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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]

The production of cloned animals by the transfer of a differentiated somatic cell into an enucleated oocyte circumvents fertilization. During fertilization, the sperm delivers a sperm-specific phospholipase C (PLCZ) that is responsible for triggering Ca2+ oscillations and oocyte activation. During bovine somatic cell nuclear transfer (SCNT), oocyte activation is artificially achieved by combined chemical treatments that induce a monotonic rise in intracellular Ca2+ and inhibit either phosphorylation or protein synthesis. In this study, we tested the hypothesis that activation of bovine nuclear transfer embryos by PLCZ improves nuclear reprogramming. Injection of PLCZ cRNA into bovine SCNT units induced Ca2+ oscillations similar to those observed after fertilization and supported high rates of blastocyst development similar to that seen in embryos produced by IVF. Furthermore, gene expression analysis at the eight-cell and blastocyst stages revealed a similar expression pattern for a number of genes in both groups of embryos. Lastly, levels of trimethylated lysine 27 at histone H3 in blastocysts were higher in bovine nuclear transfer embryos activated using cycloheximide and 6-dimethylaminopurine (DMAP) than in those activated using PLCZ or derived from IVF. These results demonstrate that exogenous PLCZ can be used to activate bovine SCNT-derived embryos and support the hypothesis that a fertilization-like activation response can enhance some aspects of nuclear reprogramming. [ABSTRACT FROM AUTHOR]

The Well of the Well (WOW) system has been developed to culture embryos in small groups or to track the development of single embryos. In the present study, we aimed to examine the effects of the microenvironment provided by the WOW system and embryo density on developmental rates, embryo quality and preimplantative gene expression profile of the resulting embryos. Embryos cultured in a group of 16 reached the blastocyst stage at a significantly lower level than zygotes cultured in a group of 50 (22.2 vs 30.3%), whereas zygotes cultured in WOW were able to compensate against low embryo densities, reaching a blastocyst rate as high as embryos cultured in a group of 50 (31.3 vs 30.3%). Moreover, embryos derived from WOW culture did not differ in terms of differential cell counts and apoptotic cell index compared with controls. The gene expression analysis revealed 62 transcripts to be upregulated and 33 transcripts to be downregulated by WOW culture. Comparing the in vivo derived blastocysts with the blastocysts derived from WOW culture, and group culture, expression of ATP5A1, PLAC8 and KRT8 was more similar to the embryos derived from WOW culture, whereas expression of S100A10 and ZP3 genes was more similar to blastocysts cultured in a group. In conclusion, microenvironment as well as embryo density significantly affected developmental rates. While subsequent blastocysts did not differ in terms of differential cell counts and apoptotic cell index, significant differences were observed in terms of the relative abundance of transcripts in the resulting embryos. [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]

Assessment of morphological stage grade is a subjective procedure. Stage grade is of vital importance to, among other things, recipient synchrony for the purpose of establishing successful pregnancies. Asynchronous embryo transfer has led to decreases in pregnancy rates (Farin et al. 1995 Biol. Reprod. 52, 676–682) and has been implicated in contributing to large offspring syndrome (Young et al. 1996 Theriogenology 45, 231). Differences in embryo kinetics based on culture conditions have been well documented (Mello et al. 2005 Reprod. Fert. Dev. 17, 221 abst). Whether such differences are the result of species, breed, metabolic stress, sire effects, or separation from an in vivo environment has yet to be determined. The correlation between oxygen respiration rates and embryo morphology as well as embryo diameter in bovine embryos produced in vitro has shown promise in the development of a more objective predictor of embryo quality and perhaps pregnancy initiation (Lopes et al. 2005 Reprod. Fert. Dev. 17, 151 abst). As well, recent examination of gene expression patterns of in vitro-derived bovine embryos seems to indicate that longer periods of in vitro culture are associated with lower rates of embryo survival (Lonergan et al. 2006 Theriogenology 65, 137–152). We hypothesize that differences do exist in the number, rate, and morphological appearance of blastocysts and that these parameters are in large part based on culture conditions in vitro. The objective of this experiment was to determine the timing and distribution of blastocyst formation of in vitro-produced bovine embryos cultured in SOF8, CR18AA, and KSOM8, under a standard incubation environment. Bovine ovaries from a local abattoir were aspirated and matured for 18-22. Oocytes were fertilized with frozen-thawed Percoll-separated semen from a Holstein bull. Presumptive zygotes were vortexed to remove cumulus cells and placed into 3 different culture media in a highly humidified atmosphere containing 20% oxygen, 5% carbon dioxide, and compressed air at 38.5C. Embryos were evaluated specifically at 168 h post-insemination (Day 7) and assigned a morphological stage grade (IETS) to determine fixed time point differences. A total of 6 complete replicates were performed. Only embryos exhibiting the presence of a blastocoel at this time were documented (early blast, mid-blast, expanded blast). At 168 h post-insemination, there were no significant differences in the total number of embryos reaching early or mid-blast stage in any of the media. However, chi-square analysis revealed an increase in the number of expanded blastocysts in SOF (n = 813) and CR1 (n = 838) treatments compared to KSOM (n = 824; P < 0.0001). Expanded blastocysts in SOF were also greater in number than in CR1 (P < 0.05). Embryo selection based on development to the expanded blastocyst stage on Day 7 may prove useful in increasing pregnancy rates, and may validate qualitative correlations based on oxygen consumption and gene expression profiles for embryos produced in vitro. [ABSTRACT FROM AUTHOR]