70 EFFECT OF CRYOPROTECTANT CONCENTRATION ON THE IN VITRO SURVIVAL AND CELL PROLIFERATION OF PORCINE BLASTOCYSTS VITRIFIED USING THE OPEN PULLED STRAW SYSTEM

2008 ◽  
Vol 20 (1) ◽  
pp. 116
Author(s):  
C. Cuello ◽  
J. Sanchez-Osorio ◽  
C. Almiñana ◽  
M. A. Gil ◽  
I. Caballero ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Survival Rate ◽  
Nuclear Antigen ◽  
Proliferation Index ◽  
Hatching Rate ◽  
Hoechst 33342 ◽  
Number Range ◽  
Significant Difference ◽  
In Vitro Survival

The objective of the present project was to study the effect of the concentrations of ethylene glycol (EG) and dimethyl sulfoxide (DMSO) during vitrification on the survival and hatching rates of porcine blastocysts. Embryos were collected by laparotomy from weaned crossbred sows (n = 18), vitrified, and warmed (one-step dilution) as described by Cuello et al. (2004 Theriogenology 62, 1144–1152). Vitrification was performed in different concentrations of EG and DMSO (15%, 16%, and 17% v/v for each cryoprotectant) or in an EG-based medium (40% v/v) using superfine open pulled straws. Fresh and vitrified blastocysts were cultured for 24 h in TCM199 and assessed by stereomicroscopy during the culture. Blastocysts that reformed their blastocoelic cavities after warming, displaying a normal zona pellucida and excellent appearance, were considered viable. The in vitro survival rate was defined as the ratio of viable embryos divided by the total number of embryos cultured. The hatching rate is determined as the ratio of the number of embryos hatched in vitro to the total number embryos cultured. Some vitrified and fresh embryos classified as viable were processed for Hoechst 33342 staining and proliferation cell nuclear antigen (PCNA) inmunolocalization. The proliferation index was defined as the number of PCNA-positive nuclei divided by the total number of nuclei stained with Hoechst 33342. Data were analyzed by ANOVA using the MIXED procedure (SPSS version 11.5; SPSS, Inc., Chicago, IL, USA). Data were expressed as mean values � SEM. The survival rate was similar for fresh and vitrified blastocysts, except for blastocysts vitrified using 15% cryoprotectants, which displayed a lower (P < 0.05) survival rate (84.2 � 4.8%) than fresh blastocysts (94.6 � 5%) and blastocysts vitrified using 40% EG, 16%, or 17% EG-DMSO (88.8 � 4.9, 96.8 � 4.9, and 96.4 � 5%, respectively). Fresh blastocysts showed a higher (P < 0.05) hatching rate (80.7 � 4.5%) than their vitrified counterparts (range: 48.4 � 7.7–55.3 � 7.8%). Vitrified and fresh blastocysts showed similar cell proliferation indexes (range: 75.8 � 3.2–83.7 � 3). When only hatched blastocysts among groups were compared, the proliferation rate decreased (P < 0.05) after vitrification with 17% EG-DMSO. Among vitrification groups, there was no significant difference in the number of total cells. However, vitrified blastocysts had a lower (P < 0.05) total cell number (range: 116.6 � 6.7–124.8 � 6.6) than fresh blastocysts (195.5 � 11.4). In conclusion, under our experimental conditions, the concentration of EG-DMSO can be decreased until 16% in the vitrification medium with no reduction of the in vitro developmental ability of the blastocysts. In addition, a 40% EG-based medium can be used for vitrification with results similar to those achieved using a medium containing 16% EG-DMSO.

scholarly journals High in vitro survival rate of sheep in vitro produced blastocysts vitrified with a new method and device

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sergio Ledda ◽  
Jen M. Kelly ◽  
Stefano Nieddu ◽  
Daniela Bebbere ◽  
Federica Ariu ◽  
...  
Keyword(s):  
Survival Rate ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
High Survival ◽  
Apoptotic Cells ◽  
Embryo Survival ◽  
New Device ◽  
In Vitro Survival

Abstract Background To advance the use of embryo vitrification in veterinary practice, we developed a system in which embryo vitrification, warming and dilution can be performed within a straw. Ovine in vitro produced embryos (IVEP) were vitrified at either early (EBs: n = 74) or fully expanded blastocyst stage (FEBs: n = 195), using a new device named “E.Vit”, composed by a 0.25-mL straw with a 50-μm pore polycarbonate grid at one end. Embryos at each stage (EBs and FEBs) were vitrified by either Two-step (TS) or Multi-step (MS; 6 different concentrations of vitrification solutions) protocol. Non-vitrified embryos (n = 102) were maintained in in vitro culture as a control. Warming consisted of placing the straws directly into 1.5 mL tubes containing a TCM-199 solution with three decreasing concentrations of sucrose. Blastocyst re-expansion, embryo survival and hatching rate were evaluated at 2, 24 and 48 h post warming. The number of apoptotic cells was determined by TUNEL assay. Results Blastocyst re-expansion (2 h) after warming was higher (P < 0.05) in FEBs group, vitrified with the MS and TS methods (77.90% and 71.25%, respectively) compared with the EBs group (MS: 59.38% and TS: 48.50%, respectively). Survival rates of vitrified FEBs after 24 h IVC were higher (P < 0.001) in both methods (MS and TS) than vitrified EBs (MS: 56.25%; TS: 42.42%) and was higher (P < 0.05) in the MS method (94.19%) compared with those in TS (83.75%). After 48 h of culture the hatching rate for FEBs vitrified in MS system (91.86%) was similar to control (91.89%), but higher than FEB TS (77.5%) and EBs vitrified in MS (37.5%) and TS (33.33%). Number of apoptotic cells were higher in EBs, irrespective of the system used, compared to FEBs. The number of apoptotic cells in FEBs vitrified with MS was comparable to the control. Conclusions A high survival rate of IVP embryos can be achieved by the new “E.Vit” device with hatching rates in vitro comparable with control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

scholarly journals Thalidomide Reduces Cell Proliferation in Endometriosis Experimentally Induced in Rats

2019 ◽  
Vol 41 (11) ◽  
pp. 668-672
Author(s):  
Luana Grupioni Lourenço Antônio ◽  
Julio Cesar Rosa-e-Silva ◽  
Deborah Juliani Machado ◽  
Andrezza Telles Westin ◽  
Sergio Britto Garcia ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Uterine Horn ◽  
Nuclear Antigen ◽  
Proliferation Index ◽  
Lesion Area ◽  
Group Iii ◽  
Group I ◽  
Significant Difference ◽  
Female Wistar Rats ◽  
Experimentally Induced

Abstract Objective To analyze the effect of thalidomide on the progression of endometriotic lesions experimentally induced in rats and to characterize the pattern of cell proliferation by immunohistochemical Proliferating Cell Nuclear Antigen (PCNA) labeling of eutopic and ectopic endometrium. Methods Fifteen female Wistar rats underwent laparotomy for endometriosis induction by resection of one uterine horn, isolation of the endometrium and fixation of a tissue segment to the pelvic peritoneum. Four weeks after, the animals were divided into 3 groups: control (I), 10mg/kg/day (II) and 1mg/kg/day (III) intraperitoneal thalidomide for 10 days. The lesion was excised together with the opposite uterine horn for endometrial gland and stroma analysis. Eutopic and ectopic endometrial tissue was submitted to immunohistochemistry for analysis of cell proliferation by PCNA labeling and the cell proliferation index (CPI) was calculated as the number of labeled cells per 1,000 cells. Results Group I showed a mean CPI of 0.248 ± 0.0513 in the gland and of 0.178 ± 0.046 in the stroma. In contrast, Groups II and III showed a significantly lower CPI, that is, 0.088 ± 0.009 and 0.080 ± 0.021 for the gland (p < 0.001) and 0.0945 ± 0.0066 and 0.075 ± 0.018 for the stroma (p < 0.001), respectively. Also, the mean lesion area of Group I was 69.2 mm2, a significantly higher value compared with Group II (49.4 mm2, p = 0.023) and Group III (48.6 mm2, p = 0.006). No significant difference was observed between Groups II and III. Conclusion Thalidomide proved to be effective in reducing the lesion area and CPI of the experimental endometriosis implants both at the dose of 1 mg/kg/day and at the dose of 10 mg/kg/day.

122 THE SURVIVAL RATE AND EMBRYONIC QUALITY OF BOVINE PARTHENOGENETIC BLASTOCYSTS POST-VITRIFICATION CRYOPRESERVATION

2010 ◽  
Vol 22 (1) ◽  
pp. 220
Author(s):  
P. Zhang ◽  
Z. W. Wang ◽  
B. Tang ◽  
J. B. Zhang ◽  
Z. Y. Li
Keyword(s):  
Survival Rate ◽  
Sucrose Solution ◽  
Polar Body ◽  
Cell Counting ◽  
Differential Staining ◽  
Hatching Rate ◽  
First Polar Body ◽  
Significant Difference

Cryopreservation of oocytes and embryos is very useful to conservation of animal genetic resources. Recently, parthenogenesis has received considerable attention as a tool for the production of stem cells. Oocytes and embryos undergo considerable morphological changes and functional damage during cryopreservation, and the survival rate is highly depending on species and developmental stage of oocytes and embryos. The aim of this study was to investigate the survival rate and embryonic quality of bovine parthenogenetic blastocysts post-vitrification cryopreservation. Cumulus-oocyte complexes (COC) from slaughterhouse ovaries were aspirated from 2-mm to 8-mm visible follicles with a 5-mL syringe. The COC were matured in vitro for 22 h in bicarbonate-buffered TCM199 media supplemented with 1 mg mL-1 of FSH, 10 mg mL-1 of LH, 1 mg mL-1 of 17-βiestradiol, and 10% FBS. After in vitro maturation, cumulus cells were removed from COC, oocytes with first polar body were activated by 5 μM ionomycin for 5 min and 2 mM 6-DMAP for 4 h. Subsequently, oocytes were co-cultured with bovine fetal fibroblast cells in SOF media supplemented with amino acids (1% NEAA and 2% EAA), 4 mg mL-1 of BSA, and 10% FBS at conditions of 38.5°C and 5% CO2 for 7 to 9 days. The good expanded blastocysts were selected and refrigerated in different vectors [glass micropipettes (GMP) and straws] and same vitrification solution (VS, 20% EG + 20% DMSO). Blastocysts were exposed to VS, loaded on vectors, and plunged into liquid nitrogen within 25 s. After two days refrigeration, vitrified blastocysts were thawed in air for 10 s and placed into 0.25 M sucrose solution for 1 min and 0.15 M sucrose solution for 5 min. Then, the blastocysts were cultured in the SOF medium same as above. Our results showed that when VS was 20% EG + 20% DMSO, the hatching rate (65%) of blastocysts loaded into GMP was significantly higher (P < 0.05) than that (19%) of blastocysts loaded into straws post-vitrification. Meanwhile, vitrified and nonvitrified blastocysts were fixed and stained for differential cell counting as described by Thouas GA et al. 2001 Reprod. Biomed. Online 3, 25-29). By the differential staining, the total cells of nonvitrified parthenogenetic bovine blastocysts were 102.7, which was higher (P > 0.05) than that (86.7) of vitrified blastocysts. Also, no significant difference (P > 0.05) was seen on ratios between vitrified blastocysts (ICM/TE = 0.22) and nonvitrified blastocysts (ICM/TE = 0.25). Our results indicated that a glass micropipette vector was much better than a straw in vitrification cryopreservation of bovine parthenogenetic blastocysts and caused less damage to blastocyst cells. This study lays the foundation for further research to increase the survival rate of vitrification cryopreservation of bovine embryos. This work was supported by the grant from national support plan, China, No. 2007BAD55B03; corresponding author: Ziyi Li.

scholarly journals The Effectiveness of Candidate Probiotic Bacteria to Control Vibriosis Disease

2017 ◽  
Vol 5 (2) ◽  
pp. 1
Author(s):  
Mulyati Mulyati ◽  
Suryati Suryati ◽  
Irfani Baga
Keyword(s):  
Survival Rate ◽  
Sea Water ◽  
Challenge Test ◽  
Probiotic Bacteria ◽  
Pathogenicity Test ◽  
Inhibitory Ability ◽  
Significant Difference ◽  
Portunid Crab

The study aims to isolate, characterize, and examine probiotic bacteria's inhibitory ability against Vibrio harveyi bacteria, both in-vitro and in vivo. Methods used in the study consist of 1) An Isolation of Candidate Probiotic Bacteria, 2) An Antagonistic Test of Candidate Probiotic Bacteria in vitro, 3) An Identification of Bacteria, 4) A Pathogenicity Test of Candidate Probiotic Bacteria, 5) An Antagonistic Test of Candidate Probiotic Bacteria against V. harveyi in vivo. According to the isolation of candidate probiotic bacteria, there are 18 isolated candidate probiotic. After being tested for its inhibitory ability in vitro, there are 8 isolates with zone of inhibition as follows: isolate MM 7 from intestine (22 mm), isolate MM 6 from intestine (12 mm), isolate MM 10 from sea water (10 mm), isolate MM 5 from intestine (9 mm), isolate MM 4 from intestine (8 mm), isolate MM 3 from intestine (7 mm), isolate MM 2.2 from intestine (7 mm), isolate MM 2.1 from intestine (7 mm). Eight genera of the candidate probiotic bacteria is derived from Portunid crab, they are Staphylococcus, Streptococcus, bacillus, vibrio, Alcaligenes, Lactobacillus, micrococcus. Before proceeding the V. harveyi bacterial challenge test in vivo, three potential isolates consisting of MM6, MM7 and MM10 as the probiotic bacteria are pathogenicity-tested against V. harveyi. The survival rate of Portunid crab on pathogenicity test using MM6, MM7 and MM10 generates 91.11-100%, while the control generates 100% survival rate. Variance analysis result through post-hoc Tukey's Honest Significant Difference (HSD) test at 95% confidence interval indicates that isolate MM7 and MM10 are significantly able to increase hatchling Portunid crab's survival rate.

scholarly journals Cell-free fat extract promotes tissue regeneration in a tissue expansion model

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Mingwu Deng ◽  
Xiangsheng Wang ◽  
Ziyou Yu ◽  
Yizuo Cai ◽  
Wei Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Collagen Type ◽  
Growth Factor Receptor ◽  
Tissue Expansion ◽  
Nuclear Antigen ◽  
Hacat Cells ◽  
Expansion Model

Abstract Background Tissue expansion techniques play an important role in plastic surgery. How to improve the quality of the expanded skin and shorten the expansion period are still worth investigating. Our previous studies found that a cell-free fat extract (CEFFE) possessed pro-angiogenic and pro-proliferative activities. However, the role of CEFFE on tissue expansion has remained unclear. The purpose of this study was to evaluate the effect of CEFFE on tissue expansion. Methods A rat tissue expansion model was used. Animals were treated with CEFFE by subcutaneous injection. After 4 weeks of tissue expansion, the skin necrosis and retraction rates were evaluated, the thicknesses of the epidermis and dermis were determined by histological analyses, blood vessel density was measured by anti-CD31 staining, cell proliferation was assessed by proliferating cell nuclear antigen staining, and the expression of specific proteins was evaluated by western blot analyses. In addition, the effects of CEFFE on the proliferation and cell cycle of cultured HaCaT cells were evaluated in vitro. Results CEFFE treatment significantly decreased the necrosis rate and retraction of the expanded skin. The thickness of the epidermal and dermal layers was higher in CEFFE-treated compared to untreated skin. The density of blood vessels and cell proliferation in the epidermis of the expanded skin was improved by CEFFE treatment. In addition, CEFFE treatment significantly increased the expression of the vascular endothelial growth factor receptor, epidermal growth factor receptor, collagen type 1, and collagen type 3. CEFFE also increased the proliferation of HaCaT cells in culture. Conclusions CEFFE improves the quality of the expanded skin by promoting angiogenesis and cell proliferation. It could be potentially used clinically for augmenting tissue expansion.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 57-61 ◽  
Author(s):  
M. Popelková ◽  
Z. Turanová ◽  
L. Koprdová ◽  
A. Ostró ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Significant Difference ◽  
Rabbit Embryos

SummaryThe aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

scholarly journals Effect of Cryoprotectant Gradient Equilibration Method on In Vitro Survival Rate and Pregnancy Rate of Bovine IVF Blastocysts.

2001 ◽  
Vol 18 (2) ◽  
pp. 67-71
Author(s):  
Yutaka Kajihara ◽  
Atsurou Hira ◽  
Ryouhei Nitta ◽  
Yoshihiko Nakanishi
Keyword(s):  
Survival Rate ◽  
Pregnancy Rate ◽  
In Vitro Survival

scholarly journals Effect of feeding management of broodstock on breeding performance of bata (Labeo bata)

2016 ◽  
Vol 1 (3) ◽  
pp. 553-568 ◽  
Author(s):  
Md Habibur Rahman ◽  
Md Anisur Rahman ◽  
Md Mer Mosharraf Hossain ◽  
Syeda Maksuda Yeasmin ◽  
Abdulla Al Asif
Keyword(s):  
Methylene Blue ◽  
Survival Rate ◽  
Lipid Level ◽  
Gonadosomatic Index ◽  
Dietary Factors ◽  
Hatching Rate ◽  
Breeding Performance ◽  
Fish Hatchery ◽  
Labeo Bata ◽  
Significant Difference

Labeo bata is one of the important minor carps in Bangladesh with great demand as good table fish. The study was conducted to observe the breeding performance at different management practices in Mafatema, Rupali, Modhumoti and Anan fish hatchery and disinfection treatments of water, eggs and fry in Jessore, Bangladesh during 10 March 2014 to 15 May 2015. An improvement in broodstock nutrition and feeding has been shown to greatly improve seed production. Protein and lipid percentage of broodstock diet have been identified as major dietary factors. Protein level was 24.77%, 23.47%, 18.08%, 17.78% and lipid level was 11.07%, 9.50%, 7.74%, 8.14% in Mafatema, Rupali, Modhumoti and Anan fish hatchery respectively. Three concentrations of four chemical-formalin (10, 20, 30 mg/L), malachite green (1, 3, 5 mg/L), NaCl (1, 2, 3 g/L) and methylene blue ( 1, 3, 5 mg/L) treatment regimes and a control were compared for efficacy in treating L. bata eggs to prevent fungus and bacterial infection and improve hatch and survival rate of fry. Highest correlation value between absolute fecundity and body weight (r=.938, p<.05) and total length (r=.891, p<.05) and gonadosomatic index (26.2%) were found in Mafatema fish hatchery among four experimental hatcheries at 24.77% protein and 11.07% lipid level. Better fertilization rate (84.2±5.17%) and hatching rate (82.0±4.30%) were found in Mafatema and Rupali fish hatchery respectively that has significant difference (P<0.05) from that of Modhumoti and Anan fish hatchery at higher protein and lipid level. Lowest deformity rate (6.05+2.65) was observed in Mafatema fish hatchery that was significantly different (P<0.05) from that of Modhumoti fish hatchery. In case of disinfection treatment, methylene blue at 1mg/L bath treatment daily for 4 days showed significantly higher hatching rate (92.33±3.51%) and survival rate (94.33±4.73%).Asian J. Med. Biol. Res. December 2015, 1(3): 553-568

83 VITRIFICATION OF IMMATURE AND IN VITRO-MATURED HORSE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 149 ◽  
Author(s):  
L. Bogliolo ◽  
F. Ariu ◽  
I. Rosati ◽  
M. T. Zedda ◽  
S. Pau ◽  
...  
Keyword(s):  
Survival Rate ◽  
Ethylene Glycol ◽  
Survival Rates ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hoechst 33342 ◽  
Nuclear Maturation ◽  
And Control ◽  
Cryopreservation Protocol

Few attempts have been carried out to cryopreserve equine oocytes, and an effective cryopreservation protocol is not defined yet. Studies were conducted to compare the viability of immature and in vitro-matured horse oocytes vitrified by the minimal volume cooling (MVC) cryotop vitrification method (Kuwayama et al. 2005 Reprod. BioMed. Online 11, 300–308). Oocytes were recovered from slaughterhouse ovaries and divided, on the basis of the morphology of cumulus cells, into cumulus-expanded (CE) and cumulus-compacted (CC) oocytes. Groups of CC and CE oocytes were vitrified immediately after recovery [germinal vesicle (GV) stage] or matured in vitro (IVM) and cryopreserved at the MII stage as follows: oocytes were incubated 30 min in TCM-199 + 20% FCS + 10% ethylene glycol (EG) + 10% DMSO, followed by 20 min in TCM-199 + 20% FCS + 20% EG + 20% DMSO + 0.25 M sucrose, loaded in cryotops (2 µL), and plunged into liquid nitrogen. Warming was performed at 38.5°C by washing the oocytes in TCM-199 + 20% FCS with decreasing sucrose concentrations (1.25 M, 0.62 M, 0.31 M). After warming oocytes cryopreserved at the GV stage were matured in vitro for 24 h (CE) or 36 h (CC) in TCM-199 + 10% FCS + FSH, LH each at (0.1 UI/mL) + cysteamine, fixed, and stained with glycerol-Hoechst 33342 to assess nuclear maturation. Oocytes vitrified at the MII stage were in vitro cultured for 2 h to evaluate their morphological survival on the basis of the presence of an intact zona pellucida and membrane. Nonvitrified oocytes undergoing the same maturation protocol were used as controls. Results (Table 1) indicated that the survival rate of oocytes vitrified at the GV stage, after IVM, was similar between CE and CC oocytes (43.6% vs 42.6%). Significantly (P < 0.01) higher numbers of vitrified CE MII oocytes (52.9%) survived, compared to CC (34.8%), after 2-h culture. The percentages of viable MII oocytes from CE and CC GV vitrified oocytes were 43.6% and 40.9% respectively and were comparable to those from vitrified MII oocytes (CE, 52.9%; CC, 34.8%) and control oocytes (CE, 56.4%; CC, 53.3%). In conclusion, the results of this study showed that vitrification by the MCV Cryotop method of horse oocytes at either the GV or the MII stage allows a similar number of viable mature oocytes to be recovered. Table 1. Maturation and survival rates of immature and mature equine oocytes vitrified by the MCV Cryotop method

52 EFFECT OF CARBOXYLATED POLY-L-LYSINE ON THE POST-THAW VIABILITY OF IN VITRO-PRODUCED BOVINE BLASTOCYST

2015 ◽  
Vol 27 (1) ◽  
pp. 119
Author(s):  
A. N. Ha ◽  
K. L. Lee ◽  
Md. Fakruzzaman ◽  
S. S. Kim ◽  
P. R. Park ◽  
...  
Keyword(s):  
Survival Rate ◽  
Dimethyl Sulfoxide ◽  
Exposure Time ◽  
High Efficiency ◽  
Apoptotic Cell ◽  
Osmotic Shock ◽  
Hatching Rate ◽  
Cell Numbers ◽  
Bovine Blastocyst

Recently, there has been development of an antifreeze polyamino acid (carboxylated poly-l-lysine: PLL) as new cryoprotectants (CPA). This compound counts as amphoteric macromolecular cationic and anionic substituents (polyampholyte) by chemically modifying poly-lysine. In addition, PLL is highly safe and frequently used as a food additive in substitution for other CPA. Other common CPA have high toxicity and caused physiological damage. In vitro-produced blastocysts were randomly assigned into 3 groups: (1) vitrified embryos with PLL vitrification solution [PLL-vit-1: 15% PLL + 15% ethylene glycol (EG); PLL-vit-2: 30% PLL + 30% EG + 0.5 M sucrose], (2) vitrified embryos with Vajta et al. solution (Conv-vit-1: 7.5% dimethyl sulfoxide + 7.5% EG; Conv-vit-2: 16.5% dimethyl sulfoxide + 16.5% EG + 0.5 M sucrose), and (3) nonvitrified blastocysts (control). All embryos were frozen by droplet vitrification method. First, the PLL-vitrified embryos were exposed to 5, 10, and 15 min in the PLL-vit-1 and then putted for 30 to about 60 s in the PLL-vit-2. Then, we compared with each group regarding exposure time of Vit-1. Post-thaw survival rate of each exposure time did not significantly differ among the 3 groups (100 v. 100 v. 100%). However, hatching rate of the 10-min exposure group was higher than that of 5- and 15-min groups (75.0 v. 25.0 v. 66.7; P < 0.05). Therefore, we confirmed that exposure time of Vit-1 was exposed for a minimum of 10 min. The post-thawed survival rate of each vitrification method was not significantly different between PLL-vit and Conv-vit groups (97.7 v. 86.4%). The total cell numbers of blastocyst did not significantly differ among groups. However, the apoptotic cell numbers of blastocyst was significantly different between the control and Conv-vit groups (0.4 ± 0.6 v. 4.4 ± 3.9; P < 0.05) but was not different in control v. PLL-vit (0.4 ± 0.6 v. 2.1 ± 2.4) and Conv-vit v. PLL-vit (4.4 ± 3.9 v. 2.1 ± 2.4). In conclusion, PLL-vit for bovine blastocyst could reduce toxicity and osmotic shock and showed high efficiency on the quality of post-thawed bovine blastocysts compared with that of Conv-vit.This work was partly supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009587022014), IPET (Grant No. 112020-3), and a scholarship from the BK21 plus program. A-Na Ha, Kyeong-Lim Lee, and Md. Fakruzzaman were supported by BK21 plus fellowships at Gyeongsang National University, Republic of Korea.

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