bluuebunny/crossref_metadata_2025 huggingface dataset:
191 TIMING OF OVULATION IN THE GONADOTROPHIN-STIMULATED SOUTHERN HAIRY-NOSED WOMBAT, LASIORHINUS LATIFRONS
Authors: Druery G.V. , Rival M.D. , Taggart D.A. , Shimmin G.A. , Horsup A.B. , Temple-Smith P.D. , Paris D.B.B.P. , Paris M.C.J.
Published: December 2004

The southern hairy-nosed wombat (SHW), Lasiorhinus latifrons, is a model species in which to develop assisted breeding techniques for the endangered northern hairy-nosed wombat, Lasiorhinus krefftii. We recently showed that anoestrus SHW respond to eutherian gonadotrophins by production of multiple ovarian follicles, but ovulation had not occurred at the time of examination 24 h post-LH (Druery GV et al. 2003 Theriogenology 59, 391 abst). This study investigated the timing of ovulation in six anoestrus captive adult female SHW (n = 3 per group) after ovarian superstimulation using porcine FSH (200 mg total, Folltropin-V, Bioniche, Belleville, Ontario, Canada) administered s.c. at 12-h intervals over 7 days. Ovulation was triggered by a single s.c. dose of porcine LH (25 mg Lutropin-V, Bioniche) 12 h after the final FSH injection. Superstimulatory response was determined by laparoscopy immediately after the final FSH injection on Day 7 prior to LH. Group 1 was re-examined at 33, 36, and 39 h post-LH, and Group 2 at 42, 45, and 48 h post-LH, for evidence of ovulations using laparoscopy and transabdominal ultrasonography. Laparoscopy on Day 7 revealed an ovarian follicular response in all six females, which coincided with the highest levels of estradiol. The reproductive tract also responded to the treatment (swollen fimbriae and enlarged, highly vascular uteri). Multiple follicles (range 16–31) up to 11 mm in diameter were observed in five females. One female had ovulated, as determined by the presence of corpora lutea. Transabdominal ultrasonographic imaging was unable to confirm the number of follicles in stimulated ovaries. Ovulation had commenced by 36 h post-LH, with the majority occurring 39–45 h post-LH. Ovulation was recorded as having occurred if a dark red, highly vascular crater on the surface of the newly formed corpus hemorrhagicum was observed. Increased circulating levels of progesterone were confirmed 9 days after the last laparoscopies. These results have important implications for the development of assisted reproductive technologies in the SHW: (1) transabdominal ultrasound imaging is ineffective for determining ovarian activity; (2) laparoscopy is a well-tolerated, repeatable minor surgical procedure that can be used for intrauterine AI in this species in which nonsurgical AI is unlikely to succeed (Paris DBBP et al. 2003 Theriogenology 59, 401 abst); and (3) knowledge of the timing of ovulation will enable insemination of spermatozoa into the uterus prior to ovulation.
Financial support was provided by Dr. M. Jacobson, and hormones were supplied by Bioniche.
197 HYSTRICOMORPH SUBPLACENTA: THE COMPARATIVE ASPECTS
Authors: Miglino M.A. , Bonatelli M. , Lima M.C. , Oliveira M.F. , Rodrigues R.F. , Carvalho R.G. , Ambrósio C.E. , Machado M.R.F. , Carter A.M.
Published: December 2004

Placentae of four hystricimorph rodents – capybara (Hydrochaerus hydrochaeris), agouti (Dasyprocta aguti), paca (Agouti paca), and rock cavy (Kerodon rupestris) – were examined by conventional histology, transmission electron microscopy, and scanning electron microscopy. In all of these animals, the subplacenta comprised cytotrophoblasts, supported on lamellae of allantoic mesoderm, and syncytiotrophoblasts. The subplacenta of paca, agouti, and rock cavy had a structure similar to that of capybara, although it was not clearly separated from the main placenta by a layer of mesenchyme. The subplacenta of agouti was examined by conventional histology and found to have a structure similar to that in capybara and paca. The cytotrophoblasts were separated by true extracellular spaces; these cells were characterized by the presence of numerous mitochondria, a large-size nucleus relative to the cytoplasm, and absence of glycogen and lipid (PAS-negative). Also, the morphology of the syncytiotrophoblasts was similar in all of these animals; these cells were PAS-positive, and were characterized by the presence of numerous spherical electron-dense granules distributed throughout the cytoplasm, presence of large accumulations of glycogen, few mitochondria, golgi apparatus and granular ER. In addition, the syncytium contained lacunae lined by microvilli, observed only by transmission electron microscopy. The subplacenta is supplied by a single fetal artery. The vessels within the subplacenta pursue a tortuous course with dilatations and constrictions as in an endocrine gland. In mid-to-late gestation, the subplacenta is supplied largely by fetal vessels. A large branch of the umbilical artery follows the central band of fetal mesenchyme to the base of the main placenta and then branches to supply the subplacenta. The subplacental vessels pursue a tortuous course with dilatations and constrictions as in an endocrine gland such as the ovary. A few branches of the maternal artery supply the subplacenta, at least in capybara and paca, where they can be demonstrated by latex injection.
This work was supported by FAPESP, Sao Paulo, Brazil, and CNPq, Brazil.
198 DISTRIBUTION OF SPERMATOZOA AND COPULATORY PLUG IN RELATION TO THE TIME OF MATING AND OVULATION IN THE FEMALE TAMMAR WALLABY (MACROPUS EUGENII)
Authors: Paris D.B.B.P. , Taggart D.A. , Paris M.C.J. , Temple-Smith P.D. , Renfree M.B.
Published: December 2004

In the monovular macropodid marsupial, the tammar wallaby (Macropus eugenii), the cervices are the primary selective barrier to spermatozoa, resulting in differential transport to the non-gravid uterus where a sperm reservoir is established (Tyndale-Biscoe CH and Rodger JC 1978 J. Reprod. Fertil. 52, 37–43). However, due to limited sample size, the dynamics of sperm transport could not be thoroughly examined. In this study, the distribution of spermatozoa, the size of the copulatory plug in the reproductive tract at various times after mating, and the timing of ovulation were characterized in 28 naturally mated female tammars. After the first postpartum (p.p.) mating, adult females were isolated and their reproductive tracts dissected at 0.5, 6, 18, 36, and 40 h post-coitum (p.c.). Each tract was ligated into 13 major anatomical sections, and spermatozoa and eggs were recovered by flushing. Mating occurred 21.7 ± 2.5 h p.p. (mean ± SEM; n = 20) in these animals that were checked frequently and lasted 7.8 ± 0.7 min (n = 15). Within 0.5 h after a single mating (n = 5) the tract contained 2.6 ± 1.0 × 107 spermatozoa and 21.6 ± 8.8 g of copulatory plug, 96 and 70% of which was lost within 6 h p.c., respectively. Spermatozoa reached the uterus, isthmus, and ampulla of the oviduct ipsilateral to the developing follicle within 0.5, 6, and 18 h p.c. respectively, and a uterine population of 2.6 ± 1.2 × 104 spermatozoa (n = 24) was maintained for over 40 h (ANOVA, P > 0.05). Sperm numbers were reduced at the cervix (up to 57-fold) and utero-tubule junction (8-fold), and only 1 in ∼7600 ejaculated spermatozoa (3.4 ± 0.9 × 103; n = 14) reached the oviduct on the side of ovulation. Although sperm numbers were reduced in the gravid uterus (n = 24), differential transport of spermatozoa was not observed (ANOVA, P > 0.05). Ovulation and recovery of sperm-covered eggs from the isthmus of the oviduct occurred 36–41 h p.c. (49–72 h p.p.) (n = 8). Like many eutherian mammals, but in contrast to polyovular dasyurid and didelphid marsupials, the tammar ejaculates large numbers of spermatozoa, but transport is relatively inefficient and sperm storage in the tract before ovulation is limited.
Research was supported by the Australian Research Council (grant No. C09930004) and the University of Melbourne.
200 IMMUNOHISTOCHEMICAL LOCALIZATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS, Flt-1 AND KDR, IN THE COLLARED PECCARY (TAYASSU TAJACU) PLACENTA
Authors: Santos T.C. , Papa P.C. , Dantzer V. , Miglino M.A.
Published: December 2004

Vascular endothelial growth factor (VEGF) is a well-known angiogenic growth factor, which has previously been localized in epitheliochorial, endotheliochorial, and hemochorial placenta types. The present study was undertaken to study the localization of VEGF and its ligands during gestation in the placenta of the Tayassu tajacu (collared peccary), which belong to the Family Tayassuidae, the super family Suoidea, and the order Artiodactyla. The 90-cm long, 30-kg collared peccary is the smallest peccary and it has a white collar around the neck. The animals are very well adapted in all Brazilian territory reproducing during all seasons of the year; it has a gestational period of 144–148 days. The placenta is diffuse and mutually folded with areola-gland subunits for absorption of uterine gland secretions. In the peccary placenta the interhemal barrier is epitheliochorial and it is andeciduated. In the present study, the VEGF and its receptors VGFR-1 and VGFR-2 or Flt-1 and KDR, respectively were immunolocalized in the epitheliochorial collared peccary placenta. The animals were obtained from Sao Pedro Farm, Sao Paulo (Brazil) and from CEMAS, Rio Grande do Norte (Brazil). Six placentae of pregnant collared peccary (60, 90, and 110 days gestation) were prepared for immunohistochemistry by routine methods. VEGF, Flt-1, and KDR exhibited intense staining in the uterine epithelium, uterine glandular epithelium, and trophoblast. The endothelial cells and smooth muscle cells of the vessels in maternal and fetal compartments were also immunoreactive. In the pig placenta the VEGF-ligand-receptor system participates in placental regulations, and we have shown here that these factors occur in peccary placenta in a similar or comparable way.
This work was supported by BIOTA FAPESP, Sao Paulo, Brazil.
204 ASSOCIATION BETWEEN SOME ULTRASONIC CHARACTERISTICS OF CORPUS LUTEUM ANDPROGESTERONE VALUES IN CASPIAN MINIATURE MARES
Authors: Gharagozloo F. , Shirazi A.
Published: December 2004

Reproductive characteristics of the Caspian miniature horse have some similarities to other equine breeds, but several differences such as follicular wave pattern, multiple ovulations, and the size of ovulatory follicle have been reported. Still there are some other characteristics to be determined. This study was carried out to investigate ultrasonic and hormonal changes of the Caspian mares during estrus cycle. Eleven clinically healthy and cycling Caspian mares, aged 3–17 years and weighing 159–236 kg L.W., were used from April to July. Using an ultrasound scanner equipped with a 5-MHz, B-mode, linear array rectal transducer, formation of the corpus luteum was observed from Day 0 (day of ovulation), and changes in plasma progesterone were investigated by daily blood collection and assay by means of an RIA method. Repeated measure ANOVA test was used for statistical analysis. The maximum size of the corpus luteum was seen at Day 3 postovulation (36.6 ± 1.91 mm), and remained up to 10 days after ovulation. At Days 10–11 a significant decrease in size was observed; thereafter gradual regression occurred and minimum size was reached by Days 16–17 (17.3 ± 1.46 and 16.9 ± 1.20 mm, respectively). The first significant increase in progesterone level was detected 24 h after ovulation and average high values of 8 ng/mL by Day 4 after ovulation. The average peak value of progesterone was detected on Days 7 to 10 of the cycle (11.6 ± 1.37 ng/mL) and remained so until Day 12. The lowest plasma progesterone concentration (less than 1 ng/mL) was observed from Day 14 till one day postovulation, coinciding with the CL regression found in sonographic observations. The results of this study revealed that the pattern of corpus luteum formation and Its changes in size and the plasma progesterone levels during the estrus cycle in this breed follow a pattern similar to that of other equine breeds.
The authors wish to thank the University of Tehran for funding and Khojir stud farm for providing animals.
208 FOLLICULAR DYNAMICS, ESTRUS, AND OVULATION IN COWS SYNCHRONIZED TO OVULATE FIRST OR SECOND WAVE DOMINANT FOLLICLES USING SHORT- OR LONG-TERM MELENGESTROL ACETATE- OR EAZI-BREED CIDR INSERT-BASED PROTOCOLS
Authors: Schafer D.J. , Bader J.F. , Busch D.C. , Kojima F.N. , Ellersieck M.R. , Lucy M.C. , Smith M.F. , Patterson D.J.
Published: December 2004

The objective of this experiment was to determine the feasibility in substituting EAZI-BREED CIDR inserts (CIDR; Pfizer Animal Health, Groton, CT, USA) for melengestrol acetate (MGA) in progestin-based protocols to synchronize estrus in beef cows. Follicular dynamics, timing of estrus, and ovulation were compared in beef cows synchronized to ovulate first or second wave dominant follicles using short- or long-term MGA- or CIDR-based protocols. The study was conducted with 48 nonsuckled, estrous cycling, crossbred beef cows assigned to one of four treatments (T1 to T4; n = 12/T) by age and body condition. Cows were synchronized to ovulate first wave (T1 and T2) or second wave (T3 and T4) dominant follicles based on assignment to treatment. Cows in T1 were fed MGA (0.5 mg h−1 d−1) for 7 days, and were injected with PGF2α (PG; 25 mg Lutalyse; Pharmacia Animal Health, Kalamazoo, MI, USA) on Day 7, GnRH (100 μg Cystorelin; Merial, Athens, GA, USA) on Day 11, and PG on Day 18. Cows in T2 had CIDR (1.38 g progesterone) inserted for 7 days, and were injected with PG on Day 7, GnRH on Day 9, and PG on Day 16. Cows in T3 were fed MGA for 14 days, and were injected with GnRH on Day 26, and PG on Day 33. Cows in T4 had CIDR inserted for 14 days, and were injected the GnRH on Day 23, and PG on Day 30. Transrectal ultrasonography was performed daily to monitor follicular dynamics from GnRH to estrus after PG; and every 4 h from 20 h after the onset of estrus until ovulation. Estrus detection was performed continuously using the HeatWatch® estrus detection system (DDx, Denver, CO, USA). Blood samples for progesterone (P4) were collected daily beginning one day prior to the initiation of treatment and continuing through ovulation following PG. Data were analyzed using the General Linear Models procedure of SAS (SAS Institute, Inc., Cary, NC, USA) and are summarized in the following table. Animals that responded to treatment and were included in the analysis were those that initiated a new follicular wave after administration of GnRH and that displayed estrus within 144 h after PG. Although estrous response was similar among treatments, there were differences in follicular dynamics, steroid secretion patterns, and timing of events that culminated in differences in timing and synchrony of estrus and ovulation among the short- and long-term groups. These differences may be important in relation to fixed-time AI programs. These data suggest that in situations that are not conducive to feeding MGA, substituting CIDR inserts into MGA-based protocols to synchronize estrus may be feasible.

This work was supported by USDA-NRI grant 2000-02163; Pfizer Animal Health, New York, NY; and Merial, Athens, GA, USA.
223 ABERRENT TIMP-2 GENE EXPRESSION IN CLONED BOVINE NEONATAL PLACENTA IS DUE TO ABNORMAL EPIGENETIC MODIFICATIONS
Authors: Kim H.R. , Kang J.K. , Yoon J.T. , Jung J.K. , Seong H.H. , Park C.S. , Wakayama T. , Jin D.I.
Published: December 2004

A global proteomics approach by 2-D gel electrophoresis and mass spectrometry was used to analyze the differential protein patterns of three placentae obtained after postnatal death of fetuses from SCNT of Korean Native cattle and three normal placentae obtained after birth of AI fetuses, as previously reported (Kim et al. 2004 Reprod. Fertil. Develop. 16, 145). One differentially up-regulated proteins in SCNT placenta was identified as TIMP-2 protein that is related to extracellular matrix degradation and tissue remodeling processes. To ensure that the identified protein was truly up-regulated in SCNT placenta, a small portion of the protein lysates were subjected to Western blotting with the antibodies against TIMP-2 and β-actin protein. Band images were scanned with a GT-6000 Scanner (Epson-Seiko, Suwa, Nagano, Japan) and densitometric analyses were performed using NIH Image (version 1.56). Proteins were normalized against the levels of β-actin protein. Indeed, Western blot analysis revealed a significant increase in TIMP-2 protein level (about 5-fold) in SCNT placenta compared with normal. Epigenetic programming of the genome by DNA methylation and histone modification ensures proper gene expression during development. To examine how the TIMP-2 gene was programmed to be active in SCNT placenta, we first investigated the status of DNA methylation in the promoter region of the TIMP-2 gene. Sodium bisulfite mapping revealed that most CpG sites in SCNT placenta cells were unmethylated in agreement with expression of TIMP-2 compared with those in normal placenta cells, suggesting that TIMP-2 gene programming by hypomethylation of CpG sites in the promoter region was responsible for the expression in SCNT placenta. We then examined histone modification of the nucleosome in the coding region of the TIMP-2 gene of SCNT placenta cells and normal placenta cells. Chromatin immunoprecipitation (ChIP) assay revealed that H3-K9/K14 acetylation of the TIMP-2 locus in SCNT placenta cells was about 2-fold higher than in normal placenta cells. Therefore, it is postulated that the SCNT placenta at the end of gestation seems to be unusual in tissue remodeling via improper expression of TIMP-2 by abnormal epigenetic modification, and that may affect placental abnormality such as enlarged and dysfunctional placentae as reported in cloned bovine and mouse.
231 TEMPORAL AND SPATIAL GENE EXPRESSION ANALYSIS OF THE BOVINE OVIDUCT EPITHELIUM
Authors: Rehfeld S. , Bauersachs S. , Blum H. , Mallok S. , Wenigerkind H. , Wolf E.
Published: December 2004

The fallopian tube plays a central role in reproduction, providing the appropriate environmental conditions for oocyte maturation as well as for sperm capacitation. Furthermore, fertilization and the first cleavage stages of embryonic development take place in the oviduct. At the molecular level, only fragmentary data are available regarding the physiological changes in the oviduct epithelium during the estrous cycle. Therefore, we performed a systematic study of gene expression changes in bovine ipsilateral oviduct epithelial cells derived from either the ampulla or the isthmus part of the oviduct at four different time points of the estrous cycle. A cDNA array consisting of approximately 400 candidate genes, primarily identified in different studies in the context of gene expression regulation in the oviduct, was designed and hybridized with 33P-labeled cDNA probes prepared from 28 different tissue samples. These tissue samples were collected from cyclic Simmental heifers at Day 0 (n = 3), Day 3.5 (n = 3), Day 12 (n = 4) and Day 18 (n = 4) of the estrous cycle. Ipsilateral epithelial cells were separately collected from ampulla and isthmus. After array evaluation (AIDA Image Analyzer, version 3.41, Raytest, Straubenhardt, Germany), the raw data were normalized to internal reference cDNAs on the arrays. Statistical analysis was done using ANOVA and the Tukey post-hoc test (GeneSpringÂ® version 6.1, Silicon Genetics, Redwood City, CA, USA). For selected genes, differential expression was verified by real-time RT-PCR. A simplified Gene Ontology was built for the genes present on the array and a pathway analysis was performed to elucidate gene networks involved in the regulation of oviduct epithelial cell function. The expression patterns of two functional groups of genes are presented here: genes that are related to immune functions and genes of the secretory pathway or encoding secreted proteins. Messenger-RNA levels of immune-related genes were higher in epithelial cells of the ampulla compared to the isthmus part of the oviduct. This implies that certain immune functions may be differentially regulated in ampulla and isthmus. Furthermore, mRNAs of genes of the secretory pathway showed highest levels mainly in the ampulla around time of estrus, which may be explained with the increase of the secretory activity in the epithelium of the ampulla beginning at pre-estrus. In general, this study shows the importance of a separate analysis of the oviduct compartments and the influence of the estrous cycle on the expression level of a variety of genes. In the context of fertilization and early embryo-maternal communication, these results could provide an insight into the physiological changes during the estrous cycle, which are the bases for these processes.
This work was supported by the Deutsche Forschungsgemeinschaft (Research Unit “Mechanisms of Embryo-Maternal Communication”; FOR 478/1).
25 IN VITRO DEVELOPMENT OF AGGREGATED NUCLEAR TRANSFERRED EMBRYOS DERIVED FROM BOVINE CUMULUS CELLS
Authors: Akagi S. , Tsuneishi B. , Watanabe S. , Takahashi S.
Published: December 2004

It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304–5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3–5 were used following culture in serum-starved medium for 5–7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264–272). NT embryos were cultured in a serum-free medium (IVD-101, Research Institute of Functional Peptide Co., Ltd., Shimojo, Yamagat, Japan). Eight-cell-stage embryos on Day 2 or 16- to 32-cell-stage embryos on day 4 were used for embryo aggregation after removal of the zona pellucida. A small depression was made in a 25-μL drop of TCM-199 with 50 μg/mL phytohemagglutinin (TCM199/PHA) or IVD-101 using a darning needle. Two or three NT embryos were placed into the depression in the drop of TCM199/PHA for 20 min. NT aggregates were then moved into the depression in the drop of IVD-101 and cultured until Day 7. In vitro development of NT aggregates was summarized in Table 1. There were no differences in the cell number and ICM ratio of blastocysts between non-aggregated zona-intact and zona-free embryos. All aggregates of three NT embryos developed to the blastocyst stage and the cell number of these blastocysts was significantly higher than that of non-aggregated NT blastocysts. These results indicate that removal of the zona pellucida does not affect the cell number and ICM ratio of blastocysts and that aggregates of three NT embryos can develop to blastocysts with high cell numbers which are equivalent to in vivo-derived embryos (166 ± 11, Knijn HM et al. 2003 Biol. Reprod. 69, 1371–1378).

Table 1.
Development, cell number, and ICM ratio of NT aggregates
251 CAMELID EMBRYO DEVELOPMENT IN VITRO: EFFECT OF PROTEIN SUPPLEMENT IN MATURATION MEDIUM AND SUBSEQUENT CULTURE IN TWO DIFFERENT MEDIA ON FERTILIZATION AND DEVELOPMENT
Authors: Nowshari M.A. , Wani N.A.
Published: December 2004

Successful IVM/IVF can be used to produce large number of embryos cheaply for transfer and for manipulations. The technology has not previously been reported for the dromedary. The objectives of this study were to determine the effect of protein supplement [BSA vs. heat inactivated estrous camel serum (OCS)] in the maturation medium and subsequent culture of in vitro-fertilized zygotes in TCM199 or G1 and G2 medium (Vitrolife, Gothenburg, Sweden) on the rate of cleavage and development of embryos to blastocysts. Cumulus-oocyte complexes (COC) were collected by puncturing the follicles excised from the slaughterhouse ovaries (Nowshari and Wernery, A.E.T.E. 19th Scientific Meeting, September 12–13th 2003, Rostock, Germany). For IVM, TCM199 supplemented with 0.33 mM pyruvate, 10 μg/mL FSH and LH, and 1 μg/mL oestradiol (maturation medium) was used. The maturation medium was supplemented with either 5 mg/mL BSA or 10% OCS. After 30 to 32 h culture, COC were fertilized with epididymal spermatozoa which was stored at 4°C in TRIS-tes-egg yolk diluent for 1 to 8 days and consisted of not less than 50% motile spermatozoa on the day of use. The spermatozoa were swim up in fertilization medium (TALP, Parrish et al. 1985 Theriogenology 24, 537). Oocytes and spermatozoa (2–4 × 106) were incubated in the fertilization medium for 14–16 h at 38°C under 5% CO2 in air. Intact oocytes were removed from the fertilization medium and washed three times in the respective culture medium. Oocytes from two of the maturation treatments were divided into two subgroups and cultured in either medium TCM199 supplemented with 0.33 mM pyruvate and 10% OCS or medium G1 plus 10% OCS at 38°C under 5% CO2, 5% O2, 90% N2. Zygotes in medium G1 were transferred to medium G2 on Day 3. Zygotes were examined for cleavage on Day 2 and further development on Day 8. The results are presented in Table 1. The results indicate that supplementation of maturation medium with BSA or OCS does not affect the rate of cleavage and development of embryos, however, culture of zygotes in sequential medium (G1-G2) affects the cleavage rate (P < 0.01) but not the further development of in vitro-produced dromedary embryos. Further studies are needed to improve the success of IVF and development during culture in this species.

Table 1.
256 COMMERCIAL-SCALE IN VITRO PRODUCTION AND TRANSFER OF JAPANESE BLACK EMBRYOS
Authors: Seizo H. , Motoharu M. , Hideki T. , Atsushi H. , Takeshi K.
Published: December 2004

In vitro fertilized (IVF) Japanese Black embryos have been preferably transferred to Holstein recipients for beef cattle production in Japan. We have been commercially producing IVF embryos, aiming at increased productivity and hereditary improvement of Japanese Black cattle. However, the use of IVF embryos has been implicated in reduced pregnancy rates as well as in increased risk of abortion and oversized newborns. In this report, we present our cumulative data that summarize the outcome of IVF embryo transfer. The IVF embryos were produced as previously described by Hamano and Kuwayama (1993 Theriogenology 39, 703) using semen collected from five stud bulls. Fertilized oocytes were developed to the blastocyst stage by co-culture with cumulus cells. Developed blastocysts were transported to farms all over Japan in two conditions: (1) non-frozen via air or ground shipping using cell transporters, or (2) cryopreserved in glycerol for direct transfer. Both methods were used at approximately the same rate. We transferred 90% of these embryos to Holstein heifers and 10% to hybrids. Although the average pregnancy rate at the farms was initially low (31%), it has exceeded 40% in recent years. After pregnancy diagnosis, abortion occurred in 9% of these cases. We compared 109 pregnancies from in vivo embryos with 824 from IVF embryos obtained during the same period: the average gestation period was 281.4 and 283.6 days, respectively, the latter being longer by 2.2 days. The average birth weight from IVF was 33.8 kg, 1.3 kg heavier than that of in vivo-derived calves (32.5 kg). In addition, oversized newborns (>50 kg) were absent with in vivo embryos, whereas it occurred in 3.1% of the IVF calves with an average gestation periods of 291.3 days. These gestation period were significantly longer (P < 0.05) than for the normal sized newborn groups. Our study suggests that the IVF technology can be effectively used to increase beef production in Japan and that oversized newborns are associated with delayed parturition. Due to the improved meat productivity obtained with calves derived from these embryos, our sales further increased with 9014, 9334, and 11,932 embryos sold in 2001, 2002 and 2003, respectively.
262 SPERM CHROMATIN STRUCTURE, OXIDATIVE STRESS AND BASIC SEMEN PARAMETERS OF MEN FROM SUBFERTILE COUPLES
Authors: Bochenek M. , Gogol P. , Janeczko J.
Published: December 2004

It is known that the mammalian sperm chromatin structure plays an important role in male fertility. In opposition to many other areas of biological research, the human sperm chromatin can be considered as a model for animal fertility investigations. This is due to the great number of males with high levels of chromatin abnormalities and the ease of tracking their fertility potential. The aim of the study was to find a relationship between sperm chromatin structure, level of reactive oxygen species (ROS) and the basic semen parameters: sperm concentration and motility. The semen from a total of 391 men from subfertile couples 22–51 years old was used. The sperm chromatin abnormalities were examined flow cytometrically according to the SCSA method (sperm chromatin structure assay; Evenson D.P. Methods In Cell Biology, vol. 33, 1990) and ROS level was examined by luminometry (Kolletis et al. 1999 Fertil. Steril.). Sperm concentration and motility were checked microscopically. Sperm concentration of the examined ejaculates ranged from 0.05 × 106/mL to 627.5 × 106/mL and progressive motility ranged from 0% to 70%. More than 30% of spermatozoa with abnormal chromatin (level considered as the infertility threshold) was found in 70 (17.9%) patients; 15–30% of spermatozoa with abnormal chromatin (level of decreased fertility potential) was found in 154 (39.4%) patients; and in 167 (42.7%) patients the number of abnormal spermatozoa did not exceed 15% (level of normal fertility potential; Evenson et al. 1999 Hum. Reprod.; Zini et al. 2001 Fertil. Steril.). High significant correlations were found between chromatin abnormality and: patients' age (0.1008, P = 0.017), sperm concentration (−0.2735, P < 0.001), progressive motility (−0.4365, P < 0.001), and ROS level (0.2709, P < 0.001). However in patients with normal sperm concentration (>20 × 106/mL, according to the World Health Organization), as many as 11.5% had a high level of chromatin abnormality (>30% of abnormal chromatin) and 29.7% of moderate chromatin abnormality (15–30% abnormal chromatin). Similarly, in patients with normal progressive sperm motility (>50%, according to the World Health Organization) 1.7% had a high level of chromatin abnormality (>30% of abnormal chromatin), and 33.9% had a moderate level of chromatin abnormality (15–30% abnormal chromatin). Contrary to the findings of many earlier investigations, a strong relationship between sperm chromatin damages and basic semen parameters was observed in this work. The sperm chromatin structure assay should be included in standard semen examination to avoid expensive and time consuming in vitro procedures for spermatozoa with damaged DNA.
264 PORCINE SPERM-HEAD RECEPTOR INTERACTION WITH PROTEINS PERIPHERALLY BOUND TO THE OVIDUCTAL LUMEN
Authors: Elliott R.M.A. , Satake N. , Watson P.F. , Fazeli A.R. , Holt W.V.
Published: December 2004

Here we show that cell–cell interaction between boar spermatozoa and the oviductal lumen surface are mediated by specific receptor–ligand binding. We have previously demonstrated increased sperm viability following incubation of boar spermatozoa with apical plasma membrane (APM) proteins from sow oviductal epithelial cells (Fazeli A et al. 2003 Reproduction 123, 509–517). Fresh intact oviducts were internally flushed with PBS and filled with Sulfo-NHS-LC-biotin (Pierce Biotechnology, Inc., Rockford, IL, USA) in PBS (pH 8.0). Each end was clamped and incubated for 30 min at RT. Unbound biotin was quenched with 50 mM ammonium chloride for 10 min, and biotinylated soluble APM (sAPM-B) preparations were prepared. Percoll-washed boar spermatozoa (25 × 106 mL−1) were incubated with sAPM-B (150 μg mL−1) for 40 min at 39°C in 5% CO2, and unbound sAPM-B was removed. The sperm pellet was resuspended in 0.5% SDS and incubated for 90 min at RT. Solubilized proteins were isolated by centrifugation at 14,000g for 5 min. The proteins were separated by SDS-PAGE alongside non-biotinylated APM and untreated “sperm-only” samples. Biotinylation was detected by NeutrAvidin/HRP. In addition, sAPM-B treated spermatozoa were smeared onto slides for the detection of biotinyl groups by anti-biotin/Alexa Fluor (Molecular Probes, Leiden, NL). NeutrAvidin/HRP Pierce detected a biotinylated sAPM band migrating to approximately 100 kDa in the sperm/sAPM-B sample. This band was not present in the “sperm-only” sample. Detection of in situ-labelled sAPM-B on spermatozoa showed that fluorescence was specific to the sperm head. We conclude that an oviductal protein of approximately 100 kDa is a potential viability-enhancing ligand for a sperm receptor that is mainly located over the acrosomal region.
This study was funded by the BBSRC (UK).
265 SPERM DNA FRAGMENTATION AND PREGNANCY OUTCOME
Authors: Evenson D.
Published: December 2004

Sperm DNA integrity is obviously important for normal embryo development and pregnancy outcome. Over the past 25 years, various methods have been developed to measure sperm DNA strand breaks in situ. The Sperm Chromatin Structure Assay (SCSA) treats sperm with low pH to denature DNA at the sites of DNA strand breaks, followed by acridine orange (AO) staining of green for native DNA and red for denatured DNA, as measured by flow cytometry (FCM), as well as % sperm with high DNA stainability (HDS: immature sperm with intact DNA related to decreased fertilization rates). FCM-sorted sperm from each SCSA-defined population (normal, moderate, and high DNA fragmentation and HDS sperm) show that the moderate DNA fragmentation index (DFI) population has the same image analysis characteristics as normal sperm without significant comets. Thus, an ICSI technician is not likely to differentiate between a normal and a moderate DFI sperm. The TUNEL assay uses an enzyme to add a fluorochrome-labeled base to a 3′-OH broken DNA strand. Both light microscopy and flow cytometry are used for measuring the % and extent of DNA fragmentation but cannot measure the level of HDS. For the COMET assay, sperm are suspended in an electrophoretic gel, placed on a glass microscope slide, digested with proteases and RNAse, subjected to an electric field, and then stained with a DNA dye. The % of comet positive sperm is scored, but the extent of fragmentation is difficult to define and the % HDS cannot be determined. Small pieces of fragmented DNA migrate in the gel forming a “comet.” All three methods have been used for both research and clinical diagnosis and as prognosis for livestock (bulls, boars, rams, stallions) and humans. Light microscope techniques suffer from a lack of statistical soundness needed for clinical decisions as well as present a potential bias in selection of sperm for measurements. Due to the thousands of sperm randomly selected for flow cytometry measurements, the data are statistically robust. Data from all three kinds of measurements in over a hundred manuscripts clearly show that sperm DNA fragmentation has a negative impact on embryo growth and pregnancy. Infertile animals may have nearly all of the sperm with fragmented DNA. Fertility ratings in bulls and boars are clearly related to the percent and extent of DNA fragmentation. Threshold levels for fertile/sub fertile/infertile differ for different species. Likewise different methods/laboratories have suggested various threshold levels to characterize a man with a highly fertile to low/very poor potential. The range of sperm with fragmented DNA is from ∼2% to 100%. The SCSA method has defined a 27–30% DFI as the point in which a man is placed into a statistical category of taking a longer time to achieve in vivo pregnancy, more intrauterine insemination and routine IVF cycles, or no pregnancy. Current data suggest that ICSI may help overcome the diminished pregnancy prognosis with high DFI over the other ART or natural methods.
267 TESTIS TISSUE XENOGRAFTING AS A BIOASSAY FOR GERM CELL DEVELOPMENTAL POTENTIAL IN EQUINE CRYPTORCHID TESTES
Authors: Rathi R. , Honaramooz A. , Zeng W. , Turner R. , Dobrinski I.
Published: December 2004

In domestic animals, spermatogenic differentiation is blocked in abdominally retained testes exposed to core body temperature. It is not known if undifferentiated germ cells are retained in long-term cryptorchid equine testes, nor is it known whether any surviving germ cells retain their ability to progress through spermatogenesis. If functional germ cells do persist in equine abdominal testes, then the possibility exists that offspring could be derived even from bilaterally cryptorchid individuals. Previously, we reported an in vivo model where completion of spermatogenesis with production of spermatozoa capable of fertilization occurred in fragments of testicular tissue from immature mice, domestic animals, and monkeys grafted under the skin of immunodeficient mice. Therefore, spermatogenic development in testis tissue xenografts can serve as an in vivo assay system for the developmental potential of germ cells. The objective of this study was to investigate if cryptorchid horse testes that had been exposed to core body temperature for 1–3 years had retained developmentally competent germ cells. Small fragments of abdominally cryptorchid testis tissue (about 1 mm3) from three donor horses (1-, 2-, and 3-year-old Quarterhorse) were grafted under the back skin of castrated male immunodeficient mice (n = 8, 6, and 3 recipient mice, respectively). At the time of grafting, donor tissue did not contain differentiated germ cells. Histological examination of the testis xenografts was performed between 5 and 45 weeks post-transplantation. Weight of the seminal vesicles in the host mouse was recorded as an indicator of bioactive testosterone produced by the xenografts. By 28 weeks after grafting, pachytene spermatocytes were observed in xenografts from all cryptorchid donor testes. While haploid gametes would be expected to be present in xenografted testis tissue from descended equine testes by 35 weeks after grafting, spermatogenesis did not progress through meiosis in the cryptorchid grafts. In all recipient animals where spermatogenic differentiation occurred, the weight of the seminal vesicles in the castrated host mice was restored to pre-castration values, indicating that xenografts were capable of releasing biologically active testosterone. These results indicate that even after 3 years of exposure to core body temperature, equine cryptorchid testes contain germ cells capable of differentiation. It remains to be investigated if supplementation of exogenous gonadotropins might support post-meiotic differentiation of germ cells in cryptorchid equine testes xenografts.
This work was supported by USDA 03-35203-13486.
277 NUCLEAR STAGE AND p34cdc2 EXPRESSION IN DIFFERENT SIZES	OF PREPUBERTAL GOAT OOCYTES
Authors: Anguita B. , Jimenez-Macedo A.R. , Izquierdo D. , Paramio M.T.
Published: December 2004

Developmental competence of prepubertal (1- to 2-month old) goat oocytes is compromised, probably because of an incomplete cytoplasmic maturation. Oocyte selection for IVM-IVF is based on morphological criteria. The main regulator of oocyte nuclear maturation is maturation promoting factor (MPF), and it could also be involved in cytoplasmic maturation. The present study tried to determined p34cdc2 (the catalytic subunit of MPF) expression in oocytes of different sizes before and after IVM. Prepubertal goat oocytes were matured in a conventional IVM medium (TCM199 with serum, hormones, and cysteamine) for 27 h. At collection time, a sample of oocytes was classified into 4 groups according to their diameter (<110 μm; 110–125 μm; 125–135 μm; and >135 μm), and nuclear stage was evaluated. After IVM, oocytes were classified by diameter (as described before) and stained to analyze nuclear stage. Before and after IVM, a sample of 10 oocytes of each diameter group was frozen at −80°C. These oocytes were used to perform the detection of p34cdc2-RNA by RT-PCR. Briefly, RNA of 10 oocytes was extracted with TriReagent (Sigma-Aldrich, Madrid, Spain), and used to perform RT using the ThermoScript kit (Invitrogen, Barcelona, Spain). The cDNA corresponding to two oocytes was amplified by PCR, and each amplification band was measured by densitometry using the Quantity One PC program. Rabbit globin mRNA was used as an extrinsic control of the whole process. Nuclear stage data were analyzed by Fisher's exact test, and RT-PCR data were analyzed by one-way ANOVA test. At collection time, a high percentage of prepubertal goat oocytes had resumed meiosis. After IVM, the percentage of MII-oocytes was higher in larger-sized oocytes. At collection time, significantly higher p34cdc2-RNA expression was found in 125–135 μm oocytes. After IVM, no differences were found among oocyte groups. During maturation, a decrease of p34cdc2-RNA was found in 125–135 μm oocytes. In contrast, an increase of p34cdc2-RNA was found in 110-125 μm oocytes. The nuclear stage in the smallest oocytes show their reduced ability to resume meiosis. IVM-oocytes of different diameters showed no difference in p34cdc2-RNA expression.

Table 1.
p34cdc2-RNA expression of prepubertal goat oocytes according to nuclear stage and oocyte sizes

This study was supported by MCYT (Spain), with the grant number AGL2000-0353.
283 DEVELOPMENT OF CAPRINE EMBRYOS PRODUCED BY ICSI AND IVF	AND CULTURED IN DIFFERENT MEDIA
Authors: Jimenez-Macedo A.R. , Anguita B. , Izquierdo D. , Paramio M.T.
Published: December 2004

Embryo development of IVM-IVF prepubertal goat oocytes is low. The ICSI techniques may allow fertilization of all IVM oocytes. The objective of this study was to assess the efficacy of ICSI compared with IVF, for embryonc development of prepubertal goat oocytes. Prepubertal (1- to 2-month old) goat oocyte were recovered from a local slaughterhouse and matured in a conventional IVM medium (TCM199 with serum, hormones and cysteamine) for 27 h. Spermatozoa from fresh ejaculates were selected by swim-up and capacitated with heparin. IVM oocytes were divided into three groups: (1) oocytes fertilized using conventional IVF methodology, (2) ICSI oocytes activated by sequential treatment with 5 mM ionomycin for 5 min and 2 mM 6-DMAP for 4 h; and (3) parthenogenetic oocyte group used as a control for chemical activation. Presumptive zygotes and activated oocytes were cultured in TALP fertilization medium for 24 h. After fertilization, all groups were divided into two groups and cultured for 192 h in: (a) sequential media G1.3/G2.3; or b) TCM199 with granulosa cells. The number of cell nuclei in embryos was assessed by Hoescht staining. Each experiment was repeated seven times, and results were analyzed using chi-square or Fisher's test. The percentage of total embryos obtained after IVF, ICSI, and chemical activation was higher (P < 0.05) for ICSI than for IVF in G1.3/G2.3 (81.4 and 67.9%, respectively) but not in co-culture (61.9 and 58.2%, respectively). The percentage of total embryos was higher (P < 0.05) in G1.3/G2.3 than in co-culture for both IVF oocytes (67.9 and 58.2%, respectively) and ICSI oocytes (81.4 and 61.9%, respectively). The highest percentage of 8- to 16-cell embryos (28.1%) was obtained in the ICSI group cultured in G1.3/G2.3 medium. We did not find significant differences among the experimental groups in the percentage of morulae. In conclusion, ICSI improved the percentage of embryos developing beyond the 8-cell stage. However, this difference could be due to the additional effect of the chemical activation. Embryo development was also improved by using G1.3/G2.3 embryo culture medium rather than TCM-199 with granulosa cells. The ICSI procedure could be a useful tool to study more accurately the oocyte competence for embryo development.

Table 1.
Embryo development of oocytes after IVF, ICSI, and parthenogenetic activation and culture in G1.3/G2.3 or co-culture in TCM199 with granulosa cells

This study was supported by MCYT (Spain), Grant AGL 2000-0353.
302 PROTEIN SUPPLEMENTATION TO IVM MEDIUM IN RELATION TO THE INCIDENCE OF APOPTOSIS IN BOVINE OOCYTES MATURED IN VITRO
Authors: Warzych E. , Matulewicz K. , Nogowska A. , Lechniak D.
Published: December 2004

Mammalian embryos derived from in vitro fertilization display lower developmental competence and quality when compared to their in vivo counterparts. The composition of culture media significantly contributes to this phenomenon. Media supplemented with FBS or the serum derivate BSA are described as biochemically undefined. Those macromolecules were shown to exert a wide range of effects on cultured embryos, dependent on batch-to-batch variability. Therefore, replacement of these protein sources with a synthetic macromolecule such as polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA) provides a possibility to use a chemically defined culture medium (Ali et al. 2002 Biol. Reprod. 66, 901–905). Apoptosis as programmed cell death naturally occurs in mammalian oocytes and embryos; however, its incidence is significantly higher in vitro. The aim of this study was to investigate whether protein supplementation (FBS, fatty acid-free (faf)-BSA, PVP40) of IVM medium affects the incidence of apoptotic oocytes. In the present study, the IVM system previously described by Makarevich et al. (2002 Biol. Reprod. 66, 386–392) was used. Briefly, follicular oocytes aspirated from slaughterhouse ovaries were matured in vitro in one of three maturation media supplemented with FBS (10%), faf-BSA (6 mg mL−1) or PVP40 (4 mg mL−1). The terminal TUNEL assay kit was used to detect the DNA fragmentation in apoptotic cells (DeadEndTM Fluorometric TUNEL system, Promega, Madison, WI, USA). The data were analyzed by chi-square test of independence. Altogether, 630 oocytes collected during 12 IVM experiments were subjected to the Tunel test, and 563 (89.4%) of them were successfully investigated: 426 after maturation in vitro and 137 follicular, non-matured. The remaining 67 cells were lost during manipulation. The rate of Tunel-positive cells differed (P < 0.001) between matured (11.8%) and follicular oocytes (1.5%). Protein supplementation of IVM media did not significantly affect the rate of apoptotic oocyte occurrence, which was 9% in the faf-BSA group, 11.5% in the FBS group, and 15% in the PVP group. No differences were observed in the rate of Tunel-positive cells between oocytes at MII and MI stages. In conclusion, protein supplementation of IVM medium used in the present study did not affect the incidence of apoptotic oocytes after maturation in vitro.
This research was supported by the State Committee for Scientific Research as a Solicited Project PBZ-KBN-084 from 2003 to 2005 year.