bluuebunny/crossref_metadata_2025 huggingface dataset:
104 EFFECT OF FROZEN MEDIA ON IGF2 EXPRESSION OF BOVINE EMBRYOS CULTURED ENTIRELY IN VITRO UNTIL DAY 14
Authors: Franco M.M. , Brandão D.O. , Pereira D.C. , Mundim T.C.D. , Ávila F.F. , Melo E.O. , Dode M.A.N. , Rumpf R.
Published: December 2004

Freezing and stocking ready to use IVP medium, including hormones and fetal serum, is a practical alternative to rationalize work and reduce costs in in vitro embryo production. In our laboratory routine, embryo culture in frozen or fresh medium (produced weekly) has shown equal Day 7 blastocyst rates. Although morphological aspects were also similar, it is known that culture environment may alter gene expression patterns and embryo development on later stages. Thus, a preliminary study of mRNA expression of IGF2 in Day 14 embryos produced in both culture conditions (frozen and fresh medium) was performed. For that, IVM (TCM 199 with hormones, antibiotics, l-glutamine and 10% fetal bovine serum) and IVC medium (SOFaaci) were split into 2-mL aliquots into Eppendorf tubes and frozen at −80°C temperature four weeks prior to use. The thawing was performed in a 4–5°C refrigerator overnight and the medium was stabilized in the incubator at least 4 h prior to use. Abattoir-derived oocytes were collected and randomly distributed into two culture groups: T1 (fresh IVM, fresh IVC) and T2 (frozen IVM, frozen IVC). On Day 7, blastocysts classified as Grades 1 and 2 (n = 12) were selected and continued in vitro culture in the Post Hatching Development system; PHD system (Brandao et al. 2004 Reprod. Fertil. Dev. 16, 123–124) under 38.5°C, 5% CO2 in air. On Day 14, elongated embryos from T1 and T2 were removed from culture and analyzed by RT-PCR. Total RNA of each group was prepared from two D14 embryos of two distinct replicates using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol with modifications. The reverse transcription (RT) was done using the EZ-First Strand cDNA Synthesis Kit (Biological Industries, Israel). The β-actin gene was used as a constitutive control and PCR reactions for the two genes were carried out in triplicate using a PTC-100 MJ Research thermocycler. PCR products were electrophoresed on a 1.5% agarose gel. The two genes were detected in both samples; the relative expression of IGF2 was higher in embryos cultivated in fresh medium (control). The consequences of IGF2 levels in embryo development are still unknown, but highlight the late effects of culture conditions on embryonic gene expression. The perspective use of the PHD system might be an alternative embryo development for monitoring until further stages. In conclusion, IGF2 appears to be a candidate marker gene for embryo development and, although frozen ready-to-use medium is a practical strategy, further studies on molecular trends are necessary to confirm its use.
110 NUCLEAR LAMIN A/C EXPRESSION IN BOVINE IVF EMBRYOS
Authors: Kelly R.D.W. , Alberio R. , Campbell K.H.S.
Published: December 2004

Lamin A/C expression is generally associated with terminally differentiated cell types; however, numerous conflicting reports in the literature demonstrate the presence of lamins A/C in pluripotent cells of pre-implantation embryos. This study characterized lamin A/C expression in bovine pre-implantation embryos using two monoclonal lamin A/C antibodies: anti-A/C IgM (A/C1) and anti-A/C IgG (A/C2) (Santa Cruz, California). Bovine embryos were produced as previously described (Fouladi-Nashta et al. 1998 Biol. Rep. 59, 255–262) and collected at various stages for immunofluorescence staining. Embryos were fixed in 100% methanol at −20°C for 20 min and then blocked for 1 h (4% goat serum in PBS) at RT. Samples were then incubated overnight at 4°C with mouse lamin A/C antibodies or with blocking solution as a control. Following the primary incubation, embryos were washed extensively in 1% BSA in PBS and then incubated with rabbit anti-mouse immunoglobulins (1:20) (DAKO, Denmark) for 1 h at RT. Unbound secondary antibody was removed by washing with 1% BSA in PBS, and embryos were counter-stained with 4',6-diamidino-2-phenylindole (2 μg/mL). Bovine fetal fibroblasts (BFF1) and human embryonic teratocarcinoma cells (EC1 and EC2) were processed identically to the embryos and used as positive and negative controls, respectively (Stewart and Burke 1987 Cell 51, 383–392). Images were viewed using epifluorescence (Leica DMR, Germany) and confocal microscopy (Leica TCS). BFF1 cells reacted with both lamin A/C1 and A/C2 antibodies. EC1 and EC2 stained positively for A/C2 whereas A/C1 was negative in both. All germinal vesicle (GV)-stage oocytes stained strongly for A/C2; however, for A/C1 only 67.5% were positive, and staining intensity was variable. Metaphase II oocytes stained negatively for both antibodies. One-cell zygotes exhibited a variable staining pattern similar to that of GV-stage oocytes. In contrast, all embryos from the 2-cell to blastocyst stage were negative for A/C1 but positive for A/C2. Our observations in embryos and EC cells indicate that the mouse anti-lamin A/C1 is specifically binding to lamin A/C whereas A/C2 is cross-reacting with other nuclear envelope proteins, possibly lamin B1/B2. The cross-reactivity of A/C2 has led to contradicting results in previous reports on lamin A/C expression in pre-implantation embryos. Our results with A/C1 show that lamin A/C is present in GV oocytes and 1-cell zygotes, suggesting that lamin A is important for pronuclear formation after fertilization. These results suggest that active remodelling of the nuclear envelope occurs during the early stages of bovine embryo development.
RK is supported by a BBSRC postgraduate studentship. RA is a Marie-Curie Fellow.
119 THE LOCALIZATION OF A METHYL BINDING DOMAIN PROTEIN (MBD4) IN MURINE AND BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS
Authors: Ruddock N. , Xue J. , Sanchez-Partida L. , Cooney M. , Korfiatis N. , Holland M.
Published: December 2004

The presence of MBD4, a member of the methyl binding domain family, was investigated in both murine and bovine oocytes and pre-implantation embryos. MBD4 is the only MBD family member that is involved in DNA repair but not active in transcriptional repression or in the formation of complexes with histone deacetylase complexes (HDACs). It contains a mismatch-specific glycosylase domain that acts to repair G:T mismatches within a CpG context. Bovine cumulus oocyte complexes were collected from abattoir-derived ovaries, matured in vitro and used for IVF as described previously (Ruddock et al. 2004 Biol. Reprod. 70, 1131–1135). Samples were analyzed at all steps in this process. Murine oocytes were collected from superovulated mice (C57BL6 × CBA) and subjected to conventional IVF. A polyclonal antibody derived in the rabbit against human peptides from specific regions of MBD4 (Imgenex, San Diego, CA, USA) was used to localize MBD4 protein. This antibody was tested at a variety of concentrations against both human HL60 leukemia cells and bovine embryos. Staining of HL60 cells was optimum at 32–64 μg/mL and embryos at 64 μg/mL. Briefly, the staining protocol consisted of fixing cells and zona-free oocytes or embryos in 4% paraformaldehyde for 15 min, followed by 15 min in 0.1% Triton X-100. Primary antibody incubation was performed overnight at 4°C. Embryos were then washed in blocking buffer for 1 hr prior to incubation at 4°C in mouse anti-rabbit IgG conjugated to FITC in blocking buffer for 30 min in the dark. Lastly, embryos were incubated in 10 μg/L Hoescht 33342 for 15 min, and then washed and mounted with Vectashield (Vector Labs, Burlingame, CA, USA). Negative controls contained no primary antibody. Mounted cells/embryos were viewed by epifluorescence microscopy. MBD4 was found to be expressed in both murine and bovine oocytes and pre-implantation embryos. In the cow, faint nuclear expression was detected at the 2-cell stage, followed by exclusion of the protein from the nucleus until the blastocyst stage of development. At this stage, staining was primarily nuclear and quite intense. In the mouse, staining was cytoplasmic at the 2 pronuclear stage, but was then concentrated in the nucleus from the 2-cell stage onward. It will be interesting to determine if this is due to the different timing of embryonic genome activation between the two species, hence implying a role for MBD4 in this important biological process. Further investigations are underway to compare the subcellular localization of the other MBD proteins in both species during preimplantation development and to identify a role for MBD4 in embryonic genome activation.
123 PROGESTERONE LEVELS DURING 20 DAYS OF PREGNANCY IN RABBIT TREATED FOR ENDOMETRIOSIS OR WITH ANTI-CD44
Authors: Illera M.J. , Bermejo P. , Natarajan A. , Willingham C. , Hernandez J.
Published: December 2004

Embryo implantation begins when the blastocyst both assumes a fixed position in the uterus and establishes a more intimate relationship with the endometrium. Successful implantation depends upon hormonal synchronization and development and the receptivity of the endometrium. CD44 is a cell surface molecule that has been implicated in the initial attachment of the embryo. The aim of this work was to study the hormonal levels of P4 in three groups of animals which have a normal pregnancy or an induced reduction in the number of implants. Twelve adult New Zealand does (n = 12) were naturally inseminated with a buck of proven fertility. Blood samples were obtained daily during 20 days of pregnancy. Hormonal determinations were performed by enzyme immunoassay. Animals were divided into three groups: group A (n = 4): control animals; group B (n = 4): endometriosis was surgically induced in the right horn a month before the animal was mated; and group C (n = 4): animals received an injection of 20 micrograms of anti-CD44 in the right horn via mid-ventral laparotomy on Day 6.5 post-coitum (0.5 mL each, from the ovarian end to the cervix). Each animal served as her own control with the left uterine horn receiving 0.5 mL of saline. Statistical analysis was performed using ANOVA.The number of corpora lutea was similar in all treatments. No statistical differences were found comparing CLs in the right/left ovary. In group A, a mean of 3.4 ± 0.47 (mean ± SE) implants was found in the right horn while the mean in the left side was 4.6 ± 0.81. In group B, a marked reduction in implantation sites was found, with 1.8 ± 0.60 and 4.66 ± 0.84 on the right and left horns, respectively. With anti-CD44 injected into the uterine horn (Group C), a mean of 0.12 implant was present in the right uterine horn compared with 3.6 implants on the left side (P < 0.001). Progesterone levels from Days 1 to 10 are shown in the following table (mean ± SE). Comparisons in day values are not statistically significant P > 0.05. After Day 10 the levels of progesterone were similar in all groups. The results showed an increase of progesterone levels in group B; this could be due to endometriosis and not to the number of implants. The results in the CD44 group reveal that progesterone profiles were similar to those in the control group, and we can conclude that the reduced number of implants found in group C did not affect the progesterone levels.

Table 1.
Progesterone levels (ng/mL) from Days 1 to 10 (mean ± SE)
165 SYNCHRONIZATION TREATMENT WITH NEW AND REUSED CIDR-B DEVICES: ESTRUS AND PREGNANCY RATES IN AN EMBRYO TRANSFER PROGRAM
Authors: Solorzano C.W. , Mendoza J.H. , Villa-Godoy A. , Galina C.S. , Romo S.
Published: December 2004

An earlier study demonstrated that in CIDRs used in adult cows on two previous occasions, there was still a sufficient amount of progesterone (P4) remaining that allowed estrus synchronization in heifers (Solorzano et al. 2004 Reprod. Fert. Dev. 16, 214). However, the diverse conditions existing in that study made a statistical comparison impossible. The objective of this study was to study the effect of new and used CIDRs, combined with estradiol benzoate (EB) and prostaglandin F2-α (PGF) treatments, on estrus synchronization rates (ESR) and pregnancy rates (PR) in an embryo transfer program in Brangus cows in a tropical climate. We used the same set of CIDRs in the same location in cows of the same breed, age, and body condition during three consecutive weeks. Cows were randomly allocated to one of three treatment groups. In Group 1, all cows (n = 44) were treated with a new 1.9-g CIDR (CIDR-B, InterAg, Hamilton, New Zealand), combined with 2 mg EB on Day 0. In Group 2, all cows (n = 43) were treated with a reused (first reuse) 1.9-g CIDR and 2 mg EB on Day 0. In Group 3, all cows (n = 42) received a reused (second reuse) 1.9-g CIDR and 2 mg EB on Day 0. CIDR devices were removed on Day 7 and all cows received PGF (0.25 μg cloprostenol) at that time. Estrus was expected to occur 24 h later. Seven days after estrus all cows showing heat were examined by rectal palpation, and those with a CL 15 mm in diameter or larger were selected to receive a frozen/thawed embryo (in 1.5 M ethylene glycol) by nonsurgical direct transfer. PR were determined by rectal palpation 60 days after estrus. In Group 1, a total of 90.9% of the cows displayed signs of estrus (40/44), and 42% of those that received a frozen embryo were pregnant (16/38). In Group 2, a total of 88.4% of the treated cows showed signs of estrus (38/43), and 37% of those recipients became pregnant (13/35). In Group 3, 88% of treated cows showed signs of estrus (37/42), and 36% of cows receiving an embryo were pregnant (13/36). ESR and PR were compared by χ2 and Fisher's tests, and no differences were found among the three groups studied. This confirms that, in 1.9-g CIDRs used on two previous and consecutive occasions, there is still a sufficient amount of P4 remaining that allows successful estrus synchronization and pregnancy rates in a third use.
175 ESTABLISHMENT OF PORCINE EMBRYONIC STEM CELL LINE DERIVED FROM IN VIVO BLASTOCYSTS
Authors: Ock S.-A. , Mohana Kumar B. , Jin H.-F. , Shi L.-Y. , Lee S.-L. , Choe S.-Y. , Rho G.-J.
Published: December 2004

A porcine embryonic stem (ES) cell line was established from an in vivo-flushed blastocyst. The present study evaluated the effectiveness of IVP, parthenotes and in vivo-produced embryos on establishment of an ES cell line. IVP blastocysts were produced from slaughterhouse ovaries based on the previously reported protocols (2000 Theriogenology 54, 787–797) with minor modifications. Parthenote blastocysts were produced by activation of oocytes matured in vitro with electric stimulation of 2 DC pulses at 2.0 kV/cm for 30 μsec in 0.3 M mannitol solution containing 100 μM CaCl2 and 100 μM MgCl2 in vivo blastocysts were recovered on Day 7 after AI (Day = 0) by flushing the uterus with D-PBS containing 10% FBS from three females. After removal of zona pellucida with 0.2% pronase, the blastocysts were subjected to immunosurgical treatment with 10% rabbit anti-pig serum to isolate the inner cell mass (ICM) as previously reported (1975 PNAS 72, 5099–5102). The ICM was seeded onto the feeder layer of STO which was inactivated by treatment with 10 μg/mL mitomycin for 2.5 h and cultured in DMEM with 0.1 mM β-mercaptoethanol, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 0.1 mM MEM non-essential amino-acid, 20 ng/mL rh-bFGF, 40 ng/mL rh-LIF, 0.03 mM adenosine, 0.03 mM guanosine, 0.03 mM cytidine, 0.03 mM uridine, 0.01 mM thymidine, and 15% FBS. The culture was maintained by changing the medium every day after initiation of ICM attachment onto the feeder layer. Any ES-like colonies were individually picked off the feed layer, dissected with 0.25% trypsin-0.02% EDTA for 3–5 min and reseeded on to new STO feed layer. Out of 140 blastocysts (25, in vivo; 55, IVF; 60, parthenotes) used, attaching rates of the ICMs onto the feeder layer were 88% (22/25, in vivo), 56.4% (31/55, IVF), and 58.3% (35/60, parthenotes). A total of 15 primary ES-like colonies was formed in in vivo (3, 12%), IVF (5, 9.1%), and parthenote (7, 11.7%). However, only one ES cell line from in vivo blastocyst was established, which was confirmed as positive by AP activity (Promega, Madison, WI, USA), and was maintained through four passages. In conclusion, for establishment of an ES cell line in pig, the in vivo blastocyst method is superior to currently available methods utilizing IVF or parthenotes.
This work was supported by grant No. 1000520040020000 from Biogreen 21, Republic of Korea.
177 GENERATION OF ES CELLS AND TRANSGENIC MICE EXPRESSING MTERT-GFP AS A MARKER OF PLURIPOTENTIAL CELLS
Authors: Ramírez M.A. , Fernández-González R. , Moreira P.N. , Pérez-Crespo M. , de la Fuente J. , Pintado B. , Gutiérrez-Adán A.
Published: December 2004

There is not a simple system that allows us to identify stem cells in adult tissues. Cells of adult tissues arise from dividing progenitor cells, which themselves are derived from multipotential stem cells. Telomerase is the enzyme that maintains the ends of linear chromosomes in eukaryotic cells. Recently, a segment of the promoter sequence of the reverse transcriptase of murine telomerase (mTert) has been characterized. mTert is expressed with greatest abundance during embryogenesis and becomes widely expressed in adult tissues at low levels. This low expression level in adult tissues may be due to the presence of pluripotent stem cells present in those tissues. To examine the relationship between telomerase activity and multipotential of adult cells we have generated three constructs (1k-, 2k-, and 5k-mTert-GFP) comprising different segments of the mTert promoter sequence coupled to the coding sequence of the green fluorescent protein (GFP). These constructs were electroporated into R1 and B6D2 (generated in our laboratory) ES cells and were used to produce transgenic mice. The generation and identification of transgenic mice (C57BL6 × CBA) has been previously described (Gutierrez-Adan and Pintado 2000 Trangenic Res. 9, 81–89). Transgenic founders were backcrossed to C57BL6 × CBA mice to obtain transgenic lines. The three constructs were able to mimic the mTert expression, which was coupled to green fluorescence. The mTert-GFP transfected ES cells were initially maintained in medium supplemented with LIF, which was subsequently removed to allow differentiation of embryoid bodies (EBs) and other cell types. GFP expression was higher during the first two days after LIF removal (period of enhanced cell proliferation), decreasing in the following days as a result of EB differentiation. Both ES cell lines showed reduced GFP expression upon differentiation, suggesting that mTert is the principal determinant of telomerase activity; moreover, different degrees of expression and down regulation were reported with the different constructs. Using these constructs we have also generated transgenic mice. Eight lines of transgenic mice carrying the 1kmTert-GFP transgene, four with the 2kmTert-GFP, and three with the 5kmTert-GFP, were obtained. There were no significant differences between the proportions of transgenic founder generates. The transgenic mice express and GFP during the fetal development, indicating their telomerase activity. We are now analyzing the expression of mTert-GFP in adults tissues. Our results suggest that telomerase-GFP transgenics are an important tool to assess the role of telomerase in adult multipotential cells as well as to select these pluripotent cells in adult tissue. It will be interesting to see if different levels of mTert-GFP expression are associated with different levels of pluripotency.
183 EFFECT OF THE ACIDIC ORGANIC BUFFER MES ON BOVINE IN VITRO EMBRYO PRODUCTION
Authors: de Haas K. , Luther I. , Gerber D.
Published: December 2004

Transferred embryos carry the risk of being vehicles of organisms causing diseases. Currently, the risk of in vitro-produced (IVP) embryos is more difficult to assess than the risk of in vivo-derived embryos, since less research has been published on the former. Foot and mouth disease virus (FMDV) is extremely sensitive to a low pH and is likely to be destroyed if embryos are exposed to a low pH for a short time. 2-(N-Morphalino)-ethanesulfonic acid (MES); an organic buffer with pKa 6.1; Sigma, South Africa, M2933) as been shown to destroy FMDV at a rate of 90% per minute at pH 6 and at a rate of 90% per second at pH 5 (Acharya et al. 1990 Vet. Microbiol. 23, 21–34; Thomson “Foot-and-mouth disease,” in Infectious Diseases of Livestock with Special Reference to Southern Africa, ed. Coetzer JAW, Thomson GR, and Tustin RC, Oxford University Press, Cape Town, 825–852). The aim of this study was to test whether exposing bovine oocytes and IVP zygotes to the organic buffer MES, buffered at pH 5.5, is detrimental to the development of bovine IVP embryos. IVM, IVF, and IVC was carried out with 1367 oocytes as described earlier [Jooste et al. 2003 Theriogenology 59, 443]. Oocytes were divided into three groups: 484 were used as controls (no MES exposure); 437 were in a maximal exposure group (MAX), i.e. MES treatment after washing of oocytes, after IVM and after IVF, and 446 had a minimal exposure (MIN), i.e. MES treatment after IVF only. To treat the oocytes with MES, 100 oocytes (from ten droplets) were drawn into a pipette in a maximal volume of 100 μL, and placed in 3 mL of MES, swirled around for 10 s, drawn up again in a maximal volume of 100 μL, and placed in 3 mL of culture medium. Oocytes or zygotes were then washed five times in culture medium before being processed through IVM, IVF, or IVC depending on their stage. Exposure of oocytes to MES varied from 30 to 60 s (10 s swirling and a variable time thereafter to pick up). A chi-square test was used to test for differences in cleavage and Day 7 blastocyst yield between control and treatment groups (P < 0.05). Cleavage (70%; 340/484) and blastocyst yield (32%; 156/484) in the control group were not different from those in MIN (68%; 304/446, and 29%; 131/446, respectively), but were significantly higher than for MAX (57%; 249/437, and 18%; 79/437, respectively). In MAX the MES had a harsh effect on the cumulus cells, making them granular and clumpy in appearance. Oocytes treated in MES solution adhered to the bottom of the dish, which made their handling difficult. Exposure time in MES was therefore variable and longer than initially planned. It is concluded that bovine IVP embryos can be exposed to MES without detrimental effect. Treatment with MAX still resulted in blastocysts but it did not yield good numbers. In future trials, treated dishes should be used to prevent oocyte and zygote adherence. Further research is needed to test whether FMDV can be removed from bovine IVP embryos with the described method.
189 PRODUCTIVE AND REPRODUCTIVE PROFILE OF HOLSTEINS KEPT IN BALOCHISTAN PROVINCE, PAKISTAN
Authors: Bilal M. , Lodhi M.Y. , Chawanakul S. , Kakar M.A.
Published: December 2004

Holstein-Friesian has been rated the number one cattle in the world as far as milk production is concerned. The productive potential is excellent in European countries. Animals of the same breed were imported to meet the milk demand of developing countries such as Pakistan. One thousand cattle from Denmark were imported in 1977–78 by the Balochistan government, and were kept at government dairy farms at seven locations (viz., Kalat, Khuzdar, Loralai, Mastung, Pishin, Quetta, and Zhob). The aim of this study was to determine the productive [age at first calving (AFC), birth weight (BWT), lactation length or milk yield (MY), milk yield per lactation (MYL), days in milk (DIM), and culling and mortality (C&L)] and reproductive potential [service period (SP), age at first service (AFS), gestation period (GP), and calving interval (CI)] of these imported animals while kept under the local conditions of Balochistan. Effect of localities with respect to the environmental data was also explored. Overall average values for AFS were 315 to 986 days with an average of 660.42 ± 12.42, GP 275 to 299 days with an average of 280.62 ± 0.25, and AFC 604 to 1299 days with an average of 944.08 ± 12.71. BWT ranged from 15 to 68 kg with an average of 30.12 ± 0.15, MY from 1471 to 7035 liters with an average of 3731.26 ± 40.52, DIM from 180 to 728 days with an average of 313.56 ± 3.83, and SP ranged from 29 to 886 days with an average of 240 ± 9.61, DP averaged 59.15 ± 20.61 days and CI ranged from 301 to 922 days with an average of 451.10 ± 5.55. The effects of year, season, age, and location were studied and the results revealed that the year influenced the AFS, AFC, BWT, and SP (P < 0.01); GP (P < 0.05) but not DIM and CI (P > 0.05). No effect of season on AFS, AFC, DIM, MY, SP or CI (P > 0.05) was observed, but GP and BWT (P < 0.05) were affected by season. Age influenced the GP and SP (P < 0.05) but not BWT, DIM, MY, and CI (P > 0.05). No effect of sex of the calves, type of birth, and calving number on GP (P > 0.05) was seen. Location of the farm did effect AFS, AFC, BWT, and MY (P < 0.01); CI (P < 0.05); but not GP, DIM, and SP (P > 0.05). In conclusion, differences in environment and seasonality had a variable impact on production parameters in Holstein-Friesian calves.
201 EFFECT OF HEPARIN AND CALCIUM IONOPHORE ON ACROSOME REACTION IN EPIDIDYMAL SPERMATOZOA OF DROMEDARY CAMEL (CAMELUS DROMEDARIUS)
Authors: Wani N.A. , Nowshari M.A.
Published: December 2004

The present study was undertaken to evaluate the effect of heparin and calcium ionophore (Ca-I) on the induction of acrosome reaction in epididymal spermatozoa of dromedary camel. Cauda epididymides were isolated from testes of mature males immediately after slaughter at a local abattoir and brought to laboratory in saline solution on ice (0–1°C). Spermatozoa were collected by aspirating the sperm-rich fluid from convoluted tubules beneath the ductus deferens with a sterile hypodermic needle attached to a 10-mL syringe containing 2–4 mL of TRIS-tes egg yolk diluent and stored overnight at 4°C. The sperm suspension was aliquoted into 3 tubes, washed twice with fertilization medium (TALP; Parrish et al. 1985 Theriogenology 24, 537); pellets were dissolved in 1 mL of fertilization medium supplemented with 10 μg/mL heparin or 10 μM/mL Ca-I or no additive (control). Aliquots (200 μL) were made in Eppendorf tubes and incubated at 38°C for 0.5, 1, 1.5, or 2 h. Sperm viability and acrosomal status was evaluated by a dual staining procedure (Didion et al. 1989 Gamete Research 22, 51–57). Briefly, spermatozoa were incubated with an equal volume of 0.2% trypan blue for 10 min and washed twice (centrifugation at 700g for 6 min) with fertilization medium. Smears were made on glass slides and dried quickly on a warm stage. Slides were stained with 10% giemsa stain for 40 min. They were rinsed under a stream of distilled water, air-dried, and covered with DPX mountant and coverslips. The experiment was replicated six times. Spermatozoa (n > 200/slide) were examined in randomly selected microscopic fields under a phase-contrast microscope at 1000×. Spermatozoa were classified as dead (stained blue in the post-acrosomal region), live non-acrosome-reacted (unstained post acrosomal region but light purple-dark pink acrosome) or live acrosome reacted (unstained acrosome and post-acrosomal region). Data were analyzed using Student's t-test and are presented as mean percent ± SEM. There was no difference in the proportion of live acrosome-reacted (LAR) and live non-acrosome-reacted (LNAR) spermatozoa in the heparin group compared to the control group (Table 1). Although Ca-I group had higher LAR sperm, the proportion of dead spermatozoa was high (P < 0.05). It may be concluded that heparin was not able to enhance the acrosome reaction, and Ca-I seems to be detrimental to the spermatozoa at this concentration. Further investigations are warranted to evaluate the appropriate concentrations and duration of exposure to both of these agents for induction of acrosomal reaction in spermatozoa of this species.

Table 1
207 OOCYTE SURVIVAL AND FOLLICULAR DEVELOPMENT IN Fas-KNOCKOUT AND KIT-DEFICIENT DOUBLE MUTANT MICE
Authors: Moniruzzaman M. , Sakamaki K.O. , Akazawa Y. , Miyano T.
Published: December 2004

Growth factors and cytokines regulate survival and growth of mammalian oocytes via their cognate receptors. Among those receptors, KIT, a receptor tyrosine kinase, has been thought of as an essential molecule for growth and survival of oocytes and for follicular development. The defect of KIT-mediated signals leads to the loss of oocytes and impairment of follicular development. Fas is a member of the death receptor family inducing apoptosis; it expresses in the ovary. In a previous study (Sakata et al. 2003 Cell Death Differ. 10, 676–86), we generated KIT-deficient and Fas-knockout double mutant (Wv/Wv:Fas−/−) mice to study the relation between Fas and KIT signaling in germ cell apoptosis. To further understand the role of KIT in oocyte survival and follicular development, we examined the ovaries of Wv/Wv and Wv/Wv:Fas−/− in comparison to those of C57BL/6 (wild type) mice. We also examined the possibility of overcoming the deleterious effects of KIT deficiency by ovarian allotransplantation. One ovary of each mouse was fixed for immediate histological examination and the other was transplanted under the kidney capsule of a female SCID (severe combined immune deficiency) mouse. Ovaries and recovered grafts were fixed, embedded, serially sectioned at 5 μm, stained with hematoxylin and eosin, and examined under a microscope. Oocytes were counted in every section where the nucleus was seen, avoiding double counting in adjacent sections. Mean (with standard deviation) numbers of oocytes per graft or ovary were compared using Student's t-test. At 13 days post-coitum (dpc), ovaries of Wv/Wv fetuses contained 1104.3 ± 118.8 (n = 4) germ cells which was significantly (P < 0.05) lower than those of wild-type mice. However, at 16 dpc (n = 6) and 2-days old (n = 6), ovaries did not contain any germ cells/oocytes. After allotransplantation of the ovaries (n = 6) from Wv/Wv fetuses (13 dpc) for 2 weeks, all of the germ cells disappeared. When the ovaries from 2-day-old Wv/Wv mice (n = 6) were allotransplanted for 12 days, no oocytes appeared. On the other hand, transplanted ovaries from C57BL/6 fetuses (13 dpc) contained 2162.0 ± 97.3 (n = 6) oocytes after 2 weeks. In those ovaries, 4.7 ± 1.6% follicles developed to secondary follicles which contained growing oocytes. Importantly, ovaries of 2-day-old Wv/Wv:Fas−/− mice (n = 4) contained 1936.0 ± 245.0 oocytes (64.0 ± 10.0% of wild-type mice), and 14-day-old mice (n = 4) still contained 911.3 ± 106.3 follicles in which 28.6 ± 6.0% and 11.4 ± 3.2% follicles developed to primary and secondary follicles, respectively. These results indicate that oocyte death due to KIT-deficiency can not be rescued by ovarian transplantation in SCID mice, and that the Fas-knockout condition partially prevents the death of oocytes induced by KIT-deficiency, and primordial follicles develop in this condition.
21 EFFECTS OF HETEROLOGOUS SEMEN PLASMA AND SEMEN EXTENDERS ON PROGRESSIVE MOTILITY OF FROZEN - THAWED RAM SPERM
Authors: Mataveia G. , Terblanche S.J. , Nöthling J.O. , Gerber D.
Published: December 2004

Frozen-thawed ram semen crosses the cervix poorly, necessitating laparoscopic insemination. Acceptable fertility can be achieved with frozen-thawed ram semen deposited at the external cervical opening if ram semen plasma (SP) is added (McPhie et al. 2000 14th ICAR 2, 78 abst). Homologous SP improves the fertility of frozen-thawed sperm of boars and dogs. Heterologous SP may have effects as well; the addition of bovine SP increased the ability of buffalo sperm (Syncerus caffer) to fertilize bovine oocytes in vitro (de Haas et al. 2003 Theriogenology 59, 392). The aim of the current study was to compare the effects of SP of rams (SPR), bulls (SPB), and dogs (SPD), protein-free TALP, Triladyl (Minitüb, Tiefenbach, Germany), and skim milk upon longevity and percentage of progressively motile frozen-thawed ram sperm. Three ejaculates from each of six rams (2 Dorpers, 2 Döhne merinos, and 2 merinos), aged 2–4 years, were extended in Triladyl, pooled and frozen as a single batch per ram at 200 × 106/mL in 0.25-mL straws. SPR was obtained from the same rams and SPB from 5 bulls by centrifugation, while the post-sperm fractions were collected from 5 dogs (SPD). Within a species, the SP from different donors was pooled and frozen in aliquots at −18°C. The 108 straws (6 rams, 6 diluents, 3 replicates) were thawed in random order. Once thawed, a straw was emptied into a tube with 0.85 mL of the appropriate fluid at 37°C and kept for 6 h. Percentage of progressively motile sperm was estimated at ×200 magnification immediately and 2, 4 and 6 h after thawing. One person thawed the semen and prepared motility specimens, while another performed all motility evaluations. Data were evaluated by means of repeated-measures ANOVA, with rams as subjects and time and fluid as fixed effects. Non-significant interactions were removed from the model. Means were compared by means of Bonferroni's test (P < 0.05). The model included ram, time, fluid, and ram × fluid, and time × fluid interactions, which were all significant (P < 0.01). Mean motility decreased from each time to the next and were 39.0% (0 h), 26.0% (2 h), 19.6% (4 h) and 12.6% (6 h), SEM 1.38%, n = 108. Mean motility was higher for skim milk (39.9%) than for all other fluids except Triladyl (27.7%), which was better than SPB (13.0%), whereas TALP (20.5%) and SPR (21.9%) were similar to Triladyl and SPB (n = 72, SEM 2.85%). The interactions (ram × fluid or time × fluid) were mainly due to SPD, SPR, Triladyl, and TALP, while milk resulted in the best and SPB in the lowest motility. This study shows that heat-treated skim milk maintains progressive motility of frozen-thawed ram sperm better than the SP of various species and protein-free TALP. In contrast to SPR, skim milk is known to result in poor fertility of frozen-thawed ram semen after cervical insemination. It would thus appear that maintenance of progressive motility in vitro may be a poor indicator of fertility after cervical insemination.
241 EFFECT OF MEIOTIC ARREST BY ROSCOVITINE AND SUBSEQUENT IVM TIME ON DEVELOPMENTAL COMPETENCE OF IMMATURE BOVINE OOCYTES
Authors: Campos-Chillon L. , Suh T. , Carnevale E. , Seidel G.
Published: December 2004

Maintaining immature bovine oocytes at the germinal vesicle stage by inhibiting M-phase promoting factor (MPF) activity is a reversible process when using roscovitine, and this can improve cytoplasmic maturation in vitro. However, optimum meiotic arrest times and subsequent IVM times have not been determined, so we evaluated the developmental competence of oocytes in relation to these times. Two by two factorial treatments consisting of 2 arrest times (8 h, 16 h) and 2 subsequent IVM times (16 h, 22 h) plus a control were replicated 6 times in this study. Semen from two bulls was used three times. Chemically defined media (CDM) were used throughout (Olson and Seidel 2000 J. Anim. Sci. 78, 152–157). Slaughterhouse-derived oocytes were arrested in meiosis in 1 mL of CDM-M without any hormones, but containing 50 μM roscovitine and 0.5% fatty acid-free (FAF)-BSA under 5% CO2 in air at 38.5°C. After 8 or 16 h of meiotic arrest, oocytes were washed and matured in 1 mL of CDM-M containing 0.5% FAF-BSA, 2 mM glucose, 50 ng/mL EGF, 15 ng/mL NIDDK-oFSH-20, 1 μg/mL USDA-LH-B-5, 1 μg/mL E2, and 0.1 mM cysteamine for 16 or 22 h under 5% CO2 in air at 38.5°C. Oocytes for the control group were matured in 1 mL of the CDM-M with hormones for 22 h. Ten oocytes from each group were fixed after IVM, stained with orcein, and evaluated for maturation to MII. For fertilization, motile sperm recovered from frozen-thawed semen were co-incubated for 18–20 h with ∼20 oocytes/group at a final sperm concentration of 0.5 × 106 sperm/mL in F-CDM. Presumptive zygotes were cultured in 0.5 mL of CDM-1 for 2.5 days and then in CDM-2 for 5.5 days in 5% CO2, 5% O2, 90% N2 in a humidified incubator at 39°C. Cleavage rates were evaluated after culture in CDM-1. Blastocyst rate, blastocyst stage (5 = early, 6 = full, 6.5 = expanding, 7 = expanded, 7.5 = hatching, 8 = hatched), and embryo quality (1 = excellent, 2 = good, 3 = fair, 4 = poor) were evaluated after CDM-2. Data were subjected to ANOVA; the arc sin transformation was used for percentage data, and least-squares means are presented. There were no significant differences in % cleavage (Cle), cell stage, or blastocyst quality among treatments (P > 0.1). However, meiotic arrest of oocytes for 16 h and subsequent IVM for 16 h improved embryo development to blastocysts compared to other roscovitine treatments (Table 1, P < 0.05). A bull effect for % blastocysts was observed, 19.9% and 25.2% for bulls 1 and 2, respectively (P < 0.08). Blastocyst production was improved by shortening oocyte maturation time from 22 to 16 h, when meiotic progression was previously inhibited for 16 h with roscovitine.

Table 1.
Effect of meiotic arrest and IVM times on oocyte maturation and embryo development
263 IDENTIFICATION OF A NOVEL MOPT GENE IN HUMAN AND MOUSE ADULT TESTIS
Authors: Choi Y.-J. , Kang S.-J. , Kim J.-H.
Published: December 2004

To discover late stage germ cell-specific transcripts we prepared a cDNA library from adult testes of 35-day old mice and subtracted it with mRNA from the testes of juvenile mice. Real-time RT-PCR analysis indicated that 42 cDNA clones in the subtracted library were expressed more intensely in the adult testes than in the juvenile testes. One clone identified by subtraction is expressed preferentially in the late spermatid and is located on chromosome 17E3 in mouse and 2p22 in human. The full nucleotide and amino acid sequences of mouse and human MOPT gene are deposited in EMBL GenBank (AY367765 And AY367766). Human MOPT is spliced by 5 exons and 4 introns and encompasses 7,000 bp of genomic DNA (from bp 355 822 to 425 511) of NT-022184.13, whereas mouse MOPT is spliced by 5 exons and 4 introns and encompasses 7,382 bp of genomic DNA (from bp 6227407 to 6235588) of NT-039658.2.
Because of the limited availability of human testis samples, development-dependent expression of MOPT mRNA was conducted using its mouse homologue and semiquantitative PCR. The number of cycles completed before entering the exponential growth, recorded by amplifier PE5700 for mouse MOPT, were 1.11 ± 0.23, 1.05 ± 0.04, 1.5 ± 0.2, 5.55 ± 0.65, 19.35 ± 0.65, 68.65 ± 2.15, and 185.15 ± 6.15 in W/W, postnatal day 5-, 8-, 12-, 15-, 18-, 22-, and 28-day mouse tissue samples, respectively. The difference among the three times was significant (P < 0.01, ANOVA). These results suggest that expression of MOPT gene increased from postnatal Day 5 to Day 28, indicating possible involvement in testicular development. The ORF encodes a protein containing 79 amino acid residues. A MORN motif, EGQFKDNMFHGLGTYTFPNG, was identified in the predicted protein sequence of MOPT; function of this motif is unknown. In situ hybridization of 12-week-old wild-type mouse testes using an antisense riboprobe and immuno-gold data indicated MOPT was expressed as a late spermatid and acrosome reaction.
This work was supported by BK21 program.
266 ANTIOXIDANT CAPACITY OF BOAR SEMINAL PLASMA
Authors: Hernandez M. , Cano A. , Arnao M.B. , Lucas X. , Vazquez J.M. , Martinez E.A. , Roca J.
Published: December 2004

It has been established that antioxidants in seminal plasma play an important role in protecting the spermatozoa against oxidative stress-induced damage. This study was conducted to measure the total antioxidant capacity (TAC) of boar seminal plasma. Fifty-four ejaculates were collected from 17 mature boars of proven fertility by the gloved-hand technique. Ejaculates were collected separately in different fractions (pre-sperm, sperm-rich, and post-sperm) according to their macroscopic (color) characteristics. After centrifugation (2400g for 3 min), the sperm pellet was discarded; the supernatant was recentrifuged and filtered through a 10-μm nylon mesh filter to remove debris or clumped spermatozoa. The seminal plasma was frozen at -20°C until further use. After thawing at room temperature, seminal plasma aliquots of 5 μL were immediately assessed for total antioxidant capacity. TAC was measured using the ABTS/H2O2/HRP decoloration method (Cano A et al. 2000 Redox Report 5, 365–370) which allows differentiation between hydrophilic and lipophilic antioxidant activity capacity. TAC units were expressed as micromolar (μM) Trolox equivalents. Data were analyzed using ANOVA. Only the hydrophilic activity was measurable, with the lipophilic activity being undetected. The overall TAC of seminal samples (mean ± SEM) was 1623.7 ± 56.28 μM, ranging from 674 to 2428 μM. Different TACs were observed among males (P < 0.05) and between ejaculates of the same male (P < 0.05). Ejaculate fraction had a significant effect (P < 0.001) on the TAC levels. The post-sperm fraction had a significantly lower TAC level (1104.09 ± 57.66 μM) than the pre-sperm and sperm-rich fractions (1611.95 ± 153.68 μM and 1356.136 ± 72.47 μM, respectively, P < 0.001). In conclusion, hydrophilic antioxidant activity represented the main contribution to the TAC in boar seminal plasma, showing differences among males, between ejaculates of the same male, and also between the different ejaculate fractions.
This work was supported by CICYT (AGF98-0533; AGL01-0471) and INIA (RZ01-019).
280 EFFECTS OF β-ENDORPHIN AND NALOXONE ON INTRACELLULAR CALCIUM LEVELS IN CUMULUS CELLS OF EQUINE OOCYTES
Authors: De Santis T. , Dell'Aquila M.E. , Maritato F. , Casavola V. , Minoia P.
Published: December 2004

Changes in intracellular calcium levels in the cumulus oocyte complex (COC) have a crucial role in oocyte maturation. In previous studies we demonstrated that the μ-opioid receptor is expressed in the bovine COC and participates in the signaling associated with oocyte maturation, by inducing an intracellular calcium increase (Dell'Aquila ME et al. 2002 Mol. Reprod. Dev. 63, 210–222). In this work we evaluated modifications of intracellular calcium induced by β-endorphin (β-end) or Naloxone (Nx) in cumulus cells of equine oocytes in relation to the time of the year and cumulus morphology at retrieval. Cumulus cells, isolated by mechanical treatment from compact (Cp, n = 120) or expanded (Exp, n = 120) COCs, recovered from the ovaries of slaughtered mares (follicles <20 mm in diameter) during anestrus, breeding season, spring transition, and autumnal transition, were cultured for 24 h and loaded with 5 μM Fura2-AM for microspectrofluorometric measurements of cytoplasmic ionized calcium (Dell'Aquila et al., 2002). The changes in β-end (30 μM)- or Nx (1mM and 10 μM)-induced calcium concentration were calculated in single cells (n = 194) and are expressed as Δ fluorescence (Fmaximal effect – Fbaseline) before and after 1-min perfusion with the drugs. The use of 1 mM Nx induced a significant increase of intracellular calcium levels in cumulus cells of oocytes recovered in all periods of the year in both Cp and Exp (P < 0.01). The addition of 10 μM Nx or 30 μM β-end significantly increased intracellular calcium only in cumulus cells from oocytes recovered in anestrus (P < 0.05). These results confirm previous observations, carried out on bovine oocytes, in which Nx behaved as a μ-receptor agonist when used at high concentration (Dell'Aquila et al. 2002). The effects of β-end and Nx may be explained in terms of a binding of the two subtances at the μ-receptor with consequent intracellular calcium increases due to extracellular calcium entry or depletion of intracellular stores. These findings could be related to differential espression and/or activation status of the μ-opioid receptor in COCs retrieved in different seasons. These substances can be used to modulate intracellular calcium in the equine COCs, and consequent effects on the stimulation/inhibition of oocyte maturation in this species need to be further investigated.
This work was supported by Grant MIUR COFIN PRIN 2003.
3 X-LINKED GENE EXPRESSION IN BOVINE PRE-IMPLANTATION EMBRYOS OBTAINED IN VIVO AND IN VITRO AS A MEASURE OF IMPACT OF EMBRYO PRODUCTION TECHNOLOGIES
Authors: Nino-Soto M.I. , King W.A.
Published: December 2004

The X chromosome provides an ideal system to study the impact of assisted reproduction technologies on gene expression because it holds a wide range of diversely functional genes and also the epigenetic features of X-inactivation are inherently susceptible to in vitro culture-mediated alterations. Using quantitative real-time RT-PCR we studied the expression of a panel of X-linked genes (ANT3, GAB3, RPS4, MECP2, XIAP, XIST) in pooled pre-attachment bovine embryos produced in vivo and in vitro. We collected pools of ten embryos in vivo (de la Fuente R. et al. 1999 Biol. Reprod. 60, 769–775) at the morula (n = 3 pools) and blastocyst (n = 3 pools) stages. Pools of ten matured oocytes (n = 10 pools), 2–4 cell (n = 10 pools), 8–16 cell (n = 10 pools), morula (n = 7 pools) and blastocyst (n = 7 pools) stage embryos were produced in LSOF (Robert et al. Biol. Reprod. 67, 1465–1472) and TCM-199/bovine oviductal epithelial cells (BOEC) co-culture (de la Fuente et al. 1999 Biol. Reprod. 60, 769–775). Total RNA was extracted with the Absolutely RNA® Microprep kit (Stratagene, La Jolla, CA, USA) and reverse transcribed using Oligo-dT12–18 primers and Superscript II RT (Invitrogen, Burlington, Ontario, Canada). Specific primers were designed for each gene and PCR products were used to build standard curves for absolute quantification in a Light Cycler™ instrument with the Light Cycler FastStart DNA Master Mix SYBRGreenI kit (Roche Diagnostics, Laval, Quebec, Canada). The data were analyzed with SAS (SAS Institute Inc., Cary, NC, USA) using a factorial ANOVA design and a log transformation. Significance level was set at α = 0.05. There were no significant differences in transcript levels between IVF systems in mature oocytes, morulae, or blastocysts. Significant differences were observed for some of the genes tested at the 2–4-cell (XIAP) and at the 8–16-cell stages (ANT3, GAB3, XIAP) but not others. There were significant differences between IVF (both BOEC and LSOF) and in vivo embryos at the morula and blastocyst stages, with IVF embryos showing an average of 8 (ANT3), 10 (XIAP), 127 (MECP2), and 593 (XIST) times more transcripts than their in vivo counterparts. GAB3 was detected in only a few samples prior to the blastocyst stage where it was consistently detected in IVF but not in vivo embryos. It was concluded that there is an effect of the IVF system on gene transcription just before and during the period of activation of the embryonic genome during the 4th cell cycle, as well as marked differences between IVF and in vivo produced embryos that are evident in the differential expression of X-linked genes. This system provides a good tool to monitor the effects of embryo production conditions and help in their improvement.
This project was supported with grants from NSERC, FSBC, and OMAF.