bluuebunny/crossref_metadata_2025 huggingface dataset:
212 EFFECT OF OVULATION STATUS ON PREGNANCY RATES IN RECIPIENT GILTS TRANSFERRED WITH CLONED EMBRYO TRANSFER
Authors: Lee J. H. , Jeung Y. W. , Hyun S. H. , Lee E. S. , Jeung E. B.
Published: December 2006

This study was conducted to determine the optimal stage of the estrus cycle to achieve the highest pregnancy rates in surrogate recipient gilts receiving somatic cell nuclear transfer (SCNT) embryos. Potential surrogate gilts over 7 months of age were checked at Day 21 of their estrus cycle for their estrous status by observing external signs: vaginal fluid, vulva redness, vulva swelling, and standing response to back pressure. Viscosity of vaginal fluid was evaluated and classified as none, medium, and strong. Vulva redness and swelling were respectively assessed as none or shrink, medium, and strong. Back pressure was estimated by an immediate move, standing less than 10 s, and standing over 10 s. Ovulation status was observed when surrogates had SCNT embryos transferred into their oviducts, and then ovulation status of each surrogate was classified depending on the follicular diameter, as follows: preovulation (PO – 17 surrogates; follicular diameter of PO was 3 to 5 mm), just prior to ovulation (JPO – 20 surrogates; follicular diameter of JPO was 6 to 8 mm), ovulating (IO – 12 surrogates; ovulation mark with JPO follicles), just after ovulation (JAO – 14 surrogates; ovulation mark without follicles), and after ovulation (AO – 24 surrogates; ovaries showed only corpora lutea). Real-time ultrasonograpy was used for pregnancy diagnosis by observing amniotic vesicles. All data were subjected to analysis of variance (ANOVA) and protected least significant difference (LSD) test using general linear models in a statistical analysis system (SAS Institute, Inc., Cary, NC, USA) program to determine differences among experimental groups. The first pregnancy diagnosis was done on Day 30 after ET and then repeated in 2-week intervals until Day 114. SCNT embryos transferred into JPO surrogates gave better pregnancy rates (9/20, 45%) than others (4% to 11%) on Day 30 after ET (P < 0.05). These results suggest that ovulation status of surrogate gilts is an important critical factor for pregnancy. This information indicates that surrogate gilts implanted with SCNT pig embryos just prior to ovulation can achieve optimal pregnancy rates. In conclusion, the JPO surrogate selected should strongly show all parameters (vaginal fluid, vulva redness, vulva swelling, and standing response to back pressure) for the preparation of surrogates.
This study was supported by the bio-organ production research grant (No.06020602) of the NLRI.
215 COMPARISON BETWEEN CONVENTIONAL DIRECT TRANSFER FREEZING AND VITRIFICATION FOR THE CRYOPRESERVATION OF IN VIVO EMBRYOS FROM BRAHMAN CATTLE
Authors: Pryor J. H. , Looney C. R. , Walker D. , Seidel, Jr G. E. , Hasler J. F. , Kraemer D. C. , Romo S.
Published: December 2006

There is a need to develop an efficient cryopreservation technique for Brahman cattle embryos that would lead to improved success in the propagation of this breed. The objective of this study was to compare the post-thaw pregnancy rates in recipient cows after nonsurgical transfer of Brahman in vivo-derived embryos frozen in ethylene glycol (EG) and by a new vitrification (VT) method. Prior to the initial in vivo study, hatching rates were recorded 72 h post-thaw on 3 treatment groups (No treatment: 43/55, 78%; EG: 39/83, 47%; and VT: 33/103, 32%) of IVF embryos to determine if development could be achieved. This warranted Phase I: In vivo embryos were produced by multiple ovulation and nonsurgical embryo collections from Brahman cows 7 days post-estrus and AI. Embryos in morula/blastocyst stage of development were graded and maintained in holding medium (Vigro; Bioniche Animal Health, Inc., Belleville, Ontario, Canada) after collection, until randomly allocated for EG or VT protocols. Vitrification was performed using experimental media provided by a commercial company (V1: Vigro + 5 M EG; V2: Vigro + 7 M EG + 0.5 M Galactose + Ficoll; and Diluent: Vigro + 0.5 M Galactose) and by direct plunging into LN2 (Walker et al. 2005 Reprod. Fertil. Dev. 17, 153). Freezing was performed at 0.5�C min-1 from -6�C to -32�C, using a commercially available medium (Vigro Freeze Plus; Bioniche). All embryos were packaged in sterile 0.25-mL plastic straws to allow for direct transfer (DT). Embryos were stored in LN2, and later thawed and nonsurgically transferred to synchronized recipient cows on Day 7 of their cycle. EG straws were air-thawed 5 s and then thawed in 30�C H2O for 10 s. VT straws were air-thawed 10 s and then thawed in 37�C H2O for 20 s prior to shaking them down to mix columns. Pregnancy results evaluated by ultrasound on Day 35 for the presence of a fetus from 4 replicates were 27% for EG (3/11) and 8% for VT (1/13) embryos. Problems with VT straws cracking during air thawing led to a second trial (Phase II) which changed the direct plunging technique for VT to a vapor freeze of 1 to 15 min prior to plunging into LN2. Pregnancy results from 16 transfers were 38% for EG (3/8) and 50% for VT (4/8) embryos, with no straws cracking. Although the number of observations is small, the results obtained showed that VT-DT is satisfactory for producing pregnancies comparable to EG-DT. Further research is required to confirm the results obtained in this preliminary study, and to test whether this method will allow the successful production of pregnancies under different environmental and field conditions.
225 TERATOMA FORMATION BY BOVINE EMBRYOS
Authors: Lim M. L. , Vassiliev I. , Verma P. J.
Published: December 2006

Teratoma formation is commonly used as a model for examining the in vivo differentiation potential of embryonic stem cells. We wanted to investigate the teratoma-forming ability of bovine ES cells; however, there are no reports of teratoma-forming ability of bovine pluripotent cells including pre-implantation embryos. In vivo-produced bovine embryos at stages earlier than Day 14 failed to develop teratomas when transplanted into one of the kidneys of immuno-deficient mice (Anderson et al. 1996 Anim. Reprod. Sci. 45, 231–240), and this prompted questions about the ability of bovine embryos to form teratomas. Bovine oocytes were cultured for 20 to 22 h after aspiration at 39�C (5% CO2/95% air) in TCM-199-bicarbonate medium supplemented with GlutaMax6" (Invitrogen Australia Pty Ltd., Mount Waverley, Victoria, Australia), penicillin/streptomycin, β-mercaptoethanol, 17β-estradiol, fetal calf serum, LH, follicle stimulating hormone, basic fibroblast growth factor, epidermal growth factor, glycine, and l-cysteine. Oocytes were fertilized with IVF media (Cook Australia, Brisbane, Queensland, Australia) and kept for 7 days at 39�C in 5% CO2/95% air to generate blastocysts. The zona pellucida of Day 7 blastocysts was enzymatically removed, and one or two zona-free embryos were injected into each testis of 5-week-old immunodeficient (SCID) mice (CB-17/ICR-Prkdcscid strain; Walter and Eliza Hall Institute, Melbourne, Australia). Eight weeks post-injection, teratomas partially expelled from testes were identified. Histological analysis has confirmed the derivatives of all 3 germ layers in teratomas. In conclusion, we report that Day 7 in vitro-produced embryos can form teratomas when injected into testes of SCID mice.
237 POTASSIUM CONCENTRATION AND mRNA LEVELS OF POTASSIUM CHANNELS DECREASED IN CYSTIC OVARIAN FOLLICLE FLUID
Authors: Kang D. , Choe C. , Kim E. S. , Yang H. Y. , Hur C. G. , Kim E. J. , Han J.
Published: December 2006

Cystic ovarian follicle (COF) is one of the most frequently diagnosed ovarian diseases and a major cause of reproductive failure in cattle. Despite an abundance of reports on this subject, the exact pathogenesis of COF still remains unclear. It is generally accepted that disruption of the hypothalmo-pituitary-gonadal axis, by endogenous and/or exogenous factors, causes cystic formation. We here examined whether ion concentration and expression of ion channels are altered in the follicle fluid derived from a Korean native cow with COF. In an ovary with a cystic follicle, granulosa cell layers were exfoliated; the theca interna was thinner than that in an ovary without cystic follicle, based on histological examination. Concentrations of K+, Na+, and Cl- in COF fluid (COFF) were 10.4 � 3.5 mM, 138 � 12 mM, and 104.9 � 7.0 mM, respectively. In COFF, K+ concentration showed a significant difference from the value observed in normal follicle fluid (NFF) (P < 0.05; NFF: 10.4 � 3.5 mM vs. COFF: 6.2 � 0.8 mM). The total numbers of follicles observed (normal, 3–5 mm in diameter vs. COF, 20–30 mm in diameter) were 200 and 20 in normal and COF, respectively. To compare mRNA expression of K+ channels, we performed semiquantitative RT-PCR using follicle fluid and ovaries with or without cystic follicles. RT-PCR showed that mRNA levels of TASK channels (TASK-1, TASK-3, and TASK-5) decreased by 50% in COFF and an ovary with cystic follicles compared to NFF and a normal ovary. TASK channels are involved in apoptosis of mammalian cells. Our results suggest that potassium may play an important role in the pathogenesis of COF.
249 INTRACYTOPLASMIC SPERM INJECTION OF ELAND (TAUROTRAGUS ORYX) AND BONGO (TRAGELAPHUS EURYCERUS)ANTELOPE OOCYTES
Authors: Wirtu G. , Pope C. E. , Gomez M. C. , Cole A. , Paccamonti D. L. , Dresser B. L.
Published: December 2006

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique applicable in cases of limited male gamete availability. Moreover, it bypasses barriers of the oocyte, thus avoiding poorly understood species-specific capacitation events affecting sperm–egg interaction. In the present study, we evaluated the application of conventional and piezo drill-assisted ICSI and whether subsequent chemical activation is required for initiating embryonic development in eland (Taurotragus oryx) and bongo (Tragelaphus eurycerus) oocytes. Oocytes were collected using transvaginal ultrasound-guided follicular aspiration after gonadotropin-induced ovarian stimulation and incubated in modified TCM-199 medium (Gomez et al. 2000 Reprod. Fertil. Dev. 12, 423) containing 10% FBS. After 3 to 24 h, the cumulus cell layers were removed either by repeated mouth-pipetting and/or by using hyaluronidase. Oocytes with an extruded first polar body were used for ICSI and the other oocytes were returned to culture and evaluated every six hours Piezo drill-assisted (Kimura and Yanagimachi 1995 Biol. Reprod. 52, 709) or conventional (Gomez et al.) ICSI were done as described previously using glass pipettes with internal tip diameters of 9–10 µm. We used frozen–thawed or freshly collected spermatozoa that were kept in HEPES-buffered Tyrode's medium (Gomez et al.) for up to 24 h. Four to 6 h after ICSI, 3 activation treatments were examined: (1) none; (2) 7% ethanol, 5 min; or (3) calcium ionophore (5 µM, 5 min) followed by DMAP (2 mM, 4 h). Then we cultured oocytes in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 at 38.5°C in one of 3 media: SOF, α-MEM, or CR1aa containing essential and nonessential amino acids and FBS. Fifty-three of 70 (76%) eland oocytes survived after piezo-ICSI, and 13 of 16 (81%) survived after conventional ICSI. For bongo oocytes, 27 of 30 (90%) survived piezo-ICSI and all (n = 8) survived after conventional ICSI. Table 1 outlines cleavage data on Day 2. Generally, embryonic development was arrested at about 10 cells. In summary, eland and bongo oocytes can survive both conventional and piezo drill-assisted ICSI. Activation treatments do not appear to be a prerequisite for initiating cleavage after ICSI in eland and bongo antelope oocytes.

Table 1.Cleavage of eland and bongo antelope oocytes after conventional or piezo-ICSI and three activation treatments
253 GROWTH OF PRIMORDIAL OOCYTES FROM INFANT AND ADULT PORCINE OVARIES IN XENOGRAFTS
Authors: Moniruzzaman M. , Lee J. , Miyano T.
Published: December 2006

Mammalian ovaries contain a lot of small oocytes in primordial follicles. A small proportion of these primordial oocytes (oocytes in primordial follicles) start to grow and reach full size, and finally they are ovulated. The mechanism regulating the activation of primordial oocytes to enter the growth phase is not understood yet. This experiment was aimed to compare the growth of primordial oocytes from infant and adult pigs. Ovarian cortical tissues containing primordial follicles collected from infant (10–20-day-old) and adult (6-month-old) pigs were xenografted under the kidney capsules of male, female (intact), and ovariectomized SCID (severe combined immune deficiency) mice. Data were subjected to one-way ANOVA, and the significance of difference among means was determined by the Tukey's multiple range test. Differences at P < 0.05 were considered statistically significant. A proportion of the primordial oocytes from the infant pigs grew to the full size at 2 months after xenografting, irrespective of the type of recipient mice. Under the same conditions, the oocytes from adult pigs did not initiate growth, although they grew after 6 months of xenografting. These results suggest that the mechanism regulating the activation of primordial oocytes in adults is different from that in infant pigs. In this regard, the involvement of FOXO3A, a member of Forkhead transcription factors, recently known as a regulator of ovarian follicular development, was studied. Western blot analysis showed stronger expression of FOXO3A in primordial oocytes from adult pigs than in those from infants. Immunolocalization was detected in the nuclei of 42 ± 7% primordial oocytes in infant pigs. In contrast, immunostaining was positive in 94 ± 2% primordial oocytes in adult porcine ovaries. Four months after xenografting ovarian tissues from adult pigs, the number of FOXO3A-positive primordial oocytes significantly declined to a proportion similar to that in the ovaries from infant pigs. These results suggest that primordial oocytes in adult pigs take a much longer time to initiate growth than those in infants and that they might be inactivated by FOXO3A.
255 EFFECT OF THE TYPE OF DIETARY FATTY ACID (α-LINOLENIC ACID OR LINOLEIC ACID) ON THE NUMBER AND THE QUALITY OF OOCYTES COLLECTED BY OVUM PICKUP AND ON IN VITRO EMBRYO PRODUCTION BY DAIRY HEIFERS
Authors: Ponter A. A. , Arnault J. , Joly C. , Guelou K. , Valy G. , Ponchon S. , Gonzales C. , Grimard B. , Humblot P.
Published: December 2006

A supplement of dietary fat can improve oocyte quality in ruminants. However, to our knowledge, the effect of the type dietary fat (differing in fatty acid profile) on oocyte and embryo production has never been reported in cattle. Therefore, in this study the effect of the type of fat supplement on the number and quality of oocytes collected by ovum pickup (OPU) and on the production of embryos was investigated in Holstein heifers. The experiment was conducted over 2 years: year 1: 8 heifers, and year 2: 10 heifers (16 to 20 months old, body weight 368 � 8.2 kg and BCS 2.3 � 0.1). Heifers were given a diet of hay (67% DM) and a concentrate (33% DM). The concentrate (130 g fat/kg DM) was formulated with either extruded linseeds (L, rich in α-linolenic acid (ω-3 fatty acid),n = 9) or extruded soybeans (S, rich in linoleic acid (ω-6 fatty acid), n = 9). Oocytes were collected by OPU for 6 weeks (2 sessions/week) and their morphological quality was assessed (Q1, excellent; Q2, good; Q3, fair; and Q4, low). The oocytes from one session/week were frozen and those from the other session were used to produce embryos (in vitro maturation and fertilization). Embryo quality was judged following IETS criteria (EQ1, excellent; EQ2, good; EQ3, fair; and EQ4, low). Blood samples were collected weekly throughout the experiment for the analysis of insulin, insulin-like growth factor-1, glucose, non-esterified fatty acids, β-hydroxybutyrate, and urea. Statistical analysis was performed on the growth rate, BCS, hormone, metabolite, and fatty acid data and on the oocyte and embryo data using Split plot ANOVA. Growth rate was not affected by diet (S, 0.93 � 0.21 kg d-1 vs. L, 0.90 � 0.21 kg d-1), and there was no effect of dietary treatment on plasma hormone and metabolite concentrations. However, treatment L increased the proportion of α-linolenic acid (P < 0.0001), and treatment S increased the proportion of linoleic acid (P < 0.0001) in the plasma. Neither the oocyte characteristics (number of oocytes collected, 5.3 � 1.0 vs. 5.7 � 1.0; their quality: Q1, 1.1 � 0.3 vs. 1.0 � 0.3; Q2, 1.8 � 0.3 vs. 1.6 � 0.3; Q3, 1.4 � 0.4 vs. 2.1 � 0.4; and Q4, 1.0 � 0.2 vs. 1.1 � 0.2; and oocytes inseminated, 4.4 � 0.8 vs. 5.1 � 0.8, and cleaved, 3.4 � 0.7 vs. 3.5 � 0.7 per heifer per session; L vs. S, respectively) nor the embryo characteristics (number of embryos, 1.1 � 0.3 vs. 0.9 � 0.3; their quality: EQ1, 0.4 � 0.1 vs. 0.3 � 0.1; EQ2, 0.4 � 0.1 vs. 0.3 � 0.1; EQ3, 0.2 � 0.1 vs. 0.3 � 0.1; and EQ4, 0.04 � 0.03 vs. 0.05 � 0.03 per heifer per session; L vs. S, respectively) were affected by dietary treatment. In conclusion, under our experimental conditions, the type of fatty acid (ω-3 vs. ω-6) does not modify significantly the numbers of oocytes and embryos produced by OPU and their quality in dairy eifers.
266 EFFECT OF 2,2′,4,4′-TETRABROMODIPHENYL ETHER ON THE EXPRESSION OF CALBINDIN-d9 k IN THE UTERUS OF IMMATURE RATS
Authors: Jeung V.-H. Dang and E.-B.
Published: December 2006

Polybrominated diphenyl ethers (PBDEs) are well known as one class of halogenated organic brominated flame retardants. They were found in the air, soil, sediments, humans, wildlife, and fish. To date, a total of 209 PBDE congeners have been reported. Among them, 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) is the dominant congener found in humans and animals. A number of studies suggest that BDE-47 possesses the potential to disrupt endocrine and reproductive function(s) via diverse mechanisms. In addition, BDE-47 has been demonstrated to act as a developmental neurotoxicant. Recently, the toxicokinetic properties of BDE-47 have been investigated in order to elucidate the health risk of BDE-47. Thus, in the present study, we employed immature rats as a developmental model to examine the potential involvement of BDE-47 in the expression of uterine calbindin-d9 k (CaBP-9 k), a novel biomarker for screening endocrine disruptors. Immature rats (n = 3) at post-natal Day 16 were treated with different doses (50, 100, and 200 mg kg-1 of BW) of BDE-47 consecutively for 3 days and euthanized at 24 h after the last injection. After treatment with this chemical, the effects on uterine weight gain and CaBP-9 k mRNA were examined by uterotrophic and RT-PCR assays, respectively. Treatment with a high dose (200 mg kg-1 of BW/day) of this chemical resulted in a significant increase in the uterine weight in immature rats, whereas a modest increase was observed with lower doses (50 and 100 mg kg-1 of BW/day). In addition, treatment with BDE-47 at all doses increased the expression level of CaBP-9 k mRNA compared to control. Taken together, these results demonstrate that BDE-47 exposure resulted in an increase of uterotrophic response and CaBP-9 k mRNA in the uterus of immature rats. A further analysis in a time response of this chemical is required to bring new insight into the adverse effect(s) of BDE-47 during the critical window stage of the developing reproductive system.
274 LOCALIZATION OF MACROPHAGE COLONY STIMULATING FACTOR (M-CSF) GENE IN THE ENDOMETRIUM OF JAPANESE BLACK COW
Authors: Oshima K. , Yoshihara K. , Kojima T. , Komatsu M. , Yamamoto N.
Published: December 2006

Macrophage colony stimulating factor (M-CSF), a member of the group of hemopoietic cytokines, plays an important role in placental physiology of humans and mice. The objective of this study was to investigate the localization of M-CSF gene in bovine endometrial tissues during pregnancy using in situ hybridization (ISH). Twelve Japanese Black cows aged between 1.4 and 14.5 years, with normal estrous cycles, were used in this study. They were observed daily for estrous behavior, and the day of estrus was considered as Day 0. Cows were artificially inseminated, and pregnancy was confirmed by presence of conceptus and/or corpus luteum using ultrasonography. They were euthanized and their uteri were collected on Days 20 to 21 (n = 2), 36 to 42 (n = 3), 59 to 64 (n = 3), 127 to 137 (n = 2), and 225 to 226 (n = 2) of pregnancy. Pregnancy was confirmed finally by the presence of a conceptus in the uterus, and the uterus was isolated by dissection, avoiding damage of the uterine artery. The uterus was perfused with 4% paraformaldehyde (PFA) solution using catheters inserted into the uterine artery, and placental tissues were isolated by dissection. Each tissue was cut into small pieces (5 mm thick) and fixed in 4% PFA solution. After being fixed in 4% PFA solution for 20 to 24 h, tissue pieces were embedded in paraffin using routine procedures. Several tissue pieces were collected from each individual cow. Sections (6 mm thick) were cut and placed on MAS-coated glass slides (Matsunami Glass, Kishiwada, Japan). The sections were dried in an oven for 3 days at 40�C. Antisense and sense biotin-labeled oligonucleotide probes for bovine M-CSF were designed from the sequence information (GenBank accession number D87917). Sense probe was used as the negative control. ISH was carried out using the GenPoint System (DakoCytomation, Glostrup, Denmark) according to the manufacturer's instructions. The sections were observed under an Eclipse 800 microscope (Nikon, Tokyo, Japan), and the positive signal for M-CSF gene was detected. Density analysis was performed by using Scion Image (Scion Corporation, Frederick, MD, USA). Data were analyzed by one-way ANOVA and Tukey-Kramer's HSD, using 0.05 as a significant level. The M-CSF gene was expressed in the stromal and luminal epithelial cells of endometrium, stromal cells of the fetal cotyledonary villus and caruncular crypt, and mono- and multi-nuclear epithelium cells of the fetal cotyledonary villus and caruncular crypt. Intensities of M-CSF gene-positive signals in all positive cells at Days 225 to 226 of pregnancy were stronger than those at other periods (P < 0.05). These results suggest that M-CSF is produced in several cell types in the endometrium and placental tissues and may play important roles in bovine pregnancy.
279 DIFFERENCES IN TRANSCRIPT ABUNDANCE BETWEEN MALE AND FEMALE BOVINE BLASTOCYSTS PRODUCED IN VITRO USING SEXED SEMEN
Authors: Rizos D. , Lonergan P. , Rath D. , De la Fuente J. , Wade M. , Parrilla-Riera I. , Gutierrez-Adan A.
Published: December 2006

There is considerable evidence in the literature indicating that, depending on the culture environment, male pre-implantation embryos develop faster than females, with, for example, a higher proportion of early developing blastocysts being male. The aim of this study was to examine gender-related differences in gene expression in bovine blastocysts produced in vitro following IVF with sex-sorted, frozen–thawed semen. For blastocyst production, immature cumulus–oocyte complexes (COCs) recovered from the ovaries of slaughtered heifers were matured in vitro for 24 h. Matured COCs were randomly split into 2 groups and inseminated with frozen–thawed sperm sorted flow cytometrically for gender. Presumptive zygotes were cultured for 7 days in synthetic oviduct fluid medium. In a preliminary experiment, blastocysts (XX: n = 61; XY: n = 47) were recovered on Day 8 post-insemination and individually snap frozen in liquid nitrogen for verification of the sorting procedure. Sexing was performed with PCR using both male-specific (BRY4.a) and bovine satellite primers. The proportion of female and male blastocysts obtained with X- and Y-chromosome-bearing sperm was 87.2 and 80.3%, respectively. In a subsequent 5 replicates, Day 8 blastocysts were snap frozen in groups of 10 for analysis of mRNA relative abundance. These developmentally important genes were selected based on microarray analysis because they showed a high sensitivity to suboptimal in vitro culture conditions (data not shown). There was no difference in the relative abundance of desmocollin II (DcII), Na/K-ATPase alpha1 subunit (Na/K), ubiquitin-activating enzyme E2 (Ube2), fibroblast growth factor 4 (FGF4), and DNA methyltransferase 1 (Dnmt1) between male and female blastocysts. X-inactive specific transcript (Xist), interferon-tau (IFN), and a Sry-related HMG box transcriptoinal factor (Sox17) were significantly up-regulated in female blastocysts, whereas DNA methyltransferase 3a (Dnmt3a), DNA methyltransferase 3b (Dnmt3b), sarcosine oxidase (Sox), and the transcription factor Oct-4 were significantly up-regulated in males. In conclusion, differences in gene expression between male and female embryos exist and may be related to the well-described differences in the kinetics of development.
28 EXPRESSION OF MHC CLASS I IN THE PLACENTA AND THE LYMPHOCYTES OF BOVINE SOMATIC CLONES
Authors: Chavatte-Palmer P. , Guillomot M. , Roïz J. , Servely J.-L. , Heyman Y. , Hue I. , Ellis S.
Published: December 2006

A major limitation for the development of somatic cloning in cattle is the low efficiency for producing live offspring because only 5 to 25% of the reconstructed embryos reach full-term development, with variation between the genotypes. Hill et al. (2002 Biol. Reprod. 67, 55–63) reported that early fetal losses were due to inappropriate expression of trophoblast major histocompatibility complex Class 1 (MHC1) antigens on the placenta of bovine somatic clones. This abnormal expression was present in all clones, with more cells expressing MHC1 in those with growth retardation. These clones, however, originated from only one genotype, and only one antibody recognizing a monomorphic determinant of bovine MHCI was used. In the present study, MHC1 expression was examined throughout gestation in bovine clone placentas originating from 3 different genotypes. Placentas were collected at Days 32, 62, 180, 260, and 280 (term) in clones (n = 8) and in controls obtained by artificial insemination (n = 6). Four different monoclonal antibodies that recognize different determinants of MHC1 molecules (ILA-A88, IL-A19, H58A, and the β2-microglobulin, i.e. the MHC1 light chain) were used. Results showed MHC1 expression in the maternal tissue but not on the fetal side, regardless of group, gestational age, or fetal growth, although patchy expression of β2-microglobulin could be found in both clones and controls in the fetal mesenchyme in some but not all samples. In conclusion, MHC1 protein expression appears normally down-regulated in the placenta of clones, but the antibodies used do not discriminate between classical and non-classical expression. The mRNA expression of classical and non-classical MHC1 genes is currently being explored. These results cast doubt on the hypothesis that the observed fetal losses would be the result of an immunologic rejection due to inappropriate expression of some placental MHC1 genes. Although early observations suggest that immune functions appear normal (Lanza et al. 2001 Science 294, 1893–1894; Chavatte-Palmer et al. 2006 Reprod. Fert. Dev. 18, 121 abst), immunological deficiencies have been reported in some bovine somatic clones after birth (Renard et al. 1999 The Lancet 353, 1489–1491), and they may be related to an abnormal expression of MHC1 genes. We therefore determined the MHC1 haplotype of cell donors from 4 genotypes and 3 of their healthy clones using the Reference Strand Conformation Analysis method with DNA extracted from lymphocytes. Results showed that the MHC1 type was identical, as could be expected in clones. Animals that died of immune deficiencies, however, may have had abnormal MHC1 expression. Further work is therefore being performed on tissues obtained postmortem from cloned animals having been diagnosed with thymic atrophy.
287 RESCUE AND IN VITRO MATURATION OF FOLLICULAR OOCYTES IN CHINCHILLA LANIGER
Authors: Aiudi G. , Cinone M. , Maritato F. , De Sandro Salvati A. , Dell'Aquila M. E.
Published: December 2006

The chinchilla is a hystricomorph rodent with a natural habitat in the Andes mountains of Chile (see review by Boussarie 2002 Proc. 27th WSAVA Congr.). For most of the chinchilla subspecies, the decline in the natural population can be attributed to human destruction of the native ecosystems and hunting for fur. Chinchillas are listed as a protected endangered species, at immediate risk of extinction. In Europe, chinchillas are reared for pets and fur production. The female has a seasonal polyestrous reproductive activity with a breeding season from November to May. The estrous cycle length is variable (28–41 days), with an estrous duration of 2 days. After a gestation of about 112 days, a litter of 1 to 6 young is born (see reviews by Morrow 1986 in Current Therapy in Theriogenology 2, W.B. Saunders; and Collot 1998 in Proc. I EVSSAR Congr.). Reproductive biotechniques in this species could play an important role in managing both captive and natural populations as well as in sustaining and improving genetic and global biodiversity. The specific aim of this preliminary work was to standardize an efficient in vitro maturation (IVM) procedure for Chinchilla laniger oocytes so that it will be possible, in the future, to perform IVF and embryo cryopreservation and transfer. Oocytes from 4 cyclic breeding females were recovered by slicing ovaries, obtained by ovariohysterectomy, and were matured in vitro according to the procedure described for bovine oocytes by Dell'Aquila et al. (2002 Mol. Reprod. Dev. 63, 210–222). Two trials of 2 estrous subjects each were performed, on the basis of behavioral signs of estrous and vaginal cytology (Harris-Schorr staining), in the early and late breeding seasons. During estrus, the vaginal smear consisted of superficial cells, further neutrophils, and small and large intermediates, whereas parabasal cells were not found. At the end of the culture time, oocytes were stained with Hoechst 33258 and evaluated for the stage of meiotic maturation. Three out of 4 oocytes recovered in November (75%) reached full meiotic maturation, showing the second metaphase plate and the first polar body (PB) extruded. The fourth oocyte, showing the first PB together with multiple pronuclear structures, was classified as activated. On the contrary, none out of 12 oocytes recovered in May reached full maturation. Of them, 7 (58%) remained at the germinal vesicle stage, 2 (17%) reached metaphase I, and 3 (25%) showed abnormally dispersed chromatin configuration. To our knowledge, this is the first study reporting that chinchilla oocytes can be matured in vitro by bovine IVM procedures. Even though the number of oocytes was poor, we can hypothesize that oocytes from C. laniger are best collected in the breeding season when subjected to an IVM technique.
29 COMPARISON OF DNA METHYLATION RATES BETWEEN CLONED CATS AND DOMESTIC CATS
Authors: Cho S. J. , Kang S. H. , Cho S. G. , Bae I. H. , Yang C. J. , Yin X. J. , Kong I. K.
Published: December 2006

Many mammalian species have recently been successfully cloned using somatic cells. We have produced cloned kittens by somatic cell nuclear transfer (SCNT). The cloning of mammals by SCNT requires epigenetic reprogramming of the differentiated state of donor cells. We recently observed that feline catus satellite DNA regions exhibit DNA methylation in the donor and somatic cells of a cloned cat. The cause of variation could lie in alterations occurring at different steps of the cloning procedure or incomplete reestablishment of DNA methylation patterns. This study was conducted to determine the methylation status of the feline catus satellite DNA region in the somatic cells of cloned cats and domestic normal cats by using a bisulfite sequencing method. Satellite sequences are heavily methylated in somatic cell chromosomes and are associated with dense heterochromatin where bundles of silent genes are wrapped up together. For the analysis of the cat satellite sequence, a 400-bp segment of the satellite genomic region, which has highly conserved 22 CpG sites, was amplified by PCR from bisulfite-treated genomic DNA, and the resulting PCR products were individually cloned and sequenced. For methylation analysis, genomic DNA was first isolated from somatic and placental cells of cloned and domestic normal cats. The DNA methylation status of the satellite DNA region was not significantly different among donor (using NT) cells (70.6, 77.0, and 79.1% in Donors A, B, and C, respectively), somatic cells of cloned cats (88.9, 82.2, and 85.9% in cloned A-1, 2, and 3; 78.0% in cloned B-1; 88.1, 78.9, 80.3, 83.2, 74.6, and 82.3% in cloned C-1, 2, 3, 4, 5, and 6, all respectively), and domestic cats (88.0, 81.6, and 81.8% in NC 1, 2, and 3, respectively). Also, the DNA methylation status of satellite DNA regions in the placenta was significantly different between cloned cats (80.5, 76.7, and 76.8% in cloned C-2, 5, and 7, respectively) and normal domestic cats (64.2, 66.7, and 74.9% in NC 1, 2, and 3, respectively). In conclusion, all of the somatic cells were highly methylated. The methylation status of the somatic cells was not different among groups, but that of placental cells was significantly different.
This work was supported by KOSEF (Grant ? M10525010001-05N2501-00110).
290 ENZYMATIC ACTIVITY LEVEL OF SOME GLYCOSIDASES IN BOVINE OVIDUCTAL FLUID AT DIFFERENT STAGES OF THE ESTROUS CYCLE
Authors: Carrasco C. , Romar R. , Marcos J. , Aviles M. , Coy P.
Published: December 2006

Carbohydrates play a key role in different reproductive events such as the sperm–oviductal cell interaction and sperm–oocyte recognition. In this way, α-d-mannosyl (Amari et al. 2006 Mol. Reprod. Dev. 59, 221–226) and α-2,3-sialic acid (Velasquez et al. 2006 Mol. Reprod. Dev. in press) residues contained in the zona pellucida have been identified as sperm receptors in bovine oocytes. The glycosidases, enzymes that remove carbohydrates, could play an important role in the reproductive tract, modulating decisive physiological events mediated by carbohydrates. However, the enzymatic activity level of these enzymes or its fluctuations throughout the estrous cycle in the bovine oviductal fluid (BOF) has not been studied. The objective of this work was to compare the enzymatic activity level of 7 different glycosidases in the oviductal fluid of cows at different stages of the estrous cycle. Oviducts were collected from the abattoir and classified according to the macroscopic aspect of the genital tract (Grippo et al. 1995 J. Reprod. Fertil. 105, 57–64) as early follicular (presence of growing follicles), late follicular (presence of a dominant follicle), early luteal phase (ovaries showing a corpus hemorrhagicum or a recent corpus luteum), and late luteal phase (old corpus luteum or corpus albicans). Oviductal fluid samples were collected by aspiration with an automatic pipette making simultaneous manual pressure from the isthmus toward the ampulla. Samples (9 per group) were centrifuged (7000g, 10 min) and supernatant was stored at −20°C until assay. Total activity levels were fluorimetrically measured at 450 nm, with the corresponding substrate conjugated to 4-methylumbelliferil for each enzyme (Abascal et al. 1998 Biochem. J. 333, 201–207) using a fluorometer Fluostar Galaxy (BMG LABTECH GmbH, Offenberg, Germany). Enzymatic assays were done in duplicate for 4 h at 37°C, and the reactions were stopped by adding glycine-calcium carbonate buffer. Fluorescences were corrected for tissue and substrate blanks. Fluorescence results of each enzyme and oviduct phase were analyzed using a 1-way ANOVA, with estrous cycle phase being the main factor. Results (mean counts of fluorescence) showed that the level of activity changes during the estrous cycle and the activity of some enzymes increases close to or after ovulation, suggesting a role of some glycosidases in the fertilization process. Preliminary assays for neuraminidase were negative for all samples. Future studies are necessary to identify the biological role played by the glycosidases present in the bovine oviductal fluid.

Table 1.Enzymatic activity level of some glycosidases in bovine oviductal fluid at different stages of the estrous cycle

This work was supported by Fundación Séneca (03018/PI/05) and MEC (Project code 3495).
293 EFFECT OF GUAIAZULENE ON IN VITRO BOVINE EMBRYO PRODUCTION
Authors: Dimitriadis I. , Rekka E. A. , Vainas E. , Amiridis G. S. , Rekkas C. A.
Published: December 2006

The substrates used in in vitro embryo production (IVP) mimic the in vivo fluids in which oocytes mature, oocytes are fertilized, and the early embryos develop (follicular and oviductal fluid). It is well established that oxidative stress negatively affects in vitro culture (IVC) outcomes. Guaiazulene (G) is a component of chamomile species oil with known antioxidant properties. In the present study, all IVP media were modified by the addition of G solutions so that the former exhibited a total protection against induced lipid peroxidation (TPaLP) similar to that of the respective in vivo environment. The IVP outcomes were then compared between G-processed and control oocytes. Bovine preovulatory follicular (BF) and oviductal (BO) fluid samples were collected from 10 Holstein 4- to 5-year-old cows in estrus. TPaLP was assessed according to the samples' ability to inhibit rat hepatic microsomal lipid peroxidation, by determination of the 2-thiobarbituric acid reactive material. TPaLP (mean % � SEM) of the BF and BO were 70.63 � 10.03 and 16.33 � 4.33, respectively, whereas those of the IVP [in vitro-matured (IVM), in vitro-fertilized (IVF), and IVC] media were lower (17.94 � 1.66, -1.82 � 0.78, and 14.57 � 1.26, respectively). TPaLP of the 0.1 mM G-modified IVP medium increased to 67.2 � 5.85, 19.98 � 2.49, and 69.19 � 6.22, respectively. A total of 2041 class A oocytes were used. The proportion of cleavage, early embryo development (embryos with more than 4 cells), or both after IVP (18 h IVM–5% CO2 in air, and 18 h IVF, 48 h IVC–5% CO2, 10% O2, 85% N) in the presence of G (n = 1237) during each of the IVP phases or any possible combination of IVP phases was compared with the respective control (C, n = 804). Statistical analysis was performed by a chi-squared test; P < 0.05 was considered significant. G improved cleavage and embryo development rates when present during IVM (79.4 and 57.8% vs. 64.5 and 38.2% for C) or both IVM and IVC (78.0 and 60.7% vs. 57.8 and 36.5%, respectively). When present only during 18 h of IVF, G had no effect on embryo production. However, an increased embryo development rate resulted from the combined exposure to G during IVF and IVM (56.4 vs. 29.6%), during IVF and IVC (55.3 vs. 35.5%), or at all IVP phases (56.6 vs. 34.9%). The latter effect resembled the one obtained after G addition only to the IVC medium (62.5 vs. 39.7%, respectively). We concluded that the addition of G to IVP substrates, at concentrations that mimic the in vivo TPaLP conditions, could promote bovine IVP efficiency.
3 HISTONE H4 ACETYLATION AT LYSINE 12 AND Cdc2a EXPRESSION ARE DECREASED IN AGED MOUSE GERMINAL VESICLE-STAGE OOCYTES
Authors: Manosalva I. , Goday C. , Esponda P.
Published: December 2006

Ageing is a phenomenon related to mammalian female infertility. One cause of ageing-induced infertility is the abnormal meiotic maturation from germinal vesicle stage oocytes (GVs). GVs are immature oocytes, which stay arrested in the ovaries during the life span. The abnormal oocyte maturation in aged animals is partially originated from molecular changes, not well defined, such as chromatin modifications and differential gene expression. Here, we analyze chromatin modifications such as histone acetylation and the corresponding gene expression changes induced by ageing in mouse GVs. We measured by immunofluorescence histone H4 acetylation at lysine residues 5 (H4-K5), 8 (H4-K8), and 12 (H4-K12) in GVs collected from young (1 month old) and aged (12–18 months old) CDC1 female mice. Immunofluorescence was analyzed with a microscope (Leica TCS SP2 AOBS) and its image analysis software. Whereas H4-K5 and H4-K8 show similar acetylation levels in both young and old mice, significant lower acetylation of H4-K12 is detected in GVs from old mice. Since H4-K12 acetylation has been related to Cdc2a expression during oocyte maturation (Akiyama et al. 2004 Mol. Reprod. Dev. 69, 222–227; Minuzzo et al. 2005 Mol. Pharmacol. 68, 1496–1503), we investigated whether Cdc2a mRNA levels change in aged mice. Cdc2a expression was measured by RT-PCR and quantified with a densitometer (BioRad GS800). We observed a decrease of Cdc2a expression in GVs of old mice. This result is further confirmed by an immunofluorescence analysis where lower levels of Cdc2a protein in old mouse GVs was observed. In conclusion, we find that the levels of H4-K12 acetylation and Cdc2a mRNA are lower in old compared to young mouse GVs. Our observations suggest that ageing affects histone modifications such as H4-K12, which might induce chromatin remodelling and gene expression changes like that of Cdc2a.
302 EFFECT OF MONOSACCHARIDES ON SPERM ABILITY TO BIND TO OVIDUCTAL EPITHELIAL CELLS
Authors: Kon Y. , Iwata H. , Shiono H. , Matsubara K. , Kuwayama T. , Monji Y.
Published: December 2006

The attachment of sperm to the oviductal epithelium is one of the major factors responsible for successful in vivo fertilization. Carbohydrates are reported to be involved in this attachment. In the present study, sperm were separated into 2 fractions based on their ability to bind to the oviductal epithelial cells (OECs; sperm that do not bind to OECs, UB-sperm; sperm that bind to OECs, B-sperm). Subsequently, the fertilization competence of these sperm fractions was examined. Furthermore, the effect of various monosaccharides on the ability of sperm to bind to OECs was investigated. Oviducts of cows whose ipsilateral ovaries demonstrated new clot formation (ovulation fossa) were collected from a slaughterhouse, and those that contained ovulated oocytes were further selected for experimentation. OECs were collected from these oviducts. OECs and frozen-thawed semen were each suspended in a medium (synthetic oviduct fluid, SOF, supplemented with 5 mg mL-1 of BSA and heparin), at a final concentration of 1 � 106/mL. These suspensions were mixed and centrifuged to separate the 2 abovementioned fractions. Immediately after the suspensions of sperm and OECs were mixed, d-mannose, d-fucose, or n-acetyl-d-glucosamine (GlcNAc) was added (final concentration of each monosaccharide, 5 mM). Next, the number of live sperm in each sperm population was estimated at 0 and 3 h. In addition, the oocytes collected from the ovaries were fertilized with the UB-sperm or non-separated (control) sperm, and the percentage of fertilization was examined. The percentage of fertilization and the number of sperm binding to the zona pellucida (ZP) were lower (P < 0.05) for the UB-sperm than for the control sperm. When sperm (106) were separated in the presence of GlcNAc, the number of motile sperm attached to OECs was significantly higher than that among sperm that were separated in the absence of monosaccharides (0.49 � 106 cells vs. 0.16 � 106 cells). The motility of B-sperm that were separated in the absence of monosaccharides was maintained for a longer time than that of the control sperm (percentage of motile sperm: 74.6% vs. 38.4%, P < 0.05). The B-sperm separated in the presence of GlcNAc or d-fucose lost their motility to a greater extent than the B-sperm separated in the absence of monosaccharaides (27.3% vs. 49.9%, P < 0.05). Further, when GlcNAc (5 mM) was added to the suspension for sperm cryopreservation, the number of sperm that bound to the OECs also increased. In conclusion, it was shown that sperm with low ability to bind to OECs have low fertilization competence, and GlcNAc increased the number of sperm binding to the OECs.
305 INCREASED MONOSPERMIC PENETRATION OF PORCINE OOCYTES USING A VISCOUS IN VITRO FERTILIZATION SYSTEM
Authors: Men H. , Si W. , Critser J. K.
Published: December 2006

The high incidence of polyspermy remains a major problem in porcine IVF systems. The high number of sperm bound to the oocyte is one of the major causes for pathological polyploidy (Funahashi 2003 Reprod. Fertil. Dev. 15, 167–177). In this experiment, we tested the hypothesis that limiting the number of competent sperm able to participate in fertilization during IVF will result in a reduction in polyspermic penetration. Combinations of various sperm (3 levels) and Percoll concentrations (3 levels) were used in a modified Tris-buffered medium (mTBM) with 3% polyvinylpyrrolidone supplementation to find the optimal combination that could result in a significant reduction in polyspermy rate compared with that resulting from the current porcine IVF system (Abeydeera and Day 1997 Theriogenology 48, 537–544). In vitro-matured gilt oocytes (n = 844) were randomly allocated into 1 of 10 treatment groups in groups of 30. Motile sperm were selected from frozen–thawed samples by density gradient centrifugation using a discontinuous Percoll gradient consisting of 3 mL of 54% Percoll in PBS on top of 1 mL of 72% Percoll in a 15-mL conical tube at 600g for 15 min. After centrifugation, the supernatant was removed as much as possible without disturbing the sperm pellet. Sperm stock solutions were made in a manner that resulted in the desired final concentration of sperm when 5 µL of the sperm solution were added to 95 µL of fertilization medium. During IVF, the sperm stock solutions were placed at one side of an IVF droplet so that most sperm were trapped inside the viscous Percoll solution and only a limited number of sperm, with high progressive motility, were capable of fertilizing the oocytes. A group of oocytes fertilized using our standard IVF protocol with a sperm concentration of 5 × 105 sperm mL−1 in mTBM served as a control. Pronuclear formation after 12 h of fertilization was used to assess sperm penetration. The data from 3 replicates were analyzed by standard ANOVA procedures using the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The treatment group of 5 × 105 sperm mL−1 + 10% Percoll resulted in a significantly higher monospermy rate than the control or the treatment group of 1 × 106 sperm mL−1 + 30% Percoll (Table 1; P < 0.05). These data support the hypothesis that the polyspermy rate in a porcine IVF system can be reduced by limiting the number of competent sperm fertilizing the oocytes.

Table 1.Monospermy rates in a viscous IVF system (mean ± SEM%)

This project was supported by NIH U42 RR 018877.