Sun-Ho Choi

Effect of dominant follicle removal before superstimulation of follicular growth, ovulation and embryo production in Holstein cows

This study was to investigate whether removing the dominant follicle 48 h before superstimulation influences follicular growth, ovulation and embryo production in Holstein cows. After synchronization, ovaries were scanned to assess the presence of a dominant follicle by ultrasonography with a real-time linear scanning ultrasound system on Days 4, 6 and 8 of the estrus cycle (Day 0 = day of estrus). Twenty-six Holstein cows with a dominant follicle were divided into 2 groups in which the dominant follicle was either removed (DFR group, n=13) by ultrasound-guided follicular aspiration or left intact (control group, n=13) on Day 8 of the estrus cycle. Superovulation treatment was initiated on Day 10. All donors were superovulated with injections of porcine FSH (Folltropin) twice daily with constant doses (total: 400 mg) over 4 d. On the 6th and 7th injections of Folltropin, 30 mg and 15 mg of PGF2alpha (Lutalyse) were given. Donors were inseminated twice at 12 h and 24 h after the onset of estrus. Embryos were recovered on Day 6 or 7 after AI. During superstimulation, the number of follicles 2 to 5 mm (small), 6 to 9 mm (medium) and > or = 10 mm (large) was determined by ultrasonography on a daily basis. At embryo recovery, the number of corpora lutea (CL) was also determined by ultrasonography and blood samples were collected for analysis of progesterone concentration. Follicular growth during superstimulation was earlier in the DFR group than in the control group. The number of medium and large follicles was greater (P < 0.01) in the DFR group than in the control group on Days 1 to 2 and Days 3 to 4 of superstimulation, respectively. The numbers of CL (9.6+/-1.1 vs 6.1+/-0.9) and progesterone concentration (30.9+/-5.4 vs 18.6+/-3.5 ng/mL) were greater (P < 0.05) in the DFR group than in the control group, respectively. The numbers of total ova (7.7+/-1.3 vs 3.9+/-1.0) and transferable embryos (4.6+/-0.9 vs 2.3+/-0.8) were also greater (P < 0.05) in the DFR group than in the control group, respectively. It is concluded that the removal of the dominant follicle 48 h before superstimulation promoted follicular growth, and increased ovulation and embryo production in Holstein cows.

Effects of genetic variants for the swine FABP3, HMGA1, MC4R, IGF2, and FABP4 genes on fatty acid composition

This study aimed to verify genetic relationships between fatty acid composition (FAC) and genotypes of several genes (FABP3, HMGA1, MC4R, IGF2, and FABP4) using pig breeds. The effects of genetic variations on FAC of the longissimus muscle were statistically significant with additive and dominance effects. The polymorphisms of FABP3 and IGF2 had the largest effects on stearic (C18:0, P=0.009) and γ-linoleic (C18:3n6, P=0.039) acids, respectively, whereas HMGA1 and FABP4 did not show significances. The analysis revealed that MC4R was significantly associated with palmitoleic acid (C16:ln7) and MUFA. Allele frequencies of the genes examined in this analysis were significantly skewed or fixed in the Korean native pig (KNP), whereas the allele frequencies of the crossbreds tended to fall between those of the purebreds except that HMGA1 and FABP4 had approximately the same allele frequencies with Duroc and KNP, respectively. The polymorphisms found in this study could be used as genetic markers in breeding programs to simultaneously change proportions of fatty acids in muscle tissues.

Integrated transcriptomes throughout swine oestrous cycle reveal dynamic changes in reproductive tissues interacting networks

Female fertility is a highly regulated process involving the synchronized activities of multiple tissues. The underlying genomic regulation of the tissue synchronization is poorly understood. To understand this better we investigated the transcriptomes of the porcine ovary, endometrium, and oviduct at days 0, 3, 6, 9, 12, 15, or 18 of the oestrous cycle. We analysed the transcriptome profiles of the individual tissues and focus on the bridging genes shared by two or more tissues. The three tissue-networks were connected forming a triangular shape. We identified 65 bridging genes with a high level of connectivity to all other genes in the network. The expression levels showed negative correlations between the ovary and the other two tissues, and low correlations between endometrium and oviduct. The main functional annotations involved biosynthesis of steroid hormones, cell-to-cell adhesion, and cell apoptosis, suggesting that regulation of steroid hormone synthesis and tissue viability are major regulatory mechanisms.

Expression and regulation of interleukin 6 and its receptor at the maternal-conceptus interface during pregnancy in pigs

It has been well established that interleukin 6 (IL6), a pleiotropic cytokine with multiple functional roles, is widely expressed in the female reproductive tract and mediates blastocyst implantation and placental development in many species. Uterine expression of IL6 during early pregnancy has been studied in pigs, but expression and function of IL6 at the maternal-placental interface throughout pregnancy have not been determined. Thus, we examined expression of IL6 and its receptors, IL6 receptor (IL6R) and GP130, in the uterine endometrium on Days 12 and 15 of the estrous cycle, and Days 12, 15, 30, 60, 90, and 114 of pregnancy, conceptus on Days 12 and 15, and chorioallantoic tissues on Days 30, 60, 90, and 114 of pregnancy in pigs. The expression of IL6, IL6R, and GP130 mRNA in the endometrial tissues increased dramatically during mid-to late-pregnancy and decreased at term. IL6, IL6R, and GP130 mRNAs were also expressed in conceptus and chorioallantoic tissues. Expression of IL6 mRNA was mainly localized to endometrial epithelial and stromal cells and chorioallantoic tissues, while IL6R and GP130 mRNAs were localized to glandular epithelial cells during pregnancy. The expression of IL6 mRNA was decreased by estrogen and progesterone treatment, whereas increasing doses of IL1β induced the expression of IL6 mRNA, but not IL6R and GP130 mRNAs, in endometrial tissue explants. These results indicate that expression of IL6 and its receptors at the feto-maternal interface is regulated in a stage- and cell-type-specific manner during pregnancy, suggesting that IL6 and its receptor signaling system may play an important role in the maintenance of pregnancy in pigs.

130 PREDICTION OF PARTHENOGENETIC DEVELOPMENTAL POTENTIAL BY POLAR BODY EXTRUSION AND FIRST CLEAVAGE ON IN VITRO MATURATION AND DEVELOPMENT OF PORCINE FOLLICULAR OOCYTES

The objective of this study was to examine the selection effects of in vitro matured porcine follicular oocytes with polar body extrusion and early cleavage as a non-invasive marker to know the developmental competence in advance. Porcine oocytes matured for 48 h and then examined for polar body extrusion. The examined oocytes were matured for an additional 16–18 h, activated with 7% ethanol, and cultured in 5 µg mL–1 cytochalasin B for 5 h for diploid formation. The treated oocytes were examined for cleavage after 48 h and continued culturing for 5 days. Each treatment was replicated by 3–4 times. Oocytes of 21.9% (70/320) were discarded in morphological selection, and 32.1% (167/520) oocytes were discarded by failure of first polar body extrusion. The selected oocytes were matured and activated, and after 48 h, the cleavage rate was examined. In morphologically selected oocytes, 15.8% (30/190) were not cleaved, 52.6% (100/190) were normally cleaved (consisted of 2–7 cells), and 31.6% (60/190) were hyper-cleaved (consisted of 8 cells or more) at 48 h after activation. However, in the first polar body extruded oocytes, 7.1% (18/253) were not cleaved, 73.1% (185/253) were normally cleaved, and 19.8% (50/253) were hyper-cleaved. From the morphologically selected oocytes, 16.7% (10/60) were developed up to blastocyst stage from those in which cleavage selection was not performed and 31.7% (19/60) from those in which cleavage selection was performed. From the polar body extruded oocytes, 39.0% (39/100) were developed up to blastocyst stage from those in which cleavage selection was not performed and 49.0% (49/100) from those in which cleavage selection was performed. Cleavage was examined within 12 h interval after activation (0 = time of activation) up to 48 h. At 0–12, 12–24, 24–36, and 36–48 h intervals, 4.1% (9/220), 68.6% (151/220), 19.1% (42/220), and 2.3% (5/220) oocytes were cleaved, respectively, and 5.9% (13/220) oocytes were not cleaved at 48 h after activation. The cleaved embryos in each interval were cultured and developed up to blastocyst with 0 (0/9), 39.1 (59/151), 9.5 (4/42), and 0% (0/5), respectively. This result suggests that the polar body extruded and cleaved at 12–36 h embryo has higher developmental potential than the others.

390 THE EFFECT OF REDUCED FSH DOSE AND NUMBER OF TREATMENTS ON SUPEROVULATORY RESPONSE IN CIDR-TREATED KOREAN NATIVE COWS

Reducing the total dose and numbers of treatments with FSH for superstimulation without decreasing embryo yield may be less stressful and more economical for bovine embryo transfer. The objective of this study was to investigate the effect of dose and the number of days of FSH treatment on superovulatory responses in CIDR-treated Korean native cows. Forty-two cows, at random stages of the estrous cycle, received a CIDR device (CIDRTM; InterAg, Hamilton, New Zealand), 1 mg estradiol benzoate (SY Esrone; Samyang, Seoul, Korea) and 50 mg progesterone (SY Ovaron; Samyang); gonadotropin treatment began 4 days later. Cows were divided into 2 groups based on the dose and numbers of days of treatment with porcine FSH (pFSH): T1 group (n = 20): a total of 28 mg pFSH (recommended dose of Antorin®; Kawasaki Pharmaceutical, Tokyo, Japan) was given in twice daily IM injections in decreasing doses over 4 days (5, 5, 4, 4, 3, 3, 2, and 2 mg); and T2 group (n = 22): a total of 24 mg pFSH given in twice daily decreasing doses over 3 days (5, 5, 4, 4, 3, and 3 mg). Otherwise, all cows received the same treatments. Twenty-five and 15 mg dinoprost (PGF2α; Lutalyse; Pharmacia & Upjohn, Puurs, Belgium) were given with the 5th and 6th injections of pFSH, respectively. CIDR devices were withdrawn with the 6th pFSH injection, and the cows received 100 µg Gonadorelin (GnRH; Fertagyl; Intervet, Boxmeer, The Netherlands) 36 h after CIDR device removal. Cows were artificially inseminated using commercial semen from 4 Korean native bulls twice, at 48 and 60 h after CIDR device removal, and embryos were recovered 6 or 7 days after the 2nd insemination. The number of CL was counted on the day of embryo recovery by transrectal ultrasonography (Sonovet 600 with 5.0 MHz linear-array transducer; Medison Co., Ltd., Seoul, Korea). The recovered embryos were evaluated according to the IETS Manual for stage of development and quality. All data between groups were compared using Students t-test. The numbers of CL (9.7 ± 1.1 vs. 9.4 ± 1.3), total ova/embryos (7.2 ± 1.1 vs. 6.3 ± 1.4), transferable embryos (4.4 ± 1.0 vs. 3.6 ± 0.9), degenerate embryos (0.9 ± 0.3 vs. 1.3 ± 0.4), and unfertilized ova (2.0 ± 0.6 vs. 1.5 ± 0.5) did not differ between groups (T1 vs. T2), respectively (P > 0.05). Data indicate that the reduced dose (24 vs. 28 mg) and numbers of treatments (6 vs. 8) of pFSH for superstimulation of Korean native cows does not affect the embryo yield.

158 EFFECTS OF APOTRANSFERRIN ON IN VITRO MATURATION OF CUMULUS–OOCYTES COMPLEXES (COCs) IN HANWOO, KOREAN NATIVE COWS

For in vitro production of embryos, animal sera have been used as energy sources, maturation promoters, vitamins, growth factors, and antioxidative compounds. However, the sera had risk of virus and mycoplasma infections which could result in too big offspring and cause dystocia in ovine and bovine. Apotransferrin (apo-Tf) is a component of mammalian sera and has played a role as an antioxidant in media. A study was conducted to investigate the effects of apo-Tf on in vitro maturation of cumulus-oocytes complexes (COCs) in Hanwoo, Korean native cows. Ovaries were collected from a slaughterhouse and COCs were taken from 2-6-mm antral follicles. The collected COCs were washed three times with 0.1M polyvinyl alcohol (PVA)-TCM199 and matured in 0, 1, 10, or 100 ¼g/mL apo-Tf with TCM-199 at 39°C, 5% CO2, 95% air for 6, 12, or 24 h. Mature COCs were fertilized with frozen-thawed Korean native cattle semen treated with BO medium (Brackett and Oliphants 1975 Biol. Reprod. 12, 260-274) containing 5 mM caffeine and 1 ¼g/mL heparin for 8 h and developed to the blastocyst stage in 5% FBS and 0.3% BSA in TCM199-IVMD (IFP, Japan). To evaluate the morphology of nuclear types, the matured COCs were fixed in 1:3 acetic acid-ethanol for 30 s and stained with 3% basic Fuchsin. IVM and IVF were replicated three times. All of the results were analyzed by ANOVA using the STATVIEW program. The maturation rates of control were 34.2%, 37.3%, and 45.8% for 6, 12, and 24 h, respectively. There were no differences among the concentrations of apo-Tf, and nuclear types at 78.3-87.0% GVBD for 6 h, 82.8-91.3% MI for 12 h, and 88.9-100.0% MII for 24 h, with 1, 10, and 100 ¼g/mL apo-Tf, respectively. Conversely, there was significant difference between 1 µg/mL and 10 ¼g/mL in terms of cleavage rates, although the others did not vary significantly (P < 0.05). There were significant differences among the concentrations of apo-Tf for blastocyst formation (P < 0.05). Blastocysts matured with 1, 10, and 100 ¼g/mL apo-TF and developed in 5% FBS and 0.3% BSA in TCM199-IVMD showed rates of 8.8-21.6%, 9.4-35.3%, and 9.1-19.1%, respectively. The control groups developed to the blastocyst stage showed rates of 8.6%, 10.8%, and 10.5% in 5% FBS and 0.3% BSA in TCM199-IVMD, respectively. These results suggest that apo-Tf is an important factor for the in vitro maturation and in vitro development of bovine COCs.