Optimization of In Vitro Maturation in Ovine Oocytes: A Comparative Study of Maturation Media

Treatment of ovine oocytes with certain water-soluble vitamins during in vitro maturation (IVM)

Heat shock may affect different aspects of oocyte maturation and its subsequent development to the blastocyst stage. A series of in vitro experiments was performed to determine whether physiologically heat shock (41°C) disrupts the progression of the ovine oocytes through meiosis, activation and blastocyst formation. Cumulus-oocyte complexes (COCs) were aspirated from 2-6-mm follicles and cultured at 38.5°C (control) or 41°C (heat shock) for the first 12 h of maturation. The oocytes were incubated at 38.5°C during the last 10 h of maturation and 8 days after activation. Results showed that most of the oocytes matured under heat-shock conditions remained at the germinal vesicle breakdown (GVBD) stage and they showed an aberrant chromatin configuration. After heat shock, oocyte diameter and time spent for zona pellucida dissolution increased (P < 0.05). The heat-shocked group had a higher percentage of oocytes with incomplete migration of cortical granules (P < 0.05). The heat-shock condition decreased (P < 0.05) cleavage rates (56.19 versus 89.28%) and morula formation (26.85 versus 37.81%). However, there was no significant difference in blastocyst formation and percentage of hatched blastocysts. At 12 h, heat shock had an adverse effect on embryo quality and reduced inner cell mass number (P < 0.05). Quantitative gene expression analysis showed greater transcripts (P < 0.05) for Na/K-ATPase mRNA in heat-shocked oocytes. To sum up, heat shock has disruptive effects on ovine oocyte maturation and can impair cellular and molecular factors that are important for embryo development.

The current study investigated the in vitro meiotic competence of ovine and caprine oocytes that underwent nuclear maturation during the breeding and nonbreeding seasons. We hypothesised that maturation rates of ovine and caprine oocyte would be significantly lower during the nonbreeding season. Ovine (Katahdin crossbred) and caprine (mainly Spanish crossbred) ovaries were collected from a local abattoir in the southern United States. Age of the animals was not determined. Cumulus-oocyte complexes (COC) were harvested by slicing the ovaries and searching using a stereomicroscope. Oocytes with more than 3 layers of unexpanded cumulus cells and with evenly granulated cytoplasm were selected for in vitro maturation (IVM). A commercial bovine IVM media (IVF Bioscience, Falmouth, United Kingdom) was used throughout the study. After 24h of IVM, ovine and caprine oocytes were denuded and oocytes with an extruded polar body (meiotic metaphase II oocytes) were considered to have reached nuclear maturation. The seasons in this study were defined as follows: breeding season=September to April and nonbreeding season=May to July. The presence of corpus hemorrhagicum or corpus luteum in at least 70% of the ovaries indicated the breeding season for the animals. Proportions of oocytes undergoing nuclear maturation were analysed using a two-tailed Chi-squared test. Statistical significance was set at P ≤ 0.05. The ovine maturation rate was 59% (65/111) and 49% (254/519) and the caprine maturation rate was 70% (39/56) and 40% (64/162) during the breeding and nonbreeding seasons, respectively. These results show a significant difference in nuclear maturation for caprine oocytes (P&amp;lt;0.001) during the breeding and nonbreeding seasons; however, there was no significant difference in nuclear maturation for ovine oocytes (P=0.06) during the breeding and nonbreeding seasons. High environmental temperatures during the nonbreeding season may have had detrimental effects on oocyte nuclear maturation in caprine but not in ovine oocytes. Why oocytes from these 2 species differ on how they are adversely affected by season remains to be elucidated.

Saving and multiplying the existing livestock population is an important strategic objective that ensures the country's food security and levelling the dependence on imports of genetically valuable highly productive animals. Obtaining farm animal embryos in vitro is one of the methods of assisted reproductive technologies (ART), which can be used both lifetime and post-mortem. This method allows to optimise breeding work with livestock in a short period of time due to the large number of offspring obtained from highly productive individuals. Aim and objectives: to investigate the effect of bovine estrous serum on meiotic oocyte maturation and early embryogenesis prior to blastocyst development, when added to oocyte-cumulus complexes (OCC) maturation medium. A blind randomised controlled trial was conducted on the effect of bovine estral serum on blastocyst development when added to OCC maturation medium. Estral serum was obtained from the blood of clinically healthy cows with pronounced signs of puberty. The study was carried out in conditions of industrial livestock breeding. The material for the study were oocyte-cumulus complexes obtained by the method of lifetime aspiration from 89 Holstein cows with milk productivity of more than 10 thousand kg per lactation. Oocyte-cumulus complexes were divided into 2 groups: Group 1 (control) - oocyte maturation was carried out in commercial IVM medium without addition of estrous serum, Group 2 (experiment) - oocyte maturation was carried out in commercial IVM medium with addition of es trous serum in concentration from 5 to 15%. The hormones, growth factors and biologically active substances contained in estrous serum have no effect on the achievement of OCC at the stage of metaphase II of meiosis, as well as at the stage of fractions; moreover, a negative effect at these stages of development can be traced. However, substances contained in estrous serum are able to saturate and accumulate in OCC at the stage of maturation to metaphase II of meiosis, thus enhancing the potential of oocytes to embryonic development and further positively affect their achievement of the blastocyst stage. The difference in the formation of blastocysts from crushed embryos reaches more than 25%, and the difference in the formation of blastocysts from viable JCCs matured in media without serum and modified with estrous serum is 37.5% in favour of the latter.

Maturation time of oocytes from alpacas is around 38 to 40h (Huanca et al. 2009) that would induce an increase in reactive oxygen species during in vitro maturation and IVF and cause cytotoxic damage to gametes. The objective of this study was to determine the optimal concentration of methionine during in vitro maturation on cleavage rate of alpacas oocytes following IVF. Cumulus-oocyte complexes were collected from slaughterhouse ovaries and transported in a thermos flask containing a saline solution 0.9% and antibiotic, antimycotic at 35°C. Cumulus-oocyte complexes were aspirated from follicles &amp;gt;2mm and evaluated with a stereomicroscope for selection. Only cumulus-oocyte complexes with a homogeneous cytoplasm and with 2 or more layers of cumulus cells were selected to be cultured in maturation medium TCM-199 supplemented with 10% FCS (v:v) plus 0.5μg mL−1 FSH, 10μg mL−1 hCG, 0.2mM sodium pyruvate, 50μg mL−1gentamycin and 1μg mL−1 oestradiol under mineral oil by 38h. Testes of mature males were collected from a slaughterhouse and transported to the laboratory. Caudal epididymide was isolated, and fluid, rich in spermatozoa, was aspirated in syringes containing 2mL of Tris-fructose-egg yolk extender. Motile spermatozoa were obtained by centrifugation at 700×g in a Percoll discontinuous gradient (22.5: 45.0%) for 10min. The supernatant was removed by aspiration, and the pellet was resuspended in TL stock and centrifuged again at 700×g for 5min. Spermatozoa and oocytes were co-incubated by 18h at 39°C with 5% CO2. Presumptive zygotes were culture in KSOMaa medium and evaluated at 72h. The treatments include 0, 14 and 21 μM of methionine in maturation and culture medium. Data were analysed by ANOVA, and results are presented in Table 1. The results suggest that addition of methionine in maturation and culture medium improve the cleavage rate in oocytes from alpacas. Table 1.Cleavage rate (%) following in vitro maturation at different concentrations of methionine Proyect 405-PNICP-PIAP-2014, INNOVATE-PERU, is acknowledged.

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.

Effects of transport method and maturation media on the maturation rate of equine oocytes

Dietary intakes of polyunsaturated fatty acids are thought to mediate a wide range of actions in reproductive tissues. This includes the effects on ovarian follicle and corpus luteum functions via improved energy efficiency as well as providing precursors for the synthesis of signalling molecules such as steroids and prostaglandins. An appropriate level of α-linolenic acid (ALA) in the oocyte maturation medium has been shown to induce molecular changes associated with oocyte maturation and embryo developmental competence. In that light, we hypothesised that supplementation of exogenous ALA to maturation media could enhance nuclear maturation and embryonic development in the goat. A preliminary experiment was executed to measure the level of ALA in antral follicles by gas chromatography/mass spectrometry analysis. Our results revealed that the concentration of ALA in follicular fluids ranged from 0.006 to 0.02mgmL–1 (21.5 to 71.8µM, with a mean of ~50µM). To test the effect of ALA on the competence of goat oocytes to complete meiotic maturation to metaphase II and sustain embryonic development, ovaries were obtained from a local abattoir. Cumulus–oocyte complexes were recovered by the slicing method followed by selection of oocytes with a homogenous cytoplasm and at least three layers of compact cumulus cells. The cumulus–oocyte complexes were placed in maturation media supplemented with 50µM ALA. Oocytes in the control group were incubated in the same maturation medium without ALA. In vitro maturation (IVM) was performed in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2 at 38.5°C for 24h. After IVM, several oocytes from the treatment (n=170) and control (n=166) groups were stained with Hoechst and were evaluated in relation to their metaphase-II rate. Other groups of oocytes from both the treatment (n=70) and control (n=61) groups were subjected to parthenogenetic activation by applying 1min of exposure to 2.5µM ionomycin followed by 2mM 6-DMAP treatment for 3h. After activation, oocytes were cultured in CR1aa medium for 7 days under the conditions stated above. Four replications were performed. Differences in developmental rates were analysed for significance by one-way ANOVA using SAS version 8.0 (SAS Institute Inc., Cary, NC, USA), considering P&amp;lt;0.05 to be significant. As a result, supplementation of the maturation media with ALA did not appear to affect cumulus expansion. In contrast, IVM of goat oocytes in the presence of ALA resulted in a significantly higher maturation rate compared with maturation without ALA supplementation (66.4% v. 57.9%). Likewise, addition of ALA to the IVM medium significantly increased the rate of cleavage (60.1% v. 52.4%) and blastocyst formation (22.6% v. 14.9%), calculated from the activated oocytes. Collectively, the results of our study show that supplementation of IVM media with 50µM ALA promotes nuclear maturation, increases cleavage rate, and results in higher blastocyst rate in goat oocytes after parthenogenetic activation. Thus, providing appropriate levels of ALA in maturation media could have beneficial effects on embryo development and reproductive efficiency in the goat.

Dietary intakes of polyunsaturated fatty acids are thought to mediate a wide range of actions in reproductive tissues. This includes the effects on ovarian follicle and corpus luteum functions via improved energy efficiency as well as providing precursors for the synthesis of signalling molecules such as steroids and prostaglandins. An appropriate level of α-linolenic acid (ALA) in the oocyte maturation medium has been shown to induce molecular changes associated with oocyte maturation and embryo developmental competence. In that light, we hypothesised that supplementation of exogenous ALA to maturation media could enhance nuclear maturation and embryonic development in the goat. A preliminary experiment was executed to measure the level of ALA in antral follicles by gas chromatography/mass spectrometry analysis. Our results revealed that the concentration of ALA in follicular fluids ranged from 0.006 to 0.02 mg mL–1 (21.5 to 71.8 µM, with a mean of ~50 µM). To test the effect of ALA on the competence of goat oocytes to complete meiotic maturation to metaphase II and sustain embryonic development, ovaries were obtained from a local abattoir. Cumulus–oocyte complexes were recovered by the slicing method followed by selection of oocytes with a homogenous cytoplasm and at least three layers of compact cumulus cells. The cumulus–oocyte complexes were placed in maturation media supplemented with 50 µM ALA. Oocytes in the control group were incubated in the same maturation medium without ALA. In vitro maturation (IVM) was performed in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2 at 38.5°C for 24 h. After IVM, several oocytes from the treatment (n = 170) and control (n = 166) groups were stained with Hoechst and were evaluated in relation to their metaphase-II rate. Other groups of oocytes from both the treatment (n = 70) and control (n = 61) groups were subjected to parthenogenetic activation by applying 1 min of exposure to 2.5 µM ionomycin followed by 2 mM 6-DMAP treatment for 3 h. After activation, oocytes were cultured in CR1aa medium for 7 days under the conditions stated above. Four replications were performed. Differences in developmental rates were analysed for significance by one-way ANOVA using SAS version 8.0 (SAS Institute Inc., Cary, NC, USA), considering P &lt; 0.05 to be significant. As a result, supplementation of the maturation media with ALA did not appear to affect cumulus expansion. In contrast, IVM of goat oocytes in the presence of ALA resulted in a significantly higher maturation rate compared with maturation without ALA supplementation (66.4% v. 57.9%). Likewise, addition of ALA to the IVM medium significantly increased the rate of cleavage (60.1% v. 52.4%) and blastocyst formation (22.6% v. 14.9%), calculated from the activated oocytes. Collectively, the results of our study show that supplementation of IVM media with 50 µM ALA promotes nuclear maturation, increases cleavage rate, and results in higher blastocyst rate in goat oocytes after parthenogenetic activation. Thus, providing appropriate levels of ALA in maturation media could have beneficial effects on embryo development and reproductive efficiency in the goat.

Assisted reproductive techniques are a valuable tool for conservation breeding of endangered species. Cryopreservation methods are the basis of gamete banks, supporting genetic diversity preservation. Unfortunately, cryopreservation of feline oocytes is still considered an experimental technique. The aim of this study was to compare two commercial kits, with our protocol for vitrification of cat oocytes (IZW), which comprises a three-step method with ethylene glycol, DMSO, fetal calf serum, trehalose and Ficoll PM-70. Furthermore, we applied slush nitrogen (SN2 ) for ultra-rapid freezing to improve survival rates. Cumulus-oocyte complexes were collected from domestic cat ovaries by slicing and vitrified at immature stage using Cryotop as storage device. Vit Kit® Freeze/Thaw (n=89) showed the lowest maturation percentage obtained after warming (10.1%). A significant difference in maturation percentage of oocytes was found between Kitazato® kit (38.7%, n=137) and IZW protocol (24.5%, n=143). The cleavage after ICSI of warmed and matured oocytes (20.7% and 28.6%, respectively) and the morula percentage (18. 2% and 22.5%, respectively), however, did not reveal any significant difference between the two methods. Application of SN2 did not result in any improvement of oocytes' cryopreservation. Maturation percentage of the oocytes vitrified by IZW method with SN2 (n=144) decreased until 6.1%, without any cleavage after fertilization. For Kitazato® (n=62), only 17.7% were able to undergo maturation and cleavage percentage dropped to 18.2%, not reaching morula stage. These data demonstrate that feline oocytes can be vitrified either by our IZW method or by commercial Kitazato® kit, but the use of SN2 is improving neither maturation nor cleavage percentages when combined with these procedures.

The objectives of the study were to evaluate the ovarian follicular response, cumulus–oocyte complex (COC) collection rate, fertilization, and culture of COC collected from alpacas after treatment with 2 different gonadotropins. Female alpacas were assigned to Group 1 (n = 8), 200 mg of FSH (Folltropin, Bioniche, Belleville, Ontario, Canada) divided b.i.d. for 3 days, plus a single IM dose of 1000 IU of hCG (Chorulon, Intervet, Salamanca, Spain) 24 h after the last FSH treatment; or Group 2 (n = 10), 750 IU of eCG (Folligon, Intervet) as a single dose, plus a single IM dose of 1000 IU of hCG on Day 3 after eCG treatment (Day 0 = start of the superstimulatory treatment). At 20 to 22 h post-hCG treatment, the ovaries were surgically exposed and COC were aspirated from follicles ≥6 mm and evaluated. The COC with a homogeneous cytoplasm and 2 or more layers of cumulus cells were transferred to plates with a 40-μL drop of TCM-199 maturation medium supplemented with 10% FCS (vol/vol) plus 0.5 μg mL–1 of FSH, 10 μg mL–1 of hCG, 0.2 mM sodium pyruvate, 50 μg mL–1 of gentamicin, and 1 μg mL–1 of oestradiol under mineral oil with 10 to 12 oocytes/drop and maturated 24 h at 39°C in an atmosphere of 5% CO2 and high humidity. After maturation, COC were removed and fertilized in vitro using epididymal sperm. Testes were collected from mature males from a slaughterhouse and transported to the laboratory. The caudal epididymide was isolated. A prick was made on the convoluted tubules with a sterile hypodermic needle and the fluid, rich in spermatozoa, was aspirated in syringes containing 2 mL of Tris-fructose egg yolk extender. Motile spermatozoa were obtained by centrifugation at 600 × g on a Percoll discontinuous gradient (45.0:22.5%) for 10 min. The supernatant was then removed by aspiration and the pellet was resuspended in TL-HEPES and centrifuged again at 300 × g for 5 min. The pellet was resuspended in TL-stock. Gametes were co-incubated for 18 h at 39°C with 5% CO2 and high humidity. Presumptive zygotes were cultured in KSOM medium supplemented with 1 mM glutamine, 0.3 mM sodium pyruvate, 50 μg mL–1 of gentamicin, EDTA, essential and nonessential amino acids, and BSA for 3 days and cultured in SOF medium for 7 days. Embryo development was evaluated at 72 h and 7 days. Data were subjected to ANOVA. The number of follicles ≥6 mm did not differ at the time of COC collection (19.3 ± 5.7 and 21.5 ± 7.3), and the number of COC collected was 16.7 ± 5.3 and 17.3 ± 6.6 for the FSH group and the eCG group, respectively. The cleavage rate was 45.2 and 42.1% for the FSH group and the eCG group, respectively, at 72 h of culture, and the blastocyst stage at Day 7 (22.2 v. 19.3) did not differ between treatments. In conclusion, the FSH and eCG treatments did not differ in the ovarian follicular response, COC collection rate, fertilization, and culture of COC. Both gonadotropins can be used in the IVF protocol for alpacas. Grant 064 FINCyT-PIBAP 2008 and Grant 032-2009 PROCYT–CONCYTEC.

In vitro maturation (IVM) of oocytes is a critical step in assisted reproductive technologies carried out in species such as cattle and swine, for generating oocytes capable of being fertilized in vitro and providing healthy offspring useful for biomedical and agricultural purposes. Cumulus-oocyte complexes (COC) collected from abattoir ovaries for IVM often respond poorly to gonadotropins, resulting in compromised oocyte competence. Our previous work demonstrated that the combination of fibroblast growth factor 2, leukemia inhibitory factor, and insulin-like growth factor 1 (FGF2, LIF, and IGF1; FLI) significantly improved nuclear maturation of porcine oocytes and their developmental competence. However, it is unclear whether the benefits of FLI are mediated through increased gonadotropin sensitivity of COC or improved downstream signalling. Here we investigated the effect of FLI supplementation of IVM medium with and without gonadotropins. The COC, collected from 2- to 6-mm follicles from prepubertal ovaries, were matured in 5% CO2/air at 38.5°C for 42 h in chemically defined TCM-199 medium (supplemented with 0.1% polyvinyl alcohol, 3.05 mM d-glucose, 0.91 mM pyruvate, 0.57 mM cysteine, 10 ng mL−1 epidermal growth factor), with or without FLI (40 ng mL−1 FGF2, 20 ng mL−1 LIF, and 20 ng mL−1 IGF1), and with or without gonadotropins (0.5 μg mL−1 LH, 0.5 μg mL−1 FSH), in a 2 × 2 factorial design experiment. After IVM, oocytes were fertilized in vitro and cultured under standard conditions until Day 6 when blastocyst formation was assessed. The experiment was replicated 4 times with a total of 792 oocytes. Percentage data were arcsin transformed and analysed by ANOVA to detect differences (significance, P &lt; 0.05). When FLI was absent from the maturation medium, oocytes matured in presence of gonadotropins demonstrated improved nuclear maturation (67.0 ± 2.2% v. 52.7 ± 5.3%) and blastocyst formation (23.7 ± 3.3% v. 10.2 ± 2.3%) relative to oocytes matured without gonadotropins, respectively. However, when FLI was present in the medium, the extent of oocyte maturation and subsequent blastocyst development was unaffected by gonadotrophin addition (maturation: 84.2 ± 1.8% with and 79.2 ± 3.4% without; blastocyst formation: 29.5 ± 4.3% with and 24.9 ± 4.3% without). In summary, these results suggest that FLI, rather than enhancing COC sensitivity to gonadotrophins, may activate certain downstream signalling pathways that are normally controlled by gonadotropins during IVM, thereby enhancing oocyte quality. Therefore, FLI appears able to substitute for gonadotropins in the maturation medium and supports porcine oocyte competence in the absence of FSH and LH. These observations will help us better understand the mechanisms whereby FLI enhances oocyte nuclear maturation and improves developmental competency. Supported by NIH R01HD69979, U42OD011140, and University of Missouri Food for the 21st Century Program.

BackgroundAlthough cystitis glandularis (CG) is a common benign urinary bladder epithelial abnormality, it remains unclear whether CG is a premalignant lesion. Cyclooxygenase-2 (COX-2) and B-cell lymphoma-2 (Bcl-2) overexpression has recently been reported as a potential tumor initiator or promoter. We evaluated and compared COX-2 and Bcl-2 expression in CG, chronic cystitis (CC), and primary vesicle adenocarcinoma (ADC) tissues.MethodsWe conducted a retrospective study to investigate COX-2 and Bcl-2 levels in CG and ADC. We obtained tissue samples from 75 patients (including 11 cases of CC, 30 typical cases of CG (CGTP), 30 cases of intestinal CG (CGIT), and 4 cases of ADC) between 1989 and 2009 from the Surgical Pathology Archives of the No. 2 People’s Hospital of Zhenjiang, affiliated with Jiangsu University. COX-2 and Bcl-2 immunohistochemical staining was performed on all tissues. Nine normal bladder epithelial specimens were evaluated as control samples. Correlations between COX-2 and Bcl-2 expression in CG were also analyzed.ResultsCOX-2 and Bcl-2 expression was higher in the ADC group compared to other groups (p < 0.05). COX-2 and Bcl-2 levels were higher in the CGIT group compared to the CGTP group (p = 0.000 for both). The CGIT and CGTP groups both showed higher COX-2 expression compared to the CC group (p = 0.000 for both). There was no difference in Bcl-2 expression between the CGTP and CC groups (p = 0.452). Additionally, the difference in COX-2 and Bcl-2 expression between the control and CC groups was also insignificant (p = 0.668 and p = 0.097, respectively). Finally, we found that COX-2 and Bcl-2 levels were positively related (r = 0.648, p = 0.000).ConclusionCOX-2 and Bcl-2 overexpression in the CG group suggests that CG, particularly the intestinal type, may be a premalignant lesion that converts into a tumor in the presence of carcinogens.

Oocyte cryopreservation will become an important tool for the creation of genetic resources bank in domestic animals. Many problems have been described following cryopreservation of MII oocytes, including spindle disorganization (Ledda et al. 2007 Reprod. Fertil. Dev. 19, 13–23) and loss or clumping of microtubules resulting in some scattered chromosomes (Sathananthan et al. 1988 Hum. Reprod. 3, 968–977). Freezing of immature oocytes at the geminal vesicle stage might circumvent these problems because at this stage the genetic material is contained within the contours of a nuclear envelope (Siqeira-Pyles and Landim-Alvarenga 2005 Anim. Reprod. Sci. 193, 176–182). However, the number of reports relating to cryopreservation of immature oocytes remains low and it has been suggested that immature oocytes are more susceptible to cryoinjuries (Ledda et al. 2007). The aim of the present work was to study the effect of CryoLoop™ (Hampton Research, Aliso Viejo, CA, USA) vitrification on survival and subsequent meiotic maturation of immature ovine oocytes. Cumulus–oocyte complexes (COCs) were isolated from ovaries of slaughtered sheep. COCs were washed three times in basal medium [BM (HEPES–TCM-199 supplemented with 10% fetal bovine serum)], and then exposed to equilibration solution (10% ethylene glycol (EG) and 0.25 m trehalose (T) in BM for 3 min. After that, COCs were exposed to vitrification solution (20% EG and 20% DMSO in BM), loaded into the CryoLoop within 1 min, and immersed in liquid nitrogen. Following cryopreservation, COCs were warmed by exposure of oocytes to (1) 10% EG and 1 mT in BM, (2) 0.5 mT in BM, and (3) BM for 3 min in each solution at 39�C. Then oocytes were matured in vitro for 24 h in maturation medium, as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). The nuclear maturation of oocytes was determined using aceto-orcein staining. Oocytes were classified into four stages: germinal vesicle (GV), metaphase-I (MI), anaphase-I + telophase-I (AI/TI), and metaphase-II (MII). Oocytes at MII stage were considered matured. Data were analyzed using chi-square test. The survival rate of oocytes after vitrification and warming based on oocyte morphology was 72.6% (61/84). Numbers of oocytes remaining at the GV stage were significantly higher in the vitrified group than in the control group (43.4 v. 12.6%, respectively; P &lt; 0.01). Maturation to MII stage was higher (63.2%, 55/87) in the control group than in the vitrified group (43.4%, 23/53); however, the difference was not significant. Also, there were no significant differences between vitrified and control groups in terms of MI and AI/TI (7.5 and 5.7 v. 10.3 and 13.8%, respectively). In conclusion, immature ovine oocytes can be vitrified using CryoLoop with high survival and maturation rates.

Cryopreservation of mature oocytes can result in damage to the metaphase spindle due to the temperature sensitivity of microfilaments and microtubules. Cryopreservation of immature oocytes may circumvent this problem because these structures have not formed yet and the genetic material is enclosed within a nuclear envelope. Because intact oocyte cumulus-oocyte complexes (COC) are essential for normal maturation, we chose to cryopreserve immature intact COC. The aim of this study was to determine if immature COC cryopreserved by slow rate freezing or vitrification would resume meiosis upon thawing. In 2 separate experiments, immature COC (n = 102 and 79) were collected from cross-bred cattle by transvaginal ultrasound-guided aspiration and divided into 2 groups. For both experiments, the first group (n = 64 and 40) was placed directly into maturation medium (TCM 199 supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 2 mM glutamine, and 5 μg mL–1 of FSH) and cultured for 22 h under 5% CO2 in air atmosphere at 39°C. In experiment 1, COC (n = 38) in the second group were cryopreserved by a slow rate freezing protocol. The COC were equilibrated for 5 min in 1.5 M ethylene glycol (EG), and 5 COC were loaded into 0.25-mL straws and placed into the cooling chamber of a Freeze Control unit at –6°C. After 5 min, straws were seeded, then cooled at 0.5°C per min to –35°C before plunging into LN. After storage in LN for 5 days, straws were removed from LN, thawed by placing straws in a 35°C water bath, and COC put into maturation as described above. In experiment 2, COC (n = 39) in the second group were cryopreserved by vitrification using a 3-step procedure. The COC were equilibrated in solutions consisting of 10% glycerol then 10% glycerol and 20% EG in PBS for 5 min each. The COC were then placed in a solution of 24% glycerol and 26% EG, and 1 to 3 COC were placed onto a cryotop device in minimal medium and plunged into LN within 45 s. After storage in LN for 2 days, COC were thawed by placing the cryotop device directly into a warmed dilution solution consisting of 0.5 M galactose in PBS. After 5 min in the dilution solution, COC were put into maturation as described above. For all groups, after 22 h of maturation cumulus cells were stripped from the oocytes by vortexing and oocytes were placed on slides for fixation in methanol acetic acid and stained with 1% orcein to determine the nuclear stage. In experiment 1, oocytes cryopreserved by slow rate freezing matured to MII at the same rate as control oocytes (45 v. 55%, P = 0.44). In experiment 2, oocytes cryopreserved by vitrification matured to MII at a lower rate than controls (49 v. 79%, P = 0.01). These results show that cryopreservation of immature intact COC is a viable alternative to cryopreservation of mature oocytes. Further studies are needed to optimize either slow rate freezing or vitrification of intact COC and determine the developmental competence of cryopreserved oocytes following fertilization.