scholarly journals Combinations of Trolox and ascorbic acid have a beneficial effect on in vitro maturation of pig oocytes

2021 ◽  
Author(s):  
Ileana Miclea ◽  
Marius Zahan
Keyword(s):  
Ascorbic Acid ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Membrane Damage ◽  
High Concentration ◽  
First Polar Body ◽  
Morula Stage ◽  
Vitro Development

Abstract: The poor in vitro development of pig oocytes and embryos has been blamed on oxidative stress. We sought to find out if combinations of Trolox (T), a synthetic and cell-permeable derivative of vitamin E, and ascorbic acid (AA) could improve the maturation rates of in vitro cultured pig oocytes. Pig oocytes underwent maturation for 44–45 h in medium M 199 supplemented with 0 μM T + 0 μM AA, 100 μM T + 250 μM AA, 300 μM T + 250 μM AA, 100 μM T + 750 μM AA or 300 μM T + 750 μM AA. These combinations were chosen based on previous research conducted in our laboratory and on the available literature. After maturation, several parameters were assessed: cumulus oophorus expansion, oocyte viability (based on the presence of metabolic activity versus membrane damage), extrusion of the first polar body, mitochondrial membrane potential (MMP), pronucleus formation, and embryo development after fertilization. All antioxidant combinations significantly improved cumulus expansion and formation of the first polar body. The best was 300 μM T + 250 μM AA for the first characteristic and 300 μM T + 750 μM AA for the second. Antioxidant presence in the maturation media increased the percentages of viable oocytes but not significantly. MMP was not significantly modified by the addition of antioxidant combinations. We also found that a low concentration of T (100 µM) mixed with a high concentration of AA (750 µM) in the oocyte maturation media led to significantly higher rates of both female and male pronuclei formation and also enhanced embryo development to the morula stage. Therefore, we recommend this combination to improve the in vitro maturation media of pig oocytes.  

212 EFFECT OF CANINE OVIDUCT CELLS AND CUMULUS CELLS CO-CULTURE ON IN VITRO MATURATION OF PORCINE OOCYTES AND EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 237
Author(s):  
S. H. Lee ◽  
H. J. Oh ◽  
G. A. Kim ◽  
M. J. Kim ◽  
Y. B. Choi ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cells ◽  
In Vitro Development ◽  
First Polar Body ◽  
Vitro Development ◽  
And Control

In oestrus stage, canine oocytes surrounded by cumulus cells undergo maturation in oviduct for 3 days after ovulation. We hypothesised that canine cumulus cells (cCC) and canine oviduct cells (cOC) in oestrus stage might affect the maturation of oocyte and embryo development. Therefore, the present study was aimed to compare the effects of cCC and cOC co-culture system on oocyte in vitro maturation and embryo in vitro development. cCC were separated from cumulus‐oocyte complex (COC) in ovary from bitches in oestrus phase. cOC were collected from oviduct flushing of bitches in oestrus phase. Both cCC and cOC were cultured and cryopreserved until use for co-culture. In the first experiment, the effect of co-culture using cCC and cOC on porcine oocyte in vitro maturation (IVM) were investigated. The porcine COC were randomly cultured in different co-culture groups as follows: 1) co-culturing with cCC for 42 h, 2) co-culturing with cOC for 42 h, and 3) culturing in absence of cCC or cOC. After IVM, extrusion of the first polar body was observed under a microscope. In the second experiment, the matured oocytes with the first polar body derived from each group were activated with electrical stimulus. Parthenotes were cultured in porcine zygote medium-5 (PZM-5) for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The embryo developmental competence was estimated by assessing the in vitro development under microscope. The third experiment was to evaluate the reactive oxygen species (ROS) levels in each supernatant medium obtained from cCC and cOC co-culture group after IVM using a OxiselectTM ROS ELISA Assay kit. Last, analysis of genes (MAPK1/3, SMAD2/3, GDF9 and BMP15) expression in cCC and cOC co-cultured with porcine COC using real-time PCR is in progress. As results, IVM rate of cOC group (91.19 ± 0.45%) was significantly higher than that of cCC and control group (86.50 ± 0.61% and 79.81 ± 0.82%; P < 0.05). Also, cOC groups expressed the highest efficiency in cleavage rate, blastocyst formation rate, and the total cell number in blastocyst (P < 0.05). In ROS levels, cOC group (555 ± 7.77 nM) were significantly lower than cCC and control groups (596.8 ± 8.52 nM and 657.8 ± 11.34 nM). The present study demonstrated that co-culture with cOC improved the in vitro oocyte maturation and the in vitro development rate of porcine embryos. The ROS level decreased in cOC co-culture would have beneficial influence on oocytes maturation. For further study, we will investigate the relation between gene expression related to oocyte maturation and the co-culture results. This research was supported by a global PhD Fellowship Program through NRF funded by the Ministry of Education (NRF-20142A1021187), RDA (#PJ010928032015), IPET (#311011–05–4-SB010, #311062–04–3-SB010), Research Institute for Veterinary Science, and the BK21 plus program.

scholarly journals Effect of Melatonin on the In Vitro Maturation of Porcine Oocytes, Development of Parthenogenetically Activated Embryos, and Expression of Genes Related to the Oocyte Developmental Capability

Animals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 209 ◽  
Author(s):  
Ling Yang ◽  
Qingkai Wang ◽  
Maosheng Cui ◽  
Qianjun Li ◽  
Shuqin Mu ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Melatonin Receptors ◽  
Melatonin Treatment ◽  
In Vitro Development ◽  
First Polar Body ◽  
Mitochondrial Distribution ◽  
Polar Body Extrusion ◽  
Vitro Development

Melatonin treatment can improve quality and in vitro development of porcine oocytes, but the mechanism of improving quality and developmental competence is not fully understood. In this study, porcine cumulus–oocyte complexes were cultured in TCM199 medium with non-treated (control), 10−5 M luzindole (melatonin receptor antagonist), 10−5 M melatonin, and melatonin + luzindole during in vitro maturation, and parthenogenetically activated (PA) embryos were treated with nothing (control), or 10−5 M melatonin. Cumulus oophorus expansion, oocyte survival rate, first polar body extrusion rate, mitochondrial distribution, and intracellular levels of reactive oxygen species (ROS) and glutathione of oocytes, and cleavage rate and blastocyst rate of the PA embryos were assessed. In addition, expression of growth differentiation factor 9 (GDF9), tumor protein p53 (P53), BCL2 associated X protein (BAX), catalase (CAT), and bone morphogenetic protein 15 (BMP15) were analyzed by real-time quantitative PCR. The results revealed that melatonin treatment not only improved the first polar body extrusion rate and cumulus expansion of oocytes via melatonin receptors, but also enhanced the rates of cleavage and blastocyst formation of PA embryos. Additionally, melatonin treatment significantly increased intraooplasmic level of glutathione independently of melatonin receptors. Furthermore, melatonin supplementation not only significantly enhanced mitochondrial distribution and relative abundances of BMP15 and CAT mRNA, but also decreased intracellular level of ROS and relative abundances of P53 and BAX mRNA of the oocytes. In conclusion, melatonin enhanced the quality and in vitro development of porcine oocytes, which may be related to antioxidant and anti-apoptotic mechanisms.

53 EFFECT OF THE NUCLEAR REPROGRAMMING TIME ON IN VITRO DEVELOPMENT OF EQUINE AGGREGATED CLONED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 174
Author(s):  
R. Olivera ◽  
C. Alvarez ◽  
I. Stumpo ◽  
G. Vichera
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Nuclear Reprogramming ◽  
Chi Square ◽  
In Vitro Development ◽  
First Polar Body ◽  
Group 3 ◽  
Group 2 ◽  
Vitro Development

The time allowed for nuclear reprogramming is considered an essential factor for the efficiency of cloning and has not been evaluated in equine aggregated cloned embryos. The aim of our work was to assess the effect of different timing of activation stimulus after fusion of adult equine fibroblast cells to enucleated equine oocytes on embryo development and embryo quality. We processed a total of 1874 equine ovaries, recovering 3948 oocytes, of which 1914 (48.5%) had extruded the first polar body after 24 h of maturation. Oocyte collection, maturation, and the NT procedure were performed as described by Lagutina et al. (2007 Theriogenology 67, 90–98). Reconstructed oocytes (RO) were activated at 3 different times after cell fusion: (1) 1 h, (2) 1.5 h, and (3) 2 h. Activation was performed using 8.7 µM ionomycin for 4 min, followed by a 4-h culture in a combination of 1 mM DMAP and 5 mg mL–1 of cycloheximide. The RO were cultured in the well of the well system, aggregating 3 RO per well. The RO were cultured in DMEM-F12 with 5% fetal bovine serum (FBS) and antibiotics. Cleavage (48 h after activation), blastocyst, and expanded blastocyst rates (8–9 days) were assessed. In vitro development was compared using the chi-square test (P < 0.05). A total of 1608 RO were cultured. Cleavage was significantly lower in group 3 with respect to the other 2 groups [(1): 396/450, 88%; (2): 540/639, 84.5%; (3): 365/519, 70.3%]. There were no significant differences in blastocyst rates within the 3 groups considering the number of total RO [(1): 19/450, 4.2%; (2): 23/639, 3.6%; (3): 15/519, 2.9%] or aggregated RO per well [(1): 12.7%; (2): 10.8%; (3): 8.7%]. However, the rate of blastocyst expansion was higher (P < 0.05) in group 2 than in group 3 [(1): 17/19, 89.5%; (2): 23/23, 100%; (3): 11/15, 73.3%]. In conclusion, the timing of nuclear reprogramming did not affect blastocyst rates but affected cleavage rates and blastocyst quality. This indicates that 1 h before activation stimulus is enough for embryo development of equine aggregated cloned embryos.

186 SUPPLEMENTATION WITH LOW DOSES OF DIMETHYL SULFOXIDE DURING IN VITRO MATURATION RESULTS IN IMPROVED IN VITRO EMBRYO PRODUCTION IN CATTLE

2017 ◽  
Vol 29 (1) ◽  
pp. 201
Author(s):  
A. E. Ynsaurralde ◽  
M. Suvá ◽  
R. Bevacqua ◽  
S. Munilla ◽  
C. Luchetti ◽  
...  
Keyword(s):  
Dimethyl Sulfoxide ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Predictive Equation ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
First Polar Body ◽  
In Vitro Embryo Production

Oocyte in vitro maturation (IVM) is crucial for subsequent in vitro embryo production. It involves acquisition of competence for fertilization and embryo development. Therefore, its optimization could have a direct impact on in vitro embryo development. Dimethyl sulfoxide (DMSO) is commonly used as solvent or vehicle, but also increases the membrane permeability and behaves as a scavenger of cytotoxic free radicals. The aim of this study was to evaluate the effect of DMSO supplementation during bovine oocyte maturation on subsequent in vitro embryo development and to determine the optimal usage dose with no toxic effect. To this aim, cumulus-oocyte complexes were collected from slaughterhouse ovaries and IVM in TCM 199 containing 10% fetal bovine serum, 10 µg mL−1 of FSH, 0.3 mM sodium pyruvate, 100 mM cysteamine, and 2% antibiotic-antimycotic. The oocytes were incubated for 24 h at 6.5% CO2 in humidified air at 38.5°C. For Experiment 1, IVM medium was supplemented with DMSO at concentrations of 0, 0.1, 0.5, 1, or 10% (vol/vol) DMSO (n = 241, 195, 42, 192, 172 oocytes) and IVM rate was determined by presence of the first polar body. For Experiment 2, 0, 0.1, 0.25, 0.5, 0.75, 1, or 10% (vol/vol) DMSO (n = 446, 322, 65, 194, 77, 250, 39 oocytes) was supplemented to IVM medium and cleavage and blastocyst rates were determined to establish the optimal usage dose. In vitro fertilization was performed according to Brackett and Oliphant (1975), with 16 × 106 spermatozoa/mL for 5 h. Afterwards, presumptive zygotes were cultured in SOF for 7 days at 38.5°C and 5% O2. Cleavage and blastocyst rates were determined on Days 2 and 7, respectively. Results were statistically analysed using Fisher’s exact test by GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Also, the percentage of blastocyst was adjusted to DMSO concentration using the R software quadratic regression model. The optimum usage dose was determined by calculating the maximum of the estimated predictive equation. In vitro maturation in 10% DMSO resulted in significantly lower first polar body extrusion rates (0% = 74%a, 0.1% = 73%a, 0.5% = 83%a, 1% = 66%a, and 10% = 8%b; different letters indicate statistical differences) and lower cleavage rates (0% = 75%a, 0.1% = 77%a, 0.25% = 80%a, 0.5% = 79%a, 0.75% = 78%a, 1% = 77%a, and 10% = 3%b) than the other treatments. Furthermore, blastocyst production was higher for the 0.25 and 0.5% (vol/vol) supplemented DMSO groups (0% = 26%b, 0.1% = 37%ab, 0.25% = 40%a, 0.5% = 41%a, 0.75% = 34%ab, 1% = 23%b, and 10% = 0%c). The predictive equation results indicate that the maximum percentage of blastocysts is obtained with a concentration of 0.458% (vol/vol) of DMSO. In conclusion, DMSO supplementation during IVM of bovine oocytes had a positive effect on in vitro development. Further studies will be carried out to elucidate its mechanism of action.

129 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES AFTER IN VITRO MATURATION AND IN VITRO CULTURE UNDER COMPARATIVE OXYGEN TENSION

2011 ◽  
Vol 23 (1) ◽  
pp. 169
Author(s):  
J. T. Kang ◽  
M. Atikuzzaman ◽  
D. K. Kwon ◽  
S. J. Park ◽  
S. J. Kim ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Oxygen Tension ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Mrna Abundance ◽  
Developmental Competence ◽  
Multiple Genes ◽  
Oxygen Concentrations

The in vitro developmental abilities of porcine oocytes are generally increasing steadily at a similar ratio to those of in vivo embryos. However, it has been suggested that the in vitro culture system for the development of porcine embryos is not optimal. In this study, we investigated the effect of 2 oxygen concentrations (5 and 20%) on porcine embryo development during in vitro maturation and in vitro culture and analyzed differences in gene expression of resulting blastocysts. Oocytes were recovered by aspiration of slaughterhouse ovaries and then matured in tissue culture medium (TCM) 199 supplemented with 10% porcine follicular fluid (pFF), epidermal growth factor (EGF), insulin, pyruvate, cystine, and gonadotropin. Matured oocytes were then activated parthenogenetically, cultured in PZM-3 media for 7 days. In vitro maturation (M group) of oocytes was carried out under two oxygen concentration (5 and 20%) in terms of nuclear maturation (polar body extrusion; Exp. 1). The developmental differences between 5% oxygen culture group and 20% oxygen culture group during in vitro culture (C group) of embryos after parthenogenetic activation was investigated in terms of first cleavage and blastocyst formation (Exp. 2). Relative mRNA abundance of multiple genes in blastocysts was analyzed for transcript abundance of genes related with metabolism (GLUT1, LDHA), oxidative response (MnSOD, GPX1), apoptosis (BAX, Bcl2), and developmental competence (CCNB1, IGF2R; Exp. 3). The results show there were no significant differences in maturation rate between 2 oxygen concentrations during in vitro maturation (83 v. 86%). It was thought that cumulus cells surrounding oocytes might have attenuated oxidative stress, but number of resulting blastocysts were (P < 0.05) increased in 5% IVC group when compared with 20% IVC group (18.67 v. 14.09%, respectively). Moreover, the M20C5 group (23.01%) had a beneficial effect on in vitro culture compared with M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. Total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each group altered the expression of genes in various patterns. Therefore, it could be concluded that high oxygen tension during in vitro maturation and low oxygen tension during in vitro culture might alter the expression of multiple genes related to oocyte competence and improve (P < 0.05) embryo development, but not blastocyst quality. This study was supported by MKE (#2009-67-10033839, #2009-67-10033805), NRF (#M10625030005-508-10N25), BK21 for Veterinary Science, IPET (#109023-05-1-CG000), and Hanhwa L&C.

287 RESCUE AND IN VITRO MATURATION OF FOLLICULAR OOCYTES IN CHINCHILLA LANIGER

2007 ◽  
Vol 19 (1) ◽  
pp. 259 ◽  
Author(s):  
G. Aiudi ◽  
M. Cinone ◽  
F. Maritato ◽  
A. De Sandro Salvati ◽  
M. E. Dell'Aquila
Keyword(s):  
Breeding Season ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Metaphase Plate ◽  
Meiotic Maturation ◽  
Current Therapy ◽  
Embryo Cryopreservation ◽  
Vaginal Smear ◽  
First Polar Body

The chinchilla is a hystricomorph rodent with a natural habitat in the Andes mountains of Chile (see review by Boussarie 2002 Proc. 27th WSAVA Congr.). For most of the chinchilla subspecies, the decline in the natural population can be attributed to human destruction of the native ecosystems and hunting for fur. Chinchillas are listed as a protected endangered species, at immediate risk of extinction. In Europe, chinchillas are reared for pets and fur production. The female has a seasonal polyestrous reproductive activity with a breeding season from November to May. The estrous cycle length is variable (28–41 days), with an estrous duration of 2 days. After a gestation of about 112 days, a litter of 1 to 6 young is born (see reviews by Morrow 1986 in Current Therapy in Theriogenology 2, W.B. Saunders; and Collot 1998 in Proc. I EVSSAR Congr.). Reproductive biotechniques in this species could play an important role in managing both captive and natural populations as well as in sustaining and improving genetic and global biodiversity. The specific aim of this preliminary work was to standardize an efficient in vitro maturation (IVM) procedure for Chinchilla laniger oocytes so that it will be possible, in the future, to perform IVF and embryo cryopreservation and transfer. Oocytes from 4 cyclic breeding females were recovered by slicing ovaries, obtained by ovariohysterectomy, and were matured in vitro according to the procedure described for bovine oocytes by Dell'Aquila et al. (2002 Mol. Reprod. Dev. 63, 210–222). Two trials of 2 estrous subjects each were performed, on the basis of behavioral signs of estrous and vaginal cytology (Harris-Schorr staining), in the early and late breeding seasons. During estrus, the vaginal smear consisted of superficial cells, further neutrophils, and small and large intermediates, whereas parabasal cells were not found. At the end of the culture time, oocytes were stained with Hoechst 33258 and evaluated for the stage of meiotic maturation. Three out of 4 oocytes recovered in November (75%) reached full meiotic maturation, showing the second metaphase plate and the first polar body (PB) extruded. The fourth oocyte, showing the first PB together with multiple pronuclear structures, was classified as activated. On the contrary, none out of 12 oocytes recovered in May reached full maturation. Of them, 7 (58%) remained at the germinal vesicle stage, 2 (17%) reached metaphase I, and 3 (25%) showed abnormally dispersed chromatin configuration. To our knowledge, this is the first study reporting that chinchilla oocytes can be matured in vitro by bovine IVM procedures. Even though the number of oocytes was poor, we can hypothesize that oocytes from C. laniger are best collected in the breeding season when subjected to an IVM technique.

scholarly journals In vitro maturation of domestic cat oocytes subjected to different incubation times

2021 ◽  
Vol 10 (3) ◽  
pp. e15710313074
Author(s):  
Denilsa Pires Fernandes ◽  
Fernanda Araujo dos Santos ◽  
Luã Barbalho de Macêdo ◽  
Roberta Gonçalves Izzo ◽  
Brenna de Sousa Barbosa ◽  
...  
Keyword(s):  
Incubation Period ◽  
Cell Expansion ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
First Polar Body ◽  
The Ideal

The aim of this study was to evaluate the effect of three different incubation times on in vitro maturation of domestic cat oocytes. Thus, ovaries (n = 42) were submitted to slicing procedure and the oocytes recovered were classified; only good quality oocytes (Grade I and II) underwent in vitro maturation for three different periods (24 vs. 30 vs. 36 h) in supplemented TCM-99 medium. After, oocytes were evaluated for cumulus cell expansion and presence of the first polar body. After six replicates (7 ± 1,7 ovaries per replicate), a total of 334 viable oocytes were recovered. Differences (p <0.05) were observed regarding the percentage of oocytes presenting expansion of the cumulus cells, where higher values were observed in the group of oocytes incubated for 36 h (84.3%), when compared to 30 (73.4%) and 24 h (71.0%). Moreover, differences were also observed regarding the presence of the first polar body (24 h: 29.7%; 30 h: 58.2%; 36 h: 69.8%). We conclude that the incubation period influenced the maturation rates, indicating 36 h as the ideal period for the in vitro maturation of domestic cat oocytes in supplemented TCM-199 medium.

scholarly journals The Number of LH Receptor could Predict the Success of Oocyte Maturity in the Process of In Vitro Maturation

2016 ◽  
Author(s):  
Adek Amansyah
Keyword(s):  
In Vitro Maturation ◽  
Clinical Laboratory ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Maturity ◽  
Lh Receptor ◽  
First Polar Body ◽  
Significant Difference

Objective: To evaluate the relationship between the number of LH receptor and the success of oocyte maturity in the process of in vitro maturation (IVM). Method: This experimental study was conducted in the Permata Hati Infertility Clinical Laboratory, Dr. Sardjito General Hospital, Yogyakarta, with the samples of 300 oocytes obtained through collecting immature cow’s oocytes from the abattoir and grouped the oocytes into 3 (three) groups based on the pattern of oocyte cumulus cells on the vesicle germinal stage 2 - 8 mm with three layers of cumulus cell. The sample of the cumulus cells from these three groups were taken and the LH receptor examination was done with immunohistochemistry. After that, the IVM process was performed to the three groups and its development for 24 hours was evaluated. Its maturation quality was evaluated with the emergence of the first polar body (1PB) and compared to the other groups and related to the number of LH receptor in the three groups. Result: The result of this study indicated that the oocyte cumulus cells showed a difference of function during IVM process. The maturity rate in this study showed that the number of LH receptor was related to the morphological pattern of oocyte cumulus cells with oocyte maturity. The maturity of the cumulus cells which 100% covered the oocyte was higher than that of the cumulus cells which > 50% and < 30% covered the oocytes, namely, 74% compared to 60% and 12%. The result of this study also showed that the average number of LH receptors in the three groups (A, B, and C) was 183.4, 78.8, and 24.0 respectively. A significant difference was found in the three groups (p < 0.0001). When related to IVM maturity, this difference showed that the bigger number of oocyte cumulus cells influenced the oocyte maturity. Conclusion: The number of LH receptor can be used as a prediction to determine the success of oocyte maturation in the process of in vitro maturation. [Indones J Obstet Gynecol 2013; 1-4:183-7] Keywords: IVM, LH receptor, oocyte cumulus cell

61 THE EFFECTS OF COLLECTION SEASON AND STORAGE DURATION IN LIQUID NITROGEN ON POST-WARMING SURVIVAL AND NUCLEAR MATURATION OF IMMATURE PORCINE OOCYTES PRESERVED BY SOLID SURFACE VITRIFICATION

2015 ◽  
Vol 27 (1) ◽  
pp. 123
Author(s):  
T. Nagai ◽  
T. Somfai ◽  
N. T. Men ◽  
H. Kabeko ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Solid Surface ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Storage Duration ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Duration Of Storage ◽  
And Storage

We investigated the effects of collection season and storage duration of vitrified porcine oocytes in liquid nitrogen (LN2) on their survival and maturation ability after warming. A total of 3338 cumulus-enclosed oocytes were vitrified using solid surface vitrification, preserved, and warmed according to previous report (Somfai et al. 2014 PLoS One 9, e97731) in 26 occasions between October 2012 and March 2014. Vitrified oocytes were stored in LN2 for various durations from 0 (vitrified but without storage) to 243 days. The date of preservation and length of storage (days) of vitrified oocytes in LN2 were recorded. Warming of vitrified oocytes was conducted on a hotplate set at 42°C. After warming, oocytes were subjected to in vitro maturation according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041). Then oocytes were denuded and their live/dead status and nuclear maturation were assessed under stereo microscope based on their morphology and the presence of the first polar body. After linear regression analysis, it was found that there was no correlation between the duration of storage of vitrified oocytes in LN2 for up to 243 days and their survival rate after warming (R = 0.254; P = 0.210) or the maturation rate of surviving oocytes (R = 0.147; P = 0.471). Vitrification during spring (March 1–May 31) resulted in significantly higher rates of survived oocytes compared with vitrification during winter (December 1–February 28; 86.9 and 73.1%, respectively; P < 0.05), whereas the mean survival rates of oocytes vitrified during summer (June 1–August 31; 79.0%) and autumn (September 1–November 31; 81.9%) did not differ significantly from those of other seasons (ANOVA). After in vitro maturation, nuclear maturation of surviving oocytes did not differ significantly among oocytes vitrified at different seasons (ranging between 59.1 and 67.8%). The results indicate that the oocyte collection season affects survival of vitrified oocytes, whereas storage duration in LN2 does not affect this parameter. Furthermore, nuclear maturation of oocytes that survive after vitrification and warming is not affected by their collection season and storage length.This work was supported by JSPS KAKENHI Grant Number 26870839.

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