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The persistence of A-type nuclear lamin in somatic cell nuclear transfer (SCNT) embryos has been proposed as a marker for incomplete nuclear reprogramming. Using monoclonal antibodies to A/C- (A/C-346 and A/C-131C3) and B-type lamin, we compared distribution during early development of bovine IVF, parthenogenetic and SCNT embryos. A/C-346 staining was observed in the pronuclei of IVF embryos and in nuclei at the two-cell stage, but was not detected in subsequent cleavage stages up to and including hatched blastocysts. In contrast, A/C-131C3 and anti-lamin B2 stained all preimplantation stage embryos. Parthenogenetic and SCNT embryos had similar staining patterns to IVF embryos for all three antibodies, demonstrating correct nuclear architecture reprogramming. Inhibiting protein synthesis with cycloheximide (CHX) in parthenogenetic and SCNT embryos did not affect lamin A/C localisation, suggesting that lamin A/C is maternal in origin. However, activation with CHX delayed lamin A/C incorporation compared with 6-dimethylaminopurine activation. In SCNT embryos, staining for both A/C- and B-type lamin was delayed compared with parthenotes, although lamin B2 incorporation preceded lamin A/C in both. In conclusion, the lamin A/C distribution in SCNT bovine embryos paralleled that of IVF and parthenogenetic controls and therefore is not a marker of incomplete reprogramming. [ABSTRACT FROM AUTHOR]

This study was designed to evaluate possible mechanisms by which colony-stimulating factor 2 (CSF2) acts during Day 5 to 7 of development to improve embryonic and fetal survival and decrease fetal losses. One hypothesis was that CSF2 causes increased secretion of interferon-τ (IFNT) by the trophoblast of elongated conceptuses. Colony-stimulating factor 2 might also affect elongation of the trophoblast and improve survival of the embryonic disc (ED) because of evidence that treatment of embryo with CSF2 caused differential regulation of a large number of genes involved in developmental processes and a decrease in apoptosis. Bovine embryos were produced in vitroin the presence or absence of 10ngmL-1of CSF2 beginning at Day 5 after fertilization. Embryos were transferred at Day 7 to lactating dairy cows using a timed embryo transfer protocol (n=20 and 15 for control and CSF2, respectively). At Day 15, embryos were recovered and assessed for length, stage, and presence of an ED. The flushings recovered from the first 60mL were stored for antiviral activity analysis. A total of 4 filamentous conceptuses were divided into 1) the ED and a small amount of adjacent trophoblast and 2) tissue containing trophoblast only. These tissues were analysed for gene expression using the Bos taurusTwo Colour Microarray Chip from Agilent (Palo Alto, CA). Quantitative real time PCR analysis of 11 differentially expressed genes and 1 housekeeping gene (GAPDH) was performed to confirm the microarray results. In addition, quantitative real-time PCR of IFNT was performed in all recovered embryos (7 and 10 for control and CSF2, respectively). Results suggest that higher pregnancy rates at Day 30 represents increased embryonic survival at Day 15 (35% for controls v.66% for CSF2; P<0.07), a greater capacity of the embryo to elongate (39mm for controls v.62mm for CSF2) and secrete IFNT (mean IFNT IU=349074 for control and 1883060 for CSF2; P<0.07) at Day 15. In addition, the amount of IFNTmRNA was 22 times higher (P=0.06) in the CSF2-treated embryos. There was no difference in the presence of an ED between groups. Analysis of gene expression between the ED and trophoblast in filamentous embryos indicated no difference in transcription among this subset of embryos. However, more than 500 genes were identified as being preferentially expressed in the ED. These genes represent candidate markers for ED that should prove useful for studying the differentiation of the bovine conceptus through the periattachment period. In conclusion, results support the idea that the increased survival of embryos exposed to CSF2 from Day 5 to 7 of development is the result of increased embryonic survival before Day 15 and a greater capacity of the embryo to elongate and secrete IFNT at Day 15. The reduction in embryonic and fetal loss after Day 30 caused by CSF2 is probably not a direct reflection of altered gene expression at Day 15. USDA Grant 2009-65203-05732 and the Southeast Milk Dairy Checkoff Program. BL was supported by a CAPES/Fulbright fellowship. [ABSTRACT FROM AUTHOR]

The use of bovine embryo transfer to increase the reproductive potential of animals with high genetic merit is unquestionable. However, studies that evaluate the response to exogenous FSH stimulation in Red Sindhi cows are rare. Therefore, the objective of this study was to compare the superovulatory response of Red Sindhi cows treated with 3 different doses of FSH (Folltropin®-V, Bioniche Animal Health, Belleville, Ontario, Canada). The animals used had body condition scores ranging from 3.5 to 4.0 (1-5 scale), were between 6 and 10 years of age, and were randomly assigned to 1 of 3 treatment groups. Three doses of FSH were administered: 100 mg (n= 16); 133 mg (n= 13), and 200 mg (n= 14). The treatments were initiated on random days of the estrous cycle. On Day 0, animals received 2 mg of estradiol benzoate i.m. (Estrogin®, Farmavet, Jaboticabal, Brazil) and an intravaginal device (CIDR; InterAg, Hamilton, New Zealand) with 1.9 g of progesterone. On Day 4, donors received FSH in 8 i.m. injections that decreased in dose during 4 consecutive days. A dose of 265 μg of cloprostenol (Ciosin®, Schering-Plough, Cotia, Brazil) was given i.m. concomitantly with the fifth FSH injection. Twelve hours after the last FSH injection, donors received 25 μg of lecirelin i.m. (GnRH; Gestran Plus®, ARSA SRL, Buenos Aires, Argentina). TheAI were performed 12 and 24 h after GnRH. Seven days after the first AI, embryos were recovered and classified as described in the IETS manual. Data were evaluated using KruskalWallis test at a 5% level of significance. Ovarian ultrasonography was also performed to count the number of CL present at the time of ova/embryo collection. Results are summarized in Table 1. The number of CL and total ova/embryos was higher in donors receiving 200 mg of FSH than in those in the other groups. However, the number of transferable or cryopreserved embryos did not differ among groups. In conclusion, the dosage of 200 mg of Folltropin®-V seemed to be the more adequate dosage for this breed of cattle. Table 1.Effect of different follicle-stimulating hormone (FSH) doses on CL number, total structures, transferable embryos, and cryopreserved embryos in Red Sindhi cows (mean ± SEM)Financial support provided by FAPERJ (E26-170.529/2007). [ABSTRACT FROM AUTHOR]

Cytokines have several functions in many cells and their appropriate balance in the maternal-fetal connections is necessary to maintain a successful pregnancy. The incidence of fetal problems reported at the delivery moment in cows with conceptus conceived by in vitroproduction (IVP) and lack of literature in the immunology gestation area in bovine prompted this study. The aim of the present study was to determine the levels of cytokines such as tumor necrosis factor a (TNF-α), interferon-y (IFN-γ), interleukin-6 (IL-6), and IL-8 in amniotic fluid of calves from IVP and conventional embryo transfer at the moment of delivery. Forty animals were used and divided into 2 groups: 1 - Twenty Nelore cross-breed cows pregnant with Nelore calves conceived by conventional embryos transfer method (ET group); 2 - Twenty Nelore cross-breed cows pregnant with Nelore calves obtained by IVP after follicular aspiration (IVP group). Around labor, cows were transferred to a maternal paddock to permit observation of delivery. During the expulsion phase the amnion was punctured and 15 mL of fluid was collected in a plastic tube and stored in a freezer for later analysis. The cytokine levels were measured by immunoenzymatic assay (ELISA). The commercial kits used were ESS0011 (Bovine TNF Alpha), ESS0026 (Bovine IFN-gamma), and ESS0029 (Bovine IL-6) from Pierce Biotechnology™ (Rockford, IL, USA) and kit D8000C (Human IL-8) from R&D Systems™ (Minneapolis, MN, USA). The protocol was performed according to the manufacturer''s recommendations. The densities were evaluated by using an automatic optical reader ELISA. The well plates were read at 450 nm. The ANOVA on ranks was used for statistical analysis because of nonparametric distribution of data, with 5% of significance. No calves in this experiment showed signs of sepsis or death during the peripartum period. There was no significant difference in the concentration of TNF-α between the animals of the ET (250.40 pg mL-1) and IVP (398.70 pg mL-1) groups (P> 0.05). There was no difference statistically for IFN-γ; the medians of this cytokine were 35.7 pg mL-1for the ET group and 48.22 pg mL-1for the IVP group (P> 0.05). Only samples 15 and 18 of the IVP group had detectable concentrations of IL-6 (2227.47 and 2686.30 pg mL-1, respectively) and for IL-8 only 15 of the samples in the same group (577.6 pg mL-1). The results for TNF-α and INF-γ in this experiment can be adopted as standards for normal pregnancy in cattle, because there are no reports of such data in the literature. However, additional research in the area of gestation immunology area is necessary to describe the role of cytokines in the maintenance of pregnancy and at the moment of delivery in cattle. Supported by FAPESP, Brazil. [ABSTRACT FROM AUTHOR]

This experiment aimed to investigate the diestrus transcriptome dynamics of endometrium that resulted in calf delivery or no pregnancy after embryo transfer. Endometrium biopsies were collected from Simmental cyclic heifers at Days 7 and 14 of estrus cycle. On the next cycle, in vivo-produced Day 7 blastocysts were transferred to all animals at Day 7 of estrous cycle. Following pregnancy diagnosis, the endometrial biopsies collected at Day 7 and 14 were categorized based on the pregnancy success. Those endometrial biopsies collected from heifers that subsequently delivered a calf were assigned to the calf-delivery group, and those collected from heifers that did not conceive were assigned to the no-pregnancy group. The endometrial temporal transcriptome profile was compared between Days 7 and 14 in both heifer groups. Total RNA was isolated from each sample in triplicate. Two rounds of RNA amplification were performed using MEGAscript®T7 Kit (Ambion, Inc., Austin, TX, USA) and GeneChip®IVT Labeling Kit (Affymetrix, Inc., Santa Clara, CA, USA), respectively. Following fragmentation, biotin-labeled cRNA samples were hybridized to Affymetrix bovine gene chip array. The microarray data normalization and background correction were performed using GCRMA, and the differentially expressed genes (DEG) (fold change >2,P< 0.05, FDR < 0.3) were identified using LIMMA written on R package integrated with Bioconductor. The result showed that in the calf-delivery group, there were 1867 DEG, among which 1015 and 852 were up- and down-regulated, respectively, in Day 7 compared with Day 14 of the estrous cycle. Some of those genes are believed to be involved in reproductive system development and function (F3, PTGER2, PTGER4, MFGE8, PTGS2, and TDGF1), embryonic development (ALDH1A1,ALDH1A3, FGF2, TGFBR2, PDGFB, and TGFBR2), and nervous system development and function (CYP3A4, CYP3A4, HSD17B4, FOXA2, MET, TDGF, WNT11). The bioinformatic analysis using KEGG revealed that those DEG were classified into several pathways including the MAPK signaling pathway. On the other hand, in the no-pregnancy group, 254 genes were found to be differentially expressed, of which 160 and 94 were up- and down-regulated, respectively, in Day 7 compared with Day 14 of the estrous cycle. Some of these genes were found to be involved in signal transducer activity (AXIN2, AGTR1, MAPK10, NTRK2, TLR2, DMBT1, IL1RN, CDK5, CHRNE), transferase activity (DGKI, TXNDC6, RPS6KA5, RIOK3, MYLK, CDK5, MET, NTRK2), receptor activity (MET,AGTR1, NTRK2, TLR2, DMBT1, CHRNE), regulation of transcription (FOS, ELF1, BHLHB2,ATF3, HOXA11), signal transduction (TLR2, AGTR1, FCNB, DGK, NOTCH2, ADAM9, PLEK), and transcription regulator activity (BHLHB2, FOS, ELF1,ATF3, HOXA11). Those DEG were found to be involved in different pathways including the focal adhesion pathway. In conclusion, the result of the current study revealed a remarkable transcriptome dynamics between Days 7 and 14 of the estrous cycle in cows resulted in calf delivery compared with those that did not support pregnancy. [ABSTRACT FROM AUTHOR]

Bovine viral diarrhea virus (BVDV) has been shown to be associated with single transferable in vivo-derived and in vitro-produced bovine embryos despite washing. Hence, the primary objective of this study was to evaluate the potential of BVDV to be transmitted via the intrauterine route at the time of embryo transfer. A total of 10 in vivo-derived Day 7 bovine embryos were nonsurgically collected from a BVDV negative and seronegative donor cow. After collection, embryos were washed in accordance with the International Embryo Transfer Society (IETS) standards. Following washing, embryos were placed into transfer media containing BVDV (SD-1; type 1a). The embryos were immediately aspirated into 0.25-mL straws and transferred into seronegative recipients (Day 0). The total quantity of virus transferred into the uterus of each recipient was 900 to 1000 cell culture infective dose 50 (CCID50)/straw. This amount of virus was previously shown to be consistent with the average amount of BVDV associated with in vivo-derived and in vitro-produced embryos following standard IETS washing procedures after in vitroexposure to virus. The positive control heifer received 1.5 × 106CCID50/straw of BVDV without an embryo. The negative control heifer received 1.5 × 106CCID50/straw of heat-inactivated BVDV without an embryo. Serum and buffy coat samples were drawn from all heifers on Days 0, 3, 4, 6, 7, 8, 9, 10, 12, 15, and 30 after inoculation and analyzed for serum neutralizing antibodies and virus, respectively. The positive control heifer and all recipients of virus-exposed embryos exhibited viremia by Day 6 and seroconverted by Day 15. The negative control heifer did not exhibit viremia or seroconvert. All recipients receiving embryos were assessed for pregnancy using transrectal ultrasonography on Day 30 and 6 of 10 heifers were pregnant. On Day 60 the pregnant heifers were again assessed for pregnancy using transrectal ultrasonography. At this time only 1 of the 6 heifers was still pregnant. However, the fetus was determined to be nonviable and was removed via colpotomy. The fetus, fetal fluids and membranes were determined to be positive for BVDV via immunohistochemistry and PCR. Additionally, 213 base pairs of the 5′ nontranslated region of this PCR product were sequenced and found to be consistent with the inoculated strain. Results demonstrate that the average quantity of BVDV associated with bovine embryos after in vitroexposure and washing can result in viremia and seroconversion of seronegative recipients following transfer into the uterus during diestrus. [ABSTRACT FROM AUTHOR]

Increasing efficiency and success of MOET continues to be a goal of researchers and practitioners. Although numerous studies report success in establishing pregnancies, fewer evaluate term development and report number of live calves born. In a previous study, Scenna FN et al.(2008 Reprod. Fertil. Dev. 20, 154) exposed embryos to 3 different medium treatments while being collected from superovulated beef donors on Day 7. Medium treatments consisted of a commercially available medium plus 1 mL of DMSO (Control), commercial medium plus 100 nmof AL-8810 (AL100), or a commercial medium plus 1000 nmof AL-8810 (AL1000). Embryos were evaluated for grade and stage according to IETS guidelines. Embryos (n= 1734 at 6 locations across 13 replicates) were transferred (fresh or frozen in ethylene glycol) by 4 experienced technicians. Pregnancy rates were determined by ultrasonography 28 to 35 days after transfer and were increased in recipients receiving embryos collected in media containing AL100 (65%) and AL1000 (60%) compared with Control (50%; P 0.10); therefore, these groups were combined (AL) for subsequent analysis of PRR. Regardless of treatment, overall PRR were 88 ± 0.01% for fresh and 85 ± 0.03% for frozen embryos (P> 0.10). Additionally, gestation length (days) was similar between recipient animals receiving Control (282 ± 1.19), AL100 (281 ± 1.30) or AL1000 (281 ± 1.17; P> 0.10). However, addition of AL to collection medium of embryos increased PRR compared with Control (90 ± 0.03% v. 83 ± 0.03%, respectively; P= 0.06). Currently, an interaction was not noted between freezing method and media treatment (P> 0.10). Although collection of data continues, addition of a prostaglandin F2αreceptor antagonist to the collection medium of embryos improves PRR of recipient animals without altering gestation length. [ABSTRACT FROM AUTHOR]

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine that has been implicated in the regulation of preimplantation embryonic development in several species. Insulin-like growth factor-1 (IGF-1) can increase the proportion of bovine embryos that develop to the blastocyst stage and increase embryo survival following transfer to heat-stressed, lactating dairy cows (Block J et al.2003 J. Anim. Sci. 81, 1590–1602; Theriogenology 67, 1518–1529). The current objective was to test whether addition of GM-CSF and IGF-1 to culture medium enhances development and post-transfer survival of in vitro-produced bovine embryos. Holstein COC shipped overnight in a portable incubator in oocyte maturation medium were fertilized with X-chromosome selected sperm from Holstein bulls. X-selected sperm were used because females were preferred. Morulae and blastocysts were collected at Day 7 after insemination and transferred at Day 7 after ovulation to lactating dairy cows subjected to a modified OvSynch protocol. In Experiment 1, conducted from June 29 to August 31, embryos were cultured in KSOM-BE2 alone, KSOM-BE2 with 100 ng mL-1of Arg3-IGF-1 or KSOM-BE2 with 10 ng mL-1of recombinant BoGM-CSF. Treatments were added at Day 1 after insemination. As compared to control embryos (17 ± 2%), the percentage of cleaved embryos that became transferable morulae or blastocysts at Day 7 was increased (P< 0.05) by GM-CSF (25 ± 2%) but not by Arg3-IGF-1 (18 ± 2%). There was no significant effect of treatment on pregnancy rate at Day 30 to 35 [34% (n= 52), 35% (n= 51), and 43% (n= 55) for control, GM-CSF, and IGF-1, respectively] or calving rate (27, 35, and 40%) although values were numerically greater for cows receiving IGF-1 treated embryos. In experiment 2, conducted from September 7 to February 1, embryos were cultured in KSOM-BE2 alone, KSOM-BE2 with 100 ng mL-1Arg3-IGF-1 added at Day 1 after insemination, or KSOM-BE2 with 10 ng mL-1recombinant BoGM-CSF added at Day 5 after insemination. GM-CSF, but not IGF-1, increased the percentage of oocytes (P< 0.03) and the percentage of cleaved embryos (P= 0.05) that became transferable morulae or blastocysts at Day 7. The percentage of cleaved embryos becoming blastocysts was 14 ± 1% for GM-CSF, 14 ± 2% for Arg3-IGF-1 (P= 0.11), and 10 ± 1% for controls. Treatment with GM-CSF increased (P= 0.056) the percentage of cows pregnant at Day 30 to 35 [34% (n= 79), 43% (n= 107), and 27% (n= 44) for control, GM-CSF, and IGF-1, respectively]. Data on calving rate are currently being collected; to date, 86% of calves were female. Results indicate that embryo competence for post-transfer survival can be enhanced by treatment with GM-CSF at Day 5 after fertilization.Research supported by USDA Grant 2006-55203-17390, BARD Grant US-3986-07 and the Southeast Milk Dairy Checkoff Program. B.L. was supported by a CAPES (Brazil)/Fulbright Fellowship. [ABSTRACT FROM AUTHOR]

The Ovsynch system (Pursley et al. 1995 Theriogenology 44, 915–923) has been effectively employed as the synchronization method of recipients for embryo transfer (ET) (Nishisouzu et al. 2004 Reprod. Fertil. Dev. 16, 212–213). To establish the most effective Ovsynch system, we experimentally assessed 3 different protocols by comparing resultant estrus-synchronization rates of Holstein heifers and pregnancy rates of the recipients after ET. Holstein heifers averaging 14–15 months old and weighing over 330 kg were used in this study. They were randomly separated into the following 3 groups: (1) Select-synch (SS) group: an intramuscular (IM) injection of 50 g GnRH analog was given to heifers at a random stage of the estrus cycle, followed 7 days later by 2 IM injections of 7.5 mg PG analog at an interval of 6 h; (2) Co-synch (CS) group: another IM injection of 50 g GnRH was given to the SS group at 48 h after the first injection of PG; and (3) Heat-synch (HS) group: an IM injection of 0.75 mg estradiol benzoate was given to the SS group at 24 h after the first injection of PG. Estrus detection was evaluated twice daily by visual observation and use of a Heat Mount Detector. All heifers were additionally injected with 1500 IU hCG 5 days after estrus, to ensure favorable luteal function (Nishigai et al. 2001 J. Reprod. Dev. 47, 283–294). Rectal palpation was used to select acceptable recipients, i.e. those having functional corpora lutea (CL) e17 mm in diameter for 7 days after showing estrus (Nishisouzu et al. 2004 Reprod. Fertil. Dev. 16, 212–213). The size of CL e17 mm was determined by rectal palpation by skillful transferors. Fresh embryos collected from super-ovulated cattle 7 days after estrus were nonsurgically transferred only into acceptable recipients that had functional CL and were properly synchronized, 7–8 days after estrus. Final diagnosis of pregnancy was carried out 60 days after ET. Data were analyzed by the chi-square test. The HS group showed significantly higher (P < 0.01) rates of estrus synchronization (87/89: 98%) at 24–72 h after PG injection, estrus detection (98%), and acceptable suitability as recipients with a functional CL (92%) compared to those of the SS (185/210: 88%, 87%, and 73%, respectively) and CS (68/81: 84%, 74%, and 69%, respectively) groups. The pregnancy rates of heifers that received an embryo were not significantly different among HS, SS, and CS groups (45/82: 55%, 78/153: 51%, and 25/56: 45%, respectively). However, because the yield rate of acceptable recipients in the HS group was higher than in the other groups, the final pregnancy rates (no. pregnant/no. synchronization treatment) of the HS group (50%) was significantly higher than those of the SS and CS groups (37%; P < 0.05, and 31%; P < 0.01, respectively). Taken together, these findings demonstrate that the HS protocol is effective for estrus–ovulation synchronization of Holstein heifers, thus improving the productivity of ET by increasing the yield rates of heifers with a functional CL which leads to acceptable pregnancy results. [ABSTRACT FROM AUTHOR]

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

Recent studies have shown that the presence of accessory or multiple corpora lutea (CL) and increased progesterone (P4) concentrations reduced early embryonic mortality in cattle. The objective of this study was to evaluate the effect of equine chorionic gonadotropin (eCG) treatment on the number of CL, the P4 concentrations, and pregnancy rates after embryo transfer (ET). Holstein heifers (n ? 120) from 7 dairy farms received an intravaginal progesterone-releasing device (CIDR; InterAg, Hamilton, New Zealand) and 2 mg IM of estradiol benzoate (EB; Gynandol; Sankyo, Tokyo, Japan) at random stages of the estrous cycle. After 7 to 9 days, CIDRs were removed and 15 mg of prostaglandin F2? (PG; Pronalgon; Pfizer Japan, Nagoya, Japan) were administered, followed by 100 g IM GnRH (Conceral; Takeda Pharmaceutical Co., Ltd., Osaka, Japan) 2 days later (Day 0). The heifers were placed at random into 3 groups for eCG treatment. The eCG was not administered in a control group (n ? 53); heifers in other 2 groups received 1000 IU eCG (Peamex; Sankyo, Japan) IM at the time (0 h group, n ? 37) or 48 h before (48 h group, n ? 30) PG injection/CIDR removal. On Day 7, heifers were examined by ultrasonography (Aloka SSD500; Aloka, Tokyo, Japan) for number of CL; heifers with at least one functional CL received an in vivo-derived frozen?thawed embryo by direct transfer. At the same time, a blood sample was collected to determine P4 concentration. Pregnancy rates were determined on Days 30 and 60 by ultrasonography and rectal palpation, respectively. The data were analyzed by ANOVA and means were compared with Fisher''s PLSD. Proportional data were analyzed by the chi-square test. P4 concentrations (mean SD) on Day 7 were 1.8 1.0, 5.6 3.3, and 2.2 1.1 ng mL-1 for the control, 48 h, and 0 h groups, respectively (48 h vs. control and 0 h; P < 0.001). The number of CL on Day 7 were 1.1 0.4, 2.5 1.4, and 1.8 0.9 for the control, 48 h, and 0 h groups, respectively (control vs. 48 h and 0 h, and 48 h vs. 0 h; P < 0.01). Pregnancy rates did not differ between 0 and 48 h groups but both were higher than in the control group (Table 1). Results suggest that the estrus synchronization protocol with administration of eCG at the time of CIDR removal or 48 h earlier significantly increased the number of CL and the P4 concentration, and improved pregnancy rates in Holstein heifers after ET. [ABSTRACT FROM AUTHOR]

Cattle embryo transfer (ET) involves the transfer of Day 7 embryos. Recent advances in molecular techniques mean that these techniques are employed in combination with ET. One example is the use of PCR to determine the sex of embryos, which requires the biopsying of blastomeres. This process, however, can compromise embryo development. The identification of embryonic markers to predict whether a pregnancy will survive to term also remains elusive. In cattle, interferon (IFN)-tau is well established as the principle factor for the maintenance of pregnancy. The transfer of Day 7 embryos a time at which minimal IFN-tau production is occurring means that using IFN-tau as a predictor of pregnancy success is limited. Compared to Day 7 embryos, Day 13 embryos are larger, with an increased number of cells that would reduce the risks of biopsy and the measurement of IFN-tau production. Although these characters are beneficial to ET, there are few reports of ET using embryos at this stage. Hence, the aims of our study were to compare the success of pregnancy maintenance after ET of Days 7 and 13 bovine embryos and determine whether IFN-tau production by Day 13 embryos prior to ET could be used as a marker to predict survival to term. Japanese Black cows (n = 10) were superovulated and inseminated (Day 0). On Days 7 (n = 5) and 13 (n = 5), embryos were nonsurgically recovered. Good quality Day 7 embryos were selected and frozen by the conventional slow freezing method until transfer. Day 13 embryos were collected, and their dimensions were recorded; they were subsequently cultured individually for 1 h in TCM-199 with 10% FBS (100 L). The amount of IFN-tau in the medium was determined by antiviral activity assay using the NIH international standard. Frozen–thawed Day 7 and fresh Day 13 embryos were transferred into synchronized recipient cows (D7ET: n = 39, D13ET: n = 37). Following ET, estrus was monitored twice daily. Ultrasound diagnosis was performed on Days 30, 40, 50, 60, 70, and 80 post-estrus. All data were analyzed by Student''s t-test. In total, 20 cows in the D7ET group and 18 cows in the D13ET group went to term. In the animals that did not maintain pregnancy, the percentage of cows that returned to estrus before Day 25 was significantly lower in the D13ET group than in the D7ET group (16.2% vs. 38.5%; P < 0.05). However, the number of cows that returned to estrus after Day 30 post-ET was significantly higher in the D13ET than the D7ET group (27.0% vs. 2.6%; P < 0.05). In the D13ET group, of the 10 cows that did not return to estrus by Day 50, half had no fetus present but fetal membranes were evident. No significant difference in IFN-tau production or the length of Day 13 embryos was determined between those that went to term and those that did not survive (432 ± 154 vs. 1448 ± 1045 pg/embryo, P = 0.36; 1.65 ± 0.34 vs. 1.40 ± 0.17 mm, P = 0.55, respectively). In conclusion, the use of Day 13 vs. Day 7 embryos in ET did not improve the pregnancy rate to term although it helped avoid early embryo death, and there was no relationship between IFN-tau production of Day 13 embryos and their pregnancy success. [ABSTRACT FROM AUTHOR]

An experiment was designed to evaluate the effect of the interval between thawing to deposition of the embryo into the uterine horn on pregnancy rates of in vivo-produced frozen–thawed embryos in 1.5 M ethylene glycol (direct transfer). Data were collected from 1122 embryo transfers performed in the same farm (Estancia El Mangrullo, Lavalle, Santiago del Estero, Argentina) during the spring and summer of 2004/05 and 2005/06 (6 replicates, ambient temperature between 20 and 40C). Recipients used in all replicates were non-lactating, cycling, multiparous Bos taurusBos indicus crossbred cows with body condition score between 3 and 4 (1 to 5 scale) that were synchronized using fixed-time embryo transfer protocols. Briefly, the synchronization treatments consisted of the insertion of a Crestar ear implant (Intervet, Sao Paulo, Brazil) or a progesterone-releasing device (DIB; Syntex SA, Buenos Aires, Argentina), plus 2 mg of estradiol benzoate (EB; Syntex) intramuscularly (IM) on Day 0, and 400 IU of eCG (Folligon 5000; Intervet, or Novormon 5000; Syntex) IM plus 150 g d-cloprostenol IM (Preloban; Intervet, or Ciclase; Syntex) on Day 5. Progestin devices were removed on Day 8 and all cows received 1 mg of EB IM on Day 9. All cows were examined by ultrasonography on Day 16 and those with a luteal area >76 mm2 (by calculating the area of the CL minus the area of the cavity) received, on Day 17, frozen–thawed embryos by nonsurgical transfer. All embryos were Grade 1, and all were frozen in 1.5 M ethylene glycol at the Embryo Plus Laboratory (Brits, South Africa). After being stored in liquid nitrogen, the embryos were plunged directly (no air thawing) in a 30C water bath for 30 s, and then transferred to the recipient cows by either one of two technicians. Based on the interval between thawing and transfer, the transfers were classified as being in one of 3 groups: Group 1: <3 min; Group 2: 3 to 6 min; and Group 3: 6 to 16 min. The main reason for delayed transfers beyond 6 min was the replacement of one recipient for another because of difficulty in threading the cervix (1% of the total transfers) or a recipient falling down into the chute or with very bad disposition and behavior. Pregnancy was determined by ultrasonography 28 to 35 days after fixed-time embryo transfer, and data were analyzed by logistic regression. There were no effects of replicate, technician, CL area, recipient body condition score, embryo stage, and time from thawing to transfer on pregnancy rates. Pregnancy rates in the 3 thawing to transfer intervals were: Group 1: 215/385, 55.8%; Group 2: 372/655, 56.8%; Group 3: 42/82, 51.2%; P > 0.6. These results may be interpreted to suggest that there is no significant effect of time from thawing to transfer (up to 16 min) in direct transfer embryos using Bos taurusBos indicus recipients transferred at a fixed time. [ABSTRACT FROM AUTHOR]

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

Two experiments were conducted to determine the effects of the addition of a progestin, equine chorionic gonadotropin (eCG), or both, in a GnRH-based protocol for timed AI (TAI) or timed embryo transfer (TET). In both experiments, Angus, Gelbvieh, and Simmental cross-bred cows were randomized by breed and postpartum interval [50 10 days (mean SD); range, 27 to 89] into 4 groups in a 2 2 factorial design. All injections were given IM. In Experiment 1, 288 cows (89.6% cycling) were given 25 mg dinoprost (PGF; Lutalyse; Pfizer Animal Health, Montreal, Quebec, Canada) on Day –11; on Day 0, they were given 100 g GnRH (Cystorelin; Merial Canada, Pointe-Claire, Quebec, Canada), with or without concomitant insertion of a CIDR (1.9 g progesterone; Pfizer Animal Health, Montreal, Quebec, Canada). On Day 7, CIDR inserts were removed and cows were given PGF, with or without concomitant injection of 400 IU of eCG (Pregnecol; Bioniche Animal Health, Belleville, Ontario, Canada). On Day 9 (54-56 h after PGF), TAI was done, with concomitant injection of 100 g GnRH. Ultrasonographic examination of 147 cows on Day 7 revealed that 62.4% had ovulated in response to the first GnRH. Pregnancy rates (ultrasonographic examination) on Day 38 did not differ between cows with or without a CIDR (52.9 and 51.4%, rspectively; P ? 0.64), with or without eCG treatment (53.5 and 50.7%, respectively; P ? 0.28), in cycling vs. anestrous cows (51.6 vs 56.7%, respectively; P ? 0.76), and in cows that had ovulated (58.1%) or did not ovulate (50.0%) after the first GnRH treatment (P ? 0.58). In Experiment 2, 151 cows were given 500 g cloprostenol (PGF; Estrumate; Schering–Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day –12, 100 g GnRH on Day 0, with or without concomitant insertion of a CIDR. On Day 3, half of the cows were given 400 IU eCG. On Day 7, CIDRs were removed and cows were given PGF; on Day 9 (54–56 h after PGF), all cows were given 100 g GnRH. On Day 15, ultrasonography was done to select suitable recipients for transfer of frozen–thawed embryos on Day 16 (part of another experiment, balanced across synchronization groups). Recipient selection rates did not differ whether cows received or did not receive a CIDR (93.4% vs 85.5%, respectively; P ? 0.27) or eCG (91.0 vs 87.8%, respectively; P ? 0.67). In addition, pregnancy rates on Day 43 did not differ whether cows received or did not receive a CIDR (32.3 vs 32.4%, respectively; P ? 0.52) or eCG (35.2 and 29.2%, respectively; P ? 0.21). In summary, the addition of a CIDR or eCG to a GnRH-based synchronization protocol initiated after PGF presynchronization in lactating beef cattle yielded no improvement in pregnancy rates following TAI, or recipient selection and pregnancy rates following TET. [ABSTRACT FROM AUTHOR]

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

An abstract of the study "Implantation of Transgenic Bovine Cloned Embryos Derived From Transfected Cells by PiggyBac Transposition," by W. J. Choi et al, is presented.

Bluetongue virus (BTV) has been categorized by the OIE as a category 1 disease agent, for which proper handling between collection and transfer is thought to be sufficient to prevent transmission through embryo transfer. For bovine viral diarrhoea virus, it was shown that effectiveness of washing procedures depends on virus strains (Waldrop et al. 2004 Theriogenology 62, 45-55). Also BTV-8 has unique characteristics in comparison with other strains (De Clercq et al. 2008 Transbound. Emerg. Dis. 55, 352-359). The aim here was to investigate whether embryo transfer of in vivo-derived bovine embryos after in vitro exposure to BTV-8 can be performed without risk for infection of the recipients if IETS washing and trypsin treatment procedures are followed. Donor cows (n = 2) were synchronized and superovulated using Stimufol® (Ulg, Liége, Belgium) and subsequently inseminated. At 6.5 days post-insemination (dpi), flushed embryos (n = 14 and n = 3) were placed in 800 μL of minimal essential medium (MEM), containing 104.9 50% tissue culture infectious doses (TCID50) of BTV-8 (Bel 2006/2 P5, VAR, Brussels, Belgium) and incubated for 1 h at 39°C in 5% CO2 in air (Vandaele et al. 2011 Vet. Res. 42, 14-21). Next, embryos were washed in pairs in 5 consecutive Petri dishes containing PBS with antibiotics and 0.4% BSA, w/o Ca and Mg. Then, embryos were exposed to 2 consecutive trypsin (Invitrogen, Carlsbad, CA, 25050-014) washes of 45 s each at 39°C in 5% CO2 in air and finally, another 5 consecutive washes in PBS with 2% FCS. Each Petri dish contained at least 2 mL of medium and was gently agitated between washes. Embryos were transferred in a maximum of 7 μL of medium and a new tip was used after every wash step. Washes 1 to 5 and washes 6 to 10 were pooled and analysed for BTV-8 (RT-qPCR). After these washes, 3 pairs of embryos (n = 6) were loaded in straws and transferred to 3 BTV-8 negative recipients. Two sentinel cows served as control. Cows were bled twice weekly and blood and serum samples were analysed for BTV-8 (RT-qPCR) and BTV-8 antibodies. Viral BTV-RNA was detected in all 3 recipient cows at 7 days after transfer and viraemia was confirmed by the establishment of high antibody titers at 14 days after transfer. Viral BTV-RNA was detected in washes 1 to 5 for each pair of embryos (Cp-value around 29), whereas washes 6 to 10 had Cp-values around the cut-off value (40), indicating that probably the last wash was BTV-8 negative. None of the recipients was pregnant at 28 days post-transfer. In conclusion, washing and trypsin treatment did not succeed in removing BTV-8 from in vitro-spiked in vivo-derived bovine embryos. These unexpected results stress the need for further in vivo research, e.g. what is the virus load in vivo embryos may be exposed to in utero during viraemia? Does BTV-8 react differently with the zona compared with other strains? Are alternative washing procedures needed to remove BTV-8 from the zona? [ABSTRACT FROM AUTHOR]

Annually, more than 400 000 in vivo-recovered bovine embryos are officially reported by members of the Canadian and American Embryo Transfer Associations. Between 65 and 70% of these embryos are cryopreserved and more than 95% are frozen in ethylene glycol (EG). Statistics on factors affecting embryo freezing are difficult to obtain because many cattle breeders/farmers no longer report pregnancy rates back to embryo transfer (ET) practitioners. Concerns are often expressed as to the optimal stage at which to freeze bovine in vivo-derived embryos. This is a retrospective analysis of results from 5 commercial ET programs (1 in the United States, 3 in Canada and 1 in the Netherlands) for which pregnancy data relative to embryo stage at freezing were made available. Embryos representing 4 stages of development, as defined by the IETS (4 = late morula, 5 = early blastocyst, 6 = mid blastocyst and 7 = expanded blastocyst) are included in the data. The number of embryos thawed and transferred ranged from 3954 to 24 827 for the 5 programs, with a total of 72 828. Embryos were frozen in either 1.5 M EG or 1.5 M EG + 0.1 M sucrose and exposure time to cryoprotectant before cooling ranged from 4 to 40 min. Pregnancy rates are shown in Table 1. Although the pregnancy rate for stage 6 embryos was only 2.6 and 3.2 percentage points lower than stages 4 and 5, respectively, these differences were highly significant and pregnancy rates for stage 6 embryos were lower than those for stages 4 and 5 in 4 of the 5 ET programs. The small decreased survival of stage 6 embryos is probably only moderately important in a commercial context. However, the pregnancy rate of stage 7 embryos was lower than all other stages for the combined dataset as well as in all 5 ET programs, with the difference between stages 5 and 7 ranging from 6.5 to 16.4 percentage points. Clearly, stage 7 embryos survive freezing at a significantly lower rate than stages 4, 5 and 6 and neither time of exposure to EG nor inclusion of sucrose in the freezing medium provided an obvious improvement. Although bovine ET practitioners routinely attempt to collect embryos on day 7 post-oestrus, recovery of stage 7 embryos cannot always be avoided. Further investigation into factors contributing to the decreased survival of stage 7 embryos is warranted. Table 1. Effect of embryo stage on pregnancy rate of bovine embryos frozen in EG [ABSTRACT FROM AUTHOR]

In conventional in vitroembryo production, oocytes are obtained from various ovaries, and donor characteristics, such as pedigree type, meat quality, or meat quantity, are not considered. The aim of this study was to examine the effects of pedigree type and meat quality and quantity of slaughterhouse donor cows on oocyte recovery, embryo development, and pregnancy rates. The ovaries of individual Korean native cows were obtained from a slaughterhouse. The ear tag numbers were obtained 24h after slaughter, and the donor data were compared according to the pedigree type (no registration to third bloodline registration), meat quantity according to the percentage of carcass weight (more than 69%, 66 to 69%, and below 66%; Grades A, B, and C, respectively), and meat quality according to the marbling grade (marbling grade 8 to 9, 6 to 7, 4 to 5, 2 to 3, and 1; Grades 1++, 1+, 1, 2, and 3, respectively). There were 390 donors in total, and each group had 30 donors. Recipients (Holstein heifers; n=222) were synchronized with a progesterone device, and a single blastocyst was transferred nonsurgically on Day 7 from the onset of oestrus. Data on embryo development were compared by Duncan’s multiple range tests, and pregnancy rate was analysed by chi-square test. Values of P<0.05 were considered to indicate a significant difference. No significant difference was detected in the average number of oocytes recovered (17.5±2.9 to 22.9±1.2%) or blastocyst development (16.4±2.6 to 19.5±3.4%) from cows of different pedigrees. A higher number of oocytes were recovered from Grade A (22.9±1.0) than from Grade B (19.8±0.7; P<0.05) cows. However, the meat quantity grade had no significant effect on blastocyst development (14.3±1.2 to 15.0±0.8%). A greater number of oocytes were recovered from the Grade 1++(25.0±1.6), 1+(27.7±2.4), and 2 (23.6±1.5) groups than from the Grade 3 group (19.7±1.0; P<0.05). The developmental rate to the blastocyst stage was significantly higher in the Grades 2 and 3 groups (22.1±2.2 and 19.4±1.7) than in the Grade 1++, 1+, and 1 groups (9.0±1.2 to 13.8±2.0). Pregnancy rate was not affected by pedigree type, meat quantity, or meat quality (41.7 to 58.6%). We concluded that 1) pedigree type had no effect on oocyte recovery and embryo development; 2) meat quantity affected oocyte recovery but did not affect embryo development; 3) high meat quality (Grades 1++and 1+) resulted in greater oocyte recovery but lower embryo development; and 4) pregnancy rate remained unaffected by pedigree type, meat quantity, and meat quality. This research was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. [ABSTRACT FROM AUTHOR]

Brazil is a leading country in the world in commercial use of in vitro-produced bovine embryos, with approximately 200000 transfers per year (IETS; Thibier, 2009). This model of large-scale commercial in vitrobovine embryo production is now available for Panamanian producers. Because of the tropical environment in Panama, the most popular breed is the Brahman, a Zebu type of cattle that has been shown to have more follicles emerging per follicular wave than Bos taurustype of cattle and, consequently, that produce more oocytes per session of follicular aspiration. This characteristic, added to the embryo production results, permits such a biotechnology to be implemented on a commercial scale and incorporated into the reproduction management of a herd. A comparison of oocyte number and quality, cleavage, and embryo production was made using the same in vitroproduction system (InVitro Brazil, Mogi Mirim, São Paulo, Brazil) for Brahman donors, both in Brazil and in Panama. Data were compared using a z-test analysis (Table 1). The percentage of cleaved zygotes was greater (P<0.001) with the Panamanian Brahman donors as compared with the Brazilian Brahman donors (73 v.69%, respectively). However, the percentage of blastocysts/cleaved zygotes was greater (P<0.01), indicating a higher blastocyst production rate from the Brazilian donors. No other differences were observed. Thus, in vitroembryo production with Brahman donors could be used as a tool to improve and spread superior genetics within a Panamanian herd and could also serve as a model for other Central American and Caribbean countries under similar management systems. Table 1.Panama and Brazil in vitroBrahman embryo production1This work was supported by BORN Animal Biotechnology, Panama City, Panama. [ABSTRACT FROM AUTHOR]

Keeping ovaries for a long time before oocyte recovery affects the maturation rate and blastocyst rate (BL rate), but a change in the temperature of the ovary at the time of transportation does not affect the BL rate (Ribeiro et al.2008 Anim. Reprod. Sci. 108, 171–179). The objective of this study was to investigate the effect of long-distance transportation of ovaries on the in vitrodevelopment of bovine oocytes into blastocysts. Ovaries in the transportation group (Group T) were collected from a slaughterhouse, placed inside a Thermos flask, and transported to the laboratory within 17 to 21h. The Thermos flask was covered with a freezer pack in a foam polystyrene box (Nakatate et al.2006 Reprod. Fertil. Dev. 18, 193). The temperature of the Thermos flask was measured with a temperature recorder. Cumulus–oocyte complexes (COC) were collected by aspirating 2- to 6-mm follicles from the ovaries. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02AUmL-1of FSH. Groups of 20 COC were incubated in 100-μL drops of IVM media at 38.5°C under an atmosphere of 5% CO2in air for 20h. After 18h of gamete co-culture (3×106spermmL-1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48h after IVF (total cleavage rates, CL rate) and on Days 7 to 9 (BL rate). The ovaries in the control group (Group C) were collected from a slaughterhouse different from that of the Group T ovaries and transported to the laboratory in saline (23°C) in a Thermos flask within 3h. These ovaries were then used for the collection of COC in the same way as for Group T. Data were analysed by chi-square test. A total of 25 experiments were performed using the ovaries from Groups C and T (n=3945 v.n=7218). The CL rate and the BL rate for Group T were significantly lower than those for Group C (41.8 v.62.0% and 16.1 v.29.7%; P<0.01). In Group T, the duration of ovary transportation did not affect the CL rate (within 19h, between 19 and 20h, and more than 20h), but the BL rate was significantly decreased with an increase in transport time (18.7 v.16.1 v.14.8%). The temperature change in the Thermos flask during transportation ranged from 12.0 to 25.1°C; the temperature was maintained virtually constant or decreased. The CL rates of the groups that underwent a temperature (t) change (t≤5.0 and 5.0

During pre-implantation embryonic development in mammals, extensive epigenetic reprogramming occurs; patterns of DNA methylation are erased during the first cell divisions and are restored at the 8- to 16-cell stage (Dean et al.2001). An important step during this developmental phase, which is controlled by epigenetic factors such as DNA methylation, is inactivation of the X chromosome (XCI). It is established in the morula stage, where the paternal X is inactivated, with immediate reactivation in the blastocyst (Ferreira et al.2010). The aim of this study was to evaluate the mRNA expression profile of the DNMT3a and DNMT3b genes, which are related to DNA methylation, and the G6PD and PGK1 genes, which are related to glucose metabolism and are subject to XCI. Cumulus–oocyte complexes (COC) were isolated from slaughterhouse ovaries from Bos taurus indicusNellore cows. Oocytes were matured in vitroand inseminated with X-sorted sperm from a Holstein bull. Embryos of each developmental stage [4-cell (44hpi), 8- to 16-cell (72hpi), morula (144hpi), blastocyst (156hpi), and expanded blastocyst (168hpi)] were produced. Three pools of 17 embryos for each stage were frozen at –80°C until RNA extraction. Total RNA was isolated using the Invisorb RNA Spin Cell Mini Kit (Invitek, Berlin, Germany) according to the manufacturer’s protocol. Complementary DNA was done using Oligo dT primers (Invitrogen, São Paulo, Brazil) and SuperScript III reverse transcriptase (Invitrogen). Relative expression of target genes was assessed by quantitative PCR using a Power Sybr®Green PCR Master Mix (Applied Biosystems, Foster City, CA) in an ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems). The cyclophilin-A gene was used as an endogenous control. To compare gene expression between treatments, we used the ΔΔCtmethod. The identity of PCR products was confirmed by the amplicon size in agarose gel and by the melt curve in quantitative PCR. Gene quantification was compared among treatments using one-way ANOVA and Tukey’s test, in the Prophet program, version 5.0 (1996, BBN Systems and Technologies, Cambridge, MA). The 4 genes showed higher expression (P≤0.05) in 4-cell stage embryos than in morulae, blastocysts, or expanded blastocysts. The 8- to 16-cell embryos showed no differences for any gene compared with other embryos, except for the DNMT3b gene, which was not tested in these embryos. We conclude that these genes have a greater quantity of mRNA molecules because of the presence of maternal mRNA and that this stock was being spent during embryo development until the expanded blastocyst stage. Financial support was provided by Embrapa and CNPq. [ABSTRACT FROM AUTHOR]

Two experiments were conducted to compare conception rates following embryo transfer (ET) of bovine embryos held and transported in Syngro®holding medium (Bioniche, Belleville, Ontario, Canada) with other 2 holding media: Emcare®(ICPbio, Auckland, New Zealand) for in vivo-derived embryos and HEPES-buffered synthetic oviduct fluid (H-SOF) for IVF-derived embryos. The first trial was performed in the period from October through December 2006 at the Curitiba farm in Poços de Caldas, Minas Gerais, Brazil. A total of 140 in vivo-derived embryos were produced from 20 Nelore donor cows and transferred fresh at the same farm. After each donor recovery, embryos were equally separated per stage (morula or blastocyst) and classification (grades 1, 2, and 3) into 2 Petri dishes, each containing either Syngro or Emcare. The embryos were held for an average of 3h after recovery, loaded into 0.25-mL straws, and transferred fresh into recipients heifers, which were all previously synchronized with the same hormonal protocol treatment and presented a corpus luteum on the day of transference. Conception rate was checked at approximately 60 days of conception by rectal palpation. The chi-square test was used for statistical analysis. The conception rate of embryos maintained in Syngro was significantly higher than those in Emcare: 64.2% (43/67) v.47.9% (35/73; P<0.05). A second experiment was performed between September and December 2008 at Embriza Biotechnology Laboratory, Campo Grande, Mato Grosso do Sul, Brazil. A total of 1689 IVF-derived embryos (stage=7, quality=1), produced from Nelore donor cows, were randomly assigned to be held and transported in either Syngro (769) or H-SOF transport medium (920). Transportation time ranged from 1 to 9h, and the recipient farms ranged from 100 to 1200km in distance from the Embriza Laboratory. Crossbred recipient heifers (Bos taurus×Bos indicus) were synchronized with prostaglandin or vaginal progesterone device protocols. Pregnancy diagnosis was performed by ultrasonography approximately 60 days after ET. Statistical comparisons were performed using the chi-square test. Conception rates resulting from embryos transported in Syngro (45.1%, 347/769) and in H-SOF (42.0%, 386/920) were not different (P=0.19). Financial support from Embriza Biotecnology, Tecnopec LTDA, and Bioniche Animal Health [ABSTRACT FROM AUTHOR]

In cattle, a low pregnancy rate directly affects calf and milk production and brings about economic loss to livestock producers. The number of repeat breeder cows in herds decreases the overall pregnancy rate. Therefore, many studies have focused on improving the cows’ reproductive performance. On the other hand, it has been demonstrated that the major embryonic loss occurs in the first or second week of pregnancy, after which mother cows recognize the presence of their embryos via a signal protein secreted by the embryos, interferon-τ. Therefore, to bypass the period with a high incidence of pregnancy loss in cattle, we used elongating embryos for embryo transfer (ET) to repeat breeder cows. Elongating embryos on Day 14 postoestrus were used for the present study because embryos of this stage have the ability to produce interferon-τ and embryos of later stages are easily damaged when they are collected from the uterus by flushing. In this study, Japanese Black cows were superovulated and inseminated when they showed standing oestrus. Elongating embryos were collected by nonsurgical uterine flushing. Collected embryos that had evident embryonic discs and intact trophectoderms were used for ET. These embryos were transferred, using plastic catheters or ET guns to ipsilateral uterine horns, to the corpus luteum of synchronized crossbred (Japanese Black×Holstein-Friesian) recipient cows. Recipient cows were divided into 2 groups according to the previous reproductive history of each cow: reproductively normal cows that were pregnant with fewer than 3 times of AI or regular ET (using Day 7 embryos) after every parturition (n=33) and repeat breeder (RB) cows (n=42). Elongating embryos were transfer on Day 14 after oestrus, and all cows were monitored for signs of standing oestrus twice a day until Day 80. Pregnancy was diagnosed with ultrasonography every 10 days from Day 30 to 80 postoestrus. The data were analysed by Fisher’s exact test. The pregnancy rate of reproductively normal cows was 54.5%. However, in repeat breeder cows, the pregnancy rate was significantly decreased (2.4%; P<0.01). When the repeat breeder cows did not become pregnant, transfer of the elongating embryos was repeated another 5 times continuously and the incidence of a return of oestrus was monitored daily. At the first ET period, the average interestrus interval in nonpregnant repeat breeder cows was 50±7.2 days. As the ET period proceeded, the interestrus intervals were gradually decreased, and at a sixth ET period, it approximated the normal oestrus cycle length (22.8±0.8). These results indicated that although transfer of elongating embryos could bypass the critical period of high incidence of embryo loss, it did not improve the pregnancy rate of repeat breeder cows. Moreover, it suggests that the uterine environment of repeat breeder cows was gradually deteriorating in a time-dependent manner. [ABSTRACT FROM AUTHOR]

A serious decline in the reproductive performance of dairy cows occurs in southern Japan in the summer period, when the total number of hot days ≥35°C numbers more than 20 days annually. Previous reports have mentioned the effectiveness of embryo transfer (ET) at 7 days after AI (AI/ET) under heat-stressed conditions. In the present study, we investigated the effect of AI/ET on conception rate (CR) under heat-stressed conditions in the summer period. Artificial insemination was performed at 13 commercial dairies in this study from August through September in 2007 and 2008. Seven days after AI, a single embryo was transferred into the uterine horn contralateral to the ovary with a corpus luteum (AI/ET, n=82). Artificial insemination at oestrus without further treatment was assigned as the control group (AI, n=367). In 2007, frozen–thawed embryos of Japanese Black cattle were transferred, and the same cattle were used for ET of fresh embryos in 2008. The temperature-humidity index [0.8×temperature+0.01×relative humidity (temperature –14.4)+46.4], rectal temperature, and diurnal highest or lowest and average ambient temperatures were measured at the time of AI and ET. Cows were diagnosed for pregnancy at 42 days after AI by palpation per rectum and were reexamined by transrectal ultrasonography at 60 days after AI. The CR was calculated as the number of cows diagnosed as pregnant 60 days after AI divided by the number of cows inseminated. Fetal loss was calculated as the number of cows that did not deliver calves after term divided by the number of cows diagnosed as pregnant. The CR, number of AI, fetal loss, and type of newborn (Holsteins, AI origin; Japanese Black, ET origin) were confirmed retrospectively. For statistical analysis, Fisher’s exact test and Student’s t-test were used for comparison of the CR, fetal loss, and body temperature by using a statistical software program for PC (Excel Statistics 2006). The CR for AI/ET was 30.4% and for AI was 13.8% in 2007 (P0.05). The fetal loss was 38.1% in AI/ET v.7.4% in AI in 2007 (P<0.05) and 12.5% v.0% in 2008 (P<0.05), respectively. The AI/ET procedure could improve CR in dairy cows during the summer period in southern Japan. However, other problems may accompany AI/ET, such as higher fetal losses. [ABSTRACT FROM AUTHOR]

The aim was to evaluate whether pregnancy rates could be improved by randomly injecting recipient mares with flunixin meglumine (FM; Banamine®, Schering-Plough, São Paulo, Brazil) on the day of embryo transfer combined or not with long-acting progesterone (P4) in commercial embryo transfer programs. One hundred sixty-five Mangalarga Marchador recipient mares from the 2006–2007 breeding season of 4 different commercial programs were used. Ages ranged from 4 to 15 years old. In 86 mares, 1.1mgkg-1of BW of FM was given intravenously immediately after embryo transfer, and in 79 mares, only saline was injected. Additionally, 59 mares receiving saline were injected with 1800.0mg of progesterone at embryo transfer and every week thereafter until 110 days of gestation. Of the 86 receiving FM, 17 received the same progesterone protocol. Embryos were transferred nonsurgically in Ham’s F-10 or Encare medium. Pregnancy was checked by ultrasonography at 15, 30, and 60 days after transfer, and only a positive diagnosis on the latter was considered pregnancy in the statistical analysis. In a 2×2 factorial design (2 levels of FM and P4), pregnancy rates were analysed using the GENMOD procedure considering a binomial distribution (SAS®, Cary NC), and means were compared by orthogonal contrasts. Pregnancy rates were lower (P<0.0001) in FM-treated (50.2±5.3%) compared with control (88.2±5.1%) mares and in P4-treated (85.9±5.0%) compared with control (52.4±5.5%) mares. An interaction effect between FM and P4was not observed (P<0.49). For mares receiving or not receiving FM, the addition of the P4protocol lowered pregnancy rates. In mares not treated with FM, pregnancy rates were 81.4±5.1% and 95.0±8.8% for P4-treated and nontreated mares, respectively (P<0.05). In FM-treated mares that did not receive P4, pregnancy rates were 76.8±4.8% compared with 23.5±9.6% of those treated with P4(P<0.0001). In conclusion, the indiscriminate use of progesterone and nonsteroidal anti-inflammatory drugs may actually decrease reproductive efficiency and even act synergistically to potentiate their effect. These results must be interpreted with caution, considering that the data are derived from field observations, although sources are from very reliable and well-controlled operations. Nevertheless, the authors consider it imperative to have a proper diagnosis before generalizing the use of medical tools to improve pregnancy rates in horses. Financial support from CNPQ–Conselho Nacional de Desenvolvimento Científico e Tecnológico, CAPES–Coordenação de Aperfeiçoamento de Pessoal de nível Superior. Schering/Plough, JOFADEL, and ABCCMM–Associação Brasileira dos Criadores do Cavalo Mangalarga Marchador. [ABSTRACT FROM AUTHOR]

The objective of this work is to describe a hands-on laboratory session for surgical embryo collection and transfer using rabbits as a model. Rabbits are characterised for their low purchase price, easy handling, and low maintenance, thus allowing their use as a tool for embryo transfer teaching and for its potential application in other species, such as the bovine, ovine, and caprine. These are important reasons in countries where the use of low-cost techniques and animal models is a must. Two females of the New Zealand breed were used as donors and one female of the Chinchilla breed was used as recipient. All females were healthy and multiparous. Three days after receiving natural service by a male, the donors were anesthetized (by injection of xylazine hydrochloride, 1.1mgkg-1IM, then sodium pentobarbital, 30mgkg-1IV), and ovariohysterectomy surgery was performed on each one. Each uterine horn was flushed 3 times with Ringer’s lactate (Hartmann) solution. Using a stereoscopic microscope, we searched for embryos and then placed them in a Petri dish for evaluation and selection. Once selected, embryos were moved to a 6-well dish containing 0.5mL of a commercially available holding solution and washed 3 times. Twenty-one grades 1, 2, and 3 embryos were recovered and evaluated, showing developmental stages 1 to 6, according to IETS. The recipient female was anesthetized, and by laparotomy, her uterine horns were exteriorized to transfer a total of 15 grade 1 embryos with development stages 4 to 6. Pregnancy diagnosis was performed by real-time ultrasound on Days 10 and 20 post-transfer, with positive results. The transferred embryos induced a 34-day pregnancy, including 3 days of embryo development within the donors. Parturition was normal and without problems; 2 live offspring were born, with normal weight and size. The technique, used for teaching purposes, was successfully performed by 3 graduate students directed by 2 professors. The use of rabbits as a teaching model was efficient and the total cost of the laboratory practice was low, as compared with the use of other domestic species. The handling of the females and their offspring was easy, the feeding costs were low, and the practical laboratory session was fulfilled. It is proposed that the rabbit species be used for teaching surgical embryo collection and transfer to graduate and undergraduate students in underdeveloped and developing countries, where low-cost teaching models are of the utmost importance. [ABSTRACT FROM AUTHOR]

Recent studies that have used eCG administration either 14 or 22 days after fixed-time AI in dairy and beef cattle have indicated a positive effect on conception rates. The aim of this study was to evaluate conception rates in embryo recipients treated with eCG 7 days after fixed-time embryo transfer (ET). A protocol for synchronization of ovulation was used in 470 Nelore and crossbred embryo recipient heifers (n=179), dry cows (n=152), and lactating cows (n=139; body condition score=3.0 to 3.5; >40 days in milk). All heifers and dry cows, but only 27.3% of lactating cows, were cycling at the beginning of the synchronization protocol. On Day 0, each female received IM injections of 2mg of oestradiol benzoate (Gonadiol, Intervet Schering-Plough Animal Health, São Paulo, Brazil) and 0.075mg of PGF2α(Preloban, Intervet Schering-Plough Animal Health). An intravaginal progesterone-releasing device (DIB, Intervet Schering-Plough Animal Health) was also inserted on Day 0. On Day 8, the DIB device was removed and cows or heifers received another IM treatment of 0.075mg of PGF2α(Preloban), 300 IU of eCG (Novormon, Syntex S.A., Buenos Aires, Argentina) and 0.6mg of oestradiol cipionate (Pfizer, Brazil). Transrectal ultrasonography was performed in all recipients on the day of ET (Day 16, 17, or 18 of the protocol) and only those with a corpus luteum greater than 15mm in diameter were selected (n=113 heifers, 114 dry cows, and 70 lactating cows). Transfers of fresh, frozen, and vitrified in vivo-produced embryos from Nelore donors were balanced between experimental groups. Seven days after ET, 127 animals received an IM injection of 400 IU of eCG (eCG group), and the 170 remaining cows or heifers were not treated with eCG, and were therefore considered the control group. Pregnancy diagnosis was performed 30 and 60 days after ovulation by transrectal ultrasound. Data were analysed using generalized linear models and results are presented as least squares means±standard error. Conception rates were similar in the eCG and control groups at Day 30 (35.9±5.5 and 33.5±4.4, respectively; P>0.10) and at Day 60 (27.6±4.9 and 26.7±3.9, respectively; P>0.10). Contrary to other results in the literature, especially in dairy cows bred by fixed-time AI, the use of eCG 7 days after ET did not improve conception rates in Bos indicuscattle used as embryo recipients. Financial support from CNPq, FAPESP, and Intervet Schering-Plough Animal Health of Brazil and Syntex S.A. of Argentina. [ABSTRACT FROM AUTHOR]

The in vitroproduction (IVP) of bovine embryos is a biotechnique that has created new perspectives in animal reproduction and improvement. However, one of the main issues in the diffusion of this technology concerns the rate of pregnancy of the recipient cows. The objective was to verify the relationship between the fertilization in vitro, the day the recipient female was in estrus, and the pregnancy rates. Two hundred sixty crossbred recipient cows were selected from July to November 2008, synchronized with 2 different protocols at a random moment of the estrous cycle. One of the groups (G1) composed of 110 females that received 2.0 mL of D-cloprostenol, i.m. in only dose. The other group (G2), with 150 females, received a norgestomet auricular implant and 2.0 mg of estradiol benzoate i.m. On the fifth day, the females received, i.m. 200 IU of equine serum gonadotrophin and 150 μg of D-cloprostenol. The implant was removed on the eighth day, and 24 h later all animals received 1.0 mg of estradiol benzoate. The recipients entered in estrus 1 day before, 1 day after, and on the day of IVF. The embryos were transferred on Day 7 after fertilization, trancervically in the ipsilateral uterine horn that contained corpus luteum. The gestation diagnosis was made by ultrasound scan 30 days after the transfer. Data were analyzed using SAS software (version 9.0, SAS Institute, Cary, NC, USA). Initially, data were analyzed by normality test using Proc Univariate; however, a normal distribution was not observed. Data were then analyzed by PROC FREQ with nonparametric method of chi-square test and a significance level of 5%. Among the recipients, 40 were observed in estrus 1 day before IVF (D - 1), 137 on the IVF day (D0) and 83 were observed in estrus 1 day after (D + 1), resulting in a total rate of pregnancy of 38.8%. For each day the resulting pregnancy rates were 42.50% in D - 1, 41.61% in D - 0, and 30.12% in D + 1. These results suggest that there is no relationship between the day of the recipient''s estrus, the day of IVF, and pregnancy rates. There was no influence of the day of estrus in relation to the day of IVF in the gestation rates of the recipients of IVP embryos. However, more studies concerning the recipients are necessary, because they assume an important role in the success of the IVP. The authors thank Brio Genetic and Technology. [ABSTRACT FROM AUTHOR]

According to the animal reproduction biotechnologies, in vitrofertilization is considered one of the most important tools to improve genetic gain as well as reduce bovine herd generations. Brazil is recognized as having the greatest commercial bovine herd in the world as well as by its use of IVP; however, this biotechnology needs more studies to improve not only the oocyte-embryo conversion rate but also the competent oocyte recovery from donor cows. Recently, one Nelore donor cow in 3 sessions was able to convert 233 (53.81%) embryos from 433 oocytes recovered, which resulted in 156 pregnancies (55.95%), which means one pregnancy per 2.7 oocytes or 3.05 pregnancies per week (when 51 weeks are admitted in one year). The COC obtained from this donor were matured (TCM-199, supplemented with FCS, LH, FSH, E2, pyruvate, and antibiotic) for 24 h and fertilized (Fert-TALP supplemented with BSA, PHE, and heparin) for 18 to 22 h (Day 0) in vitro. On Day 1, presumptive zygotes were transferred to development media (SOFaa supplemented with BSA and FCS) and on Day 7 the blastocyst production rate was evaluated. Following embryo transfer, the pregnancy diagnosis was performed at 30 days and confirmed at 60 days after ET. The possibility of identification of factors related to high performance and its selection could improve the formation of a homogeneous bovine herd, highly correlated with embryo production, which numerically could promote more than fifty calves per year (more than one calf per week). This case report refers to an animal highly superior to the average and therefore needs more investigation, mainly with molecular markers able to identify and select this class of high genetic animal. Table 1.Embryo production performance [ABSTRACT FROM AUTHOR]

The success of reproductive technologies, such as embryo transfer and IVF, greatly depend on the response to superovulation. Using hormonal therapy, it is possible to increase the reproductive rates of beef and dairy cattle for individual and herd treatment. The objective of the present study was to assess the effect of stimulating treatment with FSH or eCG on the oocytes production in Nelore (Bos indicus) donors. The experiment was carried out at Biotec (Cesumar farm, Maringá, Paraná State, Brazil). Forty-two cows that originated from the same genetic group, between 4 and 9 years old were used, with an average weight of 420 kg. The cows received an intramuscular injection of estradiol benzoate (EB, 2 mg) and an ear implant of Norgestomet®(3 mg). Then, they were divided in 3 groups: control group (n= 14); group 1 = eCG treatment (n= 14); and group 2 = FSH treatment (n= 14). In group 2, cows received on 120 IU of FSH on Days 5 and 6, divided in 4 doses that were administrated at 12-h intervals. On Day 7, all implants were removed and ovum pick-up (OPU) was performed. The results were statistically analyzed by Proc GLM (version 8.0, SAS Institute, Cary, NC, USA) and the mean differences were analyzed by Tukey test. Of 42 OPU sessions, a total of 627 oocytes were recovered, of which 502 were viable and 125 were degenerated, but there were no significant differences (P> 0.05) between the groups, as shown in Table 1. We conclude that the gonadotrophic stimulations with FSH or with eCG used in this study did not increase the overall quality of the viable oocytes. More research is needed to determine the optimal ovarian stimulation protocol in Nelore cows. Table 1.Number of oocytes recovered after OPU session in cows that were superstimulated with FSH or eCG1 [ABSTRACT FROM AUTHOR]

The necessity of greater elucidation about oocyte pathogen interactions made us examine the interaction of bovine herpesvirus type 1 (BoHV-1), Los Angeles strain, with oocytes matured in vitroto assess the potential hazard of transmission of such infectious agent through IVF. The process of capacitation and transportation of oocytes through the oviduct depends on the quality of the female gamete. Keeping in mind that the BoHV-1 causes morphophysiological modification in oocytes, the aim of this study was to evaluate, in an experimental model of in vitrooviduct, whether the pathogen interferes in the transportation of oocytes. This was determined using the percentage of oocyte recovery and assessment of the alteration in the morphology of the oviduct lumen. Oviducts and oocytes were collected from bovine ovaries derived from slaughterhouse cows. The oviducts were dissected, washed, and placed in Petri dishes (100 × 20 mm). On the previous portions of the infundibulum, in vitro-matured control oocytes or oocytes exposed to the virus (105.5 TCID50 mL-1) were introduced (20 oocytes per group) and then immersed in 100 mL of TCM-199 medium. Each dish was incubated at 38°C, 5% CO2, and 95% humidity for 24 h. Then, we introduced in the infundibulum, through intramammary infusion probe, 5 mL from a physiologic solution containing 1% of bovine fetal serum. Each oviduct effluent was collected separately and evaluated through a stereoscope for the recovery and counting of oocytes. Immediately after the recovery, both oviduct groups were sectioned longitudinally and observed through an optical microscope (100 ×) for a morphological evaluation of the luminar area. For the control group, the percentage of recovery was 14.9% (65/437), whereas in the infected group, the oocyte recovery was 23.4% (100/428). The statistical analysis was made according to Student''s t-test (P< 0.05), and presented a significant difference in the final results. The previous results show that the group of oocytes that was exposed to the virus presented a higher percentage of recovery compared with the control group. The oviducts that received the exposed oocytes presented areas where it seemed there has been cytopathic effect represented by dark and lumpy sections, in which there were round and degenerating cells mainly located in the oviducts'' peripheral area. The morphophysiological modifications in contaminated oocytes and oviducts probably complicate their transportation and also interferes in the process of their liberation. It is relevant to elucidate the importance of the studies above because of their participation in the recovery of these gametic cells in bovine oviducts, as well as the transmission during IVF processes. Vitrocel/Embriolife. [ABSTRACT FROM AUTHOR]

The aim of the work was to study the effects of year period, technical team, breed, beef cattle and dairy cattle on the pregnancy rates in fresh embryos used in bovine transfer of IVF programs. The study was carried out at the fertilization laboratory In VitroNyltta Britto de Carvalho, in partnership with In VitroBrazil, located at the Boa Vista farm, Barra do Pirai, during August 2007 to September 2008, seeking subsidies to improve the use of the technique in the field. During that period, aspirations and inovulations in 3 different periods I (August to December), II (January to April), and III (May to September) were carried out. The jobs were accomplished by 9 technical teams (A, B, C, D, E, F, G, H, and I) rendering services to the laboratory, by working with 2 beef breeds (Brahman and Nelore) and 3 dairy breeds (Gir, Girolando, and Holstein). The different breed receivers were synchronized, and in general, from 6 to 8 days after heat, they received embryo transfer, the cervical way, under low epidural anesthesia, where each female received 1 fresh embryo of IVF. All cows were submitted to gestation diagnosis by rectal palpation and ultrasonography, in general, 42 days after embryo transfer. The numbers of embryo transferred and pregnancy rates were submitted to the chi-square test, which presented significant differences (P< 0.05). There were pregnancy rates of 36.25%a(n= 960), 39.83%a(n= 1180), and 32.59%b(n= 919) in the I, II, and III periods, respectively. Among the 9 technical teams, there were verified pregnancy rates (%) of 33.51d(n= 1313), 30.30d(n= 330), 35.00cd(n= 405), 39.24cd(n= 1060), 59.25a(n= 7), 33.33d(n= 24), 53.57bc(n= 28), 43.31c(n= 157), and 58.33ab(n= 12) for A, B, C, D, E, F, G, H, and I teams, respectively. Among breeds there were rates (%) of 36.89ab(n= 412), 34.68b(n= 1286), 35.13ab(n= 74), 38.94a(n= 1140), and 37.80ab(n= 82) for Brahman, Nelore, Gir, Girolando, and Holstein, respectively. In the study, pregnancy rates (%) of 35.21b(n= 1698) in beef cattle and 38.65a(n= 1296) in dairy cattle were observed. The differences in pregnancy rates with respect to the evaluated factors, may be explained by individual, breed, and nutritional variations of the animals. There are few data in the literature with results on the embryo transfer use of IVF bovine under field conditions. [ABSTRACT FROM AUTHOR]

The in vitroembryo culture might affect epigenetic mechanisms, which are involved in controlling the expression of genes related to embryonic development and inactivation of X chromosome. Female mammals have 2 X chromosomes, and males have only 1. This has led to a particular mechanism of evolution of dosage compensation, called X-chromosome inactivation, an important epigenetic event that must occur in all mammalian female embryos. During embryogenesis, at the late blastocyst development (Xue F et al.2002 Nature Genet. 31, 216–220), 1 of the 2 X chromosomes is randomly inactivated in each cell of the inner cell mass and preferentially X paternal in trophoblast. The aim of this study was to characterize the allele-specific expression of the X chromosome-linked gene monoamine oxidase type A (MAO-A) during in vitropre-implantation embryo development in bovine. For phenotyping of the MAO-Agene, the RT-PCR restriction fragment length polymorphism technique was used. Primers were designed flanking a single nucleotide polymorphism and the sequence of forward inner primer creating a site of restriction to the RsaI enzyme, thus allowing the detection of allele-specific expression (Bos taurus Taurus× Bos taurus indicus). Oocytes were aspirated from 9 Nelore heifers homozygous for theA allele previously genotyped. The oocytes were selected, matured in vitro, and inseminated with X-sorted sperm from a Holstein bull homozygous for the G allele. Two pools of 10 heterozygous in vitroembryos of each developmental stage, 4-cell [44 h post-insemination (p.i.)], 8- to 16-cell (72 h p.i.), morula (144 h p.i.), blastocyst (156 p.i.), and expanded blastocyst (168 h p.i.), were produced and frozen until RNA extraction. Total RNA was extracted using Invisorb®Spin Cell RNA Mini Kit (Invitek, Berlin, Germany) according to the manufacturer''s protocol, and residual genomic DNA was removed with DNase I treatment. cDNA was done using Oligo dT primers (Invitrogen) and superscript III reverse transcriptase (Invitrogen). Nested PCR for each pool was performed and then the amplicons were digested with 10 U of RsaI enzyme (Promega, Madison, WI, USA). The products were separated by electrophoresis on a 3% agarose gel stained with ethidium bromide. The results showed that both alleles were expressionally represented in the 4-cell, 8- to 16-cell, and expanded blastocyst stages, with the X paternal allele disappearing in morula and blastocyst. We can conclude that both, maternal and paternal X chromosomes, are activated in the 2 earliest stages, inactivated in the morula and blastocyst stages, and reactivated in the expanded blastocyst stage. This research was supported by Embrapa Genetic Resources and Biotechnology and the Brazilian National Council for Scientific and Technological Development (CNPq). [ABSTRACT FROM AUTHOR]

Estrus synchronization and superovulation are the most widely used procedures in embryo transfer technology. However, changes in the oviduct and uterine environment due to these procedures and the subsequent influence on embryos have not yet been investigated. This study was con- ducted to investigate the effect of oviduct environment of only synchronized or superovulated cyclic heifers on the gene expression profile of blastocysts. Bovine Affymetrix array analysis was performed using 2 groups of blastocysts. The first group was bovine blastocysts produced after superovulation of Simmental heifers (n= 9) using 8 consecutive FSH injections over 4 days in decreasing doses (in total, 300-400 mg of FSH equivalent according to body weight) and flushed at Day 7 by nonsurgical endoscopic method. The second group was bovine blastocysts derived from synchronized Simmental heifers (n= 4) after transfer of 2-cell stage embryos from superovulated donor Simmental heifers (n= 9) by nonsurgical transvaginal endoscopy tubal transfer method. Total RNA was extracted from 3 pools of embryos from each experimental group (6 embryos per pool). A total of 6 biotin-labeled cRNA samples were hybridized on 6 bovine Affymetrix arrays. Data analysis was performed using LIMMA written on R package, which maintained the Bioconductor. Array data analysis revealed a total of 454 transcripts to be differen- tially expressed (P2) between the 2 groups. Of these, 429 and 25 were up- and down-regulated, respectively, in blastocysts derived from superovulated heifers compared with those derived from synchronized animals. Genes involved in response to stress (HSPA14and HSPE1), cellular and metabolic processes (CPSF3, ATPIF1, POMP, and MDH2), translation (RPS17, EEF1B2, and EIF4E), and cell commu- nication (FN1, KRT18, and DSG2) were found to be enriched in blastocysts derived from superovulated animals. On the other hand, protein metabolic processes related genes (CLGN) were found to be enriched in blastocysts derived from the synchronized group. The KEGG analysis of the differentially expressed genes showed that the ribosome and oxidative phosphorylation pathways are the dominant pathways and genes involved in these pathways are greatly abundant in the blastocysts derived from superovulated animals. Quantitative real-time PCR has confirmed the transcript abundance of 7 out of 8 genes selected for validation. In conclusion, blastocysts cultured in synchronized animals post 2-cell stage showed significant differences in transcriptome profile compared with their counterparts that remained in superovulated heifers until Day 7. Further functional analysis of some selected candidate genes could give new insights into mechanisms regulating the ability of embryos to survive after transfer. [ABSTRACT FROM AUTHOR]

Bovine diarrhea virus (BVDV) causes a variety of economically important enteric and infertility problems. For that reason, several countries have eradicated the disease and some others have schemes in progress to achieve freedom from it. Although there is a considerable amount of information about the risk of BVDV transmission through contaminated semen used for AI, there is no available evidence to indicate whether the resulting embryos, when used for embryo transfer (ET), can lead to the transmission of BVDV to recipients and offspring. For this experiment, semen from a bull persistently infected with BVDV (105TCID50/mL of NY strain) was used for insemination (2 times at estrus) of BVDV-seronegative, superovulated heifers (n= 27).All heifers seroconverted to BVDV within 10 days post-insemination. Embryos and unfertilized oocytes were collected nonsurgically (n= 92) or postmortem (n= 52) 7 days post-insemination and were either washed according to IETS recommendations (without trypsin treatment) or left unwashed. In total, out of 144 unfertilized oocytes and embryos collected, 23 (16%) were of ET quality. Most of the embryos were degenerated or unfertilized. On 17 occasions, 1 or 2 washed embryos were transferred to BVDV-seronegative recipients. After ET, all pregnant and nonpregnant recipients remained free of BVDV and antibodies. In total, 6 heifers became pregnant and 5 calves free of BVDV and BVDV antibodies, including 2 sets of twins, have been born to date. Post-insemination, BVDV was detected in 29% (12/41) of unwashed and 10% (4/40) of washed embryos, 100% (4/4) of follicular fluid samples, oviductal epithelial cells, endometrium, and corpora lutea tissues as determined by the virus isolation test. Results herein suggest that BVDV can be transmitted by AI, resulting in the production of some proportion of contaminated embryos. However, it appears that such embryos, when washed according to the IETS guidelines, do not cause BVDV transmission to recipients or their offspring. [ABSTRACT FROM AUTHOR]

Large programs of in vitroembryo production have been developed in Brazil, but the results are very variable. The objective of this study was to evaluate managing factors that influence pregnancy rate of recipients in a large embryo transfer program. Results of 1104 embryo transfers performed from November 2008 to February 2009 by Transgen Desenvolvimento e Produção Agropecuária Ltda (Uberlˆndia, Minas Gerais, Brazil) were evaluated. Embryos produced in vitrowere used: 1/2 Holstein/Gir embryos (n= 139) or 3/4 Holstein/Gir embryos (n= 961) produced from donors 1/2 Holstein/Gir and Gir with Holstein bull semen. Only excellent (grade 1) quality morulae (stage 4), early blastocysts (stage 5), mid- blastocysts (stage 6), or expanded blastocysts (stage 7) were assigned for fresh transfer using the International Embryo Transfer Society guideline for grading embryos. The heifer recipients were 1/2 crossbred Nelore/Simmental, in good health condition, 20 to 30 months of age, 330 to 400 kg of weight, and were maintained on pasture (Tifton) with mineral supplementation ad libitum. The recipients were synchronized as follows: Day 0-intravaginal device with 1.0 g of progesterone (PRIMER®, Tecnopec, São Paulo, SP, Brazil) +2 mg of estradiol benzoate (ESTROGIN®, Farmavet, São Paulo, SP, Brazil); Day 5-150 μg of D-Cloprostenol (PRELOBAN®Intervet, São Paulo, SP, Brazil) + 400IU of eCG (FOLLIGON®, Intervet); Day 8-progesterone device removed; Day 9-1 mg of estradiol benzoate. On Day 17, nonsurgical embryo transfers were performed by a trained technician and synchronization was confirmed by presence of a CL. The effects of the embryo breed, the number of transfers in each recipient (the heifers that failed to get pregnant were used in the next program), and the transfer sequence (i.e. 20 embryo transfers were performed per hour, and approximately 100 per day) on the pregnancy rate was analyzed by logistic regression with the LOGISTIC procedure of SAS (SAS Institute Inc., Cary, NC, USA). The breed of embryo (56.9% for 3/4 Holstein/Gir embryos v.62.6% for 1/2 Holstein/Gir embryos), the number of transfers in each recipient (first: 56.5% v.second: 61.8% v.third: 55.5% v.≥fourth: 55.0%), and the transfer sequence during the day of the program (first hour: 57.4% v.second hour: 60.0% v.third hour: 58.1% v.≥fourth hour: 53.8%) did not influence the pregnancy rate. Within the confines of an extensive embryo transfer program with in vitroproduced fresh embryos, a large number of embryo transfers can be executed per day without adversely affecting the pregnancy rate if high-quality embryos are used, the transfers are performed by a trained technician, and recipients are in good health condition and synchronized. [ABSTRACT FROM AUTHOR]

Reproductive efficiency has a major impact on profitability of dairy farms. Pregnancy loss in cattle is one of the major impediments for adequate reproductive performance. The aim of the present study was to evaluate factors associated with pregnancy loss between 30 and 60 days of gestation in high-producing repeat-breeder (>4 services) Holstein cows submitted to embryo transfer (ET). A retrospective analysis was performed using data from 1 commercial dairy farm, located in Descalvado, Brazil (22°01′27″S; 47°53′19″L). Overall, 2096 ET were analyzed from 2004 to 2008. High-producing Holstein cows from the same herd were used as embryo donors. Embryos were produced by superovulation using the same protocol (8 decreasing injection of FSH) and were transferred, either fresh or frozen-thawed, to repeat-breeder Holstein recipients 6, 7, or 8 days after estrus (synchrony of Day 1, Day 0, or Day 1 with the donor, respectively). Statistical analysis was performed using PROC GLIMMIX of SAS (SAS Institute Inc., Cary, NC, USA). Variables included in the model were synchrony between recipient and donor at ET day, fresh and frozen-thawed embryos, stage of embryo development, and embryo quality. All cows were examined for pregnancy by ultrasonography 23 and 53 days after ET (30 and 60 days of pregnancy). There was no effect of year. The pregnancy loss was greater (P= 0.005) for fresh (22.9%, 141/615) v.frozen-thawed embryos (19.4%, 288/1481). Also, pregnancy loss was influenced (P= 0.002) by the synchrony between recipient and donor at ET day [Day -1:18.3% (53/290); Day 0: 18.9% (233/1234); D1: 24.9% (144/578)]. However, there was no difference in pregnancy loss when the stages of embryo development [morula: 20.7% (295/1428); early blastocyst: 18.9% (67/355); blastocyst: 23.0% (59/256); expanded blastocyst: 14.0% (8/57; P= 0.31)] and embryo quality [grade 1: 17.6% (78/443); grade 2: 20.1% (216/1073); grade 3: 23.3% (135/580); P= 0.31] were compared. In conclusion, pregnancy loss was greater in high-producing repeat-breeder Holstein cows receiving fresh embryos compared to frozen-thawed embryos and when Day 1 recipients were used compared to Day -1 and Day 0. Agrindus S/A. [ABSTRACT FROM AUTHOR]

Progestagen-releasing devices are commonly used for fixed-time artificial insemination and fixed-time embryo transfer (FTET). The aim of this study was to evaluate the efficacy of a new progesterone-releasing intravaginal device made of poly(3-hydroxy)butyrate-valerate copolymer (PHB-V) and poly-ε-caprolactone (PCL; Progestar®, Innovare, São Paulo, Brazil) to synchronize crossbredcows and heifers for FTET by determining ovulation, conception, and pregnancy rates. A total of 205 recipients ranging in age from 2 to 8 years (heifers, n= 77; non-suckled cows, n= 69; suckled cows, n= 59) were used on 3 different farms during February, March, and April 2008. The experimental design was a generalized block design (cyclicity, body condition score, postpartum interval, and animal category, i.e. heifers, suckled, or non-suckled cows), with 2 different synchronization treatments for FTET (Progestar®v.Crestar®). Cows were assigned to either Crestar®(norgestomet ear implants; Intervet Schering-Plough, Kenilworth, NJ, USA; n= 103) or Progestar®(PHB-V; n= 102) treatments. All groups were treated with 2 mg of estradiol benzoate i.m. (Innovare) and received the intravaginal or auricular device according to their group (Day 0). Together with P4 device withdrawal (Day 8), all animals received 400 IU of eCG i.m. (Novormon®, Schering-Plough) and 500 μg i.m. of PGF2α, (Sincrocio®, Ouro Fino, Brasil). On Day 9, all cows were treated with 1 mg of estradiol benzoate (Innovare) and the innovulation was performed on Day 17. Embryos were maintained at 37°C during the innovulation period. The percentage of usable recipients (AR) based on the presence of a functional CL on the day of embryo transfer, conception rate (numbers of pregnant recipients of the total number transferred; CR), and pregnancy rate (numbers of pregnant recipients of the total number synchronized; PR) were determined by ultrasonography 23 days after embryo transfer. Data were submitted to ANOVA (PROC GLM, SAS Institute Inc., Cary, NC, USA) and significant differences were considered to have a P-value of <0.05. None of the interactions were significant and there was no effect of treatment on AR (72.8% v.83.3%), CR (47.9% v.42.7%), and PR (34.7% v.35.0%) for Progestar®v.Crestar®groups, respectively. This experiment showed that Progestar®was as efficacious as Crestar®to synchronize crossbred cows and heifers for FTET. FAPESP-Fundação de Amparo à Pesquisa do Estado de São Paulo and CNPq-Conselho Nacional de Desenvolvimento Científico e Tecnológico. [ABSTRACT FROM AUTHOR]

Transcriptome profiling of pre-transfer cycle endometrium in relation to its ability to sustain the upcoming pregnancy may pave the way to develop or identify molecular markers that can be utilized to detect and select receptive endometrium before embryo transfer. Here, we aim to show differential expression of genes between endometrium biopsies derived from recipients during pre-transfer cycle based on the pregnancy successes after embryo transfer. For this, endometrium biopsies were taken from 56 Simmental cyclic heifers of the same age at day 7 and 14 of the estrous cycle. On the next cycle, in vivo-produced day 7 blastocysts were transferred to all animals at day 7 of the estrous cycle. Pregnancy diagnosis was done at 28, 42, and 56 days of gestation. Thirty-two cows were returned to heat after 21 days, 7 were pregnant until day 42 but no pregnancy after that, and 15 resulted in calf delivery. Subsequently, the endometrium biopsies sampled during the pre-transfer period were categorized based on the pregnancy outcome. Those endometrial biopsies taken at days 7 and 14 during the pre-transferred period from those that resulted in successful calf delivery were named as d7CD and d14CD, respectively, and those endometrial samples collected at days 7 and 14 during pre-transferred period from those groups that resulted in no pregnancy were named as d7NP and d14NP, respectively. Total RNA was extracted from 3 pools of each experimental group in 3 replicates using RNeasy mini kit (Qiagen, Hilden, Germany). A total of 12 biotin-labeled cRNA samples were hybridized on 12 bovine Affymetrix arrays (Affymetrix, Santa Clara, CA, USA) consisting of 24128 probe sets. The microarray data normalization and background correction was performed using guanine cytosine robust multi-array analysis, and the data analysis was performed using LIMMA written on R package, which maintained the bioconductor. The results showed that 1130 transcripts were differentially expressed between d7CD and d7NP, of which 626 and 504 were up- and down-regulated, respectively, in d7CD. Genes that involve in regulation of transcription (PPARA, NR2F1, MYB, MYB, and CHF2) and the collagen families (COL1A1 and COL1A2) were enriched in d7CD. A total of 234 transcripts were differentially expressed between d14CD and d14NP, of which 94 and 140 were up- and down-regulated, respectively, in d14CD compared to d14NP. Transcripts involved in protein amino acid phosphorylation (MA2K6, GATM, AK3L1, and MAPK10) were found to be enriched in d14CD compared to d14NP. In conclusion, pre-transfer endometrium biopsies showed significant differences in transcriptome profile depending on the pregnancy outcome after transfer of in vivo-derived blastocysts and enable to identify transcripts related to pregnancy establishment. [ABSTRACT FROM AUTHOR]

Hormonal estrus synchronization followed by heat detection of recipient animals is a widely used technique in embryo transfer (ET) programs around the world. However, the successful outcomes of ET rely heavily on appropriate heat detection of recipient animals. Additionally, limited availability of personnel and time to detect heat makes ET impractical at times. Therefore, pregnancy rates (PR) following a timed embryo transfer (TET) program were evaluated taking into account different variables of recipient cows, donor embryos, and season of transfer under field conditions in Argentina. Recipient cows were synchronized utilizing a progesterone (P4) intravaginal device (DIB, Syntex, Argentina) for 7 days. On Day 0 (day of DIB insertion) 2 mg of estradiol benzoate (EB) i.m. was administered and immediately after DIB removal on Day 7, 500 μg of D(+) Cloprostenol i.m. (PG, Ciclase, Syntex), 400 IU of eCG i.m. (Novormon, Syntex) plus 1 mg of cypionate estradiol i.m. (ECP; König, Agentina) were administered to all recipient cows. Fresh (n= 1746) and frozen (n= 2047) beef bovine embryos were nonsurgically transferred into recipient cows on Day 9 after PG. The effects of body condition score (1–9, BCS I: ≤5, n= 804 and BCS II: ≥6, n= 2989), corpus luteum (CL) score (CL1: prominent papilla, n= 932, CL2: small papilla, n= 2028, and CL3: asymmetric, n= 833), location of embryo placement in uterine horn (upper, n= 3566, and lower, n= 227), and type of recipient such as heifer (n= 487), lactating (n= 1010), or dry cows (n= 2296) on PR were evaluated. Additionally, stage of embryo development (4–7), quality (1–3), and season of transfer (spring, summer, fall, and winter) were included in the analysis. Pregnancy rate was confirmed by rectal palpation 60 days after ET. Data were first screened by chi square and then analyzed by Proc Logistic of SAS (SAS Institute Inc., Cary, NC). Overall PR was 54.7%. body condition score, CL, type of recipients, stage of embryo development, and year were not significantly (P> 0.05) associated with PR. As expected, cows that received direct fresh embryos had 1.49 times greater odds of conceiving (odds ratio, OR = 1.49, 95% CI: 1.22 to 1.82) than those receiving frozen embryos. Additionally, cows receiving embryos graded as quality 1 had greater probability to conceive (OR = 1.62, 95% CI: 1.34 to 1.95, or OR = 1.64, 95% CI: 1.25 to 2.21) compared to quality 2 or 3 respectively. Moreover, embryos placed into the upper portion of uterine horn were more likely (OR = 1.48) to conceive than those placed in the lower uterine portion. Regardless of embryos and recipient cow variables analyzed, there was a greater probability for cows to conceive during winter (OR = 1.41, 95% CI: 1.13 to 1.95) as compared with summer. In conclusion, synchronized recipient animals for a TET program resulted in acceptable PR for both fresh and frozen embryos. Results suggested that the quality and location of embryos (upper portion) in uterine horn were the most important variables associated with PR in recipient cows. Therefore, TET is a viable and practical option allowing a successful outcome for many beef operations where availability of personnel is limited. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effect of environmental temperature at the time of embryo collection and transfer on embryo quality and conception rates after transfer of fresh and frozen embryos. Purebred dairy (Holstein, Czech spotted cattle, n= 174) and beef (Charolais, Blonde d''Aquitaine, Piedmontese, Hereford, Limousin and Simmental; n= 72) donors were collected during 2005 to 2007. Donor cows received one application of PGF2αand superstimulation was initiated 9 to 11 days following estrus with pFSH (Folltropin-V; Bioniche Animal Health, Belleville, ON, Canada) given twice daily for 4 days. Prostaglandin2αwas given on Day 3 of the superstimulation treatment. Donors were inseminated 3 times, with 1 unit of semen on Days 5 and 6 of treatment. Embryo recovery was carried out on Day 7 after insemination. The recipients were synchronized with PGF2αand embryos were transferred on Day 7 after estrus. The effect of temperature at the time of 246 embryo recoveries and 1338 transfers (fresh and frozen embryos) was analyzed using the GLM, assuming quasi-poisson and quasi-binomial error distribution by R software (www.r-project.org). We evaluated the total number of ova/embryos collected, embryo quality, and conception rates after transfer at the environmental temperatures shown below. Temperatures were divided into the following ranges: A) from –5 to 5°C; B) from 6 to 15°C; C) from 16 to 20°C; and D) over 20°C. The average temperature values were obtained from the Czech hydrometeorological institute and corresponded to the following seasons: winter (range A and B); spring (range B and C); summer (range C and D); fall (range C and B). Dairy cows were managed intensively in barns whereas beef cows were managed in barns during the winter and on pasture from April until October. In beef donors the mean number of total ova/embryos collected and transferable embryos were 9.1 ± 7.9 and 4.2 ± 4.5, respectively and no significant effect of temperature was detected (P> 0.05). In dairy donors the mean number of total ova/embryos collected and transferable embryos were 7.9 ± 6.0 and 4.9 ± 4.5, respectively and no significant effect of temperature was detected (P= 0.27). Conception rate was greatest (P< 0.001) in temperature range D (63.64%) compared with the other temperature ranges (A = 27.66%; B = 43.65%; C = 43.21%;) in beef cows and in the range C (65.31%) than in the other temperature ranges (A = 34.7%; B = 52.67%; D = 56.25%) in dairy cows. It was concluded that temperature at the time of embryo collection did not affect embryo production and quality. However, environmental temperature did have a significant effect on conception rates in both beef and dairy recipients. Nutritional status may also have contributed to the increased conception in beef cattle during warmer temperatures. However, the optimal period for conception in dairy cows was in temperature range from 16 to 20°C, because of the potential adverse effect of heat stress in high-yielding cows.Supported by MEYS CR MSM 2678846201, LA 330; NAZV 1B44034. [ABSTRACT FROM AUTHOR]

In cattle, reliable methods for whole genome amplification (WGA) have been implemented for DNA pre-amplification and subsequent genotyping from embryo biopsy containing 5 cells or more. In France, these methods are now tested under field conditions. Several studies report pregnancy rates after direct transfer of biopsied frozen–thawed grade 1 embryos similar to that intact frozen ones. Even so, grade 2 and 3 embryos represent 25.5% of the transferable embryos (AETE data, 2007) and may limit the use of the above mentioned techniques if results are not satisfactory. The objective of this study was to investigate the impact of the embryo quality on the pregnancy rates after single direct transfer of biopsied frozen–thawed embryos. Embryos were collected on 12 donor cows after 15 sessions of superovulation treatment using FSH injected twice daily in decreasing doses over 4 days. Cows were inseminated at 12 and 24 hours after onset of estrus. Embryos were recovered 7 days post-insemination and evaluated according to IETS standards. Biopsies were performed on stage 4 to 7 grade 1 to 3 embryos using a microblade. Embryonic cells from the biopsy were dry deposited in microtubes and frozen before WGA (QIAGEN REPLI-g®Mini kit, Valencia, CA, USA) and multi-genotyping (GeneMapper software®– Applied Biosystems Europe). Each biopsied embryo was equilibrated for 10 min in 1.5 mEthylene Glycol and then loaded into straw containing two columns of F1 medium separated by a central column of 1.5 mEG with the embryo. The freezing sequence was: –7°C directly; seeding; held for 10 min; 0.5°C min-1until –35°C before plunging into liquid nitrogen. Embryos were thawed (straws 10 s in air and 20 s in water at 20°C) and directly transferred into synchronized recipient heifers. Pregnancy was diagnosed by ultrasonography at 35 and 90 days. Effect of embryonic stage and quality on pregnancy rates were analysed by log linear models (Proc CATMOD, SAS Institute Inc). A total of 58 embryos were micromanipulated. All grade 1 and 2 embryos were successfully biopsied and frozen and, 0.8% of grade 3 (2/25) were discarded due to their low quality after biopsy. No significant effect of embryo stage and quality on pregnancy rates was found after direct transfer (Table 1). These preliminary results suggest that high pregnancy rates can be achieved after direct transfer of biopsied frozen–thawed embryos of Grade 1, 2 and 3 allowing most of the embryos to be involved in the genotyping process.Table 1.Pregnancy rates following single direct transfer of biopsied frozen–thawed G1 to 3 bovine embryosThis work has been performed through the programme TYPAGENAE (GENANIMAL 4-03) supported by FRT/ANR and Apis-Genes. [ABSTRACT FROM AUTHOR]

Hydrocephalus is a congenital defect of cattle. Births of Japanese Black calves with forehead doming were reported in Tohoku District from 1988 to 1989. The common finding on autopsy was the presence of excessive amounts of cerebrospinal fluid with expanding lateral ventricles without evidence of microbiologic infection. Breeding records for pedigree registration have been maintained by the Wagyu Registry Association in this area since 1960. A survey of the pedigrees suggested that an autosomal recessive gene might be responsible for the defect. The incidence of doming calves ceased by an advised mating system which excluded the suspected sires after 1990. Molecular genetics is useful for disclosing DNA sequences of a recessive gene and for detecting the carriers of the gene. This trial was conducted to produce congenital hydrocephalus calves for gene analysis using embryo transfer on a small number of the remaining cows that gave birth to affected calves over 10 years ago. One donor cow, 11 years old, was superovulated with 24 to 25 AU of FSH (Antrin; Kawasaki Mitaka K.K., Kawasaki, Japan) and inseminated with frozen semen of a suspected bull. Collected embryos were transferred on the day of the collection and the rest of them were frozen and transferred later. Superstimulation was repeated 4 times in this cow at an interval of 76-105 days during and 8-month period. A total of 4, 13, 17, and 18 embryos of good quality were collected. Eighteen fresh embryos were transferred into 7 (2 embryos each) and 4 (single embryo) recipients. Frozen embryos were transferred into 6 recipients (single embryo). Seven of 17 recipients became pregnant and gave birth to 8 calves. Three of 6 male calves born and one of 2 females showed symptoms of hydrocephalus. Clinically, 3 of the affected calves had a domed forehead and squint-eyes and were unable to stand. One calf underwent a fatal convulsion and ataxia 29 days after birth. Pathological findings of affected calves showed dilated bilateral ventricles with increased cerebrospinal fluid as typical lesions of hydrocephalus. Embryo transfer was demonstrated to be a useful tool for investigation of a potentially heritable disease. Gene scanning with microsatellite markers using the tissues from the calves and the related cattle was done. The suspected gene was found near the centromere of chromosome 3 (BTA3). It was possible to diagnose the mutant-carrier cattle using two DNA markers, INRA006 (17.0 cm) and BL41 (43.2 cm). [ABSTRACT FROM AUTHOR]

A precise evaluation of embryo quality is important to estimate the suitability of embryo transfer to recipient animal. Recently, an objective evaluation method was reported for bovine embryos, in which the oxygen consumption of embryos can be noninvasively determined by scanning electrochemical microscopy (SECM) (Shiku et al. 2001 Anal. Chem. 73, 3751–3758). Trimarchi et al. (2000 Biol. Reprod. 62, 1866–1874) suggested that the oxygen consumption reflects the cell number and mitochondrial activity of embryos. The objectives of this study were (1) to examine the oxygen consumption of in vivo-derived embryos by SECM, (2) to investigate the relationship between oxygen consumption and morphological estimation of embryos, and (3) to assess the correlation among the oxygen consumption, embryo viability, and pregnancy rates. Fifty-six embryos were collected from Japanese Black cattle, which were superovulated with a total dose of 20 mg porcine FSH (FSH-R; Kawasaki Pharmaceutical Co., Ltd., Tokyo, Japan) followed by AI. The qualities of collected embryos at the stage of compacted morulae (CM), early blastocysts (EB), and blastocysts (BL) on Day 7 after AI were categorized as grade 1 and grade 2, according to the IETS manual (2002). The oxygen consumption rates of embryos were evaluated by SECM, as previously described by Abe et al. (2004 J. Mamm. Ova Res. 21). Embryos were frozen by programmable freezer in Dulbecco''s PBS containing 1.5 M ethylene glycol, 0.1 M trehalose, and 20% calf serum. They were thawed by holding the straws in air for 8 s and then immersing them in a 30°C water bath for 15 s. After thawing, the embryos were examined for oxygen consumption. Twenty-eight embryos were then cultured in TCM-199 supplemented with 20% fetal bovine serum and 0.1 mM ?-mercaptoethanol for 24 h to assess the viability of embryos by re-expansion of blastocole. The remaining 28 embryos were transferred to recipients. The pregnancy rates were determined by rectal palpation on Day 70. Data were analyzed by ANOVA. The consumption rates of BL embryos on Day 7 were significantly higher (P < 0.05) than those of CM collected on the same day (0.84 vs. 1.29 × 10?14 mol s?1, respectively). A significant difference was also observed in consumption rates between grade 1 and 2 embryos at the BL stage (P < 0.05). After freezing–thawing, the average oxygen consumption rates of embryos were 0.52 × 10?14 mol s?1 for CM (n = 9), 0.67 × 10?14 mol s?1 for EB (n = 8), and 0.96 × 10?14 mol s?1 for BL (n = 11). The CM embryos with rates of < 0.5 × 10?14 mol s?1 and the EB and BL embryos with those < 0.6 × 10?14 mol s?1 did not show good morphological appearance after 24 h in culture. Pregnant animals were not obtained from embryos with rates <0.5 × 10?14 mol s?1 for CM (n = 5) and <0.7 × 10?14 mol s?1 for EB (n = 9). A high pregnancy rate (67%) was obtained from embryos with rates >1.0 × 10?14 mol s?1 for BL (n = 14). These results suggest that the measurement of oxygen consumption of embryos after embryo freezing and prior to embryo transfer may be useful for estimating embryo quality and suitability of embryo transfer. [ABSTRACT FROM AUTHOR]

An experiment was designed to evaluate alternative treatment protocols for fixed-time embryo transfer (FTET) in recipients in order to reduce cattle handling. Non-lactating, cycling Bos taurusBos indicus beef cows (n = 918), with a body condition score (BCS) of 2.5 to 3.5 out of 5, received a progesterone-releasing device (DIB; Syntex, Buenos Aires, Argentina) and 2 mg estradiol benzoate (EB; Syntex) IM on random days of the cycle (Day 0); DIB were removed on Day 8. Cows were placed randomly into 6 treatment groups in a 3 2 factorial design. The factors evaluated were time of administration of PGF and eCG [control treatment: PGF Day 5 and eCG Day 5 (Bo et al. 2002 Theriogenology 57, 53–72); PGF Days 0 and 8 and eCG Day 5; PGF Days 0 and 8 and eCG Day 8] and the time of administration of the second injection of EB (at DIB removal or 24 h later). Therefore, cows in the control treatment received 150 g of d-Cloprostenol (PGF, Ciclase; Syntex) and 400 IU eCG (Novormon; Syntex) on Day 5, whereas cows in the other 2 treatments received 75 g of PGF on Days 0 and 8 and 400 IU eCG on either Day 5 or Day 8. Each treatment group was further subdivided to receive 1 mg EB IM on either Day 8 (EB Day 8) or Day 9 (EB Day 9). All recipients, whether observed in estrus or not and with more than one CL with luteal area (total CL area minus cavity area) >76 mm2 on the day before embryo transfer, received frozen-thawed embryos (IETS Grade 1) on Day 16 (EB Day 8) or Day 17 (EB Day 9) by direct transfer. Pregnancy was determined by ultrasonography 30 days after FTET. Continuous data were analyzed by ANOVA and pregnancy rates were analyzed by logistic regression. No effects of body condition score or technician were detected (P > 0.1). Although there were no differences in conception rates between recipients treated with EB on Day 8 or Day 9 (168/321, 52.3% vs. 203/350, 58.0%, respectively; P > 0.1), treatment with EB on Day 8 resulted in fewer recipients selected/treated (321/460, 69.7% vs. 350/458, 76.4%; P < 0.05) and pregnant/treated (168/460, 36.5% vs. 203/458, 44.3%; P < 0.05) than 24 h later (EB Day 9), respectively. The mean interval from DIB removal to estrus was shorter (P < 0.05) for recipients in the EB Day 8 (28.1 7.2 h) than in the EB Day 9 (43.5 9.4 h) group, and the mean ( SEM) CL area was also less in the EB Day 8 group than in the EB Day 9 group (261.7 96.7 vs. 293.1 114. mm2; P < 0.05). The number of recipients selected/treated was higher in the group receiving PGF on Days 0 and 8 and eCG on Day 8 (243/305, 79.6%) than in the group receiving PGF on Days 0 and 8 and eCG on Day 5 (208/301, 69.1%) or the control group (220/312, 70.5%). Although the luteal area was greater (P < 0.05) in the PGF on Days 0 and 8 and eCG on Day 5 group (304.4 127.1 mm2) than in the groups receiving PGF and eCG on Day 5 (274.6 98.6 mm2) or PGF on Days 0 and 8 and eCG Day 8 (258.4 90.2 mm2), pregnancy rates did not differ between PGF and eCG treatment groups (P > 0.05). It was concluded that it may be possible to reduce animal handling by treating recipients with a half dose of PGF at the time of DIB insertion and removal, when eCG is also administered, without compromising pregnancy rates. [ABSTRACT FROM AUTHOR]

This study evaluated embryo transfers from Holstein donors in lactating Holstein recipients. It was performed at the Santa Rita Farm, Descalvado, SP, Brazil, between January 2005 and January 2006. Holstein recipients were milked 3 times a day and fed with total mixed ration. Embryos were obtained by a superstimulation protocol with two 3-mg norgestomet ear implants (Crestar; Intervet, Sao Paulo, Brazil) and an IM injection of 3 mg of estradiol benzoate (EB) on random days of the estrous cycle. Four days later, 500 IU FSH (Pluset; Laboratorios Calier de Argentina) was divided into 8 decreasing doses given IM 12 h apart. With the seventh FSH injection, 0.530 mg of sodium cloprostenol (PGF2, Ciosin; Coopers Brazil, Sao Paulo, Brazil) was given IM; implants were removed with the eighth FSH injection. Twelve hours later, 250 g gonadorelin (Fertagyl: Intervet, Brazil) was given IM, followed by two AI 12 and 24 h later. Ova/embryos were recovered 6.5 days after the first insemination. Recipients (n = 2543) received an injection of PGF2 and were observed in estrus (n = 1322). Presence of CL was evaluated by rectal palpation of cows that presented heat, and embryo transfers (n = 873) were performed with a nonsurgical method 6 to 8 days after heat. Pregnancy diagnoses were performed by ultrasonography (Aloka 500, probe 5.0 MHz; Aloka) on Days 25 and 39. Embryonic mortality was considered when any recipient pregnant on Day 25 was open on Day 39. Data were analyzed by logistic regression and variables included in the model were: donor status (lactating or non-lactating), recipient CL external size classification (1 > 2 > 3), donor–recipient synchrony (-1, 0, +1), fresh or frozen embryos, embryonic development (Mo, Bi, Bl, Bx – IETS), and embryo quality (degree 1, 2, 3 – IETS). The conception rate (CR) on Day 25 was influenced by donor–recipient synchrony (-1: 37.7%; 0: 46.9%; +1: 45.6%; P = 0.053). Embryonic development (Mo: 45.6%; Bi: 41.8%; Bl: 46.9%; Bx: 43.8%), embryo quality (1: 44.2%; 2: 47.4%; 3: 42.3%), CL external size classification (CL 1: 47.9%; CL 2: 43.5%; CL 3: 30.0%), embryo condition (fresh: 45.8%; frozen: 43.1%), and donor status (lactating: 45.5%; non-lactating: 47.08%) did not affect conception rate. The CR on Day 39 was also affected by donor–recipient synchrony (-1: 29.9%; 0: 34.9%; +1: 38.2%; P = 0.021). Embryonic mortality was affected by donor status (lactating, 16.6% vs. non-lactating, 24.0%; P < 0.10). These data suggest that donor–recipient synchrony is a major factor affecting conception rate in lactating Holstein recipients. [ABSTRACT FROM AUTHOR]

The objective of this study was to compare the calving rate after transfer of IVM/IVF/IVC embryos co cultured on both Vero and Vero/BRL cells (Duszewska 2000 Theriogenology 54, 1239?1247). Cumulus?oocyte complexes (COCs) were matured in TCM-199 supplemented with 10? FBS, 0.02 IU mL-1 FSH, 1 g mL-1 17?-estradiol, 0.2 mM Na pyruvate, and 50 g mL-1 gentamicin for 24 h at 38.5C in 5? CO2. Spermatozoa were prepared by the swim-up procedure. COCs were fertilized in Fert-TALP supplemented with 6 mg/mL-1 fatty acid-free BSA, 0.2 mM Na pyruvate, 50 g mL-1 gentamicin sulfate, 20 M penicillamine, 10 M hypotaurine, 1 M epinephrine, and 2 g/mL-1 heparin for 20 h at 38.5C in 5? CO2. The zygotes were randomly allocated to one of the co-culture systems: Vero (2 103 cells in a 40-L drop; 20 zygotes per drop), and Vero/BRL (1 103 Vero cells and 1 103 BRL cells in a 40-L drop; 20 zygotes per drop). The zygotes from Vero and Vero/BRL were cultured for 168 h post-insemination in drops of Menezo B2 supplemented with 10? FBS until 144 h and from 144 h to 168 h without FBS, at 38.5C in 5? CO2. Next, the blastocysts (Grade 1, according to IETS Manual) from Vero and Vero/BRL were transferred to recipients. The recipients were monitored daily for heat behavior, examined by ultrasound after 35 days and 65 days, and then observed monthly to confirm pregnancy. The results are presented in Table 1. Statistical significance was tested using the chi-square test. In spite of better development of cattle embryos on Vero/BRL cells than on Vero cells (P < 0.05), a lower rate of calving was obtained after transfer of these embryos to recipients than for those on Vero cells (P < 0.001). Higher loss of pregnancy after transfer of Vero/BRL embryos was observed in Days 35?65, which may indicate early fetal resorption. All calves were born naturally, healthy, and with normal weight. [ABSTRACT FROM AUTHOR]

Successful bovine embryo transfer requires synchronization of luteolysis, estrus and ovulation. The objective of the present study was to evaluate the effect of a combination of a PRID, PGF2 and eCG, on estrus synchronization and pregnancy rate in recipient heifers. A PRID (ASKA Pharmaceutical Co., Ltd., Tokyo, Japan) was inserted into the vagina at random days of the estrous cycle for 7 (n = 35) or 9 (n = 43) days. Two days before removal of the PRID, the heifers were injected with PGF2 IM (2 mL Resipron-C containing 0.25 mg mL-1 cloprostenol; ASKA). About half of the heifers in each group received 250 IU eCG IM (Serotropin; ASKA) at the time of PRID removal. Blood was collected several times from the start of treatment for 7 (n = 9) or 9 (n = 9) days and on the day of embryo transfer by jugular venipuncture; plasma was immediately separated and stored at -20C until assayed for plasma concentrations of estradiol-17? (E2) and progesterone (P4). The E2 and P4 determinations were performed by enzyme immunoassay after extraction by diethyl ether. Pregnancy was determined by ultrasonography on Day 30 (Day 0 = estrus). The rates of successful standing estrus (no. in estrus/PRID inserted), embryo transfer (no. transferred/estrus), and pregnancy (no. pregnancy/transferred) were compared between groups. Data were analyzed by chi-square analysis or Fisher''s PLSD test following ANOVA. Injection of eCG at the time of PRID removal had no significant effect on the rates of successful standing estrus, embryo transfer, or pregnancy (P > 0.05). The proportion of heifers treated for 9 days that exhibited standing estrus (93%, 40/43) was significantly higher than the proportion of heifers treated for 7 days that exhibited standing estrus (66%, 23/35, P < 0.01). Of the heifers that were treated for 9 days, the proportion of heifers exhibiting standing estrus within 2 days after the end of treatment was significantly higher (93%, 37/40) than for heifers that were treated for 7 days (65%, 15/23; P < 0.01). Pregnancy rates of heifers treated for 9 days (84%, 32/38) and 7 days (81%, 17/21) were not significantly different. The E2 : P4 ratio normally increases during follicle growth and CL regression. The plasma E2 : P4 ratio between the time of injection of PGF2? and the time of PRID removal was significantly higher for heifers that were treated for 9 days than it was for heifers that were treated for 7 days (P < 0.01). These results suggest that a combination of PRID treatment for 9 days and injection of PGF2? 2 days before PRID removal successfully synchronized estrus in recipient heifers and led to high pregnancy rates following embryo transfer. [ABSTRACT FROM AUTHOR]