured until blastocyst stage. Blastocysts were then vitrified in ESF40 cryoprotectant. Western blot was carried out to determine the expression of XIAP and S6K1 proteins. The results showed the expression of XIAP and S6K1 significantly decreased in vitrified blastocyst compared to the control. This indicates that blastocyst vitrification may impact developmental competence through the activation of apoptotic pathways.

Matched MeSH terms: Cryoprotective Agents
Preliminary studies on cryopreservation of snakehead (Channa striata) embryos

Mohd Sharifuddin M, Siti Azizah MN

Cryobiology, 2014 Aug;69(1):1-9.
PMID: 24726775 DOI: 10.1016/j.cryobiol.2014.04.001

This paper reports the findings of the ongoing studies on cryopreservation of the snakehead, Channa striata embryos. The specific objective of this study was to collect data on the sensitivity of C. striata embryo hatching rate to low temperatures at two different developmental stages in the presence of four different cryoprotectants. Embryos at morula and heartbeat stages were selected and incubated in 1M dimethyl sulfoxide (Me2SO), 1M ethylene glycol (EG), 1M methanol (MeOH) and 0.1M sucrose solutions at different temperatures for a period of time. Embryos were kept at 24 °C (control), 15 °C, 4 °C and -2 °C for 5 min, 1h and 3h. Following these treatments, the embryos were then transferred into a 24 °C water bath until hatch to evaluate the hatching rate. The results showed that there was a significant decrease of hatching rate in both developmental stages following exposure to 4 °C and -2 °C at 1h and 3h exposure in each treatment. Heartbeat stage was more tolerant against chilling at -2 °C for 3h exposure in Me2SO followed by MeOH, sucrose and EG. Further studies will be conducted to find the best method to preserve embryos for long term storage.

Matched MeSH terms: Cryoprotective Agents/pharmacology*

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