137 Comparison of two invitro maturation media on polar body extrusion of cattle oocytes

2021 ◽  
Vol 33 (2) ◽  
pp. 176
Author(s):  
S. M. Sithole ◽  
M. L. Mphaphathi ◽  
M. D. Sebopela ◽  
T. L. Nedambale
Keyword(s):  
Imaging System ◽  
Polar Body ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Significant Difference ◽  
Maturation Rate ◽  
Aspiration Technique ◽  
Level Of Significance ◽  
Polar Body Extrusion ◽  
Quality Type

The invitro embryo production technique is one of the assisted reproduction technologies that has the potential in speeding up genetic improvement in cattle. The developmental competence of invitro-matured oocytes is influenced by several factors during invitro maturation (IVM), such as maturation environment, oocyte quality, type of media, and additives. The objective of the present study was to compare two IVM media (TCM-199 and BO-IVM) on cattle oocyte maturation rate invitro. Cattle ovaries were collected from local slaughterhouse and transported to the laboratory in a thermos flask containing 0.9% saline (Adcock Ingram Critical care) at 37°C. Oocytes were retrieved from the ovaries by the aspiration technique, and then matured invitro in 500µL of TCM-199 (supplemented with 10% fetal bovine serum, FSH, LH, and E2) or in 500µL of commercially available BO- IVM (Bioscience) medium, both covered with mineral oil for 22h at 38.5°C with 5% CO2 and 5% O2. After 22h of IVM, oocyte polar body extrusion was evaluated with the aid of Oosight Imaging System (Hamilton Thorne) connected to an inverted microscope. The total number of oocytes matured in TCM-199 and BO-IVM media were 401 and 396, respectively. The experiment was replicated 19 times. Data were analysed using the GenStat® program (VSN International). Means of different treatments were separated using Fisher’s protected t-test least significant difference at 5% level of significance. No difference was recorded for polar body extrusion rates in TCM-199 (50.6±13.9) and BO-IVM (47.3±13.7) media. In conclusion, TCM-199 and BO-IVM media did not differ in terms of maturation rate; thus, both can be used for successful cattle oocyte IVM.

138 Effect of different temperatures on invitro maturation rates on cattle oocytes

2021 ◽  
Vol 33 (2) ◽  
pp. 177
Author(s):  
M. D. Sebopela ◽  
M. L. Mphaphathi ◽  
S. M. Sithole ◽  
T. L. Nedambale
Keyword(s):  
Polar Body ◽  
International Comparisons ◽  
Treatment Groups ◽  
First Polar Body ◽  
Significant Difference ◽  
Different Temperatures ◽  
Maturation Rate ◽  
Statistical Program ◽  
Polar Body Extrusion ◽  
Incubation Temperatures

Climate change represents a major stressful environmental condition that compromises the reproductive efficiency of animals. The temperature affects various components of the maturation processes in cattle oocytes, this includes disruptions of oocyte development and maturation. Therefore, the study aimed to compare the effects of different incubation temperatures (32.5, 33.5 34.5, 35.5, 36.5, 37.5, 38.5, 39.5, 40.5, 41.5, 42.5, and 43.5°C) on the maturation rate of cattle oocytes. Cattle ovaries were collected from a local abattoir and transported to the laboratory in thermo flask at 37°C. Oocytes were recovered from ovaries using the aspiration method and matured using 500 uL of TCM-199 medium supplemented with 10% fetal bovine serum, FSH, LH, and E2 covered with mineral oil. The maturation rate of oocytes was determined by the extrusion of the first polar body after 22h of IVM period. The total number of 80 oocytes were matured in each group and replicated 4 times. Data were analysed using GenStat® statistical program (VSN International). Comparisons were considered significantly different (P<0.05) using Fisher’s protected least significant difference test. The oocyte polar body extrusion at 32.5 (0±0), 33.5 (4.5±5.2), 34.5 (22.0±1.4), 35.5 (29.5±7.6), 36.5 (41.5±3.1), 37.5 (55.5±4.1), 38.5 (62.2±3.3), 39.5 (49.5±3.3), 40.5 (17.3±6.4), 41.5 (10.3±5.7), 42.5 (8.2±5.9), and 43.5°C (5.7±7.8) (P<0.05) was recorded. The highest percentages of oocytes with polar body extrusion were at 37.5°C (55.5±4.1) and 38.5°C (62.2±3.3) and differed significantly from all other treatment groups (P<0.05). Temperature lower than 37.5°C and higher than 39.5°C did not intensify oocyte polar body extrusion. We concluded that the temperatures of 37.5°C and 38.5°C were suitable for IVM of cattle oocytes with acceptable polar body extrusion rates compared with other temperatures.

178 Evaluation of polar body extrusion following exposure of cattle oocytes to different concentrations of ethylene glycol cryoprotectant

2020 ◽  
Vol 32 (2) ◽  
pp. 217
Author(s):  
M. L. Mphaphathi ◽  
M. Nkadimeng ◽  
S. M. Sithole ◽  
M. D. Sebopela ◽  
F. V. Ramukhithi ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Toxicity Test ◽  
Imaging System ◽  
Osmotic Shock ◽  
Polar Body ◽  
Positive Control ◽  
Significant Difference ◽  
Polar Body Extrusion ◽  
Selection Of ◽  
Oocyte Selection

Cryoprotectant (CPA) toxicity has been recognised as a critical barrier to further advancement of cattle oocytes. Furthermore, CPA at high concentration might be toxic to oocytes, leading to osmotic shock and cell death (Arav et al. 1993). Selection of quality oocytes among a heterogeneous pool is mostly done subjectively. The use of Brilliant Cresyl Blue (BCB) stain for oocyte selection before ethylene glycol (EG) CPA toxicity test on cattle oocytes might be a useful tool. The objectives of this study were to elucidate the toxicity of EG penetrating CPA to cattle oocytes and the effectiveness of BCB on oocyte selection. Ovaries from cows of unknown reproductive status were collected from the local slaughterhouse and transported in warmed (37°C) saline water to the laboratory within 2h of slaughter. The oocytes (n=374) were exposed to 26mM BCB for 90min at 38.5°C under an atmosphere of 5% CO2. The other oocytes (n=450) were not exposed to BCB solution or CPA (positive control: no BCB and no CPA exposure). Oocytes were classified as BCB positive (+) with a varying degree of blue cytoplasm or BCB negative (−) with no blue cytoplasm. Oocytes were exposed in EG at different CPA concentrations as follows: Toxicity test 1 (TT1) was 0, 5, 10, and 15%, followed by exposure to TT2 as follows: 10, 20, and 30% (stepwise increased CPA). The oocytes were then invitro matured (IVM) as per treatment groups for 22h. After maturation, oocytes were removed from the maturation medium and denuded of granulosa cells by vortexing. The polar body extrusion was evaluated with the aid of Oosight Imaging System (Hamilton Thorne) connected to an inverted research microscope. Treatment means were compared using Fisher protected t-test least significant difference. There was a drastic decline of oocytes with polar body extrusion as CPA concentration increased (P<0.05): 40.5% (positive control, no BCB and no CPA exposure), 33.3% [(control, CPA exposure (EG 5 + 10%)], 23.1% [BCB− with CPA toxicity test (EG 5 + 10%)], 12.5% [(BCB− with CPA toxicity test (EG 10 + 20%)] and 4.9% [(BCB− with CPA toxicity test (EG 15 + 30%)]. The BCB+ groups (EG 5 + 10% and EG 10 + 20%) had significantly more oocytes with polar body extrusion (68.9% and 51.9%) compared with the positive control (40.5%), respectively (P<0.05). In conclusion, higher EG cryoprotectant concentrations compromise oocyte polar body extrusion following IVM. We recommend that BCB be used for selection of suitable oocytes before the CPA toxicity test because of its ability to stain larger and more competent oocytes from cattle.

scholarly journals In vitro maturation of goat oocytes selected using Brilliant cresyl blue staining

2021 ◽  
Vol 52 (4) ◽  
Author(s):  
Arya T. S. ◽  
Amritha Aravind ◽  
Abhilash R. S. ◽  
Jayakumar C. ◽  
Babitha V.
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Blue Staining ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Maturation Rate ◽  
Polar Body Extrusion

The present study was conducted to assess the developmental competence of goat oocytes selected using Brilliant cresyl blue (BCB) staining. Goat ovaries were collected from the slaughtered animals with unknown reproductive history. The oocytes retrieved by aspiration technique were selected based on morphology and subjected to BCB staining. Brilliant cresyl blue staining is based on the activity of glucose-6-phospahte dehydrogenase (G6PDH) enzyme synthesised by the oocytes. The cytoplasm remains blue in oocytes that have finished the growth phase (BCB+) while the growing oocytes remain colourless (BCB-). The stained and unstained oocytes were subjected to in vitro maturation separately to assess cumulus cell expansion index and polar body extrusion. A total of 206 culture grade oocytes were subjected to study, out of which, 76.75 ± 2.38 per cent of oocytes showed positive to BCB staining and 23.21 ± 2.38 per cent were negatively stained. Significantly higher maturation rate was observed in BCB+(92.89 ± 2.37%) oocytes than BCB-(29.72 ± 2.46%). The present study concluded that BCB staining can be used for selecting goat oocytes with good cytoplasmic maturation for further in vitro embryo production

Evaluation of oocyte quality: morphological, cellular and molecular predictors

2007 ◽  
Vol 19 (1) ◽  
pp. 1 ◽  
Author(s):  
Qiang Wang ◽  
Qing-Yuan Sun
Keyword(s):  
Transforming Growth Factor ◽  
Polar Body ◽  
Morphological Characteristics ◽  
Transforming Growth Factor Β ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Meiotic Spindle ◽  
Oocyte Competence ◽  
Cumulus Oocyte Complex ◽  
Glucose 6 Phosphate Dehydrogenase

Mounting evidence that oocyte quality profoundly affects fertilisation and subsequent embryo development drives the continued search for reliable predictors of oocyte developmental competence. In the present review, we provide an overall summary and analysis of potential criteria that can be used to evaluate oocyte quality. These criteria are specifically classified as morphological and cellular/molecular predictors. Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus–oocyte complex, polar body and/or meiotic spindle. Although the use of morphological characteristics as predictors of oocyte quality is controversial, such a grading system can provide valuable information for the preselection of oocytes with higher developmental competence and, therefore, may maximise embryo developmental outcome. Several intrinsic markers (such as mitochondrial status and glucose-6-phosphate dehydrogenase l activity) and extrinsic markers (such as apoptosis of follicular cells and levels of the transforming growth factor-β superfamily in follicular fluid or serum) have also been reported as useful indicators of oocyte competence and embryo quality. Compared with the morphological parameters, these cellular and molecular predictors of oocyte quality may prove to be more precise and objective, although further studies and refinement of techniques are needed.

277 FOLLICULAR GROWTH, SUBSEQUENT OVUM PICKUP, AND DOMINANT FOLLICLE REMOVAL IN COWS

2006 ◽  
Vol 18 (2) ◽  
pp. 246
Author(s):  
K. Imai ◽  
M. Tagawa ◽  
S. Matoba ◽  
M. Narita ◽  
K. Kanayama
Keyword(s):  
Formation Rate ◽  
Cumulus Cell ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Dominant Follicle ◽  
Cleavage Rate ◽  
Significant Difference ◽  
The Mean ◽  
Follicle Aspiration ◽  
Removal Group

The present study was designed to assess the recruitment of follicles after ovum pickup (OPU) and dominant follicle (DF) removal on the follicular wave after OPU in Holstein dry cows. Cows were reared under the same feeding and environmental conditions. In Experiment 1, follicle aspiration (>2 mm in diameter) by OPU using a 7.5-MHz linear transducer with needle (COVA needle; Misawa Medical, Tokyo, Japan) connected to an ultrasound scanner (SSD-1200; ALOKA, Tokyo, Japan) was performed in four cows. Then, ovaries were observed after OPU from Day 1 (Day 0 = the day of OPU) to Day 11 to assess the number of follicles developed. In Experiment 2, two sessions of OPU were performed with a 7 day interval between sessions, with or without dominant follicle removal, to assess the quality of developing follicles and oocytes. In the DF removal group, >8-mm follicles were aspirated at Day 5 after the first OPU session, and the same cows without DF removal were designated as a control (n = 4, crossover trial). Oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. To assess the developmental competence of oocytes, Grades 1 and 2 cumulus-oocyte complexes (COCs) were collected, matured, fertilized, and cultured as described by Imai et al. (2002 J. Vet. Med. Sci. 64(10), 887-891). Embryo development was assessed by the cleavage rate on Day 2 and the blastocyst formation rate on Days 7 to 9 (the day of insemination = Day 0). Data were analyzed by ANOVA or Student t-test. In Experiment 1, a dominant follicle (>8 mm in diameter) was developed during Days 3 to 5 after OPU in each donor. The mean number of developing follicles (>2 mm in diameter) were increased from Day 1 to Day 9 (Day 1: 7.5 � 2.1, Day 3: 19.0 � 1.2, Day 5: 23.3 � 9.0, Day 7: 30.3 � 11.0, Day 9: 42.0 � 15.8 and Day 11: 41.0 � 16.7 (mean � SD), P < 0.05). In Experiment 2, there was no difference in the mean number of developing follicles on the day of OPU and collected oocytes between DF removal and control groups (follicles: 47.8 � 23.0 and 39.3 � 6.2; oocytes: 27.0 � 11.6 and 26.5 � 5.4, respectively). The number of Grades 1 and 2 oocytes for the DF removal group was significantly higher (P < 0.05) than that for the control (83.6 � 1.5 and 63.2 � 14.2, respectively), and no significant difference was found within cleavage (60.0 � 37.2, 53.6 � 23.2) and blastocyst rates (34.1 � 33.9, 34.4 � 16.8). These results indicate that populations of follicles were increased till Day 9 after OPU, and the DF removal was effective at increasing oocyte quality in the developing follicles.

Effects of a combination of EGF and IGF-I on polar body extrusion and developmental competence of bovine oocytes

1997 ◽  
Vol 47 (1) ◽  
pp. 200
Author(s):  
M. Sakaguchi ◽  
T. Dominko ◽  
M.L. Leihfried-Rutledge ◽  
T. Nagai ◽  
N.L. First
Keyword(s):  
Polar Body ◽  
Developmental Competence ◽  
Igf I ◽  
Polar Body Extrusion ◽  
Bovine Oocytes

scholarly journals 146COMPARISON OF EMBRYO CULTURE MEDIA FOR BLASTOCYST DEVELOPMENT AFTER INTRACYTOPLASMIC SPERM INJECTION OF IN VITRO-MATURED EQUINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 195
Author(s):  
Y.H. Choi ◽  
D.D. Varner ◽  
K. Hinrichs
Keyword(s):  
In Vitro Culture ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Media ◽  
Polar Body ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Significant Difference

Research on in vitro culture of equine embryos has been scant, due to failure of equine in vitro fertilization to be repeatably successful. We have recently obtained high fertilization rates of equine oocytes via intracytoplasmic sperm injection (ICSI) using a piezo drill (Choi et al., 2002 Reproduction 123, 455–465). Culture of presumptive zygotes in G1.2/2.2 medium resulted in 63% cleavage and an average of 15 cells at 4d, but only 2 to 9% blastocyst development at 7 days (Choi et al., 2003 Theriogenology 59, 1219–1229). In the present study, we evaluated the effect of two different culture media, G1.3/G2.3 v. DMEM/F-12, with or without FBS, on blastocyst development after ICSI. Oocytes were collected from slaughterhouse-derived ovaries by follicular scraping and were matured in vitro for 24h in M199 with 10% FBS and 5μUmL−1 FSH. After culture, oocytes having a polar body (198/305; 65%) were fertilized by ICSI with frozen-thawed equine sperm using a piezo drill. Presumptive zygotes were cultured in 1 of 4 media: G1.3/G2.3 (which includes 0.8% BSA) with or without 10% FBS, or in DMEM/F-12 with 0.5% BSA, with or without 10% FBS. Culture was performed in microdroplets at 5μL/zygote under oil at 38.2°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 7.5 days. In G1.3/2.3 treatments, G1.3 media were completely refreshed at 48h, zygotes were transferred to G2.3 (with or without FBS as per the first stage) at 96h, and were completely refreshed with the same media at 144h. In DMEM/F-12 treatments, media were completely refreshed every other day. Three to 5 replicates were performed in each treatment, and data were analyzed by chi-square test. There were no significant differences in cleavage rates (59–64%) among treatments. The rate of development to blastocyst, per oocyte injected, in G1.3/G2.3/BSA (1/49, 2%) was significantly lower (P&lt;0.05) than that for the other three treatments: G1.3/2.3/BSA/FBS (9/49, 18%), DMEM/F-12/BSA (9/50, 18%), or DMEM/F-12/BSA/FBS (10/50, 20%). There was no significant difference in blastocyst development among the latter three treatments. These findings indicate that G1.3/2.3 media with BSA only do not adequately support growth of equine embryos. Development of up to 20% of injected oocytes to the blastocyst stage in G media supplemented with FBS, in DMEM/F-12/BSA or in DMEM/F-12/BSA/FBS represents the highest in vitro equine blastocyst rate in medium alone (i.e. without co-culture) yet reported. The success of DMEM/F-12 as an embryo culture medium may provide a relatively simple basis for equine in vitro culture programs. To determine whether this medium was able to support further developmental competence, we cultured equine embryos resulting from nuclear transfer of in vitro-matured oocytes in DMEM/F-12+10% FBS (without BSA). We transferred 4 resulting blastocysts to recipient mares by transcervical transfer; one pregnancy is ongoing at 230d gestation at the time of this writing. This work was supported by the Link Equine Research Endowment Fund, Texas A&amp;M University.

scholarly journals 298RED DEER (CERVUS ELAPHUS) PARTHENOGENETIC BLASTOCYSTS PRODUCED USING IONOMYCIN/6DMAP ACTIVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 268 ◽  
Author(s):  
S.E. Beaumont ◽  
D.K. Berg ◽  
G.W. Asher
Keyword(s):  
Red Deer ◽  
Polar Body ◽  
Negative Control ◽  
Blastocyst Development ◽  
Cell Counts ◽  
First Polar Body ◽  
Significant Difference ◽  
Chi Square Analysis ◽  
Polar Body Extrusion

Successful activation of red deer oocytes is a necessary prerequisite for the cloning of red deer individuals with desirable genetic characteristics. To investigate this, an established biphasic protocol used for oocyte activation in sheep was investigated for suitability. The method chosen was 5μM Ionomycin for 5min followed by 2mM 6DMAP for 3h ( Loi P et al., 1998 Biol. Reprod. 58, 1177–1187). The medium used during activation and subsequent culture was Deer Synthetic Oviduct Fluid, which has been shown to support routine in vitro fertilization and blastocyst development (15%) of in vitro-matured red deer oocytes (DSOF, Berg D et al., 2003 Theriogenology 59, 189–205). Red deer abattoir-derived COCs were matured in vitro for 22h before random allocation across 3 treatment groups comprising a standard IVF group, the activation group and a negative control group exposed to medium only. Activation treatment oocytes were stripped of cumulus by vortexing in 0.1% hyaluronidase before selecting for first polar body extrusion. First-step activation was performed in medium comprising HEPES-buffered IVF-DSOF containing 4mM Ca2+. Second-step activation used 3mM Ca2+ early DSOF under 7% O2, 5% CO2, and 88% N2 at 38.5°C. Standard IVF was conducted at 23h post-IVM using 4mM Ca2+ IVF-DSOF and 0.5×106mL−1 final sperm concentration. Following activation and IVF, oocytes were washed 3 times in HEPES DSOF before culture for 7 days in sequential DSOF with late DSOF on Day 4 containing 1.5mM Ca2+. Cleavage was assessed 24h after activation, and all blastocysts were fixed for cell counts. Four replicates of each treatment were performed. Cleavage and blastocyst rates were examined by chi-square analysis and cell numbers by ANOVA. First polar body extrusion rate was 84%. Cleavage was similar between the activation treatment and IVF (P&gt;0.05 ); but a significant difference was found in blastocyst development rates (P&lt;0.05) with the Ionomycin and 6DMAP protocol being superior to the IVF treatment. Exposure to high Ca2+ media alone resulted in only 5% of the negative control oocytes cleaving to 2 cells. Results show that Ionomycin and 6DMAP are effective in activating red deer oocytes and DSOF is a suitable medium to produce parthenogenetic blastocysts.

scholarly journals 301EFFECT OF THE MII-AGE AND ACTIVATION PROTOCOL ON THE PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 270
Author(s):  
I. Lagutina ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Cytochalasin B ◽  
Average Rate ◽  
Polar Body ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Average Cell ◽  
Electric Impulse ◽  
Polar Body Extrusion

The completion of porcine oocyte nuclear maturation (MII) in vitro, characterized by the time of polar body extrusion, starts at about 32h of maturation and lasts more than 12h. This leads to the simultaneous presence in the population of matured oocytes with differing abilities to be activated. We investigated age-dependent changes in pig oocyte maturation, activation and development in SOFaa in response to electric impulse (EL) in the presence of cytochalasin B (CB) and EL in combination with cycloheximide and cytochalasin B (EL+CHX+CB). Oocytes were matured in TCM 199 with 10% FCS, cysteine, LH, FSH (Pergovet, Serono, Geneva, Switzerland) for 36h and then decumulated. Matured oocytes were activated at 40 and 44h by double pulse of 30μs DC 1, 5kVcm−1 and cultured in 5μgmL−1 CB for 4h or by EL followed by incubation in 10μgmL−1 CHX+5μgmL−1 CB for 4h. According to the MII-age before activation oocytes were divided into 2 age classes: 3–7 and 7–11h after polar body extrusion. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The rates of cleavage, blastocyst formation and cell number of BL on Day 7 (BLD7) were recorded. Our results showed that the average rate of maturation at 44h was 72% (n=1377). About 50% and 87% of oocytes, that eventually matured, extruded the polar body at 37 and 40h, respectively. The average cell number of BLD7 developed in SOFaa was 80±36 (n=52) and was not affected by activation protocol. Seventy-nine and 27% of BL had more than 50 and 100 cells per BL, respectively. Porcine oocytes activated by EL acquired their developmental competence gradually, achieving the highest rates of cleavage and blastocyst formation 7h after polar body extrusion. By contrast, oocytes activated by EL+CHX+CB showed their maximal developmental competence earlier (3–7h group). In conclusion, we demonstrate that electric impulse in combination with CHX+CB treatment permits earlier efficient activation of porcine oocytes (3–7h after polar body extrusion).

Vitrification of in vitro matured oocytes of Mangalica and Large White pigs

2008 ◽  
Vol 56 (3) ◽  
pp. 399-410 ◽  
Author(s):  
Erika Varga ◽  
Erzsébet Gajdócsi ◽  
Brigitta Petz Makkosné ◽  
Ildikó Salamon ◽  
Ágnes Bali Papp
Keyword(s):  
Embryonic Development ◽  
Developmental Competence ◽  
Large White ◽  
Normal Morphology ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Maturation Rate ◽  
Morula Stage ◽  
Potential Tool

The breeding of Mangalica, a native pig breed in Hungary, had been started in 1833, but this pig breed almost became extinct in Hungary in the past decades. In 1991, the number of sows was only 200. Although in these days the existing Mangalica population consists of more than 6000 animals representing different colour variations, the preservation of this traditional pig breed is still very important. Vitrification is a potential tool for the preservation of gametes and embryos of these animals. The aim of this study was to investigate the effects of vitrification on the developmental competence of Mangalica (M) and Large White (LW) oocytes following fertilisation. The oocytes were vitrified by the Open Pulled Straw (OPS) method using different concentrations of ethylene glycol and dimethyl sulphoxide as cryoprotectants. After rehydration the oocytes underwent in vitro fertilisation; the resultant zygotes were then cultured in vitro for four days to assess embryonic development. In the first experiment, in vitro maturation of M and LW oocytes was compared. No significant difference was observed in the nuclear maturation rate of LW and M oocytes. In the second experiment, the sensitivity of oocytes to vitrification was examined by evaluating oocyte morphology after thawing. A higher percentage of LW oocytes showed normal morphology compared to M oocytes, indicating that Mangalica oocytes are more sensitive to cryoprotectants than Large White oocytes. After warming and in vitro fertilisation, more than 50% of the oocytes started embryonic development and by the end of the incubation period morula stage embryos had developed in both groups. The results show that the OPS vitrification technique is well suited to preserve Mangalica oocytes and from these oocytes morula embryos can be produced.

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