bluuebunny/crossref_metadata_2025 huggingface dataset:
163 FUNCTIONAL COMPARISON OF BOVINE TROPHOBLASTIC VESICLES DERIVED FROM FRESHLY COLLECTED CONCEPTUSES AND SERIALLY PASSAGED TROPHOBLAST CELLS
Authors: Hashiyada Y. , Takahashi H. , Asada M. , Sakuta N. , Furuyashiki S. , Yamaya S. , Oike T. , Konishi K. , Takahashi M.
Published: December 2006

The co-transfer of trophoblastic vesicles (TVs), derived from in vivo-recovered conceptuses, is a well-known method for promoting the successful implantation of embryos through the action of bovine interferon-tau (bIFN-τ) on maternal-fetal recognition. However, the preparation of these TVs is a tedious process. Techniques have progressed for obtaining large numbers of TVs consistently from serially passaged trophoblast cells. The aim of the present study was to compare the function of TVs for co-transfer derived from in vitro production with those derived from in vivo-flushed embryos, and without co-transfer. Production of TVs from serially passaged trophoblast cells was carried out according to the method previously described by Takahashi et al. (2000, Cloned animals and placentation, 147–151, Yokendo: Tokyo, Japan). The in vitro TVs (IVP-TVs), 1–2 mm in diameter, were taken from cells passaged 48th to 52th and 96th. In vivo-derived TVs (vivo-TVs) were prepared from recovered elongating blastocysts 7 days after transfer of frozen–thawed embryos on Day 8 of the estrous cycle. Demi-embryos were produced from fresh embryos of flushed Japanese Black cows by bisection using a micromanipulator. Pairs of demi-embryos, without (control) or with 2 to 4 TVs, were transferred into the uterus ipsilateral of 65 Japanese Short Horn recipients to the functional corpus luteum. Pregnancy diagnoses were performed twice, from Day 30 to 70, by ultrasound scanning. BIFN-τ content of culture media was measured by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). Data were analyzed by chi-square test and Fisher's exact test. The pregnancy rate in the IVP-TVs group (20.0%, 5/25) had a tendency to be lower than in the vivo-TVs (46.2%, 6/13), although this did not reach statistical significance (P = 0.09). These pregnant animals were transferred TVs from passaged 48th-52nd (5/19). In the control group 33.3% (9/27) of recipients conceived. Twin pregnancy rate was also lower in the IVP-TVs (0/5) than in the other two groups (3/6 and 5/9, P = 0.09 and P = 0.04, respectively). Fetal losses occurred only in the single pregnancies of IVP-TVs (60.0%, 3/5) and vivo-TVs (16.7%, 1/6) up until Day 70. Reproductive efficiency, based on numbers of delivered offspring, was significantly lower in the IVP-TVs (8%, 2/25) compared with the vivo-TVs (61.5%, 8/13) and control (51.9%, 14/27) groups, respectively (P < 0.01). BIFN-τ levels secreted from the TVs derived from passages 46th and 48th were 0.157 ng/mL-1 and 0.113 ng/mL-1, respectively. In conclusion, compared with the in vivo TVs, those from serially passaged trophoblast cells had a negative effect on pregnancy, although the morphology of the two differently derived TVs was similar.
165 EXPRESSION OF CALCIUM TRANSPORTER 1 GENE IN THE UTERUS OF RATS DURING PREGNANCY
Authors: Lee B.-M. , Lee G.-S. , Jeung E.-B.
Published: December 2006

Calcium Transporter 1 (CaT1), which is highly expressed in calcium-absorption organs, i.e. duodenum and kidney, is a critical mediator for calcium uptake during transcellular calcium transport. It has been shown that CaT1 gene is also expressed in the placenta and uterine smooth muscle in female reproductive organs. We previously demonstrated that uterine CaT1 mRNA is highly expressed at diestrus and tightly regulated by progesterone (P4) related to calcium homeostasis for uterine functions during the estrous cycle in rats. Thus, in the recent study, we further examined the expression of CaT1 mRNA in the uterus and placenta of rats to elucidate its role during pregnancy in these tissues. Female Sprague Dawley rats (n = 2) were employed and their pregnancy days (PD) were determined by a vaginal plug every morning; the rats were euthanized daily (PD 0 to 21). The expression of CaT1 mRNA decreased from PD 0 to 4 and highly increased on PD 5 to 10. Its increased transcripts gradually decreased at the end of pregnancy. Taken together, these results indicate that the expression level of CaT1 mRNA is regulated in the uterus of rats during pregnancy, probably via sex steroid hormones and their receptors through a complex pathway. A further study is warranted to verify relevant factors which regulate CaT1 gene and to provide further insight into its role(s) in the female reproductive tissues.
166 DIFFERENTIAL EXPRESSION OF AMPHIREGULINE, EPIDERMAL GROWTH FACTOR, AND CALBINDIN-D9k IN THE UTERUS OF RODENTS AND PIGS DURING IMPLANTATION
Authors: Lee G.-S. , Kim M.-H. , Hyun S.-H. , Jeung E.-B.
Published: December 2006

Implantation in mammalian species is a complex process that involves embryo apposition and attachment to the apical surface of the uterine epithelium. This process involves a variant of molecules that are not unique in themselves but play unique roles in the process of implantation. Among them, the epidermal growth factor (EGF) family, the cytokines, cell adhesion molecules, and calcium-binding proteins appear to be important in embryonic attachment. Amphireguline (Areg), its receptor (EGFR), and calbindin-D9k (CaBP-9k) were highly expressed before implantation. Thus, in the present study, using rodent and porcine models, the expression levels of Areg, EGFR, and CaBP-9k gene were analyzed in the uterus during the estrous cycle and pregnancy by means of RT-PCR to elucidate their roles in implantation. Areg and CaBP-9k were significantly up-regulated at diestrus; however, these transcripts disappeared at proestrus in mice. In addition, EGFR mRNA was primarily observed at diestrus in mice. Although CaBP-9k and EGFR transcripts of rats increased at proestrus, no Areg mRNA was observed in this model. To clarify the regulator of the mouse Areg gene, immature mice were injected with sex steroid hormones. No transcripts of Areg were detected in these models, suggesting that mouse Areg may require other puberty factors. Porcine expressions of uterine Areg, EGFR, and CaBP-9k mRNA fluctuated during pregnancy (Days 12, 15, 30, 60, 90, and 110 of pregnancy). CaBP-9k transcripts were highly expressed on Day 12 and decreased on Days 15 to 90. On Day 110, CaBP-9k mRNA was expressed as well. Areg mRNA increased on Days 12 to 30, and decreased on Days 30 to 110. EGFR transcripts were detected on Day 12 and gradually disappeared till the end of pregnancy. Uterine Areg and CaBP-9k mRNAs were regulated by progesterone (P4) in the mouse model. Areg transcripts of porcine uteri appear to be expressed after implantation, but CaBP-9k mRNA was induced before embryo attachment. Taken together, these results indicate taht uterine Areg, EGFR, and CaBP-9k are differentially regulated during pregnancy in these species, and porcine CaBP-9k could act as an important factor in implantation. A further spatial expression of these genes will contribute to the understanding of their complex molecular actions involved in embryo attachment.
167 EXPRESSION OF INTERFERON τ IN BOVINE EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION
Authors: Minamihashi A. , Moriyasu S. , Takahashi H. , Hirayama H. , Geshi M. , Onoe S. , Sawai K.
Published: December 2006

Parthenogenetic activation (PA) is a useful technique for reproductive technologies such as somatic cell nuclear transfer. Furthermore, there is a possibility of embryonic stem cell establishment deriving from PA embryos (Cibelli et al. 2002 Science 295, 819). Understanding of the ability of development and differentiation in PA embryos is important for the application. This study was designed to assess the gene expression of octamer-binding transcription factor (OCT-4) and interferon τ (IFNτ) in bovine PA embryos at the blastocyst (BC) and elongated (EL) stages, and the protein secretion of IFNτ at the EL stage. PA embryos were produced from oocytes matured in vitro (24 h), activated with Ca-ionophore (5 min) and electric pulse, and then treated with cytochalasin B and cycloheximide (5 h). In vivo-produced (Vivo) embryos were obtained non-surgically at Day 8 (Day 0 = estrus) from superovulated donor cows. PA or Vivo embryos were transferred to recipient cows (PA: 10 embryos/cow; Vivo: 1 embryo/cow) at Day 8, and then recovered non-surgically with uterine flushings at Day 16. Total RNA in single BC and EL embryos were reverse transcribed for PCR. Quantification of mRNA abundance was performed by real-time PCR. The expression of each mRNA was normalized to the abundance of GAPDH. IFNτ secretion of uterine flushings was estimated by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). The cleavage and blastocyst developmental rates of PA oocytes were 65.8 and 29.7%, respectively. Most embryos had positive signals of OCT-4 and IFNτ regardless of the origin and stage of embryos. The relative abundance (mean � SEM) of OCT-4 expression in PA and Vivo embryos dropped to its lowest level (1.78 � 0.32 and 1.14 � 0.45, respectively) at the EL stage, and it was significantly lower than that at the BC stage (1018.87 � 148.69 and 696.29 � 151.80, respectively; P < 0.05). The transcript level of OCT-4 was not significantly different between PA and Vivo embryos at both stages. Although the transcript level of IFNτ in PA and Vivo embryos increased significantly at the EL stage (0.36 � 0.06 and 7.68 � 2.01, respectively) from the BC stage (0.03 � 0.01 and 0.01 � 0.004, respectively; P < 0.05), that in PA embryos was significantly lower than that in Vivo embryos at the EL stage (P < 0.01). The total amount (mean � SEM) of IFNτ in uterine flushings from cows with transferred PA embryos was 3.38 � 0.35 �g (the number of embryos in each uterine flushing was unknown), and it was low compared with that from cows with Vivo embryos (13.40 � 3.03 �g). Our results indicate that bovine PA embryos have the ability to secrete IFNτ in the uteri of recipient cows at the EL stage, and there is a similar expression pattern of OCT-4 for Vivo embryos.
170 EXPRESSION PROFILE AND PROTEIN SECRETION OF BOVINE INTERFERON τ ON DAYS 12 TO 28 IN EARLY PREGNANCY
Authors: Takahashi H. , Sawai K. , Minamihashi A. , Kageyama S. , Moriyasu S. , Hirayama H. , Onoe S. , Geshi M.
Published: December 2006

Bovine interferon (bIFN) τ has been implicated as a mediator of maternal recognition of pregnancy in cattle. Ovine (o) and bIFNτ expression show temporal patterns until implantation begins (Farin et al. 1990 Biol. Reprod. 43, 210–218); therefore, the expression peaks of bIFNτ are not well understood. This study aimed to investigate the expression profile of mRNA and protein secretion of bIFNτ in conceptuses obtained from cows on Days 12 to 28 in early pregnancy. Induction of superovulation and recipient synchronization were achieved by previously reported methods (Hirayama et al. 2004 Theriogenology 62, 887–896). Embryos were recovered non-surgically on Day 8 (Day 0 = estrus). Subsequently, recovered embryos were singly transferred to recipients immediately upon evaluation. Bovine conceptuses and uterine flushings were collected non-surgically from embryo-transferred cows on Days 12 (n = 2), 14 (n = 6), 16 (n = 5), 18 (n = 4), and 20 (n = 5) with MEM. On Days 22 (n = 3), 24 (n = 5), and 28 (n = 2), bovine conceptuses were recovered within 30 min after slaughter by flushing the uterus with PBS supplemented with 0.1% BSA. Uterine flushings were also collected from 6 cows in the control group on Day 16 of the estrous cycle. Conceptuses for collection of RNA were processed individually and stored at -80�C until analysis. The flushing was centrifuged at 1800g and 4�C for 30 min to remove cellular debris, and stored at -30�C for subsequent measurement of bIFNτ. Total RNA in conceptuses were reverse transcribed for PCR. Quantification of mRNA abundance was performed by real-time PCR. The expression of each mRNA concentration was normalized to the abundance of GAPDH. BIFNτ contents of the uterine flushings were measured by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). Intra-assay and inter-assay CVs were 11.0 and 8.5%, respectively. Transcripts of bIFNτ were detected in all samples from Days 12 to 28; the expression of mRNA increased remarkably from Day 16 (mean � SEM, 2.15 � 0.66) to Day 18 (9.62 � 4.09), and then declined on Day 20 (3.42 � 0.86). Protein detected on Day 16 was 13.3 � 3.02 �g. An increase of bIFNτ secretion was observed between Day 18 (39.8 � 20.62 �g) and Day 20 (71.9 � 17.36 �g); thereafter it decreased on Day 22 (27.2 � 2.83 �g). BIFNτ displayed a pattern of expression similar to that of oIFNτ, with mRNA expression peaking around Day 18 (ovine, Days 12/13) and protein secretion on Day 20 (ovine, Days 15/16), thereby reflecting the comparatively later time frame of maternal recognition of pregnancy in cattle. These results indicate that the expression and secretion profiles of bIFNτ show temporal and spatial patterns in early pregnancy.
172 DOES ESTRADIOL-17β CAUSE CYCLE-DEPENDENT MODULATIONS OF UTERINE MILK PROTEIN IN BOVINE ENDOMETRIUM?
Authors: Ulbrich S. E. , Gross K. , Schmidt S. , Blum H. , Rottmayer R. , Hiendleder S. , Fröhlich T. , Arnold G. J. , Wolf E. , Meyer H. H. D. , Bauersachs S.
Published: December 2006

Uterine secretion from the endometrial glandular epithelium provides optimal conditions for early embryonic development. The uterine milk protein (UTMP), a member of the serine proteinase inhibitor superfamily, has been demonstrated to be a major progesterone-induced glycoprotein secreted by the endometrium during pregnancy. Previous transcriptomic analysis revealed that UTMP was highly abundant at estrus in the bovine endometrium (Bauersachs et al. 2005 J. Mol. Endocrinol. 34, 889–908). Here we describe a detailed characterization of UTMP mRNA expression at several time points during the bovine estrous cycle and the pre-implantation period. Simmental heifers were monitored with respect to serum progesterone (P4) and estradiol-17β (E2), and slaughtered at estrus or 3.5, 12, 15, or 18 days after estrus, or at Day 15 or 18 of pregnancy (n = 4 per group). The uterus was divided into corpus and caudal, middle and cranial parts of the ipsilateral uterine horn for sampling of intercaruncular endometrium. In addition, effects of steroid hormones were investigated by stimulating an endometrial cell culture obtained from Day 8 animals (n = 4) with physiological doses of P4 or E2. In all cases, UTMP mRNA was quantified by real-time RT-PCR. Pronounced changes of UTMP mRNA abundance were detected during the estrous cycle. Expression was highest at estrus, followed by a remarkable decrease at Day 3.5. There was no difference between pregnant and non-pregnant animals at Day 15. Cycling animals displaying a high P4 and low E2 content (P4 > 2 ng mL-1 and E2 < 1 pg mL-1) revealed a lower expression of UTMP at Day 18 compared to the pregnant heifers, whereas animals at Day 18 progressing toward estrus (P4 < 1.5 ng mL-1 and E2 > 3 pg mL-1) exceeded the mRNA expression of the pregnant group. Following stimulation with estradiol-17β, the in vitro UTMP transcripts increased significantly. These results indicate that two different interfering stimulatory events might take place. While estradiol-17β appeared to increase UTMP mRNA expression at estrus, a second factor, most probably embryo-derived or embryo-induced, is assumed to be responsible for the UTMP rise during early pregnancy. The distinct gradient from the cranial uterine horn to the corpus at estrus was less pronounced at Day 3.5 and absent at Days 12, 15, and 18, pointing toward functional implications regarding the passing gametes, particularly sperm. An antibody raised against bovine UTMP will further validate the observed mRNA regulations on the protein level. In conclusion, bovine UTMP seems to play a decisive role for precise cyclic regulation of the bovine uterine milieu and during early embryo-maternal communication.
This work was supported by the DFG FOR 478.
176 EFFECT OF LEUKEMIA INHIBITORY FACTOR FROM HUMAN AND MOUSE ORIGIN ON THE DEVELOPMENT OF BOVINE EMBRYOS PRODUCED IN VITRO
Authors: De Frutos C. , Rodríguez A. , Díez C. , Caamaño J. N. , Facal N. , Duque P. , García-Ochoa C. , Gómez E.
Published: December 2006

Leukemia Inhibitory Factor (LIF) is a cytokine with potential to influence embryonic quality and proliferation within the inner cell mass (ICM). However, conflicting effects of LIF have been reported with in vitro-produced (IVP) bovine embryos, in spite of LIF receptor (LIFr) and gp130 transcripts being expressed at all stages during pre-implantation development (Niemann and Wrenzycki 2000 Theriogenology 53, 21–34). As there is no commercially available bovine LIF (bLIF), researchers have used human LIF (hLIF) because of its greater sequence homology compared to murine LIF (mLIF). However, mLIF has been not compared with hLIF in culture with bovine embryos; thus this was the aim of this study. Cumulus–oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro and presumptive zygotes cultured in modified synthetic oviduct fluid with 6 g L-1 BSA. At 139 h post-insemination (Day 6), a total of 423 morulae (>90%) and early blastocysts were cultured for 48 h with: (1) 100 ng mL-1 recombinant mLIF (Sigma-Aldrich Quimica SA, Madrid, Spain); (2) 100 ng mL-1 recombinant hLIF (Sigma); and (3) no LIF. Data (6 replicates) were processed by GLM and Duncan's test, and expressed as LSM � SE (ab: P < 0.05; xy: P < 0.01). Development was recorded up to the hatched blastocyst stage and cells were differentially counted in the ICM and trophectoderm (TE) following the method described by Thouas et al. (2001 Reprod. Biomed. Online 3, 25–29). There were no differences within developmental rate on Day 7, but reduced blastocyst rates were observed on Day 8 between hLIF (42.0 � 3.9a and 27.2 � 3.3a) and controls (57.7 � 3.9b and 38.9 � 3.3b) at the medium and expanded stages, respectively, whereas mLIF had no effect (47.4 � 3.9 and 32.3 � 3.3). Contrary to development, Day 8 blastocysts showed decreased cell counts in both the ICM and the ICM/total cell proportions in the presence of mLIF (19.1 � 3.1x and 13.8 � 2.4x vs. 32.6 � 3.0y and 24.8 � 2.3y for controls, respectively), whereas hLIF had no effect (29.7 � 3.1y and 20.9 � 2.4y). No changes were seen in TE and total cell counts. The disparate effects exhibited by hLIF and mLIF during blastocyst formation may reflect the fact that these compounds are inappropriate to replace bLIF, and/or endogenous LIF probably suffices during bovine development. In fact, mouse embryonic development and blastocyst cell numbers decrease in murine embryos injected with LIF antisense nucleotides (Cheng et al. 2004 Biol. Reprod. 70, 1270–1276). Furthermore, embryonic stem (ES)-like cell derivation in bovine is possible with (Saito et al. 2003 Biochem. Biophys. Res. Com. 309, 104–113) and without (Mitalipova et al. 2001 Cloning 3, 59–67) exogenous LIF. Therefore, strategies to investigate LIF signalling in bovine embryos and stem cells should be reconsidered.
This work was supported by Grant AGL2005-04479.
181 DIFFERENCES EXIST IN ZONA PELLUCIDA HARDNESS BETWEEN IN VIVO- AND IN VITRO-GENERATED BOVINE EMBRYOS
Authors: Itoi F. , Himaki T. , Kubota C. , Hirose J. , Miyamura M. , Hamano S. , Murayama Y. , Omata S. , Yoshida M.
Published: December 2006

The zona pellucida (ZP) of mammalian ova plays an important role during maturation, fertilization, and early embryonic development. The hardness of ZP of mammalian ova has been mainly evaluated by a biochemical method, such as a difference in ova dissolution speed with an enzyme or an acid reagent treatment. However, the physical hardness of ZP in bovine embryos is largely unknown. Recently, we developed a system measuring the physical hardness of ZP of mammalian ova with a micro bio-sensor (MBS). The objective of this study was to examine the physical ZP hardness of in vivo- and in vitro-derived bovine embryos using an MBS. In the measurement system, the thin glass needle was connected at the tip of a piezo-electric ceramic tactile sensor, and the sample was pushed into a sensor at fixed speed under the computer-controlled micromanipulation system. The change in frequency at the time of displacement of the sample into the sensor was recorded using the computer program. Measurement of gelatin samples of known concentration (4 to 8%) was used to determine the basic characteristic of a sensor and to make a standard curve. In vivo-generated embryos were recovered from 6 superovulated Japanese Black cows with multiple injection of FSH. On Day 7 after insemination, morulae (M), early blastocysts (EB), and blastocysts (BL) were recovered by nonsurgical flushing of uterine horns. In vitro-generated embryos were produced as described earlier (Yoshida et al. 1998 J. Vet. Med. Sci. 60, 549–554). The M, EB, and BL at Days 5 to 7 of post-insemination in vitro were used for measurement of ZP hardness. When a sensor made contact with a harder gelatin sample, the change in frequency was large; the change in frequency has the characteristic of being small for a softer gelatin sample. By comparison with a standard curve, ZP hardness converted into gelatin concentration for each stage of bovine embryos generated in vivo was 3.95% (M: n = 9), 4.14% (EB: n = 32), and 3.92% (BL: n = 14), respectively. On the other hand, ZP hardness of bovine embryos generated in vitro was 3.42% (M: n = 56), 3.33% (EB: n = 36), and 3.25% (BL: n = 23), respectively. There was a significant difference (P < 0.01) in the hardness of ZP between in vivo- and in vitro-generated bovine embryos.
184 EFFECT OF CONCENTRATION AND EXPOSURE DURATION OF FETAL BOVINE SERUM ON PARTHENOGENETIC DEVELOPMENT OF PORCINE FOLLICULAR OOCYTES
Authors: Kim H. J. , Cho S. R. , Choe C. Y. , Choi S. H. , Son D. S. , Kim S. J. , Kim Y. G. , Han M. H. , Ryu I. S. , Kim I. C. , Kim I. H. , Im K. S.
Published: December 2006

The aim of the present experiment was to examine hatching rate as a testing tool of porcine embryo viability before early-stage embryo transfer, such as zygotes or 2-cell stage embryos. We evaluated the optimal concentrations and exposure durations of fetal bovine serum (FBS) on porcine parthenotes. Ovaries were obtained from prepubertal gilts at a local abattoir and brought to the laboratory in physiological saline with antibiotics at 30–33°C. The ovaries were washed and wiped, and then cumulus–oocytes complexes (COCs) in the follicular fluid were aspirated from surface-visible follicles (2–6 mm in diameter) with a 10-mL syringe fitted with an 18-gauge needle. After being washed 3 times with modified phosphate-buffered saline (DPBS; GIBCO, Grand Island, NY, USA) containing 0.3% BSA, the COCs were suspended in maturation medium, NCSU-23 containing 10% (v/v) porcine follicular fluid, 10 ng mL−1 epidermal growth factor (EFG; Sigma-Aldrich Corp., St Louis, MO, USA), 10 µg mL−1 follicular stimulating hormone (FSH; Sigma), 35 µg mL−1 luteinizing hormone (LH; Sigma), 1 mg mL−1 cysteine (Sigma, USA), 100 IU mL−1 penicillin G, and 100 µg mL−1 streptomycin sulfate (GIBCO). After 24 h, the COCs were transferred to the same medium without hormones. The oocytes matured for 48 h were denuded. The oocytes with a visible polar body were selected and returned to the maturation medium without hormones. After 65 h of maturation, oocytes were exposed to PBS with 7% ethanol (v/v) for 7 min, and then the oocytes were washed and treated in TCM-199 containing 5 µg mL−1 cytochalasin B (Sigma) for 5 h at 38.5°C in an atmosphere of 5% CO2 and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in PZM-5 medium (IFP, Japan) and cleavage of the parthenotes was assessed at 72 h of activation. Normally cleaved parthenotes were cultured for 8 days to evaluate their ability to develop to the blastocyst and hatching stages. The FBS was added at Day 4 or 5 with concentrations of 2.5, 5, or 10%. The blastocyst rates ranged from 39.1 to 70% in each treatment. However, the hatching rate was dramatically decreased in the non-addition group. In this experiment, the developmental potential may be estimated before embryo transfer by an in vitro culture system up to the hatching stage.

Table 1.
Effect of concentration and exposure duration of FBS on parthenogenetic development of porcine follicular oocytes
19 EFFECTS OF ESTRADIOL OR GnRH ON FOLLICULAR WAVE EMERGENCE AND PREGNANCY RATES FOR A CIDR-BASED TIMED AI PROTOCOL IN REPEAT BREEDER DAIRY COWS
Authors: Suh G. H. , Kim U. H. , Hur T. Y. , Kang S. J. , Baek K. S. , Park S. B. , Kim H. S. , Kang H. G. , Kim I. H.
Published: December 2006

Repeat breeding of a high yielding cow causes enormous economic loss to the dairy producers. In this study, we examined the effect of estradiol benzoate (EB) or GnRH on follicular wave emergence and follicular development, and on pregnancy rates for a CIDR-based timed AI (TAI) protocol in repeat breeder dairy cows. Repeat breeder cows received a CIDR device (InterAg, Hamilton, New Zealand), with an injection of 1 mg EB (SY Esrone�; Samyang, Seoul, Korea) plus 50 mg progesterone (P4; SY Ovaron�; Samyang) (EB + P4 group, n = 36) or with an injection of 250 �g Gonadorelin (GnRH, Fertagyl�; Intervet, Boxmeer, The Netherlands) (GnRH group, n = 35) at the beginning of the experiment (Day 0). On Day 7, CIDR devices were removed and all cows received PGF2� (Lutalyse�; Pharmacia & Upjohn, Puurs, Belgium). Thereafter, cows in the EB + P4 group received an injection of 1 mg EB on Day 8, and TAI 30 h later; those in the GnRH group received an injection of 250 �g GnRH on Day 9, and TAI 19 h later. In a subset of cows for both groups (15 cows from each group), the ovaries of the cows were examined by transrectal ultrasonography (Sonoace 600 with 7.5 MHz linear-array transducer; Medison Co., Seoul, Korea) every 24 h from Days 0 to 8, on Day 10, and on Day 13 for the EB + P4 group, and every 24 h from Days 0 to 9, on Day 11, and on Day 14 for the GnRH group, to observe changes in ovarian structures (follicles and corpora lutea). Pregnancy was diagnosed 35 to 60 days after TAI, using both ultrasonography and rectal palpation. Data were analyzed using Fisher's exact, Student's t-, and chi-square tests. Follicular wave emergence occurred within 7 days in 12/15 EB + P4-treated and 13/15 GnRH-treated cows (P > 0.05). The mean diameters of preovulatory follicles were smaller (P < 0.05) in the EB + P4 group (12.1 � 0.1 mm) than in the GnRH group (13.8 � 0.1 mm). However, the proportion of cows with synchronized ovulation and pregnancy rates to TAI did not differ (P > 0.05) between the EB + P4 (11/15 and 19.4%, respectively) and the GnRH (13/15 and 34.3%, respectively) groups. In conclusion, treatment with either EB or GnRH for a CIDR-based TAI protocol in repeat breeder dairy cows results in synchronous follicular wave emergence, follicular development, and ovulation, and thus similar pregnancy rates to TAI.
192 THE EFFECT OF ZWITTERONIC BUFFERS AND PBS ON GENE TRANSCRIPTION OF BOVINE IN VIVO- AND IN VITRO-DERIVED EMBRYOS
Authors: Palasz A. T. , Beltrán Breña P. , Pérez-Garnelo S. , Fuentes S. , De la Fuente J. , Gutiérrez-Adán A.
Published: December 2006

Choice of buffer used for the IVF procedures affects embryo developmental rates (De la Fuente et al. 2006 Reprod. Fertil. Dev. 18, 187). Also, it has been shown that the 3 zwitterionic buffers tested in this study, TES (T), MOPS (M), and HEPES (H) (pKa values at 20°C: 7.2–7.5) interact with DNA (Stellwagen et al. 2000 Anal. Biochem. 287, 167). Our objective was to evaluate the effect of T, M, H, and PBS buffers on the expression of the following genes, Fgf-4 (fibroblast growth factor 4 precursor), Lama1 (laminin alpha 1), Ube2a (ubiquitin-conjugating enzyme), Gsta4 (glutathione S-transferase A4), Il6 (interleukin 6), Sod1 (superoxide dismutase), Prss11 (IGF binding), and Hspb1 (Heat shock protein binding 1), on bovine in vivo and in vitro embryos. Genes were selected based on their sensitivity to adverse in vitro embryo culture conditions by microarray analysis (data not shown). All buffers were prepared at a concentration of 10 mM in TALP medium and the final pH was adjusted to 7.2. Bovine follicular fluid was aspirated from abattoir-derived ovaries and evenly divided into 4 tubes. Collected oocytes (5 replicates) from each tube were processed separately through entire IVM, IVF, and IVC procedures using washing medium buffered with: PBS (n = 490), Group 1; H (n = 438), Group 2; M (n = 440), Group 3; and T (n = 394), Group 4. All buffers contained 4% BSA. Oocytes were matured in TCM-199 supplemented with 10% FCS and 10 ng mL-1 epidermal growth factor, and inseminated in Fert-TALP containing 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg mL-1 BSA-FAF, and 10 µg mL-1 heparin with 1 × 106 mL spermatozoa. After 24 h of oocytes–sperm co-incubation, presumptive zygotes were cultured in SOFaa medium with 8% BSA at 39°C under paraffin oil and 5% CO2 in humidified air. Cumulus–oocyte complexes and zygotes were held in designated buffers in vivo blastocysts exposed for P in vitro embryos irrespective of buffer used. In addition, higher expression of Hspb1 and lower expression of Ube2a and Lama 1 were identified in PBS and T than in M and H (P in vitro embryos were lower (P in vitro-derived embryos. It can be concluded that mRNA transcription of in vitro-derived embryos is affected by the choice of the buffer used for the IVF procedure.
195 SODIUM SELENITE INCREASES DEVELOPMENTAL ABILITY AND DECREASES APOPTOSIS IN PORCINE PARTHENOTES
Authors: Uhm S. J. , Gupta M. K. , Yang J. H. , Lee H. T.
Published: December 2006

Selenium is an essential component of serum required for cell proliferation and growth. It has also been shown to have a beneficial effect on in vitro maturation of oocytes. This study examined the development and apoptosis of porcine parthenotes treated with sodium selenite (SS). Immature cumulus–oocyte complexes were cultured in TCM-199 supplemented with 25 mM NaHCO3, 10% (v/v) porcine follicular fluid, 0.57 mM cysteine, 0.22 µg mL−1 sodium pyruvate, 25 µg mL−1 gentamycin sulfate, 0.5 µg mL−1 Follitropin V, 10 ng mL−1 epidermal growth factor, and 1 µg mL−1 estradiol-17β for 42–44 h. The matured oocytes were activated by electro-stimulation (single electrical pulse of 1.36 kV cm−1 for 30 µs) and treated with 10 µg mL−1 cytochalasin B for 4 h. Activated oocytes were cultured to the blastocyst stage in NCSU23 supplemented with 0.4% polyvinyl alcohol in the presence or absence of 25 ng mL−1 SS for 7 days at 39°C in a humidified atmosphere of 5% CO2 in air. Inner cell mass (ICM) and apoptosis rates in blastocysts were evaluated by differential double staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method. In addition, relative abundance of Bax/BclXL transcripts was quantified by real-time qRT-PCR. The activity of caspase-3 and MAP kinase in blastocysts was examined by western blotting. The cleavage rate, blastocyst rate, total cell number, apoptosis ratio, and ICM ratio of parthenotes in NCSU23 + PVA without/with SS is shown in Table 1. Expression level of Bax/BclXL genes in blastocysts decreased 0.4 ± 0.1-fold in the presence of SS. Furthermore, activity of caspase-3 in blastocysts decreased and activity of MAP kinase in blastocysts increased with SS. Therefore, our data suggest that SS may play a critical role in increasing the development of porcine parthenotes and in reducing their apoptosis.

Table 1.Developmental ability and apoptosis of porcine parthenotes cultured in the presence or absence of sodium selenite

This work was supported by the Research Project on the Production of Bio-organs (No. 200503030202), Ministry of Agriculture and Forestry, Republic of Korea.
20 SPERM CHARACTERIZATION OF ASTURCON PONIES AFTER COLLECTION AND EQUILIBRATION/REFRIGERATION BEFORE FREEZING
Authors: Tamargo C. , Díez C. , De La Fuente J. , Carbajo M. , Benito J. M. , Hidalgo C. O.
Published: December 2006

The need to conserve farm animal biodiversity is accepted by many countries through the ratification of the convention of biological diversity, and sperm quality is known to be an important criterion in the evaluation of breeding soundness. The aim of this work was to characterize the semen of a local breed of ponies 'Asturcon' (maintained free over the mountains all year around) before its incorporation into a germplasm bank. Semen was obtained from six stallions (6–17 years of age) using an artificial vagina, 3 days/week, during 12 weeks. Immediately after collection, gel-free semen was evaluated for volume, sperm concentration, and motility. Semen motility was again evaluated after equilibration/refrigeration. For evaluation of individual (IM) and progressive motility (PM) rates, semen was diluted (20 � 106 spermatozoa/mL) and analyzed with a CASA System (SCA; Microptic S.L., Barcelona, Spain). Five fields per sample were evaluated (minimum 500 spermatozoa/sample) under a phase contrast microscope (100�). Semen samples were subjected to a hypo-osmotic swelling test (HOS) test to detect the presence of swollen tails in a 100 mM citrate–fructose solution. Percentages of altered acrosomes and morphological abnormalities were determined by counting 100 spermatozoa (1000�). Then, semen was diluted and centrifuged for 10 min at 600g. After the supernatant was discarded, the pellet was re-suspended in freezing medium (skim milk extender containing 2% egg yolk and 2.5% glycerol) to a final concentration of 100 � 106 spermatozoa/mL, and equilibrated/cooled (60 min) to 4�C. Statistical analysis was carried out by means of the GLM and CORR procedures and Duncan test for means (SAS Institute, Inc., Cary, NC, USA). A significant effect between males (P < 0.05) on semen quality, such as volume of the ejaculate, sperm concentration, and morphological abnormalities, were detected among stallions. On the other hand, positive and significant correlations were found between the sperm motility immediately after collection and after equilbration/refrigeration (r = 0.73; P < 0.05); moreover, sperm motilities (both fresh and refrigerated) correlated with the results of the HOS test (r = 0.56; P < 0.001, and r = 0.27, P < 0.05, respectively). These preliminary results confirm that the sperm of the Asturcon ponies breed can be collected and will survive the equilibration/refrigeration procedures. Conservation and development of local breeds is important because they represent a unique source of genes for improving health and performance traits of industrial breeds. However, complementary studies on the ability of the stallion sperm to survive freezing/thawing procedures in rates higher than 30% are needed to ensure that genetic banks are correctly created.
This work was performed in collaboration with ACPRA and Dep�sito de Sementales de Santander (Spain), and supported by RZ2004-00031-C02-01.
201 FACTORS AFFECTING THE SURVIVAL OF BIOPSIED RHESUS MACAQUE EMBRYOS
Authors: Kubisch H. M. , Ratterree M. S.
Published: December 2006

The rhesus macaque (Macaca mulatta) is widely used for the study of human disease. Increasingly assisted reproductive techniques are applied to the propagation of this species to identify specific genotypes, to generate animals with genetic modifications, or to preserve valuable genetic resources. A significant advantage in this endeavor would be the ability to identify embryos with desired genetic characteristics by pre-implantation genetic diagnosis (PGD) prior to their transfer into a surrogate. The objectives of this study, therefore, were to assess the feasibility of performing blastomere biopsies on rhesus embryos and to determine the effects of (a) oxygen tension during culture, (b) transient cryopreservation, and (c) the number of blastomeres removed (one or two) on subsequent development. A total of 17 oocyte aspirations were performed, which resulted in 206 8- to 10-cell embryos that were selected for biopsy. Embryos were cultured in HECM-9 medium and cryopreserved with propylene glycol. The chi-square test was used for data analysis. For the first experiment, 160 embryos were used to assess the effect of oxygen tension and transient cryopreservation. Oxygen tension during culture had no effect on the number of embryos proceeding to the blastocyst stage (26.0 vs. 25.0%) following blastomere removal. Similarly, transient freezing and storage in liquid nitrogen did not affect subsequent development to the blastocyst stage (26.6 vs. 24.7%). For the second experiment, 46 embryos were biopsied and assigned to removal of either one or two blastomeres. Subsequent development was not affected by the number of blastomeres removed, and a similar percentage of embryos reached the blastocyst stage (38.1 vs. 20%). These results demonstrate that rhesus embryos are tolerant of biopsies and proceed to advanced stages of development at the same rate as control embryos.
Funding was provided by the Tulane National Primate Research Center base grant NIH 5P51 RR00164-44.
202 INNER CELL MASS EXCHANGE IN SHEEP EMBRYOS
Authors: Loi P. , Matsukawa K. , Galli C. , Ptak G.
Published: December 2006

The presence of a developmental axis in the mammalian oocyte/embryo is still a controversial issue (Plusa 2005 Nature 17, 391–395). However, pre-established or not, mammalian blastocysts display a clear asymmetry with distinct embryonic and abembryonic poles. The present emphasis on ‘mosaic’ development in mammalian embryos is in contrast with classical embryological work, aimed at cell lineage analysis, where manipulation procedures severely perturbed the natural blastocyst asymmetry (Gardner 2001 RBM Online 4, 46–51). However, all of the experimental work thus far has been carried out on mouse embryos. In our work, we designed experiments to determine whether sheep embryos subjected to inner cell mass (ICM) transfer retain normal developmental competence. In vitro-derived sheep blastocysts (Ptak et al. 2003 Biol. Reprod. 69, 278–285) were manipulated with a Narishige micromanipulator fitted to a inverted Nikon microscope. ICMs were dissected with a blade, and the trophoblastic vesicle and ICMs were cultured in SOFaa plus 10% FCS. After re-expansion, trophoblastic vesicles were injected with ICMs by means of a bevelled pipette and cultured overnight with SOFaa plus 10% FCS. From a total of 35 blastocysts used, 25 re-expanded following injection, and 20 of them showed ICMs adherent to the trophoblast. Seven blastocysts were transferred into synchronized ewes 7 days after estrus, and monitored every month with an Aloka linear probe (7–5 MHz; Aloka Co., Ltd., Tokyo, Japan). Twenty-one in vitro-produced (IVP) embryos were transferred as a control. Three ewes receiving ICM-exchanged blastocysts were pregnant at the first scanning, and all delivered normal offspring (two female and one male lamb; weight: 3.54 ± 0.358 kg). These data demonstrate that dramatic alteration of the blastocyst structure does not compromise its developmental potential. Our efficiency in terms of offspring is lower compared with control IVP embryos, and also compared to data obtained in mice (Papaioannou 1982 J. Embryol. Exp. Morph. 68, 199–209), but technical improvements are expected to reduce such a gap. In conclusion, we demonstrated the feasibility of ICM/trophoblastic exchange in sheep blastocysts; these results might have important application for technologies like somatic cell nuclear transfer (SCNT). Common features of SCNT clones are placental abnormalities in early (DeSousa et al. 2001 Biol. Reprod. 65, 23–30) and late pregnancies (Loi et al. 2006 Theriogenology 65, 1110–1121). The transfer of ICM from cloned embryos to normal trophoblastic vesicles, although ineffective in cattle (Murakami et al. 2006 Cloning Stem Cells 8, 51–69), might be worth trying on sheep, a species where post-natal mortality in clones is a serious issue.

Table 1.Development to term of manipulated and cloned embryos

Part of this work was supported by EUROSTELLS-European Science Foundation.
203 PRODUCTION OF ANDROGENETIC SHEEP BLASTOCYSTS
Authors: Matsukawa K. , Turco M. Y. , Ptak G. , Loi P.
Published: December 2006

The androgenetic embryo is a useful model for investigating the contribution of the paternal genome (e.g. genomic imprinting) to embryonic development. Few works on androgenetic embryo production in domestic animals exist. In this study, we compared the developmental ability of diploid, haploid, and triploid androgenetic sheep embryos. In vitro-matured metaphase II oocytes were enucleated in HEPES-buffered TCM-199 with cytochalasin B (7.5 µg mL−1) and Hoechst 33342 (5 µg mL−1) under UV light using a Narishighe Micromanipulator fitted to an inverted Nikon microscope. Enucleated oocytes were fertilized in vitro with a high sperm concentration (2.5 × 107 sperm mL−1). Fifteen hours after in vitro fertilization (IVF), embryos were centrifuged (12 000g for 10 min) to visualize the pronuclei; the number of pronuclei were scored under the inverted microscope. In Experiment 1, IVF (control), haploid (1 pronucleus), diploid (2 pronuclei), and triploid (3 pronuclei) embryos were cultured in SOFaa medium with BSA, according to the protocol in our laboratory (Ptak et al. 2002 Biol. Reprod. 67, 1719–1725). In Experiment 2, we performed pronuclear transfer to produce diploid embryos. A single pronucleus was aspirated with a bevelled pipette from haploid (haploid + haploid) or diploid (diploid + haploid) embryos and transferred into the perivitelline space of another haploid embryo. The reconstructed zygotes were electrofused in 0.27 M mannitol solution with 50 µM CaCl2 and 100 µM MgCl2 by a single DC pulse (0.8 kV cm−1 for 80 µs). As a control group for Experiment 2, IVF embryos (pronuclear stage) were centrifuged, followed by the aspiration of a small volume of cytoplasm, and fused under the same condition of diploidization. In Experiment 1, there was no significant difference in cleavage rate (91% to 98%), but there was a significant difference on blastocyst development between IVF and androgenetic embryos (IVF: 43% (26/60); haploid: 0% (0/37); diploid: 1% (1/73); and triploid: 2% (1/48)). In Experiment 2, there was no significant difference in cleavage rate (94% to 100%). However, there was a significant difference on blastocyst development (Control: 42%; haploid + diploid: 19%; and haploid + haploid: 3%; Table 1). Our results suggest that sheep androgenetic embryos show poor developmental ability compared with IVF embryos. Interestingly, diploid androgenetic embryos produced by IVF displayed very poor development; however, such poor development was rescued, for unknown reasons, by pronuclear transfer. Ongoing experiments will provide new insight into this previously uncharacterized phenomenon. In conclusion, pronuclear transfer was an effective method for producing sheep androgenetic blastocysts in vitro.

Table 1.Development of androgenetic embryos after pronuclear transfer
204 RELATIONSHIP BETWEEN RESPIRATORY ACTIVITY AND THE PREGNANCY RATE OF BISECTED BOVINE EMBRYOS IN VIVO
Authors: Moriyasu S. , Hirayama H. , Sawai K. , Kageyama S. , Aoyagi S. , Shiku H. , Matsue T. , Abe H. , Kacchi M. , Hoshi H. , Minamihashi A.
Published: December 2006

Oxygen consumption is an important indicator of the metabolic activity of living cells, which may provide valuable information for evaluating embryo quality. We have found that the bovine embryos with high oxygen consumption possess stronger potential for further development. However, the relationship between respiratory activity and the pregnancy rate of embryos is still unclear. In this study, we investigated the respiration rates of bisected bovine embryos and the pregnancy rates of demi-embryos after embryo transfer. Compact morula-stage embryos were bisected evenly by micro glass needle. One hundred bisected embryos were incubated for 24 h in embryo culture medium (IVD101; Research Institute for the Functional Peptides, Yamagata, Japan) at 39�C under 5% CO2, 5% O2, 90% N2. After the incubation, demi-embryos were classified into 2 groups: blastocoel-formed (BC) and blastocoel-not-formed (CM) embryos. Oxygen consumption rates of demi-embryos were measured by scanning electrochemical microscopy (SECM; Hokuto Denko Corporation, Tokyo, Japan). Within 3 h after the measurement, 80 demi-embryos were transferred into recipient cows (one demi-embryo/one recipient) at 7–8 days after estrus. Recipient cows were diagnosed for pregnancy by ultrasonography approximately 40 days after estrus. Statistical difference was analyzed by Tukey's post-hoc test and chi-square test. A total of 27 recipient cows became pregnant; the pregnancy rates for cows with CM and BC demi-embryos were 40.6% (13/32) and 29.2% (14/48), respectively. Mean oxygen consumption rates (� 10-14 mol s-1) in pregnant and non-pregnant cows were 0.47 and 0.39 for CM demi-embryos and 0.63 and 0.52 for BC demi-embryos, respectively. Retrospective analysis showed that the respiratory activity of demi-embryos in the pregnant group was higher than those in the non-pregnant group. In particular, the pregnancy rates for demi-embryos with respiratory activity higher than 0.35 in CM and 0.40 in BC groups were 52.0% (13/25) and 35.9% (14/39), respectively. On the other hand, cows with demi-embryos having an oxygen consumption rate under 0.35 in CM (n = 7) and 0.40 in BC (n = 9) groups did not become pregnant. These results demonstrated that bovine demi-embryos with higher respiratory activity showed a high pregnancy rate after embryo transfer. It is generally known that the pregnancy rate after the transfer of bisected embryos is lower than that of whole embryos. The measurement of oxygen consumption by SECM procedures is a useful tool to assess the quality of pre-implantation embryos and may contribute to the improvement of the success rate for bisected embryo transfer.
205 INCREASED PREGNANCY RATES OF POOR QUALITY BOVINE EMBRYOS BY ASSISTED HATCHING
Authors: Taniyama A. , Watanabe Y. , Nisino Y. , Inoue T.
Published: December 2006

Embryo transfer after superovulation is commonly used for efficient embryo and animal production and for genetic improvement in cattle. However, the quality of collected embryos varies greatly, which affects pregnancy rate. Usually, poor quality embryos are related to low pregnancy rates after embryo transfer and low viability after cryopreservation. Therefore, it is important to improve chances for survival of poor quality embryos after embryo transfer. The objective of this experiment was to improve pregnancy rates by applying the assisted hatching technique to poor quality embryos. Embryos were collected from Japanese Black cows after superovulation on Day 7 post-insemination. After being washed, embryos were morphologically classified. Embryos having more than 30% degenerated cells were assigned as poor quality embryos. The assisted hatching of embryos (cutting the zona pellucida) was performed under a stereoscope or an inverted microscope by making a cutting slit on the zona pellucida for about 20% of its circumference using a micromanipulator equipped with a cutting needle and holding pipette. After cutting, single or two embryos were transferred fresh to one uterine horn of recipient cows on Day 7 of the estrous cycle. Pregnancy and calf production rates were compared between 2 embryo transfer groups composed of fresh zona-cut embryos (ZC group) or fresh embryos with non-cut zonae pellucidae (NZC group). Pregnancy rates were determined by rectal palpation on Day 45, and calf production rates were calculated by the following formula: number of calves born/number of pregnancies. Statistical analysis was carried out using the chi-square test. Pregnancy rates of poor quality embryos in the double ET ZC group (60.3%; 44 pregnancies/73 transfers) were significantly higher (P < 0.05) than those in the single ET NZC group (25.0%; 6 pregnancies/24 transfers) and in the single ET ZC group (44.0%; 37 pregnancies/84 transfers). Calf production rates were 67.3%, 45.5%, and 35.6% for the double ET ZC group, the double ET NZC group, and the single ET ZC group, respectively. Pregnancy rates of poor quality bovine embryos after double ET were remarkably improved by assisted hatching compared with those of single ET with non-assisted hatching. These results suggest that the combined methods of assisted hatching and double ET may be beneficial to produce calves from poor quality embryos.