09.50 RESULTS OF REPRODUCTIVE RESEARCH IN FEMALE MANGALICA PIGS Klaus-Peter Brüssow, Istvan Egerszegi, Helmut Torner, Jozsef Rátky Leibniz Institute for Farm Animal Biology, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany, e-mail: bruessow@fbn-dummerstorf.de, torner@fbn-dummerstorf.de Research Institute for Animal Breeding and Nutrition, Gesztenyes ut. 1, 2053 Herceghalom, Hungary, e-mail: istvan.egerszegi@atk.hu, jozsef.ratky@atk.hu

Numerous factors determined developmental competence of the oocytes. Brilliant cresyl blue (BCB) staining has been used for selection of oocytes from several mammalian species, including pigs (Ericsson S. et al, 1993 Theriogenology, 39(1): p.214). The aim of the present study was to evaluate the developmental competence of porcine oocytes that have finished growth phase (BCB+ ) depending on diameter (d) of follicles (d 6 mm) and to detect expression of estrogen receptor (ER) in cumulus cells of BCB+ and ВСВ- oocytes. Before IVM compact cumulus oocyte complexes (COC) were incubated in BCB solution for 60 minutes. Treated oocytes were divided into BCB- (colorless cytoplasm) and BCB+ (colored cytoplasm). Only BCB+ oocytes were used in the experiments. The medium used for oocyte maturation was NCSU 23 supplemented with 10% follicle fluid (FF), 0.1 mg/ml cysteine,10 IU/ml eCG and 10 IU/ml hCG. FF was collected from follicles with 3 - 6 mm in diameter, COC cultured in maturation medium with pieces of wall (600-900 µm in length) from non atretic healthy follicles (d 3-6 mm). After 20-22 h of culture, COC and pieces of wall were washed and transferred into the same maturation medium but without hormonal supplements for another 20-22 h of culture. After IVM oocytes were fertilized in vitro and embryos were cultured by standard protocols (Stokes P. et al., Developmental Biology, 284, p.62 – 71, 2005). All chemicals used in this study were purchased from Sigma-Aldrich (Moscow, Russia). The question was: have BCB+ oocytes from follicles of different diameters the same developmental competence? We did not find significant differences between the level of cleavage and blastocyst in all groups of experiments. Percentages of cleavage and blastocyst in groups were: follicles d 6 - 48% (28/58) and 28% (16/58) (χ² test).Immunocytochemical analysis was used for detection of estrogen receptor expression (ER) in cumulus cells of 53 BCB+ and 33 ВСВ- oocytes. Immunocytochemical staining was performed using the first rabbit polyclonal anti-human ER antibodies (NCLERp, Novocastra, OOO BMS, St.Petersburg, Russia). The visualization system (ABC-universal kit) consists of avidin–biotinilated peroxidase (DakoCytomation) was applied. 3,3’-diaminobenzidine was used as it was recommended from manufacture Novocastra (OOO BMS, St.Petersburg, Russia). Hematoxylin (Abrisplus, St.Petersburg, Russia ) was used to stain cells. Antigen optical density was measured using morphometric VideoTest (Russia) computer program. Positive immunocytochemical reaction was mainly observed in the nuclei membrane and slightly on the cytoplasmic membrane of cumulus cells (probably as non-specific background). It was shown that cumulus cells of BCB+ oocytes had a significantly more pronounced expression of the ER than the cumulus cells of ВСВ- oocytes (pu003c0,001, Mann-Whitney test).

In vivo-derived embryos at a defined stage of development are often a necessary requirement for ongoing biotechnological applications. Because double fixed-time insemination after ovulation induction is commonly used in pigs to produce embryos, variations in the time of ovulation and fertilization of the ovulated oocytes by spermatozoa mainly of 1 of the 2 inseminations can cause, however, diversities in embryo development. To moderate embryo diversity and to realize a uniform outcome of porcine embryo stages, single laparoscopic fixed-time insemination can be used to minimize embryo diversity. The potential of laparoscopic intrauterine insemination (LIUI) has been demonstrated in sperm-mediated gene transfer (Fantinati et al. 2005) and evaluation of sperm migration (Brüssow et al. 2006, 2011). The aim of the present study was to analyze the development and possible diversity of embryos after LIUI. Forty-eight puberal German Landrace gilts were included in the study. Estrus of gilts was synchronized by 15-day Regu-Mate® (Intervet, Millsboro, DE, USA) feeding and follicle development was stimulated with 850 IU of eCG 24 h after Regu-Mate® treatment. Ovulation was induced by 500 IU of hCG 80 h after eCG treatment. The LIUI was performed 31 h after hCG treatment. To that, ketamine/azaperone-anaesthetized gilts were fixed in a dorsal position, a pneumoperitoneum was produced and 3 trocar cannulas were inserted into the abdomen for optics and instruments. Laparoscopic handling was observed on a television monitor. Each uterine horn was carefully fixed with an atraumatic forceps 10 to 15 cm caudal from the utero-tubal junction and the uterine wall was punctured with a 2.5-mm diameter trocar. A 2.2-mm catheter connected to a syringe was inserted about 3 cm into the uterine lumen and 20 mL of extended, fresh boar semen (32.2 × 106 sperm cells mL–1; 65% motility) was deposited in the lumen. Embryos were surgically flushed from the genital tract on Day 2 and 3, respectively. Altogether, 778 oocytes were recovered (recovery rate 68 ± 17%); 45 of 48 gilts (93.8%) revealed fertilization and 76.1% of the recovered embryos (n = 592) were at the 2- and 4-cell stage. On Day 2 (n = 22 gilts), a higher percentage of gilts displayed only 2-cell embryos compared with both 2- and 4-cell, and only 4-cell embryos (72.2 v. 22.7 and 4.6%, P < 0.05; chi-square test). On Day 3 (n = 23 gilts), there was a shift regarding the embryo stage. The proportion of gilts with 2-cell, 2- and 4-cell, and only 4-cell embryos was 4.3, 0, and 95.7%, respectively (P < 0.05). Results of the present study demonstrate high rates of fertilization and of non-diverse developed embryos after single fixed-time LIUI in gilts. Additionally, these results were achieved after inseminating a 75% lower number of sperm cells per insemination dose. Laparoscopic intrauterine insemination can be suggested as an alternative for insemination of sex-sorted semen where the number of available sperm cells after the sorting procedure is restricted.

Dynamic follicular changes occur during the equine estrus cycle, but little is known about their impact on the properties of recovered oocytes. The aim of this study was to characterize the cytoplasmic and chromatin status of equine oocytes in relation to the time of recovery during the follicle wave. Transvaginal ultrasound-guided follicle aspiration was performed two times in relation to the follicle wave: estrus-subordinate, from the subordinate follicles of mares in estrus, 24 hours after human chorionic gonadotropin stimulation of a dominant preovulatory follicle, and new-wave, from the follicles of the subsequent induced follicular wave, at the time of dominant follicle divergence (largest follicle 23 mm diameter). A total of 1011 follicles were aspirated. The oocyte recovery rate in the new-wave group was significantly lower than that for the estrus-subordinate group (12% vs. 26%, respectively); this was associated with a significantly higher proportion of oocytes with compact cumuli (44% vs. 27%, respectively). Estradiol concentrations were markedly higher in follicular fluid from new-wave follicles (885.6 ± 123.2 ng/mL vs. 54.3 ± 18.9 ng/mL, for estrus-subordinate; P < 0.001), indicating greater viability. Aspiration group did not affect glucose-6-phosphate dehydrogenase activity in recovered oocytes. Fibrillar (more juvenile) chromatin was more prevalent in new-wave oocytes, whereas estrus-subordinate oocytes showed more condensed chromatin or resumption of meiosis (P < 0.05). Mitochondrial activity was higher in oocytes with expanded cumuli in the new-wave group, but not in the estrus-subordinate group. In conclusion, our results clearly showed that the time of aspiration in relation to the follicle wave is associated with significant differences in follicle status and oocyte characteristics: new-wave oocytes were from a more viable follicle population and had more juvenile chromatin and cytoplasmic characteristics, whereas estrus-subordinate oocytes were from a more atretic follicle population and exhibited signs of atresia-related acquisition of meiotic and cytoplasmic competence. These findings will help in effective scheduling of oocyte recovery for equine-assisted reproduction techniques.

The close contact and interaction between the oocyte and the follicular environment influence the establishment of oocyte developmental competence. Moreover, it is assumed that apoptosis in the follicular cells has a beneficial influence on the developmental competence of oocytes. The aim of this study was to investigate whether bovine oocytes with varied developmental competence show differences in the degree of apoptosis and gene expression pattern in their surrounding follicular cells (cumulus and granulosa cells). Oocytes and follicular cells from follicles of 3 to 5 mm in diameter were grouped as brilliant cresyl blue (BCB)+ and BCB- based on glucose-6-phosphate dehydrogenase (G6PDH) activity in the ooplasm by BCB staining. In the follicular cells initial, early and late apoptotic events were assessed by analyzing caspase-3 activity, annexin-V and TUNEL, respectively. Global gene expression was investigated in immature oocytes and corresponding follicular cells. BCB+ oocytes resulted in a higher blastocyst rate (19.3%) compared to the BCB- group (7.4%, P < 0.05). Moreover, the analysis of apoptosis showed a higher caspase-3 activity in the follicular cells and an increased degree of late apoptotic events in granulosa cells in the BCB+ compared with the BCB- group. Additionally, the global gene expression profile revealed a total of 34 and 37 differentially expressed genes between BCB+ and BCB- cumulus cells and granulosa cells, respectively, whereas 207 genes showed an altered transcript abundance between BCB+ and BCB- oocytes. Among these, EIF3F, RARRES2, RNF34, ACTA1, GSTA1, EIF3A, VIM and CS gene transcripts were most highly enriched in the BCB+ oocytes, whereas OLFM1, LINGO1, ALDH1A3, PTHLH, BTN3A3, MRPS2 and PPM1K were most significantly reduced in these cells. Therefore, the follicular cells enclosing developmentally competent oocytes show a higher level of apoptosis and a different pattern of gene expression compared to follicular cells enclosing non-competent bovine oocytes.

Deep intrauterine insemination in pigs allows sperm deposition only into one uterine horn, but bilateral fertilization of oocytes occurs. How the sperm reach the contralateral oviduct remains disputable. The aim of this experiment was to study possible transperitoneal and/or transuterine sperm migration ways. Follicle growth and ovulation were induced in 24 peripubertal gilts with eCG and hCG 72 h after eCG. Endoscopic intrauterine insemination (IUI) was performed 32 h after hCG with 20 ml of extended semen (60 × 10(6) spermatozoa) as follows: Group CONTROL (n=8) received IUI into the right horn, and the left horn served as non-treated control; Group LIGATURE (n=8) received IUI into the right horn, and the left horn was closed by endoscopic double ligature close to the bifurcation; Group INTRAPERITONEAL (IPI; n=8) received IUI into the right uterine horn, the left horn was closed by double ligature and semen was deposited intraperitoneally at the surface of the left ovary. Genital tracts were removed 65-66 h after hCG, the oviducts were flushed and ova (n=299) were analyzed for fertilization and cleavage. Furthermore, the accessory spermatozoa count/oocyte was graded as 0, without spermatozoa, 1, <5 spermatozoa, 2, 5-50 spermatozoa, 3, 50-100 spermatozoa and 4, >100 spermatozoa. The results indicate that low dose IUI into one horn provides a lower grade of accessory spermatozoa in the contra-lateral side (1.6 vs. 2.8). No spermatozoa were found in ova flushed from oviducts of the ligated uterine horn, even after intraperitoneal insemination (P<0.05), and no fertilization occurred, respectively. Our results clearly indicate that after low dose IUI into one uterine horn, spermatozoa reach the contralateral oviduct via transuterine migration.

The objective of this study was to evaluate the role of granulosa cells (GC) and bovine prolactin (bPRL) on nuclear and cytoplasmic maturation of bovine oocytes and their developmental competence in vitro. The level of apoptosis in GC, the mitochondrial activity and the concentration of intracellular stored calcium ([Ca]is) in the matured oocytes, as well as morphology and the chromatin status of produced embryos after IVF were analyzed. Cumulus-oocyte complexes were incubated in TCM 199 containing 10% FCS (control group). Oocytes of experimental groups were culture in control medium added by 10 GC or 50 ng/ml bPRL or 50 ng/ml bPRL with 10 GC (experimental groups). The highest percentage of normal embryos at morulae and blastocyst stages was obtained from oocytes matured in the presence of bPRL and GC (47.9%). The fluorescence intensity of metabolically active mitochondria measured by intensity per oocyte (Em 570) after MitoTracker CMTM Ros Orange labeling was significantly increased in oocytes matured in the presence of 50 ng/ml bPRL and/ or GC (254,3 ± 20,2 μA vs. 119.9 ± 20.4 μA; P<0.05). In parallel, the concentration of [Ca]is in oocytes matured in bPRL and/or GC , determined by using the fluorophore chlortetracycline, was significantly lower (0,67 ± 0,06 A.U. vs. 1,0 ± 0,07 A.U., P <0.05). In addition, it was shown that bPRL decreased the level of apoptosis and the percentage of cells with pycnotic nuclei in GC. In conclusion, it was found that granulosa cells are involved in the realization of prolactin’s action on the developmental competence of bovine oocytes matured in vitro. [Journal of American Science 2010; 6(9):796-805]. (ISSN: 1545-1003).

The aim of the present study was to investigate the developmental competence, mitochondrial characteristics and chromatin status of immature follicular porcine oocytes selected for their glucose-6-phosphate dehydrogenase (G6PDH) activity by brilliant cresyl blue (BCB) staining. In Experiment 1, the oocyte parameters were determined in parallel right after BCB staining (T(0)), after 22 h of in vitro maturation (IVM) (T(22)) and after 44 h of IVM (T(44)) (n = 496). BCB-stained oocytes (BCB+) at T(0) were characterised by fibrillated chromatin filaments in their germinal vesicles (GV) and diakinesis stages whereas unstained (BCB-) oocytes at T(0) contained in their GV mainly condensed stages of chromatin (P < 0.05). After 22 h of IVM BCB+ oocytes showed a prominent chromatin configuration of metaphase I and after 44 h the majority developed a M II nuclear configuration in contrast to the BCB- group (P < 0.0001). Differences were also observed between the two oocyte populations in their mitochondrial activity (P < 0.05). At the beginning of IVM BCB+ oocytes were characterised by high mitochondrial activity in their cytoplasm. The BCB+ oocytes showed clear visible homogenous distributions of mitochondria (P < 0.005) and contained more aggregated clusters of mitochondria in contrast to BCB- oocytes (P < 0.005). In Experiment 2, 318 oocytes were tested for their G6PDH activity and introduced to IVM and IVF. Only oocytes from the BCB+ group, which were matured after 44 h up to the stage of M II (81.6%) were fertilised (17.4%), penetrated (46%) or activated (15.6%) after IVF. These results indicate a relationship between the G6PDH activity of porcine oocytes before IVM and their subsequent nuclear development, mitochondrial activity and aggregation.

The aim of this study was to evaluate the prevalence of follicles with more than one oocyte (polyovular follicles, POFs) in mouse lines selected for high fecundity. The ovaries of 18 mice, 6 each from 3 different lines, were examined to evaluate the number of POFs and the follicular histology. Polyovular follicles were observed in the two high fecundity breeds, FL1 and FL2, as well as in the unselected control line, DUKsi. The highest number of POFs per ovary (27.0 +/- 7.2) was found in the FL1 line. The FL2 and DUKsi lines had 1.9 +/- 0.7 and 0.6 +/- 0.3 polyovular follicles per ovary, respectively. Most of the POFs contained 2 oocytes (>80%), but occasionally follicles containing up to 7 oocytes were observed. Follicles with more than 2 oocytes were observed in the FL1 line only.

Progress in assisted reproduction in the horse has been rapid over the last 10 years. Efficient methods for oocyte recovery from live mares have generated clinical interest in methods for in vitro fertilization (IVF). The conventional method of IVF as used in the bovine has proved to be inefficient in the horse. Therefore, a method for more efficient production of equine zygotes has been developed - the intracytoplasmic sperm injection (ICSI). The aim of our investigation was to establish this method in our laboratory and to test its efficiency in generating developmentally competent equine embryos. Altogether, 146 COCs with different cumulus morphology were recovered. The polar body extrusion rate after IVM was between 48 and 64% depending on the cumulus morphology and the maturation time. The cleavage rate after ICSI was between 40% and 47%. Under the chosen in vitro conditions, we could obtain 16 cell stage embryos and morulae on day 5 after ICSI.

Though many factors have been shown to affect the oocyte developmental potential, it remains difficult to draw clear and reliable criteria for oocyte selection. With the urgent need for establishing non-invasive means for oocyte selection, the brilliant cresyl blue (BCB) staining test based on glucose- 6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate competent and non-competent bovine oocytes (Alm et al. 2005 Theriogenology 63, 2194-2205). Also it has been hypothesized that there is a correlation between the appearance of light atretic granulosa cells (GC) in the follicle and an increased developmental competence of the oocyte. Here we aim to investigate whether different developmental competent oocytes show differences concerning the degree of apoptosis or in the gene expression pattern of their follicular environment [GC and cumulus cells (CC)]. After follicular aspiration, the immature COCs were separately stained with 26 μM BCB for 90 minutes. Based on their colouration, oocytes were grouped into BCB- (colourless cytoplasm, low developmental competence) and BCB+ (coloured cytoplasm, high developmental competence). The corresponding CC and GC were also grouped according to the colouration of the enclosed oocytes. BCB+ oocytes were found to result in a higher blastocyst rate at Day 8 of in vitro culture (34.1%) compared to BCB- ones (3.9%) (n = 601 COCs). Apoptosis in GC was determined either by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) or by Annexin-V-staining followed by flow cytometric measurement. The degree of apoptosis in GC of BCB+ oocytes was slightly increased in contrast to the BCB- group (17.0 v. 11.0%; P < 0.05). The abundance and activity of protein kinases Akt, MAP kinase, and Caspase-3 were estimated by western blot analysis. CC, GC, and oocytes from the BCB+ group showed a higher ratio of cleaved Caspase-3/Caspase-3 in contrast to all compartments of the BCB- group (P < 0.05). Moreover, a bovine Affymetrix microarray plate form (Affymetrix Inc., Santa Clara, CA, USA) was used to analyze the gene expression profiles in oocytes, CC, and GC. The BCB+ oocytes were found to be enriched with genes regulating the oocyte maturation (EIF3F, PRKCSH) and the transition from maternal to embryonic genome activation (HMG2L1). Also BCB+ derived follicular compartments showed elevated expression of genes related to steroidgenesis, cumulus expansion and gonadotropins. In conclusion, the results demonstrate that the developmental competence of oocytes is associated with the apoptotic level and altered expression of genes in cells of their follicular environment. This work was supported financially by Deutsche Forschungsgemeinschaft (To 138/5-1).

The low developmental competence seen in in vitro cultured oocytes collected from early antral follicles may be related to their mitochondrial status. The aim of this study was to examine the chromatin configuration, pattern of mitochondrial aggregation and mitochondrial activity of non-cultured and in vitro-cultured bovine oocytes originating from early antral ovarian follicles. Cumulus-oocyte complexes with adjacent granulosa cells (COCGs) were recovered from early antral follicles of 0.4 to 0.8 mm diameter. Control (Day 0) oocytes were recovered from freshly collected COCGs and fixed and stained. Selected COCGs were placed in growth culture for 7 days (Day 7) or 14 days (Day 14). Following growth culture, COCs with normal appearance were placed in maturation medium (IVM) for 24 h and then fixed and stained with MitoTracker CMTM Ros Orange and Hoechst 33258. The percentage of oocytes with an immature meiotic configuration after growth culture decreased with the time of growth culture, being 96.7; 72.5 and 35.4% respectively for Day 0, Day 7 and Day 14 of culture; the remaining oocytes were degenerating or resuming meiosis. After subsequent IVM the highest proportion of oocytes in diakinesis or metaphase I was found in the D7+IVM group (59.4%). When growth culture was prolonged to day 14 and IVM, the number of degenerated oocytes increased dramatically after IVM. The mitochondrial distribution in the oocytes changed from homogeneous to heterogeneous as growth culture time increased. The respiratory activity as measured by fluorescence intensity increased over the time of growth culture, and was highest in oocytes that had resumed GVBD. In conclusion, for oocytes in isolated COCGs from early antral follicles, culture conditions longer than 7 days should be more adapted for a slow nuclear maturation accompanied by a decreased energy metabolism to prevent chromatin pycnosis.

Summary In vitro embryo production is not successful in the horse and only a few foals have been born by using this technology. While the rate of chromosomal maturation of in vitro cultured horse oocytes is high, fertilization rates in vitro are still not efficient. This could be due to an im paired cytoplasmic maturation in vitro. The aim of the present study was to characterize the cytoplasmic and chromatin quality of oocytes depending on different follicle populations in mares. Therefore, 14 Mecklenburger Warmblood mares underwent repeated transvaginal ultrasound guided follicle aspiration. Aspiration sessions were performed 44 times during the heat of the mare to obtain oocytes from preovulatory and subordinate follicles and 79 times oocytes were recovered from progressive follicle populations before a dominant follicle had developed. Steroid analysis of the follicle fluid confirmed clinical deviation of the follicle populations. Every single oocyte was analysed for cumulus morphology, chromatin configuration, glucose-6-phosphate dehydrogenase activity as well as the mitochondrial activity and aggregation. Our results showed no impact of the follicle population upon the glucose-6-phosphate dehydrogenase activity in the oocytes, measured by brilliant cresyl blue staining. We observed that progressive follicle populations contained significant more oocytes with a fibrillar diplotene configuration and a higher mitochondrial activity, while subordinate follicle populations had more oocytes with a condensed diplotene and a lower mitochondrial activity. In conclusion, the follicle population, originating the oocyte, significantly influences parameters of its quality. Therefore, the origin of the oocyte should be more noticed in programmes of in vitro embryo production in the future.