bluuebunny/crossref_metadata_2025 huggingface dataset:
Designing of Disubstituted Derivatives of mer-Alq3: Quantum Theoretical Study
Authors: Irfan Ahmad , Cui Ruihai , Zhang Jingping , Nadeem Muhammad
Published: August 2010

Different disubstituted derivatives of tris(8-hydroxyquinolinato)aluminium (mer-Alq3) have been designed. An electron donating group (–CH3) has been substituted at position 4; along with electron withdrawing groups (–Cl, –CN, and –F) at position 5 (i.e. tris(4-X-5-Y-8-hydroxyquinolinato)aluminium), as well as the molecules arising from reversion of these substitutions. The designed systems have been optimized at the B3LYP/6–31G* and TD-B3LYP/6–31G* level of theories, in the ground and excited states, respectively. The absorption and emission spectra have been calculated at the TD-B3LYP/6–31G* level. A significant blue shift has been observed for tris(4-methyl-5-cyano-8-hydroxyquinolinato)aluminium (2), and red shifts for each of tris(4-methyl-5-fluoro-8-hydroxyquinolinato)aluminium (3), tris(4-chloro-5-methyl-8-hydroxyquinolinato)aluminium (4), and tris(4-cyano-5-methyl-8-hydroxyquinolinato)aluminium (5). The reorganization energy in solvent best elucidates the charge transport behaviour (p- or n-type), rather than the equivalent gas phase for mer-Alq3 and its derivatives. The reorganization energies indicate that these new derivatives have better or comparable mobility when compared to the parent molecule. The electrostatic surface potential study suggested that photostability would be enhanced. On the basis of energy decomposition analysis, we have explained the distribution pattern of frontier molecular orbitals.
Crystallization and Melting of Oriented Parent - Daughter Lamellae in Sheared Isotactic Poly(propylene)
Authors: Phillips Andrew , Zhu Peng-wei , Hadinata Chitiur , Edward Graham
Published: August 2010

Alpha isotactic poly(propylene) (α-iPP) exhibits a form of lamellar branching that is unique among semicrystalline polymers, where the branches have a distinct orientation relationship with the original crystalline lamellae. This is termed a parent–daughter relationship (PD). By allowing the structure to crystallize in an oriented form, a bimodal orientation of lamellae is developed and the individual contributions of PD lamellae can be observed using wide-angle X-ray scattering (WAXS). The present study investigated oriented PD lamellae during flow-induced crystallization and subsequent melting using time resolved rheo-WAXS. During crystallization the planes of the daughter lamellae were observed to curve towards the flow direction as they grew from their parent lamellae. This was explained by the influence of neighbouring daughter lamellae confining their growth direction. Oriented daughter lamellae were found to melt ~5°C lower than oriented parent lamellae, which provides a new explanation for the multiple melting behaviour observed in the melting thermograms of sheared α-iPP.
A Computational Study of the Radical Ring-Opening Polymerization of Diphosphetanes
Authors: Coote Michelle L. , Namazian Mansoor , Wild S. Bruce
Published: August 2010

Radical ring-opening of four-membered 1,3-diphosphetanes appears to be a facile process of similar propensity and mechanism to that of the parent phosphetanes, but the greater kinetic stability of the diphosphetanes could make them more attractive precursors for polymerization.
Jan Romuald Zdysiewicz, FRACI, 1943 - 2010
Authors: Bennett Jennifer M. , Cameron Donald W.
Published: August 2010

This Obituary gives an account of the life of Jan Zdysiewicz, known to most of us as John Z, former Managing Editor of the Australian Journal of Chemistry, who died in the Royal Melbourne Hospital on 10 March 2010.
Challenge testing of gametes to enhance their viability
Authors: Callesen Henrik
Published: August 2010

Embryos, oocytes and spermatozoa undergo several manipulations during the in vitro procedures that are an integral part of assisted reproductive technologies (ART) in mammals. Consequently, some of the gametes are damaged irreparably, whereas others react to these challenges with some sort of survival mechanism that enables them to come through the process. The details of the mechanism remain unknown but, if identified, it could have immense potential as a new way to improve the viability of embryos produced by ART. However, few publications describe systematic ways to challenge test gametes and then to use the results as a basis for improving gamete viability. Furthermore, new methods to monitor the reactions of gametes to such challenge tests are needed. In the present review, these two issues are discussed, as are some of the conditions necessary before a challenge test protocol can be part of future work with ART.
Pregnancy recognition and abnormal offspring syndrome in cattle
Authors: Farin C. E. , Farmer W. T. , Farin P. W.
Published: August 2010

Development of the post-hatching conceptus in ruminants involves a period of morphological expansion that is driven by complex interactions between the conceptus and its intrauterine environment. As a result of these interactions, endometrial physiology is altered, leading to establishment of the pregnancy and continued development of the placenta. Disruption of normal fetal and placental development can occur when embryos are exposed to manipulations in vitro or when inappropriate endocrine sequencing occurs in vivo during the pre- and peri-implantation periods. The present review addresses the development of the post-hatching bovine conceptus, its interactions with the maternal system and changes in development that can occur as a result of in vivo and in vitro manipulations of the bovine embryo.
Coordinated regulation of follicle development by germ and somatic cells
Authors: Binelli Mario , Murphy Bruce D.
Published: August 2010

The continuum of folliculogenesis begins in the fetal ovary with the differentiation of the oogonia and their isolation within the primordial follicles. Primordial follicle activation is an enigmatic process, whereby some follicles enter the growing pool to become primary follicles, thereby embarking on an irreversible progression towards ovulation or atresia. This process is under the coordinated regulation of factors from the oocyte itself, as well as from the somatic cells of the ovary, in particular the theca and granulosa cells, which are structural components of the follicle. These two influences provide the principal stimuli for the growth of the follicle to the late preantral or early antral stage of development. The endocrine effects of the gonadotrophins FSH and LH are essential to the continued progression of the follicle and most atresia can be attributed to the failure to receive or process the gonadotrophin signals. The peri-ovulatory state has received intensive investigation recently, demonstrating a coordinated role for gonadotrophins, steroids, epidermal growth factor family proteins and prostaglandins. Thus, a complex programme of coordinated interaction of governing elements from both germ and somatic cell sources is required for successful follicle development.
Towards the use of microfluidics for individual embryo culture
Authors: Krisher R. L. , Wheeler M. B.
Published: August 2010

Mammalian embryo development is still relatively inefficient in vitro. Much research has been conducted on the chemical environment, or culture medium, surrounding the embryo, but little attention has been given to the actual physical culture environment, which has changed very little over the years. The application of microfluidics to embryo production in vitro is a tantalising approach that may alleviate some of the limits that traditional microdrop culture places on embryo development and research into gamete and embryo physiology. These devices may lead to enhanced in vitro embryo development and quality by more closely mimicking the in vivo environment. Initial work in this area is promising and gives us proof-of-principle that these unique microfluidic systems may indeed be applicable to in vitro culture of gametes and embryos. The present paper reviews the advantages of microfluidics for in vitro embryo production: how the platforms are manufactured, the current uses of microfluidics in assisted reproduction, static v. dynamic culture environments, individual gamete and embryo culture and the future directions of microfluidic application to in vitro embryo production and manipulation. Finally, preliminary data from our laboratory using a new microfluidic well insert for porcine, bovine and murine embryo culture is discussed.
Factors affecting fertilisation and early embryo quality in single- and superovulated dairy cattle
Authors: Sartori Roberto , Bastos Michele R. , Wiltbank Milo C.
Published: August 2010

Data on fertilisation and embryo quality in dairy cattle are presented and the main factors responsible for the low fertility of single-ovulating lactating cows and embryo yield in superovulated dairy cattle are highlighted. During the past 50 years, the fertility in high-producing lactating dairy cattle has decreased as milk production increased. Recent data show conception rates to first service to be approximately 32% in lactating cows, whereas in heifers it has remained above 50%. Fertilisation does not seem to be the principal factor responsible for the low fertility in single-ovulating cows, because it has remained above 80%. Conversely, early embryonic development is impaired in high-producing dairy cows, as observed by most embryonic losses occurring during the first week after fertilisation. However, in superovulated dairy cattle, although fertilisation failure is more pronounced, averaging approximately 45%, the percentage of fertilised embryos viable at 1 week is quite high (>70%). Among the multifactorial causes of low fertility in lactating dairy cows, high feed intake associated with low concentrations of circulating steroids may contribute substantially to reduced embryo quality. Fertilisation failure in superovulated cattle may be a consequence of inappropriate gamete transport due to hormonal imbalances.
New approaches to superovulation and embryo transfer in small ruminants
Authors: Menchaca A. , Vilariño M. , Crispo M. , de Castro T. , Rubianes E.
Published: August 2010

The present paper reviews the current state of knowledge in multiple ovulation and embryo transfer (MOET) technology in small ruminants, focusing on recently reported information. Major new findings are related to follicular wave patterns in small ruminants, the elucidation of follicular dominance and the integration of this information into ovarian superstimulatory treatment protocols. Follicular dynamics determine steroid and gonadotrophin secretion, follicular responses to FSH, ovulatory responses and embryo yield. Protocols that control follicular dominance have been designed to allow the initiation of superstimulation at the beginning of a follicular wave. New approaches consist of (1) synchronisation of ovulation for superstimulation during Wave 1 (i.e. Day 0 protocol), (2) pretreatment with a gonadotrophin-releasing hormone (GnRH) antagonist from 10 days prior to FSH treatment to avoid follicular dominance and (3) progesterone–oestradiol cotreatment to synchronise follicle wave emergence. These protocols provide a homogeneous pool of small follicles that are gonadotrophin responsive, enhancing the superovulatory response and embryo yield with a reduction in the incidence of unovulated follicles and early regression of corpora lutea. In addition, the rate of fertilisation failure has been reduced by using an inducer of ovulation (i.e. GnRH) associated with intrauterine insemination. In summary, the application of recently acquired knowledge has resulted in relevant improvements in MOET programmes in small ruminants.
105 CRYOPRESERVATION OF VENDA COCK SPERMATOZOA: EFFECT OF CRYOPROTECTANT FOLLOWING ANALYSIS BY COMPUTER-ASSISTED SPERM ANALYSIS
Authors: Mphaphathi M. L. , Makhafola M. B. , Munyai P. H. , Nedambale T. L. , Luseba D.
Published: August 2010

The choice of ideal permeable cryoprotectant for cock semen equilibration and freezing is critical. The aim of this study was to compare three different cryoprotectants [dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propanediol (PND)] following cryopreservation. The abdominal massaging technique was used for semen collection from 5 Venda cocks. Individual ejaculates were diluted with modified Kobidil+ (mK+) extender (fraction A) at a ratio of 1:2 and equilibrated for 2 h at 5°C. Semen samples of 5 μL were taken at 0, 60, and 120 min and evaluated by CASA for spermatozoa motility parameters (rapid, medium slow, static, NPM and PM) and velocity. Semen was further diluted at 1:1 volume ratio with mK+ supplemented with 8% DMSO, EG and PND (fraction B), and equilibrated for additional 2h at 5°C and evaluated at 60 and 120 min for motility and velocity. Cooled semen were then transferred into 0.25-mL straws and placed into a programmable freezer. The temperature of the chamber was decreased in a stepwise manner, from 5°C to -20°C, at the rate of 1°C/min until it reached the target temperature. The straws were exposed to liquid nitrogen (LN2) vapor and then plunged into LN2 (-196°C). The semen straws were stored into LN2 tank at -196°C. After 3 months of storage, semen straws were thawed at 5°C and evaluated by CASA for spermatozoa motility and velocity. Data were analyzed by ANOVA. There were no significance differences between DMSO and EG regarding the survival and motility rate of frozen/thawed semen; however, these parameters were lower compared with the fresh semen. The PND was not a suitable cryoprotectant to cryopreserve Venda spermatozoa. In conclusion, ethylene glycol was found to be a suitable cryoprotectant to cryopreserve spermatozoa of South African Venda cocks.

Department of Agriculture Forestry and Fisheries, NRF,ARC, Department of Science & Technology.
107 MOTILITY PARAMETER PATTERNS OF NGUNI BULLS: EFFECT OF VARIOUS GYCEROL CONCENTRATIONS FOLLOWING CRYOPRESERVATION
Authors: Nedambale T. L. , Mphaphathi M. L. , Munyai P. H. , Tshabalala M. , Malusi P. , Dinnyes A.
Published: August 2010

The Nguni breed of South Africa is small, hardy, disease-tolerant, thrives on poor pasture, and was regarded as an inferior breed in the past. For optimizing routine fresh and freezing of Nguni bull semen analysis, 3 different concentrations of glycerol (7, 10, and 14%) were examined. Ten ejaculates were collected from each 6 Nguni bulls using electro-ejaculator at ARC, Irene, South Africa. Following semen collection, semen was examined for macroscopic (volume, pH, and concentration) and microscopic (motility) parameters. The semen was extended with Tris + 10% egg yolk diluent at a ratio of 1 : 2 (v/v) and frozen at different concentrations of glycerol (7, 10, and 14%). The semen was then evaluated using the sperm class analyzer (SCA; CASA system) for progressive motility parameters. Fresh and frozen-thawed were fixed and stained with Nigrosin-Eosin for morphology (dead and live). Data were analyzed by ANOVA. There was a significant difference among individual Nguni bull spermatozoa volume and concentration. Analyzed frozen-thawed Nguni spermatozoa resulted in a significant (P < 0.05) difference of spermatozoa motility parameters frozen in 10% glycerol (68%) compared with 7 (41%) and 14% glycerol (30%). In conclusion, Nguni spermatozoa can be cryopreserved successfully when 10% of glycerol concentration is used. The results of this study will improve the viability of cryopreserved Nguni bull spermatozoa following the development of a South African semen cryo-gene bank.

This study was supported by grants from National Research Foundation (NRF), Hungarian, South African Bilateral Scientific and Technological (TETNo. OMFB-00302/2008, RT24000) collaborative project. Department of Agriculture Forestry and Fisheries (DAFF, RPPP15).
11 FERTILITY EVALUATION OF THE GOAT SEMEN DILUTED IN CITRATE-EGG YOLK EXTENDER WITH HIGH OR LOW EGG YOLK CONCENTRATION, COOLED AND STORED AT 5°C FOR 24 HOURS
Authors: Bispo C. A. S. , Palhão M. P. , Coelho P. G. B. , Carvalho G. R. , Costa S. L. , Machado T. P. , Pugliesi G. , Ker P. G.
Published: August 2010

This experiment was designed to evaluate, in vitro and in vivo, the cooling resistance of the goat semen diluted in citrate-egg yolk extender with high (20%) or low (2.5%) concentration of egg yolk. The experiment was conducted at Dairy Goat Station of the Animal Science Department (Federal University of Viçosa, MG, Brazil), during the natural breeding season (March to June). Four bucks of the Alpine (n = 2) and Saanen (n = 2) breeds were used as semen donors. The bucks did not have apparent abnormalities of the reproductive tract and the fertility was proved based on andrologic examination and the records from the previous breeding season. The semen was collected by the artificial vagina method and a fresh sample was analyzed for the sperm cell motility and concentration. Therefore, the semen was diluted in citrate-egg yolk extender with high (T1 = 20%) or low (T2 = 2.5%) concentration of egg yolk, cooled and stored at 5°C for 24 h, before being used either for quality analysis or for AI. Reproductive-age female Saanen and Alpina goats (n = 68) were inseminated via cervix with cooled semen from T1 (n = 32) or from T2 (n = 36). The parametric data from cooled semen quality (sperm cell motility, vigor, hyposmotic swelling test, morphology, and supravital staining) were analyzed by ANOVA, for the main effect of the treatment, using the SAEG 9.0 program (UFV, 2005). The differences between means were accessed by the Student-Newman-Keuls test. The qualitative data from pregnancy rate were tested by chi-square (Ayres et al. BioEstat 2.0, 2000). The mean ± SEM for each quality test of the cooled semen, as well the P-value for comparisons between treatments were as follows: motility, 68.6 ± 15.5 and 78.0 ± 5.6% (P < 0.05); vigor, 2.5 ± 0.6 and 3.2 ± 0.3 (P < 0.05); supravital staining, 59.4 ± 17.3 and 71.2 ± 10.6% (P < 0.05); and hyposmotic test, 42.3 ± 15.8 and 56.3 ± 11.6% (P < 0.05), respectively, for T1 and T2. The pregnancy rate was higher (P < 0.05) in goats inseminated with cooled semen from T2 (66.7%) than in those inseminated with T1 (31.25%). The number of the offspring did not differ (P > 0.05) between treatments (1.3 ± 0.5 and 1.4 ± 0.5 kids, forT1 andT2, respectively). The interpretation was that the goat semen diluted, cooled, and stored in citrate-egg yolk extender prepared with a low (2.5%) concentration of egg yolk produced higher pregnancy rate and the tests of semen quality were sensitive to assess this difference.

Financial support was provided by CnPQ (Brazil).
112 EFFECT OF EGG YOLK CONCENTRATION IN TEST BUFFERED EXTENDER ON SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION
Authors: Saenz J. R. , Dumas C. , Dresser B. L. , Gómez M. C. , Godke R. A. , Pope C. E.
Published: August 2010

Our purpose was to examine the effect of egg yolk concentration (EY; 2, 5, or 10%) on in vitro survivability and functionality of cat epididymal spermatozoa cryopreserved in TEST-buffered extender (TYB). Testes were transported in HEPES saline; epididymes were dissected in HEPES 199 medium (He199) and repeatedly sliced. The sperm suspension was filtered (40 μ), layered onto a density gradient column (Isolate®, Irving Scientific, Santa Ana, CA, USA), and centrifuged at 650 g for 20 min. Aliquots of the sperm pellet were extended in TYB containing 2, 5, or 10% EY. After cooling to 4°C, samples were diluted 1:1 with TYB containing 2, 5, or 10% EY + 12% glycerol in 4 steps as modified from Gao DY et al. Hum. Reprod. 1995 10, 1109-1122. Then, samples were loaded into 0.25-mL straws, sealed, and frozen on a dry ice block (-80°C) for 20 min before storage in LN2. Straws were thawed by exposure to air (˜22°C) for 5 s and immersion in a 60°C water bath for 5 s. Samples were diluted by addition of He199 in 7 steps as modified from Gao DY et al. Hum. Reprod. 1995 10, 1109-1122 centrifuged at 200g for 10 min, and pellets resuspended in He199. Motility (Mot, phase contrast, 37°C), membrane integrity (M.I., SYBR 14-PI), and acrosomal status (A.S., FITC-PNA) were evaluated at 0 h, after gradual cooling to 4°C and after freezing at 0 and 3 h post-thaw (37°C). Ten replicates were done. Cumulus oocyte complexes (COC) were placed in modified TCM-199 and cultured for 24 h in 5% O2, 5% CO2, and 90% N2 at 38°C (IVM). For IVF, COC were co-incubated with spermatozoa frozen in TYB + 2% egg yolk or HSPM (no egg yolk) in droplets (1 million sperm/mL) of IVF medium under 5% CO2 in air at 38°C. After 18 h, oocytes were rinsed and cultured until blastocyst development was evaluated (Day 8). There were no treatment differences at any time or temperature point for the 3 sperm characteristics evaluated (one-way ANOVA; P > 0.05). As shown in the Table 1, at 0 h post-thawing, sperm in each group retained ˜70% of their initial pre-freeze motility. After 3 h of post-thaw incubation, motility decreased to ˜50% of the pre-freeze value. Cooling to 4°C did not affect membrane integrity or acrosomal status, but post-thaw values were reduced by 30-35% as compared with pre-freeze. Cleavage frequency and blastocyst development of 284 IVM oocytes after IVF using sperm frozen in TYB + 2% EY and HSPM were 53 v. 52% and 42 v. 38%, respectively (P > 0.05). In summary, we have shown that cat epididymal spermatozoa can be frozen successfully in a cryoprotectant solution containing minimal egg yolk (2%).

Table 1.Motility, membrane integrity, and acrosomal status of cat epididymal sperm after cryo-storage
114 INFLUENCE OF COOLING TIME IN THE VIABILITY OF POST-THAW RAM SEMEN
Authors: Sedenho T. , Cadamuro V. H. A , Oliveira L. A. , Abreu C. O. , Martinez A. C.
Published: August 2010

The objective of the present experiment was evaluated the influence of different cooling time in the viability of post-thaw ram semen. The experiment was conducted in Umuarama, Paraná, Brazil. A pool of 9 ejaculates from Saint Inês rams was analyzed. Motility (mot, in percent), vigor (vig, in percent), and concentration were evaluated. The samples were diluted in Tris-egg-yolk extender, in an insemination dose of 30 million spermatozoa/mL. The straws were taken into a refrigerator for cooling in 5°C. The tested times of cooling were 1 h (T1), 2 h (T2), 3 h (T3), 4 h (T4), and 5 h (T5). After each one of these periods, motility and vigor were evaluated and the semen were freezing in a polystyrene container with liquid nitrogen. After 10 days, the straws were thawed, motility and vigor were evaluated again, and the samples were submitted to thermo-resistence test. The results were analyzed by Proc GLM of SAS software (SAS Institute, version 8.0, Cary, NC, USA). For fresh semen, the motility and vigor were 78.89 ± 8.75 and 4.00 ± 0.47, respectively. After cooling, the results were mot T1 (47.78 ± 10.30), vig T1 (3.00 ± 0.00); mot T2 (45.56 ± 8.31); vig T2 (2.67 ± 0.47); mot T3 (45.56 ± 14.23), vig T3 (2.78 ± 0.42), mot T4 (53.33 ± 9.43), vig T4 (3.11 ± 0.31), mot T5 (45.56 ± 13.43), vig T5 (2.78 ± 0.63). For thermo-resistance test, the results of each cooling time were T1 motility = 14.44 ± 11.65; T1 vigor = 1.78 ± 1.03; T2 motility = 16.67 ± 9.43; T2 vigor = 2.22 ± 0.92; T3 motility = 23.33 ± 9.43; T3 vigor = 2.33 ± 0.47); T4 motility = 26.67 ± 8.16; T4 vigor = 2.78 ± 0.42; T5 motility = 27.78 ± 7.86; T5 vigor = 2.56 ± 0.50). There were no statistical differences for the cooling time results of motility, vigor and thermo-resistance test (P < 0.05). In the conditions of this research, the cooling time had no influence in the post-thaw viability.
136 EARLY EMBRYONIC LOSSES USING PROGESTERONE SUPPLEMENTATION IN CYCLIC AND NONCYCLIC RECIPIENT MARES
Authors: Araujo G. H. M. , Moya-Araujo C. F. , Rocha A. N. , Burns C. M. , Burns S. D. , Meira C.
Published: August 2010

The aim of the present study was to determine the effectiveness of the commercial progesterone to maintain the pregnancies in noncyclic recipient mares and if it helps to decrease the early embryonic death when used in cyclic pregnant recipient mares. A total of 199 mares were used during February to April of the 2008 breeding season at Burns Ranch. The mares were assigned to groups according to their reproductive status. Noncyclic (n = 42) mares were prepared as embryo transfer recipients using our standard protocol with estradiol cypionate (3 days, 10/6/4 mg, Wickliffe, USA) and long action progesterone (P4LA, 1500 mg, BET Labs, Lexington, KY, USA), and 157 naturally cyclic mares were used from Day 3 to 8 after ovulation as embryo transfer recipients. After embryo transfer, 134 of the 157 cyclic mares got pregnant at 15 days. Some of the pregnant mares (n = 43) were assigned to a progesterone-supplemented group; these mares were administered P4LA (1500 mg) after the first pregnancy diagnosis at around 15 days and repeated 7 days later. The remaining mares were placed in a control group (n = 91), with no treatment, in order to compare the embryonic loss rates for the 3 groups. Recipients were evaluated for the presence of the embryonic vesicle between 13 and 16 days (embryo age), and pregnancies were monitored at 30 and 45 days to determine the early embryonic loss. Statistical analyzes were performed using the chi-square test with 5% defined as significance. Pregnancy rates at 13 to 16 days were not different (P = 0.336) for noncyclic and cyclic recipients (39/42, 90.7% and 134/157, 85.4%, respectively). The embryonic loss was evaluated in the 3 groups as follows: control group; 7 of 91 pregnancies were lost between 15 and 30 days (7.69%) and 2 of 84 were lost between 30 and 45 days (2.38%); the progesterone-supplemented group had lost 4 of the 43 pregnancies between 15 and 30 days (9.30%); and the noncycling progesterone mares had lost 2 of the 39 between 30 and 45 days (5.13%). Rates of embryo loss before 45 days of gestation were not different for the control mares with no progesterone supplementation (9.89%), progesterone-supplemented cyclic mares (9.3%), and noncyclic recipients (5.13%). The noncyclic recipients maintain gestation with the supplemental progesterone with no influence on embryo loss. These results demonstrate no advantage in the use of exogenous progesterone to supplementation for cyclic embryo recipients.

Burns Ranch Inc., Menifee, California, USA.
140 INTERFERON-τ SECRETION BY IN VIVO DERIVED BOVINE TROPHOBLASTIC VESICLES
Authors: Hashiyada Y. , Takahashi H. , Geshi M.
Published: August 2010

In ruminants, interferon-τ (IFN-τ) is a major pregnancy factor, secreted by the embryonic trophoblast cells during the pre-implantation period, being important for the maternal-fetal recognition. The co-transfer of bovine trophoblastic vesicles (bTVs) derived from in vivo recovered conceptuses is known to promote the successful implantation of embryos with expected lower viability, such as in vitro handled embryos, through the effects of IFN-τ secreted by bTVs. We have also reported that the pregnancy rate was improved using this technique in early pregnancy phase (Hashiyada et al. 2005 J. Reprod. Dev. 51, 749-756). However, the IFN-τ secretion level from bTVs has not been well known. Therefore, the objective of the present study was to measure concentration of IFN-τ released from individually cultured bTVs in vitro. Furthermore, we also investigated the transition of IFN-τ level in continuous culture of bTVs. Blastocysts were produced by artificial insemination of Japanese black cows following superstimulatory treatment and were recovered on 16 or 18 days post-estrus. Sixty-eight bTVs were prepared from 23 elongating blastocysts, 3 to 20 mm in length, by dissection using a surgical blade. Each trophoblastic fragment, 1 to 1.5 mm in width, was cultured in a well of 96-well plates using TCM-199 supplemented with 20% (v/v) fetal bovine serum and 0.1 mM β-mercaptoethanol at 38.5°C in a humidified atmosphere of 5% CO2 in air. After 24 h of culture, fragments of unformed vesicles were re-cultured for an additional 24 h; 10 bTVs from this group were continuously cultured until Day 24 (the day of insemination was defined as Day 0). The volume of culture medium was 100 μL/well/day until Day 2 and thereafter changed to 200 μL/well/2 days to terminate. The viability of bTVs was assessed based on maintained spherical shape of vesicle, morphologically. Exchange and collection of culture media, morphological observation of bTVs were performed on Days 1, 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, and 24. Culture fluids were stored at -30°C. IFN-τ was measured by RIA (Takahashi H et al. 2005 Theriogenology 63, 1050-1060). Data were analyzed by Student’s τ-test Initial IFN-τ secretion did not differ between groups that had formed and unformed vesicles on Day 1, 89.8 ± 7.1 (mean ± SEM, n = 41) and 76.6 ± 7.2 ng mL-1 (n = 27), respectively. On Day 2, in the unformed group, all of the fragments had made vesicles and the IFN-τ increased to 99.4 ± 11.8 ng mL-1. In the extended culture group (n = 10), IFN-τ secretion tended to increase from Day 2 (66.9 ± 14.2 ng mL-1) to Day 8 (166.0 ± 46.7 ng mL-1) (P = 0.06). However, this large amount of IFN-τ on Day 8 significantly decreased from Day 10 (32 ± 4.9 ng mL-1, P < 0.05) to Day 24 (9.2 ± 1.0 ng mL-1, P < 0.05) gradually. The survival rate of these bTVs decreased to 90% (9/10) on Day 10 and then to 60% (6/10) during Days 18 to 22. These results indicate that bTVs cultured for a long term in vitro might decrease IFN-τ secretion.
144 USE OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT AND SURVIVAL FOLLOWING VITRIFICATION
Authors: Block J. , Bonilla L. , Hansen P. J.
Published: August 2010

The objective of the present study was to determine whether culture of bovine embryos in a proprietary serum-free culture medium, Block-Bonilla-Hansen-7 (BBH-7), could improve development to the blastocyst stage and enhance survival following vitrification. For Exp. 1, embryos were produced in vitro and cultured in BBH-7 or modified synthetic oviductal fluid (mSOF; as in zygote 10:341 except with 10 μL mL-1 of nonessential amino acids, 20 μL mL-1 of essential amino acids, and 1 mg mL-1 of polyvinyl alcohol instead of albumin) in 5% (v/v) oxygen. Grade 1 expanded blastocysts were harvested at Day 7 post-insemination and vitrified using the open-pulled straw method (Vagta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Vitrified embryos were thawed and cultured in vitro in either mSOF or BBH-7 supplemented with 10% fetal bovine serum and 50 μM dithiolthreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h post-thaw. There was no effect of culture medium on cleavage rate. The proportion of oocytes that developed to the blastocyst and advanced blastocyst stages (expanded, hatching, and hatched) at Day 7 was higher (P < 0.001) for embryos cultured in BBH-7 than for embryos cultured in mSOF (41.9 ± 2.0 v. 14.7 ± 2.0% and 31.1 ± 1.3 v. 6.4 ± 1.3%, respectively). There was no effect of culture medium on re-expansion rates at 24, 48, and 72 h post-thaw or on hatching rates at 48 or 72 h. However, the proportion of embryos that were hatching or had hatched by 24 h post-thaw was higher (P < 0.001) for BBH-7 than for mSOF (59.0 ± 0.5 v. 26.7 ± 0.5%). For Exp. 2, late lactation and/or repeat breeder, lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol. Embryos were produced in vitro and cultured in BBH-7 in 5% (v/v) oxygen. Vitrified embryos were produced as for Exp. 1. Fresh embryos were grade 1 expanded blastocysts harvested at Day 7 after insemination. A single embryo was transferred at Day 7 after putative ovulation to all cows with a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at Day 28-30 of gestation by ultrasonography. There was no difference in the proportion of recipients that became pregnant after receiving either a fresh (7/18 = 39%) or vitrified (10/27 = 37%) embryo cultured in BBH-7. The results of the present study indicate that BBH-7 can be used to increase the proportion of oocytes that develop to the blastocyst stage. Moreover, the results demonstrate that vitrified embryos produced after culture in BBH-7 can achieve pregnancy rates similar to those obtained using fresh embryos.

Support: USDA 2006-55203-17390 and Southeast Milk Checkoff Program.
150 DEVELOPMENT OF ZONA-FREE AND WITH ZONA PARTHENOGENETIC GOAT EMBRYOS IN DIFFERENT MEDIA
Authors: Jena M. K. , Malakar D. , De A. K. , Garg S. , Akshey Y. S.
Published: August 2010

The present study was carried out to see the developmental efficiency of zona-free and with zona parthenogenetic goat embryos cultured in Research Vitro Cleave from Cook Australia (RVCL), Embryo Development Media (EDM), modified synthetic oviductal fluid (mSOF), and modified Charles Rosenkrans media (mCR2a). Zona-free embryos were cultured in 4 media, whereas with zona embryos were cultured in 3 media except mCR2a. Ovaries were collected from slaughterhouse and oocytes were isolated by puncturing the follicles in medium containing Dulbecco’s phosphate-buffered saline, 3% BSA, and 50 μg mL-1 gentamicin. Oocytes were matured in maturation medium containing TCM-199 (HEPES modified), 0.05 mg mL-1 Na pyruvate, 0.003 mg mL-1 L-glutamine, 5.5 mg mL-1 glucose, 3 mg mL-1 BSA, 5 μg mL-1 FSH, 10 μg mL-1 LH, 1 μg mL-1 estradiol-17β, 50 μg mL-1 gentamicin, and 10% FBS in 5% CO2 in air at 38.5°C. The COC (15 to 20 oocytes) were placed in 100-μL droplets of maturation medium and incubated in a CO2 incubator (5% CO2 in air) with maximum humidity at 38.5°C for 27 h. Matured oocytes were made cumulus free by treatment with hyaluronidase (0.5 mg mL-1) and zona-free by pronase (2 mg mL-1) in zona-free parthenogenesis. Then the oocytes were activated by 5 μM Ca ionophore for 5 min in a CO2 incubator and then treated with 2 mM 6-DMAP for 4 h. Activation was also done by electrical activation with DC 1.78 kV cm-1, 20 μs, and 2 pulses. Then the zona-free oocytes were kept for in vitro culture in 4 types of media such as RVCL, EDM, mSOF, andm CR2a for 7 days in 5% CO2 in air at 38.5°C. The cleavage rate andmorulae formation were observed in RVCL 40.95%, 13.95%, in EDM 46.92%, 14.75%, in mCR2a 56.66%, 5.88%, and in mSOF 48.23%, 14.63%, respectively. The cleavage rate and morulae formation were also found 55.9%, 14.63% during chemical activation and 32%, 12.5% in electrical activation. Hence, better result was found in chemical activation than electrical activation. For with zona parthenogenesis, the matured oocytes were chemically activated by 5 μM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h. Then the oocytes were cultured in RVCL, EDM, and mSOF in 100-μL micro-drops media for 7 days. The cleavage, morulae, and early blastocyst production rate were as follows: cleavage rate 75.68%, 72.03%, and 57.11%; morulae 44.61%, 30.29%, and 40.22%; and early blastocyst 17.49%, 11.88%, and 25.01% in RVCL, EDM, and mSOF, respectively. Hatched blastocyst formation rate was 6.75%, 5.48%, and 1.15% in RVCL, EDM, and mSOF, respectively. It could be concluded that zona-free parthenogenetic embryos were produced better in EDM medium and with chemical activation. With zona parthenogenetic embryo development was significantly (P < 0.05) higher in RVCL and EDM media.
158 SUCCESSFUL TRANSFER OF FRESH AND VITRIFIED BIOPSIED EQUINE EMBRYOS
Authors: Cullingford E. L. , Stokes J. E. , Bouma G. J. , McCue P. M. , Seidel Jr G. E.
Published: August 2010

In horses, determination of certain genetic traits/alleles (e.g. HYPP, SCID, sex, or color) before transfer of embryo often would be advantageous due to the costs of these pregnancies. An attractive option is pre-implantation genetic diagnosis, but to date, few biopsied equine embryos have resulted in pregnancies. In the current experiment, 10 embryos ranging in diameter from 160-575 μm were biopsied. To obtain embryos, donor mares were monitored using transrectal ultrasonography. When a follicle >35 mm in diameter was observed, 2500 IU of hCG or 1.5 mg of deslorelin acetate was administered, and mares were inseminated daily until ovulation was detected. Embryos were recovered nonsurgically on Days 6.5-7 (Day 0 = ovulation). Trophoblast biopsies were collected in a 30-μL droplet of Syngro Holding Media (Bioniche, Belleville, Ontario, Canada) using a Piezo drill and beveled injection pipette (7-10 μm outer diameter). Biopsy samples ranged from 4-10 cells by visual assessment. After removal of the embryo, the droplet containing the biopsied cells was moved into an Eppendorf tube and centrifuged at 11 000g for 10 min. Supernatant was removed leaving 5 μL of sample, which was snap frozen for later genetic testing. Embryos were immediately transferred nonsurgically into the uteri of recipient mares synchronized to ovulate 0 to 2 days after the donor. Pregnancy examinations were performed via ultrasonography on Days 11, 12, 14, 16, 20, and 25. Pregnancies were confirmed on Day 16 for 6 of the 10 transfers. Five embryos (175-240 μm) had confirmed heartbeats on Day 25; one recipient mare lost her pregnancy (160 μm) between Days 16 and 20. The two largest embryos, 390 and 575 μm, did not result in pregnancies. As a preliminary experiment, 7 embryos (150-300 μm) were vitrified according to Eldridge-Panuska et al. (2005) after biopsying and later warmed and transferred directly. Three (all 200 μm) Day 16 pregnancies resulted from the transfer of vitrified, biopsied embryos. Two pregnancies were maintained to a heartbeat; one pregnancy resulted in the formation of an empty trophoblastic vesicle. All pregnancies were terminated on or after Day 25 to collect embryos for further genetic testing. For pre-implantation genetic testing, a duplex-nested PCR was developed for amplification of the DNA from the biopsied cells using primers for sex chromosome-linked zinc finger protein genes (ZFX/ZFY; 445 bp), and 2 pairs of primers for equine-specific sex-determining region on the Y-chromosome (SRY; 217 bp, 121 bp). Experiments on XX and XY genomic DNA from white blood cells revealed accurate genetic testing on as little as 9 pg of DNA, which equals 1 cell. Preliminary results revealed successful sex genotyping on biopsied material, and current experiments are underway to confirm and expand on these results.
161 MODULATION OF TELOMERASE ACTIVITY IN BOVINE EMBRYOS USING CYTOPLASMATIC PLASMID INJECTION
Authors: Garrels W. , Kues W. , Baulain U. , Niemann H.
Published: August 2010

Telomeres are repetitive, noncoding sequences at the ends of linear chromosomes that shorten with each cell division. They play an important role in aging and affect the regenerative capacity of cells. The holoenzyme telomerase rebuilds telomeres and is composed of 2 components, i.e. the catalytic protein component telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC). In mammals, telomerase is active during embryogenesis, in germ cells and a subset of stem and progenitor cells. In the present study, we set out to express the TERC component alone and then in combination with TERT, the human telomerase complex, in bovine embryos. The human telomerase components are highly homologous to bovine telomerase genes. Here, 3 different expression constructs encoding hTERC, hTERT, and a green fluorescent protein (GFP) reporter were co-injected into bovine zygotes cytoplasm, and three groups of 528, 1865, and 110 zygotes were constituted; hTERC/GFP (Group 1), hTERT/hTERC/GFP (Group 2), and GFP alone (Group 3), respectively. GFP fluorescence was used to identify successfully injected embryos. This method has recently been established in our laboratory. Injected and control embryos were cultured for 7 days to the blastocyst stage in vitro and the impact on early embryonic development and the physiological consequences of an ectopic overexpression of telomerase in early bovine embryos were assayed. We obtained 45 blastocysts with green fluorescence in the first, 192 in the second, and 28 in the third group. Embryos with GFP fluorescence were frozen for subsequent PCR analysis and telomerase activity measurement. Some blastocyts were analyzed using quantitative fluoresence in situ hybridization to monitor telomere length. Control groups were analyzed for the endogenous levels of TERC and TERT. Results indicate that endogenous TERC and TERT are up-regulated in morulae and blastocyts. In this study, we show that human TERC and TERT can be expressed in blastocysts by cytoplasmic plasmid injection in bovine zygotes. Statistical analyses were performed using the JMP 7.0.1 for Windows software (SAS Institute Inc., Cary, NC, USA). The Tukey-Kramer test was applied to compare the group means. The expression of hTERC alone resulted in a significant extension of telomere length of 280 telomere fluorescence units. Expression of both components also resulted in a significant extension of telomere length. In conclusion, TERC component alone is sufficient to elongate telomere length. The activity measurement showed that telomerase activity in the hTERT and hTERC injected group is 1.77 times higher than in the control group. Findings from this study will allow a comprehensive analysis of the functions of TERT and TERC in early embryogenesis. The ectopic expression of telomerase components in bovine embryos could pave new avenues for generating stem cells and for the development of novel regenerative therapies.

Funded by DFG.
166 COMPARISON OF TWO METHODS TO SYNCHRONIZE CROSSBRED BEEF HEIFERS FOR EMBRYO TRANSFER
Authors: Borges J. B. S. , Thedy D. , Mendes D. , Kawata R.
Published: August 2010

Estrous synchronization with prostaglandin in commercial MOET programs frequently results in insufficient numbers of recipients for embryo transfer. The aim of this trial was to compare the efficiency of 2 different methods of estrous synchronization in crossbred beef heifers. A total of 235 cycling heifers (Brangus and Braford), with a mean age of 24 months (range 22-26 months), body condition score of 3.5 ± 0.1 (1-5 scale), and mean weight of 340 ± 22 kg were assigned to 2 treatment groups stratified according to body weight and genotype. In Group 1 (n = 157), heifers received 2 injections of prostaglandin [Croniben, 150 μg of D(+) cloprostenol, i.m.; Biogenesis-Bágo, Buenos Aires, Argentina] with an interval of 14 days. After the second injection, detection of estrus was monitored for 5 days. In Group 2 (n = 78), heifers received an intravaginal device of progesterone (Day 0; 0.558 g; Cronipres Mono Dose M-24, Biogenesis-Bágo) and at the same time received an i.m. injection of estradiol benzoate (2 mg; Cronibest, Biogenesis-Bágo). On Day 7, devices were removed and heifers were injected with prostaglandin. Twenty-four hours later (Day 8), 1 mg of estradiol benzoate was injected i.m. All recipients had estrus detected for 72 h after the end of treatment to determine the estrous synchronization rate. Before embryo transfer 7 days after the detected estrus, heifers were examined by transrectal ultrasonography (Chison 600Vet, 5-MHz transducer, National Ultrasound, Duluth, GA, USA) to confirm the presence of a CL and its size or an unovulated follicle on the ovaries. Those with a CL of 1.7 cm2 or greater were selected to receive a frozen-thawed embryo. The frequencies of synchronous estrus, ovulation, unovulated follicles, and use of recipients were compared by chi-square test. Group 2 had a higher estrous synchronization rate (70%) and proportion of selected recipients (51.2%) compared with Group 1 (55% and 35.6%, respectively; P < 0.05). Ovulation rate (64% v. 72%) and incidence of unovulated follicles (20.6% v. 21.8%) for Groups 1 and 2, respectively, did not differ (P > 0.05). In summary, treatment with a 0.558-g progesterone intravaginal device, plus injections of estradiol benzoate and PGF, was more efficient than 2 PGF injections alone in synchronizing estrus and increased the use of crossbred beef heifer recipients in MOET programs. The ovulation rate and incidence of unovulated follicles were similar in both methods of estrous synchronization.
167 EVALUATION OF SUPEROVULATORY RESPONSE IN Santa Inês EWES BY ULTRASONOGRAPHY AND LAPAROSCOPY
Authors: Brandão F. Z. , Arashiro E. K. N. , Henry M. , Figueira L. M. , Souza J. M. G. , Viana J. H. M. , Fonseca J. F.
Published: August 2010

The development and use of non-invasive techniques would reduce risks of surgery sequels on the same animal and use of the donor could be optimized. The aim of this study was to evaluate the accuracy of ultrasonography performed prior to embryo collection to estimate superovulation response in sheep. Fifteen pluriparous Santa Inês sheep, 2 to 5 year old, with an average body weight of 46.79 ± 6.00 kg and body condition score of 2.96 ± 0.32 (1 to 5 scale) were allocated into 3 groups (GI, GII, GIII) in a cross-over design. In GI, intravaginal sponges (60 mg of medroxyprogesterone acetate; Progespon®, Schering Plough Animal Health, São Paulo, Brazil) were inserted (Day 0) and maintained for 14 days, and the superovulatory (SOV) protocol started on Day 12. In GII and GIII, intravaginal sponges were inserted (Day 0) and maintained for 6 days. On Day 5, the animals were treated with 300 IU of eCG (Novormon 5000®, Schering Plough Animal Health) and 5 mg of dinoprost (Lutalyse® Pfizer Animal Health, São Paulo, Brazil) i.m. Animals in GIII received 0.025 mg of gonadorelin acetate (Gestran-Plus®, Tecnopec, São Paulo, Brazil) i.m. 12 h after sponge withdrawal. In GII and GIII, the SOV protocol started 48 h after sponge removal and a new sponge was inserted immediately after its removal. The SOV protocol in all groups consisted of 200 mg of pFSH (Folltropin-V®, Tecnopec) administered in 6 decreasing doses given every 12 h (50/50, 30/30, and 20/20 mg). At the time of the fifth dose of pFSH, 5 mg of dinoprost was administered i.m. and the sponges were removed. Animals were bred by a sexually mature ram twice a day until the end of estrus. Prior to embryo collection, an ultrasonographic evaluation (5 MHz, Aloka SSD-500, Tokyo, Japan) was performed to determine the number of CL present on both ovaries. The number of CL was further evaluated by laparoscopy, when it was possible to detect poor responders or even non-ovulating animals. Statistical analysis was performed using all tests at the 95% confidence interval by SAEG program. Results are presented as mean ± SE. The number of CL was not different among all groups. The total number (GI, GII, and GIII) of CL determined by ultrasonography (9.09 ± 5.01) was not different (P > 0.05) from that observed by laparoscopy (8.87 ± 5.25). A significant correlation (r = 0.56, P < 0.0005) between evaluation performed by ultrasonography and laparoscopy was observed. These results suggest that ultrasonography can be used to determine the response to the superovulatory protocol. As embryo collection in sheep is performed mainly by surgical techniques, the implementation of non-invasive techniques such as ultrasonography could avoid unnecessary surgeries on animals that did not respond to the SOV protocol, therefore preventing early culling of embryo donors.

Financial support: Fopesq/Proppi and Faperj (E26/171.065/2006).