Cleavage Rate Of Oocytes Research Articles

Prolactin affects bovine oocytes through direct and cumulus-mediated pathways

The available evidence points to participation of PRL in regulation of mammalian oocyte maturation. The aim of the present study was to characterize pathways of PRL action on bovine oocytes. We analyzed (1) the presence of the PRL receptor and its mRNA isoforms in oocytes and cumulus cells; (2) the effect of PRL on meiosis resumption and the role of cumulus cells, the NO/NO synthase system, protein kinase C, and tyrosine kinases in this effect; and (3) PRL effects in the presence of gonadotropins on the developmental capacity of cumulus-free and cumulus-enclosed oocytes. The transcript and protein expression of the PRL receptor in the cells were detected by reverse transcription polymerase chain reaction and immunocytochemistry, respectively. The nuclear status of oocytes was assessed after culture of cumulus–oocyte complexes (COCs) and denuded oocytes (DOs) with or without PRL (5–500 ng/mL) for 7, 14, or 24 hours. Besides, DOs were incubated for 7 hours in the absence or the presence of PRL (50 ng/mL) and/or L-NAME (an inhibitor of NO synthase), genistein (an inhibitor of tyrosine kinases), or calpostin C (a protein kinase C inhibitor). After IVM in 2 different systems containing PRL (50 ng/mL) and/or gonadotropic hormones, a part of oocytes underwent IVF and IVC and the embryo development was tracked until the blastocyst stage. Messenger RNA of long and short isoforms of the PRL receptor was revealed in both oocytes and cumulus cells. Immunocytochemistry confirmed the presence of the PRL receptor in oocytes and the cumulus investment. In the absence of gonadotropins (system 1), PRL retarded meiosis resumption in DOs but not in cumulus-enclosed oocytes, with this effect being short term, dose dependent, suppressed by L-NAME and genistein, and unaffected by calpostin. In systems containing gonadotropins, PRL did not affect nuclear maturation and the cleavage rate of cumulus-free and cumulus-enclosed oocytes. However, in the case of COCs, it raised the blastocyst yield both in system 2 (from 20.5%–40.9%, P < 0.01) and in system 3 (from 21.7%–33.9%, P < 0.05). The findings show for the first time the functioning of the direct pathway of PRL signaling into bovine oocytes, as confirmed by the expression of receptors of PRL and its direct meiosis-retarding effect involving activation of tyrosine kinases and NO synthase. Furthermore, this is the first demonstration that the beneficial effect of PRL on the oocyte developmental capacity is achieved via cumulus cells containing PRL receptors.

IGF-I slightly improves nuclear maturation and cleavage rate of bovine oocytes exposed to acute heat shock in vitro.

An in vitro model of embryo production was used to examine the effects of insulin-like growth factor (IGF)-I on maturation and developmental competence of oocytes exposed to heat shock. Cumulus-oocyte complexes were matured at 38.5°C or exposed to acute heat shock (HS; 41.5°C), with or without 100 ng/ml IGF-I, for 22 h through in vitro maturation. The experimental groups were control (C), C + IGF-I, HS, and HS + IGF-I. Oocytes were fertilized at the end of maturation, and the proportion of cleaved embryos was recorded 44 h later. HS during maturation increased the proportion of TUNEL-positive oocytes (P < 0.05). HS did not have any effect on cortical granule translocation but impaired resumption of meiosis, expressed as a decreased proportion of oocytes with nuclei in metaphase I (P < 0.05) and metaphase II (MII; P < 0.05). HS decreased the proportion of oocytes that cleaved (P < 0.05), in particular those oocytes that further developed to 4-cell-stage embryos (P < 0.05). IGF-I alleviated, to some extent, the deleterious effects of HS on the oocytes as reflected by a reduced proportion of TUNEL-positive oocytes (P < 0.03). While not significant, IGF-I tended to increase the proportion of MII-stage oocytes (P < 0.08) and 4-cell-stage cleaved embryos (P < 0.06). Further examination is required to explore whether IGF-I also affects the developmental competence of oocytes exposed to HS.

Effect of vitrification and slow freezing on in-vitro matured prepubertal goat oocytes

The present study was conducted to study the post thaw recovery rate of morphologically normal matured oocytes subjected to vitrification and slow freezing method. Oocytes were matured in TCM-199, which comprises sodium pyruvate (0.25 mM), L- glutamine (1 mg/10 ml), gentamycin (50 μg/ml) and supplemented with 10% fetal bovine serum at 38.5°C in CO2 incubator under humidified air. After 27 h of in-vitro maturation oocytes were assigned for freezing, viz. vitrification, slow cooling and control cooling. The percentage of oocytes recovered after thawing was significantly higher for control than those for vitrification (69.67%) and slow freezing (85.00%), respectively. Moreover, the cleavage rate in vitrification, slow freezing and control were 1.14, 3.92 and 13.37%, respectively. Thus, the cleavage rate of oocytes in vitrification and slow freezing method did not differ significantly. However, there was a significant difference in cleavage rates of freezing groups and the control groups.

In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up–derived oocytes have different kinetics and requirements regarding maturation media

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived oocytes.

156 EFFECTS OF MORPHOLOGY TYPE OF POLAR BODY ON PORCINE OOCYTE QUALITY AND DEVELOPMENTAL POTENTIAL AFTER IN VITRO FERTILIZATION

Evaluation of morphology of first polar body (1st PB) could be a method for the oocyte's quality and developmental competence. The developmental potential of oocyte with fragmented PB after in vitro maturation (IVM) is a controversial issue. The aim of this study is to investigate the effects of PB morphology type on oocyte quality and developmental competence after IVF. Porcine ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to the laboratory within 2 h in physiological saline supplemented with 100 IU mL–1 penicillin G and 100 mg mL–1 streptomycin sulfate. The cumulus–oocyte complexes (COC) were aspirated using an 18-gauge needle attached to a 10-mL disposable syringe from superficial follicles 3 to 6 mm in diameter followed by IVM. After IVM, oocytes were classified into 3 types as follows, oocytes with normal PB (A type), oocytes with a little of fragmented PB (B type), and oocytes with separated 2 PBs (C type), respectively. As classification of PB types, we analysed the distribution ratio of each PB type after IVM, and then performed IVF for analysis of fertilization rate and developmental potential. The ratio of oocyte with A type (73%) was significantly (P &lt; 0.05) higher than that of B type (24.5%) or C type (2.5%) after IVM. Only mature oocytes were selected from A and B type and were subjected to IVF because of a small number of oocytes with C type. In the IVF experiment, the efficiency of monospermy and fertilization were significantly higher in oocytes of A type (46.7%) than those of type B (20.0%). The cleavage rate of oocytes with A type (63.9%) was significantly (P &lt; 0.05) higher than the oocytes with B type (43.8%). Embryonic developmental competence to the blastocyst stage after IVF was significantly (P &lt; 0.05) higher in the A-type oocytes (26.3%) than in the B-type oocytes (16.9%). The levels of glutathione and reactive oxygen species were not affected by the morphological classification of the PB. In summary, these results suggest that polar body morphology could be a marker of oocyte quality after IVM. We are currently studying gene expression of each oocytes and blastocysts. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

Beneficial Effect of an Oral Antioxidant Supplementation (Fertimax2) on IVF-ICSI Outcomes: A Preliminary Clinical Study

Background: Considerable evidence points towards a significant role of oxidative stress (OS) in the pathogenesis of sperm dysfunction. OS as a result of an inappropriate balance between oxidants and antioxidants in the semen can cause DNA damage and lipid peroxidation leading to failure of conception, miscarriage or potentially even childhood cancer. The objective of this study was to investigate whether a male antioxidant therapy can improve semen parameters and the results of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Methods: A total of forty eight infertile couples were considered. Male participants were administrated Fertimax2 antioxidant treatment for at least two months prior to their partner’s IVF-ICSI cycle. Sperm parameters (volume, concentration, progressive motility) and the IVF-ICSI outcomes were compared before and after the antioxidant treatment. The primary outcome measures were oocyte fertilization, cleavage and good embryo quality rates; the secondary outcomes were biochemical pregnancies, clinical pregnancies and implantation rates. Results: The principal finding that emerged from this study was that antioxidant therapy resulted in significant improvements in fertilization (p = 00.2), cleavage (p = 0.004) and good-embryo quality (p = 0.002) rates accompanied by a marked increase in clinical pregnancy (18.7% versus 2.5%) and implantation (11.8% versus 1.02%) rates. No significant changes in routine sperm parameters were observed. Conclusion: The Fertimax2 antioxidant therapy appears to influence favorably chances of conception in couples undergoing assisted reproduction treatment (ART).

13 MOTILITY CHARACTERISTICS OF SPERMATOZOA FROM BULLS GRAZING TALL FESCUE PASTURES

Fertilisation is less than expected with spermatozoa from bulls consuming toxic endophyte-infected (E+) tall fescue. The objective of this study was to evaluate motility characteristics of spermatozoa from bulls grazing tall fescue pastures using computer-assisted sperm analysis (CASA). Semen was collected from six Angus bulls (average age = 15.1 ± 0.04 months) during a three-month grazing study. Bulls grazed Kentucky 31 tall fescue (Festuca arundinacea Schreb.) infected with Neotyphodium coenophialum, an ergot alkaloid-producing endophyte (n = 3), or Jesup tall fescue with Max-Q™ (NTE), a non-ergot alkaloid producing endophyte (n = 3), and grouped by body weight and scrotal circumference to graze pastures from April 18 to June 26. Semen was collected once per week between 0600–0800 h beginning in mid-May and ending the last week of June. Gross motility and morphology was evaluated before extending with Bioxcell® animal protein-free formula (IMV, Aigle, France) and antibiotics (CSS 100, 2% of total volume). Extended semen was then evaluated using CASA to determine final dilution and packaged into straws (20 million sperm/straw), where equilibration occurred over 3 h in a cold room at 4°C. Straws were frozen for 7 min in static vapor of liquid nitrogen and plunged into goblets filled with liquid nitrogen. Semen was thawed and assessed using CASA at 0 and 3 h post-thaw. Data were analysed as a randomised block design with the fixed effects of treatment, blocking on semen collection date, utilising the mixed models procedure of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). Data were tested for normality (Shapiro-Wilk W ≥ 0.90), and treatment differences were determined using F-protected least significant differences. Path velocity (P = 0.001) and progressive velocity (P = 0.003) were lower in spermatozoa from bulls grazing E+ during the last 2 weeks of collection in June independent of time of assessment post-thaw. Sperm head area decreased in size in spermatozoa from E+ grazing bulls at 3 h post-thaw (P = 0.04) compared with NTE grazing bulls. Percent of rapid (progressive % with path velocity &gt;50 μm s–1) and medium (progressive % with path velocity &lt;50 μm s–1 but &gt; 30μm s–1) velocity spermatozoa was decreased for E+ grazing bulls compared to NTE grazing bulls (P &lt; 0.0001 and P = 0.004, respectively) and was accompanied by an increase in static (immobile) spermatozoa from E+ bulls (P &lt; 0.0001). These findings indicate that spermatozoa movement and velocity are impaired in bulls grazing E+ tall fescue pastures compared to bulls grazing NTE tall fescue pastures after the freeze and thaw process, which may explain decreased fertilisation and cleavage rates of oocytes co-incubated with these spermatozoa.

Prematurational Culture with 3-Isobutyl-1-methylxanthine Synchronizes Meiotic Progression of the Germinal Vesicle Stage and Improves Nuclear Maturation and Embryonic Development in &lt;i&gt;In Vitro&lt;/i&gt;-grown Bovine Oocytes

The objective of this study was to clarify the effects of prematurational culture (pre-IVM) supplemented with 3-isobutyl-1-methylxanthine (IBMX) on nuclear and cytoplasmic maturation of in vitro-grown bovine oocytes. In experiment 1, oocytes (95 μm in diameter) derived from early antral follicles (0.5–1 mm in diameter) were cultured for 12 days for in vitro growth (IVG). IVG oocytes with a normal appearance were subjected to examinations of diameter and chromatin structure in the germinal vesicle (GV) before IVM. In addition, percentages of metaphase II (M II) were examined after IVM. Regardless of pre-IVM, the mean diameters of IVG oocytes were about 115 μm. The proportions of GV3 (50.0%) and M II stages (80.1%) of IVG oocytes with pre-IVM were higher than those without pre-IVM (28.0 and 49.4%, respectively). In experiment 2, the fertilizability and developmental competence of IVG oocytes were examined. Regardless of pre-IVM, the normal fertilization rates of IVG oocytes were similar (around 70%) but were lower than that of in vivo-grown oocytes (88.0%). Cleavage and blastocyst rates of IVG oocytes with pre-IVM (63.0 and 26.1%, respectively) were higher than those without pre-IVM (45.8 and 12.7%, respectively). The blastocyst rate based on cleaved IVG oocytes with pre-IVM (41.7%) was similar to that of in vivo-grown oocytes (48.7%), although the cleavage rate of IVG oocytes with pre-IVM was lower than that of in vivo-grown oocytes. In conclusion, pre-IVM with IBMX improved the maturational and developmental competences of IVG oocytes, probably due to promotion of their chromatin transition and synchronization of meiotic progression.

The effects of meiotic stage on viability and developmental capability of goat oocytes vitrified by the Cryoloop method

In order to evaluate the effects of meiotic stage on survival of vitrified goat oocytes, the Cryoloop method was used to cryopreserve goat immature germinal vesicle (GV) and meiosis II (MII) oocytes following in vitro maturation (IVM). The oocytes collected from local slaughter house were divided into two parts. In the first part, the immature cumulus oocyte complexes (COCs) were further divided into three groups: (1) untreated (control), (2) exposed to the vitrification and dilution solutions but without being plunged into liquid nitrogen (toxicity), or (3) vitrified by the Cryoloop method (vitrification). In the second part, the MII oocytes produced by IVM of immature COCs were also divided into three groups as the above immature COCs. Oocytes survival was assessed by morphological appearance, nuclear maturation, and developmental capability following parthenogenetic activation (PA). The experiment was repeated three times. Our data indicated the rates of oocytes with normal appearance and developmental capability in the toxicity or vitrification group were decreased as compared to the control oocytes. In the toxicity group, the rate of GV oocytes with normal morphology was 76.25%±3.37% and significantly less than that of MII oocytes (92.18%±1.94%, P<0.05). However, the cleavage rate of MII oocytes was not significantly different from that of GV oocytes in the toxicity group (68.23%±1.71% vs 67.59%±3.51%, P>0.05). Additionally, the percentage of GV oocytes developing to blastocyst was significantly less than that of MII oocytes in the toxicity group (13.84%±2.81% vs 29.78%±4.17%, P<0.05). The rate of vitrified/thawed GV oocytes with normal morphology was 60.37%±2.91% and significantly less than that of MII oocytes (82.91%±3.01%, P<0.05). Additionally, the cleavage rate of GV oocytes was also significantly less than that of MII oocytes in the vitrification group (42.81%±2.94% vs 57.91±1.06%, P<0.05). However, the blastocyst rate of vitrified/thawed GV oocytes was not significantly different from that of MII oocytes (8.51%±1.46% vs 12.41%±3.74%, P>0.05). In conclusion, although vitrification can greatly damage the structure and development capability of goat oocytes, the Cryoloop method can result in acceptable levels of survival and development of goat oocytes. Additionally, compared to immature GV goat oocytes, mature MII oocytes may be more tolerant to the vitrification process.

Dietary α-linolenic acid from flaxseed oil improved folliculogenesis and IVF performance in dairy cows, similar to eicosapentaenoic and docosahexaenoic acids from fish oil

The objectives of this study were to determine the differential incorporation of various omega-3 (n-3) fatty acids (FAs) supplemented to dairy cows into ovarian compartments and assess the effects on IVF. Forty-two 256-day pregnant cows were supplemented with encapsulated fats, in treatments designated as i) SFA - saturated fat at 240 and 560 g/day per cow, prepartum and post partum (PP) respectively; ii) FLX - flaxseed oil at 300 and 700 g/day per cow prepartum and PP respectively; and iii) FO - fish oil at 300 and 700 g/day per cow prepartum and PP respectively. Commencing at 60 days in lactation, ovum pickup (OPU) was performed twice weekly (20 sessions; five cows per group) and in vitro maturation and IVF were conducted. The proportion of α-linolenic acid (ALA) was greater in follicular fluid (FF), granulosa cells, and cumulus-oocyte complexes (COCs) of FLX cows than in other groups (P<0.001). The proportion of docosahexaenoic acid (DHA) was 6.7 times as great in FF of FO as in other groups (P<0.001); docosapentaenoic acid n-3 and DHA were detected in COCs of FO but not in others. The follicle number during OPU was higher in FLX and FO than in SFA (P<0.05), and the oocyte cleavage rate was higher in FLX and FO than in SFA (P<0.01). Also, the percentage of oocytes that developed to blastocysts tended to be higher in both n-3 groups than in SFA (P<0.1). In conclusion, both dietary n-3 FAs similarly improved folliculogenesis and IVF performance; therefore, ALA-rich botanical n-3 seems to be a satisfactory approach to improve oocyte quality.

Comparison between different combinations of chemical treatment on parthenogenetic activation of mouse oocytes and its subsequent embryonic development

Combination of different chemicals has been an effective method to activate oocytes. The objectives of this study were to evaluate the optimal: (1) concentration (Experiment 1) and duration (Experiment 2) of strontium chloride (SrCl2) in the presence of cytochalasin B (CB), (2) secondary activation agent [6-dimethylaminopurine (6-DMAP), cycloheximide (CHX) and CB] in combination with calcium ionophore (CaI) (Experiment 3) and (3) ethanol (EtOH) concentration in combination with 6-DMAP (Experiment 4). There were no significant (P>0.05) differences in cleavage (86.82–89.06%) and blastocyst (33.42–46.83%) rates when oocytes were treated with various concentrations of SrCl2 (2–10 mM). Three-hour duration generally showed the highest cleavage (93.83%) and blastocyst rates (60.42%) compared to other durations. Comparing with various combinations, CaI +6-DMAP showed the highest cleavage at all stages of development and significantly (P<0.05) higher from other combinations at 8-cell (50.58%), morula (30.29%) and blastocyst (23.28%) stages. When comparing the EtOH concentrations, 8% EtOH gave significantly (P<0.05) highest cleavage rates at 8-cell stage (39.70%), morula (21.50%) and blastocyst (13.86%) stages followed by 9% and 7% EtOH. In conclusion, 10 mM SrCl2+5 µg/ml CB in calcium-free Chatot Ziomek Bavister medium for 3-h incubation duration gave the highest cleavage rates of parthenotes mouse oocytes.

In vitro development of ovine preantral follicles and oocyte cleavage rate are not affected by long-term ingestion of detoxified castor meal

In the present study, we evaluated the effect of long-term ingestion of detoxified castor meal on in vitro development of sheep preantral follicles as well as on the developmental competence of oocytes from antral follicles. Ovarian fragments were cultured for one or seven days and further analyzed by histology and fluorescence microscopy (experiment 1). The cumulus oocyte complexes (COCs) obtained from antral follicles were matured in vitro and activated by parthenogenesis or fertilization in vitro (experiment 2). Even after 1 day of culture, in both tested groups, a significant reduction in the percentage of primordial follicles and a concomitant increase in the percentage of intermediate follicles were observed when compared to non cultured tissue. After 7 days of culture, the percentage of primary follicles in both tested groups was significantly higher when compared to the non cultured tissue or the tissue cultured for 1 day (P<0.05). The number of in vitro embryos produced was similar between the tested groups. However, for those animals fed with detoxified castor meal, the parthenogenesis method resulted in a higher number of embryos (morulae) when compared to in vitro fertilization (IVF) activation. In conclusion, detoxified castor meal can be used as an alternative protein source without affecting the ovine preantral and antral folliculogenesis.

144 EFFECTS OF CO-CULTURE WITH FIBROBLASTS AND OVIDUCT CELLS ON IN VITRO PRODUCTION OF PORCINE EMBRYOS

Cell co-culture during in vitro maturation or embryo culture has been reported as a method to improve the efficiency of maturation or embryo development (Kidson et al. 2003 Theriogenology 59, 1889; Romar et al. 2005 Anim. Reprod. Sci. 85, 287). Here, we present the impact of different methods of co-culture with mouse embryonic fibroblasts or oviduct epithelial cells on in vitro embryo production in pigs. Cumulus–oocyte complexes (COC) were collected from follicles with diameter larger than 3 mm and used for in vitro embryo production based on the method of Kikuchi et al. 2002 (Biol. Reprod. 66, 1033) with minor modifications. There were 8 groups; group 1: maturation and embryo culture without cell co-culture (control group); group 2: maturation in the presence of fibroblasts; group 3: embryo culture in the presence of fibroblasts; group 4: both maturation and embryo culture in the presence of fibroblasts; group 5: maturation in the presence of oviduct cells; group 6: embryo culture in the presence of oviduct cells; group 7: both maturation and embryo culture in the presence of oviduct cells; group 8: both maturation and embryo culture in the presence of both fibroblast and oviduct cells. In vitro maturation (IVM) was carried out at 39oC under 5% CO2 in air for 44 h using NCSU-37 as basic medium. Matured oocytes were inseminated using epididymal frozen semen in IVF medium modified Pig-FM supplemented with 2 mM caffeine and 5 mg mL–1 bovine serum albumin (Kikuchi et al. 2002). The percentage of cleaved embryos and percentage of cleaved embryos which developed to the compact morula and early blastocyst stage were recorded. Results were analysed by one-way ANOVA followed by Dunnett’s test. To investigate the effects of co-culture with mouse embryonic fibroblasts and oviduct epithelial cells on oocyte maturation, some COCs cultured in groups 1, 2, 5, and 8 were fixed to assess their nuclear maturation to the metaphase II stage. The rate of matured oocytes in the groups 2, 5, and 8 was 76.85 ± 3.39% (n = 102), 79.11 ± 3.75% (n = 64), and 81.84 ± 3.93% (n = 66), respectively; these rates were increased significantly compared to the group 1 (55.87 ± 1.88%, n = 94; P &lt; 0.05). The effect of co-culture on the fertilization and embryo development is shown in Table 1. Our results indicate that co-culture increases the rates of embryonic cleavage in all groups by comparison with the control group. However, a significant increase in the rate of morula-blastocyst was only observed when embryos were co-cultured with fibroblasts or when both maturation and culture were performed in co-culture with either fibroblasts or oviduct cells (groups 3, 4, 7, and 8). The most important increase in morula-blastocyst rate was recorded for the group of embryos co-cultured with fibroblasts (group 3). In conclusion, the co-culture with fibroblast or oviduct cells during maturation can improve oocyte maturation and cleavage rate, while co-culturing the embryos with fibroblasts seems sufficient to improve both the cleavage and the morula-blastocyst rates. Table 1.Effect of co-culture on the fertilization and embryo development in pig Supported by a grant from the NAFOSTED VN.

185 PARTHENOGENETIC ACTIVATION OF SIKA DEER (CERVUS NIPPON TEMMINCK) OOCYTES WITH CHEMICALS

Parthenogenetic activation of the oocyte represents an important step in the somatic cell nuclear transfer. The aim of the present study was to establish optimizing conditions for parthenogenetic activation of Sika deer oocytes necessary for cloning Sika deer. Sika deer ovaries were collected from a slaughter house during oestrus season (October and November), placed into saline (25°C) supplemented with 1% (v/v) penicillin and streptomycin and transported into the laboratory within 4 h. The small vesicular follicles (diameter, 2–5 mm) on the ovarian surface were incised with a scalpel in a Petri dish containing PBS to release the cumulus–oocyte complexes (COC). Only COC with uniform cytoplasm and at least 3 layers of compact cumulus cells were cultured in vitro for 24 h. The media of in vitro maturation (IVM) was TCM-199 supplemented with 10% fetal bovine serum, 10 μg mL–1 FSH, 1 μg mL–1 LH, 0.2 mM cysteamine and 50 ng mL–1 epidermal growth factor. After IVM, the cumulus cells were denuded with 0.2% hyaluronidase in TCM-199 at 38.5°C by pipetting. The cumulus-free Sika deer oocytes were stimulated by 1 of the following treatments: 1) ethanol + 6-DMAP, treated with 7% ethanol for 7 min and 2 mM 6-dimethylaminopurine (6-DMAP) in DSOF for 4 h; or 2) ionomycin + 6-DMAP, treated with 5 μM ionomycin for 5 min and 2 mM 6-DMAP in DSOF for 4 h. Then, oocytes were transferred into culture media for 7 days [Day 0 (D0) = activation]. On D3, embryos were transferred into fresh DSOF drops supplemented with 10% (v/v) fetal bovine serum. All cultures were overlaid with mineral oil and kept in a humidified modular incubation chamber gassed with 5% CO2. Effects of these chemicals on oocyte activation were then examined and compared with the controls, in which oocytes were cultured in TCM-199 for 4 h without chemical supplement. Our results showed that rates of cleavage, morula and blastocyst were 72.7, 43.9 and 32.4% (n = 139), respectively, by treatment with ionomycin + 6-DMAP. And rates of cleavage, morula and blastocyst were 61.1, 29.7 and 17.8% (n = 134), respectively, by treatment with ethanol + 6-DMAP. However, the rates of cleavage, morula and blastocyst were 5, 0 and 0% (n = 101) in the control group. Meanwhile, the rates of oocyte cleavage (72.7% vs 61.1%), morula (43.9% vs 29.7%) and blastocyst (32.4% vs 17.8%) between 2 treatments of ionomycin + 6-DMAP and ethanol + 6-DMAP were significantly different (P &lt; 0.05). In conclusion, parthenogenetic activation of Sika deer oocytes with ionomycin + 6-DMAP is more effective than that with ethanol + 6-DMAP. These results have begun to elucidate parameters important for animal modeling and cloning with the Sika deer and should facilitate the development of genetically defined animal models in this species. This work was supported by the grant from the China Postdoctoral Science Foundation (No. 20090451135).

Construction of Ipr1 expression vector and development of cloned embryos in vitro

The purpose of this study was to prepare intracellular pathogen resistance 1 (Ipr1) transgenic donor cells for somatic cell nuclear transfer (SCNT). Based on our current understanding of Ipr1, a macrophage special expression vector pSP-EGFP-Ipr1was constructed. Bovine fetal fibroblasts were transfected with pSP-EGFP-Ipr1. The green fluorescent protein (GFP)-expressing cells were selected and transferred into enucleated bovine oocytes. Then, the rates of oocyte cleavage and blastocyst formation of transgenic cells and non-transgenic cells were observed, respectively. The results showed that reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive. This study may provide cloned embryos for the production of anti-tuberculosis transgenic animals.

Comparison of two in vitro gonadotrophin priming protocols in maturation treatment of patients with polycystic ovary syndrome

Objective To compare the therapeutic effect of two Methods of in vitro oocyte maturation (IVM) technology with polycystic ovary syndrome (PCOS) patients in infertility treatment.Methods Retrospective analysis was performed on patients with PCOS infertility who were stimulated with two different kinds of stimulating method of the IVM cycle in our center during April 2010 and August 2010.There were 20 patients in the temporary FSH stimulating group (group A) and 31 patients in the HCG stimulating group (group B).The number of oocytes maturation rate,fertilization rate,cleavage rate,excellent embryo rate,implantation rate,pregnancy rate per oocyte recycle,pregnancy rate per transfer and abortion rate of transfer were compared.Results There was no significant difference between the two groups of age,duration of infertility,body mass index,based sex hormone levels by t test(P＞0.05).There was no significant difference between the two groups of the number of oocytes retrieved,oocyte maturation rate,cleavage rate,and abortion rate of transfer(P＞0.05).In group B,the fertilization rate was 80.3%,good-quality-embryo rate was 49.8%,implantation rate was 31.5%,pregnancy rate per oocyte recycle was 54.8%,which was significantly higher than in group A (73.9%,35.4%,12.5% and 25.0%,respectively).Conclusions If IVM was used to treat PCOS patients with infertility,HCG stimulating will be better than FSH stimulating,and it could result in a higher clinical pregnancy rate. Key words: Follicle stimulating hormone/AD; Chorionic gonadotropin/AD; Polycystic ovary syndrome/DT; Fertilization in vitro

In vitro assessment of sperm from bulls of high and low field fertility

The aim of this study was to investigate the reasons for differences in field fertility of bulls following insemination with frozen-thawed semen. The study was carried out in two separate parts over two years and comparisons were made between 5 high and 4 low fertility Holstein Friesian bulls as determined by their either 90 day non-return rate (Year 1) or calving rate (Year 2). Two high fertility Limousin bulls were included in Year 1 for comparative purposes. The ability of sperm from each bull to penetrate artificial mucus was assessed (Year 1 = 7 replicates; Year 2 = 5 replicates). Glass capillary tubes (2 per bull per replicate) were filled with artificial mucus and incubated with sperm stained in 1% Hoechst 33342 for 30 min at 37 °C. The number of sperm were subsequently counted at 10 mm intervals along the tube between 40 and 80 mm markers. Sperm mitochondrial activity of each bull was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay (4 replicates in each year). Sperm were incubated with MTT for 1 h at 37 °C following which the absorbance of formazan was read using a spectrophotometer. Sperm viability after thawing was assessed for each bull using a live/dead sperm viability kit (Year 1 = 3 replicates; Year 2 = 4 replicates). A minimum of 250 cells were assessed per bull in each replicate and classified as either live or dead. Finally, the ability of sperm to fertilize oocytes in vitro and their ability to develop to blastocyst stage embryos were assessed (5 replicates in each year involving 220 to 306 oocytes per bull). Data transformation to normalize residuals was required for mucus sperm penetration (square root) and IVF (cleavage and blastocyst rate) results (arcsin). The mean number of sperm counted at each 10 mm mark between 40 and 80 mm was higher in the high fertility (56.0; 95% CI 39.5 to 75.3) compared to the low fertility (42.9; 95% CI 29.3 to 59.1) Holstein Friesian bulls but the difference did not reach formal significance (P = 0.09). Fertility status had no effect on the ability of sperm to reduce MTT to formazan (mean absorbance 0.34 ± 0.051 and 0.30 ± 0.044) or on the percentage of live sperm per straw (mean 47.3 ± 5.47 and 32.4 ± 4.66) for high and low fertility Holstein Friesian bulls respectively. Oocyte cleavage rate following insemination with sperm from high fertility Holstein Friesian bulls was significantly higher than with sperm from low fertility Holstein Friesian bulls [76.7% (95% CI 60.9 to 89.4) and 55.3 (95% CI 40.4 to 69.7) respectively, P = 0.04]. There was no significant effect of bull fertility on blastocyst rate [34.7% (95% CI 21.1 to 49.6) and 24.2 % (95% CI 14.1 to 36.0) for the high and low fertility Holstein Friesian bulls, respectively; P = 0.2]. In conclusion, sperm from high fertility bulls tended to be more effective in penetrating artificial mucus and to have an increased ability to fertilize oocytes in vitro; however, once fertilization occurred subsequent embryo development was not significantly affected by fertility status.

Participation of the sperm proteasome during in vitro fertilisation and the acrosome reaction in cattle

In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertilisation (IVF) and the acrosome reaction (AR). Motile spermatozoa, obtained by a swim-up method in Sperm-Talp medium, were capacitated for 3.5 h and incubated in the presence or absence of the specific proteasome inhibitor epoxomicin for 30 and 60 min. Then, the spermatozoa were co-incubated with mature bovine cumulus oocytes and after 48 h the cleavage rate of inseminated oocytes was evaluated. In addition, we evaluated the participation of the sperm proteasome during the progesterone-induced AR. Capacitated spermatozoa were incubated for 30 min with or without epoxomicin, then progesterone was added and the ARs were evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. The results indicate that the proteasome inhibitor decreased the cleavage rate of oocytes inseminated with treated spermatozoa. In addition, acrosomal exocytosis levels were statistically significantly higher in the samples treated with the AR inducer progesterone than in control samples in the absence of the inducer. However, the progesterone-induced AR was significantly reduced by previous treatment of the spermatozoa with epoxomicin (P < 0.001). These observations indicate that the bovine sperm proteasome participates in the IVF and AR processes.

74 MECHANICAL DELIPIDATION IMPROVES CRYOSURVIVAL AND IN VITRO DEVELOPMENT OF VITRIFIED CAT OOCYTES

Although considerable progress has been made in the development of successful methods for cryopreservation of embryos, oocytes are much less cryotolerant. There appears to be an inverse relationship between cryosurvival and intracellular lipid levels. For example, cat oocytes, which appear microscopically as coffee-coloured, nearly opaque spheres due to their high lipid content, are extremely sensitive to cryopreservation. Oocyte delipidation thus represents a potential approach to improving cryosurvival. The objectives of the present study were to examine 1) the effects of calcium (Ca2+, 0 v. 10 nM), FBS (0 v. 10%), and cytochalasin B (CB, 7.5 v. 20.0 μg mL–1) during mechanical delipidation by high-speed centrifugation on in vitro development of IVM cat oocytes, and 2) the influence of centrifugation, degree of lipid polarization (partial v. full), and co-culture with cat fetal fibroblasts (CFF) on in vitro development of vitrified IVM cat oocytes. In Experiment 1, oocytes were randomly allocated to each centrifugation medium and centrifuged at 12 000 × g for 20 min. Oocytes were then fertilized with epididymal sperm (motile sperm mL–1) and cultured until Day 8 (Pope et al. 2006 Theriogenology 66, 59–71). In Experiment 2, oocytes were centrifuged with the optimal centrifugation medium obtained in experiment 1, allocated to each treatment and vitrified in a solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose (2008 Reprod. Fertil. Dev. 20, 188). Liquified oocytes were fertilized and cultured until Day 8. In both experiments, cleavage and degeneration rates were determined on Day 2 and blastocyst development on Day 8. Data were analysed by 2-way ANOVA and chi-square tests. In Experiment 1, of 939 oocytes that were centrifuged and fertilized, 40% of those treated in 0 nM Ca2+ cleaved and 22% developed into blastocysts, v. 33 and 6%, respectively, in 10 nM Ca2+ (P &lt; 0.05). The respective cleavage and degeneration frequencies for oocytes treated in 10% FBS were 43 and 19% v. 19 and 3% in 0% FBS (P &lt; 0.05). Cleavage and blastocyst development after treatment with 7.5 and 20.0 μg mL–1 CB were 36 and 15% v. 42 and 22%, respectively. In Experiment 2, 493 oocytes were vitrified/liquified and fertilized. The degeneration, cleavage, and blastocyst rates of non-centrifuged oocytes were 49, 21, and 0% v. 31 (P &lt; 0.05), 38 (P &lt; 0.05), and 7%, respectively, of centrifuged oocytes. Of centrifuged oocytes with partially extruded lipids, 34% degenerated, 34% cleaved, and 4% developed into blastocysts v. 29, 42, and 10%, respectively, of oocytes with fully extruded lipids. Degeneration, cleavage and blastocyst rates of co-cultured v. control oocytes were 18, 36, and 10%, v. 26 (P &lt; 0.05), 34, and 3%, respectively. In summary, cryotolerance of domestic cat oocytes to vitrification was 1) affected by their lipid content, and 2) improved by mechanical reduction of intracellular lipids. When oocytes were fully delipidated in Ca2+-free medium containing 10% FBS and 20.0 μg mL–1 CB before vitrification and co-cultured after IVF with CFF, blastocyst development was similar to that of control, non-vitrified oocytes.

252 EFFECT OF FOLLICLE-STIMULATING HORMONE ADDITION ON IN VITRO MATURATION AND CLEAVAGE OF ALPACA (VICUGNA PACOS) EMBRYOS

We have previously reported that alpaca oocytes require between 38 and 42 h of maturation time (Huanca et al. 2010 Reprod. Fertil Dev. 22(1), 327). The objective of this study was to evaluate the effect of the addition of FSH in the maturation medium on nuclear maturation and cleavage rate. Ovaries were collected from a slaughterhouse and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. Cumulus–oocyte complexes (COC) were obtained by slicing of ovaries with a scalpel and were pooled in a conical tube for sedimentation before evaluation. The 476 COC with homogeneous cytoplasm and 2 or more layers of cumulus cells were transferred to plates with a 40-μmL drop of maturation medium TCM-199 supplemented with 10% FCS (vol:vol), 10 μg mL–1 of hCG, 0.2 mM sodium pyruvate, 50 μg mL–1 of gentamicine, and 1 μg mL–1 of oestradiol plus 0.5 μg mL–1 of FSH (Folltropin, Bioniche Animal Health, Belleville, Ontario, Canada) according the following treatments: T1 (Control): FSH by 42 h, T2: 21 h with FSH + 21 h without FSH, T3: 21 h without FSH + 21 h with FSH. The COC were maintenance under mineral oil with 10 to 12 oocytes per drop and maturated 42 h at 39°C in an atmosphere of 5% CO2 and high humidity. After the maturation time, part of the COC were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mg/mL of hyaluronidase and fixed in ethanol: acetic acid (3:1). Oocytes were placed on the slide with minimum medium and stained with 1% orcein for 5 min. The slides were examined under a phase contrast microscope at ×400 to evaluate the status of nuclear maturation and classify as germinal vesicle (GV), metaphase I (MI), anaphase–telophase, metaphase II (MII), and degenerated. The other part of oocytes was fertilized in vitro using epididymal sperm. Motile spermatozoa were obtained by centrifugation at 600 × g on a percoll discontinuous gradient (22.5:45.0%) for 10 min. After the supernatant was removed by aspiration, the pellet was resuspended in TL HEPES and centrifuged again at 300 × g by 5 m. The pellet was resuspended in TL Stock. Gametes were coincubated for 18 h at 39°C with 5% CO2 and high humidity. Presumptive zygotes were culture in KSOM supplemented with 1 mM of glutamine, 0.3 mM of sodium pyruvate, 50 μg mL–1 of gentamicine, ethylenediaminetetraacetic acid, amino acids essentials and nonessentials, and BSA by 3 days and cultured in SOF medium for 7 days. Cleavage rate was evaluated at 72 h. Proportional data were compared by chi-square test. The proportions of oocytes reaching MII stage were 64.9 ± 8.1, 49.2 ± 9.4, and 53.8 ± 7.5% for T1, T2, and T3, respectively. Cleavage rates were 39.1, 36.3, and 33.1% and blastocysts rates were 13.6, 16.1, and 14.5% for T1, T2, and T3, respectively. The results suggest that the moment of addition of FSH does not have an effect on the maturation and cleavage rate of alpaca oocytes. This work was supported by Grant 064 – FINCyT – PIBAP 2008 and Grant 032 – PROCYT – CONCYTEC.