bluuebunny/crossref_metadata_2025 huggingface dataset:
Circadian clock genes in reproductive tissues and the developing conceptus
Authors: Dolatshad Hamid , Davis Fred C. , Johnson Martin H.
Published: December 2008

The circadian (near 24-h) clock is involved in the temporal organisation of physiological and biochemical activities of many organisms, including humans. The clock functions through the rhythmic transcription and translation of several genes, forming an oscillatory feedback loop. Genetic analysis has shown that the circadian clock exists in both a central circadian pacemaker (i.e. the suprachiasmatic nucleus of the hypothalamus), as well as in most peripheral tissues. In particular, the circadian clockwork genes are expressed in all female and male reproductive tissues studied so far, as well as in the conceptus itself. The current data clearly show a robust rhythm in female reproductive tissues, but whether rhythmicity also exists in male reproductive tissues remains uncertain. Although the conceptus also expresses most of the canonical circadian genes, the rhythmicity of their expression is still under investigation. Published data indicate that environmental and genetic manipulations influence reproductive function and fecundity, suggesting an important role for the circadian clock in reproduction, and possibly early development.
Telomeres and reproductive aging
Authors: Keefe David L. , Liu Lin
Published: December 2008

Infertility, miscarriage and aneuploid offspring increase with age in women, and meiotic dysfunction underlies reproductive aging. How aging disrupts meiotic function in women remains unclear, but as women increasingly delay having children, solving this problem becomes an urgent priority. Telomeres consist of a (TTAGGG)n repeated sequence and associated proteins at chromosome ends, mediate aging in mitotic cells and may also mediate aging during meiosis. Telomeres shorten both during DNA replication and from the response to oxidative DNA damage. Oocytes do not divide in adult mammals, but their precursors do replicate during fetal oogenesis; eggs ovulated from older females have traversed more mitotic cell cycles before entering meiosis during fetal oogenesis than eggs ovulated from younger females. Telomeres also would be expected to shorten from inefficient DNA repair of oxidative damage, because the interval between fetal oogenesis and ovulation is exceptionally prolonged in women. We have tested the hypothesis that telomere shortening disrupts meiosis by shortening telomeres experimentally in mice, which normally do not exhibit age-related meiotic dysfunction. Interestingly, mouse telomeres are much longer than human telomeres, but genetic or pharmacological shortening of mouse telomeres recapitulates in mice the human reproductive aging phenotype as the mouse telomeres reach the length of telomeres from older women. These observations led us to propose a telomere theory of reproductive aging. Moreover, chronological oxidative stress increases with reproductive aging, leading to DNA damage preferentially at (TTAGGG)n repeats. Finally, if telomeres shorten with aging, how do they reset across generations? Telomerase could not play a significant role in telomere elongation during early development, because this enzyme is not active until the blastocyst stage, well after the stage when telomere elongation takes place. Rather, telomeres lengthen during the early cell cycles of development by a novel mechanism involving recombination and sister chromatid exchange. Telomere dysfunction resulting from oxidative stress, a DNA damage response or aberrant telomere recombination may contribute to reproductive aging-associated meiotic defects, miscarriage and infertility.
10 POSTCERVICAL INSEMINATION OF SOWS WITH REDUCED SPERM NUMBERS IN FIELD CONDITIONS
Authors: Hernandez-Caravaca I. , Izquierdo-Rico M. J. , Garcia-Vazquez F. A.
Published: December 2008

In the current procedures for AI in pigs, billions of spermatozoa are used (2.5–4.0 × 109 spermatozoa per insemination) in a large volume of liquid (70–100 mL), which is deposited in the cervix at the moment of AI. Approximately 90% of the spermatozoa inseminated cannot be recovered from the uterus at 2 h after AI (Viring S et al. 1980 Acta Vet. Scand. 21, 145–149). Several technical methods are used in the pig production industry for semen application using AI. To reduce the number of spermatozoa, several methods have been developed. The present study was performed to evaluate the reproductive effects under field conditions of postcervical AI (post-CAI) in southeast Spain with multiparous sows. In total, 509 sows were inseminated and divided in 3 groups: sows in the first group were inseminated using cervical AI (CAI, control group) with 3 × 109 spermatozoa in 80 mL of extender (group 1, n = 168); sows in the second group were inseminated by post-CAI using 1.5 × 109 spermatozoa in 40 mL of extender (group 2, n = 175); and sows in the third group were inseminated by post-CAI but using 1 × 109 spermatozoa in 26 mL of extender (group 3, n = 166). The catheter used for post-CAI consisted of a conventional AI catheter with an additional inner catheter that could extend past the catheter tip (Soft & Quick®, Import-Vet, Barcelona, Spain). Day 28 of pregnancy rates, farrowing rates, total pigs born, total live piglets, and stillborns were evaluated. Data were analyzed by ANOVA. Pregnancy and farrowing rates after post-CAI with 1.5 × 109 or 1 × 109 spermatozoa did not differ from those of the control group (group 1: 87.50%; group 2: 88.57%; group 3: 86.14%; P = 0.367). The number of spermatozoa inseminated did not significantly affect the number of live (12.27 ± 4.99; 12.18 ± 5.20; 12.64 ± 5.31; P = 0.946) or stillborn pigs (1.60 ± 1.79; 1.80 ± 2.27; 1.78 ± 1.84; P = 0.386) or the total number of piglets born (13.87 ± 5.39; 14.02 ± 5.75; 14.42 ± 5.65; P = 0.865). The present study shows that post-CAI with a low number of sperm cells does not negatively influence the reproductive parameters of multiparous sows compared with conventional AI. In conclusion, post-CAI in sows is simple, effective, and safe, and allows the sperm dose to be reduced to one billion spermatozoa.
102 EVIDENCE FOR PRE-IMPLANTATION ORIGIN OF PLACENTAL FAILURE IN BOVINE CLONE PREGNANCIES
Authors: Bauersachs S. , Zakhartchenko V. , Ulbrich S. E. , Sinowatz F. , Reichenbach H.-D. , Blum H. , Wolf E.
Published: December 2008

Placental abnormalities account for a high proportion of pregnancy loss after transfer of cloned (SCNT) bovine embryos. The high rate of pregnancy failure has been linked to the finding of abnormal placental formation and function. Because bovine SCNT embryos were shown to exhibit altered trophoblast differentiation (Arnold DR et al. 2006 Reproduction 132, 279–290), we hypothesized that placental abnormalities in bovine clone pregnancies may originate from disturbed embryo-maternal communication in the pre-implantation period. To test this hypothesis, we evaluated the response of the endometrium to SCNT v. IVF embryos. The SCNT embryos were produced from fibroblast cultures derived from 4 different fetuses to exclude specific effects of a particular donor cell culture. After SCNT or IVF, embryos were cultured under identical conditions. Two SCNT or IVF blastocysts (grade 1) were transferred per recipient heifer (Day 8 of estrous cycle). Ten days later the recipients were slaughtered, the uteri were recovered, and pregnancy was verified by the presence of at least one normally developed embryo. Endometrium samples of 9 SCNT and 10 IVF pregnancies were used for transcriptome profiling with a custom cDNA microarray (BOE array; Bauersachs S et al. 2007 J. Dairy Sci. 90, 4420–4423). In total, 58 transcripts were found to be differently abundant between endometrium samples from SCNT v. IVF pregnancies (SAM, FDR 5.24%). Interestingly, for some of them an important role in implantation and/or placentation has already been shown. NR2F2, encoding the orphan nuclear receptor superfamily member NR2F2 (COUP-TFII), was downregulated in endometrium from SCNT pregnancies (1.5-fold; P < 0.01). Uterine-specific Nr2f2 mutant mice are infertile due to implantation failure (Kurihara I et al. 2007 PLoS Genetics 3, e102). Another interesting candidate is GJA1, encoding connexin 43 (Cx43), with 1.8-fold (P < 0.001) downregulated transcript levels in SCNT pregnancies. A striking increase of stromal Cx43 has been observed in the ovine intercaruncular and caruncular endometrium during intensification of the feto-maternal contact (Gabriel et al. 2005 Placenta 25, 287–296), suggesting that reduced GJA1 mRNA expression in bovine clone pregnancies may negatively affect placentation. In view of the well-orchestrated spectrum of transcriptome changes in endometrium during the peri-attachment period (Bauersachs S et al. 2006 Reproduction 132, 319–331), these findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo-maternal communication already in the pre- or peri-implantation period.

This study was supported by the Deutsche Forschungsgemeinschaft (FOR 478).
105 DETECTION OF PLACENTAL LACTOGENS IN SWAMP BUFFALO BY RADIOIMMUNOASSAY TECHNIQUE
Authors: Hanh N. V. , Huu Q. X. , Uoc N. T. , Sulon J. , Sausa N. M. , Nguyen B. X. , Beckers J.-F.
Published: December 2008

Ruminant placental lactogens (PL) are members of the growth factor/prolactin (GH/PRL) family. They are synthesized by trophectodermal binucleate cells. There is evidence to suggest that PL is involved in control of fetal growth, through actions in both the maternal and fetal compartments, as well as in influencing mammary growth during pregnancy (Byatt JC et al. 1992 J. Anim. Sci. 70, 2911–2923). The structure and biology of PL have been studied in the cow, sheep, goat, human, and mice. The maternal concentration of PL is 100- to 1 000-fold greater in pregnant sheep and goats than in cows but no information exists about PL concentration in buffalo. The aim of the present study was to evaluate the ability to detect PL in buffalo fluids by using bovine PL antibody. Samples were collected in the slaughterhouse immediately after animal slaughter. The fetuses were measured after heart blood collection. A bPL RIA system was used to determine the bPL concentrations in the buffalo samples (Alvarez-Oxiley AV et al. 2007 Reprod. Fertil. Dev. 19, 877–885). The rbPL molecules were radio-iodinated with [125]I-Na by using the lactoperoxidase method (Thorell JI and Johansson BG 1971 Biochim. Biophys. Acta 251, 363–369). Concentrations of buffalo PL are presented in Table 1. In this RIA system, the minimum detected value was 0.068 ng mL–1, and the binding competition curves of bovine PL standard and buffalo fluids dilution using bovine PL antibody were paralleled in all kinds of samples. The lowest concentration was detected in allantoid fluid and the greatest concentration in fetal plasma (P < 0.05). Study of the biology of PL in buffalo has proved difficult because the concentration of PL in all buffalo fluids is very low. Furthermore, the research concerning buffalo PL function required in vivo experiments. Existing data suggest that at least the concentration of buffalo PL is different from cattle and other smaller domestic ruminants. In conclusion, our results provide preliminary information about concentrations of PL in buffalo fluids.

Table 1.Concentration of placental lactogen in buffalo fluids

This work was supported by a grant from the Belgian Technical Cooperation.
109 EFFECTS OF LIPOLYTIC AGENTS FORSKOLIN, EPINEPHRINE AND CAFFEINE ON EMBRYONIC DEVELOPMENT AND LIPID CONTENT OF BOVINE EMBRYOS PRODUCED IN VITRO
Authors: Barcelo-Fimbres M. , Seidel G. E.
Published: December 2008

The objective of this experiment was to evaluate lipid accumulation and embryonic development of bovine morulae treated with different chemicals. A total of 2619 slaughterhouse oocytes from heifers and mature cows were matured in CDM medium (similar to SOF) plus 0.5% fatty acid-free BSA and hormones (M-CDM) for 23 h at 38.5°C in 5% CO2 in air. Frozen–thawed sperm were centrifuged through a Percoll gradient and co-cultured with matured oocytes for 18 h in F-CDM (CDM+heparin). Zygotes were cultured at 38.5°C in 5% CO2/5% O2/90% N2 in CDM-1 with nonessential amino acids, 10 μm EDTA, 0.5% fatty acid free BSA, and 0.5 mm fructose. After 60 h, resulting 8-cell embryos were cultured 120 h in CDM-2 (CDM-1+essential amino acids and 2 mm fructose). A factorial design was used with 7 treatments, 2 ovary sources (cows v. heifers), and 3 bulls (A, B and C) replicated twice for each bull (6 replicates). At Day 2.5 embryo cleavage and 8-cell rates were evaluated, and on Day 6 a total of 755 morulae were randomly assigned to the 7 treatments (control, 2 and 8 mm caffeine, 1 and 4 μm epinephrine, and 10 and 40 μm forskolin). To quantify lipid accumulation, Day 7 blastocysts were fixed and stained with 1 μg mL–1 Nile red dye, after which a digital photograph of the equatorial part of the embryo (including the inner cell mass) was taken at 200×, and fluorescence intensity was measured with Image Pro software from 0 to 255 shades for each pixel (0 = no lipids; 255 = greatest lipid accumulation), as previously reported (Biol. Reprod. 2007 (Suppl. 1), 87–88). Data were analyzed by ANOVA. No differences in cleavage rates (75 v. 68 ± 3.6%) or eight cell rates (61 ± v. 57 ± 2.8%) were found for heifer v. cow oocytes (P > 0.1); however, blastocyst rates per oocyte and per 8-cell embryo were greater for cows than heifers (20 v. 10 ± 2.1%, and 68 v. 35 ± 3.8%, respectively; P < 0.05). Treatments: 2 and 8 mm caffeine produced fewer blastocysts per morula than 1 and 4 μm epinephrine, 10 and 40 μm forskolin and the control (39, 5 v. 54, 49, 48, 54 and 52 ± 5.8%; respectively) (P < 0.01). More lipid content was found in whole embryos and trophoblast of heifer-derived than cow blastocysts (P < 0.05), and forskolin resulted in less lipid content than control, caffeine- and epinephrine-treated morulae in whole embryos, embryonic mass and trophoblasts (P < 0.05; Table 1). In conclusion, mature cows were a better source of oocytes than feedlot heifers for embryonic development. High doses of caffeine were detrimental to embryos, and the addition of the lypolitic agent forskolin reduced lipid content relative to control, caffeine and epinephrine-treated embryos.

Table 1.Main effect treatment means of lipid content (arbitrary fluorescence units)
113 EFFECT OF PLANT PROTEIN ON DEVELOPMENT AND QUALITY OF CULTURED IN VITRO PORCINE ZYGOTES
Authors: Gajda B. , Grad I. , Smorag Z.
Published: December 2008

Basic culture media are usually supplemented with serum albumin or serum, which contain amino acids that play an important role as energy sources, osmoregulators, and pH stabilizers. However, the presence of undefined serum in culture media introduces a variation from batch to batch and increases viral or prion contamination risk. The aim of the present study was to investigate the possibility of using plant protein substitute (PP) in place of bovine serum albumin (BSA) during in vitro culture of porcine zygotes. The PP is a mixture of several plant proteins and soya lecithin prepared using a high pressure homogenization process. The experiment was done on pig zygotes obtained surgically from superovulated gilts at 24–26 h after insemination. Morphologically normal zygotes were cultured in vitro in 5% CO2 in air at 39° in NCSU-23 medium supplemented with: 0.002 g mL–1 (group 1), 0.004 g mL–1 (group 2), 0.008 g mL–1 (group 3) PP or 0.004 g mL–1 BSA (control group). Embryo quality criteria were developmental competence (cleavage, morula and blastocyst rates), total cell number per blastocyst and degree of apoptosis as assessed by TUNEL method. Results were analyzed by Chi-square test. There were no differences in cleavage rates on Day 2 between zygotes cultured in NCSU-23 medium supplemented with PP (86.0, 88.0, 84.8; group 1 to 3, respectively) and BSA (91.0%, control group). Culture with 0.008 g mL–1 PP increased morula (85.7%) and blastocyst (69.2%) production as compared with control (75.0% and 56.3%, respectively; P < 0.05) and 0.002 g mL–1 PP (79.5% and 51.8%, respectively; P < 0.05). The mean number of cells in Day 7 blastocysts cultured in NCSU-23 medium + 0.004 g mL–1 BSA was lower (P < 0.05) than in NCSU-23 + 0.004 g mL–1 PP (39.1 v. 43.7, respectively). The blastocysts cultured in NCSU-23 medium + 0.002 g mL–1 PP had higher average number of apoptotic nuclei (13.0) as compared with the control (6.5) and 0.004 g mL–1 PP (6.9). In conclusion, this study suggest the positive effect of PP on development in vitro of porcine zygotes to the morula/blastocyst stage. However, further studies are required to determine the quality of the embryos cultured with PP.

This study was supported by Scientific Net of Animal Reproduction Biotechnology.
123 LONG-TERM PRODUCTION OF IN VITRO-MATURED PORCINE OOCYTES AND EMBRYOS
Authors: Paprocki A. M. , Syverson C. M. , Koppang R. W. , Dobrinsky J. R.
Published: December 2008

Although in vivo matured, ovulated, or both, oocytes provide the finest genetic material for use in assisted reproductive technologies (ART), their en masse production requires livestock production facilities, staff and associated overhead, is expensive and labor intensive, their harvest involves surgical or laparoscopic expertise, and large yields needed for en masse daily embryo production are cumbersome and very costly. In vitro-matured (IVM) oocytes have long been a practical gamete source for ART, including in vitro fertilization, ICSI and cloning. Rather than using conventional IVF to produce embryos, we employ in vitro oocyte activation for the production of diploidized parthenogenetic embryos, removing problems associated with variable embryo production due to polyspermic inseminations. In this way, we can produce a repeatable and consistent supply of mature oocytes, advanced embryos, or both, used in product testing, quality control, transgenic or cloned (or both) embryo production, in vitro development controls, as well as in-house culture control embryos for customer scientific data sharing. In this study, we observe mature oocyte and parthenogenetic embryo production over a complete year as control information for our laboratory. Additionally, colleagues may use these data for comparison in their own scientific mission. At least 3 times a month for 12 consecutive months, ovaries were collected from mature females at an abattoir and transported to our laboratory. Cumulus–oocyte complexes were aspirated from 4–6 mm follicles with an 18-gauge needle fixed to a vacuum pump system. Only COC surrounded by two or more layers of compact cumulus investment and containing oocytes of equal size were placed into a commercial TCM-199-based IVM system (Minitube of America Inc., Verona, WI, USA). After 42 h IVM, mature oocytes were isolated from their expanded cumulus and subjected to chemical (ionomycin/DMAP) parthenogenetic activation based on US Patent 5,496,720. Embryos were cultured 120 h in NCSU-23, then cultured for an additional 48 h in NCSU-23 (no BSA) supplemented with 10% FBS. A minimum of 1504 premium and 4604 standard oocytes (Minitube of America Inc.) were placed into IVM. Both premium (1364, 90.7%) and standard (4061, 88.2%; P > 0.05) oocytes are used to produce mature oocytes (MO). Of 781 premium MOs made into diploidized parthenogenetic embryos, 459 (58.8%) developed into blastocysts (61.3 cells/embryo). Of 2068 standard MO made into diploidized parthenogenetic embryos, 914 (44.2%; P < 0.05) developed into blastocysts (64.7 cells/embryo). En masse in vitro maturation of oocytes can supply a repeatable and consistent supply of mature oocytes for use in assisted reproductive technologies. These MO have the developmental potential to form blastocysts in vitro and enable scientists to infer developmental competence of in vitro-produced embryos for research and commercial use.
124 SUPPLEMENTATION OF IN VITRO MATURATION MEDIUM WITH GROWTH HORMONE DID NOT AFFECT THE TIMING OF THE FIRST ZYGOTIC CLEAVAGE BUT SIGNIFICANTLY IMPROVED THE QUALITY OF RESULTING BOVINE BLASTOCYSTS
Authors: Pers-Kamczyc E. , Warzych E. , Peippo J. , Lechniak D.
Published: December 2008

It is well documented that growth hormone (GH) supplemented to maturation medium positively affects in vitro oocyte maturation and embryonic development in cattle. Conditions of in vitro maturation significantly influence oocyte competence for successful fertilization and preimplantation embryo development. It has been shown that bovine zygotes that cleave early are characterized by better quality than their later cleaving counterparts. In this context, the aim of this study was to determine whether supplementation of maturation media with GH affects the timing of the first zygotic cleavage and quality of produced blastocysts. Oocytes were matured in TCM-199 supplemented with fatty acid free BSA and hormones (FSH and GH) and then fertilized (Parrish et al. 1998 Biol. Reprod. 38, 1171–1180), whereas embryos were cultured in sequential media (Lane et al. 2005 Theriogenology 60, 407–419). All embryos that cleaved by 30hpi (early cleavers, EC) were selected and cultured separately. The remaining embryos cleaved by 48hpi (non early cleavers, NEC) were also incubated in separate drops. Blastocysts of proper morphology, collected at 176hpi, were subjected to TUNEL assay and differential staining (TE:ICM ratio). Due to technical problems only expanded blastocysts were analyzed by differential staining. The significance of GH supplementation in relation to apoptotic index and blastomere number (total, TE, ICM) was analyzed by ANOVA. This experiment included 250 blastocysts obtained from 1092 oocytes. The presence of GH did not influence the basic developmental parameters: cleavage rate, percentage of EC embryos and blastocysts (P > 0.05). Blastocysts derived from oocytes matured with GH more often hatched regardless of the timing of the first zygotic cleavage when compared with blastocysts that developed from the control group (P < 0.05). GH supplementation to IVM medium significantly reduced apoptotic index in EC blastocysts (2.9 and 4.8% for control, P < 0.05). Interestingly, blastocysts containing none or one apoptotic blastomere were observed only among EC embryos (20.6%). Although exogenous GH did not affect the cell count in TE and ICM of expanded EC blastocysts, it increased total cell number and reduced the TE:ICM ratio in embryos derived from oocytes culture with GH. In summary, although exogenous GH was previously shown to accelerate the kinetics of oocyte maturation, it has no effect on the timing of the first zygotic cleavage in cattle. The results of the present work support previously published data on GH as growth stimulating and antiapoptotic factor. Its stimulatory effect was especially evident in NEC blastocysts. Those embryos exhibited even better quality (higher cell count, lower apoptotic index) than control EC blastocysts.

This work was supported by the project No. 3780/P01/2006/31 of the Ministry of Science and Higher Education, Poland.
130 FLAVANOID INCREASES THE IN VITRO DEVELOPMENTAL ABILITY OF CLONED AND PARTHENOGENETIC PORCINE EMBRYOS
Authors: Uhm S. J. , Gupta M. K. , Das Z. C. , Kim K. T. , Yang J. H. , Kim J. H. , Lee H. T.
Published: December 2008

Flavonoid has anti-oxidant properties and has been shown to protect cells against oxygen radical damage. This study therefore, evaluated the effect of flavonoid on the in vitro development ability of porcine embryos produced by parthenogenesis (PA) or somatic cell nuclear transfer (SCNT). Porcine embryos were produced from abattoir-derived prepubertal oocytes either by PA or SCNT of fetal fibroblast into enucleated oocytes essentially as we described earlier (Gupta et al. 2008 Mol. Reprod. Dev. 75, 588–597). One-cell embryos were subsequently cultured in NCSU23 + 0.4% polyvinyl alcohol supplemented with or without 10 μm flavanoid for 7 days at 39°C in a humidified atmosphere of 5% CO2 in air. Results showed that the presence of flavonoid in the culture medium increased (v. controls) the rate of blastocyst in both PA (31.7 ± 4.0 v. 20.4 ± 2.0%) and SCNT (20.6 ± 2.5 v. 12.2 ± 2.9%) groups, respectively (P < 0.05). These blastocysts also had higher ability to hatch (PA: 53.7 ± 3.6 v. 34.0 ± 2.4%; SCNT: 70.9 ± 2.5 v. 45.7 ± 2.9%) and contained higher cell number (PA: 38.9 ± 2.0 v. 31.3 ± 2.1; SCNT: 37.5 ± 2.0 v. 29.7 ± 2.5) than those of control groups (P < 0.05). Western blot analysis of parthenogenetic blastocysts showed that, flavonoid also reduced the expression of caspase-3 and p38 mitogen-activated protein kinase (MAPK) proteins by 3.1 ± 0.1 and 7.7 ± 0.2 fold, respectively while expression of ERK1/2 protein was increased by 4.7 ± 2.3 fold. The 2′,7′-dichlorofluorescene fluorescence staining of embryos further revealed that the activity of reactive oxygen species (ROS) was significantly reduced in flavonoid treated embryos by 2 fold(P < 0.05). These data therefore, suggest that flavonoid may improve the in vitro development rate and quality of porcine embryos by reducing ROS activity and change in protein expression.
134 VIABILITY OF EQUINE BLASTOCYSTS SUBJECTED TO BIOPSY FOR PRE-IMPLANTATION GENETIC DIAGNOSIS
Authors: Choi Y. H. , Hartman D. L. , Hinrichs K.
Published: December 2008

Pre-implantation genetic diagnosis of embryos is commonly performed in humans but has not been developed in the horse. To our knowledge, only one previous report of transfer of equine embryos after biopsy is available (Huhtinen et al. 1997 Theriogenology 48, 361); in that study, three pregnancies were achieved after transfer of 14 embryos. Failure of pregnancy after biopsy of equine embryos has been attributed to difficulty in penetrating the equine embryonic capsule, and possible effects of capsule damage on subsequent embryo survival. Recent developments in intracytoplasmic sperm injection (ICSI) and embryo culture make it possible to produce equine blastocysts in vitro. In vitro-produced equine embryos lack a capsule, but form one when transferred to the uterus. This study was designed to examine the viability of equine blastocysts subjected to biopsy. In Study 1, in vitro- and in vivo-produced embryos were vitrified after biopsy, as would be needed while awaiting results of genetic testing. In vitro-produced blastocysts (n = 5) were obtained from slaughterhouse-derived oocytes that were matured, fertilized by ICSI, and cultured 7 to 8 days. In vivo-recovered blastocysts (n = 9) were obtained by uterine flush of inseminated mares 6.5 days after ovulation. The biopsy procedure was conducted using a Piezo drill in modified PBS without calcium and magnesium. Two to three biopsy manipulations were conducted on each embryo to ensure that an adequate number of cells was obtained. After biopsy, embryos were vitrified as described by Eldridge-Panuska et al. (2005 Theriogenology 63, 1308). Four vitrified in vivo-produced blastocysts were warmed and cultured for 2 days. Of these, two appeared viable, showing a 50- to 100-μm increase in diameter, and two did not grow. The remaining 5 in vivo- and 5 in vitro-produced vitrified blastocysts were shipped to a transfer facility, warmed and transferred transcervically to recipient mares. None of these embryos established pregnancy. In Study 2, three Day-6 in vivo-recovered blastocysts were biopsied then shipped by air (6 h transport time) for transfer to recipient mares. All three embryos established pregnancies and showed a normal heartbeat at 25 days. These results indicate that cells may be obtained from in vitro and in vivo-produced equine blastocysts by biopsy with the Piezo drill. In vivo-produced blastocysts transferred directly after biopsy were viable (3/3). While some blastocysts vitrified after biopsy showed growth in vitro, vitrified embryos did not produce pregnancies after transfer. To our knowledge, this is the first report of normal pregnancy rate after transfer of biopsied equine embryos. Further studies are needed to improve survival of embryos vitrified after biopsy.

This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.
150 THE ROLE OF THE PRE-IMPLANTATION EMBRYO IN THE VERTICAL TRANSMISSION OF NATURAL SCRAPIE INFECTION IN SHEEP
Authors: Low J. C. , Chambers J. , McKelvey W. A. C. , Jeffrey M.
Published: December 2008

The objective of this study was to examine whether or not the pre-implantation embryo can act as a carrier of scrapie. The study was carried out on quarantined premises with sheep of highly susceptible scrapie genotypes. Fifty-seven lambs were produced from embryos collected from donor ewes. These donor ewes were in a scrapie-infected flock and became clinically sick with scrapie or developed clinical scrapie after embryo collection. Fifty of the generated lambs were confirmed as having scrapie-susceptible genotypes with 48 animals identified as ARQ/ARQ and 2 as ARQ/ARH. The respective mean and median ages for the 57 scrapie-susceptible experimental offspring in the study were 1565 and 1826 days. Forty-one of these animals survived to the end point of the study at 5 years of age. In a negative control group the mean and median ages at death for the 17 sheep were 1500 and 1826 days, respectively, and 12 survived to 5 years of age. Post-mortem examinations were carried out on all animals derived by embryo transfer and in none was histological or immunohistochemical evidence of scrapie found. In contrast, in the originating infected flock the majority of scrapie cases occurred in ARQ/ARQ genotyped animals with 56% mortality from scrapie in animals of this genotype. Thus, the study provides no evidence for transmission of scrapie and reinforces published evidence that vertical transmission of scrapie may be circumvented by embryo transfer procedures.

This study was commissioned and financially supported by Defra. Amongst the many involved in the study the authors particularly wish to acknowledge Jon Hunton, James Mylne, Tony Wrathall and Chris Lewis. Keith Chalmers Watson is thanked for his interest and for the land rental that made the work feasible.
164 CHARACTERIZATION OF THE COMMON ELAND (TAUROTRAGUS ORYX) ESTROUS CYCLE
Authors: Pennington P. M. , Gentry L. R. , Pope C. E. , MacLean R. A. , Paccamonti D. L. , Dresser B. L. , Bondioli K. R. , Godke R. A. , Wirtu G.
Published: December 2008

Three species of Tragelaphine (spiral horned) antelopes are endangered including the Eastern Bongo. Though most antelope species are represented as captive herds, a detailed characterization of their estrous cycles has not been described. In a crossover experimental design utilizing two groups of four females (n = 8), two estrus synchronization treatments were applied to each female. The treatments were: 1) PGF2∝ (Lutalyse®, 25 mg IM) repeated 11 days later and 2) administration of a progestin (altrenogest, 5 mL, 2.2% p.o.) for 7 days followed by PGF2∝ (25 mg IM) on 7th day. Blood samples were collected three days (Monday, Wednesday and Friday) per week until 2 days before expected estrus when ovaries were examined via transrectal ultrasound once daily and blood samples were collected twice daily, until the dominant follicle was absent. Blood samples were then collected three days per week until two days before the predicted subsequent natural estrus, when intensive ultrasound and blood collection was resumed. Plasma progesterone levels were determined via competitive radioimmunoassay utilizing a double antibody system from a commercially available kit (Diagnostic Systems Laboratories, Webster, TX, USA). Progesterone levels declined in an equal number of females (n = 6 or 75%) following prostaglandin injection in treatments 1 and 2, suggesting both methods to synchronize the estrous cycle or induce estrus are equally effective. Five of the eight females ovulated at least once during the study. A total of eight (25%) of the 32 possible ovulations were detected by ultrasound observation and confirmed by progesterone levels. Basal progesterone levels for the ovulating females around the days of ovulation were approximately 0.5 ng mL–1 and peaked at approximately 2 ng mL–1 during the luteal phase. Ultrasound observed ovulations occurred as frequently on the right ovary as the left. Two animals from treatment 2 ovulated during both the induced and natural cycles on alternate ovaries. An interovulatory interval of 21 days was observed and agrees with data previously reported by Durrant (1983 Zoo Biol. 2, 191–197). The average time to ovulation after PGF2∝ injection was 69 h (range 58–82 h). The average follicular size 12 to 24 h prior to ovulation was 9 and 7 mm for the induced and natural ovulations, respectively, suggesting that eland follicles ovulate at 10 mm. The described technology allows hormonal and ultrasonographic characterization of the estrous cycle in the eland antelope.
167 FAECAL STEROID ANALYSES FOR MONITORING REPRODUCTIVE FUNCTION IN POLAR BEARS (URSUS MARITIMUS)
Authors: Stoops M. A. , Vollmer L. , Roth T. L.
Published: December 2008

Although there has been only limited research on polar bears (Ursus maritimus), it is known that they are seasonal breeders that mate in early spring and experience delayed implantation in the fall, giving birth to cubs in early winter. A non-invasive method for monitoring reproductive function in polar bears would facilitate the management of captive and wild polar bears while expanding the physiological database on this species. Longitudinal faecal steroid analyses were conducted on a captive female polar bear (8 years old) to identify suitable enzyme immunoassay (EIA) techniques for monitoring reproduction. Faecal samples were collected over 23 months (July 2006 to June 2008). In January 2007, the female was introduced to an intact male (17 years old) for breeding. For the first 6 months of the study, faecal samples were collected weekly. However, prior to introducing the male, collection frequency was increased to thrice weekly. Animal keeper staff recorded behaviour daily. Steroid hormones were extracted from 0.1 g of lyophilized faeces by mechanically shaking samples overnight in 3 mL 90% v/v ethanol. Samples were centrifuged at 500g for 10 minutes and the supernatant stored at –20°C until assayed. Immunoreactive progesterone (P4), estrogen (E2), progesterone metabolite (PdG) and androgen metabolite concentrations were measured in the faecal samples. Due to the established reproductive seasonality of the species, differences in faecal steroid excretion between breeding (January to June) and non-breeding (July to December) seasons were examined with ANOVA. Values are presented as mean ± SEM dry mass faeces (dmf). Seasonal differences in faecal androgen and PdG excretion were significant (P < 0.05). Baseline androgen metabolite concentrations during the breeding season (124.7 ± 4.8 ng g–1 dmf) were higher (P < 0.05) than during the non-breeding season (105.6 ± 4.7 ng g–1 dmf). Peak androgen metabolite concentrations during the breeding season averaged 1108.8 ± 171.2 ng g–1 dmf and coincided with oestrus behaviour and mating. There was no correlation (P ≥ 0.05) between faecal androgen and E2 patterns. In addition, there were no increases in faecal E2 coinciding with mating behaviour or copulation. Therefore, E2 was deemed unsuitable for oestrous cycle monitoring. Faecal P4 and PdG results were significantly correlated (P < 0.05). Prior to breeding, PdG concentrations were 167.5 ± 22.5 ng g–1 dmf. After a March 2007 breeding, the faecal PdG profile appeared to demonstrate a luteal phase (282.5 ± 25.8 ng g–1 dmf) followed by a PdG spike in mid-October (coinciding with expected implantation) lasting approximately 60 days (1025.5 ± 142.6 ng g–1 dmf). Concentrations of PdG declined precipitously to baseline in early December, but cubs were never observed with the female in her den. These preliminary data suggest that non-invasive faecal steroid analyses can be used to monitor reproductive function in female polar bears.
168 POST-THAW VIABILITY OF EPIDIDYMAL SPERM FROM WHITE-TAIL DEER THAT WAS FROZEN USING GLYCEROL OR DMSO AS THE CRYOPROTECTANT
Authors: Stout M. A. , Saenz J. S. , Gentry G. T. , Leibo S. P. , Bondioli K. R. , Godke R. A.
Published: December 2008

Cryoprotectants are compounds that protect and maintain the viability of cells when being subjected to cold environments. In addition to glycerol, DMSO has been evaluated for post-thaw epididymal sperm viability in various endangered species, but has not yet been assessed in White-tail deer. Testicles (in the scrotum) were collected from hunter harvested sexually mature White-tail bucks (n = 7) during the peak rutting season. Testicles within the scrotum, once removed from the postmortem buck, were placed in a plastic Ziploc bag and then into a Styrofoam ice chest (at ambient temperature) and transported to the Embryo Biotechnology Laboratory (EBL). Upon arrival at the EBL, sperm were flushed in a retrograde flow out of a small incision at the medial section of the cauda epididymides. Epididymal sperm were flushed from the epididymis using a non glycerol extender. Sperm from both epididymides of each buck were pooled and allowed a slow cool to 4°C. Upon reaching 4°C, the pooled epididymal sperm were equally divided and either 6% glycerol (A) or 6% DMSO (B) was added to each sperm sample. After the addition of the cryoprotectant sperm were loaded into 0.5-mL plastic straws and cryopreserved using liquid nitrogen vapor. Total motility (TM) was determined subjectively using an inverted Nikon Diaphot microscope. Membrane integrity (MI) was determined using SYBR 14 and propidium iodide staining under a microscope equipped with epiflurescence. Morphology was determined using an eosin-nigrosin staining. A paired t-test was used for statistical analyses. There were no differences between the glycerol and DMSO treatments for any of the three parameters measured (Table 1). However, post-thaw total motility and membrane integrity were significantly lower for both treatment groups (A and B) when compared with pre-freeze values. Furthermore, the DMSO treatment group had significantly lower normal morphology when compared with pre-freeze values, but was not different than the glycerol treatment group. Our results indicate that cryopreservation of epididymal sperm from White-tail deer with either 6% glycerol or 6% DMSO yield similar results for total motility, membrane integrity, and normal sperm morphology.

Table 1.Pre-freeze and post-thaw total sperm motility, membrane integrity and normal sperm morphology for White-tail deer epididymal sperm

Tony Vidrine of the Louisiana Department of Wildlife and Fisheries.
177 EMBRYOS PRODUCED IN VITRO FROM PREPUBERTAL LAMB AND ADULT SHEEP OOCYTES DISPLAY DIFFERENT GENE EXPRESSION PATTERNS
Authors: Bebbere D. , Bogliolo L. , Ariu F. , Fois S. , Leoni G. , Ledda S.
Published: December 2008

Breeding from prepubertal females reduces the generation interval and increases the rate of genetic gain in animal breeding programs. Despite considerable interest in this technology, its efficiency remains too low. Reduced in vitro and in vivo developmental competence of oocytes derived from prepubertal animals have been reported in association with morphologic, metabolic, and biochemical differences. The objective of this study was to compare the relative transcript abundance of a panel of developmentally important genes in embryos produced in vitro from prepubertal lamb and adult sheep oocytes. Cumulus–oocyte complexes derived from ovaries of regularly slaughtered 1-month-old prepubertal and adult sheep were matured in vitro in TCM-199 with 10% heat-treated oestrus sheep serum (OSS), 10 μL mL–1 of FSH/LH and 100 μm cysteamine, in 5% CO2 in air at 38.5°C for 24 h. Matured oocytes were fertilized with frozen–thawed ram semen in SOF medium + 2% OSS for 22 h at 38.5°C and 5% CO2, 5% O2, and 90% N2 atmosphere. Zygotes were cultured in SOF + AA + 0.4% BSA in 5% CO2 and 5% O2 up to blastocyst stage. Three groups of 10 blastocysts for each class (4 replicates) were used to quantify the relative expression of 15 genes by reverse transcription followed by real-time PCR. The relative quantification of the transcripts was performed with the 2-ddCt method (Livak and Schmittgen 2001 Methods 25, 402–408), after normalization against the β-actin expression levels. The analysis of gene expression evidenced higher relative abundance for Aquaporin 3, P34Cdc2, cyclin B, Oct4, H2A.Z, and Nanog transcripts in sheep embryos than in prepubertal-derived ones (ANOVA; P < 0.05), while interferon τ and insulin-like growth factor (IGF) 2 mRNAs were significantly more abundant in lamb-derived embryos (ANOVA; P < 0.01). No differences were observed for the remaining analyzed transcripts (BAX, IGF2R, heat shock protein 90, NaKATPase, E-cadherin, PAP, and glyceraldehyde 3-phosphate dehydrogenase). Overall, results show that embryos produced in vitro from prepubertal and adult oocytes display different patterns of expression at the blastocyst stage. Such difference may be related to the generally observed reduced in vitro and in vivo developmental competence. Increased understanding of the gene expression status during pre-implantation development may provide valuable insights into the molecular basis underlying the very early stages of life and an opportunity for optimizing in vitro embryo production systems.
184 ALTERNATIVE SPLICING OF DNA METHYLTRANSFERASE 1 IN PORCINE OOCYTES
Authors: Giraldo A. M. , Mendicino M. V. , Vaught T. D. , Bondioli K. R. , Ayares D. L.
Published: December 2008

In human, mouse, and some marsupials, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (Dnmt1) enzyme. The identification and characterization of a similar oocyte transcript variant in farm animals would greatly contribute to the understanding of the methylation processes that occur during nuclear remodeling of in vivo embryos as well as of cloned embryos. The objective of this study was to identify and sequence isoforms of Dnmt1 expressed in porcine oocytes. Total RNA was isolated from pools of 50 denuded mature oocytes as well as from fibroblast cells using the Trizol method. RNA was co-precipitated with glycogen, and residual genomic DNA was removed with DNase I. A RACE System (Invitrogen, Carlsbad, CA, USA) was used to amplify the 5′ cDNA end of Dnmt1. Briefly, the first strand of cDNA was synthesized using SuperScript II and a Dnmt1-specific primer followed by degradation of the RNA strands and incorporation of TdT and dCTP tails to the 3′ ends of the cDNA. Tailed cDNA was amplified by PCR using a forward anchor primer and a reverse Dnmt1-specific primer. PCR products were separated by electrophoresis on an agarose gel. Resulting PCR products were subcloned into a cloning vector, and the cDNA inserts were sequenced. PCR primers capable of amplifying all possible alternatively spliced isoforms of Dnmt1 were used to identify the presence of the RNA sequences found in fibroblasts and oocytes of pigs. Two distinctive bands (290 and 390 bp) were observed after 5′RACE, PCR, and electrophoresis of oocyte Dnmt1 cDNA. Only 1 band of 290 bp was observed after amplification of fibroblast cDNA. The location of exons and introns of every transcript variant was determined by aligning the 5′RACE-derived sequences to the Sus scrofa Dnmt1 genomic sequence located on chromosome 2 (CU462940). The smaller 290-bp band, amplified from both oocytes and fibroblasts, had an identical DNA sequence (EU908731). Structure analysis of the larger 390-bp band indicates that this oocyte Dnmt1 isoform has an additional exon located between exon 1 and 2 of the somatic form of Dnmt1 (EU908730). PCR products amplified using primers specific for the oocyte or somatic transcript verified the presence of the additional exon in the oocyte Dnmt1 splicing variant. In conclusion, this study shows that porcine oocytes express an alternative isoform of Dnmt1 in addition to the somatic transcript. The 2 identified isoforms are produced by alternative splicing of the Dnmt1 gene. Analysis of the oocyte and somatic Dnmt1 isoforms in pre-implantation embryos will determine the expression pattern of this transcript during genomic methylation and its involvement during nuclear reprogramming and cellular differentiation.
190 CELL CYCLE REGULATION IN RHESUS MONKEY OOCYTES AND EMBRYOS OF DIFFERENT DEVELOPMENTAL POTENTIAL
Authors: Mtango N. , Latham K.
Published: December 2008

After fertilization, cell division is required for development during the transition from a zygote to an embryo. Degradation of oocyte transcripts, transcriptional activation of the nucleus, and chromatin remodeling occur during early cleavage divisions. Defects in cell cycle regulation decrease the ability of embryo to grow and can be detrimental. In the rhesus monkey, embryos derived by fertilization of oocytes from prepubertal females or oocytes collected during the non-breeding season undergo cleavage arrest (Schramm and Bavister 1994; Zheng et al. 2001). We employed the Primate Embryo Gene Expression Resource (PREGER; www.Preger.org) to examine the expression pattern of 70 mRNAs involved in cell cycle regulation in rhesus monkey oocytes and embryos derived from different stimulation protocols (non-stimulated, FSH stimulated-in vitro matured, and FSH and hCG stimulated-in vivo matured; Mtango and Latham 2007, 2008; Zheng et al. 2005). The resource encompasses a large, biologically rich set of more than 170 samples with 1 to 4 oocytes or embryos which were constructed using the quantitative amplification and dot blotting method. This method entails the direct lysis of small numbers of oocytes or embryos in a reverse transcription buffer supplemented with nonionic detergent, thereby avoiding RNA losses associated with organic extractions (Brady and Iscove 1993). We find that aberrant regulation of cell cycle regulatory gene mRNAs is a prominent feature of oocytes and embryos of compromised developmental potential (FSH stimulated-moderate reduced potential and NS-severely compromised potential). Of the 56 mRNAs for which expression was detected, there was significant aberrations related to oocyte and embryo quality in the expression of more than half (n = 30), P < 0.05), 26 of 30 display significant differences in metaphase II stage oocytes, 20 being altered in FSH stimulated females and 24 of 30 being altered in NS females. The comparison between monkey and previously reported mouse array expression data (Zeng et al. 2004) revealed striking differences between 2 species. These data provide novel information about disruptions in the expression of genes controlling the cell cycle in oocytes and embryos of compromised developmental potential.

We thank Bela Patel, Malgorzata McMenamin, and Ann Marie Paprocki for their technical assistance. We also thank R. Dee Schramm for his contribution to the development of the PREGER resource. This work was supported by National Centers for Research Resources Grant RR-15253.