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.

292 OOCYTES FROM FOLLICLES OF DIFFERENT DIAMETERS: EMBRYO IN VITRO DEVELOPMENTAL POTENTIAL AND QUALITY

2007 ◽  
Vol 19 (1) ◽  
pp. 261
Author(s):  
T. H. C. de Bem ◽  
K. R. L. Schwarz ◽  
L. G. Mesquita ◽  
P. R. Adona ◽  
C. L. V. Leal
Keyword(s):  
Apoptotic Cell ◽  
Total Cell ◽  
Sperm Cells ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Visual Evaluation ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Follicle Diameter

The present study aimed to assess the developmental potential and quality of embryos produced from oocytes originating from follicles of different sizes. Ovaries were collected at a slaughterhouse and follicles of &lt;3, 3 to 6, and &gt;6 mm were aspirated. Follicle diameters were estimated based on the size of their exposed surface on the ovarian cortex using a ruler as reference for the first aspirations and were then based on visual evaluation. Aspirated oocytes, separated by their follicular origin, were matured in vitro in TCM-199 supplemented with 10% FCS, 5.0 �g mL-1 of LH, 0.5 �g mL-1 of FSH, 0.2 mM pyruvate, and 10 �g mL-1 of gentamicin for 22 h. After in vitro maturation, oocytes were in vitro-fertilized (IVF) using frozen–thawed semen prepared by Percoll gradient. Sperm cells were co-cultured with the oocytes at a final concentration of 2 � 106 sperm cells mL-1 in TALP-IVF medium supplemented with 2 �M penicillamine, 1 �M hypotaurine, 250 �M epinephrine, and 20 �g mL-1 of heparin. After 20 h, presumptive zygotes were partially denuded and transferred to in vitro culture (IVC) medium (TCM-199 supplemented with 10% FCS, 2.0 mM pyruvate, and 10 mg mL-1 of gentamicin). All cultures were incubated at 38.5�C under 5% CO2 in air and maximum humidity. The cleavage rate was assessed after 48 h of IVC, and blastocyst development was assessed on Day 7 (D7). On Day 9 (D9), the hatching rate was assessed and the hatched embryos were fixed for 1 h (3% paraformaldehyde in PBS), permeabilized for another h (0.5% Triton-X, 0.1% sodium citrate, and 0.1% plyvinyl alcohol in PBS), and evaluated by the TUNEL technique (in situ cell death detection kit). Total cell number and TUNEL-positive cells in embryos were counted under an epifluorescence microcope. Data of 4 replicates were analyzed by ANOVA and comparisons among groups were made by the Tukey test. The level of significance used was 5%. From the oocytes used (&lt;3 mm = 100; 3–6 mm = 99; &gt;6 mm = 88), cleavage rates increased with increasing follicular diameter. Oocytes originating from &lt;3-, 3- to 6-, and &gt;6-mm follicles resulted in 75, 93.6, and 95.5% cleavage rates, respectively (P &gt; 0.05). For blastocyst rates on D7, &lt;3-mm follicles showed 21.5% blastocyt development, which was lower (P &lt; 0.05) than for the other follicle diameter groups (3–6 mm = 35.4% and &gt;6 mm = 41.1%; P &gt; 0.05). Regarding the hatching rate, total cell numbers, and TUNEL-positive cells on D9, &lt;3 mm (20.4%, 268, and 0.37%, respectively), 3–6 mm (29.1%, 248, and 0.32%, respectively), and &gt;6 mm (32.3%, 237, and 0.23%, respectively) were not different (P &gt; 0.05). The results suggest that oocytes from larger follicles are more competent for cleavage and blastocyst development on D7; however, when oocytes reach the blastocyst stage, hatching, total cell numbers, and apoptotic cell numbers are not influenced by follicle diameter. Financial suport for this work was provided by the FAPESP, Brazil.

scholarly journals In vitro Induced Mitotic Polyploidy in Drosera capensis L.

2013 ◽  
Vol 46 (4) ◽  
pp. 107-110 ◽  
Author(s):  
Pavla Zahumenicka ◽  
Barbora Sysova ◽  
Ales Holik ◽  
Eloy C. Fernandez
Keyword(s):  
Flow Cytometry ◽  
Survival Rate ◽  
Exposure Time ◽  
Leaf Segments

Abstract The objective of this study was to induce mitotic polyploidization in Drosera capensis. Tetraploid plants of D. capensis were induced successfully by treating leaf segments in vitro with oryzalin solution with four different concentrations (20, 40, 60 or 80 μM) for 12, 24 or 48 hours. Three tetraploid (2n = 4x = 80) plants were obtained in three treatments (20 μM for 48 h, 60 μM for 24 h and 80 μM for 12 h). Tetraploidy was confirmed by flow cytometry. The survival rate of these plants was not significantly influenced by oryzalin concentration or exposure time.

335 EFFECT OF VITAMIN E ON THE DEVELOPMENT OF BOVINE OOCYTES AFTER CHEMICAL ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 283
Author(s):  
J. I. Park ◽  
Y. Jang ◽  
E. S. Lee
Keyword(s):  
Vitamin E ◽  
Apoptotic Cell ◽  
Chemical Activation ◽  
Calf Serum ◽  
Cell Number ◽  
Total Cell ◽  
Chi Square ◽  
Cell Numbers ◽  
Humidified Air

Oxidative stress is known to induce apoptotic cell death by reactive oxygen species (ROS) generated from in vitro culture systems. This study was conducted to evaluate the effect of Vitamin E (VitE), as antioxidant, on development of bovine embryos activated in vitro. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles 3-8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL-1 l-cysteine, 20 mg/mL-1 sodium pyruvate, gonadotropins (250 IU each of eCG and hCG/mL), 10 mg/mL-1 epidermal growth factor, and 100 �M VitE. Oocytes were cultured at 38.9�C in 5% CO2 in humidified air. After 22 hours of culture, oocytes with polar bodies were selected and subjected to activation treatments. Oocytes were exposed to calcium ionomycin (5 �M for 5 min), followed by incubation with 6-DMAP (2 mM) for 3.5 hours in medium supplemented with or without VitE (100 �M). After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9�C in 5% CO2, 5% O2 in humidified air for 7–8 days. Cell numbers were counted by the number of nuclei of blastocysts stained with Hoechst 33342, and apoptosis was detected by TUNEL assay using a MK500 kit (Takara Bio, Inc., Otsu, Shiga, Japan). Total cell and apoptotic cell number were determined under a fluorescence microscope. Data were analyzed using Student&apos;s t-test and chi-square test. The cleavage and blastocyst rates were significantly higher (P &lt; 0.05) after activation with VitE (78.1&percnt; and 16.3&percnt;, n &equals; 80) than without VitE (66.7&percnt; and 11.0&percnt;, n &equals; 60). Total cell numbers were also significantly higher (P &lt; 0.05) in blastocysts after activation with VitE (143.0 &plusmn; 34.02, n &equals; 21) than in those without VitE (127.63 &plusmn; 40.25, n &equals; 20). However, the percentage of TUNEL-positive (apoptotic) cells was similar between blastocysts activated with VitE (5.38 &plusmn; 2.22) and those without VitE (6.76 &plusmn; 1.98). The results of the present study demonstrate that vitamin E added to activation medium promoted further development of activated embryos, although its role in the alleviation of apoptosis remains unclear.

125 EXAMINING CRITERIA FOR EXTENDING BOVINE BLASTOCYST SURVIVAL IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 143
Author(s):  
F. N. T. Cooke ◽  
T. M. Rodina ◽  
P. J. Hansen ◽  
A. D. Ealy
Keyword(s):  
Conditioned Medium ◽  
Culture System ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Cell Numbers ◽  
Bovine Blastocyst

Most of the current culture procedures used for bovine in vitro embryo production terminates at the blastocyst stage. Developing procedures for extending embryo lifespan beyond this phase will provide a valuable tool for understanding events that occur during the second week of pregnancy in cattle. The overall objective of the present studies was to identify culture conditions required to support bovine blastocyst development beyond its initial formation. In the first study, individual day 8 blastocysts (day 0 = day of IVF) were cultured until day 11 in 30 µL microdrops of Potassium Simplex Optimized Medium-Bovine Embryo 2 containing 0.1 mm non-essential amino acids or Tissue Culture Medium 199 (M199). Both media were supplemented with 5% [v/v] fetal bovine serum (FBS) and incubations were in an atmosphere of either 5 or 21% (v/v) oxygen. A medium by oxygen interaction (P = 0.007) occurred when assessing cell number on day 11. Blastocysts cultured in M199 and in a 5% O2 environment had greater (P < 0.002) cell numbers (536 � 49) than blastocysts incubated in other conditions (339 � 28). Conditioned medium from blastocysts incubated in 21% O2 contained greater (P < 0.05) concentrations of bioactive interferon-tau (IFNT) than blastocysts incubated in 5% O2 regardless of medium type (70.5 � 28 v. 17.2 � 2.6 ng mL–1). In a follow-up study, blastocysts could remain morphologically viable through day 11 in M199 containing at least 2.5% FBS. To examine whether oxidative stress was responsible for the increase in IFNT production under 21% O2, blastocysts were incubated under a 5% O2 atmosphere in M199 containing 2.5% FBS and increasing concentrations of tert-butylhydroperoxide (tBH), a membrane-permeable oxidative agent. Addition of e3 nm tBH decreased cell numbers but did not increase IFNT concentrations in conditioned medium. To examine whether blastocysts could survive beyond day 11 in culture, day 11 blastocysts were transferred to 400 �L of M199 with 20% FBS under 5% oxygen and cultured from day 11 to 20–21 post-IVF. Half of the medium was replaced every 2–3 days. On day 13–14, 16.6 � 6.1% of blastocysts showed initial signs of degeneration. A portion of blastocysts (32.9 � 9.6%) began attaching to plates on days 13–15 and produced outgrowths that appeared viable on days 20–21. All of the non-attached blastocysts degenerated by day 17–18. No blastocyst elongation was detected. In conclusion, a culture system was developed that sustains blastocyst viability and IFNT production in vitro to day 11. Although this culture system allowed blastocyst survival until day 14, normal conceptus development (i.e. elongation/filamentation) was not achieved. Nonetheless, the culture system provides a useful tool for examining the initial stages of blastocyst development and IFNT production from individual bovine embryos.

47 A New Device and Method for Successful Vitrification of In Vitro-Produced Ovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 163
Author(s):  
S. Ledda ◽  
J. M. Kelly ◽  
S. K. Walker ◽  
Y. Natan ◽  
A. Arav
Keyword(s):  
Survival Rate ◽  
Embryo Transfer ◽  
Blastocyst Stage ◽  
Embryo Cryopreservation ◽  
Control Group ◽  
Hatching Rate ◽  
High Survival ◽  
Embryo Survival ◽  
New Device

To advance the use of embryo vitrification technology in veterinary practice, we developed a system in which embryo vitrification, warming, and dilution can be performed within a straw. An in-straw embryo cryopreservation method reduces the need for equipment and technical skills and can facilitate direct embryo transfer to the uterus. This study proposes the use of a new device named “Sarah” that is designed to permit all in-straw embryo cryopreservation procedures. Ovine in vitro-produced (IVP) embryos were vitrified at either early blastocyst stage (EB, n = 65, 6 days post-IVF) or fully expanded blastocyst stage (FB, n = 168, 7 days post-IVF). The vitrification procedure using Sarah constituted a 0.25-mL straw with a capsule having 50-µm pores inserted at one end. Embryos at each stage (EB and FB) were divided into 2 subgroups and vitrified by 1 of 2 methods: (1) multi-step (MS) group-a straw containing 2 embryos was sequentially loaded vertically into 1.5-mL tubes containing 6 different vitrification solutions: 10, 20, 40, 60, 80, or 100% ES (with 100% ES being 7.5% DMSO +7.5% EG + 20% FCS in TCM-199; 90 s each step) followed by 30 s each in 75 and 100% VS (100% VS being 18% DMSO +18% EG + 0.5 M trehalose + BSA in TCM-199); and (2) two-step (TS) group-the straw (2 embryos/straw) was loaded with 100% of ES (5 min), followed by 100% VS solution for 30 s. For both methods, at the end of the preparation steps, the straws were plunged directly into liquid N2. Non-vitrified embryos were maintained in in vitro culture as a control group (n = 102). The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, 25% WS (WS = 1 M sucrose in TCM-199+ 20% FCS) at 38.6°C (for first solution) and at room temperature for all the rest (5 min each), before being placed into the holding medium. Embryos were recovered from the straws, incubated at 38.6 C in 5% CO2 in air in TCM 199 + 5% FCS, and evaluated for blastocoel re-expansion, embryo survival, and hatching rate at 2, 14, 48 h post-warming. Blastocyst re-expansion (2 h) after warming increased as the developmental stage progressed and was not affected by the vitrification method. In fact, it was significantly (P < 0.05) higher for FB vitrified in the MS and TS methods (77.90% and 71.25%, respectively) compared with the EB method (62.5% and 48.50%, respectively). At 24 h, survival rate of vitrified FB was significantly higher (P < 0.05) in the MS system (95.35%) compared with those in TS (86.25%). Survival rates of FB embryos for both methods (MS and TS) were significantly higher (P < 0.001) than EB embryos vitrified in MS (56.25%) and TS (56.55) methods. After 48 h of culture, the hatching rate for FB vitrified in the MS system (87.21%) was comparable with TS (77.5%) and control (85.3%) groups but significantly higher (P < 0.001) than vitrified EB in MS (43.75%) and TS (36.36%). In conclusion, we showed that a high survival rate of IVP embryos can be achieved by this new in-straw vitrification and warming device (“Sarah”), with hatching rates in vitro comparable with that of control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

scholarly journals 276INFLUENCE OF SPERM TREATMENTS ON BLASTOCYST DEVELOPMENT IN VITRO AND CELL NUMBER IN CATTLE

2004 ◽  
Vol 16 (2) ◽  
pp. 258
Author(s):  
B.G. Jeon ◽  
J.S. Moon ◽  
K.C. Kim ◽  
G.J. Rho
Keyword(s):  
Silicone Oil ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Glass Wool ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Serum Free ◽  
Cell Numbers ◽  
Bull Sperm

Experiments were designed to examine the effects of developmental rate and cell numbers in embryos produced by in vitro fertilization (IVF) using sperm from 2 bulls (sperm A and B purchased from commercial sale) isolated by three methods. Cumulus-oocyte-complexes collected from ovaries harvested from a local slaughter house were matured in 50μL droplets of serum-free M199 medium supplemented with 1μgmL−1 estradiol-17β, 10μgmL−1 LH and FSH under silicone oil at 39°C in a humidified atmosphere of 5% CO2 in air. After 24h of culture, oocytes were fertilized with the sperm treated by three different methods of isolation;; percoll gradient, swim-up and glass wool filtration;; a final concentration of 2×106 cells mL−1 was used. At 16h after fertilization, presumptive zygotes were co-cultured in serum-free M199 with BOEC for up to 192h post-insemination. At 48h and 120h post-insemination, the cultures were fed with 25μL of serum-free M199. The embryos were compared for their rates of cleavage at 48h post-insemination, development to the blastocyst stage, and hatching, and also the cell number at 192h post-insemination. Differences between treatments were analyzed using one-way ANOVA after arc-sine transformation of the proportional data of cleavage, development into blastocyst stage and hatching. Comparisons of means among treatments were performed using Tukey-Kramer multiple comparisons test. The results are summarized below. The rates of cleavage in embryos produced by IVF using sperm from 2 bulls isolated by percoll, swim-up and glass wool were not significantly different. The blastocyst development and hatching rates between sperm treatment were not significant within bull sperm A and within sperm B. However, although the hatching rate in percoll treatment of bull sperm A was higher than in bull sperm B, the difference was not statistically significant. The mean cell numbers in percoll treatment of bull sperm A (176.5±7.1) were significantly higher (P&lt;0.001) than the others. In bull sperm B the cell numbers in percoll treatment were higher than the other two treatments, but the differences were not statistically significant. In conclusion, these results support the concept that sperm preparation using percoll has beneficial effects on blastocyst cell number. Table 1 Developmental rates of in vitro embryos using sperm from 2 bulls isolated by three methods with 4 replicates

scholarly journals Salinity-Induced Alterations in the Growth, Membranes and Osmolytes of Musa spp. cv. Caipira (AAA) During Micropropagation

2019 ◽  
Vol 11 (6) ◽  
pp. 171
Author(s):  
Yuri Lima Melo ◽  
Isabele Aragão Gomes Trindade ◽  
Monique Cristina Simão Lopes ◽  
Cibelley Vanúcia Santana Dantas ◽  
Josemir Moura Maia ◽  
...  
Keyword(s):  
Survival Rate ◽  
Plant Height ◽  
Exposure Time ◽  
Root Length ◽  
Growth Traits ◽  
In Vitro Rooting ◽  
Ms Medium ◽  
Musa Spp ◽  
Number Of Leaves

The aim of this study was to determine the concentration and exposure time to NaCl suitable for the micropropagation of banana, through the analysis of growth traits. Banana propagules were inoculated in MS medium with different concentrations of NaCl (0; 50; 75 and 100 mM) for 120 days (multiplication and rooting, 60 days each), with monthly subcultures. These propagules were measured for plant height, number of leaves, sprouting rate, average number of formed propagules, rooting rate, root length and survival rate. After 30 days, NaCl reduced sprouting rate at multiplication; the number of leaves, rooting rate and root length in rooting; and the height and propagules number in both phases. After 60 days, the NaCl affected the sprouting rate and propagules number in the multiplication; length of root in rooting; and the height and number of leaves in both phases. After 120 days, the reduction in the survival rate was proportional to the increase of NaCl in the medium. Thus, it is concluded that NaCl reduces most of the growth traits and the treatments with 75 and 100 mM NaCl affected multiplication and in vitro rooting more intensely.

scholarly journals Leukemia Inhibitory Factor Stimulates Primitive Endoderm Expansion in the Bovine Inner Cell Mass

2021 ◽  
Vol 2 ◽  
Author(s):  
Lydia K. Wooldridge ◽  
Alan D. Ealy
Keyword(s):  
Leukemia Inhibitory Factor ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Inhibitory Factor ◽  
Primitive Endoderm ◽  
Total Cell Number ◽  
Cell Numbers ◽  
Bovine Blastocyst

Previous work determined that bovine interleukin-6 (IL6) increases inner cell mass (ICM), primitive endoderm (PE), and total cell number in in vitro produced (IVP) bovine blastocysts. Another IL6 family member, leukemia inhibitory factor (LIF), has the potential to produce the same effects of IL6 due to the presence of its receptor in bovine blastocysts. We compared the abilities of LIF and IL6 to increase ICM cell numbers in day 7, 8, and 9 IVP bovine blastocysts. Supplementation with 100 ng/ml LIF from day 5 onward improved blastocyst formation rates on days 7 and 8 similar to what was observed when supplementing 100 ng/ml IL6. However, LIF supplementation did not cause an increase in ICM numbers like was observed after supplementing IL6. On day 9, increases in PE cell numbers were detected after LIF supplementation, but 300 ng/ml LIF was required to achieve the same effect on PE numbers that was observed by providing 100 ng/ml IL6. Collectively, these results show that LIF can mimic at least some of the effects of IL6 in bovine blastocyst.

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.

209 EFFECT OF HEAT STRESS ON DEVELOPMENT OF IN VITRO-FERTILIZED AND PARTHENOGENETIC BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 203
Author(s):  
F. Paludo ◽  
M. M. Pereira ◽  
C. C. R. Quintao ◽  
L. T. Iguma ◽  
M. M. Gioso ◽  
...  
Keyword(s):  
Heat Stress ◽  
Bos Taurus ◽  
Apoptotic Cell ◽  
Chemical Activation ◽  
Bos Indicus ◽  
Bovine Embryos ◽  
Cell Numbers ◽  
Bovine Reproduction ◽  
Blastocyst Rate

Heat stress has been a challenge for bovine reproduction in tropical and subtropical environments. Although the role of the oocyte in thermotolerance has been studied, little attention has been paid to the contributions of sperm to embryo resistance to heat shock. The current study aimed to evaluate the development of fertilized and nonfertilized (parthenogenetic) bovine embryos undergoing heat stress during the pre-implantation stage. Cumulus–oocyte complexes obtained from ovaries collected from Bos indicus × Bos taurus crossbred cows at slaughter were in vitro matured with TCM-199 supplemented with 20 μg mL–1 of FSH, under 5% CO2 at 38.5°C for 24 h. Afterward, oocytes were randomly allocated into 2 groups: 1) IVF and 2) PART (chemical activation for parthenogenesis induction). In vitro-fertilized oocytes were cultured with 2.0 × 106 Holstein sperm mL–1 in Fert-TALP medium supplemented with heparin, for 20 h. For chemical activation, oocytes were activated with calcium ionomycin for 4 min, followed by 6-DMAP for 4 h, both in CR2aa medium supplemented with 0.1% BSA. Presumptive IVF (n = 1 262) or PART (n = 1 206) zygotes were denuded by vortexing and cultured in CR2aa medium with 2.5% of FCS under 5% CO2, 5% O2, and 90% N2 at 38.5°C. At 44 h post-insemination or chemical activation, embryos were exposed to 38.5 or 41°C for 12 h in an atmosphere of 5% CO2, 5% O2, and 90% N2. After that, embryos were cultured at 38.5°C under 5% CO2, 5% O2, and 90% N2 until Day 8 post-insemination. Blastocyst rates were evaluated at Day 7 and Day 8 post-insemination and were calculated based on the total number of presumptive zygotes. Blastocysts at 192 h post-insemination or activation were fixed and permeabilized for TUNEL assay (DeadEndTM Florimetric TUNEL System, Promega, Madison, WI) according to the manufacturer’s instructions. The effect of heat stress was compared within groups (IVF or PART) and the data were analysed by ANOVA. As expected, heat stress reduced the blastocyst rate of IVF embryos at Day 7 (24.3 ± 2.0% and 17.4 ± 2.2% for nonstressed and stressed IVF embryos; P < 0.05) and at Day 8 (32.4 ± 1.9% and 23.0 ± 2.1% for nonstressed and stressed IVF embryos; P < 0.01). However, the effect of heat stress on blastocyst rate of PART embryos was observed only at Day 8 post-insemination (30.0 ± 1.7% and 22.6 ± 2.0% for nonstressed and stressed PART embryos; P < 0.05), with no difference in blastocyst rate at Day 7 (21.6 ± 1.5% and 18.2 ± 1.8% for nonstressed and stressed PART embryos; P > 0.05). There was no difference in total cell numbers between nonstressed and stressed IVF or PART embryos. Apoptosis cell numbers and the apoptotic cell index were higher (P < 0.05) for stressed IVF (18.45 ± 1.24 and 0.16 ± 0.00) and PART (16.40 ± 5.20 and 0.17 ± 0.00) embryos than for nonstressed IVF (13.70 ± 0.75 and 0.13 ± 0.00) and PART (14.15 ± 0.86 and 0.13 ± 0.00) embryos. In conclusion, heat stress can induce apoptosis in both IVF and PART embryos, but its effect on pre-implantation development may occur at earlier stages in IVF embryos when compared with PART embryos. Financial support from Fapemig and CNPq.

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